Acropat

FACULTY OF ME D IC N E UA ISSN 0045 - 3803 NLM (PubMed) Id: 0362256 Since 1967.
Gr Id: 28312 Input of the IMEMR (WHO) Since 1986.
www.medicaljournalofcairouniversity.com
Vol. 79, No. 1, September 2011
EDITORIAL BOARD
EDITOR IN CHIEF
LAMIS RAGAB
(Faculty Dean)
ASSISTANT EDITORS
MOHAMAD RIFAAT ADEL F. RAMZY NADER A. ABULATTA MOHAMED S. EL-TAMAWY
EDITING SECRETARY
SUZAN EL SHAFEI
ADVISORY BOARD
(Department Chairmen, Cairo University) ABDEL FATTAH MARIE (Pharmacology) AHMED ATEYA (Andrology) AHMED EISSA (Neurosurgery) AHMED EL-SEIFY (Clinical & Chem. Path.) AHMED SAMY SAEED (Radiology) ALY EL-HENDAWY (Pathology) AMAL EL-SAFTY (Industrial Med.) AMR SALAH EL-DIN (Ophthalmology) AYMAN EL-GARF (Rheumatology) DINA SHOKRY (Forensic Med.) EMAD HAMDY GHOZ (Psychiatry) FAWZAN SHALTOUT (Pediatrics) HASSAN EISSA (Physiology) HASSAN KHALED (Critical Med.) HODA RASHEED (Dermatology) HOSSAM BADRAWY (Obst. & Gynec.) HUSSEIN RIZK (Cardiology) IBRAHIM HEGAZY (Community Med.) IMAN SADEQ (Histology) KHALED EL-BOROLOSY (Anaesth Esiology) MAHASEN ABDEL RAHMAN (Tropical Med.) MAYSA SOBHY (Parasitology) MEDHAT ABDEL KHALEQ (Chest Med.) MOHAMED ABOUL EZZ (Cardiosurgery) MOHAMED KADDAH (Orthopedics) MOHAMED KHATTAB (General Med.) MOHAMED RIFAIE (Otorhinolaryngology) MONA SARKEES KARASS (Bio-Chemistry) MOSTAFA ABOU EL-NASR (Gen. Surgery) NABILA YOUSSEF (Anatomy) NAGWA EID (Family Med.) SALAH EL-MESIDY (Radio-Nucl. Med.) SOLIMAN EL-TAMAWY (Neurology) SOMAYA EISSA (Micro. & Immuno.)
III
PEER REVIEWERS
ABDALLAA EL-MUHAMED (Cardio., UK.) ABDEL MAGID EL-NAHASS (Med., UK.) ADEL LOTFY (Paed. Surg., Egypt) AHMED NASSEF (Gen. Surg., UK.) CHRISTOPHER CHEN (Obst. & Gyn., Singapore) ESSAM EL-SAHWY (Pediat. Surg., Egypt) HAMDY BADRAWY (Obst. & Gyn., Egypt) HANY LASHIN (Obst. & Gyn., UK) HAYDAR GHALEB (Pharm., Egypt) HAZEM EL-MEHERY (E.N.T., Egypt) HOSSAM KAMEL (Oncology, Egypt) HUSSEIN ABDEL DAYEM (Radio., USA) HUSSEIN KHALED (Oncology, Egypt) JOHN PAUL CHIGOT (Endocrine Surg., France) KAMAL IBRAHIM (Orthop. Surg., USA) KAMAL KHALIL (Cardiothorac. Surg., USA) KAMAL MANSOUR (Thoroc. Surg., USA) KAZOU SHIMIZU (Surg., Japan) KEICHI S. DOBSON (Psych., Canada) KOICHI TANAKA (Transplant., Japan) MAHER HALAWA (Orthop., UK.) MAHER RAMZY (Nephrology, Egypt) MAHMOUD EL-MENAWY (Obst. & Gyn., Egypt) MAKRAM MILAD (Pathology, Egypt) MOHAMED ABOU EL-GHAR (Obst. & Gyn., Egypt) MOHAMED EL-GUINDY (Gen. Surgery, Egypt) MOHAMED GAZAYERLI (Laparoscopic Surg., USA) MOHAMED GHONEIM (Urology, Egypt) MOHAMED HABIB (Dermat., Egypt) MONA SALEM (Pediat., Egypt) MOUNIR AGEEB (Gen. Surg., Egypt) MOUNIR HANNA (Paed. Urol., USA) NABIL FAHMY (Anaesth., USA) NADEY HAKIM (Transplant., UK.) NAGY HABIB (Hepat. Surg., UK.) PAULO MICCOLI (End. Surg., Italy) RAOUF SALLAM (Gen. Surg., Egypt) RASHAD BARSOUM (Nephrology, Egypt) ROCCO MARUOTTI (Gen. Surg., Italy) SHERIF MOKHTAR (Cardio., Egypt) YEHYA EL-RAKHAWY (Psychiatry, Egypt)
IV
NOTES TO CONTRIBUTORS
The Medical Journal of Cairo University welcomes original papers. Review articles, book reviews, abstracts from current literature and historical notes are also accepted. Particular priority is given to works presented to the Kasr El Aini clinical society by the staff of Kasr El Aini Faculty of Medicine and by its guess speakers. Papers are accepted on the clear understanding that the subject matter has not and will not be published in any other journal. Manuscripts should be typewritten in double spacing on one side of the paper only. The title of the paper should be written in block letters. Name of the author or authors should appear as first name, initials for second name followed by the surname (family name) and the initials of the highest university degree. Finally the name and address of the hospital or laboratory where the work was performed. An abstract should be written at the beginning of the article. Photographs and photomicrographs should be printed on glossy paper. They should be cellar and unmounted. Captions to the illustrations should be typed on a separate sheet. Drawings and diagrams should be done boldly in black ink. These should be numbered, the top should be indicated and the authors name, all written on the back. Long tables should be avoided and preferably replaced by graphs. No more than three tables, submitted in a form of photographs, are accepted. References in the text are to be arranged to the Vancouver style, i.e. references must be indentified by Arabic numerials between parentheses in order of their mention. The list of references is to be typed in numeric order as follows: For Journals, authors name, followed by initials, the title of the article, the name of the journal, the volume number in arabic numerals, the numbers of first and last pages of article followed by year of publication. For books, authors name, title, edition, publisher, town, page and year of publication. Reprints: Thirty reprints will be supplied. Editorial Address: Two manuscripts should be handed or sent to Dr. ADEL F. RAMZY, assistant editor, Clinical Society office. Manyal University Hospital, Cairo, Business communications, advertisements, subscriptions should be addressed to Dr. NADER A. ABULATTA, assistant editor. Exchange of periodicals and book reviews should be addressed to: Dr. SUSAN EL SHAFEI, editing secretary. Notice to subscribers: Annual subscription rate for individuals and institutions in Egypt L.E. 200 payable to: The Clinical Society Office Manyal University Hospital Cairo, Egypt. Annual subscription rate for all countries overseas is $ 400 payable to: The Medical Journal of Cairo University through The Bank of Egypt, Kasr El-Aini Branch. Cairo, Egypt.
Address for mail: The Medical Journal of Cairo University, the Clinical Society, El-Kasr El-Aini Hospital Telefax: (202) 236 55 768 www.medicaljournalofcairouniversity.com info@medicaljournalofcairouniversity.com
CONTENTS
Page
Topiramate for Prophylaxis of Basilar Migraine in Children and Adolescents, SAYED A. AMIN
and WALEED R. ABOU SEREE
355
Physical Inactivity among Adult Saudis Attending Primary Health Care Centers: Magnitude, Determinants and Barriers, OSSAMA A. MOSTAFA, FA TEN M.R. ISMAEIL and AHMED S.
KHASHABA
361
Response to Combined Pegylated Interferon and Ribavirin among Patients with Chronic Hepatitis C in Upper Egypt, SAAD ZAKY, MAGDA SHEHATA, HOWAIDA ESMAEL, NASR K. KHALIL, HANY
A. ALLAM and EKRAM M. ABD EL-KHALEK
369 377 383
Maternal Weight and the Success of Trial of Vaginal Delivery after Caesarean Section, WALEED
A.S. AHMED and AYMAN MORSY
Impact of Adjuvant Chemotherapy Delay on Outcome in Cancer Colon Patients, ZEINAB M. ABD
EL-HAFEEZ, AZZA M. ADEL and SHERIF ABD EL-WAHAB
Detection of Coronary Artery Stenoses with Thin Slice Multi Detector Row Spiral Computed Tomography Angiography, SHERIF MOHAMED MAHER, IHAB ISMAIL AL Y, NASHWA ABED
MOHAMED and MOHAMED ABD EL-FATTAH HASSAAN
389 407 415 421
Ocular Trauma Visual Outcomes during the 2011 Egyptian Revolution, HANY E. EL-MEKAWEY Effect of Treadmill Exercise on Intraocular Pressure in Normal Subjects, AZZA F. ISMAIL,
NESREEN G. EL-NAHAS and DALIA M. MOSAAD
Comparative Study of the Effect of Allopurinol and Nabumetone either Alone or Combined on Freunds Adjuvant-Induced Arthritis in Rats, SOHA S. ESSAWY Comparative Study between Three Different Protocols for Prevention of Hypertensive Disorders Associating Pregnancy, AHMED M. MAGED, GHADA ABD EL-FATTAH and ABD EL-RAHMAN
A. ABD EL-RAZEK
429
Impact of Successful Balloon Mitral Valvotomy on the Severity of Functional Tricuspid Regurgitation in Patients with Rheumatic Mitral Stenosis, MOHAMED A. ORABY and REDA
MAHFOUZ
435
Descriptive Study of Cases of Respiratory Distress in NICU in Ahmed Maher Teaching Hospital,
MOHAMMED HESHAM ZAAZOU, MAHMOUD M. KAMAL, RAGHDAA M. ALI, NAGI A. EL-HUSSIENY and MANAL EL-SAYED
441 449 457 465
Overexpression of C-KIT in Schistosomal Urinary Bladder Carcinoma, TAHANY SHAMS and

MOKHTAR METAWEA
The Value of Ovarian Reserve Tests in the Prediction of Poor Response t o Controlled Ovarian Hyperstimulation, IMAN A. KHALIL Expression of Apoptosis Related Proteins, P-Cadherin and Claudin-3 in Different Endometrial Lesions, AHLAM A. ABD EL-MAKSOUD and AHMED M. FAHMY Ultrastructural Study of Hepatic Changes after Human Umbilical Cord Blood Stem Cell Transplantation in Chronic Murine Schistosomiasis, HALA N. HOSNI, HODA A. YEHIA and
RANYA M. EL-SHEIKH
475 485
Impact of Intrauterine Growth Restriction on Neonatal Frontal Lobe Dimensions (Transcranial Sonographic Measurments), EMAN A. AHMED and NAFISA H.R. ABD EL-AZIZ Carotid Intima Media Thickness (CIMT) in Nondiabetic Patients with non Alcoholic Fatty Liver Disease (NAFLD), NOUMAN H. ALGAREM, MONA A. AMIN, MAY M. FAWZI and ASHRAF H.
AIAD
491
The Diagnostic Utility of Third Generation TSH Electro-Chemilumenescence Immunoassay in Detecting the Incidence of Thyroid Dysfunctions in Saudi Arabia, ABDULAZIZ A.A.
ALGHAITHY and ASMAA A. EL-REWENY
497
(B)
Page Comparison between Body Image and Self-Esteem Among Female Nursing Students in Three Different Arab Countries, NABILA TAHA, ESSMAT MANSOUR, AMAL SOBHY and ENTISAR M.
YOUNESS
509 519 525 529 533 537 541
Effect of Cinnamon Beverage on Some Biochemical Aspects of Diabetic Male Albino Rats,
MAGDA M. KHALIL, SHADIA A. FREIG and EMILY T. HANNA
Patterns of Seizures Among Children in Aseer Region, Saudi Arabia, MANSOUR Y. OTAIF, AYED
A. SHATI, ALI M. ALSUHEEL, MOHAMED A. HUNEIF and MOHAMED E. ELAWAD
Pattern of Infertility Among Couples in Gezira Area, Sudan, NEZAR M. ABDALLA Leishmaniasis in Western Sudan: Prevalence and Clinical Picture, AMANI A. OSMAN Profile of Idiopathic Pulmonary Arterial Hypertension in High Altitude Aseer Region, Saudi Arabia, ABDULLAH S. ASSIRI Sources of Stress as Perceived by Nursing Students at King Saud University, MOFIDA YOUNIS
AL-BARRAK, MONA TALAT EL-NADY and ELHAM ABDELKADER FAYAD
Med. J. Cairo Univ., Vol. 79, No. 1, September: 355-360, 2011 www.medicaljournalofcairouniversity.com
Topiramate for Prophylaxis of Basilar Migraine in Children and Adolescents

SAYED A. AMIN, M.D.* and WALEED R. ABOU SEREE, M.D.**
The Departments of Pediatrics* and Otorhinolaryngology**, Faculty of Medicine, Fayoum University
Abstract
Objective: The aim of this study is to assess the efficacy and safety of topiramate for prophylaxis of basilar type of migraine in children and adolescents. Methods: A prospective outpatient dose comparison study of patients with BM as defined by the International Classification of Headache disorder (second edition) where patients with 4 migraines/month were randomized to receive either 25mg or 75mg/day topiramate. Results: Fifteen children (5 boys, 10 girls) completed the study (8 in the 25-mg group and 7 in the 75-mg group). During the prospective baseline, the mean headache frequency of the combined group all migraines per month was 4.5/month (25mg) and 4.8/month (75mg). Average duration of migraine was 5.5 hours (25mg) and 5.0 hours (75mg) and average mean pain (5-point faces scale) was 3.3 for both (25mg 75mg). The reduction in median monthly migraine rate relative to baseline was 2.9 (64.4%) and 3.6 (75.0%) for the 25-mg and 75-mg topiramate-treated groups, respectively (p<.001). The reduction in median monthly BM rate relative to baseline was 2.5 (74.24%) and 2.3 (82.8%) for the 25-mg and 75 mg topiramate-treated groups, respectively. The overall reduction in BM attacks reduced from 2.84/month to 0.59/ month (79.2%; p<.0042). Overall, 86% of patients responded with a greater than 50% reduction in migraine frequency (100%, 25mg and 71%, 75mg). Mean reduction in migraine duration was 18 minutes (25mg) and 89 minutes (75mg). There was no significant difference in migraine severity between the 2 groups. There were no serious adverse events in both groups. Conclusions: Preventive therapy with topiramate resulted in reducing the overall migraine frequency and the frequency of attacks of BM at both 25mg and 75mg doses relative to the historical baseline and prospective baseline periods. The 2 treatment groups resulted in comparable outcomes. Key Words: Topiramate Migraine Prophylaxis Children. Correspondence to: Dr. Waleed R. Abou Seree, The Department of Otorhinolaryngology, Faculty of Medicine, Fayoum University.
Introduction IN the last 20 years the International Headache Society (IHS) has fostered the development of high quality research in headache including migraine. The 2004 (2nd) edition of the International Classification of Headache Disorders [1] provides a framework for headache research, including clinical trials which has become rather more child relevant. Migraine is in essence a familial episodic disorder whose key marker is headache with certain associated features. These features, according to HIS, include any two of the following; unilateral headache, moderate to severe, worsened with exercise and throbbing in association with one of the following; photophobia or phonophobia and nausea or vomiting. Approximately 30 million people in the United States suffer from migraines, with 18% of women and 6% of men experiencing at least 1 migraine headache annually [3]. Because of its significant impact on quality of life and interpatient variability in treatment response, migraine management remains challenging for many health care professionals. Migraine has a significant societal burden resulting in costly medical bills and lost productivity, averaging about 64 to 150 million work days lost each year [4]. Consequently, migraine headache has been classified by the World Health Organization as one of the 19 most disabling diseases worldwide [1]. Acute or abortive migraine management encompasses specific and nonspecific migraine therapeutics, including nonopioid and opioid analgesics, triptans, and ergotamines. Prophylactic migraine management data span the pharmacological spectrum from antiepileptic and antihypertensive agents to botulinum toxin type A [5]. Special considerations for migraine management also must be applied in various populations, including children, pregnant women, and the elderly [6].
355
356
Topiramate for Prophylaxis of Basilar Migraine in Children & Adolescents
The clinical course of migraine is especially difficult to predict in children. Migraine will come for weeks, months or a few years then remit for months or years, sometimes returning unpredictably later on. Long term follow-up is difficult and studies have demonstrated this variability [2]. Basilar-type migraine (BM) is a common clinical entity estimated to represent 3-19% of migraine in children and adolescents. The onset of BM tends to be in young children, with mean age being 7 years, although the clinical entity probably appears as early as 12-18 months as episodic pallor, clumsiness, benign paroxysmal vertigo and cyclical vomiting. Attacks are characterized by episodes of intense dizziness, vertigo, visual disturbances, ataxia, and diplopia. These early, transient features may last for several minutes, or up to 1 hour, and are then followed by the headache phase [7]. Material and Methods A prospective outpatient controlled study to assess the efficacy and safety of topiramate, a novel antiepileptic agent that has been established as effective for the prevention of migraine in adults and children, in the prophylaxis of basilar migraine in children and adolescents by comparing two doses, 25mg and 75mg/day. Fifteen patients aged less than 16 were collected among attendants of the pediatric and neurology outpatient clinics in the Fayoum University Hospital and other private clinics from October 2008 through June 2010. Selection of the patients was done according to the following criteria: - The age is less than 16 years. - The headache fulfills the criteria of migraine with or without aura with two or more of the following symptoms: Vertigo. Dysarthria. Diplopia or double vision. Tinnitus. Decreased hearing. Simultaneous bilateral parasthesia. Decreased level of consciousness. - Four attacks of migraines headache per month. - Exclusion of other associated causes of headache as chronic sinusitis, disturbed sleep disorders (due to central causes as other neurological diseases or obstructive causes as adenoid, deviated
septum, nasal, oral or laryngeal space occupying lesions) or general medical diseases. According to a prospective baseline headache characteristic period of 4 weeks, patients where divided into two groups; group (A) included 8 patients and received topiramate at 25mg/day, and group (B) included 7 patients who received topiramate at 75mg/day. These baseline headache characteristics were the following; average monthly migraines days, average severity of migraines, average duration of migraines in hours and proportion of headaches requiring abortive medicines. Collection of these data was achieved through the patient him or her self when possible or through the parents. After a period of regular intake of topiramate for 11-14 weeks (average 12 weeks) in both groups, the efficacy outcomes were compared between the two groups. These efficacy outcomes included the following: Reduction in frequency, severity, and duration of basilar symptoms. Reduction in migraine pain severity and duration. Reduction in migraine episode and headache episode frequency. Reduction in total headache days. Proportion of responders (i.e., the proportion of subjects who experience a 50% reduction in migraine days and migraine episodes). Reduction in the use of acute/abortive medications. Adverse events. A grading scale as a measurement of the headache disability was used as follows; grade I (non to mild), grade II (mild), grade III (moderate) and grade VI (severe). The proportion of each grade at the baseline was reported. Statistical analysis was conducted with SAS v. 9.1 (SAS Institute, Cary, NC, USA) using intentto-treat principles. A 2-sided level of significance, where =0.10, was set. Results Fifteen patients enrolled in this study were divided into 25mg group (A) and 75mg group (B). The baseline patient demographics is presented in Table (1). Before entry into the prospective baseline period, history of the headache was recorded and this is presented in Table (2).
Sayed A. Amin & Waleed R. Abou Seree

Table (1): Baseline demographics. Characteristic Age, years, Mean (SD) Gender: Male, n (%) Females, n (%) Weight (lb) 25mg/day, n=8 13.57 (1.72) 2 (25) 6 (75) 130 75mg/day, n=7 13.29 (2.93) 3 (42.9) 4 (57.1) 108 p-values t-test, p=.82 Fisher's exact test, p=.56 Wilcoxon rank-sum test, p=.13
357
Table (2): Headache history at screening. Headache history Migraine with aura, n (%) Migraine without aura, n (%) Age at migraine onset, mean (SD) Years with migraines, mean (SD) Average number of migraines per month, median Average headache duration, in minutes, median Headache pain severity over past 3 months, n (%): None (aura only) Mild Moderate Severe Excruciating Dizziness or vertigo: None (aura only) Mild Moderate Severe I- Little to no disability II- Mild disability III- Moderate disability IV- Severe disability 25mg/day, n=8 7 (88.5%) 3 (42.9) 10.1 (3.1) 3.4 (1.9) 8 240 0 (0) 0 (0) 2 (25) 3 (37.5) 3 (37.5) 0 (0) 0 (0) 3 (28.6) 5 (71.4) 0 (0) 3 (42.9) 3 (42.9) 2 (15.2) 75mg/day, n=7 7 (100%) 7 (100%) 5 (71.4) 8.3 (4.3) 5 (2.6) 5 240 0 (0) 0 (0) 1 (14.3) 5 (71.4) 1 (14.3) 1 (14.3) 0 (0) 1 (14.3) 5 (71.4) 0 (0) 3 (42.9) 2 (28.6) 2 (28.6) p-values Fisher's exact test, p=1.0 Fisher's exact test, p=.59 t-test, p=.38 t-test, p=.23 Wilcoxon rank-sum test, p=.124 Wilcoxon rank-sum test, p=.23
Fisher's exact test, p=.13
Fisher's exact test, p=1.0
Fisher's exact test, p=1.0
The mean age of onset of migraine was 8 years (25-mg group) and 10 years (75-mg group) and the average number of all migraine attacks per month (both migraine with aura, including BM, and migraine without aura) was 8 in the 25-mg group and 5 in the 75-mg group. The average duration of attacks was 4 hours and 12 of 15 patients described the pain as severe or excruciating. Nine of 15 rated the disability caused by their migraine as producing moderate-to-severe disability. There was no significant difference between the two groups at screening. The patients then entered a 4-week prospective baseline period to capture the headache frequency and severity for later comparison with the treatment phase (Table 3). The patients were permitted to use acute (abortive) medicines to treat attacks but were permitted no other preventive therapies. The average monthly migraine days were 4.5 (25mg) and 4.8 (75mg) and the average duration and severity were similar between the 2 groups. About
half of the 25mg group used abortive medicines and 75% of the 75mg group took acute treatments during the prospective baseline.
Table (3): Prospective baseline period. Prospective baseline 25mg, headache characteristics n=8 Average monthly headache days Average monthly migraine days Average severity of headache Average duration of migraine, in hours Average severity of migraine 4.52 4.52 3.37 (0.53) 5.48 (3.51) 3.37 (0.53) 75mg, n=7 6.76 4.83 p-value Wilcoxon ranksum test, p=.49 Wilcoxon ranksum test, p=1.0
t-test p=.77 3.26 (0.79) p=.76 5.02 (1.80) p=.91 3.4 (0.67)
Proportion of headaches 28/58 42/56 Fisher's exact requiring abortive (48.3%) (75%) test, p=.004 medicines (%)
358
Table (4) demonstrates the primary outcome for all migraines showing a reduction of headache frequency "overall" (groups combined) and in both the 25- and the 75-mg group. A reduction of 3.97 migraine days/month was observed from the prospective baseline to treatment maintenance phase (p<.001) without significant difference between the 2 groups (p=.86). The reduction in mean monthly migraine rate relative to baseline was 2.9 (64.4%) and 3.6 (75.0%) for the 25-mg and 75-mg topiramate-treated groups, respectively. For the target headache subtype, BM, the overall combined reduction of monthly attacks was 2.49 (p<.0042) with a reduction of 2.96/month in the 25-mg group and 1.93/month in the 75-mg group (Table 5). The reduction in mean monthly BM rate to baseline was 2.5 (74.24%) and 2.3 (82.8%) for the 25-mg and 75-mg topiramate-treated groups, respectively. Again, no significant difference was noted between the 2 groups, both showing reduction in headache frequency. For BM, average monthly migraine days reduced from 3.41 (25mg) and 2.8 (75mg) during
Table (5): Primary outcomes: Basilar-type migraines. Difference in Average Monthly Basilar Migraine Days During the Following Periods Prospective baseline Vs, maintenance, mean (SD) (min, max) Combined treatment group Overall 2.49 (2.55) (+2.40, 7.51) p<.0042 p-value
the prospective baseline to 0.88 (25mg) and 0.48 (75mg) during the treatment maintenance phase. Overall, the BMs reduced from 2.84/month to 0.59/ month during the treatment phase. All of the patients (8/8) in the 25mg group and 5 of 7 patients in the 75-mg group were categorized as responders, as defined by a >50% reduction in migraine days/month. Overall, 86% of patients responded with a greater than 50% reduction in migraine frequency. There were no serious adverse events reported. Numbness and tingling in the face and hands were the most common adverse events, but no patient withdrew from the study because of these symptoms. One patient in the 75-mg group reported learning difficulty but demonstrated no objective deterioration in school performance.
Table (4): Primary outcome: All migraines combined. Primary Outcome: All Baseline Maintenance Overall 4.67 1.56 25-mg group 4.52 1.56 75-mg group 4.82 1.22
Each treatment group 25mg/day, n=8 2.96 (2.46) (0, 7.51) 75mg/day, n=7 1.93 (2.75) (+2.40, 5.66) t-test, p=.83 p-value
Discussion The treatment of migraine headache in children and adolescents requires an individually tailored regimen of acute therapies (i.e., ibuprofen, acetaminophen, triptan agent), biobehavioral strategies (ie, sleep hygiene, regular exercise, biofeedback, stress management), and preventive agents. Daily preventive agents should be limited to those patients whose headaches occur with sufficient frequency or severity so as to warrant a daily treatment program. Most study designs require a minimum of three headaches per month to justify a daily agent. A clear sense of functional disability must be established before committing to a course of daily medication [8]. A diverse group of medications, including antihistamines, antidepressants, antihypertensives, and antiepileptics, are used to prevent attacks of migraine in children. Encouraging data are, how-
ever, emerging regarding the use of antiepileptic agents including disodium valproate, levetiracetam, and topiramate for the prevention of pediatric migraine. Migraine preventive medications, when given, should decrease the number, intensity, and duration of headaches, improve how patients respond to acute treatment, and improve the quality of life [9]. Topiramate is a novel antiepileptic agent that has been established as effective for the prevention of migraine in adult [10]. This study, specifically targeting patients with a migraine variant, observed that preventive therapy with topiramate was effective in reducing overall migraine frequency and also in reducing the frequency of attacks of BM at both 25-mg and 75mg doses relative to the prospective baseline period. Regardless of treatment dose (25mg Vs. 75mg), patients had significantly fewer monthly migraine
Sayed A. Amin & Waleed R. Abou Seree
359
days and experienced significant improvement in migraine-associated disability and quality of life. Overall, 86% of patients responded with a greater than 50% reduction in migraine frequency (100%, 25mg and 71%, 75mg). However, the duration and severity of individual migraine attacks were not significantly different (mean reduction in duration was 18 minutes [25mg] Vs. 89min. [75mg] and there were no other statistical differences noted between the 2 treatments groups. Given the disabling nature of BM, the design of this study incorporated a 1-month prospective baseline period following which the patients were divided into a high-dose (75mg) or low-dose (25 mg) comparison groups. This design chosen follows epilepsy models (high dose Vs. low dose). No placebo used in this study because of the vulnerable nature of the enrolled patients who suffering from disabling symptoms so the decision was made not to expose these patients to duration off treatment. Winner et al. [11] , conducted a multicenter, parallel-group, double-blind trial comparing topiramate with placebo in 162 children, aged 6 to 15 years. Patients were randomized in a 2:1 fashion, topiramate to placebo. A total of 54.6% of patients taking topiramate and 46.9% of patients taking a placebo experienced a 50% reduction of mean migraine days per month. No difference was found between groups. However, 32.4% of patients in the topiramate group experienced 75% reduction in migraine days, whereas only 14.3% of patients taking a placebo had the same effect (p=.02). Campistol et al. [12], in a 24-patient case series also found topiramate to be beneficial for migraine prophylaxis in children (aged 6-14 years) for whom other prophylaxis medications had failed. Topiramate was titrated up to 200mg daily or 6mg/kg/day. The mean monthly migraine episodes decreased from 3.6 at baseline to 2.9 at 2 month (p<.0001) and 2.7 at 4 months (p=.001), in addition, 90% of children experienced a shorter migraine than previously. Other trials [13-15] evaluated topiramate in older adolescents and adults and found beneficial results as well. Topiramate may be considered in pediatric patients who require migraine prophylaxis. The results of this study suggest that when choosing to begin a patient on topiramate for migraine prophylaxis, a low starting dose is used and a slow titration method (incremental dose
increases every 2-3 weeks) is employed using clinical symptomatology as the endpoint rather than a dose/kg target. Conclusion: This study found topiramate to be effective for decreasing migraine frequency, duration, and severity in children, as well as reducing the school days missed and improving quality of life in the child. References
1- Headache Classification Subcommittee of the International Headache Society. Cephalalgia. 2. Suppl 1. Vol. 24. The international classification of headache disorders; pp. 1160, 2004. 2- BILLE B.: 40-year follow-up of school children with migraine. Cephalalgia, 17: 488-491, 1997. 3- National Headache Foundation. NHF headache facts. 4- RAMADAN N.M., SILBERSTEIN S.D., FREITAG F.G., GILBERT T.T. and FRISHBERG B.M.: Evidence-based guidelines for migraine headache in the primary care setting: Pharmacological management for prevention of migraine. U.S. Headache Consortium. www.aan.com/ professionals/practice/pdfs/g10090.pdf. Accessed February 2008. 5- SILBERSTEIN S.D.: Migraine. Lancet, 363: 381-391, 2004. 6- KIMBERLY A. PESATURO, PHARM D., FAE G. WOODING and PHARM D.: Modern Management of the Migraine Headahe. Am. J. Lifestyle Med., 3 (2): 147159, 2009. 7- DONALD L. and ERIKA P.: Double-Blind, Dose Comparison Study of Topiramate for Prophylaxis of BasilarType Migraine in Children: A Pilot Study. Headache, 47 (10): 1409-1417, 2007. 8- DAMEN L., BRUIJN J., VERHAGEN A.P., BERGER M.Y., PASSCHIER J. and KOES B.W.: Prophylactic treatment of migraines in children (Part 2: A systematic review of pharmacological trials). Cephalgia, 26: 497504, 2006. 9- LEWIS D., ASHWAL S., HERSHEY A., HIRTZ D., YONKER M. and SILBERSTEIN S.: Practice parameter: Pharmacological treatment of migraine headache in children and adolescents. Neurology, 63: 2215-2224, 2004. 10- HERSHEY A., POWERS S., VOCKELL A., LECATES S. and KABBOUCHE M.: Effectiveness of topiramate in the prevention of childhood headaches. Headache, 42: 810-818, 2002. 11- WINNER P., PEARLMAN E., LINDER S., JORDAN D., FISHER A. and HULIHAN J.: Topiramate for migraine prevention in children: A randomized, double-blind, placebo-controlled trial. Headache, 45: 1304-1312, 2005. 12- CAMPISTOL J., CAMPOS J., CASAS C. and HERRANZ J.: Topiramate in the prophylactic treatment of migraine
360

in children. Journal of Child Neurology, 20: 251-253, 2005. D.: Topiramate in migraine prevention. Archives of Neurology, 61: 490-495, 2004. 15- SHAYGANNEDJAD V., JANGHORBANI M., GHORBANI A., ASHTARY F., ZAKIZADE N. and NASR V.: Comparison of the effect of topiramate and sodium valproate in migraine prevention: A randomized blinded crossover study. Headache, 46: 642-648, 2006.
13- BRANDES J.L., SAPER J.R., DIAMOND M., COUCH J.R., LEWIS D.W., SCHMITT J., et al.: Topiramate for migraine prevention. Journal of the American Medical Association, 291: 965-973, 2004. 14- SILBERSTEIN S., NETO W., SCHMITT J. and JACOBS
Physical Inactivity among Adult Saudis Attending Primary Health Care Centers: Magnitude, Determinants and Barriers
OSSAMA A. MOSTAFA, Dr.Ph.*; FATEN M.R. ISMAEIL, Dr.Ph.* and AHMED S. KHASHABA, M.D.**
The Departments of Public Health* and Physiology**, King Khalid College of Medicine* and Riyadh Colleges of Dentistry & Pharmacy**
Abstract
Aim of Study: To assess the grades and determinants of physical activity and barriers against it among adult Saudis. Subjects and Methods: A total of 400 adult Saudi were consecutively selected from attendants of primary health care (PHC) centers in Abha City. The validated Arabic version of the International Physical Activity Questionnaire-long form has been used to assess physical activity of participants. Results: Two thirds of participants had low activity (65%), one fourth performed moderate activity (25%), while only 10% of participants had high activity. More than two thirds correctly answered 8-10 questions (69.5%), while 16.5% answered correctly <8 questions and 14% answered all questions correctly. All participants reported having a variable number of barriers against performing physical activities, 28.3% had only one barrier, while 71.8% had two barriers or more. Significant characteristics related to high physical activity were being within the age group 30-44 years, with secondary/diploma levels of education, governmentally employed; and married with high monthly income. Significant characters related to increased number of barriers against physical activity were younger age group, having secondary/ diploma levels of education, unemployment and earning a low monthly income i.e., <5000 Saudi Riyals (SR). Conclusions: The prevalence of high physical activity among adult Saudis is quite low, especially among females. The main barriers against physical activity are the unavailability of physical activity centers, having no leisure time due to familial commitments, and having no money for practicing physical activity. Significant characters related to high physical activity are being within the age group 30-44 years, with secondary/diploma levels of education, governmentally employed, single with high monthly income (>10,000 SR). Significant characters related to increased number of barriers against physical activity are being young (19-29 years), having secondary/diploma levels of education, unemployment and earning low monthly income (<5000 SR). Recommendations: Physical activity equipment can be made available at institutions; to encourage different sports and physical activities among school children; to provide the necessary motivation and social encouragement to increase Correspondence to: Dr. Ossama A. Mostafa, The Department of Public Health, King Khalid College of Medicine.
peoples involvement in physical activity; allocating a reserve off-day during which everyone can play his own sports, or attend a gymnasium; advise people to choose a physical activity that they would feel comfortable to perform; and to encourage indoor physical activities. Key Words: Physical activity Epidemiology Magnitude Determinants Barriers.
Introduction THERE is a worldwide significant increase in the global burden of non-communicable diseases (NCD), which is closely related to corresponding changes in lifestyles mainly in tobacco use, physical inactivity and unhealthy diet [1]. The World Health Organization (WHO) Report [2] emphasized the major contribution of these risk factors, especially physical inactivity, to the overall global burden of NCD. The trend in global physical inactivity is alarming. Despite all the advantages of exercise, at least 60% of the global population are physically inactive [3]. During the past 25 years, rapid developments in standards of living in the Kingdom of Saudi Arabia (KSA) and increased mechanization have touched all aspects of peoples lives. As a result, great changes in physical activity and eating habits have occurred in our society and low levels of physical activity and sedentary living are becoming increasingly prevalent among the Saudi population [4-7]. The prevalence of physical inactivity in KSA is reported to be high [8,9]. This is coupled with the high prevalence of chronic diseases in the Kingdom compared with other parts in the world [9]. The proportion of Saudi adults who are at risk due to inactivity is much higher than those at risk due to any of the other CHD risk factors. Therefore, health promotion strategies aiming at decreasing
361
362
Physical Inactivity among Adult Saudis Attending Primary Health Care Centers
the proportion of inactive Saudi adults should be a priority public health concern [8]. Moreover, with progressively massive urbanization and increased reliance on computers and telecommunication technology, further reductions in physical activity are projected for the coming years. These lifestyle changes that are rapidly occurring in Saudi Arabia (as well as in the rest of the Gulf Cooperation Council countries) have a considerable impact on the health of society. In fact, such lifestyle transformation is thought to be responsible for the epidemic of non-communicable diseases, and their complications, in this part of the world [7,8]. The WHO [10] has recognized physical inactivity as a major threat to worldwide population health. WHO recommended some possible goals and priority actions aimed at promoting active living. Included in these actions is the need to assess the level of physical activity among various segments of the population. Thus, early identification and prevention of lifestyle-related disease is an important public health priority. A recent statement from the American Heart Associations Council on Cardiovascular Disease in the Young has recommended that primary health care (PHC) physicians should incorporate physical activity counseling into medical practice as a way of promoting physical activity [11]. This study aims to assess the grades of physical activity its determinants and barriers against it among adult Saudis. Material and Methods A total of 400 adult (i.e., aged 18 years) attendants of PHC centers in Abha City were included in the present study. Abha City is the capital of Aseer Region, which lies in the southwestern part of the Kingdom of Saudi Arabia. It has 6 PHC centers, which provide both preventive and curative services to a total of 94250 persons [12]. The validated Arabic version of the long form of the International Physical Activity Questionnaire (IPAQ) has been used. It was developed by the IPAQ organization to be well-developed instruments that can be used internationally to obtain comparable estimates of physical activity. The IPAQ assesses physical activity undertaken across a comprehensive set of domains including: Leisure time physical activity, domestic and gardening (yard) activities, work-related physical activity
and transport-related physical activity. The items in the long IPAQ form were structured to provide separate domain specific scores for walking, moderate-intensity and vigorous-intensity activity within each of the work, transportation, domestic chores and gardening (yard) and leisure-time domains [14]. Three levels of physical activity are proposed, as follows [14]: - Low physical activity: This is the lowest level of physical activity. Those individuals who do not meet criteria for categories 2 or 3 are considered physically inactive. - Moderate physical activity: Any one of the following 3 criteria: 3 or more days of vigorous activity of at least 20 minutes per day. 5 or more days of moderate-intensity activity or walking of at least 30 minutes per day. 5 or more days of any combination of walking, moderate-intensity or vigorous intensity activities achieving a minimum of at least 600 METmin/week. (One MET is defined as the energy cost of sitting quietly, and is equivalent to a caloric consumption of 1kcal/kg/hour). - High physical activity: Any one of the following 2 criteria: Vigorous-intensity activity on at least 3 days and accumulating at least 1500 MET-minutes/ week. 7 days of any combination of walking, moderateintensity or vigorous intensity activities achieving a minimum of at least 3000 MET-minutes/ week. Results Table (1) shows that among participants, men were slightly more than females (55.5% and 44.5%, respectively). More than half of participants aged 19-29 years (52%), while 22.3% aged 30-44 years and 25.8% of them aged 45+ years. Almost twothirds of participants had secondary or diploma level of education (63.8%), 17.5% had primary or intermediate level, 17% had university education, while a minority of them were just able to read and write (1.8%). About one fourth of participants were governmentally employed (25.2%), about one fifth were students (21.2%) or military personnel (21.8%), 14.5% worked in private sectors, 10.2% were free laborers, 5.8% were retired and 1.2 were unemployed. Almost half of participants
Ossama A. Most afa, et al.
363
were married 48.8%. Smokers constituted 13% of participants. The monthly income of almost one half of participants was less than 5000 SR (49.8%), while 43.5% had a monthly income of 5,000-10,000 SR and 6.8% had more than 10,000 SR monthly income. Table (2) shows that almost two thirds of participants had low activity (65%), one fourth performed moderate activity (25%), while only 10% of participants had high activity. More than two thirds of participants correctly answered 8-10 questions (69.5%), while 16.5% answered correctly less than 8 questions and 14% answered all questions correctly. All participants reported having a variable number of barriers against performing physical activities, 28.3% had only one barrier, while 71.8% had two barriers or more. Table (3) shows that the unavailability of physical activity centers was the most frequently stated barrier against practicing physical activity (51.8%), followed by having no free time due to their tight familial and social commitments (35.5%) and having no extra-money allocated for practicing physical activity (32%). Table (4) shows that low physical activity was significantly higher among females than men (71.9% Vs. 59.5%, respectively, p<0.001). Age was a significant variable related to grading of physical activity (p<0.001). The age group (30-44 years) had the highest proportions for high physical activity. Difference in distribution of physical activity according to age was statistically significant (p<0.001). Educational level was a significant variable related to grading of physical activity (p=0.007). All those who could only read and write were physically inactive. Occupation was a significant variable related to grading of physical activity (p<0.001). Those who were retired or unemployed had the highest proportion of physical inactivity (87% and 100%, respectively). Marital status was a significant variable related to grading of physical activity (p=0.041). Those who were married practiced less physical activity than single participants. Cigarette smokers were significantly more physically inactive than non-smokers (67.3% Vs. 64.7%). Moreover, none of the smokers had high physical activity. Differences in grades of physical activity according to smoking status were statistically significant (p=0.024). Monthly income was a significant variable related to grading of physical activity (p<0.001). Those who had a monthly income exceeding 10,000 SR had the highest proportion of high physical activity (22.2%), compared with participants with less monthly income.
Table (5) shows that females reported more barriers than males. However, difference was not statistically significant. Age was a significant variable related to number of barriers stated by participants (p<0.001). The age group 19-29 years had the highest proportion of participants who stated two or more barriers (88.5%). Educational level was a significant variable related to number of barriers stated by participants (p<0.001). Those who had secondary/diploma levels of education had the highest proportion of those who stated two or more barriers (82.0%). Occupation was a significant variable related to number of barriers stated by participants (p<0.001). All of those who had no work stated two or more barriers (100%). Married participants expressed significantly more barriers than single participants (p<0.001). Cigarette smoking was not a significant variables related to the number of barriers stated by participants. Monthly income was a significant variable related to number of barriers stated by participants (p= 0.005). Those who had a monthly income less than 5,000 SR had the highest proportion of those who stated two or more barriers (87.9%).
Table (1): Socio-demographic characteristics of primary care centers attendants. Socio-demographic characteristics Gender: Males Females Age in years: 19-29 30-44 45+ Educational level: Read and write Primary or intermediate Secondary or diploma University Occupation: Governmental Private sector Student Free labor Retired Military No work Marital status: Married Single Smoking status: Nonsmoker Smoker Monthly income in Saudi Riyals (SR): <5000 SR 5000-10000 SR >10000 SR No. 222 178 208 89 103 7 70 255 68 101 58 85 41 23 87 5 205 195 348 52 199 174 27 % 55.5 44.5 52.0 22.3 25.8 1.8 17.5 63.8 17.0 25.2 14.5 21.2 10.2 5.8 21.8 1.2 51.2 48.8 87.0 13.0 49.8 43.5 6.8
364
Table (3): Most frequently stated barriers against physical activity. Barriers Unavailability of physical activity centers Having no free time due to the tight familial and social commitments No extra-money to practice physical activity Doing other recreational activities other than physical activity Feeling no energy to practice physical activity Having poor general health No time due to studying hours No tools/equipment for practicing physical activity at home It is difficult to practice physical activity Fear of laughing or teasing by others No. 207 142 128 63 59 33 23 20 17 11 % 51.8 35.5 32.0 15.8 14.8 8.2 5.8 5.0 4.2 2.8
Table (2): Distribution grades of physical activity and barriers to physical activity among attendants. Variables Grades of physical activity: Low activity Moderate activity High activity Number of barriers against performing physical activity: No barrier One barrier Two barriers or more 0 113 287 0.0 28.3 71.8 260 100 40 65.0 25.0 10.0 No. %
Table (4): Distribution of grades of physical activity according to socio-demographic characteristics. Socio-demographic characteristics Gender: Males Females Age in years: 19-29 30-44 45+ Educational level: Read and write Primary/Intermediate Secondary/Diploma University Occupation: Governmental Private sector Student Free labor Retired Military No work Marital status: Married Single Cigarette smoking: Nonsmoker Smoker Monthly income (SR): <5000 5000-10000 >10000 Low activity No. 132 128 142 49 69 7 48 166 39 62 41 60 22 20 50 5 137 123 225 35 133 121 6 % 59.5 71.9 68.3 55.1 67.0 100 68.6 65.1 57.4 61.4 70.7 70.6 53.7 87.0 57.5 100 66.8 63.1 64.7 67.3 66.8 69.5 22.2 Moderate activity High activity p-value No. 56 44 56 16 28 0 19 55 26 18 14 20 16 3 29 0 55 45 83 17 53 32 15 % 25.2 24.7 26.9 18.0 27.2 0.0 27.1 21.6 38.2 17.8 24.1 23.5 39.0 13.0 33.3 0.0 26.8 23.1 23.9 32.7 26.6 18.4 55.6 No. 34 6 10 24 6 0 3 34 3 21 3 5 3 0 8 0 13 27 40 0 13 21 6 % 15.3 3.4 4.8 27.0 5.8 0.0 4.3 13.3 4.4 20.8 5.2 5.9 7.3 0.0 9.2 0.0 6.3 13.8 11.5 0.0 6.5 12.1 22.2
<0.001
<0.001
0.007
<0.001
0.041
0.024
<0.001

Table (5): Distribution of number of stated barriers according to socio-demographic characteristics. One barrier (n=113) No. Gender: Males Females Age in years: 19-29 30-44 45+ Educational level: Read and write Primary/Intermediate Secondary/Diploma University Occupation: Governmental Private sector Student Free labor Retired Military No work Marital status: Married Single Cigarette smoking: Smoker Non-smoker Monthly income (SR): <5000 5000-10000 >10000 67 46 24 36 53 4 35 46 28 43 23 12 16 10 9 0 24 89 % Tow or more barriers p (n=287) value No. %
365
Socio-demographic characteristics
30.2 155 69.8 25.8 132 74.2 11.5 184 88.5 40.4 53 59.6 51.5 50 48.6 57.1 50.0 18.0 41.2 42.6 39.7 14.1 39.0 43.5 10.3 0.0 3 35 209 40 58 35 73 25 13 78 5 42.9 50.0 82.0 58.8 57.4 60.3 85.9 61.0 56.5 89.7 100.0
These extraordinarily high prevalence rates for physical inactivity among Saudis can be explained by the rapidly occurring lifestyle changes in Saudi Arabia have their own considerable impacts on the health of the society. In fact, such lifestyle transformations are thought to be responsible for the epidemic of non-communicable diseases, and their complications in KSA [3]. In Saudi Arabia, Al-Nozha et al. [9] studied 17,395 Saudis aged 30-70 years. They reported that prevalence of inactivity was very high (96.1%). There were significantly more inactive females than males (98.1% Vs. 93.9%, respectively, p<0.001). The World Health Organization [2] stated that physical inactivity in the Saudi population seems to be among the highest in the world. It is this high prevalence of inactivity and the high calorie diet intake that has led to the epidemic of overweight and obesity, diabetes mellitus type 2, and coronary heart disease. Oldridge [12] estimated that the prevalence of physical inactivity among adults in different countries can range from 17% to 91%. The present study revealed a variable number of barriers against performing physical activities that were stated by participants. All participants expressed at least one barrier that would stop them from practicing physical activities. More than one fourth of participants had only one barrier, while almost three fourths of them had two barriers or more. The unavailability of physical activity centers was stated as the most frequent barrier against performing physical activity, followed by having no time due to familial and social commitments and having no money devoted for practicing physical activity. These findings are in agreement with those of Al-Quaiz and Tayel [3], who stated that in Riyadh, Saudi Arabia, the main barriers to adherence to physical activity were lack of resources, lack of willpower, lack of social support and lack of energy. Lack of resources was the most common barrier reported. This was not surprising as in KSA there is limited access, especially for females, to join sport clubs, jogging trails, swimming pools or exercise facilities at work. Time constraint was stated as the major contributing factor to inactivity among male Saudis by Al-Rafaee and Al-Hazzaa [5]. Prasad and Das [15] emphasized the negative role played by different barriers against physical activity. They stated that time seems to be the main barrier to an active life style. Physical activity needs to be a part of our everyday life like house-
0.339
<0.001
<0.001
<0.001
11.7 181 88.3 45.6 106 54.4
<0.001
9 17.3 43 82.7 104 29.9 244 70.1 24 80 9 12.1 175 87.9 46.0 94 54.0 33.3 18 66.7
0.060
0.005
Discussion The present study showed that prevalence of physical inactivity among the study sample is quite high. Almost two thirds of participants had low physical activity, mainly among females, while only 10% of participants claimed to have high activity. These findings are similar to those of Al-Rafaee and Al-Hazzaa [5], who reported that over 53% of Saudi males in Riyadh are physically inactive, and another 27.5% are irregularly active, while only 19% are physically active on a regular basis. Moreover, Al-Hazzaa [8] reported that prevalence of physical inactivity in KSA ranges from 43.3% to 99%. Al-Nozha et al. [9] reported that the prevalence rate of regular physical activity among adult Saudis is as low as 3.9%.
366
hold, occupational, commuting and recreational activity. They recommended restructuring worksites by parking farther away, utilizing steps, encouraging walking/activity breaks, etc. The present study showed that significant characteristics related to high prevalence of physical inactivity were female gender, participants within the older age group (above 45 years), read and write, unemployed; and married participants with low monthly income (<10,000 SR). Al-Rafaee and Al-Hazzaa [5] described a curvilinear relationship between age and physical inactivity, with the middle age group the least active. Physical activity was lower among those who were married, working in the private sectors, and less educated. Al-Nozha et al. [9] noted that prevalence of physical inactivity increases with increasing age category and decreases with increasing education levels. The present study revealed that significant characters related to increased number of barriers against physical activity were younger age group (19-29 years), having secondary/diploma levels of education, unemployment and earning a low monthly income (<5000 SR). These findings are in agreement with those of Alquaiz and Tayel [3], who noted that younger age and those with lower education (less than university level) face more barriers against physical activity. Participants whose monthly income is less than 10,000 SR believe that lack of resources is a barrier because it is expensive for them to join a club or to by equipment for exercise. Based on the results of this study, it has been concluded that prevalence of low physical activity among adult Saudis is quite high, especially among females. Several barriers against practicing physical activity are universally present in the Saudi community, most frequently are the unavailability of physical activity centers, having no leisure time due to familial and social commitments, and having no money allocated for practicing physical activity. Significant characteristics related to high physical activity are being within the age group 30-44 years, with secondary/diploma levels of education, governmentally employed participants, married with high monthly income (>10,000 SR). Significant characters related to increased number of barriers against physical activity are being young (19-29 years), having secondary/diploma levels of education, unemployment and earning low monthly income (<5000 SR).
To increase practice of physical activity, it is recommended that equipment be made available at institutions, so that affiliated staff can use during their breaks. In addition, different sports and physical activities must be encouraged among school children, so as to build up a healthy habit that would grow with them. It is also advised to provide the necessary motivation and social encouragement to increase peoples involvement in physical activity. Moreover, a reserve off-day should be allocated during which everyone can play his own sports, or attend a gymnasium. It is also advised that people should choose a physical activity that they would feel comfortable to perform and to encourage indoor physical activities. References
1- SALOMON J.A. and MURRAY C.J.L.: The epidemiological transition revisited: New compositional models for mortality by age, sex and cause. Geneva, World Health Organization 2000; (GPE Discussion Paper No. 11, revised edition). 2- World Health Report: Reducing Risks, Promoting Healthy Life. WHO, Geneva, 2002. 3- AL-QUAIZ A.M. and TAYEL S.A.: Barriers to a healthy lifestyle among patients attending primary care clinics at a university hospital in Riyadh. Ann. Saudi Med., 29 (1): 30-5, 2009. 4- AL-HELALI N.S., ABOLFOTOUH M.A. and GHANEM H.M.: Pattern of erectile dysfunction in Jeddah City. Saudi Medical Journal, 22: 34-8, 2001. 5- AL-RAFAEE S. and AL-HAZZAA H.M.: Physical activity profile of Saudi males: Implications for health. Saudi Medical Journal, 22: 784-9, 2001. 6- AL-HAZZAA H.M.: Physical activity, fitness and fatness among Saudi children and adolescents: Implications for cardiovascular health. Saudi Medical Journal, 23: 14450, 2002. 7- AL-RUKBAN M.O.: Obesity among Saudi male adolescents in Riyadh, Saudi Arabia. Saudi Medical Journal, 24: 27-33, 2003. 8- AL-HAZZAA H.M.: Prevalence of physical inactivity in Saudi Arabia: A brief review. Eastern Mediterranean Health Journal, 10 (4-5): 663-70, 2004. 9- AL-NOZHA M.M., AL-HAZZAA H.M., ARAFAH M.R., AL-KHADRA A., AL-MAZROU Y.Y., AL-MAATOUQ M.A., KHAN N.B., AL-MARZOUKI K., AL-HARTHI S.S., ABDULLAH M. and AL-SHAHID M.S.: Prevalence of physical activity and inactivity among Saudis aged 3070 years. A population-based cross-sectional study. Saudi Med. J., 28 (4): 559-68, 2007. 10- World Health Organization (WHO): Health and Development Through Physical Activity and Sport Non-communicable Diseases And Mental Health. Non-communicable Disease Prevention and Health Promotion. The WHO Document Production Services, Geneva, Switzerland, 2003.

11- WILLIAMS C.L., HAYMAN L.L., DANIELS S.R., ROBINSON T.N., STEINBERGER J., PARIDON S., et al.: Cardiovascular health in childhood. A statement for health professionals from the committee on atherosclerosis, hypertension, and obesity in the young (AHOY) of the council on cardiovascular disease in the young, American Heart Association. Circulation, 106: 143-60, 2002. 12- Highlights Demographic Survey 1428 H.: Population by age groups, nationality (Saudi/Non-Saudi), and sex in Aseer Area. Kingdom of Saudi Arabia, Ministry of Economics and Planning. General Statistics and Information Administration, Table (8), p. 68, 2007.
367
13- OLDRIDGE N.B.: Economic burden of physical inactivity: Healthcare costs associated with cardiovascular disease. Eur. J. Cardiovasc. Prev. Rehabil, 15: 130-9, 2008. 14- CRAIG C.L., MARSHALL A.L., SJSTRM M., BAUMAN A.E., BOOTH M.L., AINSWORTH B.E., PRATT M., EKELUND U., YNGVE A., SALLIS J.F. and OJA P.: International physical activity questionnaire: 12-country reliability and validity. Med. Sci. Sports Exerc., 35 (8): 1381-95, 2003. 15- PRASAD D.S. and DAS B.C.: Physical inactivity: A cardiovascular risk factor. Indian J. Med. Sci., 63: 33-42, 2009.
Response to Combined Pegylated Interferon and Ribavirin among Patients with Chronic Hepatitis C in Upper Egypt
SAAD ZAKY, M.D.*; MAGDA SHEHATA, M.D.*; HOWAIDA ESMAEL, M.D.*; NASR K. KHALIL, M.D.**; HANY A. ALLAM, M.D.** and EKRAM M. ABD EL-KHALEK, M.D.***
The Departments of Tropical Medicine & Gastroenterology*, Pathology**; Public Health and Community Medicine***, Faculty of Medicine, Assiut University, Assiut**, Fever and El-Eyman Hospitals-M.O.H.-Assiut-Ministry of Health
Abstract
Background and Aim of the Study: Egypt has one of the highest prevalence of hepatitis C in the world. Combination therapy using pegylated interferon alfa 2a or alfa 2b plus ribavirin is now the established standard therapy for chronic hepatitis C. However, non-response to this therapy remains common and is associated with several factors. We aimed to assess the response rate and the factors affecting the response to pegylated interferon and ribavirin among patients with chronic hepatitis C in Upper Egypt. Patients and Methods: A clinical trial study was conducted in the center for treatment of chronic hepatitis C in Assiut Governorate included 400 patients with chronic HCV infection fulfilling the inclusion and exclusion criteria for treatment with pegylated interferon (PEG-INF) plus ribavirin. All the included patients were subjected to detailed medical history and complete clinical examination, detailed abdominal ultrasonogaphic examination, laboratory investigations including complete blood picture, complete liver function tests, blood sugar, serum creatinine, prothrombin time and INR, FP, HBsAg, ANA, T3, T4, TSH, quantitative PCR for HCV-RNA, fundus examination, ECG and liver biopsy with grading and staging with METAVIR Score. Results: The responses to PEG-INF plus ribavirin at 12, 24, 48 weeks and SVR were 292/400 (73.0%), 289/400 (72.2%) and 257/400 (64.2%) and 96/233 (41.2%), respectively. The high pretreatment viral load is associated with poor response at 12 and 48 weeks of therapy and sustained virological response (SVR). The high pretreatment weight and body mass index (BMI) were associated with poor response at 12 weeks while high pretreatment AST and lower WBC count were associated with poor response at 24 weeks of therapy. Early virological response (at 12 weeks of therapy) can significantly predict the response at 24 and 48 weeks and SVR (p=0.000, p=0.03 and p=0.000, respectively). Conclusion: This study concluded that, SVR after combined PEG-INF plus ribavirin for patients with chronic HCV infection in Upper Egypt was 41.2%. The pretreatment viral load was the only significant predictor for SVR among those Correspondence to: Dr. Saad Zaky, The Department of Tropical Medicine and Gastroenterology, Faculty of Medicine, Assiut University, Assiut.
patients. Early virological response significantly predicted the response at 24, 48 weeks of therapy and SVR. Key Words: HCV PEG INF SVR Predictors of response.
Introduction HEPATITIS C virus (HCV) is considered as a global health problem with approximately 3% of the world population, roughly 170-200 million people are infected [1-3]. Approximately 70-80% of them develop chronic hepatitis, 20-30% of them develop liver cirrhosis [4]. In Egypt, the situation is quite worse. Egypt has one of the highest prevalence of hepatitis C in the world. According to government figures, about 10-15% of the Egyptians (8-10 millions people) are carrying hepatitis C antibodies, meaning that they either have or at one time had the virus. About 90% of them are genotype 4 [5]. Combination therapy using pegylated interferon alfa 2a or alfa 2b plus ribavirin is now the established standard therapy for chronic hepatitis C [6-9] . However, non-response to this therapy remains common and is associated with several factors [10]. Because of these therapies have important side effects and high cost, it is important to identify whom patients have the best chance to respond to this therapy [11]. Accordingly, the ability to accurately predict the response of patients to antiviral therapy is of great interest. In general, predictors of response may be clinical, biochemical or histological [3]. There are several patterns of response to therapy; non responders: Patients who are virologically non responders showing detectable HCV RNA levels throughout the treatment period, null responders: Patients who achieved less than 2 logs reduction of the baseline HCV RNA at weeks 12 of treatment, partial responders: Patients who achieved more than 2 logs reduction of HCV RNA at 12 weeks but detectable at 24 weeks, responders
369
370
Response to Combined Pegylated Interferon & Ribavirin among Patients
(R): Those patients with negative HCV RNA at the end of therapy (after 48 weeks), sustained virological response (SVR): Those who have undetectable HCV RNA 6 months after discontinuation of therapy, relapse: Patients who have negative PCR for HCV RNA at the end of treatment and became positive at 6 month after discontinuation of therapy and breakthrough: Undetectable HCV RNA at some point of treatment but became detectable while on treatment [9,12]. There is great controversy regarding the response rate and the predictors of response to pegylated interferon and ribavirin among patients with genotype 4. In addition, there is insufficient data about the response to therapy in Upper Egypt. Aim of the study: The present study aims to assess the response rate and the factors affecting the response to antiviral combination therapy with pegylated interferon and ribavirin among patients with chronic hepatitis C in Upper Egypt. Patients and Methods A clinical trial study was conducted in the period from November 2007 to May 2010, in the center for treatment of chronic hepatitis C in Assiut Governorate which is sponsored by the National project for treatment of hepatitis C, Ministry of Health. Out of 825 patients with chronic HCV infection attending the center during the period of the study, 400 patients were selected randomly fulfilling the inclusion and exclusion criteria for treatment with pegylated interferon plus ribavirin. The drop out was due to several reasons; stop of treatment without cause, stop because of side effects, shift of the patient file to another center, lack of the PCR results at one or more stages during the treatment course. Inclusion criteria: Patients aged between 18 and 60 years, both sex, with positive PCR for HCV RNA, persistently elevated alanine aminotransferase (ALT) levels above the normal limit, liver biopsy consistent with chronic hepatitis C, negative HbsAg and antinuclear antibody (ANA), normal thyroid function tests, normal alfa fetoprotein (FP), normal blood glucose, normal ECG and normal fundus examination were included in this study. Exclusion criteria: The following patients were excluded from the study: Women who were pregnant or breast-feeders, those with concomitant infectious or metabolic liver disease, alcohol consumption more than 50g/day within the last year,
any cause of liver disease other than HCV, decompensated liver disease or presence of hepatocellular carcinoma, any immunologically mediated diseases (systemic lupus erythromatosis and rheumatoid arthritis), poorly controlled diabetes mellitus, retinal abnormalities, neutrophil count <1500/mm 3 or platelet count <100 000/mm3, serum creatinin >1.5 times the upper normal limit, and medical history of psychiatric disease or uncontrolled depression at screening. Patients who stopped treatment during the study without reason or due to development of side effects were also excluded. Ethical consideration: Patient participation was voluntary and informed written consent was obtained from all patients. The study proposal was approved by the Ethical and Technical Review Committee. Formal administrative approvals were taken before the start of the work. All the included patients were subjected to the followings: 1- Detailed medical history taking and complete clinical examination. 2- Recording of the body weight, height and estimation of body mass index (BMI) according to WHO guidelines (1995) [13]. 3- Detailed abdominal ultrasonogaphic examination. 4- Laboratory investigations (complete blood picture, complete liver function tests, blood sugar, serum creatinine, prothrombin time and INR, FP, HBsAg, ANA, T3, T4 and TSH). 5- Quantitative PCR for HCV RNA. 6- Fundus examination. 7- ECG examination. 8- Liver biopsy and histopathological examination and scoring using METAVIR score [14,15]. Different personal, biochemical, virological and histopathological variables are evaluated as factors affecting the response to therapy. Enrolled patients were treated with pegylated interferon and ribavirin for 48 weeks. Pegylated INF-2a (180mcg) (Pegasys; Roche, Switzeland) or INF-2b (1.5mcg/kg) (PEG-Intron; ScheringPlough) subcutaneously weekly, plus ribavirin at a daily dose of 1000mg (if their body weight is 75kg or less) or 1200mg (if their weight exceeds 75kg) [9] divided into two oral doses. Treatment was discontinued or reduced in case of intolerance to the drugs due to development of severe side effects. Clinical and biochemical parameters were evaluated at the start of therapy, monthly during therapy and 6 months after the end of the treatment. Serum levels of HCV RNA were assayed at the start of therapy, after 12, 24, 48 weeks and 6 months after discontinuation of therapy. Patients who are still viremic (not achieved 2 logs reduction) after 3 months of treatment, therapy was discontinued and they were considered non-responders (NR).
Saad Zaky, et al.
371
Early virological responders (EVR) are those who have undetectable serum HCV RNA or a minimum of 2 logs decrease of the baseline level at week 12. Responders (R) are those patients with negative PCR for HCV RNA at the end of therapy. SVR: Those who have undetectable HCV RNA 6 months after discontinuation of therapy. Relapsed patients: Those who have negative PCR for HCV RNA at the end of treatment and became positive 6 month after discontinuation of therapy. Breakthrough: Patients with undetectable HCV RNA at some point of treatment but became detectable while on treatment [9,12]. Liver biopsy: Ultrasound guided liver biopsies were obtained from all patients prior to therapy; all samples fixed in formalin and embedded in paraffin were stained with hematoxylin and eosin. Hepatic necroinflammation (activity) and fibrosis (staging) were assessed by the METAVIR scoring system. Fibrosis was staged on a scale of F0-F4 [14,15]. Liver steatosis was graded in accordance with Brunt et al. (1999) [16]. Statistical analysis: Data were collected and analyzed by using computer program SPSS (version 16). The frequencies, percentages, the mean and standard deviation were computed. To compare results between groups, Student's t-test was used for a Gaussian distribution; otherwise, the Mann-Whitney U-test was applied. An ANOVA was used to compare data with a Gaussian distribution from more than 2 groups. The chi square (2) test was used as a test of significant. p value <0.05 was considered to be statistically significant. Results The baseline characteristics of 400 patients including personal, biochemical, virological and histopathological are shown in Tables (1&2). The mean age of the studied sample was 41.8 yeas. Male respondents represented as 89.5%. The mean of BMI was 27.4. The responses to PEG-INF plus ribavirin at 12, 24, 48 weeks and SVR were 292/400 (73.0%), 289/400 (72.2%) and 257/400 (64.2%) and 96/233 (41.2%), respectively (Fig. 1). The results of response at 6 month after discontinuation of therapy of 167 patients were not available, so the SVR was calculated from the results of 233 patients only. The high pretreatment viral load is associated with poor response at 12, 48 weeks of therapy and SVR. The over weight and BMI were associated with poor response at 12 weeks while
high pretreatment AST and lower WBC count were associated with poor response at 24 weeks of therapy (Tables 3&4). Following the response to therapy, 50 patients (12.5%) developed breakthrough at 24 or 48 weeks of therapy (reappearance of HCV-RNA in the serum at 24 or 48 weeks of therapy after being absent at 12 weeks). Early virological response (at 12 weeks of therapy) can significantly predict the response at 24 and 48 weeks and SVR (p=0.000, p=0.03 and p=0.000, respectively). Out of 292 patients with negative PCR for HCV-RNA at 12 weeks, 271 patients (92.8%) had also negative PCR for HCV-RNA at 24 weeks. Similarly out of 272 patients showing negative PCR for HCV-RNA at 12 weeks, 244 (90.1%) also had negative PCR for HCV-RNA at 48 weeks. Out of 134 patients with negative PCR for HCV-RNA at 12 weeks, 90 patients (67.2%) developed SVR (Table 5).
% 80 70 60 50 40 30 20 10 0 12 weeks 24 weeks 48 weeks SVR
41.2 73 72.2 64.2
Response Fig. (1): Response to combined pegylated interferon/ribavirin among patients with chronic HCV, Assiut HCV Center, 2010. Table (1): Personal characteristics of patients with chronic HCV, Assiut HCV Center, 2010. Variable Age: (Mean SD in years) Gender: Male Female Weight: (Mean SD in Kg) Height: (Mean SD in cm) BMI: Mean SD Normal Overweight Obese 122 168 110 358 42 75.712.7 166.25.7 27.44.4 30.5 42.0 27.5 358 10.5 No. = 400 41.89.1 %
372

Table (2): Laboratory characteristics of patients with chronic HCV, Assiut HCV Center, 2010. Variable AST: Mean SD Normal (12 U/L and less) Abnormal (>12 U/L) ALT: Mean SD Normal (12 U/L and less) Abnormal (>12 U/L) WBC: (Mean SD x13/cc) Hb: (Mean SD in gm/dl) Platelets: Mean SD x13/cc) HCV-RNA: Mean SD Liver biopsy Steatosis grade: 0 1 2 Fibrosis stage: 0 1 2 3 Inflammation grade: 1 2 3 No. = 400 14.611.2 214 186 14.311.9 239 161 6.02.0 14.31.5 210.851.1 557517.1617303.9 59.8 40.2 53.5 46.5 %
318 67 15 23 194 126 57 190 149 61
79.5 16.8 3.8 5.8 48.5 31.5 14.3 47.5 37.3 15.3
Table (3): Relation between personal characteristics and the response to combined pegylated interferon/ribavirin among patients with chronic HCV, Assiut HCV Center, 2010. At 12 weeks Responders 292 (73.0%) No. Age: Mean SD Gender Male Female Weight: Mean SD Height: Mean SD BMI: Mean SD Normal Overweight Obese
Unpaired t-test.
Variable
At 24 weeks Responders 289 (72.2%) No. 41.69.3 %
At 48 weeks Responders 257 (64.2%) No. 41.79.3 %
SVR Yes 96 (41.2%) No. 41.98.9 %
% 41.6 9.3
264 28
90.4 9.6
259 30
89.6 10.4
228 30
88.4 11.6
87 9
90.6 9.4 75.613.5 165.77.0 26.64.1
72.612.5* 166.45.6 26.44.3* 84 122 86 28.8 41.8 29.5 81 119 89
76.913.0 166.25.8 26.73.3 28.0 41.2 30.8 74
76.913.1 166.15.9 27.84.6 28.7 41.5 29.8 32 39 25 107 77
33.3 40.6 26.0
* Significant.
Saad Zaky, et al.
373
Table (4): Relation between pre-treatment laboratory data and the response to combined pegylated interferon/ribavirin among patients with chronic HCV, Assiut HCV Center, 2010. At 12 weeks Responders 292 (73.0%) No. % 14.510.7 156 53.4 136 46.6 14.712.4 167 57.2 125 42.8 6.02.1 14.21.5 209.551.2 518760.6593182.8* No. 16 146 92 38 147 99 46 232 60 51 9
Unpaired t-test
Variable
At 24 weeks Responders 289 (72.2%) No. % 14.310.5* 157 54.3 132 45.7 14.611.9 161 55.7 128 44.3 6.12.1* 14.21.5 209.649 539111.6617498.3 No. 20 144 88 37 144 103 42 229 60 52 8 % 6.9 49.8 30.4 12.8 49.8 35.6 14.5 79.2 20.8 86.7 13.3
At 48 weeks Responders 257 (64.2%) No. % 14.19.8 54.3 45.7
SVR Yes 96 (41.2%) No. % 14.810.8 49 47 14.511.7 48 48 6.62.7 14.41.6 213.549.9 495428.9544021.7* No. 8 49 25 14 46 36 14 74 22 18 4 % 8.3 51.3 26.0 14.6 47.9 37.5 14.6 77.1 22.9 81.8 18.2 50.0 50.0 51.0 49.0
AST: Mean SD Normal Abnormal ALT: Mean SD Normal Abnormal WBC: Mean SD Hb: Mean SD Plt: Mean SD HCV-RNA: Mean SDo Liver biopsy Fibrosis stage: 0 1 2 3 Inflammation grade: 1 2 3 Steatosis: No (no=318) Yes (no=82) Steatosis grade: 1 (n=67) 2 (n=15)
Chi-square test
140 118
14.210.9 56.6 146 43.4 112 6.02.1 14.21.5 211.551.6 502652.5568632.8* No. 19 129 79 31 133 86 39 205 53 48 5 % 50.0 30.6 12.0 51.6 33.3 15.2 79.5 20.5 90.6 9.4
% 5.5 50.0 31.5 13.0 50.3 33.9 15.7 79.5 20.5 85.0 15.0
o Mann-Whitney test
Table (5): Relation between the early treatment response and the response at 24, 48 weeks and SVR, Assiut HCV Center, 2010. Variable At 12 weeks r NR p-value
Discussion Early studies reported that, patients infected with HCV genotype 4 responded poorly to conventional interferon alfa and to combined regular interferon alfa plus ribavirin therapy [17-20]. More recently, few trials on the treatment of HCV genotype 4 using PEG-INF alfa plus ribavirin showed highly variabl SVR rates ranging between 33% and 69% [21-30] . There are major controversies among different studies regarding the factors affecting the response to combined PEG-INF plus ribavirin. In the present study, 400 patients are evaluated for their response to pegylated interferon plus ribavirin. Their response rates were 73.0%, 72.2%, 64.2% and 41.2% at 12, 24, 48 weeks of therapy and SVR respectively. The low SVR in this study may be attributed to lack of the results of PCR for HCV RNA at 6 months after discontin-
At 24 weeks of treatment: r (no=289) 271 (92.8%) 18 (66.7%) 0.000* NR (n =30) 21 (7.2%) 9 (33.3%) Total 292 27 At the end of treatment: r (no=257) 244 (90.1%) 13 (72.2%) 0.03* NR (no=32) 27 (9.9%) 5 (27.8%) Total 271 18 SVR: Yes (no=96) No (no=137) Total
R: Responders.
90 (67.2%) 44 (32.8%) 134

NR: Non responders.
6 (6.1%) 0.000* 93 (93.9%) 99
374
uation of therapy (167 patients out of 400) and possible irregular use of ribavirin (some of the patients reduced the dose by their self or stopped treatment for some days due to financial causes). Several factors were identified as causes of this drop out, shift of the patient file to another center, lack of financial support, lack of interest, frustration following positive results. In the present study, the factors associated with poor virological response at variable stages of treatment course were high pretreatment viral load, weight, BMI, AST and lower pretreatment WBC count. No impact for age, gender, hieght, ALT, ALP, Hb level, platelet count, grading, staging and steatosis in liver biopsy on the response to therapy at any stage during the treatment course. Bressler et al., (2003) reported that, high body mass index but not steatosis was an independent risk factor for response to antiviral treatment [31] . A high BMI was also inversely correlated with sustained virological response to therapy [32]. El Makhzangy et al., (2009) concluded that, end treatment response to combined PEGINF plus ribavirin and SVR were 69.5% and 61.1% respectively which is nearly comparable with end treatment response in our study but higher SVR. However, they identified different predictor factors for SVR including steatosis and fibrosis and no impact of the age, gender, weight, BMI, pretreatment ALT and viral load on the SVR [30]. Al Ashgar et al., (2009) reported response to combined PEG INF and rebavirin at 12, 48 and 72 weeks of 92.3%, 63.8% and 50.7%, respectively. They added that, the independent predictors of SVR were younger age, lower serum AST and being treatment nave [33]. Similarly, researchers performed multivariate analysis of baseline factors and have identified lower baseline viral level as an independent predictor of sustained virological response regardless of genotype [32,34]. Cross et al., (2010) showed that, infection with genotype 1 or 4 and pretreatment weight were the only variables associated independently with sustained virological response [35]. In contrast to our results, Fried et al., (2002) found a significant negative correlation between male gender and sustained virological response on univariate analyses [8] while Shiffman et al., (2007) reported that younger age correlated significantly with a SVR [36]. Assessment of response after 12 weeks of treatment are used for many years and defined as early virologic response, viral load decline 2 logs or undetectable HCV-RNA at week 12, and is used to be the mainstay of HCV on treatment decision making. According to Fried et al, up to 65% of patients responding at week 12, go on to develop a SVR which is similar to our result (67.2%). Conversely, if an early virologic
response is not present, treatment should be stopped, because the chance of developing a sustained response of HCV eradication is less than 3% [8]. A study by Gad and his colleagues in 2008 about predictors of a sustained virological response in patients with genotype 4 chronic hepatitis C concluded that severe firosis, severe steatosis, treatment with standard interferon and a high serum alpha fetoprotein level were all negatively associated with sustained virological response [37]. Breakthrough was reported among 12.5% of the patients of this study. Similarly, Hoofnagle and Seeff et al., (2006) reported 10% breakthrough [9]. This major controversy among studies evaluating the predictors of response to combined PEGINF plus ribavirin stimulated the search for another underlying causes of the variable response in different geographic and sometimes in the same geographic areas. Several studies evaluated the underlying genetic predisposition for response or resistance to interferon and identified that 3 genes encoding IL29, IL28A, and IL28B [38-40], have been implicated in the response to PEG-IFN/RBV among patients infected with HCV genotype 1. The important genetic findings related to treatment response in patients with chronic hepatitis C will suggest the possibility of personalized medicine of treatment of this disease. Conclusion: This study concluded that SVR after combined PEG-INF plus ribavirin in Upper Egypt was 41.2% for patients with chronic HCV infection. The pretreatment viral load was the only significant predictor for SVR among those patients. Early virological response significantly predicted the response at 24, 48 weeks of therapy and SVR. References
1- World Health Organization: Hepatitis C: Global prevalence. Wkly Epidemiol. Rec. (72): 341-344, 1997. 2- LAUER G.M. and WALKER B.D.: Hepatitis C virus infection. N. Engl. J. Med., 345: 41-52, 2001. 3- SAMUEL S.L. and AYMAN A.A.: Predicting antiviral treatment response in chronic hepatitis C: How accurate and how soon? Journal of Antimicrobial Chemotherapy; 51: 487-491, 2003. 4- SVITLANA G. and ANNA G.: Biochemical markers of fibrosis in patients with hepatitis C1. Journal of Iinterferon and Cytokines Research; 70: 731, 2007. 5- DERBALA M., AMER A., BENER A. et al.: Pegylated interferon alpha 2b in combination with ribavirin in Egyptian patients with genotype 4 chronic hepatitis. Journal of Viral Hepatitis; 12 (4): 380-385, 2005. 6- MANNS M.P., MCHUTCHISON J.G., GORDON S.C., et al.: Peginterferon alfa-2b plus ribavirin compared with
Saad Zaky, et al.

interferonalfa-2b plus ribavirin for initial treatment of chronic hepatitis C: A randomised trial. Lancet; 358 (9286): 958-965, 2001. 7- NIH.: NIH. Consensus Statement on Management of Hepatitis C. NIH Consensus State Sci Statements; (19): 1-46. 8- FRIED M.W., SHIFFMAN M.L., REDDY K.R., et al.: Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med.; 347: 975-982, 2002. 9- HOOFNAGLE J.H. and SEEFF L.B.: Peginterferon and ribavirin for treatment of hepatitis C. The new England Journal of Medicine; 335: 2444-2451, 2006. 10- IDREES M. and RIAZUDDIN S.: A study of best positive predictors of sustained virologic response to interferon alfa plus ribavirin tyerapy in nave chronic hepatitis C patients. BMC Gastroenterology; 9: 5, 2009. 11- RODRIGUEZ-IIGO E., LPEZ-ALCAROCHO J., BARTOLOM J., et al.: Percentage of hepatitis C virus infected hepatocytes is a better predictor of response than serum viremia levels. JMD; 7 (4): 535-543, 2005. 12- MAUSS S., BERG T., ROCKSTROH J., et al.: Patterns of response to treatment in chronic hepatitis C. Hepatology-A Clinical Textbook. Eds, Mauss S., Berg T., Rockstroh J., Sarrazin C and Wedemeyer H., Flying Publisher, p. 199, 2009. 13- World Health Organization: Physical status: The use and interpretation of anthropometry. Report of WHO experts of a WHO Expert Committee. WHO Technical Report Series 854. Geneva, 1995. 14- BEDOSSA, P.: Intraobserver and interobserver variation in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Comparative Study Group. Hepatology; (20): 15-20, 1994. 15- BEDOSA P. and POYNARD T.: An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Comparative Study Group. Hepatology; (24): 280-293, 1996. 16- BRUNT E.M., JANNEY C.G., DI BISCEGLIE A.M., et al.: Non-alcoholic steatohepatitis: A proposal for grading and staging the histolocial lesions. Am. J. Gastroenterol.; 94: 2467-2474, 1999. 17- EL-ZAYADI A., SIMMONDS P., DABBOUS H., et al.: Response to interferon-alpha of Egyptian patients infected with hepatitis c virus genotype 4. J. Viral. Hepat.; 3: 261264, 1996. 18- EL-ZAYADI A., SELIM O., HADDAD S.P., et al.: Combination treatment of interferon aplhn-2b and ribnvirin in comparison to interferon monotherapy. J. Gastroenterol. Hepatol.; 31: 472-475, 1999. 19- KAMAL S.M. MADWAR N.A. PETERS T., et al.: Interferon therapy in patients with chronic hepatitis C and schistosomiasis. J. Hepatol.; 32: 172-174, 2000. 20- KOSHY A., MADDA J.P., MARCELLIN P., et al.: Treatment of hepatitis C virus genotype 4-related cirrhosis. Ribavirin and interferon combination compared with interferon alone J., Cli. Gastroenterol.; 36: 82-85, 2002. 21- ESMAT G., ABOUZIED A., ABDEL-HUMID M., et al.: Subjects with chronic hepatitis c and genotype 4 have a similarly effective response to standard or pegylated
375
interferon in combination with ribavirin. Hepatology; 38: 324A, 2003. 22- SHOBOKSHI O., SEREHOUR E., SHAWKI L., et al.: Combination therapy of piginterferon alfa2a (40KD) (Pegasys and ribavirin significantely enhance sustained virological and biochemical response rate in chronic hepatitis C genotype 4 patients in Saudi Arabia (Abstract). Hepatology; 38: 636A, 2003. 23- THAKEB F., OMER M., AWADY M., et al.: Randomized controlled trial of peginterferon alfa 2a plus ribavirin for chronic hepatitis C virus genotype 4 among Egyptain patients. Hepatol; 38: 237A, 2003. 24- ALFALEH F.Z., HADAD Q., KHUROO M.S., et al.: Peginterferon alpha-2b plus ribavirin compared with interferon alpha-2b plus ribavirin for initial treatment of chronic hepatitis C in Saudi patients commonly infected with genotype 4. Liver Int.; (24): 568-574, 2004. 25- HASAN F., ASKER H., AL-KHALDI J., et al.: Pegylated interferon alpha 2b plus ribavirin for treatment of chronic HCV genotype 4. American Journal of Gastroentrology; 99: 1733-1737, 2004. 26- KAMAL S.M., EL-TAWIL A.A. NAKANA T., et al.: Peginterferon (nlpha)-2b and ribavirin therapy in chronic hepatitis C genotype 4. Impact of treatment duration and viral kinetics on sustnine virological responss. Gut.; 54: 858-860, 2005. 27- KAMAL S.M., EL-KAMARY S.S., SHARDELL M., et al.: Pegylated interferon alpho 2b plus ribuvirine in patients with genotype 4 chronic hepatitis C: The role of rapid and early virology response Hepatology; 4G: 1732-1740, 2007. 28- ROULOT D., BOURCIER V., GRANDO V., et al.: Epidemiological characteristics and response to peginterferon plus ribavirin treatment of hepatitis C virus genotype 4 infection. J. Viral. Hepat.; 14: 406-407, 2007. 29- KAU A., VERMEHREN J. and SARRAZIN C.: Treatment predictors of a sustained virologic response in hepatitis B and C. Journal of Hepatology; 49: 634-651, 2008. 30- EL-MAKHZANGY H., ESMAT G., SAID M., et al.: Response to pegylated interferon alfa-2a and ribavirin in chronic hepatitis C genotype 4. J. Med. Virol; 81: 15761583, 2009. 31- BRESSLER B. L., GUINDI M., TOMLINSON G., et al.: High body mass index is an independent risk factor for non response to antiviral treatment in chronic hepatitis C. Hepatology; 38: 639-644, 2003. 32- BERG T., VON WAGNER M., NASSER S., et al.: Extended treatment duration for hepatitis C virus type 1: Comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin. Gastroenterology; 130 (4): 1086-1097, 2006. 33- AL-ASHGAR H., HELMY A., KHAN M.Q., et al.: Predictors of sustained virological response to a 48-week course of pegylated interferon alfa-2a and ribavirin in patients infected with hepatitis C virus genotype 4. Ann. Saudi Med.; 29 (1): 4-14, 2009. 34- ZEUZEM S., BUTI M., FERENCI P., et al.: Efficacy of 24 weeks treatment with peginterferon alfa-2a plus ribavirin in patients with chronic hepatitis C infected with
376

genotype 1 and low pretreatment viremia. J. Hepatol.; 44: 97-103, 2006. with genotype 4 chronic hepatitis C. Liver Int.; 28 (8): 1112-1119, 2008. 38- GE D., FELLAY J., THOMPSON A., et al.: Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature; 461: 399-401, 2009. 39- TANAKA Y., NISHIDA N., SUGIYAMA M., et al.: Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat. Genet.; 41: 1105-1109, 2009. 40- SUPPIAH V., MOLDOVAN M., AHLENSTIEL G., et al.: IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat. Genet.; 41: 1100-1104, 2009.
35- CROSS T.J.S., QUAGLIA A., NOLAN J., HUGHES S. and HARRISON P.M.: Do steatosis and steatohepatitis impact on sustained virological response (SVR) rates in patients receiving pegylated interferon and ribavirin for chronic hepatitis C infection? J. Med. Virol.; (82): 958964, 2010. 36- SHIFFMAN M.L., GHANY M.G., MORGAN T.R., et al.: Impact of reducing peginterferon alfa-2a and ribavirin dose during retreatment in patients with chronic hepatitis C. Gastroenterology; 132 (1): 103-112, 2007. 37- GAD R.R., MALES S., EL-MAKHZANGY H., et al.: Predictors of a sustained virological response in patients
Med. J. Cairo Univ., Vol. 79, No. 1, September 377-381, 2011 www.medicaljournalofcairouniversity.com
Maternal Weight and the Success of Trial of Vaginal Delivery after Caesarean Section
WALEED A.S. AHMED, M.D., MRCOG*,** and AYMAN MORSY, MRCOG*
The Department of Obstetrics Gynecology, Ulster Hospital, Dundonald, Belfast, Northern Ireland* and Faculty of Medicine, Suez Canal University**
Abstract
Objective: To study the effect of maternal weight on the success of trial of vaginal delivery in women with previous one transverse lower segment caesarean section. Design: Prospective analytic study. Setting: The study is carried out in Ulster Hospital, a district general hospital in Northern Ireland. Material and Methods: A total of 218 women with a previous one caesarean section eligible for trial of labour were identified in the year 2008. All data were collected including height, weight, BMI, antenatal course, intrapartum and postpartum events. Women were classified into normal weight (BMI 20-24.9kg/m2), overweight (BMI 25-29.9kg/m2) and obese (BMI >30kg/m2). Results: Overall vaginal delivery rate was 64.2% (67.6%, 67.8% and 52.9% in the normal, overweight and obese groups respectively). Induction of labour (IOL) carried out a higher risk for caesarean section (CS) compared to the spontaneous onset of labour (45.6% versus 32.3% CS rate respectively). Hypertensive disorders complicating pregnancy complicated 71.4% of obese women. Diabetes mellitus/impaired glucose tolerance test complicated 19.6% of cases in the obese group. Notably in the post partum period, wound infection complicated 37.5% of cases in the obese group. Neonatal weight was significantly higher in the obese group. Discussion: Complicated antenatal course and certain complications in the peripartum period are more common in obese women. Counselling these women who had a previous caesarean section regarding the likelihood of achieving a vaginal delivery is crucial. Obese women whom their pregnancy is complicated by a medical problem are less likely to successfully have a vaginal delivery especially if labour has been induced. Key Words: Obesity Previous caesarean section and induction of labour.
men are overweight and 11% obese [1]. In the UK, 56% of all women are over the normal body mass index (BMI), with 33% of them classified as overweight (BMI>25) and 23% obese (BMI>30). Although the exact incidence of obesity in pregnant women is not known, the Confidential Enquiry into Maternal and Child Health 2004 reported that 35% of all maternal deaths occurring in the triennium 2000-2002 were in obese women with a BMI >30kg/m2 [2]. The association between pre-pregnancy body mass index and caesarean delivery have been reported in several population based cohort studies from the USA, Weiss, et al., [3] Canada Joseph, et al., [4], and England Sebire, et al., [5] as well as from several tertiary care hospitals. The aim of this study is to specifically look at vaginal birth after one caesarean section in the different subsets of body mass indices, identifying trends and complications with a view to better planning of management and counselling for the obese group of patients. Patients and Methods This is a prospective hospital based cohort study. We looked at all deliveries in 2008 and analysed in detail the outcome in the vaginal birth after caesarean delivery (VBAC) group. All women who had a previous caesarean section and were counselled to a trial for vaginal delivery either spontaneously or after induction of labour were included in the study. BMI was calculated from the weight in kilograms divided by the height in Meters Square that were recorded at the first antenatal booking appointment. We used the WHO standard definitions of underweight (BMI<18.5kg/m 2), normal BMI
Introduction THE rising rate of obesity is a major health concern throughout the world, where 28% of pregnant woCorrespondence to: Dr. Waleed A.S. Ahmed, E-mail: waleed_asa@hotmail.co.uk
377
378
Maternal Weight & the Success of Trial of Vaginal Delivery after CS
(18.5-24.9kg/m2), overweight (BMI 25-29.9kg/m2) and obese (BMI >30kg/m2). The obese group was then further divided into Class 1 (BMI 30-34.9 kg/m2), Class 2 (BMI 35-39.9kg/m2) and Class 3 (BMI >kg/m2) [6]. Women were admitted to labour ward if they were in spontaneous labour. Continuous fetal monitoring with close observation for signs of uterine rupture e.g. fetal distress, abnormal per vaginal bleeding or sharp pains in the uterine scar. If labour is to be induced, prostaglandin is the usual agent for induction given vaginally as 1mg gel. If labour pains have not started within 6 hours, another 1mg gel can be given again. Prostaglandin should not be repeated if the uterus was contracting or artificial rupture of fetal membranes was feasible. In cases of spontaneous rupture of membranes, either prostaglandin or Syntocinon would be used to induce women. Monitoring would be as described earlier. We looked at the different demographic data including age, parity, smoking and socio-economic status. Antenatal management and complications are recorded. Details of labour and the immediate postpartum period are also recorded and all the relevant informations were compared in the three groups divided according to the BMI. Statistical analysis was conducted using Statistical Package for Social Scientists (SPSS version 14). Univariate analysis was done using ANOVA or Mann-Whitney test for continuous variables and chi-square test for categorical variables. A p-value of less than 0.05 was regarded as statistically significant. Results Two hundreds and eighteen women who had a previous one caesarean section and were eligible for trial of scar were identified in the year 2008. The total deliveries in that year were 2300 i.e. the study group comprised 0.94% of all deliveries. Maternal height and weight and hence the BMI were calculated at the booking antenatal visit which took place between 12 to 16 weeks gestation in all cases. No women in the underweight category were identified. Out of the 218, 105 were of normal weights, 59 overweight and 51 obese women. In the obese group, 26 were class 1 obesity (BMI 3035), 16 were class 2 obesity (BMI 35-40) and 9 were morbidly obese (BMI >40). In the normal weight group, the age ranged between 21-33 years with a mean of 26.32.3 years. There was no statistically significant difference between the mean age in this group compared
to the other 2 groups (mean age S.D. 25.63.1 and 26.44.2 in the overweight and obese groups respectively. Despite the tendency of obese women to be of higher parity, this did not reach statistical significance. The median parity in obese women was 3 compared to 2 in the normal and overweight groups. Two women in the obese group were known diabetics (5/51=9.8%) as compared to one in each of the other 2 groups. The past medical history was no different among the study groups. No significant difference was found in the socio-economic status or the smoking habit among the groups. The indications of the previous caesarean sections were as follows: Abnormal cardiotocography (CTG)/suspected fetal compromise (23%), failure to progress (21%), breech presentation (18%), severe pre-eclampsia (12%), ante partum haemorrhage (10%), intrauterine growth restriction (7%) and miscellaneous (9%). Antenatally, pregnancy induced hypertension/ pre-eclampsia was the most common medical problem complicating pregnancy. It complicated about 10% of cases (21 women). Fifteen of these women (71.4%) were in the obese group, three in the normal weight and three in the overweight groups respectively. Type 1 diabetes mellitus and impaired glucose tolerance test complicated 10 cases (5 each) in the obese group compared to three in the other groups. Overall, the vaginal delivery rate in the study group was 64.2%. Looking at the different subgroups, it was found that 64.8% (70/108) in the normal weight group achieved vaginal delivery. A slightly higher success was gained in the overweight group 67.8% (40/59). Women in these 2 groups had significantly higher vaginal delivery rates compared to women in the obese group 52.9% (27/51). One hundred and sixty one women were in spontaneous labour and the rest (57 women) were induced. The induction of labour (IOL) rate in the study group was 26.1%. The indications for IOL were postdates (25/57=43.9%), pre-labour spontaneous rupture of membranes (15/57=26.3%), raised blood pressure/mild pre-eclampsia (11/57=19.3%) and maternal diabetes mellitus (6/57=10.5%). Women in the spontaneous labour group had a significantly higher vaginal delivery rates compared to women in the IOL group (67.7% versus 54.4%
Waleed A.S. Ahmed & Ayman Morsy
379
respectively). Obese women in the IOL group had the lowest vaginal delivery rate (33.3%) Table (1). Women who went into spontaneous labour did so between 31 and 41 weeks with a mean gestational age at delivery of 36+4 weeks. In the IOL group, the mean gestational age at delivery was 38+4 weeks ranging between 34 and 41+3 weeks. There was no significant difference in preterm labour i.e. delivery before 37 completed weeks among the different groups. Post term delivery was also not significantly different in the study groups.
The mean neonatal weights in the study groups were 3524, 3605 and 3889 grams in the normal, overweight and obese groups respectively and the neonatal weight in the obese group was significantly higher compared to the other two groups. The postpartum complications were higher in the obese group especially those who delivered by caesarean sections. It is noted that two scar dehiscence were found in our study in the normal and overweight women categories. Table (2) shows the peripartum complications in the study groups; wound infection was calculated only against those who had caesarean sections.
Table (1): Vaginal delivery rates in the study groups and in relation to onset of labour. Normal weight Number Spontaneous Labour I.O.L. Total 59/84 14/24 73/108 % 70.2 58.3 67.6 Overweight Number 31/45 9/14 40/59 % 68.9 64.3 67.8 Obese Number 19/32 8/19 27/51 % 59.3 42.1 52.9 Total Number 109/161 31/57 140/218 % 67.7 54.4 64.2
Table (2): Peripartum complication and the maternal BMI. Normal weight Number Scar Dehiscence P.P.H. U.T.I. Wound infection Chest infection D.V.T. 1/108 8/108 16/108 4/35 4/108 1/108 % 0.9 7.4 16.7 11.4 3.7 0.9 Overweight Number 1/59 3/59 12/59 3/19 3/59 0/59 % 1.7 5.1 20.3 15.8 5.1 0 Obese Number 0/51 5/51 12/51 9/24 3/51 2/51 % 0 9.8 23.5 37.5 5.9 3.9 Total Number 2/218 16/218 40/218 16/78 12/218 3/218 % 0.9 7.3 18.3 20.5 5.5 1.37
Discussion Our study adds to the increasing body of literature showing the adverse effects of maternal obesity on the outcome of pregnancy and labour [7]. In this prospective study, the antenatal, intrapartum and postpartum events in women who had a previous one caesarean section were compared in normal, overweight and obese women. This is a subgroup of pregnant women who already had an obstetric risk factor of previous caesarean section to which the risks of obesity are also added. Obesity can affect women (and indeed men!) at any age. At the age of around 25 years, 24% of the study population were obese. This is in keeping with the prediction assuming that by the year 2010, 25% of all adult UK populations will be obese [8]. All women in our study underwent physical examination including height, weight and hence
BMI at the first antenatal booking visit so the bias resulting from obtaining the weight related information at different gestational ages or prepregnancy was eliminated. Seventy percent of women who had pregnancy induced hypertension/pre-eclampsia were in the obese group. Previous research had found strong correlation between blood pressure problems and obesity which is confirmed again in our study. Bhattacharya and associates found a 3 times higher risk of pre-eclampsia in obese (BMI 30 to 39.9 kg/m2) and a 7 times higher risk in morbidly obese (BMI >40kg/m2) primgravid women [9]. In our study, obesity was an independent risk factor for IOL and emergency caesarean section; 46.1% of inductions were in the obese group of those only 33.3% achieved a vaginal delivery. These results agree with the previous reports and
380
Maternal Weight & the Success of Trial of Vaginal Delivery after CS
studies linking increased BMI to different obstetric interventions [10-12]. Some studies attributed the high surgical intervention including instrumental deliveries and emergency caesarean section to the high rates of IOL, as indeed was the case in our study. IOL in the obese group was related primarily to presence of medical complications including pregnancy induced hypertension and diabetes mellitus. The combination of medical problem and previous caesarean section in an obese woman is highly likely to end up by emergency caesarean section and there is a point in the tendency towards performing elective caesarean section in these women. The success rate of VBAC (vaginal birth after caesarean section) was 64%, which is in keeping with the different studies demonstrating a success rate of between 60-80% [13,14]. Women who went in spontaneous labour had significantly higher vaginal delivery rate compared to those induced. This was in agreement with Durnwald and Mercer whom in their study on 768 women concluded that spontaneous labour is associated with successful VBAC in women with a single prior low transverse caesarean delivery and no prior vaginal deliveries [10]. Obese women in our study had 45.8% vaginal delivery rate significantly lower women in the normal and overweight groups. Landon, et al. had shown that BMI >30, among other factors, significantly lowered the chance for successful trial of labour in women with previous caesarean section [13]. Other investigators have shown reduction in VBAC rates in obese women [15,16]. Our resultand those from literature-highlights the importance of counselling obese women who had a previous caesarean section regarding the likelihood of achieving a vaginal delivery in subsequent pregnancies. Despite the tendency of having bigger babies in the obese group in our study compared to the normal and overweight groups, this did not reach statistical significance. Several studies had shown that being obese is a risk factor for fetal macrosomia. Jensen, et al. also found an association between women in the overweight category and fetal macrosomia in a population of 2,459 glucose-tolerant Danish women. These data suggest that being overweight or obese can influence the nutrition or growth of the fetus. It is clear that in women with diabetes mellitus fetal macrosomia can be secondary to poor diabetic control and maternal hyperglycaemia [17].
Intra and postoperative complications are known to increase in obese women. Difficult operation due to poor access, limited mobility postoperatively and the tendency for blood or serous fluid to accumulate in the subcutaneous fat layer would predispose to complications. Notably wound infection was significantly high in obese women in our study. Useful surgical technique while operating on obese women include the avoidance of making the incision directly below the pannaliculus, good haemostasis is essential and preferably aided by the use of diathermy, antibiotic prophylaxis, sheath closure with looped PDS is ideal, closure of the subcutaneous tissue and adequate thromboprophylaxis. No difference in infection and haematomas was found with or without a drain [18]. Our study does have some limitations. Some women had a history of successful vaginal delivery following caesarean section before enrolling in the study. This is known to increase the success rate of attempted vaginal delivery. The physicians attitude in managing women in labour will differ especially in high risk cases with some will recourse to early surgical intervention to prevent a more challenging surgical exercise in an emergency situation. However, our results represents the routine clinical practice that is practiced in most hospitals. In conclusion, obesity tends to adversely affect the chances of successful vaginal birth in women with a previous caesarean section. Pre-pregnancy health education and counselling for this category of patients is important to optimize both the antenatal and delivery outcome. Management of such obese pregnant women should be supervised by experienced obstetricians as intrapartum events can be challenging with a higher tendency for complications. References
1- RCOG, 5TH October 2006 Press Releases [http://www. rcog.org.uk/index.asp?PageID=97&PressReleaseID=126]. 2- Report of the Confidential Enquiry into Maternal and Child Health. Why mothers die 2000-2002 Edited by: Lewis G. RCOG: London 2004. 3- WEISS J.L., MALONE F.D., EMIG D., BALL R.H., NYBERG D.A., COMSTOCK C.H., et al.: Obesity, obstetric complications and caesarean delivery rate-a population-based screening study. Am. J. Obstet. Gynecol., 190: 1091-7, 2004. 4- JOSEPH K.S., YOUNG D.C., DODDS L., OCONNELL C.M., ALLEN V.M., CHANDRA S., et al.: Changes in maternal characteristics and obstetric practice and recent increases in primary caesarean delivery. Obstet. Gynecol., 102: 791-800, 2003.
Waleed A.S. Ahmed & Ayman Morsy

5- SEBIRE N.J., JOLLY M., HARRIS J.P., WADSWORTH J., JOFFE M., BEARD R.W., et al.: Maternal obesity and pregnancy outcome: A study of 287, 213 pregnancies in London. Int. J. Obs. Relat. Metab. Disord., 25: 1175-82, 2001. 6- WHO. Obesity: Preventing and managing the global epidemic. Report of a WHO consultation. WHO Technical Report Series 894. Geneva: World Health Organization, 2000. 7- WAX J.R.: Risk and management of obesity in pregnancy: Current controversies. Curr. Opin. Obstet. Gynecol. 2009 Apr.; 21 (2): 117-23. Review. 8- Department of health. Health survey for ENGLAND 2003. London. HMSO, 2003. 9- BHATTACHARYA S., CAMPBELL D.M., LISTON W.A. and BHATTACHARYA S.: Effect of Body Mass Index on pregnancy outcomes in nulliparous women delivering singleton babies. BMC Public Health., 24; 7: 168, 2007. 10- DURNWALD C. and MERCER B.: Vaginal birth after Cesarean delivery: Predicting success, risks of failure. J. Matern. Fetal. Neonatal. Med., 15 (6): 388-93, 2004. 11- EL-SAYED Y.Y., WATKINS M.M., FIX M., DRUZIN M.L., PULLEN K.M. and CAUGHEY A.B.: Perinatal outcomes after successful and failed trials of labor after cesarean delivery. Am. J. Obstet. Gynecol. Jun., 196 (6): 583.e1-5, 583.e5, 2007. 12- ATHUKORALA C., RUMBOLD A.R., WILLSON K.J. and CROWTHER C.A.: The risk of adverse pregnancy
381
outcomes in women who are overweight or obese. BMC Pregnancy Childbirth. Sep. 17; 10: 56, 2010. 13- LANDON M.B., LEINDECKER S., SPONG C.Y., HAUTH J.C., BLOOM S., VARNER M.W., MOAWAD A.H., CARITIS S.N., HARPER M., et al.: The MFMU Cesarean Registry: Factors affecting the success of trial of labor after previous cesarean delivery. Am. J. Obstet. Gynecol., 193 (3 Pt 2): 1016-23, 2005. 14- GUISE J.M., EDEN K., EMEIS C., DENMAN M.A., MARSHALL N., FU R.R., JANIK R., NYGREN P., WALKER M. and MCDONAGH M.: Vaginal birth after caesarean: New insights. Evid. Rep. Technol. Assess. (Full Rep). 2010 Mar.; (191): 1-397. Review. 15- EHRENBERG H.M., DIERKER L., MILLUZZI C. and MERCER B.M.: Prevalence of maternal obesity in an urban center, Am. J. Obstet. Gynecol. 187, 1189-1193, 2002. 16- CARROLL C.S., MAGANN E.F., CHAUHAN S.P., KLAUSER C.K. and MORRISON J.C.: Vaginal birth after cesarean section versus elective repeat cesarean delivery: Weight-based outcomes, Am. J. Obstet. Gynecol. 188., 1516-1522, 2003. 17- JENSEN D.M., DAMM P., SRENSEN B., MLSTEDPEDERSEN L., WESTERGAARD J.G., OVESEN P. and BECK-NIELSEN H.: Pregnancy outcome and prepregnancy body mass index in 2459 glucose-tolerant Danish women. Am. J. Obstet. Gynecol., 189 (1): 239-44, 2003. 18- COYLE M.E., SMITH C.A. and PEAT B.: Cesarean section in the morbidly obese. Cochrane Database Syst. Rev., CD003928, 2005.
Impact of Adjuvant Chemotherapy Delay on Outcome in Cancer Colon Patients

ZEINAB M. ABD EL-HAFEEZ, M.D.; AZZA M. ADEL, M.D. and SHERIF ABD EL-WAHAB, M.D.
The Departments of Radiation Oncology and Nuclear Medicine, Faculty of Medicine, Ain Shams University
Abstract
Background: When adjuvant chemotherapy is indicated in colorectal carcinoma (CRC) it is usually prescribed within two months after curative surgery. The effect of delay in adjuvant chemotherapy is unclear. The aim of this retrospective study was to assess the impact of adjuvant CT delay beyond 8 weeks (56 days) in stage III and high risk stage II colon cancer patients on DFS and OS. Patients and Methods: This retrospective study was conducted through revision of medical records of patients diagnosed with stage II-III colon adenocarcinoma who received adjuvant CT after complete surgical resection at the department of Clinical Oncology, Ain Shams University Hospitals. Results: Between January 2003 and August 2008, 108 patients were included. Patients were divided into 2 groups: Group I including 62 patients who started adjuvant CT <56 days from surgery and group II which included 46 patients who had CT delay 56 days. Both groups were matched demographically. Disease free survival was found to be significantly worse in group II patients compared to group I patients. Median DFS was 189.3 and 2714.13 in the two groups respectively (p=.000). There was no significant difference in OS between the two groups. Analysis of other prognostic factors (age, sex, T, N, CT type) were done with only nodal status predicted for worse DFS (p=0.05) and OS (p= 0.006). Conclusion: Delay in starting adjuvant chemotherapy for high risk stage II, and stage III colorectal cancer patients was associated with lower DFS but no difference in OS. Key Words: Colorectal cancer Adjuvant Delay.
analysis of randomized controlled trials [1]. The addition of oxaliplatin to a 5-fluorouracil-based regimen improved overall survival rates at 6 years by a further 4% (from 69% to 73%) in the MOSAIC trial [2] . The role of adjuvant chemotherapy in patients with stage II colon cancer remains controversial. However, in the group of stage II colon cancer patients with high risk features such as T4 tumors, poor differentiation, perforation, and inadequate number of sampled lymph nodes, adjuvant chemotherapy is recommended [3]. Adjuvant CT is believed to increase cancer cure by eradicating micrometastases after surgical resection. Although initiation of adjuvant therapy soon after recovery from surgery is desirable, the optimal timing of adjuvant therapy after surgery is still not known and it is usually accepted that it should begin within two months after surgery [4] . Rationales for prompt initiation of adjuvant therapy argue that this approach is superior in deterring growth of residual cancer cells, preventing spread of micrometastases, and reducing developmenof resistant clones [5,6]. However, several reasons may delay the initiation of adjuvant CT like post-operative complications and longer recovery from surgery. Delays may also be related to poor compliance of some patients. When delayed, does adjuvant CT bring the same benefit in terms of overall survival (OS) or disease free survival (DFS)? Most adjuvant chemotherapy trials defined a particular time from surgery to the start of chemotherapy beyond which patients were no longer eligible to participate. Strictly speaking, the benefits of a treatment described by a clinical trial are only applicable to patients treated within the same time frame as in the trial. Whether equivalent benefit can be ascribed when chemotherapy is started beyond the time window specified is not known [7].
Introduction ADJUVANT chemotherapy (CT) is the standard treatment for patients with stage III colon cancer. Fluorouracil-based regimen improved overall survival rates at 8 years by 10% (range: 43% to 53%) for patients with stage III disease in a recent metaCorrespondence to: Dr. Azza M. Adel, The Departments of Radiation Oncology and Nuclear Medicine, Faculty of Medicine, Ain Shams University.
383
384
It is unclear from the published literature to what extent the time to initiating adjuvant CT for colon cancer correlates with patients' survival. Various analyses of prospective clinical trials and a few retrospective studies have reported conflicting results [8-14]. However, it can be postulated from these published studies that delay of adjuvant chemotherapy for more than 56 to 60 days could have a negative impact on survival. The aim of this retrospective study was to assess the impact of adjuvant CT delay beyond 8 weeks (56 days) in stage III and high risk stage II colon cancer patients on DFS and OS. Patients and Methods This retrospective study was conducted through revision of medical records of patients diagnosed with stage II-III colon adenocarcinoma who received adjuvant CT after complete surgical resection at the department of Clinical Oncology, Ain Shams University Hospitals. Eligibility criteria: All patients had to be 18 years old and started adjuvant CT no more than 180 days after complete surgical resection. Patients with rectosigmoid carcinoma, metastatic disease, another primary tumor, patients who had palliative resection of the tumor, and who were lost to follow-up or died within 6 months of diagnosis were excluded. In addition, patients who had inconclusive information about the number of treatment cycles were not included in this study. Data collected from patients' medical records included: Age, sex, date of surgery, pathology information (TNM staging), adjuvant CT data (time between surgery and adjuvant CT start, type, number of cycles, and date of last cycle, cause of CT delay), follow-up data (date and status at last follow-up). A cut-off date of 8 weeks (56 days) was chosen to separate patients into 2 groups: Group I in which patients received adjuvant CT within 56 days of surgery, and group II which included patients who had adjuvant CT 56 days. Study end points: The study end points included DFS and OS. Disease free survival was defined as the time between curative resection of the tumor and the first relapse (locoregional or metastatic), death from any cause when no evidence of relapse was recorded, or the last date at which the patient was known to be free of the disease. Overall survival was defined as time between curative resection of the primary tumor and death or the date at which patient was last confirmed to be alive. Follow-up
information was collected and the disease status on the last follow-up was recorded. Relapse was documented as either a clinical or biopsy-proven disease relapse. Statistical analysis: Statistical analysis was performed using IBM SPSS statistics (V. 19.0, IBM Corp., USA, 2010). Cumulative survival rate was calculated according to the Kaplan-Meier method, and differences between survival curves were tested by the log-rank test. Various clinicopathological variables were examined with respect to their prognostic significance for DFS and OS. A p-value 0.05 was considered statistically significant. The probability of error at 0.05 was considered significant; while at 0.01 and 0.001 are highly significant. Results Between January 2003 and August 2008, 108 patients fulfilled the eligibility criteria for inclusion in this study. Mean age was 44.9513.03. The male:female ratio was 2.3:1.3. Sixty two patients (57.4%) had T3 tumors while 76 patients (70%) had N+ve disease. Time to adjuvant CT ranged between 14 and 148 days with a mean of 59.535.2 days. Adjuvant CT was delayed beyond 8 weeks in 46 patients. Causes of this delay were related to surgery (postoperative complications or late recovery) in 12 patients (11%), system related causes (late referral, time needed to get governmental insurance support) in 10 patients (9%), patient related reasons (time needed to decide on CT, missed appointments) in 7 patients (4.5%), presence of co-morbidities in additional 7 patients (4.5%). In 10 patients (9%) cause of delay was unknown. Demographic and clinical characteristics of the study patients are outlined on Table (1). Patients were divided into 2 groups: Group I including 62 patients who started adjuvant CT <56 days and group II which included 46 patients who had CT delay 56 days (Table 2). Follow-up duration ranged between 6 and 70 months (mean duration 25.9313.45 months). Disease free survival was found to be significantly worse in group II patients compared with group I patients. Median DFS was 189.3 and 2714.13 in the two groups respectively (p=.000) (Fig. 1). There was no significant difference in OS between the two groups (Fig. 2). Analysis of other prognostic factors (age, sex, T, N, CT type) were analyzed. Only nodal status predicted for worse DFS (p=0.05) and OS (p=0.006).
Zeinab M. Abd El-Hafeez, et al.

Survival Functions 1.0 0.8 Cum Survival 0.6 0.4 0.2 0.0 40 60 DFS Fig. (1): Disease free survival for group I receiving treatment within 56 days and group (2) receiving treatment after 56 days. Survival Functions 1.0 0.8 Cum Survival 0.6 0.4 Adjuvant treatment Adjuvant ttt <56 Adjuvant ttt >56 0 20 Adjuvant treatment Adjuvant ttt <56 Adjuvant ttt >56 Age: <60 60 Sex: Male Female Tumor depth: T2 T3 T4 LNs: N0 N1 N2 CT type: 5FU/L (deGramont) 5-FU/L (Mayoclinic) FOLFOX
385
Table (2): Difference between clinical data of the patients based on delay in adjuvant treatment. Variable Group I % (n=61) 55 7 39 23 6 33 23 23 33 6 49 8 5 88.7 11.3 62.9 37.1 9.7 53.2 37.1 38.7 51.6 9.7 79.02 12.9 8.08 Group II % (n=46) 39 7 30 16 6 29 11 9 29 8 31 8 7 p value
84.8 0.755 15.2 65.2 0.964 34.7 13.04 0.337 63.04 18.02 19.6 0.111 63 17.4 67.4 0.357 17.4 15.2
CT: Chemotherapy. FOLFOX: 5FU/leucovorin/oxaloplatin. LN: Lymph node metastases.
Discussion
0.2 0.0
40 60 OS Fig. (2): OS in both groups receiving chemotherapy within 56 days (group I) and after 56 days group (2). Table (1): Demographic and clinical characteristics of the study patients (n=108). Characteristic Age Sex: Male Female Tumor depth (T): T2 T3 T4 Nodal status (N): N0 N1 N2 Time to adjuvant CT start: <56 days 56 days CT type: 5-FU/L (deGramont) 5-FU/L (Mayoclinic) FOLFOX No. Range: 20-74 Mean: 44.9513.03 69 39 12 62 34 32 62 14 62 46 80 16 12 63.9 36.1 11.1 57.4 31.5 29.6 57.4 13 57.4 42.6 74.1 14.8 11.1 %
20
One of the most important advances of medical oncology over the past 30 years has been the gradual refinement through large scale randomized trials of the use of adjuvant systemic therapy after surgical resection for stage III and high risk stage II colon cancer [15]. In earlier trials which have a surgery-alone group, the treatment was generally initiated within 35 days, and the maximum number of days allowed was 56 [16-18]. Few studies have assessed the prognostic impact of timing of adjuvant therapy [4,8,9]. In the Short Adjuvant Fluorouracil and Folinic Acid Study (SAFFA) trial, which was permissive in allowing patients to receive adjuvant therapy up to 12 weeks after surgery, early initiation of chemotherapy was associated with a better survival [4]. In a subset analysis of this trial, patients who received treatment between 8 to 12 weeks after surgery had a 37% increased mortality compared with those who received treatment within 8 weeks after surgery. Likewise in the pooled analysis of Nordic Gastrointestinal Tumour Adjuvant Therapy Group (NGTATG) trials, patients who started treatment beyond 8 weeks after surgery had worse survival compared with those who started adjuvant therapy within 8 weeks [19]. In the current study, it was found that delay in initiation of adjuvant CT for 56 days was associated with worse DFS (p=.000). On the other hand,
386
current data failed to demonstrate significant decline in OS with the same delay. This finding is in contrast to the results of a recent meta-analysis which included 8 studies on 13,158 patients with stage III colon cancer who received adjuvant CT. In this meta-analysis it was found that delaying the initiation of adjuvant CT for more than 8 weeks after surgery significantly decreased OS but not RFS. They attributed this discrepancy to factors not directly related to cancer like postoperative complications, social status, or to a more accurate appraisal of death. The point of accurate documentation of death is lacking in the current study as cancer registration is still a problem in developing countries and death due to causes other than cancer may be missed. On the other hand, many studies did not find a relationship between early initiation of treatment and a better survival in colon cancer [8,9]. Other smaller retrospective studies reported conflicting results, 2 studies reported inferior outcome with treatment delay beyond 8 weeks, whereas another reported no difference in outcome if treatment was started beyond 8 weeks [14,20,21]. The contradictory results reported in different studies may reflect the presence of other non modifiable confounding factors. Thus, adverse outcome noted in patients who received adjuvant therapy at later dates could be due to the presence of other factors that resulted in delayed treatment, including poor performance status or presence of co-morbidities, and these factors may also increase mortality independent of adjuvant CT delay. Analysis of other possible prognostic factors in the current study revealed that the presence of lymph node metastases was associated with worse OS (p=0.006) and DFS (p=0.05). Regional LN metastasis is one of the strongest prognostic factors in early stage CRC [22]. The local extent of tumor and regional LN metastases has been classified as category I in the College of American Pathologists consensus statement [23]. The present study has certain limitations. It is a retrospective study with lack of control over confounding variables. However, for obvious ethical reasons, a randomized controlled trial comparing the effect on survival of two different delays of initiation of adjuvant CT after surgery would not be feasible. Therefore, the question of timing to CT cannot be answered prospectively. Second, the number of study patients' was relatively small for detailed analysis of other prognostic factors. Another important point is lack of data on patient comorbidities and their impact on survival. Lastly, the relatively short follow-up period, a longer followup may reveal the impact of CT delay on OS.
In conclusion, delay of adjuvant CT for more than 8 weeks after surgical resection was found to be associated with poor DFS in stage II-III colon cancer patients. Further extensive retrospective studies with longer follow-up period and larger number of patients are needed to confirm the impact of adjuvant CT delay on outcome and survival of colon cancer patients. Meanwhile, effort should be made to start adjuvant CT within 8 weeks after surgery. References
1- SARGENT D., SOBRERO A., GROTHEY A., et al.: Evidence for cure by adjuvant therapy in colon cancer: Observations based on individual patient data from 20, 898 patients on 18 randomized trials. J. Clin. Oncol., 27 (6): 872-7, 2009. 2- ANDRE T., BONI C., NAVARRO M., et al.: Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J. Clin. Oncol., 27 (19): 3109-16, 2009. 3- BENSO A.B. III, SCHRAG D., SOMERFIELD M.R., et al.: American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon caner. J. Clin. Oncol., 22 (16): 3408-19, 2004. 4- CHAU I. and CUNNINGHAM D.: Adjuvant therapy in colon cancer-what, when and how?. Ann. Oncol., 17: 1347-59, 2006. 5- GOLDIE J.H. and COLDMAN A.J.: Theoretical considerations regarding the early use of adjuvant chemotherapy. Cancer Res., 103: 30-5, 1986. 6- FISHER B., GUNDUZ N. and SAFFER E.A.: Influence of the interval between primary tumor removal and chemotherapy on kinetics and growth of metastases. Cancer Res., 43: 1488Y92, 1983. 7- LOHRISH C., PALTIEL C., GELMON K., et al.: Impact on survival of time from definitive surgery to initiation of adjuvant chemotherapy for early-stage breast cancer. J. Clin. Oncol., 4888-94, 2006. 8- TAAL B.G., VAN TINTEREN H. and ZOETMULDER F.A.: Adjuvant 5FU plus levamisole in colonic or rectal cancer: Improved survival in stage II and III. Br. J. Cancer, 85: 1437-43, 2001. 9- ANDRE T., COLIN P., LOUVET C., et al.: Semimonthly versus monthly regimen of fluorouracil and leucovorin administered for 24 or 36 weeks as adjuvant therapy in stage II and III colon cancer: Results of a randomized trial. J. Clin. Oncol., 21: 2896-903, 2003. 10- ARKENAU H.T., BERMANN A., RETTIG K., et al.: 5Fluorouracil plus leucovorin is an effective adjuvant chemotherapy in curatively resected stage III colon cancer: Long-term follow-up results of the adjCCA-01 trial. Ann. Oncol., 14: 395-9, 2003. 11- BERGLUND A., CEDERMARK B. and GLIMELIUS B.: Is it deleterious to delay the start of adjuvant chemotherapy in colon cancer stage III? Ann. Oncol., 19: 4002, 2008. 12- BIAGI J.J., BARNES C. and OCALLAGHAN C.J.: Time to adjuvant chemotherapy in colorectal cancer. Paper
Zeinab M. Abd El-Hafeez, et al.

presented at: 2007 Gastrointestinal Cancers Symposium, Orlando, FL, January 19, 2001. 13- AHMAD I., ANAN G. and ALVI R.: Impact of timing of initiation of adjuvant chemotherapy on survival after resection of colorectal cancer. J. Clin. Oncol., 25 (18 Suppl): 4054, 2007. 14- CARLSSON G., ALBERTSSON P. and GUSTAVSSON B.: Delayed start of adjuvant 5-FU/leucovorin based chemotherapy in colon cancer is safe up to 12 weeks postoperatively. J. Clin. Oncol., 25 (18 Suppl): 4056, 2007. 15- GILL S., LOPRINZI C.L., SARGENT D.J., et al.: Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: Who benefits and by how much. J. Clin. Oncol., 22: 1797-806, 2004. 16- LAURIE J.A., MOERTEL C.G., FLEMING T.R., et al.: Surgical adjuvant therapy ofmlarge-bowel carcinoma: An evaluation of levamisole and the combination of levamisole and fluorouracil. J. Clin. Oncol., 7: 1447-56, 1989. 17- International Multicentre Pooled Analysis of Colon Cancer Trials (IMPACT) investigators. Efficacy of adjuvant fluorouracil and folinic acid in colon cancer. Lancet, 345: 939-44, 1995. 18- MAMOUNAS E., WIEAND S., WOLMARK N., et al.: Comparative efficacy of adjuvant chemotherapy in patients
387
with Dukes B versus Dukes C colon cancer: Results from four NSABP adjuvant studies. J. Clin. Oncol., 17: 1349-55, 1999. 19- GLIMELIUS B., CEDERMARK B., DAHL O., et al.: Adjuvant chemotherapy in colorectal cancer: A joint analysis of randomised trials by the Nordic Gastrointestinal Tumour Adjuvant Therapy Group. Acta. Onco., 44: 90412, 2005. 20- BAYRAKTAR U.D., BAYRAKTAR S. and HERNA S.: Does delay of adjuvant chemotherapy affect the clinical outcome in patients with colon cancer [abstract]? J. Clin. Oncol., 27: 4046, 2009. 21- ZAYKOWSKI P., GILL S., KENNECKE H., et al.: Adjuvant chemotherapy (AC) for stage 3 colon cancer: Does timing matter [abstract]? Paper presented at the American Society for Clinical Oncology Gastrointestinal Cancers Symposium, January 19-21, 2007; Orlando, FL. 22- CHANG G.J., RODRIGUEZ-BIGAS M.A. and SKIBBER J.M.: Lymph node evaluation and survival after curative resection of colon cancer: Systematic review. J. Natl. Cancer Inst., 99: 433-41, 2007. 23- COMPTON C.C., FIELDING L.P., BURGART L.J., et al.: Prognostic factors in colorectal cancer. College of American Pathologists Consensus Statement 1999. Arch. Pathol. Lab. Med., 124: 979-94, 2000.
Med. J. Cairo Univ., Vol. 79, No. 1, Sep. 389-406, 2011 www.medicaljournalofcairouniversity.com
Detection of Coronary Artery Stenoses with Thin Slice Multi Detector Row Spiral Computed Tomography Angiography
SHERIF MOHAMED MAHER, MBBCh*, IHAB ISMAIL ALY, M.D.*, NASHWA ABED MOHAMED, M.D.** and MOHAMED ABD EL-FATTAH HASSAAN, M.D.*
The Departments of Radiodiagnosis* and Critical Care**, Faculty of Medicine, Cairo University
Abstract
Background: Multislice CT coronary angiography is a recent technique used to diagnose coronary artery diseases with better spatial and temporal resolution. Objectives: To detect stenosis in the coronary arteries using thin slice multidetector row CT coronary angiography. Methods: A total number of 15 patients scheduled for elective conventional coronary angiography for known or suspected coronary artery disease were enrolled for multislice CT angiography of the coronary arteries 5 patients with previous CABG operation and other 3 patients with percutaneous trans-luminal coronary angioplasty (PTCA) and stenting were included. Results: CT coronary angiography detected significant and non-significant stenoses in the native arteries and evaluated the patency of the grafts, conduits and stents in post interventional cases. Conclusion: The improved spatial and temporal resolution of the CT coronary angiography makes it capable of detecting stenosis in the coronary arteries, diagnosing coronary artery anomalies and proper evaluation of the grafts and stents. Key Words: Coronary artery disease (CAD) Multislice computed tomography (MSCT) Computed tomography angiography (CTA).
performed only for diagnostic purposes (American Heart Association 2002). Recent insights into the patho-physiology of atherosclerotic CAD suggest that coronary arterial wall structure has a crucial role in these disorders. Since coronary angiography depicts only the intraluminal morphology but not the wall, much research effort has been focused on other imaging modalities. MSCT, unlike conventional angiography, enables assessment of the vessel wall abnormalities [1]. Until recently, CT applications for the assessment of CAD per segment were almost exclusively directed at the detection and quantification of coronary arterial calcium. In recent years, considerable interest has accordingly been directed at the beneficial use of high-spatial-resolution contrast enhanced CT angiography for noninvasive interrogation of the coronary arterial tree. To date, the central rationale of this application has been the noninvasive detection and grading of coronary artery stenosis and follow-up after coronary bypass surgery, with the ultimate goal of replacing diagnostic invasive conventional coronary angiography [2]. Challenges in evaluating the coronary arteries at CT are the small size of the vessels, their tortuous courses and fast continuous movement. Possible solutions are imaging on scanners with an increasing number of detector rows, faster rotation speeds and reconstructing multiple sets of images obtained in different phases of the cardiac cycle (ECG gating) from a volume acquisition. The challenges of CT angiography of the coronary arteries have been partially met and will likely be addressed with the continued evolution of technology [3]. However, CT angiography, among the noninvasive methods for assessment of the coronary
Introduction CORONARY artery disease (CAD) remains a leading cause of death all over the world. The standard reference for diagnosis of CAD is still the conventional coronary angiography. The greatest advantage of conventional angiography is the perfect spatial resolution and the option of direct performance of interventions such as balloon dilatation or coronary stent placement. However, only one-third of all conventional coronary angiographic examinations are performed in conjunction with an interventional procedure, while the rest are
Correspondence to: Dr. Nashwa Abed Mohamed, The Department of Critical Care, Faculty of Medicine, Cairo University.
389
390
Detection of Coronary Artery Stenoses
arteries, currently appears to be the best owing to its combination of unprecedented acquisition speed, high spatial resolution, and robustness of use [4]. Since the clinical outcome of the patients is closely related to the patency rate of their bypass grafts, it would be important for the patients to have the patency rate of their grafts assessed on time to detect any graft occlusion before the majority of the grafts get occluded. Recently, multislice computed tomography offers an attractive tool for this purpose. Bypass grafts are ideal vessels for evaluation by MSCT because of their greater diameter and their relative spatial fixation [4]. The aim of this study is to evaluate the accuracy of the multislice CT coronary angiography, as a non-invasive imaging tool for morphological assessment of the coronary arterial tree to detect stenosis and also to detect calcification, location and severity of obstruction of the coronary plaques. Patients and Methods Study population: A total number of 15 patients (12 males and 3 females) with a mean age 56 years old (ranging between 43 and 65 years old) were scheduled for elective conventional coronary angiography for known or suspected coronary artery disease were enrolled for multislice CT angiography of the coronary arteries. These patients presented with ischemic chest pain (defined as a retro-sternal or precordial diffuse burning, heaviness, or squeezing sensation that may radiate to the left arm, neck or lower jaw and is precipitated by effort and relived by rest or nitrates) or suspected progression of known coronary artery disease. Some of them had history of previous percutaneous trans-luminal coronary angioplasty (PTCA) and stenting (n=3) or coronary artery bypass graft (CABG) surgery (n=5). In the group with previous history of CABG surgery (n=5); a total number of 13 grafts were to be evaluated, with an average of three grafts per patient. The major inclusion criterion was scheduling for conventional coronary angiography. Patients enrolled in this study had to fulfill the following criteria; sinus heart rhythm, hear rate below 70beats/min, able to hold breathe for accepted time (30 seconds) and normal serum creatinine. Exclusion criteria were; irregular heart beats (arrhythmias), contra-indications to iodinated contrast material including known allergy and renal insufficiency (serum creatinine more than 1.4mg/dl), contra-indications to radiation exposure i.e. pregnancy, respiratory impairment (inability to withhold breathing), and unstable clinical status or marked heart failure.
Patient preparation: All patients are instructed to fast 6 hours prior to the examination and medications are not to be discontinued. Before the examination; the heart rate was evaluated. Patients with pre-examination heart rates above 65-70beats per minute were given cardioselective beta-blocker; 100mg of Metoprolol or atenolol orally 1 hour before the study to obtain a stable low heart rate, provided that contra-indications to B-blockers are excluded. If the heart rate was still above 75beats per minutes, the examination was postponed to anther setting. Nitroglycerine was not administrated prior to the study because, in spite of its coronary-dilator effect that enhances visualization; the drug has the potentiality to increase the heart rate and also falsely increases the estimated diameter reduction of the stenotic lesions. All steps of the study were explained in details for each patient. To evaluate patients ability of breath-withholding for the time of examination; they were required to perform a deep inspiration and to continue to hold their breath without pushing (i.e.,Valsalva maneuver). During this trial, the patient was observed for compliance and the electrocardiogram for significant changes. Contrast material: A bolus of 120ml of non-ionic contrast (Ultravist 370 Schering, Berlin, Germany), was injected through 18-gaugc canula into an upper limb vein (right antecubital vein is preferred when available) with a flow rate of 4-5mL/sec using a programmed power injector pump. Another bolus of 30ml saline is injected following the contrast as a chaser bolus used to wash the contrast from the right side of the heart during visualization of the RCA. Scan protocol: CT scans were done at Cairo radiology center using a sixteen channel multi-detector row CT scanner (CT sensation; siemens medical systems). Scanning parameters were: 16 x 0.75mm collimation, pitch 2 according to the estimated heart rate (decreased at higher HR to increase the image segments overlap yet on the expense of increased total scanning time and breath hold), tube rotation time of 370msec, tube voltage of 120KV (increased to 140KV in obese patients and in cases with previous CABG, owing to presence of dense metallic issues near the grafts) and current of 350mA. The field of view is 250mm with an image matrix of
Sherif M. Maher, et al.
391
256 x 256 pixels. Scanning direction; cranio-caudal. The patient is then positioned supine on the CT table. ECG leads are fixed at the four corners of the pericordium. All reconstructions are performed using the retrospective ECG gating. For this technique; an ECG must be recorded simultaneously through out the duration of the scanning. Each stage of acquisition was preceded by the same gentle hyperventilation protocol, which took 20 seconds. First; a localization scan (scanogram) is performed that yields an antero-posterior view of the chest. It is used to position the imaging volume of the coronary arteries that extends from the level of the carina down to about 1cm below diaphragm. The center of the field of view is 2cm to the left of the dorsal spine on the AP scout. In cases with previous CABG operation; the scanning range was extended to include the entire course of the arterial and venous grafts. An initial step of non-contrast CT examination of the heart was performed for all patients in order to detect and quantify coronary calcifications through the volume extended from below the carina to the apex of the heart. Acquisition parameters were ECG gated at 60 % of the RR interval, 370ms gantry rotation, 16 x 0.75mm collimation, 350mA, and 120kV. To minimize the total effective patient radiation dose, this stage of the scanning was conducted with a relatively low tube current. Cases with high coronary calcium score (above 1000) were shifted directly to conventional coronary angiography. In a third step, semi-automated determination of the starting time using the Bolustracing technique was utilized in all patients. It entails injection of the whole volume of the utilized contrast material as a one bolus at the pre-determined rate. After a delay of about 10 seconds from the start of injection (time estimated for the contrast to reach the great vessels of the chest, being variable according to the site of the canula, rate of injection, body built and heart rate); series of axial images at the level of the origin of the left main coronary artery is acquired with an interval of 1 second between subsequent images. The density within the ascending aorta is monitored in each axial image on a real time base while the region of interest (ROI) carefully avoiding the athermanous calcifications. Time-attenuation curves were generated. When the density within the ascending aorta exceeds 100HU (i.e. the contrast started to arrive), the scanning is triggered with a delay of further 3 seconds (time needed for the table movement to the cranial start position while the patient is instructed to hold breathing). This time delay
also allows for increase in the contrast concentration at the ascending aorta and coronary arteries. Finally; the volume data set for coronary artery visualization is acquired in a spiral mode with simultaneous acquisition of 16 parallel slices at certain scan parameters. During the helical scan; the ECG signal was recorded digitally. Patients were automatically instructed to maintain an inspiratory breath hold while the CT data and the ECG trace were acquired. The volume between the level of the carina and the base of the heart (120mm) was covered in 18+2 seconds in the 16 detector-row CT scanner. In cases with known previous CABG surgery; the mean scanning time was increased to 30+2 seconds in the 16 detector-row CT scanner. Mean heart rate during time of examinations was 65beats per minutes. Bolus timing procedures and main acquisitions were performed in all patients with no clinically important adverse reactions to contrast material. Despite that the CT scan is completed within a couple of minutes; the total examination time (including the patient preparation and instruction) was around 20 minutes. Scan parameters: All CT angiographic examinations were done on CT sensation 16-detector row CT scanners (n= 15; 10 cases for evaluation of the native coronary arteries and 5cases for evaluation of the bypass grafts as well) (Siemens Medical Systems, Germany). Scanning parameters were: 16 x 0.75mm collimation, pitch 2 according to the estimated heart rate (decreased at higher HR to increase the image segments overlap yet on the expense of increased total scanning time and breath hold), tube rotation time of 370msec, tube voltage of 120KV (increased to 140KV in obese patients and in cases with previous CABG, owing to presence of dense metallic issues near the grafts) and current of 350mA. The field of view is 250mm with an image matrix of 256 x 256 pixels. Scanning direction; cranio-caudal. Image reconstruction: Cardiac phase pre-selection in this study was influenced by the results obtained by Achenbach, et al., 2000 [5] with regard to coronary artery motion on transverse electron-beam CT images and also those obtained by Kopp A.F., et al. 2001 [6]. They found that LMT could be clearly visualized at almost all time points in the cardiac cycle. For most patients, LAD was best visualized at 60%70% of the cardiac cycle. The time point for best
392
image quality of the RCA was early in diastole at 40% of the cardiac cycle. The LCX showed optimal image quality at 50% of the cardiac cycle with considerable inter-patient variation. And so, for each patient; 6 data sets were created during different time instants of the cardiac cycle (30%, 40%, 50%, 60%, 70% and 80% of the R wave to R wave interval). Time needed to reconstruct an axial image is about 2 seconds; and so it takes about 30min to create this large number of images. For each individual coronary artery; the data set containing the fewest motion artifacts (on the bases of cross sectional images) was used for further creation of the reconstructed images and evaluation of the coronary artery. The average time of the study was 20 minutes. Another 60 minutes were spent for result evaluation at the workstation. Data evaluation: The reconstructed axial images at different points of the cardiac cycles are sent to an off-line workstation. Dedicated cardiac reconstruction software was used to evaluate the coronary arteries. Total calcium scores of all patients were calculated with the dedicated software and expressed as Agatston scores. Using the sequential axial images; any tissue above the 130HU occupying a minimum of 0.5 mm2 could be identified along the anatomical course of a coronary artery is considered as coronary calcification, and hence, highlighted and analyzed using the software. The Agatston score is a commonly used scoring method that calculates the total amount of calcium on the basis of the number, areas, and peak Hounsfield units of the detected calcified lesions. Visibility of the coronary artery segment was considered good when there is sharp delineation from the surrounding structures, nearly artifact-free course of the segment, with less blurring even in its peripheral sections and
sufficient contrast detected between the vessel lumen and wall. Visualization was considered adequate in presence of image-degrading artifact that didnt interfere with evaluation with moderate confidence and poor in the presence of imagedegrading artifacts when the evaluation is possible yet only with low confidence. The examined segment was considered non-assessable when the image-degrading artifacts were severe enough to prevent differentiation between the significant stenosis and occlusion on one hand and the normal segment or mildly atherosclerotic lesions on the other hand. These segments were excluded from the study. When there is doubt regarding the image quality, it was determined by measuring the mean CT density within the artery lumen and the mean CT density in the connective tissue immediately next to the coronary artery. These values are referred to the standard density at the ascending aorta. Better image quality was achieved with larger difference between the arterial lumen and the surrounding tissues and with higher contrast density within the ascending aorta. Identification of coronary artery segments was based on the model suggested by the American Heart Association (AHA) (Fig. 1), where the RCA shows proximal (1), middle (2) and distal (3) segments and the PDA branch (4). The left coronary system is formed by the left main trunk; LMT (5) that bifurcates to LAD and left circumflex. The LAD has proximal (6), middle (7) and distal (8) segments. It gives off at least two sizable diagonal branches (9) and (10). The left circumflex artery has proximal segment (11) before it gives off the first obtuse marginal artery (12). The distal segment (13) turns on the inferior surface of the heart. LCX may give additional obtuse marginal branches (14) and (15) that are not included in statistics of this study owing to their usual small size.
2 3
RCA Circumflex Fig. (1): Segmental anatomy of right coronary artery (RCA) (lateral view), and left coronary artery (right anterior oblique view) with left main trunk (LMT), left anterior descending (I.AD), and left circumflex (LCX) according to American Heart Association (AHA) [6].
393
The coronary artery segments are further classified into proximal [RCA 1, LMT 5, LAD 6], middle [RCA 2, LAD 7, LCX 11], distal [RCA 3, LAD 8, LCX 13] and branches [including the diagonals of LAD (9 and 10), obtuse marginal of the left circumflex (12) and posterior descending artery (4). Results Multislice CT coronary angiography was performed to all patients without complications. In our study, 15 patients were included and here are the following results. The total number of examined segments was 195 considering 13 segments in each of the examined 15 coronary systems according to the model suggested by the American Heart Association (AHA). However, in 4 patients; the second diagonal artery was not identified at CT. Hence; the total number of examined segments is reduced to 191 (Table 1). Each segment was evaluated at the axial images at proper R-R interval, and then different postprocessing techniques, including multi-planner and curved reformations, maximal intensity projection and volume rendering, some or all of them were employed in an integrated manner to reach the diagnosis. The coronary segments were classified as evaluable or non-evaluable depending on the image quality (being mainly assessed at the sequential axial images). The non-evaluable arteries are discarded from the statistical analysis. The evaluable arteries are categorized as being either (A) Normal (smooth parallel or tapering arterial walls), (B) Showing either coronary artery wall irregularities or non-significant stenotic lesion(s) (with less than 50% diameter reduction) (C) Showing significant stenotic lesion(s) (defined as equal to or more than 50% diameter reduction; according to AHA classification), or (D) Occluded. The reconstruction window: For each patient; 6 data sets were created during different time instants of the cardiac cycle (30%, 40%, 50%, 60%, 70% and 80% of the R wave to R wave interval). For each individual coronary artery; the data set containing the fewest motion artifacts (on the bases of cross sectional images) was used for further processing of the reconstructed images and evaluataon of the coronary artery. The distribution of the image reconstruction window with least motion artifacts relative to the cardiac cycle is given in (Table 1).
The left main and LAD arteries were best visualized in mid-diastole at 60-70% for most patients. The time point for best image quality of the right coronary artery was different being at 40%. The left circumflex artery showed optimal image quality most frequently at 50-60%. The total number of examined segments was 195 considering 13 segments in each of the examined 15 coronary systems according to the model suggested by the American Heart Association (AHA). However, in 4 patients; the second diagonal artery was not identified at CT. Hence; the total number of examined segments is reduced to 191. To start with, each segment was evaluated at the axial images at proper R-R interval, and then different post-processing techniques, including multi-planner and curved reformations, maximal intensity projection and volume rendering, some or all of them were employed in an integrated manner to reach the diagnosis. Diseased vessels per case: Among the 15 patients; the CT coronary angiography showed one vessel disease (i.e. significant stenotic lesion or occlusion) in 3, and three vessel disease in 12 patients. Distribution of the diseased vessels: CT coronary angiography revealed a total number of 50 stenotic segments, 39 of them show significant stenosis while the other 11 segments show non significant stenosis and other 5 segments show complete occlusion. The total number of diseased segments is 55 segments. Their distribution is tabulated in (Table 2). Evaluation of coronary stents: Seven coronary stents in 3 patients were evaluated for patency, in-stent re-stenosis and occlusion. The stents were distributed as 2 in the RCA, 3 in the LAD and 2 in the Cx arteries. According to the CT angiographic findings; 5 stents were patent while the other two showed in-stent re-stenosis depending on the indirect sign of faint contrast opacification distal to the stent rather than actual detection of lumen narrowing. Group of patients with previous CABG surgery: The total number of examined patients with previous CABG surgery is 5 having a total number of 12 conduits with an average of 2 conduits per patient. In 4 of these conduits; the left internal mammary artery (LIMA) was used to enforce the blood supply in the left coronary system being inserted at the distal LAD segment. One patient has right internal mammary artery (RIMA) used
394
to enforce the blood supply in the left coronary system being inserted at the circumflex artery. 7 arterial and venous grafts were also studied; 2 of them were inserted distally at the LAD, 2 at the RCA, one at the PDA, one to the circumflex artery and finally, another one to the OM. Evaluation of the LIMA and RIMA: There were 4 cases of LIMA and one RIMA case. The LIMA anastomosis was to the distal LAD artery while the RIMA anastomosis was to the circumflex artery. The evaluation consists of evaluation of the body and the distal anastomosis to the native coronary arteries. In our study, the LIMA
Table (1): Showing distribution of the image reconstruction window with least motion artifacts relative to the cardiac cycle. Cardiac cycle 30% LAD Cx RCA 40% 6 50% 1 7 4 60% 8 7 5 70% 5 1 80% 1 Total 15 15 15
and RIMA conduits were patent as well as their distal anastomotic sites (Table 3). Evaluation of the bypass (arterial and venous) grafts: The 7 bypass grafts included in the study were evaluated regarding their proximal anastomotic site at the ascending aorta, body and the distal anastomotic sites. 6 of the 7 grafts showed patent body as well as proximal and distal anastomotic ends while the other case shows some degree of stenosis in its proximal anastomotic end with the ascending aorta and patent body and distal anastomotic end with the RCA (Table 4).
Table (2): Showing the distribution of the diseased coronary artery segments. Segments Sig. stenosis 1 2 3 4 5 6 7 8 9 10 11 12 13 6 5 6 1 3 5 5 0 0 0 0 0 4 1 1 3 1 1 1 0 1
Nonsig. stenosis 1 1 2 0 1 4 0 0 0 Occlusion 1 0 0 0 0 1 0 0 0
Table (3): Showing the CT angiographic findings for each coronary segment for all the examined 15 cases where. seg. case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 RCA 1 SS A P A O SS SS P SS P NSS P SS SS P 2 SS A P A ST SS SS ST A P P P SS SS NSS 3 SS A SS A A SS SS NSS A P P P SS SS NSS 4 P P P A A SS A P P P P P A A P 5 P SS P SS P SS NSS P P P P P P P P 6 SS SS A O ST SS NSS A NSS NSS P NSS SS SS ST LM and LAD 7 SS P SS P P SS P ST P P P P SS SS P 8 P P A P P P P P P P P P A P P 9 P A P P A P P P P P P P A P P 10 P NA NA P A P P P P P P P A NA NA 11 P SS O NSS ST SS A SS P P P P SS A A Cx 12 SS P O P A SS A ST NSS P P P SS P A 13 SS P O P A P A P P P P P A P A
P = Patent. A = Atherosclerotic.
NSS = Nonsignificant stenosis. SS = Significant stenosis.
ST = Stent. NA = Nonassessable.
Table (4): Showing the CT angiographic findings for the five LIMA and RIMA conduits used to enhance the coronary circulation. Case 1 LIMA 2 RIMA 3 LIMA 4 LIMA Body Patent Patent Patent Patent Distal anastomosis Patent Patent Patent Patent
Table (5): Showing the CT angiographic findings for each of the arterial and venous grafts used to enhance the coronary circulation. Case 1 2 To LAD LAD OM RCA RCA CX PDA Proximal end Patent Patent Patent Patent Stenosis Patent Patent Body Patent Patent Patent Patent Patent Patent Patent Distal end Patent Patent Patent Patent Patent Patent Patent
3 4
395
Case (1): Male patient 51 years old, not known to be hypertensive or diabetic, presented for the last two months by intermittent attacks of mild chest pain and so he came to perform CT coronary angiography. There is no history of previous cardiac operations. The total estimated calcium score was 23.2. The LAD artery shows two atheromatous plaques, a soft and another mixed one in its proximal segment. They cause about 30 and 40% lumen reduction respectively.
The RCA and the Cx arteries are patent and of normal caliber.
396
Case (2): Male patient 52 years old, known to be diabetic and hypertensive, presented for the last one month by intermittent attacks of staggering chest pain and so he came to perform CT coronary angiography. There is no history of previous cardiac operations. The total calcium score for the coronary arteries was 272.7 represented by calcific plaques seen at the RCA, LAD and Cx arteries. The RCA is a dominant artery that gives PDA branch. It shows a soft plaque that arises shortly after its origin and it causes significant lumen reduction more than 60% Two other mixed plaques are seen in the middle segment of the artery that causes significant about 80% lumen reduction The distal segment of the artery as well as the PDA is seen diffusely attenuated.
The Cx artery is diffusely attenuated and its distal half shows multiple consecutive atheromatous calcific plaques markedly encroaching on the lumen and causing significant lumen reduction.
The distal portion of the LM coronary artery shows a mixed plaque that is seen extending in the proximal portion of the LAD and causes less than 50% lumen reduction.
397
Case (3): Male pt 61yrs old is known to have IHD & he has a history of graft operation to the coronary arteries 2yrs ago. Diabetic & on treatment. He is performing CT coronary angiography as a check up. A large atheromatous mixed soft & calcific plaque is seen at the distal part of the LM coronary artery & it is seen extending to the proximal portion of the LAD subtotally obliterating it. A venous graft is seen connected to the ascending aorta while its other end is seen anastomosed to the LAD artery, which shows adequate opacification by contrast material distal to the graft.
The Cx artery shows a mixed atheromatous plaque at its ostium markedly encroaching upon the vessel lumen
The RCA is a dominant artery that shows subtle atherosclerotic changes of no significance.
398
Case (4): A male patient 59 years old is known to be diabetic but not hypertensive. He is complaining of intermittent attacks of mild anginal pain in the form of discomfort. His doctor advised him to perform a conventional cardiac catheterization, but he refused and appreciated to have a CT Coronary angiography performed to him. He has no history of cardiac operations. The left and right coronary arteries are seen arising from the ascending aorta by a short common trunk. The LM coronary artery then bifurcates into LAD and Cx arteries.
The RCA shows small soft atheromatous plaques in its proximal portion causing wall irregularities and insignificant lumen reduction.
The LAD and Cx arteries are patent and of normal caliber showing tiny calcific foci in their walls causing no significant lumen reduction. The Cx artery is a dominant artery giving OM and PDA branches.
399
Case (5): Male pt 55yrs old is known to have an ischemic heart disease. He had an operation of stent application done for him 5 months ago, & recently he has a classic anginal chest pain that is only relieved on nitrates. He is diabetic & under controlled treatment. The RCA is a dominant artery. A hypodense thrombus is seen at the mouth of the RCA causing almost total occlusion of the artery. This lesion is followed by multiple consecutive metallic stents. Distal to these stents, the artery and its PDA branch are diffusely attenuated.
The Circumflex artery shows a mixed osteal lesion that causes significant stenosis. Distal to this stenosis, there is a metallic stent in the OM branch which failed to opacify by contrast distal to the stent.
The LM coronary artery is patent and it bifurcates into LAD and Cx arteries. The LAD shows a proximal stent within with distal patent well opacified artery with contrast.
400
Case (6): Male pt 57yrs old is known to be a heavy smoker, diabetic & hypertensive, came complaining of a classic anginal chest pain. He experienced the pain several times in the last month that was only relieved by sublingual nitrates. He has a conventional cardiac catheterization done for him & the RCA couldnt be selectively injected by the doctor so, he sent him to perform a multislice CT coronary angiography. The total calcium score for the coronary arteries was 425.7 represented by multiple calcific plaques in the RCA, LAD & Cx arteries. The RCA is a dominant artery. It shows an abnormal origin where it arises from the left aortic cusp, just anterior to the LM coronary ostium, then it courses a malignant course between the root of the pulmonary artery & the ascending aorta, where it is seen markedly attenuated at this area. The middle segment of the RCA shows mixed atheromatous plaques predominantely soft causing more than 60% lumen reduction.
The LM coronary artery is patent and of normal caliber. It bifurcates into LAD & Cx arteries. The LAD artery shows two mixed atheromatous plaques in its proximal segment causing >60% lumen reduction.
The Cx artery is patent and it shows atheromatous wall calcification with no significant lumen reduction.
401
Discussion While selective conventional coronary angiography will remain vital for planning and guiding catheter-based and surgical treatment of significantly stenotic coronary lesions or occlusions, the comprehensive and serial assessment of asymptomatic or minimally symptomatic stages of coronary artery disease (CAD) for preventive purposes will eventually need to rely on noninvasive imaging techniques. Cardiovascular imaging with tomographic modalities, including computed tomography (CT) and magnetic resonance imaging (MRI), has great potential for providing valuable information [4]. The first generations CT scanners used a sequential acquisition pattern, also known as stepand-shoot. These scanners produced an axial image while the table remained motionless and for each other slice the table moved to a different position. This sequence of events was repeated throughout the scan range. CT scanning was both time consuming and extremely sensitive to respiratory movements and was therefore not suitable for cardiac imaging. The total scan time was significantly reduced with the introduction of spiral CT scanners. The scan is performed while the patient is continuously moving at a pre-defined speed through the scanner. These scanners acquire volumetric data and cross sectional images can be reconstructed later for any anatomic region. This configuration significantly reduced the total scan time, but still was not fast enough for cardiac scanning. Electron beam CT (EBCT) was originally developed as a research togl to study the cardiac physiology. These scanners use no moving parts for generation of the tomographic images. This technology allows very short scanning times of 50 to 100 milliseconds per image, and with this high temporal resolution, tomographic images of the beating heart can be obtained with minimal blurring or motion artifacts. Typical cardiac studies with EBCT include coronary calcium scoring, CT angiography of the coronary arteries, studying cardiac anatomy, wall motion abnormalities and myocardial perfusion [7]. Although a significant correlation between the amount of calcium score and the degree of atherosclerotic wall changes has been demonstrated, the diagnostic value of this technique is still controversial. And so, the mere identification of high grade coronary calcification alone does not inevitably deliver significant information concerning the localization of coronary plaques and the degree
of stenosis. Further more, it has been often demonstrated that calcified plaques are at less risk of spontaneous rupture than the non-calcified plaques and thus bear no higher cardiac risk. On the contrary, it is mostly the soft plaque that ruptures spontaneously causing an acute cardiac incident. Multislice CT is a recent development in the spiral CT. MSCT scanners are equipped with multiple and thinner detector rows, and has a faster xray tube rotation speed. These technical advances have an important impact on image quality [8]. The ability of MSCT coronary angiography to assist in the identification of asymptomatic, sub-occlusive CAD, above and beyond coronary calcium evaluation, is clear. Identifying disease in the asymptomatic phase, in low-and intermediate-risk patients, allows the targeting of therapies including statins and antiplatelets to secondary prevention goals, resulting in reduction of coronary ischemic events. It is also clear that MSCT angiography has the capability to assist in the detection of obstructive disease in native coronary arteries and bypass grafts [9]. Challenges in evaluating the coronary arteries at CT are their small size and tortuous courses and location adjacent to the moving heart. The vessels are typically 2-4mm in diameter and are parallel, oblique, or perpendicular to the axial plane in portions. In addition, they are adjacent to both atria and ventricles and therefore may be affected by cardiac motion in different phases of the cardiac cycle. Possible solutions are imaging on scanners with an increasing number of detector-rows, faster rotation speeds and reconstructing multiple sets of images obtained in different phases of the cardiac cycle [10]. The duration of the diastolic phase with little cardiac motion is inversely related to the heart rate, and hence, the heart rate plays an important role regarding the image quality during CT coronary angiography. Studies based on four-detector row CT found that the upper limit of the heart rate at which motion artifacts can be consistently minimized ranges between 65-75beats per minute [11-15]. In this study; the pre-examination heart rate was evaluated, those with rates above 65beats per minute were given cardio-selective (B-blocker orally 1 hour before the study to obtain a stable low heart rate, provided that contra-indications to B-blockers are excluded, that agree with [16]. If the heart rate was still above 75beats per minutes, the examination was postponed to another setting. Nitroglycerine was not administrated prior to the
402
study, in contrast to [17,18] because, in spite of its coronary-dilator effect that enhance their visualization; the drug has the potentiality to increase the heart rate and also falsely increases the estimated diameter reduction of the stenotic lesions as it dilates the healthy segments according to [15]. Regarding the distribution of the image reconstruction window with least motion artifacts relative to the cardiac cycle; the left main and LAD arteries were best visualized in mid-diastole at 60-70% for most patients. The time point for best image quality of the right coronary artery was different being early in diastole at 40%. The left circumflex artery showed optimal image quality most frequently at 50-60%. This agrees with Kopp A.F., et al., 2002 [17] who found the same distribution. Motion artifacts were significantly more frequent in patients with heart rates of more than 70bpm than in patients with lower heart rates. The analysis, interpretation, and documentation of coronary CT examinations are complex and not sufficiently standardized. Reviewing the axial images is an important step in interpretation of the CTCA, but because of the tortuous course of coronary arteries, reviewing the acquired transverse sections alone is often not sufficient. Multi-planner image reformation at the sagittal, coronal and oblique planes can be displayed. Reformatted images following the major cardiac axes or the course of individual coronary artery (curved plane) are also possible. Coronary arteries are typically divided into smaller segments according to accepted angiographic classifications. These segments are then individually analyzed in longitudinal and cross-sectional planes. Diagnosis of luminal stenosis and vessel wall changes relies on these two-dimensional projections. Advanced image procession permits display of the entire three-dimensional data set of coronary arteries. Volume-rendered images facilitate the assessment of spatial orientataon but provide only limited information about the arterial lumen and the vessel wall [4]. In this study, there was no attempt to determine the relative contributions of the different image post-processing tools to the final diagnosis. To start with, each segment was evaluated at the axial images at the proper R-R interval, and then different post-processing techniques, including multi-planner and curved reformations, maximal intensity projection and volume rendering, some or all of them were employed in an integrated manner to reach the diagnosis.
The published studies revealed high negative predictive value and high specificity of CT coronary angiography. The high NPV means low false negative results and the high specificity means high true positive results. In other words; when a segment is interpreted as negative at CT (meaning normal or mildly atherosclerotic), it is unlikely to show significant stenotic lesion or occlusion at conventional angiographic examination, hence the CT angiography is a good negative test. The importance of this fact is to determine the potential role of the multislice CT angiography as a noninvasive tool for initial evaluation of the coronary system in patients with clinically moderate probability for coronary artery disease who show equivocal results at the stress-tests (stress ECG, stress perfusion scan and stress echo) before proceeding to the conventionad (invasive) angiography. In such cases, negative CT examination would help the patient to avoid the unnecessary invasive test. The comparatively lower values for the positive predictive value are attributed to the relatively high incidence of the false positive results, meaning that some of the apparently significant lesions at CT may actually be not more than mild atherosclerotic plaques or even artifacts caused by dense calcification, metallic material (stent or clips) or respiratory/cardiac motion. The high false positive results at this study may be attributed to the tendency to appreciate rather than under-estimate coronary plaques in order not to miss lesions. Lepor N.E. and Madyoon H. 2005 [19], stated that from their own experience using both the 16and 64-slice CT scanners, they have observed an improved ability to obtain coronary artery images in patients that are hard to image (e.g. the obese, those unable to maintain long breath holds). These scanners have also proved useful in coronary arteries that are calcified or had metal stents placed within and in patients with faster heart rates. It is now clear that non-invasive CT angiography of the coronary arteries requires the use of, at minimum, the 16-slice scanner, with improved imaging observed with the 64-slice CT. The significant improvement in sensitivity and specificity in studies using the 64-rather than the 16-slice scanners could be attributed to the reduced tube-rotation time (330ms instead of 420ms) with consequent improvement in the temporal resolution (165ms instead of 210ms). Another important factor is the improved spatial resolution owing to the use of the least available slice thickness (0.5 and 0.6mm instead of 0.75mm). However; the positive predictive value is still low with the 64-slice scanners. This, again, would be attributed to the tendency
403
to consider the coronary lesions whose CT interpretation was uncertain, resulting in a number of false-positive outcomes, rather than underestimating these lesions and thereby "missing" lesions, which may have serious consequences in a symptomatic patient population. Coronary artery bypass grafts (CABG) are important issues being suitable for imaging with MSCT angiography. Patency or occlusion of grafts can be established by the presence or absence of contrast material within, respectively. Because of their relatively large size and less motion with heart beats; the grafts are generally better evaluated for the presence and severity of stenosis. Abeamhardening artifact from metallic surgical clips may obscure the adjacent portion of a bypass graft [4]. To evaluate the role of MSCT in assessment of the coronary bypass grafts, 20 performed a prospective comparison of MSCT with invasive coronary angiography in 65 patients with CABG, using a scanner able to acquire 4 parallel slices of the heart at 1-mm collimation. Accuracy of MSCT in the detection of occluded conduits was very high, with sensitivity and specificity rates of 97% and 98%, respectively; however, 38% of 124 patent grafts couldnt be evaluated by MSCT angiography because of insufficient breath holding with respiratory motion artifacts (11 cases), arrhythmias (5 cases), poor opacification owing to improper amount or timing of contrast (7 cases), and metallic clips (17 cases). Within the limits of conduits judged evaluable, sensitivity and specificity in the detection of significant stenosis were 75% and 92%, respectively. When conduits judged unevaluable were included in the analysis, the overall accuracy for detection of stenoses was quite low, with a sensitivity of 48%. In this study, the total number of examined patients with previous CABG surgery is 5 having a total number of 12 conduits with an average of 2 conduits per patient. In 4 of these conduits; the left internal mammary artery (LIMA) was used to enforce the blood supply in the left coronary system and in one case a RIMA was used, while the remaining 7 conduits were venous and arterial grafts. During their courses; all patent LIMA, venous and arterial grafts were correctly identified at the CT angiographic examinations. In a study performed by Willmann J.K., et al. 2004, to evaluate the influence of different reconstruction intervals of MSCT coronary angiography on image quality of different segments of various types of coronary artery bypass grafts, they found that the best image quality of all segments was
obtained at a reconstruction interval of 50%-70% of the cardiac cycle. Image quality of the proximal segment did not vary significantly with different reconstruction intervals, whereas image quality of the graft body and distal anastomosis changed significantly with varying reconstruction intervals. Accuracy of CT angiography for detection of graft patency was 95%. In our study, all the axial sets of images that showed best image quality ranged between the 50 and 70% of the cardiac cycle in agreement with William, et al. In-stent restenosis attributable to intimal hyperplasia occurs at a relatively high rate. This problem has led to the routine use of the invasive and costly angiography for surveillance of stent patency. Highattenuation metallic coronary stents precludes confident detection and grading of in-stent restenosis with CT due to blooming effect. However, differentiation between stent patency and occlusion and evaluation of stenosis at the leading or trailing ends of a stent are possible. In addition, stent patency can be inferred on the basis of presence or absence of coronary luminal enhancement distal to the stent [4]. In a study performed by Hong C., et al., 2004 the authors investigated the ability of the MSCT coronary angiographic examinations to assess the coronary stent patency as well as detection and quantification of the in-stent restenosis. They also tested the CT ability to accurately measure the in-stent luminal diameter. Nineteen patients with 26 coronary stents underwent ECG-gated CT angiography with a 16-detector row scanner 1-3 weeks after stent placement. CT images depicted the lumina of 20 stents in 14 patients. CT attenuation measured in the stented lumen was higher than the attenuation in the proximal and distal unstented lumen (owing to the beam-hardening artifacts by the metallic stents). Estimated values for in-stent luminal diameter were lower with CT than with conventional angiography. Finally; they concluded that the visualization of the in-stent lumen at CT angiography with a 16-detector row scanner allows assessment of coronary artery stent patency yet the accurate evaluation of the in-stent restenosis is quite challenging.
[11] ,
This study included 7 coronary stents. According to the angiographic findings; 5 stents were patent and two showed in-stent re-stenosis. All of the five patent stents were diagnosed at CT while the two cases of in-stent re-stenosis were successfully detected at CT. A well-recognized limitation in the assessment of coronary stenosis with CT angiography is related
404
to dense coronary arterial calcification. The resulting "blooming" effect causes difficulties in assessing adjacent plaque structures and patent lumen, potentially resulting in a false-positive evaluation of stenosis (overestimation of the degree of narrowing) [4]. However, the presence of high calcium score itself has its own diagnostic value, identifying patients who may benefit from stress nuclear myocardial perfusion and conventional angiography to assist in detection of obstructive CAD [21]. In our study; all cases underwent coronary calcium score prior to CT angiography. Cases with high calcium score (1000 or more) were shifted directly to conventional angiography. In a study done by Choi H.S., et al., 2004 [22] to identify artifacts and pitfalls that commonly degrade image quality and that could result in misinterpretation; CT coronary angiography using MSCT scanner with four detector rows in 110 consecutive patients were analyzed. The problems identified were classified into four broad categories: (A) Motion-related artifacts due to cardiac, respiratory$ or other body motions; (B) Beam-hardening effects caused by metallic implants, severe calcifications, or air bubbles in the pulmonary artery that obscured the underlying coronary arterial lumen; (C) Structural artifacts produced by adjacent contrast material-filled structures and overlying vessels; and (D) Artifacts that resulted from technical errors or limitations. The most frequently observed artifacts were those related to cardiac motion. The most effective methods for minimizing cardiac motion artifacts are pre-medication with B-blockers to maintain optimal heart rate during scanning and optimal selection of the reconstruction window. In order to overcome these pitfalls; [23] recommended the usage of contrast agent with the highest iodine concentration available to improve the opacification of the smaller vessels and the proper visualization of the coronary side branches and collateral pathways [16]. Stated that the choice of a proper reconstruction window for each coronary artery is the key factor for reliable evaluation of the coronary lesions in CT studies, which was also pointed out by Zhang S.Z., et al., 2005, Hamoir X.L. et al., [24,25]. Radiation dose: Radiation exposure at cardiac CT has received special attention recently, particularly, in light of the current and potentially expanding future use of this modality as a screening tool for CAD in apparently healthy asymptomatic individuals. Relatively high radiation exposure occurs with retrospectively
ECG-gated cardiac CT because of continuous Xray exposure and overlapping data acquisition at a slow table feed. However, the radiation dose at retrospectively ECG-gated cardiac CT can be substantially reduced by means of reduction of tube output in each cardiac cycle during phases that are of less importance for image reconstruction i.e. systole "ECG-gated dose modulation" [26]. Pitfalls and artifacts in multislice CT coronary angiography: Various artifacts can degrade image quality at CT coronary angiography. Artifacts at CT angiography included cardiac pulsation, rhythm disorders, respiratory issues, partial volume averaging effect; high attenuation entities, inappropriate scan pitch, contrast material enhancement and patient body habits are important factors hampering accurate diagnosis [27]. Rhythm disorders involve apparent arrhythmia such as atrial fibrillation of recent technical advances, reliable coronary imaging can be performed only in patients with normal sinus rhythm at the time of examination. Even in these patients, however alteration of the heart rate during data scan creates data sets in a slightly different cardiac cycle, leading to a section gap and coronary pseudostenosis [13]. Section gaps exaggerate the rapid moving segment and higher heart rate, although a significant heart rate increase leading to a section gap cannot be determined, the association of section gaps and heart rate increase is frequently recognized. Nonassessable segments are frequently observed in arrhythmia, although successful coronary CT angiography can be performed even in atrial fibrillation, when the average heart rate is very low. Apparent multiple section gaps in imaging data sets are called banding artifacts. Banding artifacts are observed even in motion free imaging data sets. The most frequent causes of banding artifacts arc arrhythmia, no breath hold, and alteration in heart rate during acquisition. However, the occurrence of banding artifacts cannot be predicted prior to scanning in most cases. Long acquisition time is associated with increased frequency of alteration in heart rate during data acquisition. Thus 16-detector row CT, with its fast scanning capability, reduces the prevalence and degree of alterations in heart rate data acquisition [27]. Motion artifacts other than those from cardiac motion result either from respiratory or voluntary motions that is generally preventable with careful instruction of the patient. Artifacts caused by
405
respiratory or voluntary movement affect both multi detector row and single detector row CT equally, have unpredictable patterns, and cannot be corrected with image data reconstruction methods. These artifacts usually cause a severe degradation of image quality in two ways; first, physical motion causes blurring and severe gaps and overlaps between sections. Second, respiratory or voluntary movement produces a phase mismatch artifact similar to that produced by acceleration of the heart rate toward the end of a long breath hold. Respiratory artifacts frequently affect depiction of the inferior aspect of the heart toward the end of the scanning performed over a relatively long time period. This is a common problem in the evaluation of coronary artery bypass graft, because of the border anatomic coverage needed for bypass evaluation than for evaluation of the native coronary artery. However, artifacts related to pulmonary or voluntary motion can be distinguished from cardiacmotion related artifacts in that stepladder effect in the former group is apparent in the anterior chest wall, as well as along the heart border [28]. The most commonly encountered beam hardening and structure related artifacts are those produced by surgically implanted high attenuating materials or by contrast enhancement in natural structures. Beam hardening effects are usually caused by metallic objects such as clips, markers, and wires used in coronary artery bypass surgery or by coronary stents, but they may also be caused by naturally occurring coronary calcifications. The use of nonmetallic surgical materials also helps to ensure the accuracy of coronary bypass graft with multi-detector row CT. The nature and extent of artifacts caused by intra-coronary stents depend largely on the material of which the stent is made, the lumen visibility varies depending on the type of stent; stent made of or coated with gold causes the most severe artifacts [29]. Both high and low-attenuating artifacts may be exacerbated by motion or by inappropriate selection of the reconstruction, or they may be minimized with reduced motion and optimal reconstruction window. Any negative effects of beam hardening or structure-related artifacts on the accuracy of image interpretation can be avoided with a review of axial source images [30]. Artifacts that result from technical errors in image data acquisition and interpretation may be avoided with appropriate planning and execution of the scanning procedure, including instruction and practice of the patient in breath holding, as well as the optimal selection of anatomic coverage,
scanning delay, pitch, and reconstruction window. To improve interpretations, it is essential to use reconstructions tailored to each case. Insufficient anatomic coverage in the evaluation of bypass grafts can be avoided with an awareness of surgical records and with scout imaging of the entire thorax to determine the area that should be included in CT angiography. Inaccurate estimation of the circulation time, which may result in a useless study, can be easily avoided with a 5-second scanning delay after peak enhancement of the aorta, because graft vessels enhance later than do native coronary arteries. Artifacts that are related to flow dynamics also limit the usefulness of multi-detector row CT coronary angiography. Because multi-detector row CT coronary angiography does not depict the temporal course of contrast enhancement, it cannot be used to detect sluggish retrograde, or antegrade flow. Competitive flow in a coronary artery bypass graft has been reported as an important cause of graft failure, particularly in bypass grafts that are connected to a moderately stenotic coronary artery [31]. References
1- FLOHR T., OHNESORGE B. and SCHALLER S.: Design, technique and future perspective of MSCT. Springer, New York. Pp. 3-16, 2005. 2- ROPERS D., BAUM U., POHLE K., el al.: Detection of coronary artery stenoses with thin-slice multi-detector row spiral computed tomography and multi-planner reconstruction. Circulation, 107: 664, 2003. 3- PANNU H.K., FLOHR T.G., CORL F.M. and FISHMAN E.K.: Current Concepts in Multi-Detector Row CT Evaluation of the Coronary Arteries: Principles, Techniques, and Anatomy. Radiographics, 23; 111, 2003. 4- SCHOEPF U.J., BECKER C.R., OHNESORGE B.M. and YUCEL E.K.: CT of coronary artery disease. Radiology, 232; 18, 2004. 5- ACHENBACH S., ROPERS D., REGENFUS M., et al.: Diagnostic value of 2-and 3-dimensional image reconstruction techniques for the detection of coronary artery stenosis by contrast enhanced electron beam CT. J. Am. Coll. Cardiol., 35; 416, 2000. 6- KOPP A.F., SCHROEDER S., KUETTNER A., et al.: Non invasive coronary angiography with high resolution multidetector-row computed tomography. Eur. Heart J., 23; 1714, 2002. 7- HILL D.G.: CT of the heart; principles and applications. Chapter 2; Electron beam CT of the heart. Humana Press, Inc. 1st edition. 15, 2004. 8- MOLLET N.R., CADEMARTIRI F., KRESTIN G.P., et al.: Improved diagnostic accuracy with 16 row multislice computed tomography coronary angiography, J. Am. Coll. Cardiol., 45; 128, 2005. 9- SCHLOSSER T., KONORZA T., HUNOLD P., et al.: Noninvasive visualization of coronary artery bypass grafts using 16-detector row computed tomography. J. Am. Coll. Cardiol., 44; 1224, 2004.
406
10- PANNU H.K. and FLOHR T.G.: Corl F\l FishmanEK. Current concepts in multi-detector row CT evaluation of coronary arteries: Principles, techniques, and anatomy, Radiographicsx, 23 (special! issue): Sl11-S125, 2004. 11- HONG C., BACKER C.R., HUBER A., et al.: ECG-gated reconstructed multi-detector row CT coronary angiography: Effect of varying trigger delay on image quality. Radiology, 220; 712, 2001. 12- BACKER C.R.: Assessment of coronary arteries with CT. Radiol. Clin. N. Am., 40; 773, 2002. 13- NIEMAN K., RENSING B., VAN GEUNS R., et al.: Non-invasive coronary angiography with multislice spiral computed tomography: Impact of heart rate. Heart, 88; 470, 2002. 14- GIESLER T, BAUM U., ROPERS D., et al.: Noninvasive visualization of coronary arteries using contrast-enhanced multidetector CT: Influence of heart rate on image quality and stenosis detection. Am. J. Roentgenol., 179; 911, 2002. 15- GERBER T.C., KUZO R.S., KARSTAEDT N., et al.: Current results and new developments of coronary angiography with use of contrast-enhanced computed tomography of the heart. Mayo. Clin. Proc., 77; 55, 2002. 16- PUGLIESE F., MOLLET N.R., RUNZA G., et al.: Diagnostic accuracy of noninvasive 64-slice CT coronary angiography in patients with stable angina pectoris. Eur. J. Radiol., 16; 575, 2006. 17- KOPP A.F., OHNESORGE B., BACKER C.R., et al.: Reproducibility and accuracy of coronary calcium measurements with multi-detector row versus electron beam CT. Radiology, 225; 113, 2002. 18- SATO Y., KANMATSUSE K., INOUE F., et al.: Non invasive coronary artery imaging by multislice spiral computed tomography-A novel approach for a retrospectively ECG-gated reconstruction technique. Circulation, 67; 107, 2003. 19- LEPOR N.E. and MADYOON H.: The emerging use of 16-and 64-slice computed tomography coronary angiography in clinical cardio-vascular practice. Rev. Cardiovasc. Med., 6 (1); 47, 2005. 20- ROPERS D., ULZHEIMER S., WENKEL E., et al.: Investigation of aorto coronary artery bypass grafts by multislice spiral computed tomography with electrocardiographic-gated image reconstruction. Am. J. Cardiol., 88, 792, 2001.

21- BERMAN D.S., WONG N.D., GRANSAR H., et al.: Relationship between stress-induced myocardial ischemia and atherosclerosis measured by coronary calcium tomography. J. Am. Coll. Cardiol., 44; 923, 2004. 22- CHOI H.S., CHOI B.W., CHOE K.O., et al.: Pitfalls, Artifacts, Remedies in Muli-detector row CT coronary angiography. Radiographics, 24; 787, 2004. 23- CADEMARTIRI F., MOLLET N.R., LEMOS P.A., et al.: Impact of coronary calcium score on diagnostic accuracy for the detection of significant coronary stenosis with multislice computed tomography angiography. Am. J. Cardiol., 95; 1225, 2005. 24- ZHANG S.Z., HU X.H., ZHANG Q.W. and HUANG W.X.: Evaluation of computed tomography coronary angiography in patients with high heart rates using 16slice spiral computed tomography with 0.37 gantry rotation time. Eur. J. Radiol., 15; 1105, 2005. 25- HAMOIR X.L., FLOHR T., HAMOIR V., et al.: Coronary arteries: Assessment of image quality and optimal reconstruction window in retrospective ECG gated multislice CT at 375ms gantry rotation time. Eur. J. Radiol., 15; 296, 2005. 26- MORIN R., GCRBCR T. and MCCOLLOUGH C.: Radiation dose in computed tomography of the heart. Circulation, 107; 917, 2003. 27- NAKANISHI T., KAYASHIMA Y., INOUC R., et al.: Pitfalls in 16-Detector row CT of coronary arteries. Radiographics, 25: 425-440, 2005. 28- ENZWEILER C.N., KIVELITZ D.E., WIESE T.H., et al.: Coronary artery bypass grafts: Improved electronbeam tomography by prolonged breath holds with preoxygcnation. Radiology, 217: 278-283, 2000. 29- MAHNKCN A.H., BUECKCR A., WILDBERGCR J.E., et al.: Coronary artery stents in multislice computed tomography: In vitro artifact evaluation. Invest. radiol., 39: 27-33, 2004. 30- KNEZ A., BCCKER C.R., LEBER A., et al.: Usefulness of multislice spiral computed tomography angiography for determination of coronary artery stenosis. Am. J. Cardiol., 1191-1194, 1988. 31- HASHIMOTO H., ISSHIKI T., IKARI Y., et al.: Effects of competitive blood flow on arterial graft patency and diameter: Medium-term postoperative follow-up. J. Thoracic. Cardiovasc. Surg., 111: 399-407, 1996.
Ocular Trauma Visual Outcomes during the 2011 Egyptian Revolution

HANY E. EL-MEKAWEY, M.D.
The Department of Ophthalmology, Faculty of Medicine, Cairo University
Abstract
Purpose: To report the pattern of ocular injuries attending the ophthalmic casuality room and outpatient clinics of Kasr El-Aini Hospital in Egypt, during the period of the 2011 Egyptian revolution (25th January revolution) as well as to predict the visual prognosis of these injuries. Methods: Retrospective, non-comparative study using medical records in Cairo University (Kasr El-Aini) Hospital. Statistical analysis was done using SPSS11 software. Prediction of best-corrected visual acuity (BCVA) was categorized using the ocular trauma score (OTS) grading system. Results: 192 patients were included in the study. Mean age was 28.48.98 (range from 12 to 51). Males represented 96.9% (186 cases). Bilateral eye injuries occurred only in 9.4% (18 cases). Gunshot was the leading cause of globe injury; 84.4% (162 cases). 79.2% (152 cases) of patients were admitted due to open globe injury, with 82 (42.7%) patients showed retained intraocular foreign body (IOFB). Patients with retinal detachment represent 51% (98) of all cases. grade 1 OTS was found in 36 cases (18.8%), grade 2 in 123 cases (64.1%), grade 3 in 26 cases (13.5%), grade 4 in 7 cases (3.6%), and no cases in grade 5 OTS (0.0%). Conclusions: Ocular injuries due to plastic bullets used in civil uprising fights lead to huge number of devastating ocular injuries with poor visual prognosis and bad impact on the patients, their families, and the society, necessitating prohibition of the use of such gunshots in civil situations. Key Words: Open globe injury Revolution Plastic bullets Retinal detachment IOFB Perforation.
There were many deaths reported, and a large number of people were injured. The capital city of Cairo was described as a war zone, and the port city of Suez was the scene of frequent violent clashes. For many years, police forces around the world have used rubber and plastic bullets to control rioting during political demonstrations and civil conflicts. These terms can be misleading, as most consist of a metallic core surrounded by a coating of rubber or plastic [1,2]. These fire weapons of reduced power aim to cause pain but not serious injury. They are expected to produce contusions, abrasions, and hematomas. They are not intended to kill. Initially wooden projectiles were used in the 1950s and 1960s. In 1970 single shot rubber bullets were introduced in Northern Ireland [2]. These baton rounds are generally directed to the lower part of the body, but due to flight instability their aim has shown to be inaccurate [3-6]. Impacts upon vulnerable body parts such as the face, head and neck causing severe injuries or even death have occurred. Rubber bullets have since been replaced by hard plastic projectiles which were designed to offer more accurate aim without causing more damage than their predecessors. However, as a number of projectiles have been developed and used in different countries there have been a varied array of subsequent injuries reported. Injuries to vulnerable upper body structures have proven to be the most serious [6-11]. Despite comprising as little as 0.1% of the total body surface area and 0.27% of the frontal silhouette, the proportion of eye injuries in nonfatal casualties has been escalating in recent conflicts. Several reasons account for the increasing risk of eye injuries: Preferential exposure of the eyes during combat and improved munitions, which create more and
Introduction THE 2011 Egyptian revolution (Revolution of 25 January) took place following a popular uprising that began on 25 January 2011. The uprising was mainly a campaign of non-violent civil resistance, which featured a series of demonstrations, marches, acts of civil disobedience, and labour strikes. Despite being predominantly peaceful in nature, the revolution was not without violent clashes between security forces and protesters.
Correspondence to: Dr. Hany E. El-Mekawey, The Department of Ophthalmology, Faculty of Medicine, Cairo University.
407
408
smaller fragments that can cause severe, even blinding, injuries [1,12-15]. Material and Methods This retrospective, non-comparative study, examined all casualty patients injured during the 2011 Egyptian revolution (Revolution of 25 January) who were evacuated from Tahrir square to Kasr Eini Hospital. Information then was recorded onto a standardized sheet that was used for data entry into a structured database program for analysis (SPSS software version 11.0; SPSS, Inc., Chicago IL). Data obtained included the demographic profile of the patients, the circumstances surrounding the
injuries and the ocular findings on ophthalmoscopy (both direct and indirect). Slit lamp biomicroscopy where possible and visual acuity using the Snellen system in meters were also carried. The nature of injuries, interventions and the outcome were recorded. Injuries were classified as closed or open globe in accordance with the Birmingham Eye Trauma Terminology (BETT) (Table 1). The variables specified in the International Ocular Trauma Classification (IOTC) were analyzed, including: (1) visual acuity on admission, (2) zone of injury, and (3) type of injury (Table 2), which are responsible for the ultimate functional success of open globe injuries. Unfortunately, the presence or the absence of relative afferent pupillary defect was not documented [16-19].
Table (1): The Birmingham Eye Trauma Terminology (BETT) sytem for open globe injuries [19]. Open Globe Injury (The eye wall a full-ticknes wound) Rupture (Full tickness wound caused by a blunt object) Laceration (Full tickness corneal or scleral wound caused by a sharp object)
Penetrating injury (Single, full tickness wound of the corneosclera, usually caused by a sharp object)
Intraocular foreig body injury (The retained foreign object causes a single entrance wound)
Perforating injury (Two full tickness woundsentrance and exit-of the corneosclera, usually caused by a missile)
Table (2): Open globe injury classification [19]. Type: A- Rupture B- Penetrating C- Inaocular foreign body D- Perforating Grade: Visual acuity: 1- 0.5 2- 0.1 to 0.4 3- 0.05 to 0.025 4- Light perception to 0.02 5- No light perception Pupil: Positive (Relative afferent pupillary defect present in affected eye) Negative (Relative afferent pupillary defect absent in affected eye)
Zone: I- Isolated to corneal (including the corneoscleral limbus). II- Corneoscleral limbus to a point 5mm posterior into the sclera. III- Posterior to the anterior 5mm of sclera.
Injuries not involving the globe were categorized as oculoplastic or neuro-ophthalmologic injuries. (6) The ocular trauma score (OTS) was calculated retrospectively as described by Kuhn et al. [17,18].
The OTS provides a single probability estimate that an eye trauma patient will obtain a specific visual range by 6 months after injury. The OTS uses initial visual acuity and injury type to predict an outcome based on findings at the time of pre-
Hany E. El-Mekawey
409
sentation. Higher OTS indicates a better prognosis. The OTS can be used as an aid in patient counseling of eye injury patients and can assist in identifying severe ocular injuries with a poor visual prognosis [19]. To calculate the Ocular Trauma Score, first, determine the patients initial visual acuity after the injury and their tissue diagnoses. Second, assign a raw point value for initial visual acuity from row A from Table (3). Then subtract the appropriate raw points for each diagnosis from rows B-F. (For example, a patient with an initial visual acuity of 1/200, scleral rupture, and retinal detachment would receive a raw OTS score of 80-23-11=46). Higher OTS scores tend to indicate a better prognosis. To provide an estimate of the patients probability of attaining a specific visual acuity range at a sixmonth follow-up, locate the row in Table (4) corresponding to the patients OTS. (A patient with a raw OTS score of 46 would have an OTS category score of 2). Table (4) shows the estimated probability of all potential visual outcomes vision after six-months [20,21].
Table (3): Computational method for deriving the OTS score. Initial visual factor A- Initial visual acuity category Raw points NLP = LP to HM = 1/200 to 19/200 = 20/200 to 20/50 = 20/40 = 60 70 80 90 100 23 17 14 11 10
Most of these patients were young with mean age 28.48.98 (range from 12 to 51). Males represented 96.9% (186 cases) of them while female were 3.1% (6 cases). Patients from Urban governorates (Great Cairo-Alex-Suez-Port said) represented 81.3% (156 cases), those from lower Egypt were 11.5% (22 cases), and those from upper Egypt were 7.3% (14 cases), while no patient from Frontier governorates (North, south Sinai, Red sea, New valley and Matrouh) was admitted. Bilateral eye injuries occurred only in 9.4% (18 cases), while the majority of injuries were unilateral 90.6% (174 cases), with almost equal distribution between both eyes (right; 49% and left; 41.7%). Gunshot was the leading cause of globe injury; 84.4% (162 cases), while stone was the offending agent in 10.4% (20 cases). In 5.2% (10 cases) the cause of ocular injury was unknown. 79.2% (152 cases) of patients were admitted due to open globe injury, whether alone or associated with oculoplastic injuries (Tables 5,6). 3.1% (6 cases) of these patients showed uveal tissue prolapse. Those admitted for removal of subconjunctival foreign body represented 6.3% (12 cases), while those with subconjunctival hemorrhage admitted for exploration surgery were 4.2% (8 cases). 20 cases (10.4%) showed closed globe injury with intraocular hemorrhage (hyphema or vitreous hemorrhage). 82 (42.7%) patients with open globe injury showed retained intraocular foreign body (IOFB), all have associated retinal detachment except for 18 cases (9.4% of all cases and 22% of IOFB cases) that didnt show any associated retinal detachment. 22 (11.5%) patients with open globe injury showed ocular perforation, 18 of these patients (9.4%) are associated with both intraocular and orbital foreign bodies, while 4 cases (2.1%) are associated with only orbital foreign bodies. Patients with retinal detachment represent 51% (98) of all cases, those who are associated with enophthalmitis and perforation (worst OTS prognosis) represent 5.2% (10 cases) of all patients, and those associated with perforation only represent 10.4% (20 cases) of all patients. Initial visual acuity was no perception of light (NO PL) in 12 cases (6.3%), PL or hand motion
B- Globe rupture C- Endophthal mitis D- Perorating injury E- Retinal detachment F- Afferent pupillary defect (Marcus Gunn pupil) Raw score sum = sum of raw points
Table (4): Estimated probability of follow-up visual acuity category by the OTS score. Raw score OTS 1/200- 20/200 to NLP LP/HM 20/40 sum score 19/200 20/50 0-44 45-65 66-80 81-91 92-100 1 2 3 4 5 73% 28% 2% 1% 0% 17% 26% 11% 2% 1% 7% 18% 15% 2% 2% 2% 13% 28% 21% 5% 1% 15% 44 74 92%
Results 192 patients with ocular trauma resulted from the conflict between police and the protesters during the 2011 Egyptian revolution were admitted to Kasr El-Aini Hospital (casualty ward and outpatient clinics).
410
in 122 (63.5%) cases, counting fingers to 5/60 (1/ 200 to 19/200) in 44 cases (22.9%), and better than 6/60 (>20/200) in 14 cases (7.3%).
Table (5): Zones of globe injury. Zone 1 (corneolimbal) Zone 2 (corneoscleral) Zone 3 (wound extend more than 5mm from limbus) 17.2% (33) 37.5% (72) 45.3% (87)
Evacuation of hyphema present in closed globe injuries was done in 8 cases (4.2%). Ten cases (5.2%) required initial intravitreal injection of antibiotics for treatment of endophthalmitis. Eight cases (4.2%) escaped before any intervention was done. One hundred and twelve patients (58.3%) required further secondary surgery, including secondary cataract extraction with or without intraocular lens implantation, and/or vitreoretinal procedures for removal of IOFB, cleaning of dense vitreous hemorrhage, and repair of retinal detachment. Discussion This is a retrospective, non-comparative study which examined all casualty patients injured during The 2011 Egyptian revolution (Revolution of 25 January) who were transported from Tahrir square to Kasr Eini Hospital which is the largest referral center for casualty cases in Egypt. To my knowledge, this is the first study to examine the visual outcomes of civil uprising combat ocular trauma (COT) that mostly occurred due to police use of rubber pullet against the protesters. Disruption of the ocular anatomy in cases of gunshot injuries often result in poor visual acuity, globe perforation and retained foreign body. Recent articles have focused on the incidence and type of injuries sustained in ordinary day activities. Such a retrospective review has potential drawbacks, including incomplete records, lack of followup for all patients, and inability to contact some of the patients (no consent). This contributes to a larger problem of poor longitudinal patient data tracking from the point of injury to the final outcome. Moreover, the inability to document consistently initial visual acuity early in the course after injury makes analysis and interpretation of the visual acuity outcomes and OTS difficult [22]. This study was done on 192 patients with ocular injury, with average age 28.48.98 which constitute the major workforce and the future of any society. Male patients represented the majority of cases (96.9%), male-to-female ratio of 31:1 in contrast to noncombat civilian ocular trauma, in which the male-to-female ratio is 4:1.23. Regarding residential area, most of the patients came from urban governorates (Great Cairo-AlexSuez-Port said), in contrast to non-COT in which Upper Egypt presented the highest number of ocular emergencies and admissions [24].
On calculating the ocular trauma score (OTS), grade 1 OTS was found in 36 cases (18.8%), grade 2 in 123 cases (64.1%), grade 3 in 26 cases (13.5%), grade 4 in 7 cases (3.6%), and no cases in grade 5 OTS (0.0%).
Table (6): Distribution of admitted ocular injuries (No; number of cases, %; percentage). Ocular emergency No. Open Globe injuries Vitreous hemorrhage Hyphema Cataract Intraocular foreign Body Closed globe injury Oculoplastic injuries: Orbit foreign body Ecchymosis & lid contusion Lid wound Neuro-ophthalmology Endophthalmitis Perforation Retinal detachment APD 152 116 42 78 82 20 26 24 42 0 10 44 98 0 Total % 79.2 60.4 21.9 39.6 42.7 10.4 13.5 12.5 21.9 0.0 5.2 22.9 51 0.0
The most common primary surgery is concerned with reconstitution of the globe integrity with reposition or excision of ocular contents. The majority of primary intervention involved globe exploration, removal of extraocular foreign bodies, suture of the wound, anterior chamber wash, with or without excision of prolapsed intraocular contents (83.6%). Seventy six patients (39.6%) with open globe injuries required secondary procedures, after receiving a primary repair for their condition; whether this primary repair was performed in our casualty operating room or referred from other hospitals. Primary cataract removal; for patients with open anterior lens capsule with cortical material in the anterior chamber, was performed in eighteen cases (9.4%). Forty-two cases (21.9%) required additional lid wound repair. Three primary eviscerations (1.6%) were performed for severely ruptured globes.
Hany E. El-Mekawey
411
Most of the cases are unilateral (90.6%), while 9.4% of cases showed bilateral globe injury, which is similar to other study of combat ocular trauma but higher than other study on Egyptian ocular trauma [23,30]. This can be attributed to the nature of the plastic bullets gunshot which is the most common cause of globe injury in the present study, as it is formed 35 hexagonal PVC-cylinders of 11g each, wrapped in a plastic foil. After leaving the rifle with a muzzle velocity of 200m/s, the plastic foil ruptures and the projectiles reach their goal as buckshot. At an operational distance of 20m these projectiles are scattered almost randomly over a surface area of 2m in diameter (and for operational distances of 10 and 5m, 1.5 and 1.0m in diameter, respectively). Due to the scatter of these plastic bullets, it is impossible to avoid hits to the head and neck with frequent bilateral globe injury [25]. The socio-economic impact of injury occurring to both eyes simultaneously is usually enormous to the individual, their families and the society. The situation is worse especially when the injury occurred in young adults and middle age individuals [26,27]. In the present study, gunshot injury was the most frequent cause accounting for 84.4% of all cases. In Egypt gunshot injuries occur in 2%, while in the united states it accounts for 6% of all cases of ocular trauma while blunt trauma is the most common cause of non-combat ocular injury (30%) [23,28]. Most of the patients were admitted due to open globe injury (79.2%), which is similar to other studies [23,29]. 82 patients have retained IOFB with additional 26 patients with orbital foreign bodies. All such cases were admitted during a month period, and are almost equal in number to those admitted during a year in other study done on casualty records in kasr elini hospital, which showed 110 patients with IOFB and 8 patients with orbital foreign bodies. In a recent study on ocular trauma in the Mediterranean area, IOFB occurred in 16.8% of cases [30,31]. All the cases of the present study with IOFB are in the posterior segment and are due to plastic bullets gunshot which is non magnetizable and require removal through pars plana vitrectomy. In a large series of 158 patients analyzed transscleral magnetic extraction versus pars plana approach found the latter approach to be a manage-
ment of choice and that final visual acuity did not depend on the interval between injury and IOFB removal [32]. Retinal detachment is a common sequel of traumatic globe injury with IOFB. In the present study 51% of patients have retinal detachment which represent a large percentage that could be explained partly due to the posterior extension of scleral rupture beyond ora serrata in 45.3% of patients (87 cases); zone 3 injury, and partly due to perforating injuries in 30 patients (15.6%). This result is higher than other reports of traumatic retinal detachment [23,30,33,34]. Infectious endophthalmitis following penetrating eye injuries is a potentially catastrophic complication due to the underlying eye trauma and the frequency of more virulent organisms such as Bacillus species. Risk factors for infection include retained intraocular foreign body, a rural injury setting, delay in primary wound closure, and disruption of the crystalline lens. Although endophthalmitis is difficult to distinguish from traumatic changes, recognition of early clinical signs of endophthalmitis, such as hypopyon, vitritis, or retinal periphlebitis, is important and early treatment is recommended [33]. Incidence of infectious endophthalmitis after penetrating injury with retained foreign body was reported to be from 2.5% to 13.3% [34]. In the present study 5.2% of cases have neglected rupture globe with perforation, IOFB and endophthalmitis for which intravitreal antibiotic injection was administrated. Such a higher percentage could be attributed to the delay of patient presentation to hospital for fear of being arrested by the police force. Oculoplastic injuries represented 47.9% of patients in the present study which is closely similar to other studies [22,35-37]. Orbital foreign bodies were found in 26 (13.5%) of patients, which is higher than other studies from Egypt [23,30]. The most important factors in the prognosis of postoperative visual acuity in open globe injuries are: Type of injury, preoperative condition of the eye (in strong relationship with initial visual acuity), localization and extent of the wound, presence of foreign bodies, their localization, size and nature, and as early as possible definitive treatment. Kuhn and Pieramici et al. (The Ocular Trauma Classification Group) have found these parameters to be statistically significant for final visual outcome [16,17].
412

6- BALOURIS C.A.: Rubber and plastic bullet eye injuries in Palestine. Lancet, 335: 415, 1990. 8- COHEN M.A.: Plastic bullet injuries of the face and jaws. S. Afr. Med. J., 68: 849-52, 1985. 9- Geschftsprfungskommission des Gemeinderates, Einsatz der Stadtpolizei bei den Auseinandersetzungen vom 1. 1996. Stadt Zrich, 144, 1997. 10- METRESS EKSP METRESS: The anatomy of plastic bullet damage and crowd control. Int. J. Health Serv., 17: 333-42, 1987. 11- MISSLIWETZ J., WIESER I. and DENK W.: Medical and technical aspects of weapon effects. IV. Plastic bullets reduce the risk of the military assault rifle (StG 77). Beitr Gerichtl Med., 49: 361-6, 1991. 12- RITCHIE A.J.: Plastic bullets: Significant risk of serious injury above the diaphragm. Injury, 23: 265-6, 1992. 13- ROCKE L.: Injuries caused by plastic bullets compared with those caused by rubber bullets. Lancet, 1: 919-20, 1983. 14- SHAW J.: Pulmonary contusion in children due to rubber bullet injuries. Br. Med. J., 4: 764-6, 1972. 15- SHERIDAN SMRI WHITLOCK: Plastic baton round injuries. Br. J. Oral Surg., 21: 259-67, 1983. 16- PIERAMICI D.J., STERNBERG P. JR., AABERG T.M. Sr., et al.: Ocular Trauma Classification Group. A system for classifying mechanical injuries of the eye (globe). Am. J. Ophthalmol., 123: 820-31, 1997. 17- KUHN F., MORRIS R. and WITHERSPOON C.D.: Birmingham Eye Trauma Terminology (BETT): Terminology and classification of mechanical eye injuries. Ophthalmol. Clin. North Am., 15: 139-43, 2002. 18- KUHN F., MORRIS R., WITHERSPOON C.D., et al.: A standardized classification of ocular trauma. Ophthalmology, 234: 399-403, 1996. 19- IVNA PLESTINA-BORJAN, MARIA MEDVIDOVICGRUBISIC, IGOR ZULJAN, VENERA LAKOS, SNJEZANA MILJAK, IRENA MARKOVIC and MILAN IVANISEVIC: Wartime open globe eye injuries. Graefes Arch. Clin. Exp. Ophthalmol., 248: 305-12, 2010. 20- KUHN F., MAISIAK R., MANN L., et al.: The ocular trauma score (OTS). Ophthalmol. Clin. North Am., 15: 163-5, 2002. 21- PIERAMICI D.J., AU EONG K.G., STERNBERG P. Jr. and MARSH M.J.: The prognostic significance of a system for classifying mechanical injuries of the eye (globe) in open-globe injuries. J. Trauma, 54: 750-4, 2003. 22- ERIC D. WEICHEL, MD, LTC, MARCUS H. COLYER, MD, CPT, SPENCER E. LUDLOW, MD, CPT, KRAIG S. BOWER, MD, COL and ANDREW S. EISEMAN, MD, COL.: Combat Ocular Trauma Visual Outcomes during Operations Iraqi and Enduring Freedom. Ophthalmology Volume 115, Number 12, December, 2235-45, 2008. 23- MAHMOUD M.S. and TAMER A.M.: Pattern of ocular trauma in Egypt. Graefes Arch. Clin. Exp. Ophthalmol. Feb., 246 (2): 205-12, 2008. Epub 2007 Dec. 11. 24- KHALED G.A., HANY E.E., MOHAMMAD A., AMR K. and EMAN M.E.: Epidemiology of ocular emergencies
Family members of injured patients universally want a prognosis for their sons or daughters ocular injury on arrival to hospital. The OTS is a useful tool in predicting outcomes to better counsel the family and the patients. In the present study most of the patients (159 patients) have poor visual prognosis (grade 1 OTS in 18.8% and grade 2 OTS in 64.1%). Their expected final visual outcome at 6 months post injury is no light perception in 73% and 28% and hand motion in 17% and 26% in both grades respectively. The rest of the patients (33 patients) have better visual prognosis (grade 3 OTS in 13.5% and grade 4 OTS in 3.6%). Their expected final visual outcome at 6 months post injury is from 6/60 to 6/12 in 28% and 21% and better than 6/12 in 44% and 74% in both grades respectively. Complete follow-up of all patients to verify the predicted visual outcome is beyond the scope of this retrospective cross sectional study. The poor visual prognosis in the present study can be explained by the devastating nature of the injuries, large number of ocular perforation, large number of zone 3 rupture globes, and the poor initial visual acuity which may have been measured days or weeks after the initial injury and very frequently occurred after primary globe closure. Despite the lack of complete records, accurate registration of patients visual acuity, and their follow-up to verify the predicted visual outcome, however, the present study demonstrated the devastating nature of plastic bullets injury, with its tremendous effect on the patients, their families, and the society, necessitating the prohibition of its use against the peaceful civilians even during their uprising. References
1- DUKE-ELDER S. and MACFAUL P.A.: War injuries. In: Mechanical Injuries. Part1. In: Injuries. Vol 14. In: Duke-Elder S., ed. System of Ophthalmology. St Louis, Mo: C. V. Mosby, 49-56, 1972. 2- STEINDORF K.: Die Kreigschirurgie des schorgans. Berlin Klin Wochensch, 51: 1787-9, 1914. 3- PARSONS J.: Protection of the eyes from war injuries. Trans. Ophthalmol. Soc. UK, 61: 157-78, 1941. 4- BOZEMAN WILLIAM P. and WINSLOW JAMES E.: Medical Aspects of Less Lethal Weapons. The Internet Journal of Rescue and Disaster Medicine, 2005. 5- MILLAR R., RUTHERFORD W.H., JOHNSTON S. and MALHOTRA V.J.: Injuries caused by rubber bullets: A report on 90 patients. British Journal of Surgery, 62 (6): 480-6, 1975.
Hany E. El-Mekawey
among the Egyptian population: A five year retrospective study. Kasr Eini Medical Journal (under print). 25- FLORIAN K.P. SUTTER: Ocular injuries caused by plastic bullet shotguns in Switzerland. Injury, Int. J. Care Injured, 35: 963-7, 2004. 26- KUHN F., MESTER V., BERTA A. and MORRIS R.: Epidemiology of severe eye injuries. United States Eye Injury Registry (USEIR) and Hungarian Eye Injury Registry (HEIR) [in German]. Ophthalmologe, 95: 332-43, 1998. 27- BIEHL J.W., VALDEZ J., HEMADY R.K., et al.: Penetrating eye injury in war. Mil. Med., 164: 780-4, 1999. 28- DONALD R. MAY, FERENC P. KUHN, ROBERT E. MORRIS C., DOUGLAS WITHERSPOON, RONALD P., DANIS G., PHILIP MATTHEWS and LORETTA MANN: The epidemiology of serious eye injuries from the United States Eye Injury Registry. Graefes Arch. Clin. Exp. Ophthalmol., 238: 153-7, 2000. 29- MERIH SOYLU, SELCUK SIZMAZ and SIBEL CAYLI: Eye injury (ocular trauma) in southern Turkey: Epidemiology, ocular survival, and visual outcome. Int. Ophthalmol., 30: 143-8, 2010. 30- RASHA A.E. and RANIA A.: Eye trauma with foreign body (FB) is not that rare: A retrospective study of cases
413
admitted to the cairo university hospital in the year 2006. Bull Ophtalmol. Soc., 100 (5): 767-71, 2007. 31- CILLINO S., CASUCCIO A., DI PACE F., PILLITTERI F. and CILLINO G.: A five-year retrospective study of the epidemiological characteristics and visual outcomes of patients hospitalized for ocular trauma in a Mediterranean area. BMC Ophthalmol., 8 (6): 1471-2415, 2008. 32- KAREL I. and DIBLIK P.: Management of posterior segment foreign bodies and long-term results. Eur. J. Ophthalmol., 5: 113-8, 1995. 33- REYNOLDS D.S. and FLYNN H.W. JR.: Endophthalmitis after penetrating ocular trauma. Curr. Opin. Ophthalmol., 8: 32-8, 1997. 34- ROWSEY J.J., NEWSOM D.L., SEXTON D.J. and HARMS W.K.: Endophthalmitis: Current approaches. Ophthalmology, 89: 1055-65, 1982. 35- JANKOVIC S., ZULJAN I., SAPUNAR D., et al.: Clinical and radiological management of wartime eye and orbit injuries. Mil. Med., 163: 423-6, 1998. 36- HOEFLE F.B.: Initial treatment of eye injuries: First Corps Area of Viet Nam, 1966. Arch. Ophthalmol., 79: 335, 1968. 37- LA PIANA F.G. and HORNBLASS A.: Military ophthalmology in the Vietnam War. Doc. Ophthalmol., 93: 2948, 1997.
Effect of Treadmill Exercise on Intraocular Pressure in Normal Subjects

AZZA F. ISMAIL, P.T.D.*; NESREEN G. EL-NAHAS, P.T.D.* and DALIA M. MOSAAD, P.T.D.**
The Departments of Cardiovascular/Respiratory Disorder & Geriatrics* and Basic Sciences**, Faculty of Physical Therapy, Cairo University
Abstract
Purpose of this study was to investigate the influence of treadmill exercise on intraocular pressure in normal subjects, Fifteen normal subjects were enrolled in this study (10 women and 5 men) with age ranges between 30-40 years. They performed a supervised treadmill exercise program (3 sessions/ week, 40 minutes/session for 3-months) where intraocular pressure was measured before exercise and 30 minutes after exercise, at baseline and after 3-months of training. Results; showed that there was a significant decrease of the intraocular pressure which scoped in the maintenance of the good nourishment for the eye tissues. Conclusion; It was concluded that regular treadmill exercise was of great benefit in the preservation of normal intraocular pressure in response to age-related changes of ocular health. Key Words: Treadmill exercise Intraocular pressure Ocular health.
or the eye whose vasculature is compromised by a pre-existing systemic state, may not [1]. The normal intraocular pressure is about 10 to 20mmHg. For Caucasian people, the average eye pressure is 16mmHg; this varies slightly with each heartbeat and with respiration. Many factors affect IOP as variation during the course of the day as pressure is highest in early morning before waking but the peak may occur at other times where the difference is usually between 3 and 4mmHg as well as the age changes in the trabecular meshwork and other disease such as Diabetes, Hypertension and glaucoma [2]. Snell and Lamp [3] stated that, there are three chief factors responsible for maintaining a normal intraocular pressure: - The rate of formation of aqueous humor by the cells of the ciliary processes. - The rate of drainage of aqueous humor through the trabecular meshwork. - The pressure in the episcleral veins into which the sinus venosus sclerae (canal of Schlemn) drains. There are two ways to measure the intraocular pressure either manometrically or tonometrically. Tonometry is the most utilized method in clinical practice as well as in the experimental studies. It is an important test in the evaluation of ocular conditions such as glaucoma as well as conditions such as phthisis bulbi, and iritis. Most tonometers are calibrated to measure pressure in mmHg. [4]. An elevated IOP is the main risk factor for glaucoma, with the degree of risk increasing as the level of IOP increases. Elevated intraocular pressure is known to reduce retinal blood flow, although the effect of intraocular pressure on retinal
Introduction EXERCISE induces blood flow changes in all organs in the body, including the eye and the brain that are autoregulated in order to maintain a constant blood supply in the face of metabolic stress or imbalance. Blood flow autoregulation refers to the ability of the vascular bed to maintain a relatively constant flow despite moderate alterations in the perfusion pressure, or in terms of eye, ocular perfusion pressure. Exercise, by its very nature, leads to increase in systolic blood pressure and decrease in the intraocular pressure (IOP). These two components are strongly influenced by the autonomic nervous system, and the result is an increase in ocular perfusion pressure. A normal healthy eye can cope with the stress and altered ocular perfusion pressure, while the diseased eye,
Correspondence to: Dr. Nesreen G. El-Nahas, The Department of Cardiovascular/Respiratory Disorder and Geriatrics, Faculty of Physical Therapy, Cairo University.
415
416
vascular pressures is unknown. Researches investigate this relation by direct measurements of intravascular pressures in the cat retina at various intraocular pressures. The results showed that retinal artery pressure depends on both intraocular pressure and mean systemic blood pressure [5]. Many studies explained that the factors associated with high IOP included age, baseline IOP, hypertension, blood pressure as well as wearing a tight necktie could raise IOP. Incidence increased with age, with rates 2.5 times higher at 70 years or older than at ages 40 to 49 years. In addition, older individuals with a higher baseline IOP were more likely to develop elevated IOP after 4 years. Although blood pressure was also associated with high IOP, the relationship may be nonlinear [6]. According to, Koss, [7]; the functional role of nitric oxide in regulation of ocular blood flow is an important subject to be studied. Nitric oxide generated by three distinct enzyme systems appears to play a critical role in many diverse physiological processes. Using both conventional and immunohistochemical techniques, nitric oxide synthases have been identified throughout the body, including all regions of the eye. Nitric oxide is implicated in a variety of ocular pathophysiological states including uveitis, retinal ischemic disease, diabetes and glaucoma. Several Studies confirm that regular exercise can enhance the release of nitric oxide (NO) that leads to a functional consequence of the good nourishment of the delicate and sensitive tissues to blood deprivation such as EYE and normalize intraocular pressure that will save it from occular complications as diabetic retinopathy (DR) which is the major cause of blindness in adults aged 2574 years [8]. The endothelium plays a primary role in the modulation of vascular tone and structure through production of the relaxing factor nitric oxide (NO), which acts by protecting the vessel wall from the development of atherosclerosis and thrombosis. A dysfunctioning endothelium, characterized by reduced NO availability induced by oxidative stress, can in the presence of most of the cardiovascular risk factors, including aging, be a promoter of atherosclerosis [9]. Nitric oxide (NO) is an important regulator of basal choroidal blood flow [10]. Exercise has been repeatedly shown to improve endothelial vasomotor function in healthy subjects and in disease states including hypertension, congestive heart failure, Coronary artery disease (CAD) as well as Diabetics. These effects appear to be
mediated in large part by increased NO bioavailability and may be greatest in vascular beds exposed to repetitive increases in blood flow during exercise which includes the coronary circulation for all types of exercise [11]. According to, Singleton et al., [12] the autonomic nervous system contributes to blood flow control to vital organs through autoregulation. Autoregulation involves local myogenic, metabolic, and circulating humoral agents to maintain relatively constant blood flow to tissues despite perfusion pressure fluctuations. Several studies search the effect of different modes and types of exercises on IOP in healthy and diseased eyes and its effect on the ocular health and overall quality of life on those subjects. The results came to be hopeful that certain exercises not all have good effect on IOP not only in the healthy subjects but also on patients. Intraocular pressure is known to be responsive to the effects of physical exercise. This is usually reflected as a decrease in IOP immediately following exercise, with gradual return to the pre-exercise level over an hour period post-exercise. That was confirmed with a study on the effect of cycling on 10 hypertensive patients that revealed decrease in IOP with exercise which indicates better perfusion of the retina [13]. The effects of physical fitness on intraocular pressure were reviewed in several studies that concluded that physical fitness reduces intraocular pressure in both trained and sedentary life subjects. It would seem reasonable at present not to discourage patients who have glaucoma from light exercise, perhaps; on the contrary, it should be encouraged [14]. The effects of intensity, duration and quantity (intensity x duration) of exercise on the reduction of (IOP) in healthy and physically fit individuals were studied. Five minutes after 15 minutes of exercise at 70%, 55% and 40% of maximum exercise load (%HRmax) decreased IOP by 4.30.7 mmHg, 2.20.7mmHg and 0.60.5mmHg, respectively. The magnitude of IOP reduction increased with exercise load. Also running exercises for 7.5 minutes at 70% HRmax decreased IOP comparable to 15 minutes of running at the same exercise load (4.40.6mmHg) and also compared by 25 minutes of running at 40% HRmax is almost the same quantity of exercise as 15 minutes of running at 70% HRmax. However, the former did not result in IOP reductions to equal the latter (2.30.5 Vs. 4.40.6mmHg). The amount of IOP reduction after short-term exercise seems to depend on the intensity
Azza F. Ismail, et al.
417
of exercise, not on the duration of exercise or the quantity of exercise [15]. Exercise proved to reduce intraocular pressure in healthy subjects. A study designed to evaluate the effect of moderate exercise over a short duration on intraocular pressure and pulsatile ocular blood flow. Thirty-one subjects (20 males and 11 females) ranging in age from 20-60 years had measurements of intraocular pressure and pulsatile ocular blood flow before and after pedaling on a stationary bicycle for 10 minutes. The results gave the evidence that exercise can decrease intraocular pressure and increase pulsatile ocular blood flow [16]. In a study to examine the effects of jogging on intraocular pressure (IOP), blood pressure (BP), and heart rate (HR) on twenty-nine healthy individuals-25 athletes and 4 untrained-were studied. IOP, systolic and diastolic BP, and HR were measured before and just after 20 minutes of jogging (submaximal--70%--aerobic exercise). The results showed that IOP decreased (1 to 8mmHg). BP increased (systolic: 0 to 60mmHg, diastolic: 0 to 15mmHg). HR increased as well (15 to 80pulses/ min). IOP decreases all after the jogging [17]. Dynamic exercise changes Ocular perfusion pressure (OPP), and produces increased tissue blood flow in the retina in the immediate postexercise period, while blood flow increases more persistently in the choroid-retina. Difference in control of blood flow in these two regions may be related to stronger autoregulatory mechanism of blood flow in the retina. Nitric oxide may play a role in the regulation of blood flow [18]. Researchers found that regular physical exercise can reduce IOP by as much as 4mmHg; where this reduction may be enough to protect the retinal ganglion from damage. In addition to the acutephase IOP lowering effect of exercise, consistent exercise programs decrease ocular pressure in long term [1]. In a recent study, it was observed that people with glaucoma who exercised regularly for three months reduced their IOPs an average of 20%. These people rode stationary bikes 4 times per week for 40 minutes. Measurable improvements in eye pressure and physical conditioning were seen at the end of three months. These beneficial effects were maintained by continuing to exercise at least three times per week; lowered IOP was lost if exercise was stopped for more than two weeks [19]. Hilton (2003) [1] mention that there are two primary responses occur in the eye as a result of
exercise: 1) IOP decreases; 2) Ocular blood flow alters. In the eye, exercise is associated with a decrease in IOP and this has been demonstrated by a number of studies. Material and Methods Fifteen normal subjects were enrolled in this study, they were chosen from the employees in the Police Hospital in Assuit. (10 women and 5 men), All subjects were with age ranges between 30-40 years, with normal Body Mass Index (BMI) <30 Kg/m2. All the subjects underwent evaluation procedures that include: (BMI) using Weight and Height Scale, Complete eye examination done by ophthalmologist to exclude any ophthalmic condition that might interfere with (IOP) measures. Also measuring (IOP) by using Goldman applanation tonometer. Procedures: The IOP was measured at baseline and after 3-months of treadmill training in the periods before the exercise and 30 minutes after exercise, where the training program was in the form of controlled walking exercise on the treadmill (Enraf Nonius, EN-TRED) with frequency of 3 sessions/week for 12-weeks. Each training session lasted for 40 minutes; 10 minutes warming up and actual training period for 20 minutes followed by 10 minutes cooling down. The actual training period was in the form of walking with a speed that were be increased slowly on an individual basis until the heart rate reached 140beats/minute(by using pulsometer)-that was proved to induce ocular perfusion changes [20]. For each patient, matters to be taken in consideration that the exercise will be stopped when any symptom limiting exercise appears as chest pain, leg pain, fatigue, dyspnea, disturbance in breathing or heart rates. Results
Table (1): Subject criteria at baseline. Mean SD Number/Sex 15 healthy subjects 10 women and 5 men Age (yrs) Weight (Kg) Height (cm.) BMI (Kg/m2)
BMI: Body mass index. SD : Standard deviation.
36.32.2 78.49.1 162.84.7 28.422.6
418

Table (2): Statistical analysis for Intraocular pressure of the right and left eyes throughout the study. Pre-exercise Right eye At Baseline After 3-months of training t-value p-value
Min.: Minutes.
After 30min. Right eye 132.9 12.61.9 1.913 0.001* Left eye 13.23.6 12.22.4 1.329 0.001*
Left eye 14.83 13.73 2.402 0.056
t-value 4.35 2.009
p-value 0.016 0.001*
15.43.4 13.32.3 3.993 0.000*
*: Significant.
15.5 15 14.5 mmHg
Rt. eye Lt. eye
13.5 13 12.5 12 Pre-ex. After 30min.
mmHg
14
13.2 13 12.8 12.6 12.4 12.2 12 11.8 11.6 11.4
Rt. eye Lt. eye
Pre-ex.
After 30min.
Fig. (1): Intraocular pressure measuring for both eyes at the baseline
Fig. (2): Intraocular pressure measuring for both eyes after 3 months of training.
Discussion Here came the importance of (Prevention is better than cure). In a study by Green et al., [21], findings showed the effect of exercise training on endotheliumderived nitric oxide function in humans where its results clarified that the vascular endothelial function is essential for maintenance of health of the vessel wall and for vasomotor control in both conduit and resistance vessels. Exercise training has been shown to augment endothelial, niticoxide-dependent vasodilatation in both large and small vessels. Recent studies also indicate that exercise training may improve endothelial function by up-regulating endothelial nitric oxide (eNOS) protein expression and phosphorylation. While improvement in NO vasodilator function has been less frequently found in healthy subjects, a higher level of training may lead to improvement [21]. On comparing the findings of the present study about the effect of exercise on mean arterial blood pressure and its direct relation with IOP it came in consistent with the findings of Ismail et al., 1998 [13] that declared that exercise lead to significant decrease in IOP in normal and hypertensive
patients which in turn returned to its initial value within 60 minutes. In the present study, the impact of 3-months treadmill exercise is the most important variable that plays an important role in adjusting intraocular pressure. That was in consistent with the results of a study by Silver and coworkers, [22] that reported an increase in blood flow of the opthalamic artery, which is accompanied by a decrease in IOP following acute exercise. In contrast, a separate study by Nemesure, et al., [6] which used color Doppler ultrasound imaging to measure blood flow velocities of the ophthalmic artery and central retinal artery in healthy volunteers before and after exercise, showed decreased blood flow in the ophthalmic artery, while blood flow in the central retinal artery remained stable. The authors concluded that efficient autoregulatory mechanisms existed for retinal blood flow, which was kept constant during the altered cardiac state, but not for the ophthalmic artery. It was evident that there were many factors that determine the decrease in intraocular pressure (IOP) that occurs during dynamic exercise as mentioned by Harris et al., [23] in a study about correlated factors that involved in the decrease in IOP during acute dynamic exercise and with comparison
Azza F. Ismail, et al.
419
2- THEELEN T., MEULENDIJKS C.F., GEURTS D.E., VAN LEEUWEN A., VOET N.B. and DEUTMAN A.F.: Impact factors on intraocular pressure measurements in healthy subjects. Br. J. Ophthalmol., 88 (12): 1510-1 (ISSN: 0007-1161), 2004. 3- SNELL R.S. and LEMP M.A.: Clinical anatomy of the eye, 2nd ed., ch. 6, the eyeball, pp. 132-210, 1998. 4- CLINE D., HOFSTETTER H.W. and GRIFFIN J.R.: Dictionary of Visual Science. 4 th ed . ButterworthHeinemann, Boston. 5- BONOMI L., MARCHINI G., MARRAFFA M. and MORBIO R.: The relationship between intraocular pressure and glaucoma in a defined population. Data from the Egna-Neumarkt Glaucoma Study. Ophthalmologica.; 215 (1): 34-8, 2001. 6- NEMESURE B., WU S.Y., HENNIS A. and LESKE M.C.: Factors related to the 4-year risk of high intraocular pressure: The Barbados Eye Studies. Arch. Ophthalmol., 121 (6): 856-62, 2003. 7- KOSS M.C.: Functional role of nitric oxide in regulation of ocular blood flow. Eur. J. Pharmacol., 18; 374 (2): 16174, 1999. 8- SCHMIDT K.G., RCKMANN A. VON, MATTHES B., and HAMMES H.P.: Ocular pulse amplitude in diabetes mellitus. Br. J. Ophthalmol., 84 (11): 1282-4. 9- WIDLANSKY M.E., GOKCE N., KEANEY J.F. and VITA J.A.: STATE-OF-ART PAPER The clinical implications of endothelial dysfunction Am. Coll. Cardiol., 42: 1149-60, 2003. 10- LUKSCH A., POLSKA E., IMHOF A., SCHERING J., FUCHSJGER-MAYRL G., WOLZT M. and SCHMETTERER L.: Role of NO in choroidal blood flow regulation during isometric exercise in healthy humans. Invest. Ophthalmol. Vis. Sci., 44 (2): 734-9, 2003. 11- VITA J.A. and KEANEY J.F. JR.: Exercise--toning up the endothelium? N. England J. Med. Feb. 17; 342 (7): 503-5. Comment on: N. Engl. J. Med., 2000 Feb. 17, 342 (7): 454-60, 2000. 12- SINGLETON C.D., ROBERTSON D., BYRNE D.W. and JOOS K.M.: Effect of Posture on Blood and Intraocular Pressures in Multiple System Atrophy, Pure Autonomic Failure, and Baroreflex Failure. Circulation., 108: 2349-54, 2003. 13- ISMAIL A.F., ABDEL HADY A., GAWDAT I.M. and BADR N.M.H.: The effect of leg exercise on the systemic blood pressure and intraocular pressure in normal individuals and patients suffering from essential hypertension. Master thesis, Faculty of Physical Therapy, Cairo University. 14- QURESHI I.A.: Does physical fitness influence intraocular pressure? J. Pak. Med. Assoc., 47 (3): 81-4. 15- KIUCHI Y., MISHIMA H.K., HOTEHAMA Y., FURUMOTO A., HIROTA A. and ONARI K.: Exercise intensity determines the magnitude of IOP decrease after running. Jpn. J. Ophthalmol., 38 (2): 191-5, 1994. 16- BROWNLEE PATRICK O.D., LIZ FLATT O.D. and JEFF MILLER O.D.: Effects of moderate exercise on intraocular pressure and ocular blood flow. Br. J. Ophthalmol., 90: 175-88.
of the IOP response of trained and sedentary subjects, where the results declared that: Acute dynamic exercise lowers IOP in a graded fashion proportional to relative, not absolute, work load. The IOP decline is correlated with blood lactate but not with PCO2 or plasma osmolarity changes. On the other hand, many studies regarded the relationship between physical fitness and IOP as claimed by Dane et al., [24] that acute exercise increased IOP in male athletes, but had no effect in sedentary men. Also, it decreased IOP in sedentary women, but had no effect in female athletes. Sex and physical fitness both were significant factors influencing the changes in IOP due to exercise. These results suggest that acute dynamic exercise is useful to decrease IOP in sedentary women, but not in male athletes. Hilton, [1], claimed that the reason why exercise results in reduced IOP in terms of the eye, this correlation offers three explanations for the reduced IOP. 1- Ocular dehydration may occur through osmotic changes in the retinal and uveal vasculature. 2- An increase in colloid osmotic pressure may reduce aqeous formation via reduced ultrafilteration and hence IOP. 3- Altered colloid osmotic pressure could act directly on the hypothalamus resulting in IOP reductions through an unspecified reflex response. In one study, jogging for 20 minutes lowered IOP by 1mmHg to 8mmHg. In another, weight lifting also led to decreases in IOP, with IOP dropping by 14.5% after the third set of chest presses and 13.2% after the third set of leg presses. While the jogging and weight training studies were conducted in healthy, athletic people without glaucoma, exercise has also been found to benefit sedentary people with ocular hypertension. For instance, three months of moderate exercise for nine sedentary people suspected of having glaucoma decreased mean IOP by 4.6mmHg (20% for these particular patients). Simply going for a walk three or more times a week is all you need to protect against glaucoma progression [25]. Conclusion: Overall, exercise has been found to lower IOP. Studies also have found that it improves blood flow to the retina and optic nerve. References
1- HILTON E.: Exerc-eyes; Effects of exercise on the ocular health, Clinical OT, 45-9, 2003.
420

21- GREEN D.J., MAIORANA A., ODRISCOLL G. and TAYLOR R.: Effect of exercise training on endotheliumderived nitric oxide function in humans Topical review, The Journal of physiology, 561.1, pp.1-25, 2004. 22- SILVER D.M., FARRELL R.A., LANGHAM M.E., et al.: Ocular Blood-Flow in Response to Acute Exercise. Investigative Ophthalmology and Visual Science, 32 (4): 864-8, 1991. 23- HARRIS A., MALINOVSKY V. and MARTIN B.: ARTICLES AND REPORTS Correlates of acute exerciseinduced ocular hypotension Investigative Ophthalmology and Visual Science, Vol. 35, 3852-7, 1994. 24- DANE S., KOER I., DEM1REL H., UCOK K. and TAN U.: Effect of acute submaximal exercise on intraocular pressure in athletes and sedentary subjects. Int. J. Neurosci., 116 (10): 1223-30, 2006. 25- JOHNS HOPKINS HEALTH ALERT: Exercise and Glaucoma: Staying fit benefits your eyes, 2007.
17- KARABATAKIS V.E., NATSIS K.I., CHATZIBALIS T.E., LAKE S.L., BISBAS I.T., KALLINDERIS K.A. and STANGOS N.T.: Correlating intraocular pressure, blood pressure, and heart rate changes after jogging. Eur. J. Ophthalmol., 14 (2): 117-22. 18- OKUNO T., SUGIYAMA T., KOHYAMA M., KOJIMA S., OKU H. and IKEDA T.: Ocular blood flow changes after dynamic exercise in humans. Eye, 20 (7): 796-800, 2006. 19- KLEIN B.E., KLEIN R. and KNUDTSON M.D.: Intraocular pressure and systemic blood pressure: Longitudinal perspective: The Beaver Dam Eye Study. Br. J. Ophthalmol., 89 (3): 284-7, 2005. 20- LOVASIK J.V., KERGOAT H., RIVA C.E., PETRIG B.L. and GEISER M.: Choroidal Blood Flow during Exercise-Induced Changes in the Ocular Perfusion Pressure. Investigative Ophthalmology and Visual Science; 44: 2126-32, 2003.
Comparative Study of the Effect of Allopurinol and Nabumetone either Alone or Combined on Freunds Adjuvant-Induced Arthritis in Rats
SOHA S. ESSAWY, M.D.
The Department of Pharmacology, Faculty of Medicine, Suez Canal University
Abstract
Background: Non steroidal anti-inflammatory drugs (NSAIDs) partially alleviate symptomatic manifestation of rheumatoid arthritis (RA) but do not provide long term protection from articular damage. Nabumetone possesses a powerful anti-inflammatory profile as convential NSAIDs, with little gastric and renal side effects due to selective blocking of Cyclooxygenase-2 (COX-2), thus prevent formation of prostaglandins (PGs) responsible for the cardinal signs of inflammation. Over expression of XO enzyme, overproduction of free radicals plus release of inflammatory cytokines has been shown to be involved in the inflammation-induced tissue damage. Xanthine oxidase (XO) inhibitors as allopurinol may retard disease progression most probably due to an antioxidant effect. Hence, XO inhibitors/NSAIDs combinations may help to achieve both long term prevention of disease progression as well as rapid onset of symptomatic control. Aim of the Work: This study was designed to compare the biochemical and pharmacological effects of allopurinol, nabumetone and their combination in RA. Material and Methods: Forty male rats were used, divided into five groups (8 rats each): Control group, Complete Freund's Adjuvant (CFA) induced RA group-not treated-, arthritic group treated with allopurinol, arthritic group treated with nabumetone and arthritic group treated with allopurinol plus nabumetone. After 6 weeks of treatment, the anti-inflammatory and antioxidant effects of the tested drugs on the severity of arthritis was evaluated by ELISA assay of serum TNF-, a critical inflammatory mediator in this condition, calorimetric assay of serum uric acid due the evidence of increased XO activity in serum and tissue in patients with RA and by calorimetric assay of the oxidative stress parameters, lipid peroxides (LPO) and superoxide dismutase (SOD), due to overproduction of reactive oxygen species (ROS) during inflammatory process. Results: In the arthritic non-treated group, the serum levels of TNF-, uric acid and LPO were significantly higher while the activity of SOD was significantly lower than the control group; also there was a significant difference in right hind paws thickness between the two groups. In the allopurinol Correspondence to: Dr. Soha S. Essawy, E-mail: sohaessawy@yahoo.com.
treated group, the activity of SOD was significantly increased while the serum levels of TNF-, LPO, uric acid and right hind paws thickness were significantly decreased in comparison with the arthritic non-treated group. Nabumetone induced a significant decrease in the levels of TNF- and LPO and a significant increase in SOD activity accompanied by a significant decrease in right hind paws thickness in comparison with the arthritic non-treated group. Combination of allopurinol and nabumetone induced also a significant improvement of all serum bio-indices and right hind paws thickness compared to other treated group, this improvement was still significantly different from the non-arthritic control group. Conclusion: Both allopurinol and nabumetone showed anti-inflammatory and antioxidant effects in CFA-induced arthritis in rats. Addition of allopurinol to nabumetone induced a synergistic effect evidenced by reducing paw edema, free radical generation and improves antioxidant status. Key Words: Rheumatoid arthritis Allopurinol Nabumetone Antioxidants.
Introduction RHEUMATOID arthritis is polyarticular autoimmune disease affecting 1-2% of the population; it is characterized by joint swelling, hyperplasia of synoviocytes, mainly of synovial fibroblasts resulting in progressive destruction and disability [1]. Immunization of rats with Complete Freund's Adjuvant (CFA) leads to development of adjuvantinduced arthritis as a model of chronic inflammation that bears resemblance to RA. Freund's Adjuvant is an antigen solution emulsified in mineral oil that is effective in stimulating cell-mediated immunity and may lead to the potentiation of the production of certain immunoglobulins [2]. The pro-inflammatory cytokines TNF-, IF, IL-1, 2, 6, 8 PGs are shown to play an important role in the pathophysiology of arthritis development in animal models and in humans [3]. Cyclooxygenase (COX) enzyme is the key enzyme in the bio-
421
422
Comparative Study of the Effect of Allopurinol
synthesis of prostanoids from arachidonate. Therapeutic and side effects of NSAIDs resulted mainly from inhibition of the COX enzyme activity [4]. Cyclooxygenase-2 (COX-2) has been shown to be induced in vivo under inflammatory conditions. Selective inhibitors of COX-2 are potential therapeutic agents expected to have anti-inflammatory effects similar to those of convential NSAIDs as they decrease formation of PGs mainly PGE2; the key prostaglandin mediating the cardinal signs of inflammation, but with improved side effects profile on the gastric and renal systems [5]. Nabumetone-a selective COX II inhibitor-is converted in liver to an active metabolite. Its half life is about 24 hours, so it is suitable for once daily dosing. Also, it is not renally excreted; this gives it an advantage in many rheumatic diseases that are associated with renal pathology [6]. Overproduction of free radicals has been shown to be involved in the inflammation-induced tissue damage [7] . The triggering mechanisms for the production of free radicals are intracellular enzymes like xanthine-oxidase (XO), microsomal NADPHdependent cytochrome P-450 reductase and mitochondrial membrane bound dehydrogenases. All these enzymes can initiate the production of oxygen radicals which are mainly responsible for further initiation of lipid peroxidation [7]. A variety of well known endogenous enzymes such as SOD, catalase and glutathione peroxidase act as scavengers for free radicals, in contrast other endogenous enzymes such as XO can produce free radicals [8]. Allopurinol, a well known XO inhibitor, has been shown to prevent ischemia-reperfusion injury-induced tissue damage [9]. It has been shown that allopurinol has a preventive effect on rejection in organ transplantation, in chronic gastric ulceration, in chronic inflammation and in focal cerebral ischemic injury [10]. There is an evidence for increased circulating levels of XO in human plasma samples from patients with RA. Allopurinol ameliorates the symptoms of arthritis in animal models of RA; at least, some of these effects are likely related to the antioxidant actions of the compound [8,11]. Allopurinol may be a novel strategy in the treatment of various cellular autoimmune disorders due to antioxidant effect [10]. The aim of the present study was to determine the possible antioxidant and anti-inflammatory effects of allopurinol and to compare these effects
with those of nabumetone. The effect of their combination was also studied. Material and Methods Animals: Forty adult male albino rats, weighing 150-170 g each, were used in this study. Rats were purchased from the National Research Center (NRC), Cairo, Egypt and kept in the Department of Pharmacology, Faculty of Medicine, Suez Canal University where the experiment was conducted. Each animal was left alone in a polyethelene cage and allowed for acclimatization before the start of the study for 7 days. They were kept on a standard rodent chow and water was freely provided. The transportation, care and use of animals were harmless and directed by well trained and experienced person, taking in consideration the avoidance or minimization of discomfort, distress, and pain. The treatment protocol was approved by the National Research Center Animal Rights Committee. Drugs and chemicals: Allopurinol: It was supplied as a white powder from Sigma pharmaceuticals. Nabumetone: It was supplied as white crystalline powder from Smithkline. Beecham pharmaceuticals. Complete Freund's Adjuvant (CFA): It was provided from Sigma biosciences, Egypt as 10ml amber viscous emulsion. Each milliliter contains 1mg heat killed and dried mycobacterium tuberculosis emulsified in 0.85ml mineral oil and 0.15ml mannide monooleate, dissolved in 85 percentage saline. It was stored at 2-8C. Kits: TNF- (using cytoscreen immunoassay Kit for rat TNF- Catalog No. KRC3012, bioscience International, Inc., Cmarillo, USA). Uric acid, LPO and SOD kits were obtained from biodiagnostic company (Cairo, Egypt). Induction of arthritis: Arthritis was induced by a single subcutaneously injection of 0.1ml CFA corresponding to 100 microgram mycobacterium T.B. for each animal in the right hind paw [12]. Study design and animal groups: Forty animals were used, divided into the following 5 groups (8 animals each); treatment was
Soha S. Essawy
423
given once daily orally (P.O.) by oral gavage for 6 weeks starting with the day of CFA injection to investigate the effect of tested drugs on induction of arthritis. Control group: Non-arthritic that received no medication. Arthritic control group: Injected once by 0.1ml CFA and received 0.5ml water P.O. Allopurinol group: Arthritic group received allopurinol alone P.O. suspended in 0.5ml water in a dose of 50mg/Kg. The dose of allopurinol was selected as the optimal dose that does not cause nephrotoxicity [11,13]. Nabumetone group: Arthritic group received nabumetone alone P.O. suspended in 0.5ml water in a dose of 300mg/Kg. The dose represented the average human therapeutic dose [14]. Combination group: Arthritic group received allopurinol + nabumetone P.O. in the same above mentioned doses. All rats were sacrificed by decapitation after 6 weeks from the onset of the experiment. The degree of severity of the induced arthritis and the effects of the tested drugs were evaluated by two parameters; blood testes for some biomarkers and the thickness in the hind paws. Blood tests: Five ml blood was collected from retro-orbital plexus of each rat in a clean sterile tube inserted at the inner canthus of the eye. The blood was left to be clotted, blood samples were centrifuged and serum separated to be stored at 20C until analyzed. Serum level of the inflammatory mediator (TNFa): These kits depend on solid-phase sandwich Enzyme-linked immunosorbent assay (ELISA) [15]. Serum levels of uric acid: Using uric acid Kits, colorimetric method [16]. Serum levels of lipid peroxides (LPO): By thiobarbituric acid (TBA) test. The sample under test is treated by TBA and a pink chromogen is measured. In TBA reaction, one molecule of malondialdehide (MDA) reacts with 2 molecules of TBA with the production of pink pigment detected by spectrophotometer with an absorption maximum of 532nm [17]. Superoxide dismutase (sod) activity: This assay relies on the ability of the enzyme to inhibit
the phenazine methosulphate (PMS)-mediated reduction of nitroblue tetrazolium dye by spectrophotometer with an absorption maximum of 560nm [18]. Evaluation of the rat paw edema: The thickness of right hind paw of each rat was measured in m.m. by using hand screw micrometer [14,19]. The reading was taken when the metal facets of the micrometer made firm contact with the skin without compressing the soft tissues. Statistical analysis: The data was coded and entered using the statistical package SPSS version 15. The results were expressed as mean + S.E.M. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc multiple comparison; bonferroni test, to test the significance among group means. p value 0.05 was considered statistically significant at confidence interval 95% [20]. Results Serological studies: As seen in Table (1 and Fig. (1, panel A), induction of arthritis by CFA in rats showed a significant increase in the serum level of TNF- (129.93.1 pg/ml) compared to non-arthritic control rats (33.1 2.6). Administration of allopurinol, nabumetone or their combination resulted in a significant decrease in serum level of TNF- (84.42.9, 67.32.8 and 43.81.7 respectively) (p<0.05) compared to arthritic untreated group. Combination of the two drugs resulted in a significant decrease in TNF- serum level compared to the other treated groups and the control group. Also, serum level of uric acid was significantly increased in the arthritic untreated rats (2.70.8 mg/dl) compared to non-arthritic control rats (1.3 0.8) as shown in Table (1 and Fig. (1, panel B). Oral administration of allopurinol either alone or combined with nabumetone resulted in a significant decrease in uric acid serum level (1.90.4, 1.70.9 respectively) compared to arthritic untreated group. Combination of allopurinol and nabumetone resulted in a significant decrease in uric acid serum level compared to nabumetone alone treated group and the non-arthritic control group. Regarding LPO, as shown in Table (1 and Fig. (1, panel C), there was a significant increase in its serum level in the arthritic untreated rats (1.70.04 nmol/ml) compared to non-arthritic control rats (0.620.06). Oral administration of allopurinol, nabumetone or their combination induced a signif-
424
icant decrease in LPO serum level (0.760.04, 0.80.04 and 0.590.02 respectively) compared to arthritic untreated group. Combination of the two drugs resulted in a significant decrease in LPO serum level compared to the other treated groups and the non-arthritic control group. As illustrated in Table (1 and Fig. (1, Panel D), SOD activity was significantly decreased in arthritic untreated rats (116.15.3%) when compared to control non-arthritic rats (341.47). This activity was significantly increased again after oral administration of allopurinol, nabumetone or their combination for 6 weeks (238.66.7, 211.17.1 and 277.59.1 respectively) when compared to arthritic untreated rats. The highest increase was observed in the combination group that was significantly
150.0
different from the other treated groups and the non-arthritic control group. Evaluation of the rat paw edema: As presented in (Table 2), induction of arthritis induced a significant increase in the right hind paw thickness by about 9 folds compared to non-arthritic control group. Administration of allopurinol, nabumetone or their combination resulted in a significant decrease in the hind paw thickness by about 37.5%, 46% and 56% respectively comparing to arthritic untreated group. Combination of allopurinol and nabumetone resulted in a significant decrease in right hind paws thickness compared to the other treated groups, but this decrease still significantly different from the control group. 3.0 2.5
Uric acid (mg/dl) $
# *
* # *
TNF- (pg/ml) $
# * @
* @ # *
100.0
2.0 1.5 1.0 0.5
50.0
# *
0.0 CON ART ALLO (A) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
*
NABU
COMB
0.0
CON
ART
ALLO (B)
NABU
COMB
LPO (nmol/ml)
400 300
SOD %
# *
# *
# *
$ # *
@ $ # *
# *
200
*
100 0
CON
ART
ALLO (C)
NABU
COMB
CON
ART
ALLO (D)
NABU
COMB
Fig. (1): The effect of the tested drugs on serum biomarkers at the end of week 6. Rats were injected once with CFA (0.1ml subcutaneously in the right hind paw) to induce experimental arthritis. Rats were treated orally once daily with either allopurinol (50mg/Kg), nabumetone (300m/Kg) or their combination starting from the 1st day of CFA injection. Arthritic control rats showed significant increase in serum TNF-, LPO, uric acid and significant decrease in SOD activity; treatment with allopurinol, nabumetone or their combination improved these parameters significantly, the best effect was observed with the combination group. Results are mean SEM and analyzed using one-way ANOVA and Bonferroni post-hoc test.
* = Significantly different from control group at p0.05, # = Significantly different from arthritic group at p0.05, $ = Significantly different from allopurinol group, @ = Significantly different from nabumetone group. CON = Normal control. ART = Arthritic control. ALLO = Allopurinol. NABU = Nabumetone. COMB = Combination.
Soha S. Essawy
Table (1): The effect of allopurinol, nabumetone or their combination on serum biomarkers. Parameter TNF- (pg/ml) Uric acid (mg/dl) LPO (nmol/ml) SOD% Control group 33.12.6 1.30.8 0.620.06 341.47 Arthritic group Arthritic control 129.93.1* 2.70.8* 1.700.04* 116.15.3* Allopurinol 84.42.9*# 1.90.4*# 0.760.04*# 238.66.7*# Nabumetone 67.32.8*#$ 2.50.14*# 0.800.04*# 211.17.1*#$
425
Combination 43.81.7*#$@ 1.70.9*#@ 0.590.02*#$@ 277.59.1*#$@
Data are expressed as mean S.E. n=8. * : Significant difference from control group. # : Significant difference from arthritic control group. $ : Significant difference from allopurinol group. @: Significant difference from nabumetone group. Data are analyzed by using one way ANOVA followed by bonferroni test at p<0.05.
Table (2): The effect of allopurinol, nabumetone or their combination on the right hind paws thickness (in mm). Parameter Right hind paw thickness Control group 0.90.05 Arthritic group Arthritic control 8.30.13* Allopurinol 6.10.06*# Nabumetone 4.50.12*#$ Combination 3.70.24*#$@
Data are expressed as mean S.E. n=8. * : Significant difference from control group. # : Significant difference from arthritic control group. $ : Significant difference from allopurinol group. @: Significant difference from nabumetone group. Data are analyzed by using one way ANOVA followed by bonferroni test at p<0.05.
Discussion The present study was designed to find out the possible role of allopurinol on induction of arthritis. An animal model of RA was induced by CFA which was proved as a useful model of RA [2,21]. Also the study aimed to compare the effect of allopurinol with nabumetone and to illustrate the effects of their combination. It was proved that increase oxidative stress is one of the major hypotheses purposed to explain the development and progression of arthritis. Once formed, reactive oxygen species (ROS) induce lipid peroxidation and deplete antioxidant defenses such as SOD, which converts superoxide radicals to hydrogen peroxide, rendering the affected cells and tissues more susceptible to oxidative damage [22]. In the present study, treatment with allopurinol (50mg/kg/d P.O.) and nabumetone (300mg/kg/d P.O.) either alone or combined led to significant reduction in serum level of TNF- and LPO and significant increase in SOD activity compared to arthritic control group. The combined treatment was superior in the beneficial effect and induced a significant benefit comparing to other treated groups. These results were in agreement with Bae, et al. [23] who proved that oxidative injury and
inflammatory reactions in RA were associated with increased levels of inflammatory mediators like nitric oxide (NO), PGs, TNF-, IL-6 and blocking of these mediators formation can improve the clinical condition. Also, Inglis, et al. [24] proved that treatment with TNF- antagonists is of benefit in RA patients; in addition to exerting an antiinflammatory effect and slowing the progression of RA, anti-TNF- therapy produces a profound and rapid analgesia. Decreasing lipid oxidation is an important line of treatment of inflammatory conditions because oxidized lipids enhance endothelial cell superoxide anion production and stimulate the expression of several genes and their dependent cytokines and adhesion molecules in the synovial membrane. In addition oxidized lipids are chemotactant for monocytes and lymphocytes and enhance macrophages to produce toxic ROS, cytokines, proteases and growth factors [25]. Similarly, Kuzkaya and Stone [26] found that SOD activity was significantly lower in arthritis as inflammatory cytokines reported to have a suppressant effect on the gene expression and activity of SOD and blockage of xanthine oxidase (XO) enzyme significantly elevated this level [27] . On the other hand, the SOD activity in the current study was in contrast with Edmonds, et al.
426
and Schimdt, et al. [29] who showed that antioxidant therapies had no effect on RA disease activity or indices of inflammation, but only improve pain, probably due to central analgesic mechanism.
[28]
immune effector cells [37]. Allopurinol is reported to interfere with adenosine metabolism [37]; yet, allopurinol might share the adenosine release/ adenosine receptor activation hypothesis on immune-effector leucocytes. Allopurinol pretreatment prevented the increased synthesis of leukotriene B4 (LTB4) in hind limb ischemia-reperfusion in rabbits and LTB4 has been shown to be increased in synovial fluid during arthritis [38]. So there is a possibility that allopurinol may provide an additional beneficial effect by inhibiting the synthesis of LTB4 in adjuvant arthritis. The effect of nabumetone can be explained by the fact that lipid peroxidation and ROS can be formed as by-products of COX pathway, during PGG2 to PGH2 conversion, which generated hydrogen peroxide [39]. So there is increasing evidences suggest that COX-II inhibitors may exert a beneficial effects in oxidative stress. In the present study, the bio-indices results were confirmed by the direct physical evidence. Injection of CFA led to more that 89% increase in hind paw thickness compared to the control. These results are in agreement with Mileti et al. [40] c who showed that immunization with CFA induced an increased expression of heat shock protein (HSP) 47, a molecular chaperone involved in the synthesis and assembly of collagen molecules in adjuvant arthritis, in joints of rats, which exhibited severe clinical signs of arthritis at the time of disease peak. Oral administration of allopurinol, nabumetone or their combination induced as significant decrease in the right hind paws thickness by about 37.5%, 46% and 56% respectively compared to arthritic control rats. The combined regimen resulted in a significant decrease compared to other treated groups. The anti-inflammatory effect of allopurinol can be explained also as it suppresses the production of TNF- and down-regulates the expression of Intracellular Adhesion Molecule-1 (ICAM-1) and P2X7 receptors on monocyte/macrophages. ICAM1 serves as a ligand for Leukocyte Functional Antigen-1 (LFA-1) on T lymphocytes, allowing proper antigen presentation. P2X 7 receptors are thought to be involved in IL-1 release, mitogenic stimulation of T lymphocytes and the probable cytoplasmic communication between macrophages and lymphocytes at inflammation sites [41]. These results are in agreement with Pacher, et al. [37] who approved that allopurinol may be useful
The effect of allopurinol was explained by Zhang, et al. [30] who suggested that arthritis is associated with increased articular formation of nitrotyrosine, which may contribute to injury. Nitrotyrosine is formed by nitration of tyrosine by reactive nitrogen species, the formation of which may be enhanced by XO that can generate NO from nitrite/nitrate, and O free radicals during xanthine metabolism. So XO inhibitors may have anti-oxidant and anti-inflammatory properties by decreasing nitrotyrosine formation. Meanwhile, oral administration of allopurinol alone or combined with nabumetone induced significant decrease in uric acid serum level. These results were supported by the findings of Nemeth, et al. [31] who described that uric acid is considered one of the non-enzymatic antioxidants, but increased production of uric acid means increased free radical production due to the activation of the XO enzyme system. Nabumetone alone induced a non-significant decrease in uric acid serum level compared to arthritic untreated group. This can be explained as its mechanism of action is not through XO enzyme [5]. The underlying mechanism by which allopurinol exerts a protective effect on free radicalmediated pathological conditions as RA has been studied. It is a scavenger of the highly reactive hydroxyl radicals [32]. The drug also prevents the formation of free radicals through XO inhibition [11]. As an intracellular enzyme, two forms of XO, a dehydrogenase (type D) and an oxidase (type O) have been described. In normal conditions, type D of the enzyme converts the xanthine to uric acid; during inflammation, this form of enzyme transforms into type O which is responsible for the production of free radicals [33,34]. Most interestingly, the anti-inflammatory and antioxidant effects of allopurinol may be not related to ROS generation [35] . In addition to blocking uric acid production, inhibition of XO causes an increase in hypoxanthine and xanthine, which are converted to adenosine and guanosine monophosphates [35]. Adenosine is an intrinsic anti-inflammatory mediator involved in several immunoregulatory cascades [36] . Other researchers attributed the immunomodulatory effect of allopurinol to its affinity to bind adenosine receptors; particularly A2 receptors which are found on the leucocytic
Soha S. Essawy
427
7- OZKAN Y., YARDYM A. S., SEPICI A., KESKIN E., SEPICI V. and SIMSEK B.: Oxidative status in rheumatoid arthritis. Clin. Rheumatol., 26: 64-8, 2007. 8- DAS D.K., ENGELMAN R.M., CLEMENT R., OTANI H., PRASAD M.R. and RAO P.S.: Role of xanthine oxidase inhibitor as free radical scavenger; A novel mechanism of action of allopurinol and oxypurinol. Biochem. Biophys. Res. Commun., 148: 314-9, 1987. 9- GARCIA J., MARTIN ROLLAN C., REFOYO ENRINQUEZ M.A., HOLGADO MADRUGA M., MARINO HERNANDEZ E. and MARCIAS NUEZ J.F.: Improved survival in intestinal ischemia by allopurinol not related to xanthine-oxidase inhibition. J. Surg. Res., 48: 144-6, 1990. 10- PL P., ALEX N. and CSABA S.: Therapeutic effects of Xanthine Oxidase inhibitors. Pharmacol. Rev., 58: 87114, 2006. 11- KLOCKE R., MANI A.R., MOORE K.P., BLAKE D.R. and MAPP P.I.: Inactivation of xanthine oxidoreductase is associated with decreased joint swelling and nitrotyrosine formation in acute antigen-induced arthritis. Clin. Exp. Rheumatol., 23: 345-50, 2005. 12- KAKLAMANIS P.M.: Experimental models of arthritis in rats. Clin. Rheumatol., 11: 41-7, 1992. 13- YOSSIF A.M., IBRAHIM T.M., SALEM H.A., GAMIL N.M. and EL-SAYED L.M.: Effect of high lipid diet and allopurinol on the development of experimentally induced arthritis in rats. Pharmacology. 51 (3): 160-4, 1995. 14- SOMIA M., ATEF A., OSAMA M. and SHADIA A.: Nabumetone versus endomethacin on experimentally induced polyarthritis in rats. J. Egypt. Soc. Pharmacol. Exp. Ther., 13: 215-32, 1994. 15- MAINI R.N., ELLIOTT M.J., BRENNA F. M. and FELDMAN N.: Beneficial effect of tumor necrosis alpha blockade in rheumatoid arthritis. Clin. Exp. Immuno., 101 (2): 207-12, 1995. 16- SPINCER K.: Analytical reviews in clinical biochemistry. Ann. Clin. Biochem., 23: 1-25, 1986. 17- DRAPER W. and HADLEY M.: Assessment of Malondialdehyde. Method. Enzymol., 186: 421-31, 1991. 18- NISHIKIMI M., ROA N.A. and YPGI K.: The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem. Biophys. Res. Commun., 46: 849-54, 1972. 19- CHILLINGWORTH N.L. and DONALDSON L.F.: Characterization of a Freund's complete adjuvant-induced model of chronic arthritis. J. Neurosci. Methods, 128: 45-52, 2003. 20- DAWSON-SAUNDERS B. and TRAPP R.G.: Determination of sample size, estimation and comparing means. In: Basic and clinical biostatistics. Appleton and Lang, Norwalk, Connecticut., 1: 118-9, 1990. 21- PEARSON C.M. and WOOD F.D.: Development of arthritis following injection of adjuvant. Arthritis and Rheum., 2: 440, 1959. 22- BAUEROVA K. and BEZEK A.: Role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis. Gen. Physiol. Biophys., 18: 15-20, 1999.
as additive therapy in RA due to its antioxidant proprieties. The effect of nabumetone of hind paws thickness was supported by the results of Somia, et al. [14] and Chang, et al. [42] who demonstrated that nabumetone induced significant reduction in the mean arthritic score of hind paws of rats. This can be explained due to inhibition of COX-2, the inducible enzyme that can be expressed in inflamed tissues and in synovial tissues from patients with RA and experimental animals respectively. The COX-2 plays a pathophysiological role by production of prostaglandins involved in inflammation, pain and pyrexia [43]. Inhibition of COX-2 also decreased production of ROS formed during prostaglandin synthesis [39] . In a short-term double blind comparative study, nabumetone was proved to be equally effective to indomethacin in improvement of arthritis but with improved side effect profile on ulcers [44]. Again, Cardin, et al. [45] demonstrated that nabumetone was more effective (70%) than indomethacin (55%) in reducing paw edema. Other selective COX-2 inhibitors also improved the arthritic index in mice with collageninduced arthritis [46]. Conclusion: Both allopurinol and nabumetone showed anti-inflammatory and antioxidant effects in CFA-induced arthritis in rats. Addition of allopurinol to nabumetone reduced free radical generation and improved antioxidant status. The combine regimen resulted in a significant improvement of arthritis compared to each drug individually. Combination of allopurinol and nabumetone may effectively enhance the impaired oxidant/antioxidant system and may be useful in delaying the complication of RA by different mechanisms. References
1- SANGAHA O.: Epidemiology of rheumatic diseases. Rheumatology (Oxford) 39 (Suppl 2): 3-12, 2000. 2- BARSANTE M.M., ROFFE E., YOKORO C.M., TAFURI W.L., SOUZA D.G., PINHO V., CASTRO M.S. and TEIXEIRA M.M.: Anti-inflammatory and analgesic effects of atorvastatin in a rat model of adjuvant-induced arthritis. Eur. J. Pharmacol., 516 (3): 282-9, 2005. 3- NISHIMOTO N., YOSHIZAKI K. and MAEDA K.: Toxicity, pharmacokinetics, and dose-finding study of repetitive treatment with the humanized anti-interleukin 6 receptor antibody MRA in rheumatoid arthritis. Phase I/II clinical study. J. Rheumatol., 30: 1426-35, 2003. 4- SMITH W.L.: Prostanoids biosynthesis and mechanism of action. Am. J. Physiol., 268: 181-9, 1992. 5- HARRIS R.C. and BREYER M.D.: Update in cyclooxygenase II inhibitors. Clin. J. Am. Soc. Nephrol., 1: 23645, 2006. 6- BROOKS P.M. and DAY R.O.: Non-steroidal anti-inflammatory drugs, differences and similarities. N. Engl. J. Med., 324: 1716-25, 1991.
428
23- BAE C.K., KIM S.J. and SUNG M.K.: Inadequate antioxidant nutrient intake and altered plasma antioxidant status of rheumatoid arthritis patients. J. Am. COII. Nutr., 22: 311-5, 2003. 24- INGLIS J.J., NISSIM A., LEES D.M., HUNT S.P., CHERNAJOUSKY Y. and KIDD B.L.: The differential contribution of tumor necrosis factor alpha to thermal and mechanical hyperalgesia during chronic inflammation. Arthritis Res., 7: 807-16, 2005. 25- MAHAJAN A. and TANDON V.R.: Antioxidants and rheumatoid arthritis. J Indian Rheumatol. Assoc., 12: 13942, 2004. 26- KUZKAYA L.G. and STONE T.W.: Antioxidants ameliorate symptoms of rheumatoid arthritis and related disorders. Br. J. Nutr., 85: 251-69, 2005. 27- YOSHIKAWA T., TANAKA H.M. and KONDO M.: The increase of lipid peroxidation in rat adjuvant arthritis and its inhibition by superoxide dismutase: Role of xanthine oxidase. Biochem. Med., 33: 320-6, 1985. 28- EDMONDS S.E., WINYARD P.G. and GUO R.: Putative analgesic activity of repeated oral doses of vitamin E in the treatment of rheumatoid arthritis: Results of a prospective placebo controlled double blind trial. Ann. Rheum. Dis., 56: 649-55, 1997. 29- SCHMIDT A.P., BHMER A.E., LEKE R., SCHALLENBERGER C. and ANTUNES C.: Anti-nociceptive effects of intracerebroventricular administration of guanine-based purines in mice: Evidences for the mechanism of action. Brain Res., 1239: 50-8, 2008. 30- ZHANG Z., NAUGHTON D., WINYARD P.G., BENJAMIN N., BLAKE D.R. and SYMONS M.C.: Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: A potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. Biochem. Biophys. Res. Commun., 249: 767-72, 2004. 31- NEMETH I., TALOSI G., PAPP A. and BODA D.: Xanthine oxidase activation in mild gestational hypertension. Hypertens. Pregnancy, 21: 1-11, 2002. 32- NAMAZI M.R.: Cetirizine and allopurinol as novel weapons against cellular autoimmune disorders. Int. immunopharmacol., 4: 439-53, 2004. 33- GOMEZ-CABRERA M.C., BORRS C., PALLARD F.V., SASTRE J., JI L.L. and VIA J.: Decreasing xanthine oxidase-mediated oxidative stress prevents useful cellular adaptations to exercise in rats. J. Physiol. 567 (Pt 1): 11320, 2005. 34- VESKOUKIS A.S., NIKOLAIDIS M.G., KYPAROS A., KOKKINOS D., NEPKA C., BARBANIS S. and KOURETAS D.: Effects of xanthine oxidase inhibition on oxidative stress and swimming performance in rats. Appl. Physiol. Nutr. Metab., 33 (6): 1140-54, 2008.

35- FORREST C.M., HARMAN G. and MCMILLAN B.: Purine modulation of cytokine release during therapy of rheumatoid arthritis. Nucleosides Nucleotides Nucleic Acids, 23: 1107-10, 2004. 36- AKDEMIR H., ASIK Z., PASAOGLU H., KARAKUCUK I., OKTEM I.S. and KOC R.K.: The effect of allopurinol on focal cerebral ischemia: The role of adenosine, an experimental study in rabbits. Neurosurg. Rev., 24: 1315, 2001. 37- PACHER P., NIVOROZHKIN A. and SZAB C.: Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol. Rev., 58: 87-114, 2006. 38- GOLDMAN G., WELBOURN R. and VALERI D.: Oxygen free radicals are required for ischemia-induced leukotriene B4 synthesis and diabetes. Surgery, 111: 28793, 1992. 39- ADDERLEY S.R. and FITZGERALD D.J.: Oxidative damage of cardiomyocites is limited by extracellular regulated kinases 1, 2-mediated inhibition of COX-2. J. Biol. Chem., 374: 5048-42, 1999. 40- MILETIC T., KOVACEVIC-JOVANOVIC V. and STA NOJEVIC S.: Strain Differences and the Role for HSP47 and HSP70 in Adjuvant Arthritis in Rats. Scand J. Immun., 64: 623-32, 2006. 41- YAMADA I., FUKUNARI A., OSAJIMA T., KAMEZAWA M., MORI H. and IWANE J.: Pharmacokinetics/ pharmacodynamics of Y-700, a novel xanthine oxidase inhibitor, in rats and man. Nucleosides Nucleotides Nucleic Acids, 23: 1123-5, 2004. 42- CHANG D.M., BAPTISTE P. and SCHUR P.H.: The effect of antirheumatic drugs on interleukin 1 (IL-1) activity and IL-1 and IL-1 inhibitor production by human monocytes. J. Rheumatol. 17: 1148-57, 1990. 43- KENNDY B.P.: Cloning and expression of rat prostaglandin endoperoxide synthase. Biochem. Biophy. Res., 197: 494-8, 1993. 44- FLEISHMAN R.M.: Clinical efficacy and safety of nabumetone in rheumatoid arthritis and osteoarthritis. J. Rheumatol., 19 (36): 32-40, 1992. 45- CARDIN S., SOLL A. and TACHE W.: Different effects of indomethacin and nabumetone on prostaglandin mediated gastric response in rats. J. Pharmacol. Exp. Ther., 275 (2): 667-73, 1995. 46- AHMED M., JULIA J., SALWA M., AMANI E., RICHARD O., WAFAA I. and ASHRAF B.: Nimesulide improves the disease modifying anti-rheumatic profile of methotrexate in mice with collagen-induced arthritis. Euro. J. Pharma., 644: 245-50, 2010.
Comparative Study between Three Different Protocols for Prevention of Hypertensive Disorders Associating Pregnancy
AHMED M. MAGED, M.D.*; GHADA ABD EL-FATTAH, M.D. and ABD EL-RAHMAN A. ABD EL-RAZEK, M.D.**
The Departments of Obstetrics & Gynecology* and Pediatrics**, Kasr El-Aini Hospital, Cairo University
Abstract
Objective: To compare the role of low-dose aspirin, antioxidants (vitamin C and E) and calcium in preventing hypertensive disorders of pregnancy and their maternal, fetal and neonatal sequelae. Study Design: 400 pregnant women less than 12 weeks' gestation were divided into 4 equal groups. Group I received low-dose aspirin 75mg, group II received vitamin C 1000mg/d plus vitamin E 400mg/d, group III received calcium 1000mg/d and group IV was a control group. Each group was further divided into high and low risk patients. Results: The rate of gestational hypertension, preeclampsia and IUGR was significantly reduced more in group I and II compared to group IV. There was a significant difference between group I and II versus group III and IV regarding development of severe pre-eclampsia. There was insignificant difference regarding GA at delivery, eclampsia, HELLP, placental abruption, neonatal deaths and congenital anomalies between all study groups. Conclusion: Use of anti-oxidants is superior to either low-dose aspirin or calcium in reduction of the risk of gestational hypertension, preeclampsia and eclampsia. Use of lowdose aspirin has better results than either anti-oxidants or calcium in reduction of the rate of severe preeclampsia, HELLP and IUGR. Key Words: Hypertensive disorders of pregnancy Low-dose aspirin Anti-oxidants Calcium Neonatal outcome.
in which reduced placental perfusion (stage 1) leads to the maternal syndrome (stage 2) is likely to provide a simplified yet largely accurate description of the origin of severe early onset disease but may be less relevant for the later onset milder disease [3].
[2]
As an inhibitor of cyclo-oxygenase enzymes, aspirin inhibits thromboxane A2 and reduce vasoconstriction. Systematic reviews show that the use of low dose aspirin has a moderate consistent reduction of PE but numbers needed to treat were very high [4]. Oxidative stress is considered to be a key factor in PE process [5]. Maternal plasma concentrations of ascorbic acid are low and in the placenta synthesis of reactive oxygen species are reduced [6]. Activities of antioxidant enzymes are reduced [7]. Therefore antioxidant supplementation may offer potential for prevention or amelioration of PE [8]. Epidemiologic data suggest an invert correlation between dietary calcium intake and incidence of PIH in diverse population [9]. In this study, we aimed at showing the role of different protocols (antioxidants, low dose aspirin and calcium) in preventing hypertensive disorders of pregnancies and their complications. Material and Methods This prospective case-control study was performed on pregnant women in their first trimester that came to Kasr El-Aini maternity hospital during the period starting from January 2009 to December 2010. The study protocol was approved by our Investigational Review Board and was conducted in accordance with the University by laws for human
Introduction ABOUT 10-15% of pregnancies are complicated by hypertensive disorders such as pregnancy induced hypertension (PIH) and preeclampsia (PE) making them one of the most important causes of maternal and neonatal morbidity and mortality [1]. The cause of preeclampsia remains largely unknown, but poor placentation is an important predisposing factor. The proposed 2 stage model
Correspondence to: Dr. Ahmed M. Maged, Email: prof.ahmedmaged@gmail.com
429
430
Comparative Study between Three Different Protocols
research and the study and possible outcomes were explained and informed consent was obtained from 400 pregnant women with the following conditions: Maternal age between 18 and 41 years, gestational age <12 weeks (from LMP or ultrasound), all were singleton living pregnancy. Pregnant females were randomly assigned to one of four groups. Group I included 100 pregnant women who received acetyl salicylic acid 75mg/day (Aspocid infantile, CID pharmaceutical. Egypt). Among this group 6 women aborted and 4 were lost during follow-up and 90 completed the study. Group II included 100 pregnant women who received Vitamin C 1000mg/day (Vitacid effervescent tablets, CID pharmaceutical. Egypt) plus Vitamin E 400mg/day (Vitamin E 400, pharma pharmaceutical. Egypt). Among this group 7 women aborted and 5 were lost during follow up and 88 completed the study. Group III included 100 pregnant women who received Calcium 1000mg/day (Calcimate 500mg, Misr pharmaceutical. Egypt). Among this group 8 women aborted and 6 were lost during follow-up and 86 completed the study. Group IV included 100 pregnant women as control. Among this group 6 women aborted and 7 were lost during follow-up and 87 completed the study. Pregnant women in the study were then subdivided into: a- Women at high risk of developing pre-eclampsia. b- Women at low risk of developing pre-eclampsia. Women at high risk of developing pre-eclampsia were identified as those who had essential hypertension prior to pregnancy, insulin-treated diabetes or history of pre-eclampsia in previous pregnancies. Women at low risk for pre-eclampsia were considered to be those without any risk factor for preeclampsia. Blood pressure at randomization was measured with a mercurial sphygmomanometer blood pressure measuring instrument, GOH industries LTD, Tokyo, Japan. After the participant had been seated for 5 minutes, the average of two measurements
Table (1): Basic characteristics of the study groups. GI Mean Age Primigravida (N & %) BMI Smokers (N & %) Ch HTN (N & %) Previous H/O PIH (N & %) SBP DBP 29.8 43 27.7 9 10 19 117.9 76.6 SD 5.7 47.7 5.1 10 11.1 21.1 14.3 8.7 Mean 28.7 45 28.1 7 9 16 113.5 72.3
taken 3 minutes apart was recorded. Clinical or biochemical grounds by identification of one additional feature of pre-eclampsia (e.g. HELLP syndrome, eclampsia) follow-up every 4 weeks till 28 weeks then biweekly for blood pressure, proteinuria and ultrasonography were done. Primary outcomes for the women included gestational hypertension which was defined as two diastolic blood pressure readings of 90mmHg or more at least 4 hours apart, or one reading of at least 110mmHg, occurring after 20 weeks' gestation or up to 48 hour postnatal and excluding labor and pre-eclampsia which were defined as gestational hypertension with proteinuria in accordance with the International Society for the Study of Hypertension in Pregnancy guidelines. Proteinuria was defined as a result of at least 1+ for dipstick analysis of a midstream specimen on two or more occasions or more than 300mg urinary protein per 24 hour. Secondary outcomes included development of eclampsia, HELLP syndrome, placental abruption. For the newborn, primary outcome variables are preterm birth (Liveborn infants delivered before 37 weeks from the-1 st day of the last menstrual period are termed premature by the World Health Organization) [10] and IUGR (The term intrauterine growth restriction (IUGR) indicates fetuses with birth weight below 10th percentile for their gestational ages with clinical evidence of stunting, but born at term) [11]. Congenital anomalies, NICU admission, neonatal complications and stillbirth or neonatal deaths were the main secondary outcome variables. Results Demographic data for all study groups is represented in Table (1). There were no significant differences (p value >0.05) regarding age, parity, BMI, number of smokers, patients with chronic hypertension, and history of PIH in previous pregnancy between the four study groups. It shows also no statistically significant difference between the study groups regarding baseline systolic and diastolic blood pressure.
GII SD 6.1 51.1 5.4 7.95 10.2 18.18 12.9 8.2 Mean 32.1 43 27.6 7 11 16 110.1 69.9
GIII SD 5.7 50 4.6 8.13 12.7 18.6 13.1 8.1 Mean 29.3 46 26.8 8 8 13 111.4 68.6
GIV SD 5.5 52.8 4.9 9.19 9.19 14.9 12.2 9.1
Ahmed M. Maged, et al.
431
Table (2) shows a statistically insignificant difference regarding abortion rate, GA at delivery, incidence of eclampsia, HELLP, placental abruption, neonatal deaths, congenital anomalies, RDS and neonatal enterocolitis between all study groups (p value <0.05). There was a statistically significant difference between group IV and other 3 groups regarding incidence of gestational hypertension, pre-eclampsia, IUGR (higher incidence among group III more than group I and II), neonatal birth weight, NICU admission and neonatal jaundice (p value <0.05). There was a statistically significant difference
Table (2): Outcome parameter among study groups. GI No GA at delivery (mean SD) Abortions Gestational HTN Preeclampsia Severe preeclampsia Eclampsia HELLP Placental abruption IUGR Neonatal BW (mean SD) NICU admission Neonatal deaths Congenital anomalies Neonatal complications: RDS Jaundice Enterocolitis 37.9 6 6 9 4 1 2 2 3 3356 22 1 1 5 9 0 % 1.4 6.66 6.66 10 4.4 1.1 2.2 2.2 3.3 789 24.4 1.11 1.11 5.55 10 0 No 38.1 7 7 9 4 0 2 2 4 3176 19 1 0 5 8 0
between group I and II versus group III and IV regarding development of severe pre-eclampsia (p value <0.05). Regarding high risk women among the study groups there was no statistically significant difference regarding the age, parity, BMI, SBP and DBP (p value >0.05). There was a statistically significant difference between GI and the other 3 groups regarding number of smokers with higher number of smokers in group I (10 Vs. 6, 6, and 7 respectively) (p value <0.05) (Table 3).
GII % 1.6 7.95 7.95 10.2 4.54 0 2.27 2.27 4.54 803 21.59 1.13 0.0 5.68 9.09 0 No 37.4 8 7 8 7 1 3 3 4 3245 28 2 1 4 12 1
GIII % 1.2 9.3 8.13 9.3 8.13 1.16 3.48 3.48 4.65 815 32.55 2.32 1.16 4.65 13.95 1.16 No 36.3 6 9 13 8 2 4 3 9 2695 37 2 2 4 20 3
GIV % 1.9 6.8 10.34 14.94 9.19 2.29 4.59 3.44 10.34 982 42.52 2.29 2.29 4.59 22.98 3.44
Table (3): Basic characteristics among high risk study groups. GI Total number Age Primigravida no (%) BMI Smokers no (%) SBP DBP 49 32.1 25 27.9 10 121.3 76.8 5.8 51.02 5.7 20.4% 15.1 8.5 47 30.4 21 28.7 6 120.6 71.9 5.9 44.68 5.9 12.76% 13.7 8.2 GII 44 32.3 20 28.4 6 112.6 68.8 6.1 45.45 5.2 13.63% 13.2 8.1 GIII 40 31.2 22 28.2 7 116.5 72.4 6.1 55 5.4 17.5% 14.1 9.6 GIV
There was a statistically significant difference between IV and the other 3 groups as regards the gestational age (33.22.9 Vs. 37.42.1, 37.42.4 and 37.61.9 respectively) and the development of gestational HTN (8 Vs. 5, 6 and 5 respectively) and pre-eclampsia (12 Vs. 9, 9 and 8 respectively) (p value <0.05) with higher incidence of preterm labour, incidence of gestational HTN and preeclampsia in group IV (the control group) compared with the other 3 groups (group IV).
There was a higher incidence of severe preeclampsia in group III and IV (7 and 7 respectively) compared to group I and II (4 and 4respectively) with no difference between group III and IV. There was a statistically significant difference between group IV and the other 3 groups as regards the development of IUGR (9 Vs. 3,3 and 4 respectively) and neonatal birth weight (2068927 Vs. 2994807, 2869870 and 2902908 respec-
432
tively) (p value <0.05) with higher incidence of IUGR and lower birth weight in the control group (group IV). There was a highly significant difference between group IV and the other 3 groups as regards the admission to NICU (21 Vs. 12, 10 and 14 respectively) (p value <0.05) with higher number of neonates admitted to the NICU in the control group (group IV), while group II showed the least number of neonates admitted to NICU as compared to groups I and III. There was a statistically significant difference between group IV and the other 3 groups as regards
Table (4): Outcome parameter among high risk study groups. GI 49 No GA at delivery (mean SD) Gestational HTN Preeclampsia Severe preeclampsia Eclampsia HELLP Placental abruption IUGR Neonatal BW (mean SD) NICU admission Neonatal deaths Congenital anomalies Neonatal complications: RDS Jaundice Enterocolitis 37.4 5 9 4 1 2 2 3 2994 12 1 0 4 5 0 % 2.1 10.2 18.36 8.16 2.04 4.08 4.08 6.12 807 24.4 2.04 0 8.16 10.2 0 47 No 37.4 6 9 4 0 2 2 3 2869 10 1 0 3 4 0
the development of neonatal jaundice with higher incidence in group IV (the control group) compared to the other 3 groups (11 Vs. 5, 4, 7 respectively) (p value <0.05). There was no statistically significant difference among the 4 groups as regards the development of eclampsia (1, 0, 1, 2 respectively), HELLP (2, 2, 3, 4, respectively), placental abruption (2, 2, 2, 3 respectively), number of neonatal deaths (1, 1, 1, 2 respectively), congenital anomalies (0, 0, 1, 0 respectively), RDS (4, 3, 3, 3 respectively) and enterocolitis (0, 0, 1, 1 respectively) (p value >0.05) (Table 4).
GII % 2.4 12.76 19.14 8.51 0 4.25 4.25 6.38 870 21.27 2.12 0 6.38 8.51 0 44 No 37.6 5 8 7 1 3 2 4 2902 14 1 1 3 7 1
GIII % 1.9 11.36 18.18 15.9 2.27 6.81 4.54 9.09 908 31.8 2.27 2.27 6.81 15.9 2.27 40 No 33.2 8 12 7 2 4 3 9 2068 21 2 0 3 11 1
GIV % 2.9 20 30 17.5 5 10 7.5 22.5 927 52.5 5 0 7.5 27.5 2.5
Discussion PIH can develop into pre-eclampsia, eclampsia or even HELLP syndrome, these constitute an important cause of maternal and neonatal morbidity and mortality [1]. In our study, we found a significant lower incidence of gestational hypertension and preeclampsia in women treated with low dose aspirin. Askie and colleagues in 2007 [12] and James and coworkers [4] reported moderate but consistent reduction in pre-eclampsia in patients taking aspirin. Our findings were also in line with Hermida and colleagues [13] who reported a highly statistically significant blood pressure reduction in women receiving aspirin in comparison with placebo group. However, the CLASP trial [14] failed to show a significant decrease in the incidence of gestational hypertension but noted a significant decrease in the incidence of pre-eclampsia.
Our study showed a significant decrease in the incidence of IUGR, neonatal birth weight, NICU admission and neonatal jaundice between the group taking aspirin and the control group. A systematic review done by Duley and others in 2001 [15] , showed that there was a significant difference in the risk of small for gestational age in the aspirin group, however, there were no significant difference between treatment and control groups in the risk of birth weight <2500gm or admission to NICU. In this study, there was a statistically insignificant difference between the aspirin group and the control group as regards the abortion rate, GA at delivery, incidence of eclampsia, HELLP syndrome, placental abruption, neonatal deaths, congenital anomalies, RDS and neonatal enterocolitis. This result goes with what have been reported by Duley and others [15] that there was no significant difference between the aspirin group and the control
Ahmed M. Maged, et al.
433
group as regards the risk of eclampsia or placental abruption. In our study, we have found a much lower incidence of gestational hypertension, pre-eclampsia, IUGR, neonatal birth weight, NICU admission and neonatal jaundice in the antioxidant group (vitamin C and vitamin E) in relation to the control group. Maarten and colleagues [16] have stated that a combination of vitamin C and vitamin E, which act in synergy to prevent lipid peroxidation, may be effective in the prevention of pre-eclampsia. Also, Lucy and others in 2002 [17] have found that supplementation with vitamin C and vitamin E in women at increased risk of pre-eclampsia was associated with improvement of some relevant indices of oxidative stress and placental dysfunction towards values that were observed in a group of healthy women. However, Beazley and colleagues [18] reported that, according to their findings, antioxidant supplementation with vitamin C and vitamin E had no effect on the rate of pre-eclampsia in a high risk patient population. However, this can be due to the fact that they have terminated the study prematurely because of inadequate funding and also due to their small sample size (220 pregnant women). We found that calcium supplementation during pregnancy significantly decreases the risk of gestational hypertension, preeclampsia and IUGR. This goes with Hofmeyr and colleagues [19] who found that calcium supplementation have halved the risk of pre-eclampsia, reduced the risk of preterm birth and reduced the rate of fetal death. Also Lorrene and Janet [20] have stated that there is insufficient evidence to support routine calcium supplementation of all pregnant women, however, high risk groups may benefit from consuming additional dietary calcium. Also, a large prospective RCT, done by Belizan and colleagues [21], of elemental calcium 2g/day versus placebo in nulliparous women started after 20 weeks' gestation showed a significant reduction in the prevalence of pre-eclampsia. In support of our study, Bucher and others [22] have found that calcium supplementation did not significantly reduce the incidence of preterm delivery. So we can conclude that the use of low-dose aspirin, anti-oxidants (Vitamin C and E) and calcium separately is of significant importance in reducing the risk of gestational hypertension, preeclampsia and IUGR. However, the use of anti-oxidants (Vitamin C and E) is better than either low-dose aspirin or calcium in preventing the development of gesta-
tional hypertension, pre-eclampsia and eclampsia while the use of low-dose aspirin is the best in reducing the risk of severe pre-eclampsia, HELLP syndrome and IUGR (better than either anti-oxidants or calcium). The role of anti-oxidants, low dose aspirin and calcium is highly evident in the high risk groups for hypertensive disorders of pregnancy than in those with lower risk. References
1- SCHUTTE J.M., SCHUITEMAKER N.W., VAN ROOSMALEN J. and STEEGERS E.A.: Substandard care in maternal mortality due to hypertensive disease in pregnancy in the Netherlands. BJOG, 115: 732-6, 2008. 2- ROBERTS J.M. and HUBEL C.A.: Is oxidative stress the link in the two-stage model of pre-eclampsia? Lancet., 354: 788-9, 1999. 3- REDMAN C.W.G. and SARGENT I.L.: Placental debris, oxidative stress and pre-eclampsia. Placenta, 21: 597602, 2000. 4- JAMES A.H., BRANCAZIO L.R. and PRICE T.: Aspirin and reproductive outcomes. Obstet. Gynecol. Surv., 63: 49-57, 2008. 5- HUBEL C.A.: Oxidative stress in the pathogenesis of pre-eclampsia. Proc. Soc. Exp. Biol. Med., 222: 222-35, 1999. 6- SIKKEMA J.M., VAN RIJN B.B., FRANX A., BRUINSE H.W., DE ROOS R. and STROES E.S.G.: Placental peroxide is increased in pre-eclampsia. Placenta, 22: 304-8, 2001. 7- WANG Y. and WALSH S.W.: Antioxidant activities and mRNA expression of superoxide dismutase, catalase and glutathione peroxidase in normal and pre-eclamptic placentas. J. Soc. Gynecol. Invest., 3: 179-84, 1996. 8- CHAPPELL L.C., SEED P.T., KELLY F.J., BRILEY A., HUNT B.J., JONES S.C., MALLET A. and Poston L.: Vitamin C and E supplementation in women at risk of pre-eclampsia is associated with changes in indices of oxidative stress and placental function. Am. J. Obstet. Gynecol., 187: 777-84, 2002. 9- MARCOUX S., BRISSON J. and FABIA J.: Calcium intake from dairy products and supplements and the risk of pre-eclampsia and gestational hypertension. Am. J. Epidemiol., 133: 1266-72, 1991. 10- STOLL B.J.: Prematurity and intrauterine growth retardation, in Kliegman RM,Behrman RE, Jenson HB and Stanton BF (eds), Nelson textbook, 18th ed. 93, p 671, 2008. 11- ARIAS F.: Fetal growth retardation. In: Practical Guide to High-Risk Pregnancy and Delivery, 2nd ed. St. Louis; Mosby year book Inc., 301-4, 1993. 12- ASKIE L.M., DULEY L., HENDERSON-SMART D.J. and STEWART L.A.: Antiplatelet agents for prevension of pre-eclampsia: A meta-analysis of individual patient data. Lancet, 369: 1791-8, 2007.
434
13- HERMIDA R.C., AYALA D.E. and IGLESIAS M.: Administration time-dependent influence of aspirin on blood pressure in pregnant women. Hypertension, 41: 651-6, 2003. 14- CLASP: A randomized trial of low dose aspirin for the prevention and treatment of pre-eclampsia among 9364 pregnant women: Collaborative Low-Dose Aspirin Study in Pregnancy Collaborative Group. Lancet., 343: 619-29, 1994. 15- DULEY L., HENDERSON-SMART D.J., KNIGHT M. and KING J.: Antiplatelet drugs for prevention of preeclampsia and its consequences: systematic review. BMJ, 322: 329-33, 2001. 16- MAARTEN T.M.: Raijmakers, Ralf Dechend and Lucilla Poston. Oxidative stress and pre-eclampsia: Rationale for antioxidant clinical trials. Hypertension, 44: 374-80, 2004. 17- LUCY C., PAUL T., FRANK J., ANNETTE BRILEY, ANTHONY MALLET and LUCILLA POSTON: Vitamin C and E supplementation in women at risk of pre-eclampsia is associated with changes in indices of oxidative stress and placental function. Am. J. Obstet. Gynecol., 777-84, 2002.

18- BEAZLEY D., AHOKAS R., LIVINGSTON J., GRIGGS M. and BAHA M.: Vitamin C and E supplementation in women at high risk for pre-eclampsia: A double-blind, placebo-controlled trial. Am. J. Obstet. Gynecol., 192: 520-1, 2005. 19- HOFMEYR J., THERESA A. LAWRIE, ALVARO N. ATALLAH and DULEY L.: Calcium supplementation during pregnancy for preventing hypertensive disorders and related problems. Cochrane Database of Systematic Reviews, Issue 8 Art. No. CD001059, 2010. 20- LORRENE D. RITCHIE and JANET C. KING: Dietary calcium and pregnancy-induced hypertension: Is there a relation? Am. J. Clin. Nutr., 71 (suppl): 1371S-4S. 21- BELIZAN J.M., VILLAR J., GONZALEZ L., CAMPODONICO L. and BERGEL E.: Calcium supplementation to prevent hypertensive disorders of pregnancy. N. Engl. J. Med., 325: 1399-405, 1991. 22- BUCHER H.C., GUYATT G.H., COOK R.J., HATALA R., COOK D.J. and LANG J.D.: Effect of calcium supplementation and pregnancy-induced hypertension and pre-eclampsia. A meta-analysis of randomized controlled trials. JAMA, 275: 1113-9, 1996.
Impact of Successful Balloon Mitral Valvotomy on the Severity of Functional Tricuspid Regurgitation in Patients with Rheumatic Mitral Stenosis
MOHAMED A. ORABY, M.D. and REDA MAHFOUZ, M.D.
The Department of Cardiology, Faculty of Medicine, Suez Canal University
Abstract
Background: Significant rheumatic mitral stenosis is frequently associated with functional tricuspid regurgitation (TR) and when it is severe it adversely impact morbidity and mortality in patients undergoing mitral valve surgery. However, the effect of successful balloon mitral valvotomy (BMV) on significant TR is not fully established, so we sought to evaluate the effect of BMV on the severity of associated functional TR. Methods: This study included 46 consecutive patients with moderate-severe rheumatic mitral stenosis (valve area <1.5cm 2), and at least moderate pre-procedural functional TR (grade II/IV) by Doppler echocardiography, who had been underwent successful BMV by either Inoue or multitrack system at our center. Post-procedural Doppler echocardiography was performed at hospital discharge, 3 months, and 18 months later. Results: Significant regression of TR (1 grade) was reported in 24 (31%) patients at 3 months follow-up with no further significant regression at 18 months. There was a parallel reduction in pulmonary artery systolic pressure (PASP) starting immediately after the procedure and continued through 18 months (from 6422 to 2514mmHg, p<.002). Predictors of significant TR regression in this study included: Younger age, higher preprocedural PASP, absent atrial fibrillation, and larger post-procedural mitral valve area. Conclusions: In patients with moderate-severe symptomatic rheumatic mitral stenosis and significant functional TR, successful BMV could lead to significant regression of TR severity paralleled with reduction in PASP. Key Words: Balloon mitral valvotomy Tricuspid incompetence Pulmonary hypertension.
hypertension. Significant TR in this situation is occasionally associated with an adverse impact on morbidity and mortality in patients undergoing mitral valve surgery [1,2]. TR in the setting of mitral valve disease is expected to regress after replacement of the diseased mitral valve. Although earlier reports suggested that TR can resolve once the diseased mitral valve is replaced, [1,2] the results of later studies are contradictory [3,4]. Percutaneous balloon mitral valvotomy (BMV) has proved to be an effective technique for the relief of obstruction in patients with mitral stenosis. Several studies reported the immediate responses of pulmonary artery pressure to BMV [5,6]. However, the long-term effects of this technique on the associated TR have not been extensively studied [7]. Accordingly, the purpose of this study was to evaluate the effect of successful BMV on the severity of associated functional TR and PASP at 18 months follow-up. Material and Methods Data were analysed from 46 patients with symptomatic moderate-severe rheumatic mitral stenosis (mitral valve area <1.5cm 2 ) with at least grade II/IV functional TR who underwent successful BMV. Procedure: All procedures were performed by the anterograde trans-septal approach. An Inoue balloon system was used in 34 (75%) patients, and the multi-track system in 12 (25%) patients, according to the standard stepwise technique, and under echocardiographic guidance. Contraindications to BMV were: Severe calcification of both commissars, left atrial thrombus on transesophageal
Introduction SIGNIFICANT tricuspid valve regurgitation (TR) is a common finding in patients with severe mitral stenosis, and in the majority of cases, it is functional, resulting from right ventricular and tricuspid annular dilation caused by long standing pulmonary
Correspondence to: Dr. Mohamed A. Oraby, E-mail: maoraby@yahoo.com
435
436
Impact of Successful Balloon Mitral Valvotomy
echocardiography, and mitral regurgitation of grade II/IV. A successful immediate result was considered with doubling of the preprocedural mitral valve area (MVA) or absolute post-procedural MVA 1.5cm2 (whichever larger) with no regurgitation II/IV [8]. Echocardiography. Two-dimensional and color Doppler echocardiographic examination was performed before BMV, immediately after the procedure, predischarge from hospital and at 3 and 18 months follow-up, using commercially available equipment (Hewlett-Packard Unit Sonos 2500) and standard techniques. TR was assessed by Doppler color flow mapping of the regurgitant jet. Careful Doppler evaluation of the jet was performed in all obtainable views of the right ventricle and atrium, including the parasternal short-axis view at the aortic valve level, the right ventricular inflow view, the apical 4-chamber view, and subcostal views. The color flow mapping display of reversed or mosaic signals originating from the tricuspid valve and extending into the right atrium during systole identified the presence of TR. The area of disturbed flow that was traced included the aliased signals as well as the immediately contiguous non-turbulent velocities that were moving in the same direction as the jet. Right atrial area was traced from the same frame as the maximal jet area. The severity of regurgitation was graded as grade I if the regurgitant jet area occupied 20% of the right atrial area, grade II if this value was between 20% and 30%, grade III if this value was between 30%-40% and grade IV if it was >40% [9-11]. TR was defined as organic if thickening, doming, or restricted motion of the valve leaflets were present. Patients with organic TR were excluded from the study. Significant regression in TR was considered with at least one grade reduction in TR severity [12]. The right ventricular systolic pressure was estimated by continuous wave Doppler, using the modified Bernoulli equation (4 X [peak TR velocity]2), with 10mmHg added for the estimated right atrial pressure. The right ventricular systolic pressure was considered to be equal to the PASP unless there was pulmonary stenosis [9]. The right ventricular end-diastolic diameter was measured from the septum to the free right ventricular wall at the mid-cavitary level in enddiastolic frames. Clinical follow-up: Patients were assessed at the same sitting of the echocardiographic followups for heart failure symptoms (assessed by NYHA
functional class). Other clinical endpoints included death, cerebral strokes, repeat BMV; and mitral valve replacement. Statistical analyses: The statistical analyses were performed with the use of commercially available software (SAS version 8 of Statistical Analysis System, SAS Institute Inc, Corp, Cary, NC). The over-time changes from baseline to follow-up were evaluated by the paired Student t test for continuous variables. Baseline clinical, echocardiographic, and hemodynamic characteristics were examined with multivariate regression analysis to determine their predictive value for reduction in TR. All results are expressed as mean + SD. Differences were significant at a value of p<0.05. Results Baseline characteristics: The mean age of the patients was 289 years (range 22 to 41 years), 40 (86%) were females, and 33 (72%) were in AF. Most of the patients were in NYHA functional class III/IV. A previous commissurotomy had been performed in 2 (3%) patients. In all patients, 9 (19%) had grade II/IV TR, 24 (53%) had grade III/IV and 13 (28%) had grade IV/IV. The mean PASP was 6422mmHg. The baseline pulmonary artery systolic pressure measured invasively at the procedure time was found to be highly correlated to the Doppler value (r=0.67). Baseline characteristics of the study population are summarized in Table (1). Immediate changes after percutaneous BMV: BMV was considered successful in all patients; resulting in a significant increase in mitral valve area (from 0.920.31 to 1.720.46 cm2 p<.001), with a significant decrease in mean transmitral diastolic gradient (from 116 to 64 mmHg, p<.05). Immediately after the procedure, PASP decreased significantly (from 6422 to 459mmHg, p<.002), while TR severity did not change significantly. Significant mitral incompetence (grade II/IV) were induced in 5 (11%) patients. Significant ASD (>5mm) was detected in 1 (2%) patient. Long-term results: TR: At 3-month follow-up the regurgitant jet area decreased significantly from 5.53.2 to 3.22.8 cm2; p<.001. Significant regression in TR severity (1 grade) was noted in 31 (67%) with no further significant regression in TR severity by 18 months (Table 2, and Fig. 1). Using multivariate regression analysis; younger age, higher preprocedural PASP, absent atrial fibrillation, and larger post-procedural
Mohamed A. Oraby & Reda Mahfouz
437
Table (2): Clinical and echocardiographic parameters at 3 and 18 months after BM. 3 months 18 months p NYHA class: Class I Class II Mitral valve area cm2 TR: Grade I Grade II Grade III Grade IV PASP (mmHg) 35 (75%) 32 (70%) 11 (25%) 14 (30%) NS NS
mitral valve area were all significant predictors of significant regression in TR severity. PASP: At 3-month follow-up; significant drop in PASP as compared with the immediate results was reported (from 459 to 3012mmHg p< 0.001). All patients with significant drop in PASP showed significant regression of TR severity at 3-month follow-up. At 18-month follow-up; further drop of PASP was reported (from 3012 to 2514mmHg, p<.05) (Table 2). Normalization of PASP (<30 mmHg) was achieved in 14 (31%) patients. Predictors of drop in PASP were the same predictors of TR regression (younger age, higher preprocedural PASP, absent atrial fibrillation, and larger post-procedural mitral valve area). RV dimensions: Significant reduction in the RV end-diastolic dimension paralleled the drop in PASP at 18-month follow-up (from 286 to 2111 mm, p<.002). Clinical outcome: Significant improvement in the functional status (by one NYHA class) was observed in 44 (95%) patients at 3-month followup. At 18-month follow-up 32 (70%) patients were in NYHA class I and 14 (30%) patients in NYHA class II. One patient had underwent mitral valve replacement for procedure-induced severe mitral regurgitation.
Table (1): Clinical characteristics of the study population.
1.720.46 1.690.49 NS 6 (14) 24 (53%) 10 (22%) 6 (11) 3012 5 (11%) 27 (58%) 10 (22%) 4 (9% 2514 2111 NS NS NS NS 0.01 NS
RV end-diastolic diameter (mm) 249

NS: Non-significant.
Grade IV 13
Grade IV 3 6 16
N 4
III
24
III
10
II
1 4
II
27
Before BMV Age (years) Sex (Females) Atrial fibrillation Baseline NYHA class: Class II Class III Class IV Mitral valve area (cm2) Mitral valve score TR: Grade II Grade III Grade IV PASP (mmHg) RV end-diastolic diameter (mm)
NYHA : New York Heart Association; TR : Tricuspid regurgitation; PASP : Pulmonary artery systolic pressure; RV : Right ventricle.
18 months after BMV
289 years 40 (86%) 33 (72%)
Fig. (1): Regression of TR after BMV.
Discussion Although earlier reports suggested that TR can resolve once the diseased mitral valve is replaced [1,2] the results of later studies are contradictory [12,13]. This study was designed to determine the impact of successful BMV on the severity of concomitant functional TR in patients with moderatesevere rheumatic mitral stenosis and clinically indicated BMV. This study showed that successful BMV could lead to significant regression in the severity of TR that parallels the drop in the PASP. Similarly Breyer and Song showed regression of significant TR after successful BMV in relatively young patients (mean age 2510 years) with severe MS and concomitant significant pulmonary hypertension (7022mmHg) [4,14]. On the other hand, Sagie et al. reported no regression of TR in relatively older patients (mean age 5715 years) with severe MS and mild pulmonary hypertension (4615mmHg) [15].
4 (8%) 33 (72%) 9 (20%) 0.920.31 6.52.3
9 (19%) 24 (53%) 13 (28%) 6422 286
438
Impact of Successful Balloon Mitral Valvotomy
Shafie et al. [13] found that despite successful closed commissurotomy for severe mitral stenosis in 8 of their patients with initially severe TR, no improvement in TR was observed at 1-year followup. Repeat catheterization in 4 of these patients for persistent right ventricular failure demonstrated an adequate mitral valve area and good left ventricular function but persistent severe TR. One of the potential mechanisms for their observations was persistently elevated PASP, which maintains the driving pressure for TR and could also play a role in maintaining the right ventricular and tricuspid annular dilation. In the current study older patients and patients with atrial fibrillation were less likely to have significant regression of TR and PASP. The same observation was reported by Sagie et al. who studied 32 patients with different demographic characteristics from ours; being much older (age, 5715 years), and had very high prevalence of atrial fibrillation (75%) and their results showed failure of TR regression in these patients subgroup [15]. This is understandable as older age and atrial fibrillation both are associated with more advanced and possibly irreversible pathophysiological changes at the level of pulmonary vasculature leading to persistence of pulmonary hypertension and failure of TR regression [16]. Recoil of the tricuspid annulus after BMV may take sometime even after drop in PASP. This could explain the time lag between the procedure and the regression in TR severity (observed by 3 month) in the current study, while significant drop in PASP was reported immediately after the procedure [17,18]. In this study no further significant regression in TR was observed after the first 3 months despite continuing drop in PASP at 18 month. This observation was not reported in any of the available studies, and it could suggest a maximum for the recoil of the tricuspid annulus that can not be exceeded by further drop in PASP. However, because of the small sample size of this study we suggest investigating this observation on a larger population scale. Conclusion: In patients with moderate-severe symptomatic rheumatic mitral stenosis and significant functional TR, successful BMV could lead to significant regression of TR severity paralleled with reduction in PASP. Younger age, higher preprocedural PASP, absent atrial fibrillation, and larger post-procedural mitral valve area are all predictors of TR regression in these patients.
References
1- BRAUNWALD N.S., ROSS J. JR. and MORROW A.G.: Conservative management of tricuspid regurgitation in patients undergoing mitral valve replacement. Circulation, 35 (Suppl 1): 63-69, 1967. 2- PLUTH J.R. and ELLIS F.H. JR.: Tricuspid insufficiency in patients undergoing mitral valve replacement: Conservativemanagement or replacement. J. Thorac. Cardiovasc. Surg., 55: 299-308, 1968. 3- CARPENTIER A., DELOCHE A., HANANIA G., FORMAN J., SELLIER P., PIWNICA A., DUBOST C. and MCGOON D.C.: Surgical management of acquired tricuspid valve disease. J. Thorac. Cardiovasc. Surg., 67: 53-65, 1974. 4- BREYER R.H., MCCLENATHAN J.H., MICHAELIS L.L., MCINTOSH C.L. and MORROW A.G.: Tricuspid regurgitation: A comparison of non-operative management, tricuspid annuloplasty, and tricuspid valve replacement. J. Thorac. Cardiovas. Surg., 72: 867-874, 1976. 5- LEVINE M., WEINSTEIN J.S. and DIVER D.J.: Progressive improvement in pulmonary vascular resistance after percutaneous mitra] valvuloplasty. Circulation, 79: 1061-1067, 1989. 6- HANNOUSH H., FAWZY M.E., STEFADOUROS M., MOURSI M., CHAUDHARY M.A. and DUNN B.: Regression of significant tricuspid regurgitation after mitral balloon valvotomy for severe mitral stenosis. Am. Heart J., 148: 865-870, 2004. 7- SKUDICKY D., ESSOP M. and SARELI P.: Efficacy of mitral balloon valvotomy in reducing the severity of associated tricuspid regurgitation. Am. J. Cardiol., 73: 209-211, 1994. 8- FAWZY M.E., RIBEIRO P.A. and DUNN B.: Percutaneous mitral valvotomy with the Inoue balloon catheter in children and adults: Immediate results and early followup. Am. Heart J., 123: 462-465, 1992. 9ZOGHBI W.A., ENRIQUEZ-SARANO M., FOSTER E., GRAYBURN P.A., KRAFT C.D., LEVINE R.A., NIHOYANNOPOULOS P.C., OTTO M., QUINONES M.A., RAKOWSKI H., STEWART W. WAGGONER J.A. and WEISSMAN N.J.: Recommendations for evaluation of the severity of native valvular regurgitation with twodimensional and Doppler echocardiography. J. Am. Soc. Echocardiogr., 16: 777-802, 2003.
10- COOPER J.W., NANDA N.C. and PHILPOT E.: Evaluation of valvular regurgitation by color Doppler. J. Am. Soc. Echocardiogr., 22: 56-66, 1989. 11- MUGGE A., DANIEL W.G. and HERMANN G.: Quantification of tricuspid regurgitation by Doppler color flow mapping after cardiac transplantation. Am. J. Cardiol., 66: 884-887, 2002. 12- CHOPRA H.K., NANDA N.C. and FAN P.: Can twodimensional echocardiography and Doppler color flow mapping identify the need for tricuspid valve repair. J. Am. Coll. Cardiol., 14: 1266-1274, 2005. 13- SHAFIE M.Z., HAYAT N. and MAJID O.A.: Fate of tricuspid regurgitation after closed valvotomy for mitral stenosis. Chest, 88: 870-873, 1985. 14- SONG J.M., KANG D., SONG J.K., JEONG HOON KIM M.D., PARK K.M., CHOI K.J., HONG M.K.,
Mohamed A. Oraby & Reda Mahfouz

CHUNG C.H., LEE J.W., PARK S.W. and PARK S.J.: Outcome of significant functional tricuspid regurgitation after percutaneous mitral valvotomy. Am. Heart J., 145: 371-376, 2003. 15- SAGIE A., SCHWAMMENTHAL E., PALACIOS I.F., KING M.E., LEAVITT M., FREITAS N., WEYMAN A.E. and LEVINE R.A.: A significant tricuspid regurgitation does not resolve after percutaneous balloon mitral valvotomy. J. Thorac. Cardiovasc. Surg., 108: 727-735, 2003. 16- PALACIOS I.F., SANCHEZ P.L., HARRELL L.C., WEYMAN A.E. and BLOCK P.C.: Which patients enefit from
439
percutaneous mitral balloon valvuloplasty? Prevalvuloplasty and postvalvuloplasty variables that predict longterm outcome. Circulation, 105: 1465-1471, 2002. 17- RIBIERO P.A., A1 ZAIBAG M. and ADULLAH M.: Pulmonary artery pressure and pulmonary vascular resistance before and after mitral balloon valvotomy in 100 patients with severe mitral valve stenosis. Am. Heart J., 125: 1110-1114, 1993. 18- DEV V. and SHRIVASTAVA S.: Time course of changes in pulmonary vascular resistance on the mechanism of regression of pulmonary arterial hypertension after balloon mitral valvoplasty. Am. J. Cardiol., 67: 439-442, 1991.
Descriptive Study of Cases of Respiratory Distress in NICU in Ahmed Maher Teaching Hospital
MOHAMMED HESHAM ZAAZOU, M.D.; MAHMOUD M. KAMAL, M.D.; RAGHDAA M. ALI, M.D.; NAGI A. EL-HUSSIENY, M.D. and MANAL EL-SAYED, M.D.
The Department of Pediatrics, Ahmed Maher Teaching Hospital, Cairo
Abstract
Respiratory distress is one of the most common causes of admission in NICU. The aim of this work was to study the commonest causes of respiratory distress in NICU in Ahmed Maher Teaching Hospital, analysis of each cause, and to determine the strategic plan needed to improve the outcome of these cases. Patients and Methods: A retrospective study was conducted through the period from 1st Jan. to 31st Dec. 2009. Data were collected from all patients files admitted in the unit during this period. Results: Total number of admissions was 233 cases, 206 cases were due to respiratory distress representing 88.4% of cases. The commonest causes of respiratory distress in our study were transient tachypnea of newborn (TTN) 78 cases (37.9%), respiratory distress syndrome (RDS) 64 cases (31%), meconium aspiration syndrome (MAS) 21 cases (10.2%), and perinatal asphyxia 15 cases (7.3%). Discussion: Ceserean section was the most common predisposing factor associated with the development of TTN and RDS (the most 2 common causes of respiratory distress in our study). The overall mortality rate of cases of respiratory distress in our study was 18.9%. Cases with perinatal asphyxia were associated with the highest mortality rate (40%), RDS (39%), and MAS (19%). Of the 4 most common causes, (178 cases) ventilatory support was needed in 76 cases (42.7%). 41 were put on nasal CPAP with survival rate of 85.4%, while 35 cases were put on IMV with a mortality rate of 80%. Recommendations: 1-Proper antenatal care 2-Use of surfactant 3-Use of high-frequency ventilators. Key Words: Respiratory distress NICU.
The spectrum of respiratory distress in neonates includes transient tachypnea of newborn, respiratory distress syndrome, meconium aspiration syndrome, congenital pneumonia, congenital heart disease, perinatal asphyxia, and congenital anomalies as tracheo-oesophageal fistula, and congenital diaphragmatic hernia [2]. The aim of this study was to study the commonest causes of respiratory distress in NICU in Ahmed Maher Teaching Hospital, and to determine the strategic plan needed to improve the outcome of these cases. Patients and Methods A retrospective study was conducted in NICU in Ahmed Maher Teaching Hospital through the period from 1st of January 2008 till 31st of December 2009. Data were collected from all patient files admitted in the unit during this period. Inclusion criteria: All patients presented with respiratory distress on admission. Data collected included: 1- Obstetric history: Maternal diseases (diabetes mellitus-hypertension), premature rupture of membranes and mode of delivery. 2- Clinical examination data on admission included: Weight, sex, gestational age, vital signs, degree of respiratory distress, in addition to any positive clinical findings reported. 3- Radiological: Plain chest X-ray (routine). Plain X-ray abdomen, abdominal Ultra-sonography, ECHO, cranial Ultra-sonography if available.
Introduction RESPIRATORY distress is one of the commonest disorders encountered within the first 48-72 hours of life. It occurs in approximately 0.96%-6% of live births, and is responsible for about 20% of neonatal mortality [1].
Correspondence to: Dr. Mohammed Hesham Zaazou, The Department of Pediatrics, Ahmed Maher Teaching Hospital, Cairo.
441
442
Descriptive Study of Cases of Respiratory Distress in NICU
4- Laboratory evaluation; complete blood count with differential, reticulocytic count, blood culture and senstivity, electrolytes, blood sugar, blood gases. Results Total number of admissions during the studied period was 233 cases. Number of cases presented with respiratory distress was 206, representing 88.4% of all cases admitted. Number of cases with
respiratory distress who died was 39 cases representing mortality rate of 18.9% of cases with respiratory distress. 121 cases were males while 85 cases were females representing male to female ratio 1.4:1. Weight of studied groups ranged from 600gm to 4500gm with mean of 2392gm. Gestational age of all neonates included in the study range from 2542 weeks with mean of 35.1 weeks.
Table (1): Spectrum of diagnosis and mortality rate of cases with respiratory distress. Cases Diagnosis Number TTN TTN with sepsis TTN with CHD TTN with ICH RDS RDS with CHD RDS with ICH RDS with pneumothorax RDS with NEC RDS with sepsis RDS with HIE MAS MAS with HIE MAS with pneumothorax MAS with CHD MAS with CHD and HIE Perinatal Asphyxia Perinatal asphyxia with pneumonia Perinatal asphyxia with ICH Perinatal asphyxia with CHD Congenital pneumonia Congenital pneumonia with CHD Congenital pneumonia with ICH CHD Tracheo-oesphageal fistula Congenital diaphragmatic hernia IDM (hypoglycemia) Metabolic acidosis Acute hemolytic anemia Total
TTN : Transient tachypnea of newborn. RDS : Respiratory distress syndrome. CHD : Congenital heart disease. ICH : Intra-cranial hemorrhage. NEC : Necrotizing enterocolitis. MAS : Meconium aspiration syndrome. IDM : Infant of a diabetic mother.
Mortality Number 0 0 0 0 16 1 1 4 2 0 1 1 1 1 1 0 4 1 0 1 0 1 0 2 Referred Referred 0 1 0 39 0 2.55 0 100 Percent (%) 0 0 0 0 41.15 2.55 2.55 10.2 5.1 0 2.55 2.55 2.55 2.55 2.55 0 10.2 2.55 0 2.55 0 2.55 0 5.1
Percent (%) 35.9 1.0 0.5 0.5 20.9 1.9 1.9 1.9 2.4 1.5 0.5 7.8 1.0 0.5 0.5 0.5 5.8 0.5 0.5 0.5 3.8 1 0.5 3.3 1.9 1.5 0.5 0.5 0.5 100
74 2 1 1 43 4 4 4 5 3 1 16 2 1 1 1 12 1 1 1 8 2 1 7 4 3 1 1 1 206
Mohammed H. Zaazou, et al.

Table (2): Clinical criteria-history-of the commonest causes of respiratory distress. RDS* Clinical criteria GA (w) Range Mean Number of each group Percentage (%) Sex : ratio Weight (gm) Range Mean Mode of delivery Number (%) CS VD Maternal risk factors No (%) PROM Hypertension DM Stained liquor Twin 1.2: 1 1800-4200 2893 66 (84.6) 12 (15.4) 10 (12.8) 5 (6.4) 3 (3.9) 0 5 (6.4) 1.5: 1 500-1700 1256 25 (52) 23 (48) 12 (25) 6 (12.5) 3 (6.3) 0 10 (20.8) 1.7: 1 1100-2500 2037 10 (62.5) 6 (37.5) 6 (37.5) 3 (18.8) 1 (6.3) 0 2 (12.5) 1.6: 1 2500-3600 2969 3 (14.3) 18 (85.7) 0 2 (9.5) 0 21 (100) 0 2.7:1 2250-3500 2900 7 (46.6) 8 (53.4) 3 (20) 1 (6.7) 0 5 (33.3) 0 TTN Group I 34-42 37.6 78 37.9 25-<32 30 48 23.3 Group II 32-36 34.2 16 7.8 37-40 38.6 21 10.2 30-42 36.8 15 7.5 MAS Perinatal asphyxia
443
*RDS was categorized into two groups according to gestational age; group I 25-<32 weeks and group II 32-36 weeks. PROM= Premature rupture of membranes. DM= Diabetes mellitus.
Table (3): Clinical examination of the commonest causes of respiratory distress. RDS TTN Group I Grades of RD No (%) - Grade I - Grade II - Grade III - Grade IV Auscultation No (%) - Fair air entry - Mild decrease air entry - Severe decrease air entry Crepitations - Bronchial breathing 31 (39.7) 41 (52.6) 6 (7.7) 0 72 (92.3) 6 (7.7) 0 27 (35.5) 0 1 (2.1) 7 (14.6) 30 (62.5) 10 (20.8) 0 0 48 (100) 6 (12.5) 0 Group II 2 (12.5) 4 (25) 8 (50) 2 (12.5) 6 (37.5) 10 (62.5) 0 1 (6.3) 0 2 (9.5) 11 (52.5) 4 (19) 4 (19) 3 (14.3) 6 (28.6) 12 (57.1) 11 (52.4) 0 4 (26.7) 3 (20) 6 (40) 2 (13.3) 12 (80) 3 (20) 0 0 0 MAS Perinatal asphyxia
Table (4): Radiological findings no. (%) of the commonest causes of respiratory distress. RDS Radiological findings Increase bronchovascular markings Linear streaking at hilum Air bronchogram Fine reticulations Ground glass appearance Atelectasis, hyperinflation Patchy infiltrates TTN Group I 31 (39.7) 51 (65.4) 0 0 0 0 0 0 0 0 8 (16.7) 40 (83.3) 0 0 Group II 0 0 6 (37.5) 10 (62.5) 0 0 0 0 0 0 0 0 13 (61.9) 8 (38.1) 5 (33.3) 0 0 0 0 0 1 (6.7) MAS Perinatal asphyxia
444

Table (5): Echo findings of congenital heart disease cases. No. Transposition of great arteries. Total anomalies pulmonary venous return. Hypertrophic obstructive cardiomyopathy, pulmonary stenosis. Pulmonary hypertension, patent ductus arteriosus, patent foramen ovale. Ventricular septal defect, pulmonary hypertension. Ventricular septal defect, patent ductus arteriosus. Ventricular septal defect, patent ductus arteriosus, pulmonary hypertension. Persistent pulmonary hypertension of newborn. Pulmonary hypertension, patent ductus arteriosus, atrial septal defect. 1 1 1 2 2 2 2 3 3 17 (%) 5.9 5.9 5.9 11.8 11.8 11.8 11.8 17.55 17.55 100
Table (6): Ventilatory support no. (%) of commonest causes of respiratory distress. RDS TTN Group I Nasal CPAP IMV 9 (11.5) 0 15 (31.2) 25 (52) Group II 10 (62.5) 2 (12.5) 4 (19) 3 (14.3) 3 (20) 5 (33.3) MAS Perinatal asphyxia
IMV: Intermittent mechanical ventilation.
CPAP: Continuous positive airway pressure.
Table (7): Mortality of cases on ventilatory support no. (%). RDS TTN Group I Nasal CPAP IMV 0 0 2 (13.3) 19 (76) Group II 2 (20) 2 (100) 2 (50) 3 (100) 0 4 (80) MAS Perinatal asphyxia
Table (8): Outcome of commonest causes of respiratory distress no. (%). RDS TTN Group I Discharged Died 78 (100) 0 27 (56.2) 21 (43.8) Group II 12 (75) 4 (25) 17 (81) 4 (19) 9 (60) 6 (40) MAS Perinatal asphyxia
Discussion The importance of Respiratory distress in neonates can be realized from the fact that the neonates with respiratory distress are 2-4 times more likely to die than those without respiratory distress [3]. Knowledge of the causes of respiratory distress is important for plan and provision of basic facilities for sick and low birth weight newborns [4]. In our study, respiratory distress constituted 88.4% of all cases admitted in the unit during the studied period. On the contrary, many other studies reported a much lower percentage of cases with respiratory distress in the admission of their NICU. Mathur el al., 2001 [5] found in their study that 29% of all the admissions to their neonatal unit were due to respiratory distress. This discrepancy could be explained on the basis that our unit is tertiary referral unit receiving mostly complicated
cases, while in the study of Mathur in India, their neonatal unit was only a primary care unit and 51% of their neonates were delivered at home. The commonest causes of respiratory distress in our study were TTN (37.9%), RDS (31%), MAS (10.2%) and Perinatal Asphyxia (7.3%). Similar data were reported by Kumer and Bhat who found that TTN was the most common cause of RD in their unit. It constituted >40% of their cases. Also Kasp et al., [7] stated that TTN represents 33-50% of RD in neonates.
[6]
In our study, most causes of TTN were full terms with their mean GA 37.6 weeks, and their mean weight was 2893gm. There was nearly equal sex affection with to ratio of 1.2:1. Similarly; Raweling and Smith, [8] found that male sex and macrosomia have been associated with increased risk of TTN.
445
By studying the predisposing factors, we found that cesarean section was the most common factor associated with development of TTN in neonates as 84.6% of cases in our study were delivered by CS. Milner et al., [9] noted that infants born by elective CS had much higher incidence of development of TTN. They attributed this mainly to that these infants had higher volumes of interstitial and alveolar fluid compared with those born vaginally. Other factors as premature rupture of membranes, maternal hypertension, twins or maternal diabetes mellitus had a much decreasing role in precipitating the occurrence of TTN. In our study most cases of TTN (92.3%) presented with mild degree of respiratory distress. 52.6% of cases presented with RD grade II, and 39.7% presented with RD grade I, and they had fair air entry on both lungs, while the remaining 7.7% of cases had only mild decrease in air entry. Bland [10] described TTN as quiet tachypnea with variable grunting, flaring and retractions. 65.4% of cases in our study had characteristic radiological findings of linear streaking at hilum. This coincides with the findings of Jain and Eaten [11] who reported that chest radiography is the diagnostic standard of TNN with prominent perihilar streaking that correlates with engorgement of lymphatic system with retained lung fluids. 11.5% of cases in our study needed ventilator support in the form of nasal CPAP; no single case needed IMV, all cases survived with no single mortality. Similarly, Helve et al., [12] stated that TTN is self resolving disorder with excellent prognosis and that rarely develops a worsening of picture of respiratory distress that may require more aggressive support including the use of CPAP or mechanical ventilation. Respiratory distress syndrome was the second most common respiratory distress in our study. It constituted 31% of cases. 75% of cases occurred in those with gestational age ranging from 26<32weeks, their mean weight was 1256gm, while the remaining 25% of cases occurred in those with gestational age ranging from 32-36weeks, and their mean weight was 2037gm. There is preponderance of male affection in cases with respiratory distress syndrome with to ratio >1.5:1. In the United States, respiratory distress syndrome has been estimated to occur in 20000-30000 newborn infants each year.
Similar to our data, Hirtz et al., 2007 [13] reported that approximately 80% of neonates born at <32 weeks gestation develop respiratory distress syndrome, while it occurred in 50% of those born between 26-28 weeks, and in <30% of those born at 30-31 weeks gestation, and that the incidence rate of respiratory distress syndrome was 42% in infants weighing 501-1500gm. 54.7% of cases were delivered by cesarean section, premature rupture of membranes >24hrs occurred in 25% of cases, twin delivery in 18.7% of cases, maternal hypertension in 14% of cases, while only 6.3% of cases had a history of maternal diabetes mellitus. Gerten et al., [14] reported that in addition to prematurity, the other risk factors for development of respiratory distress syndrome included maternal diabetes, cesarean section delivery, second born twins and asphyxia. Respiratory distress grade III was the most common clinical presentation of respiratory distress syndrome. It occurred in 62.5% cases with gestational age <32 weeks, and in 50% of those with gestational age between 32-36 weeks. Fanaroff el al., [15] stated that neonates weighing <1000gm may have lungs so stiff, that they are unable to initiate or sustain respiration in delivery room as symptoms and signs include rapid, labored, grunting respirations appear immediately or within a few hours after delivery, with supra-sternal and sub-sternal retractions and flaring of ala nasai. As atelectasis and respiratory failure progress, symptoms worsen with cyanosis, lethargy, irregular breathing and apnea. Ground glass appearance was the most common radiological finding in cases with respiratory distress syndrome. It occurred in 62.5% of all cases of respiratory distress syndrome, and in 83.3% of cases with gestational age <32 week while a fine reticulation was the second most common radiological finding. It occurred in 28.1% of all cases, and 62.5% of cases with gestational age (32-37 weeks). In our study, ventilator support was needed in 83.2% of cases with gestational age <32weeks. 31.2% of cases needed nasal CPAP, while 52% of cases needed intermittent mandatory ventilation. Cases put on nasal CPAP had a survival rate of 86.7% while cases put on IMV had mortality rate of 76% while in those with gestational age (32-37 weeks), 75% of cases needed ventilator support.
446
62.5% of cases needed nasal CPAP with survival rate of 80% while the remaining 12.5% of cases needed to be put on IMV, with a mortality rate of 100%. The over all mortality rate of cases with respiratory distress in our study was 39.5% (43.8% in cases <32 weeks gestation, and 25% in those with 32-36 weeks gestation). This high mortality rate could be attributed to many factors; the most important of them is lack of surfactant replacement therapy in our unit. Steven el al., [16] reported that mortality rate of respiratory distress syndrome decreased by approximately 50% over the last decade with the advent of surfactant therapy. CPAP is adjuvant therapy given after surfactant if prolonged assistanted ventilation is not required. In a retrospective study, bubble nasal CPAP was successful in 76% of infants who weighed less than 1250gm and in 50% of infants who weighed less than 750 grams [17]. Kirbyetal, [18] several decades ago demonstrated that assisted ventilation further decreased respiratory distress syndrome-related morbidity and mortality. However, early ventilators were associated with complications such as air leak, bronchopulmonary dysplasia and intracranial haemorrhage. New ventilation techniques have been introduced to minimize the complications of conventional intermittent mandatory ventilation. Early use of high-frequency oscillatory ventilation was clearly superior to conventional ventilation [19]. Meconium aspiration syndrome (MAS) was the third most common cause of respiratory distress in our study (10.2%). Males were more affected with male/female ratio of 1.6:1. All cases (100%) had stained liqour at birth and 85.7% of cases were delivered vaginally. However, Mathur et al. [5], in their study of the causes of respiratory distress in neonates found that meconium aspiration syndrome represented only 4% of their cases. This could be explained that in developing countries with less availability of prenatal care, and where home births are common the incidence of meconium aspiration syndrome is thought to be higher than in industrialized world [20].
In our study, all cases were full term. Their mean GA was 38.6 weeks, and their mean weight was 2969 grams. This coincides with Singh et al., [21], who reported that meconium aspiration syndrome is exclusively a disease of newborns, especially those who are delivered at or after their due date. More than 50% of cases had RD grade II while 38% had severe RD (grade III, IV). Most cases (57%) had severe decrease in air entry and 52.4% had fine crepitations allover the chest. Dargaville and Copnell [22] stated that meconium aspiration syndrome may be presented with severe respiratory distress. Barrel chest, is associated with air trapping. Auscultatory rales and rhonchi are found in some cases. 61.9% of our cases had radiological findings in the form of areas of hyperinflation and atelectasis, while the remaining 38.1% had patchy infiltrates in both lungs. Similar findings were reported by Mellinda et al. [23]. All over mortality rate of cases of meconium aspiration in our study was 19%. Mellinda [23] stated that mortality rate for meconium aspiration syndrome resulting from severe parenchymal pulmonary disease and pulmonary hypertension is as high as 20%. 33.3% of cases needed ventilator support. 19% were put on nasal CPAP with mortality rate of 50% and 14.3% were put on IMV with mortality rate of 100%. Wiswell et al., [24] reported that mechanical ventilation is required by approximately 30% of infants with meconium aspiration syndrome. Collins et al., [25], added that although conventional ventilation commonly is initially used, high frequency oscillation and jet ventilation are alternate effective therapies. Perinatal asphyxia was the fourth most common cause of respiratory distress in our study. It constituted 7.3% of cases. Their mean gestational age was 36.8 weeks, and their mean weight was 2900 gm. Males are affected mainly with male to female ratio 2.7:1. Oswyn et al. [26] reported that the incidence of perinatal asphyxia in resource poor countries is high (5-10/1000 live births). However Jones et al., [27] stated that the true community incidence of perinatal asphyxia in resource poor countries is poorly estimated and understood.
447
Grow and Barks [28] found that perinatal asphyxia most often occur in infants who are term at birth. In our study, fetal distress presenting with stained liquor was the most common predisposing factor for the development of perinatal asphyxia. It occurred in 33.3% of cases and premature rupture of membranes >24hs occurred in 20% of cases. Ellis et al., [29] stated that numerous causes of perinatal asphyxia include placental abruption, cord compression, intrauterine pneumonia or severe meconium aspiration. In our study >50% of cases presented with severe form of respiratory distress (grade III-IV). The mortality rate of perinatal asphyxia was 50%. Lawn et al., [30] reported that mortality rate in cases of perinatal asphyxia is as high as 25-50% of cases. 53.3% of cases in our study needed ventilator support. 20% of cases were put on nasal CPAP and were discharged. However, 33.3% of cases were put on intermittent mandatory ventilation. 80% of these cases died. This study does not reflect the true incidence of perinatal asphyxia as respiratory distress is not a common presentation of perinatal asphyxia. In putting the strategy for dealing with cases of respiratory distress in our NICU, we are interested in two factors: a- Cases that represent the higher incidence of occurrence and their main predisposing factors b- Cases associated with the higher mortality rate, and their management protocols. TTN, and RDS were the most two common diagnoses (68.9%) of respiratory distress in our study, cesarean section was the most common predisposing factor associated with development of these conditions. Indication of ceserean section that represent most deliveries associated with development of TTN, and RDS should be re-evaluated. Cases with perinatal asphyxia were associated with the highest mortality rate (40%), RDS (39%), and MAS (19%). Of the four most common causes 178 cases (86.4%) of all cases of respiratory distress, ventilatory support was needed in 76 cases (42.7%). 41 cases were put on nasal CPAP with survival rate of 85.4%, while 35 cases were put on IMV with a mortality rate of 80%.
Conclusion: Respiratory distress was the major cause of admission in our NICU. The most common causes of respiratory distress were TTN, RDS, MAS, and perinatal asphyxia. Ceserean section was the most common predisposing factor associated with the development of respiratory distress in neonates. Mortality rate of cases of respiratory distress was 18.9%. Of the commonest causes of respiratory distress in our study, nasal CPAP was needed in 23% of cases with survival rate of 85.4%, while IMV was needed in 19.7% of cases with mortality rate of 80%. Recommendations: 1- Proper antenatal care to decrease the incidence of premature labour, evaluation of indication of ceserean section, and antenatal steriods for expected premature delivery. 2- Use of surfactant immedietly after delivery for all premature infants especially <32 weeks gestation. 3- Use of high-frequency oscillatory ventilators to counteract the high mortality rate associated with the use of conventional ventilators. References
1- Neonatal morbidity and mortality. Report of the national neonatal perinatal database. Indian Pediatrics, 34: 103942, 1997. 2- Am. Fam. Physician American Academy of Family Physicians, 76: 987-94, 2007. 3- MISRA P.K.: Rrspiratory distress in newborn. Indian Pediatr., 24: 77-80, 1987. 4- KHATUA S.P., GANGWAL A. and PATODHI P.K.R.: The incidence and etiology of respiratory distress in newborn. Indian Pediatr., 16: 1121-6, 1979. 5- N.B. MATHUR, K.GARG and S. KUMAR.: Respiratory distress in neonates with special reference to pneumonia. Indian Pediatrics, 39: 529-37, 2002. 6- KUMAR A. and BHAT B.V.: Epidemiology of respiratory distress of newborns. Indian J. Pediatrics, 63: 93-8, 1996. 7- KASAP B., DUMAN N., OZER E., TATLI M., KUMAR A. and OZKAN H.: Transient tachypnea of the newborn: Predictive factor for prolonged tachypnea. Pediatr. Int. Feb., 50 (1): 545-8, 2008. 8- RAWLINGS J.S. and SMITH F.R.: Transient tacypnea of the newborn. An analysis of neonatal and obstetric risk factors. Am. J. Dis. Child. Sep.,138 (9): 869-71, 1984. 9- MILNER A.D., SAUNDERS R.A. and HOPKIN I.E.: Effects of delivery by ceserean section on lung mechanics and lung volume in the human neonate. Arch. Dis. Child., 53 (7): 545-8, 1978. 10- BLAND R.D.: Lung fluid balance during development. Neo. Reviews, 6 (6): e2555-670, 2005.
448

decreasing incidence of meconium aspiration syndrome. Obstet. and Gynecol. May, 99 (5 pt 1): 731-9, 2002. 21- SINGH B.S., CLARK R.H., POWERS R.J. and SPITZER A.R.: Meconium aspiration syndrome remains a significant problem in the NICU: Outcomes and treatment patterns in term neonates admitted for intensive care during a tenyear period. J. Perinatol. Jul., 29 (7): 497-503, 2009. 22- DARGAVILLE P.A. and COPNELL B.: The epidemiology of meconium aspiration syndrome: Incidence, risk factors, therapies, and outcome. Pediatrics, May, 117 (5): 171221, 2006. 23- MELLINDA B. CLARK: Meconium Aspiration Syndrome. (print)-eMedicine Pediatrics, Mar. 30, 2010. 24- WISWELL T.E., TUGGLE J.M. and TURNER B.S.: Meconium aspiration syndrome: Have we made a difference?. Pediatrics. May, 85 (5): 715-21, 1990. 25- COLLINS M.P., LORENZ J.M., JETTON J.R. and PANRTH N.: Hypocapnia and other ventilation-related risk factors for cerebral palsy in low birth weight infants. Pediatr. Res. Dec., 50 (6): 712-9, 2001. 26- OSWYN G., VINCE J.D. and FRIESEN H.: Perinatal asphyxia at Port Moresby General Hospital: A study of incidence, risk factors and outcome. PNG Med. J., 43: 110-20, 2000. 27- JONES G., STEKETEE R.W., BLACK R.E., et al.: How many child deaths can we prevent this year? Lancet: 362: 65-71, 2003. 28- GROW J. and BARKS J.D.: Pathogenesis of hypoxicischemic cerebral injury in the term infant: Current concepts. Clin. Perinatol. Dec., 29 (4): 585-602, 2002. 29- ELLIS M., MANANDHAR N., MANANDHAR D.S., et al.: Risk factors for neonatal encephalopathy in Kathmandu, Nepal,a developing country; unmatched casecontrol study, B.M.J., 320: 1229-36, 2000. 30- LAWN J., SHIBUYA K. and STEIN C.: No cry at birth: Global estimates of intrapartum stillbirths and intrapartumrelated neonatal deaths. Bull. World Health Organ. Jun., 83 (6) 409-17, 2005.
11- JAIN L. and EATON D.C.: Physiology of fetal lung fluid clearance and the effect of labor. Semin. Perinatol. Feb., 30 (1): 34-43, 2006. 12- HELVE O., ANDRESSON S., KIRJAVAINEN T. and PITKANEN O.M.: Improvement of lung compliance during post natal adaptation correlates with airway sodium transport. American Journal of Respiratory and Critical Care Medicine, 173: 448-52, 2006. 13- HINTZ S.R., VAN MEURS K.P., PERRITT R., et al.: Neurodevelopmental Outcomes of Premature Infants with Severe Respiratory Failure Enrolied in a Randomized Controlled Trial of Inhaled Nitric Oxide. Journal of Pediatrics. July, 151e 1-3, 2007. 14- GERTEN K.A.,COONROD D.V., BAY R.C., et al.: Ceserean delivery and respiratory distress syndrome: Does labor make a difference? Am. J. Obstet. Gynecol. Sep., 193 (3pt 2): 1061-4, 2005. 15- FANAROFF A.A., STOLL B.J., WRIGHT L.L., et al.: Trends in Neonatal Morbidity and Mortality for Very Low Birth Weight Infants. American Journal of Obstetrics and Gynecology. Feb., 147: e1-e8, 2007. 16- STEVENS T.P., BLENNOW M. and SOLL R.F.: Early surfactant administration with brief ventilation Vs. selective surfactant and continued mechanical ventilation for preterm infants with or at risk. Rev., CD003063, 2004. 17- NARENDRAN V., DONOVAN E., HOATH S., et al.: Early Bubble CPAP and Outcomes in ELBW Preterm Infants. Journal of Perinatology. May, 23: 195-9, 2003. 18- KIRBY R., ROBISON E., SCHULZ J. and DELEMOS R.A.: Continuous-flow ventilation as an alternative to assisted or controlled ventilation in infants Aneth Analg. Nov.-Dec., 51 (6): 871-5, 1972. 19- COUTNEY S.E., DURAND D.J., ASSELIN J.M., et al.: High-frequency oscillatory ventilation versus conventional mechanical ventilation for very low birth weight infants. N. Engl. J. Med. Aug. 29., 347 (9): 643-52, 2002. 20- YODER B.A., KIRSCH E.A., BARTH W.H. and GORDON M.C.: Changing obstetric practices associated with
Overexpression of C-KIT in Schistosomal Urinary Bladder Carcinoma

TAHANY SHAMS, M.D.* and MOKHTAR METAWEA, M.D.**
The Departments of Pathology* and Urology**, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
Abstract
Background and Aim: In Egypt, urinary bladder squamous cell carcinoma is most prevelant. It is attributed to chronic urinary infection with schistosoma haematobium (schistosomiasis). The proto-oncogene c-KIT encodes a tyrosine kinase receptor, it has been implicated in the development of a number of human malignancies but there is no data concerning its expression in Schistosomal urinary bladder squamous cell carcinoma (SBSCC). Material and Methods: We examined the expression of c-KIT on paraffin embedded tissue blocks from 60 radical cystectomies of squamous cell carcinoma (SCC) were obtained from pathology department of Suez Canal University (Ismailia, Egypt). Each slide was evaluated for staining extent of >10% of cells was considered positive. Results: C-KIT overexpression was found in 78.3% (47/60) of the studied patients, the staining extent in the tumor cells were 11-50% and >50% in 20 (42.6%) and 27 (57.4%) respectively. The staining intensity for the positive cases were 14.9%, 63.8%, 21.3% as weak, moderate and strong intensity respectively. Patients with positive bilharzial ova had significantly higher c-KIT expression than patients without (95.2% Vs. 38.9%, p=0.000). Our results needs to be confirmed by other molecular and genetic studies, the high rate of positivity in SBSCC was one of the striking findings, however, CD117 may be a potential target for site specific immunotherapy to improve the outcome of this tumor. Key Words: C-KIT Clinicopathological Egypt Immunostaining Schistosomiasis Squamous cell carcinoma Urinary bladder.
34.26% of male malignant tumors; it is characterized by high frequency of squamous cell carcinoma due to schistosomiasis which induces squamous metaplasia of the urothelium [1]. Muscle invasion is the usual presentation of schistosomal bladder cancer; this invasive behavior is secondary to the aggressive nature of the disease [2]. The proto-oncogene c-KIT encodes a 145-160 KDA, type III transmembrane tyrosine kinase receptor known as c-KIT or CD117 [3,4], which belongs to the same family of receptors as plateletderived growth factor and colony-stimulating factor [3,5]. Expression of c-KIT is essential in the development of some cell types, including melanocytes, germ cells, mast cells, erythrocytes, and interstitial cells of Cajal [3,6,7]. In addition, expression of this receptor may be seen in other histologically normal cell types, such as breast epithelial cells, renal tubule cells, astrocytes, Purkinje cells, parotid acini, and endometrial cells [8-10]. Steel factor (SLF), also known as KIT ligand or stem-cell factor, is the cognate ligand for KIT. Binding of SLF to KIT results in receptor homodimerization, activation of KIT tyrosine kinase activity, and resultant phosphorylation of a variety of substrates. In many cases, these substrates are themselves kinases and serve as effectors of intracellular signal transduction [11-13]. Three general mechanisms of KIT activation in tumor cells have been described: (1) Autocrine and/or paracrine stimulation of the receptor by its ligand, SLF, (2) Cross-activation by other kinases and/or loss of regulatory phosphatase activity, and (3) Acquisition of activating mutations [4,14-16]. KIT expression has been detected in a variety of different tumor entities. In gastrointestinal stromal tumors (GISTs), the frequency of KIT positivity is so high (90% to 95%) that entities immunohistochemical KIT detection is considered a prerequisite for the histologic diagnosis of GISTs. A
Introduction URINARY bladder carcinoma (UBC) is the first ranking tumor in Egyptian males and it represents
Abbreviations: SCC : squamous cell carcinoma, SBSCC : Schistosomal urinary bladder squamous cell carcinoma, UBC : Urinary bladder carcinoma, IHC : Immunohistochemistry, LVSI : Lympho-vascular space invasion, TIL : tumor infiltrating lymphocyte. Correspondence to: Tahany Shams, The Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt, E-mail: tahanishams@hotmail.com
449
450
smaller fraction of KIT-positive specimens has been described in at least 46 additional tumor entities [7,8,17-23]. In terms of therapeutic strategies, it has become important to evaluate the frequency of c-KIT expression and mutation in malignancies since the emergence of the targets of the tyrosine kinase inhibitor imatinib mesylate (STI571; Gleevec); a potential therapeutic agent which inhibits the action of BCL-ABL in chronic myeloid leukemia and cKIT in gastrointestinal stromal tumor (GIST) and mast cell disease [24]. In fact, its excellent effect against these tumors has been reported in clinical trials [25-27]. Imatinib mesylate is also effective against small cell carcinoma of lung in vitro [28]. The aim of the present study was to evaluate the expression of c-KIT in urinary bladder squamous cell carcinoma in the Egyptian patients and its relation with schistosomiasis and various clinicopathologic variables, since this could provide us with useful information concerning whether imatinib mesylate might be effective against this aggressive malignancy. Material and Methods A total of 60 patients with urinary bladder squamous cell carcinoma were treated by radical cystectomy and five cystoscopic controls of normal urinary bladder mucosa were taken from patients not harboring tumors were diagnosed at Suez Canal University Hospital, Ismailia, Egypt from 2002 to 2007 were included in this study. Formalin-fixed, paraffin embedded tumor tissues of these cases were retrieved from the archives of the pathology department, demographic data on this group could be summarized from patient's files from the urology department, Faculty of Medicine, Suez Canal University. Histopathological methods: Representative H & E stained sections were examined to evaluate the histo-pathological type, grade, stage and nodal metastasis. Schistosomiasis infection was confirmed by the presence of ova on histological sections and according to this; the cases divided into 42 tumors (70.0%) were positive for bilharziasis and the rest of the tumors were negative. According to WHO grading system [29], three tumors (5.0%) were low grade (grade 1), thirty one tumors (51.7%) were intermediate grade (grade 2) and twenty six tumors (43.3%) were high grade (grade 3). Pathologic staging was performed according to the 2002 TNM (tumor, lymph nodes, and metastasis) classification system [30] into 2
(3.3%) T1, 37 (61.7%) T2, 13 (21.7%) T3 and 8 (13.3%) T4. Twenty two patients (36.7% of cases) had lymph node infiltration on pathological examination. Also the presence of hyperplasia, metaplasia, and dysplasia in adjacent mucosa were assessed. Immunohistochemical staining: Immunohistochemical staining was performed with the avidin-biotin complex method of Hsu et al. [31]. In brief, serial 5m-thick sections of formalin-fixed, paraffin-embedded tissue samples were used for the studies. Slides were deparaffinized twice in xylene for 5min and rehydrated through graded ethanol solutions to distilled water. Endogenous peroxidase activity was inactivated by incubation in 0.3% hydrogen peroxide diluted in methanol for 30min. Nonspecific binding sites were blocked using Protein Block (DAKO) for 20min. Antigen retrieval was carried out by microwave heating sections in citrate buffer (DAKO Target Retrieval Solution S1699, DAKO Corporation, Carpintera, CA, USA) for 15min. IHC was performed with anti-CD117 antibody from DAKO (A4502). In a comparison of multiple antibodies, A4502 had previously yielded the highest specificity and the least background [32]. A4502 was applied at a dilution of 1:300 at room temperature for 2.5h followed by biotinylated secondary antibody (DAKO) at room temperature for 30min and then with streptavidin-biotin peroxidase complex kit (Dako) for 30min. Sections were exposed to 3,3-Diaminobenzidine for 3-5min as the chromogen, the slides were counterstained with Mayer's hematoxylin and mounted. Sections from gastrointestinal stromal tumors were used as positive controls. A preabsorption experiment using CD117 peptide stock solution (Neomarkers PP1518; NeoMarkers, Freemont, CA) was used as a negative control. Assessment of immunostaining: All slides were evaluated under the light microscopy without knowledge of the patient's clinical status. The entire section was scanned at low magnification, and hot spots were preferentially scored. At least 1000 cells were analyzed. The cutoff value of 10% were accepted as positive staining is based on the criteria used in the literature for c-KIT staining, staining was considered true positive if the reaction product was localized to the cell membrane alone or to the cell membrane and cytoplasm simultaneously, cytoplasmic staining alone proved to be false-positive in all preabsorption control samples [32] . Staining extent was scored as (1) When 10-50% of the cells were stained, and (2) When >50% of the cells were stained. Staining intensity was evaluated only in positive cases and
Tahany Shams & Mokhtar Metawea
451
scored as (1) For weak staining (faint, light yellow), (2) For moderate staining (brown), and (3) For strong staining (dark brown) [33]. Statistical analysis: Statistical methods: Pathological stage was grouped into two subgroups: T2 and >T2. Tumor grade was categorized into two subgroups: GII and >GII. Lymph node metastases were recorded as negative or positive. Bilharzial ova were recorded as present or absent. Data analyses were conducted to assess a) the association between c-KIT expression and clinicopathological parameters including patients age, gender, tumor grade, stage, size, lymph node metastasis, Distant metastasis, LVSI, TIL and presence of bilharzial ova. Data were analyzed using SPSS 11.0 software. The 2 test or Fisher's exact test were used to compare categorical variables between two groups. Significance was defined as p<0.05 [34]. Results I- Clinicopathological features of the studied cases: Totally, 60 patients were analyzed, the age of the studied cases were ranged from 39 to 72 years with (median, 49.6 years). Thirty seven patients (61.7%) of tumors occurred 50 years, followed by 38.3% in the age of >50 years. Out of sixty patients, forty eight (80%) were males and twelve (20%) were females with male to female ratio of 4:1. Tumor size ranged from 2.0 to 7.5cm (average 4.2cm), forty patients out of sixty (66.7%) showed tumor size >4cm. All patients except two had advanced disease (T2 or above) at presentation. Pathologic stage was identified into the lamina propria T1 (2 patients, 3.3%), muscularis propria T2 (37 patients, 61.7%), perivesical fat T3 (13 patients, 21.7%), and adjacent structures T4 (8 patients, 13.3%), respectively. Among patients, 36.7% (22/60) had lymph node metastases and (LVSI) in 28 of 60 patients (46.7). Four patients had distant metastases at presentation Most tumors were moderately (51.7%) or poorly (43.3%) differentiated, whereas only 3 tumors were well differentiated (5.0%). Regarding the tumor site, it represented (40.0%) in the lateral walls of the bladder, (26.7%) in the bladder trigone, (18.3%) were multicentric and followed by the dome and bladder neck (15.0%). The mucosa surrounding the tumor showed squamous metaplasia (86.7%), squamous cell carcinoma in situ (16.7%), squamous metaplasia with dysplasia (13.3%), urothelial hyperplasia (18.3%).
II- Immunohistochemical analyses of c-KIT expression: C-KIT immunostaining in squamous cell carcinomas of the urinary bladder were shown in (Table 1) and (Figs. 1-3). Staining expression (Table 1): C-KIT expression was localized to the cell membrane alone or to the cell membrane and cytoplasm simultaneously. Its expression was not demonstrated in all specimens of normal urothelium. According to our scoring criteria, positive cKIT expression was demonstrated in 47 of 60 (78.3%) of patients with squamous cell carcinomas of the urinary bladder. 13 samples (21.7%) were negative (10 samples with no staining, three with less than 10% staining). Staining pattern (Figs. 1-3): C-KIT did not expressed in specimens of histologically normal or hyperplastic urothelium and stromal cells surrounding the tumor. Cases of squamous metaplasia showed surface epithelial staining in 26/52 (50.0%). The dysplastic urothelium showed basal and suprabasal expression of c-KIT, but the full thickness of carcinoma in situ was positive. C-KIT was expressed homogeneously and intensely in the cell membrane of most cases of SCC (Fig. 1); the heterogeneous expression pattern was seen in few cases (Fig. 2). In some cases, the staining was detected in the cell membrane and cytoplasm simultaneously (Fig. 3). Staining intensity and extent (Table 1): In the present study, staining intensity was evaluated. Weak staining intensity: (1) Was observed in 7/47 (14.9%) of cases and moderate intensity. (2) In 30/47 (63.8%), while the remaining 10 (21.3%) cases demonstrated strong (score 3) staining intensity. Regarding the extent of staining, we found that, 42.6% (n=20) cases demonstrated c-KIT staining in >10-50% of the tumor cells. While 27/47 (57.4%) cases were expressed staining in >50%. III- Comparisons with clinicopathological parameters: Relations between c-KIT expression and various clinicopathologic variables of SCC are listed in Table (2). Patients with positive bilharzial ova demonstrated significantly higher rates of c-KIT expression as compared to patients without (95.2% Vs. 38.9%, p=0.000). Most of the c-KIT positive immunostaning were belonging to tumor sized >4cm in diameter; statistically there is significant association between c-
452

Table (1): C-KIT staining in urinary bladder squamous cell carcinoma regarding expression, intensity, and extent of staining. Squamous cell carcinoma Staining expression No. (%): Positive Negative Staining intensity No. (%): 1 (weak) 2 (moderate) 3 (strong) Staining extent No. (%): 1 (>10-50%) 2 (>50) 47 (78.3) 13 (21.7) 7 (14.9) 30 (63.8) 10 (21.3) 20 (42.6) 27 (57.4)
KIT positive immunostaining and the increased size of tumor (p=0.004). Patients with distant metastasis demonstrated significantly higher rates of c-KIT expression as compared to patients without (63.8% Vs. 21.2%, p=0.0005). There was no correlation of c-KIT expression with studied clinicopathological characteristics including age (p=1.0), gender (p=0.71), tumor grade (p=0.30), pathologic T stage (p=0.35), LVSI (p=0.79), TIL (p=0.07) and lymph node metastasis (p=0.12).
Table (2): C-KIT expression according to clinicopathological characteristics of the squamous cell carcinoma patients. Characteristics All cases Age: 50 years >50 years Gender: Male Female Tumor size: 4cm >4cm Stage: T2 >T2 Bilharzial ova: Present Absent Grade: GII >GII LVSI**: Positive Negative TIL***: Present Absent Node metastasis: Positive Negative Distant metastasis: Positive Negative Patients No. (%) 60 37 (61.7) 23 (38.3) 48 (80.0) 12 (20.0) 20 (33.3) 40 (66.7) 39 (65.0) 21 (35.0) 42 (70.0) 18 (30.0) 34 (56.7) 26 (43.3) 28 (46.7) 32 (53.3) 28 (46.7) 32 (53.3) 22 (36.7) 38 (63.3) 4 (6.7) 56 (93.3) c-KIT positive No. (%) 47 (78.3) 1.0+ 29 (78.4) 18 (78.3) 0.71+ 38 (79.2) 9 (75.0) 0.004+* 10 (50.0) 37 (92.5) 0.35+ 32 (82.1) 15 (71.4) 0.000+* 40 (95.2) 7 (38.9) 0.30++ 25 (73.5) 22 (84.6) 0.79++ 22 (78.6) 25 (78.1) 0.07++ 19 (67.9) 28 (87.5) 0.12+ 20 (42.6) 27 (57.4) 0.0005+* 3 (63.8) 1 (21.2) p-value
* : p-value <0.05 is significant. ** : LVSI, lymphovascular space invasion. *** : TIL, tumor infiltrating lymphocytes. + : Fisher exact test. ++ : Chi-square test.
453
biology of SBSCC, one of these studies demonstrates mutations in exons 5-8 of the p53 gene [35]; another study demonstrates alteration of p15 and p16 genes [36]. The c-KIT gene encodes a tyrosine kinase receptor related to platelet-derived growth factor (PDGF)/colony-stimulating factor 1 (CSF-1) [5]. The c-KIT pathway has been shown to be involved in a number of physiological and pathological processes, including hematopoiesis, spermatogenesis, melanogenesis, and oncogenesis [8,37]. Two mechanisms of c-KIT activation exist: Autocrine and/or paracrine activation by its ligands and ligand-independent activation by mutation. Liganddependent activation of c-KIT depends on the engagement of its ligand (the stem cell factor); the binding of c-KIT by stem cell factor induces phosphorylation and activation of the c-KIT signal transduction pathway. This type of activation occurs during some normal physiological processes, and during oncogenesis of some cancers [15,38]. It has been shown that some tumor cells express both cKIT and stem cell factor which appears to be capable of protecting the cells against apoptosis [38]. Ligand-independent constitutive activation of cKIT involves mutations, mainly in exon 11, the region between the transmembrane and tyrosine kinase domains. C-KIT protein derived from mutation is constitutively activated without engagement by its ligand. Stable transfection of mutant c-KIT complementary DNA has been shown to induce malignant transformation, suggesting that the mutations contribute to tumor development [39]. For more validity of our results, the study of gene mutation is recommended. To the best of our knowledge, the current study is the first study that looks for CD117 expression in SBSCC, we used c-KIT antibody of A4502 because the data suggested the highest sensitivity and no background problems for this antibody. Moreover, A4502 had the advantage that a specific peptide was available for negative control experiments, which blocked specific reactions by adding a surplus of KIT antigen [32]. In previous studies, several cutoff levels of c-KIT expression were used, varying from 10 [18,33,40,41], 20% [42], 25% [43] and even to 50% [21]. We followed most authors and used a cutoff level of 10%. In our study, 47/60 (78.3%) showed c-KIT expression by IHC, patients with positive bilharzial ova were expressed c-KIT higher than negative patients (95.2% versus 38.9% respectively, p= 0.000), there are no similar data for comparison. In our study, the normal urothelium was negative
Fig. (1): Strong membraneous immunoreactivity of c-KIT in grade II squamous carcinoma of the urinary bladder (Hematoxylin & DAB, X 400).
Fig. (2): Heterogeneous c-KIT expression of grade III squamous cell carcinoma (Hematoxylin & DAB, X 200).
Fig. (3): Simultaneous membranous and cytoplasmic immunostaining of c-KIT of intermediate intensity (Hematoxylin & DAB, X 400).
Discussion Schistosomal bladder squamous cell carcinoma (SBSCC) is felt to be an aggressive carcinoma de novo; this is suggested by high frequency of mutant p53 expression (73%) and high proliferation index (87%) [2]. This has led to numerous studies in an effort to understand the cytogenetic and molecular
454

human disease. J. Allergy Clin. Immunol., 100: 435-40, 1997. 4- TIAN Q., FRIERSON J.R. H.F., KRYSTAL G.W., et al.: Activating c-KIT gene. Mutations in human germ cell tumors. Am. J. Pathol., 154: 1643-7, 1999. 5- YARDEN Y., KUANG W.J., YANG-FENG T., et al.: Human proto-oncogene c-KIT: A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J., 6: 3341-51, 1987. 6- ZSEBO K.M., WILLIAMS D.A., GEISSLER E.N., et al.: Stem cell factor is encoded at the S.l. locus of the mouse and is the ligand for the c-KIT tyrosine kinase receptor. Cell., 63: 213-24, 1990. 7- TSUURA Y., HIRAKI H., WATANABE K., et al.: Preferential localization of c-KIT product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: Immunohistochemical study on formalin-fixed, paraffinembedded tissues. Virchows. Arch., 424: 135-41, 1994. 8- NATALI P.G., NICOTRA M.R., SURES I., et al.: Expression of c-KIT receptor in normal and transformed human nonlymphoid tissues. Cancer Res., 52: 6139-43, 1992. 9- ELMORE L.W., DOMSON K., MOORE JR., et al.: Expression of c-KIT (CD117) in benign and malignant human endometrial epithelium. Arch. Pathol. Lab. Med., 125: 146-51, 2001. 10- NATALI P.G., NICOTRA M.R., SURES I., et al.: Breast cancer is associated with loss of the c-KIT oncogene. product. Int. J. Cancer., 52: 713-7, 1992. 11- HUANG E., NOCKA K., BEIER D.R., et al.: The hematopoietic growth factor K.L. is encoded by the S.l. locus and is the ligand of the c-KIT receptor, the gene product of the W. locus. Cell., 63: 225-33, 1990. 12- BLUME-JENSEN P., CLAESSON-WELSH L., SIEGBAHN A., et al.: Activation of the human c-KIT product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J., 10: 4121-8, 1991. 13- LEV S., GIVOL D. and YARDEN Y.: A specific combination of substrates is involved in signal transduction by the kit-encoded receptor. EMBO J., 10: 647-54, 1991. 14- TURNER A.M., ZSEBO K.M., MARTIN F., et al.: Nonhematopoietic tumor cell lines express stem cell factor and display c-KIT receptors. Blood., 80: 374-81, 1992. 15- DIPAOLA R.S., KUCZYNSKI W.I., ONODERA K., et al.: Evidence for a functional KIT receptor in melanoma, breast, and lung carcinoma cells. Cancer Gene., 4: 17682, 1997. 16- INGRAM D.A., YANG F.C., TRAVERS J.B., et al.: Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor. Suppressor gene modulates melanocyte and mast cell fates in vivo. J. Exp. Med., 191: 181-8, 2000. 17- ARBER D.A., TAMAYO R. and WEISS L.M.: Paraffin section detection of the c-KIT gene product (CD117) in human tissues: Value in the diagnosis of mast cell disorders. Hum. Pathol., 29: 498-504, 1998. 18- SELA G.B., KUTEN A., ELIEZER S.B., ARI E.G. and IZHAK O.B.: Expression of HER2 and c-KIT in nasopha-
for c-KIT immunostaining which is consistent with previous results [32] , suggested that c-KIT was upregulated in urinary bladder carcinoma. Regarding c-KIT expression in the urinary bladder carcinoma, There was one study on small cell carcinoma of the urinary bladder demonstrated c-KIT overexpression in 27% of studied cases [41], another study including 3000 human tumors tissues were studied for c-KIT expression, positive tumor entities included GIST (100%), seminoma (84%), malignant melanoma (36%), follicular carcinoma of the thyroid (23%), large-cell carcinoma (17%), and smallcell carcinoma of the lung (7%). Urinary bladder, transitional cell carcinoma, invasive (pT2-4), noninvasive (pTa), sarcomatoid carcinoma, squamous cell carcinoma were 4%, 15%, 12%, 16% respectively [32]. In this last study, the expression of cKIT in urinary bladder SCC was 16% which is comparatively lower but the comparison is not precise because the study included 6 cases only of SCC. Therapy with KIT tyrosine kinase inhibitors (TKIs) will be effective only against tumors for which growth is driven almost entirely by KIT activity. Therefore, in selecting cancer targets that will respond to therapy with KIT TKIs, the most attractive targets will be tumors for which there are biologic data indicating that the cells of interest are partially or completely dependent on KIT activation for proliferation and/or survival. In this regard, the greatest wealth of preclinical data exists for tumor types that have activating mutations of c-KIT [44]; so we will recommend the confirmation of our results by study of gene mutation. Conclusion: In this study, c-KIT over-expression was detected in seventy eight percent of patients with SBSCC but the possible use of targeted therapy to inhibit the kinase activity of KIT will need further studies regarding c-KIT gene mutation. In this group of patients we will recommend the study of gene mutation and then the randomized clinical trials assessing the role of TKIs to improve their outcome. References
1- MOKHTAR N., GOUDA I. and ADEL I.: Cancer Pathology Registry 2003-2004 and Time trend analysis; Ch.3. P.: 24-39, 2007. 2- BADR K.M., NOLEN J.D., DEROSE P.B. and COHEN C.: Muscle invasive schistosomal squamous cell carcinoma of the urinary bladder: Frequency and prognostic significance of p53, BCL-2, HER2/neu, and proliferation (MIB1). Hum. Pathol., 35 (2): 184-9, 2004. 3- VLIAGOFTIS H., WOROBEC A.S. and METCALFE D.D.: The protooncogene c-KIT and c-KIT ligand in

ryngeal carcinoma: Implication for a new therapeutic approach. Mod. Pathol., 16 (10): 1035-40, 2003. 19- YAMASAKI T., SHIMAZAKI H., AIDA S., et al.: Primary small cell (oat cell) carcinoma of the breast: Report of a case and review of the literature. Pathol. Int., 50: 914-8, 2000. 20- HORNICK J.L. and FLETCHER C.D.: Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am. J. Clin. Pathol., 117: 188-93, 2002. 21- HOLST V.A., MARSHALL C.E., MOSKALUK C.A., et al.: KIT protein expression and analysis of c-KIT gene mutation in adenoid cystic carcinoma. Mod. Pathol., 12: 956-60, 1999. 22- JENG Y.M., LIN C.Y. and HSU H.C.: Expression of the c-KIT protein is associated with certain subtypes of salivary gland carcinoma. Cancer Lett., 154: 107-11, 2000. 23- BOKEMEYER C., KUCZYK M.A., DUNN T., et al.: Expression of stem-cell factor and its receptor c-KIT protein in normal testicular tissue and malignant germcell tumours. J. Cancer Res. Clin. Oncol., 122: 301-6, 1996. 24- SAVAGE D.G. and ANTMAN K.H.: Imatinib mesylatea new oral targeted therapy. N. Engl. J. Med., 346: 68393, 2002. 25- DEMETRI G.D., VON MEHREN M., BLANKE C.D., VAN DEN ABBEELE A.D., EISENBERG B., ROBERTS P.J., et al.: Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N. Engl. J. Med., 347: 472-80, 2002. 26- PARDANANI A., ELLIOTT M., REEDER T., L.I. C.Y., BAXTER E.J., CROSS N.C., et al.: Imatinib for systemic mast-cell disease. Lancet, 362: 535-6, 2003. 27- DRUKER B.J., TALPAZ M., RESTA D.J., PENG B., BUCHDUNGER E., FORD J.M., et al.: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med., 344: 1031-7, 2001. 28- HEINRICH M.C., BLANKE C.D., DRUKER B.J. and CORLESS C.L.: Inhibition of KIT tyrosine kinase activity: A novel molecular approach to the treatment of KITpositive malignancies. J. Clin. Oncol., 20: 1692-703, 2002. 29- MOSTOFI F.K., SOBIN L.H. and TORLONI H.: International histological typing of urinary bladder tumors. Geneva, WHO, 1973. 30- GREENE F., PAGE D., FLEMING I., et al.: AJCC Cancer Staging Manual, 6th ed, Springer-Verlag, New York, N.Y., 2002. 31- HSU S.M., RAINE L. and FANGER H.: Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled
455
antibody (PAP) procedures. J. Histochem Cytochem, 29: 577-80, 1981. 32- WENT P.T., DIRNHOFER S., BUNDI M., et al.: Prevalence of KIT expression in human tumors. J. Clin. Oncol., 22: 4514-22, 2004. 33- AYDIN O., YILDIZ L., KEFELI M. and KANDEMIR B.: CD117 expression in normal,neoplastic, inflammatory, and reactive lesions of the thyroid. Pathology research and practice, 204: 359-65, 2008. 34- DAWSON B. and TRAPP R. EDITORS: Basic and clinical biostatistics, 3rd ed. Oxford, London and Boston Lange Medical Books, 87-93, 2000. 35- WARREN W., BIGGS P.J., EI-BAZ M., et al.: Mutations in the p53 gene in schistosomal bladder cancer: A study of 92 tumours from Egyptian patients and a comparison between mutational spectra from schistosomal and nonschistosomal urothelial tumours. Carcinogenesis, 16 (5): 1181-9, 1995. 36- SWELLAM M., ABD EL-AAL A.A. and ABU-GABEL K.H. M.: Deletions of p15 and p16 in schistosomal bladder cancer correlate with transforming growth factor- expression. Clinical biochemistry 37 December: 1098-104, 2004. 37- ULLRICH A. and SCHLESSINGER J.: Signal transduction by receptors with tyrosine kinase activity. Cell, 61: 203-12, 1990. 38- KRYSTAL G.W., HINES S.J. and ORGAN C.P.: Autocrine growth of small cell lung cancer mediated by coexpression of c-KIT and stem cell factor. Cancer Res., 56: 370-6, 1996. 39- HIROTA S., ISOZAKI K., MORIYAMA Y., et al.: Gainof-function mutations of c-KIT in human gastrointestinal stromal tumors. Science, 279: 577-80, 1998. 40- SABAH M., LEADER M. and KAY E.: The problem with KIT: Clinical implications and practical difficulties with CD117 immunostaining. Appl. Immunohistochem. Mol. Morphol., 11: 56-61, 2003. 41- PAN C.X., YANG X.J., LOPEZ-BELTRAN A., et al.: CKIT expression in small cell carcinoma of the urinary bladder: Prognostic and therapeutic implications. Mod. Pathol., 18: 320-3, 2005. 42- LONARDO F., PASS H.I. and LUCAS D.R.: Immunohistochemistry frequently detects c-KIT expression in pulmonary small cell carcinoma and may help select clinical subsets for a novel form of chemotherapy. Appl. Immunohistochem. Mol. Morphol., 11: 51-5, 2003. 43- MAKHLOUF H.R., REMOTTI H.E. and ISHAK K.G.: Expression of KIT (CD117) in angiomyolipoma. Am. J. Surg. Pathol., 26: 493-7, 2002. 44- MICHAEL C. HEINRICH, CHARLES D. BLANKE, BRIAN J. DRUKER, et al.: Inhibition of KIT Tyrosine Kinase Activity: A Novel Molecular Approach to the Treatment of KIT-Positive Malignancies: Journal of Clinical Oncology, 20: 1692-703, 2002.
The Value of Ovarian Reserve Tests in the Prediction of Poor Response t o Controlled Ovarian Hyperstimulation
IMAN A. KHALIL, M.D.
The Department of Obstetrics & Gynecology, Faculty of Medicine, Cairo University
Abstract
Background: To evaluate endocrine tests (FSH, LH, E2, Inhibin-B) and ovarian volume with respect to their ability to assess ovarian response in patients undergoing IVF/ICSI. Material and Methods: A total of 90 regularly menstruating patients undergoing their first IVF/ICSI cycle by standard long protocol, among them 60 patients were 35 years and 30 patients were <35 years. Measurement of cycle day 3 (CD3) FSH, LH, E2, Inhibin-B and ovarian volume. Controlled ovarian hyperstimulation (COH) started by 300IU HMG in young age group and 450IU in old age group and adjusted according to the ovarian response. Results: Univariate linear regression analysis showed that FSH, FSH/LH, Inhibin-B, stimulated cycle day 7 (sCD7) E2 and (sCD7) follicles >10mm were independent predictors of ovarian reserve. Multiple linear regression analysis showed that sCD7 follicles >10mm (100% accuracy in all age group), Inhibin- (96.67%-88.33% accuracy among the young and old age group) and FSH/LH ratio (86.67-71.64% accuracy in young and old age group respectively) were the best predictors. Conclusions: Among all the investigated predictors stimulated sCD7 follicles >10mm was the best predictor of ovarian reserve tests (ORT) in all patients with the highest predictive accuracy. It could be considered a simple dynamic test identifying ovarian reserve. Key Words: Ovarian reserve FSH LH Inhibin B Ovarian response.
success rate of IVF [1]. It would be plausible to be able to accurately assess oocyte quality and quantity prior to performing IVF in order to estimate the prognosis. As the ultimate assessment is achieved by direct microscopic examination of oocytes, we are left with indirect means of assessment. Tests of functional reserve of the ovaries include baseline serum concentrations of hormones such as FSH, LH, E2, Inhibin-B and anti mullerian hormone AMH [2,3]. Dynamic tests of ovarian reserve include Clomiphene citrate challenge test, Gonadotrophin agonist stimulation test and exogenous follicle stimulating hormone ovarian reserve test. Ultra-sonographic tests have also been suggested to predict ovarian response, however, despite the validity of all these testes, there still remain patients who respond poorly to stimulation despite having normal tests of OR [4,5] this supports the idea that ovarian reserve is not a simple static anatomic number of follicles but rather a dynamic process, the mechanism of which is not yet fully understood. It has been recently stressed that the ideal ovarian reserve test is the response of the ovaries to a normal or standard stimulation protocol [6]. The aim of this is to find a prospective parameter that is easily measurable, minimally invasive, inexpensive and with good predictive value for counseling in IVF. Also early identification of women with markedly reduced ovarian reserve before embarking on expensive and invasive treatment, thus reducing unnecessary procedures, cycle cancellation, wasting resources and emotional stress to the patients. Material and Methods Ninety women undergoing their first IVF/ICSI cycle between March 2007 to January 2010 at Egyptian International fertility center were included
Introduction ASSISTED reproduction is an expensive, time consuming and emotionally stressful process for the patients. Women may react differently to the dosage of gonadotrophins resulting in different ovarian responses and different IVF pregnancy outcomes. Poor ovarian response to gonadotrophin stimulation results in a small number of oocytes collected and thus a smaller number of embryos available for transfer, which therefore reduces the
Correspondence to: Dr. Iman Abd El-Mohsen Khalil, The Department of Obstetrics & Gynecology, Faculty of Medicine, Cairo University.
457
458
The Value of Ovarian Reserve Tests in the Prediction of Poor Response
in this cohort study. Institutional review board approval was attained before beginning of the study. 60 patients were 35 years and 30 patients were <35 years. All participants gave their written consent on the first visit. Inclusion criteria were as follow: Undergoing their first IVF/ICSI treatment cycle (to avoid the possible bias from previous experience), regular menstrual cycles, BMI ranging from 18-25Kg/m2, patients were excluded if they were known to have past history of partial or complete ovarian resection or laparoscopic ovarian drilling, exposure to radiation or chemotherapy, clinical signs of hyper-androgenism, thyroid disorders or hyperprolactinemia. The upper limit of FSH was <15mIU/mL and E2 <80pg/ml on (CD3) in unstimulated cycle. LH level must be always below FSH level. Causes of infertility were male factor, tubal abnormalities, unexplained or mixed factors. Study protocol: Upon the onset of menstrual bleeding in spontaneous cycle (1-3 months) preceding GnRH-a treatment, on cycle day 3, all patients underwent transvaginal ultrasound scan to rule out the presence of ovarian cysts and determine the ovarian volume. Serum blood sample was also taken to assess bFSH, b-LH, b-E2 using electro-chemiluminescence immunoassay (ECCLIAS kit) and inhibin- levels using (ELISA) kit with a range 10-531pg/ml. Ovarian volume was calculated using the simplified formula for a prelate ellipsoid V= (0.5 X D1 X D2 X D3) [7] . Total ovarian volume was measured as a summed volume of both ovaries. Protocol of treatment was mid-luteal long protocol using (Decapeptyl; 0.1mg-Ferring-Denmark) for downregulation, ovarian stimulation was initiated on CD2-3 using (Merional 75IU, IBSA-Suisse) or (Menogon 75IU Ferring-Denmark) ranging from 300-450IU according to the age and BMI. On sCD7 vaginal US was done for detection of number and size of the follicles with measurement of serum E2 concentration, the dose of HMG was adjusted accordingly. Serial ultrasounds together with E2, LH and progesterone testing were done every 23 days, when at least 3 leading follicles >17mm in diameter were reached, human chorionic gonadotrophin (HCG) 10.000I.U. (choriomon IBSASuisse) was given I.M, trans-vaginal oocytes retrieval was performed 36 hours later, embryo transfer was carried out on day 2-5 and luteal phase support by progesterone in oil (gestone, 100mg
IM-IBSA. Egypt) for 2 weeks. At present, there is no consensus on the definition of poor ovarian response. The most widely used classification is based on the number of collected oocytes. Poor response in the current study was defined as less than 3 follicles 16mm or peak E2 <500pg/ml on the day of HCG injection or cancellation of the cycle of COH at any time [8]. Fortunately no cycle was cancelled due to hyper response (OHSS). Statistical analysis: Data were statistically described in terms of mean standard deviation (SD), frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was done using Student t test for independent samples in comparing 2 groups when normally distributed and Mann Whitney U test for independent samples when not normally distributed. For comparing categorical data, Chi square (2) test was performed. Exact test was used instead when the expected frequency is less than 5. Accuracy was represented using the terms sensitivity, specificity, +ve predictive value, ve predictive value, overall accuracy, the likelihood ratio of a positive test and the likelihood ratio of a negative test. Receiver operator characteristic (ROC) analysis was used to determine the optimum cut off value for the studied diagnostic markers. p values less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2007 and SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows. Results Ninety patients were enrolled in the study, divided into two groups according to the age, 60 patients 35 years (old age group) and 30 patients <35 years (young age group). Each group is subdivided into poor and normal responders. In the old age group 17 (28.3%) patients were diagnosed as poor responders among them 6 were cancelled while in the young age group 6 (20.0%) patients were diagnosed as poor responders and 2 were cancelled. Table (1) showed baseline patient's characteristics, the treatment and outcome variables of the two age groups, CD3 FSH, LH, FSH/LH ratio were significantly higher in the old age group. CD 3 Inhibin-B and ovarian volume were significantly higher in the young age group, the only factor that
Iman A. Khalil
459
was not significant was CD3 E2. The number of days of stimulation and the amount of HMG used were significantly higher in the old age group, CD7 E2 and follicles >10mm, the total number of mature follicles yielded and serum E 2 on day of HCG were significantly higher in the young age group. p<0.001. Table (2) showed comparison of the baseline patient's characteristics, treatment and outcome variables between poor and normal responders in women 35 years. Age, CD3 FSH, FSH/LH ratio were significantly higher in the poor responders. CD3 inhibin-B was significantly higher in normal responders whereas CD3 LH, E2 and ovarian volume were similar in both groups. The sCD7 follicles >10mm and serum E2, mature follicles and E2 on day of hCG were significantly higher in normal responders p<0.001. Table (3) showed comparison in the baseline patient's characteristics, treatment and outcome variables between poor and normal responders in women <35 years. Age, duration of infertility, CD3 LH, E2 and ovarian volume were similar in both groups, CD3 FSH, FSH/LH ratio were significantly higher in poor responders, inhibin-B, sCD7 follicles
>10mm and E2, mature follicles 16mm and peak E2 at day of hCG, were significantly higher in the normal responders. Table (4) The Cut of value and accuracy of all significant variables in the two age groups. In the young age group sCD 7 Follicle Count >10mm and sCD7 serum E2 concentration had the highest predictive accuracy 100%. In the old age group sCD7 FC >10mm and sCD7 E2 still had the highest prognostic value by (ROC)c analysis. The 3 rd predictor with the best predictive accuracy was inhibin-B in each group. The COV and the predictive accuracy still higher in the younger age group (38.034pg/ml with 96.67% accuracy) versus (32.063 pg/ml with 88.33% accuracy) in old age group. The 4th predictive variable was FSH/LH ratio, but with a higher accuracy in the young age group. Tables (5,6) Show the multiple regression analysis of the all variables using number of follicles 16mm or serum E2 concentration on the day of HCG as a dependent variables. sCD7 FC >10mm was the best predictor of ovarian response followed by inhibin-B, age and FSH among the overall patients while the rest of the predictors, were deleted.
Table (1): Baseline patient's characteristics and outcome between women <35yrs and those 35yrs. Less than 35 years (n=30) Age (yrs) Duration of infertility (yrs) Type of infertility: Primary Secondary CD3 FSH (mIU/ml) CD3 LH (mIU/ml) CD3 FSH/LH CD3 E2 (pg/ml) CD3 Inhibin-B (pg/ml) CD3 Ovarian vol. CD7 E2 CD7 FC>10mm Stimulation days Ampoules used D.hCG E2(pg/ml) D.hCG FC >16mm Response: Poor responders Normal responders 283.15 4.572.21 25 (83.3%) 5 (16.7%) 7.362.07 4.81.04 1.60.36 53.518.96 54.7724.02 11.682.21 333.3157.3 9.875.1 10.670.96 49.3313.25 2729.31670.3 16.279.92 6 (20.0%) 24 (80.0%) 35 years or more (n=60) 38.22.69 7.052.84 38 (63.3%) 22 (36.7%) 10.162.47 5.571.04 1.840.35 54.9419.91 41.3718.37 9.781.84 214.89109 5.923.34 11.350.99 75.910.76 1256.5832.1 8.034.66 17 (28.3%) 43 (71.7%) p value <0.001 <0.001 0.041 <0.001 0.001 <0.001 0.744 0.004 <0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 0.278
Results are represented by mean (SD) or number of cases (%) when appropriate; CD : Cycle day. D.hCG : Day of hCG administration. FC : Follicular count.
460

Table (2): Baseline patient's characteristics, treatment and outcome variables of the normal and poor responders in women 35yrs. Poor responders (n=17) Age (yrs) Duration of infertility (yrs) CD3 FSH (mIU/ml) CD3 LH (mIU/ml) CD3 FSH/LH CD3 E2 (pg/ml) CD3 Inhibin-B (pg/ml) CD3 ovarian vol. sCD7 E2 (pg/ml) sCD7 FC >10mm Stimulation days hMG ampoules used D.hCG E2 (pg/ml) D.hCG FC >16mm 39.942.63 8.183.36 12.121.86 5.731.06 2.140.27 62.1223.42 20.225.65 9.291.33 106.0728.98 2.530.72 11.121.69 78.9413.53 386.65195 3.060.97 Normal responders (n=43) 37.512.4 6.62.52 9.382.25 5.51.03 1.720.3 52.117.86 4222.11 9.971.99 257.9198.4 7.263.0 11.44(0.5 74.79.37 1600.43(728.6 104.02 p value 0.001 0.053 <0.001 0.445 <0.001 0.079 <0.001 0.198 <0.001 <0.001 0.256 0.171 <0.001 <0.001
Results are represented by mean (SD) or number of cases (%) when appropriate; CD : Cycle day. sCD : Stimulation cycleday. D.hCG : Day of hCG administration. FC : Follicular count.
Table (3): Baseline patient's characteristics and outcome of the normal and poor responders in women <35yrs of age. Poor responders (n=6) Age (yrs) Duration of infertility (yrs) CD3 FSH (mIU/ml) CD3 LH (mIU/ml) CD3 FSH/LH CD3 E2 (pg/ml) CD3 Inhibin-B (pg/ml) CD3 ovarian vol. sCD7 E2 (pg/ml) sCD7 FC >10mm Stimulation days hMG ampoules used D.hCG E2 (pg/ml) D.hCG FC >16mm 29.832.64 62.28 8.882.86 4.71.37 1.920.37 62.6819.36 25.633.94 10.72.13 113.7232.56 2.830.75 11.171.72 69.3313.78 439.83216.7 2.51.97 Normal responders (n=24) 27.543.15 4.212.08 6.981.58 4.830.98 1.450.22 51.2118.55 59.4824.5 11.922.2 388.24123.5 11.637.04 10.540.66 44.337.04 3301.721341 19.717.84 p value 0.112 0.075 0.034 0.798 <0.001 0.190 0.002 0.232 <0.001 <0.001 0.157 <0.001 <0.001 <0.001
Results are represented by mean (SD) or number of cases (%) when appropriate; CD : Cycle day. sCD : Stimulation cycle day. D.hCG : Day of hCG administration. FC : Follicular count.
Table (4): Cut of value (COV) and accuracy of all significant variables in the two age group. Group Cases <35 years Item CD3 FSH FSH/LH ratio CD3 Inhibin-B sCD7 E2 sCD7 FC >10mm CD3 FSH FSH/LH ratio CD3 Inhibin-B sCD7 E2 sCD7 FC >10mm COV 8.3 1.5 38 162 4 9.5 1.66 32 152 3 Sensitivity 83.33 100.00 100.00 100.00 100.00 88.24 66.67 94.12 94.12 94.12 Specificity 66.67 83.33 95.83 100.00 100.00 74.42 74.42 86.05 95.35 93.02 (+)ve PV 38.46 60.00 85.71 100.00 100.00 57.69 59.26 72.73 88.89 84.21 ()ve PV 94.12 100.00 100.00 100.00 100.00 94.12 80.00 97.37 97.62 97.56 Accuracy 70.00 86.67 96.67 100.00 100.00 78.33 71.64 88.33 95.00 93.33
Cases 35 years
Results are represented as percentage.
CD: Cycle day.
sCD: Stimulation cycle day.
Iman A. Khalil
Table (5): Regression summary for dependent variable: D-HCG-FC 16mm. Start. multiple regress No=90 Intercept sCD7 FC >10mm CD3 INH B AGE CD3 FSH Regression summary for dependent variable: D-HCG-FC 16mm Beta 0.786323 0.159170 0.136519 0.089873 St. Err. Of Beta 0.044277 0.35941 0.041697 0.032766 B 1.176167 1.402285 0.058792 0.191346 0.644512 St. Err. of B 2.392479 0.078961 0.013275 0.058442 0.230672 t (85) 0.49161 17.75927 4.42863 3.27412 2.79407
461
p-level 0.624262 0.000000 0.000028 0.001534 0.006431
Table (6): Regression summary for dependent variable: D-HCG_E2. Start. multiple regress No=90 Intercept sCD7 FC >10mm CD3 INH B AGE CD3 FSH Regression summary for dependent variable: D-HCG_E2 Beta 0.794253 0.145397 0.128552 0.073207 St. Err. Of Beta 0.046571 0.037803 0.43857 0.33832 B 147.4738 245.6754 9.3149 31.2515 91.0585 St. Err. of B t (85) 0.33788 17.05469 3.84613 2.93117 2.16381 p-level 0.736288 0.000000 0.000231 0.004337 0.033285
436.4702 14.4052 2.4219 10.6618 42.0824
Discussion Decreased ovarian reserve is defined as poor ovarian follicular response to gonadotropin stimulation during ART, probably secondary to a reduced number and quality of ovarian follicles available for stimulation. This results in much higher rate of cycle cancellation, fewer eggs retrieved, fewer embryos and a lower pregnancy rate [9]. In this study age, b-FSH, LH, FSH/LH ratio, E2, inhibinB and ovarian volume were investigated prior to stimulation with gonadatropins, and sCD7 follicle count >10mm and sCD7 E2 concentration after the start of COH. The study revealed that poor ovarian response was heterogeneous and highly variable in its onset, no age was immune against poor ovarian response. Although the mature follicles and E 2 concentration were higher in the young age group, still we have patients showed poor response and cancellation occurred indicating that chronological age does not always equal biological age of the ovary [1,10-14]. In the current study the age 38 years old was a cut of point in the old age group whereas age was not significant in young age group and this in agreement with other studies [15,16]. b-FSH is the most commonly used and the most widely investigated test for predicting success of IVF treatment, it is a measure of the of inhibin-B and estradiol that a cohort of follicles is producing and the feedback effect at the pituitary [17]. A wide range values have been used for abnormal level of
b-FSH. In this study a cut of value of 8.3mIU/ml with accuracy 70% in the young age group and COV 9.5 mIU/ml and accuracy 78.33% for old age group in agreement of others [17,18]. FSH should be used for screening patients with decreased ovarian reserve and so one must accept a low COV to have high sensitivity but at the expense of specificity. b-LH measurement may also have predictive value for ovarian reserve. As menopause approaches, FSH and LH levels increase, there is a monotropic rise in FSH sooner than LH and this attributable to diminished production of inhibin by the ovaries [19]. As a result the first intimation of a diminished ovarian reserve may be an elevated FSH/LH ratio. In this study the mean value of FSH/LH ratio using was 1.50, 1.66 in young and old age groups respectively, the mean value of LH was 4.80 and 5.57 in the young and old age groups respectively and the high FSH/LH ratio was attributed more to higher FSH in contrast to others which showed increase FSH/LH ratio with normal FSH and low LH [20]. b-E2 level was used as a marker of ORT. The finding varied widely and no clear COV. Elevation of b-E2 may suppress otherwise elevated FSH into the normal range thus masking what might have an abnormal test result, patients with elevated E2 levels 80pg/ml had increased risk of cycle cancellation and distinctly diminished chance of pregnancy even if they had normal FSH levels [20-23]. The upper limit of the b-E2 in this study was 80 pg/ml.
462
Inhibin-B was suggested as an early sensitive, direct indicator of ovarian reserve as it produced by the granulosa cells of developing follicles [24-28] . In the current study the mean value of inhibinB was 55.77pg/ml and 41.37pg/ml in young and old age group respectively indicating decrease serum Inhibin-B concentration with age and this is in agreement of other studies [12,29,30]. The COV and the predictive accuracy still higher in the younger age group. (96.67% versus 88.33). Ovarian volume measurement was another marker used as a predictor of OR. In the current study the mean value of the total ovarian volume was significantly higher in the young age group than the old age group and was accompanied by higher estradiol concentration and number of mature follicles 16mm on day of HCG with less number of HMG ampoules used and less days of stimulation, this in agreement with other studies [31,32]. Sanja Kupesic and Kurjak [12] used 3D ultrasonography for measurement of (AFC, ovarian volume and mean ovarian stromal flow index) he founded that decreased total ovarian volume on 3D ultrasonography was associated with fewer oocytes retrieved lower fertilization and pregnancy rates, however no predictive value should be attributed to the measurement of ovarian volume, although we believe that, because of its easy execution, it could be included in the preparatory protocols and providing data for the continuity of research. In the current study the patients were also evaluated by measuring sCD7 E2 level and follicles >10mm as a predictors of ovarian response. E 2 response to stimulation reflects the function integrity of ovarian follicles and a poor E2 response can be regarded as a consequence of dwindling cohorts of secretory follicles [33]. sCD7 E2 level and follicles >10mm in diameter were highly predictive of poor ovarian response. As a conclusion sCD7 FC is the best predictor of ORT, by far exceeded other hormonal static or dynamic markers with the highest predictive accuracy (100% in all age groups) in agreement with others [30,34,35]. This may help clinicians and women to cancel cycles earlier and decrease the psychological, financial, and medical burden of a later cancellation. Two points in this study still a current of debate and need to be clarified and furtherly investigated. First although sCD 7 E 2 in the study offered an easily dynamic test of OR, it directly reflected
follicular activity to the ongoing regimen of gonadotropin stimulation. As a single predictor it had one of the highest predictive accuracy but surprisingly in multivariate analysis it was deleted and became insignificant. Second antral follicle count was not considered in the current study although data in the literature indicate that b-AFC is the best predictor of ovarian reserve [36]. But the way AFC is performed differs between centers, and depends on the clinicians experience and technical properties of the ultrasound used. There is high variability of counting follicles, 2-5mm or 2-10mm, considering that the endocrine function of a follicle is related to its size [37]. sCD7 follicle >10mm was chosen in this study instead of AFC and it was the best predictor of ORT. AMH was not included in the current study as the hormonal assay of the test was pricey and technically difficult at the time of the set up of the study. It should be remembered that these tests predict the gonadal response and do not predict pregnancy since the last one depends on variables such as characteristics of the gametes, the fertilization technique and the endometrium. The question still arises; which group to be investigated? All population, sub fertile group, age >35 years, those who postpone pregnancy, smokers; family history of premature ovarian failure and premature menopause, before ART or previous poor response to ART. References
1- BANCSI L.F., BROEKMANS F.J., EIJKEMANS M.J., DE JONG F.H., HABBEMA J.D. and TE VELDE E.R.: Predictors of poor ovarian response in in vitro fertilization: A prospective study comparing basal markers of ovarian reserve. Fertil Steril, 77 (2): 328-36, 2002. 2- LA MARCA A., SIGHINOLFI G., RADI D., ARGENTO C., BARALDI E., ARTENISIO A.C., STABILE G. and VOLPE A.: Anti-Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum. Reprod Update, 16 (2): 113-30, 2010. 3- LEE J.R., KIM S.H., KIM S.M., JEE B.C., KU S.Y., SUH C.S., CHOI Y.M., KIM J.G. and MOON S.Y.: AntiMullerian hormone dynamics during controlled ovarian hyperstimulation and optimal timing of measurement for outcome prediction. Hum. Reprod, 25 (10): 2597-604, 2010. 4- FARHI Y., HOMBURG R., FERBER A., ORVIETO R. and BENRAFAEL Z.: Non response to ovarian stimulation in normogonado-trohic, normogonadal women: A clinical sign of impending onset of ovarian foli failure preemptying the rise in basal follicle stimulating hormone levels. Hum. Reprod, 12, No. 2, PP: 241-3, 1997. 5- RANIERI D.M., PHOPHONG P., KHADUM I., MEO F., DAVIS C. and SERHAL P.: Simultaneous evaluation
Iman A. Khalil
of basal FSH and oestradiol response to GnRH analogue (F-G-test) allows effective drug regimen selection for IVF. Hum. Reprod, 16 (4): 673-5, 2001. 6- AL-AZEMI M., KILLICK S.R., DUFFY S., PYE C., REFAAT B., HILL N. and LEDGER W.: Multi-marker assessment of ovarian reserve predicts oocyte yield after ovulation induction. Hum. Reprod, 26 (2): 414-22, 2011. 7- ADAM H.B., JOOP S.E.L., SEANG-LIN T. and DIDIER D.: Ultrasound assessment of the polycystic ovary: International consensus definitions. Hum. Reprod Update, 9, No. 6, PP: 505-14, 2003. 8- RAGA F., BONILLA-MUSOLES F., CASAN E.M. and BONILLA F.: Recombinant follicle stimulating hormone stimulation in poor responders with normal basal concentrations of follicle stimulating hormone and oestradiol: Improved reproductive outcome. Hum. Reprod., 14, 14311434, 1999. 9- BRADLEY Y. VAN VOORHIS: Outcomes from assisted Reproductive technology, obstet. Gynecol., 107: 183-200, 2006. 10- RICHARD T. SCOTT. and GLEN E. HOFMAN: Prognostic assessment of ovarian Reserve. Fertil Steril, 63, No. 3, PP: 1-11, 1995. 11- AMIR LASS, CAROLYN CROUCHER, SUZANNE DUFFY, KARIN DAWSON RAUL MARGARA and ROBERT M.L.W.: On thousand initiated cycles of in vitro fertilization in women 40 years age. Fertil Steril, 70, No. 6, PP: 1030-4, 1998. 12- SANJA KUPESIC and KURYAK: Predictors of IVF outcome by three-dimensional ultrasound. Hum. Reprod., 17, No. 3, PP: 950-5, 2002. 13- EL-TOUKHY T., KHALAF Y., HART R., TAYLOR A. and BRAUDE P.: Young age does not protect against the adverse effects of reduced ovarian reserve-an eight year study. Hum. Reprod, 17 No. 6, PP: 1519-24, 2002. 14- AKANDE A., FLEMING C.F., HUNT L.P., DEAY S.D. and JENKINS J.M.: Biological versus chronological ageing of oocytes, distinguishable by raised FSH level in relation to the success of IVF treatment. Hum. Reprod., 17. No. 8. PP: 2003-8, 2002. 15- NIKOLAOU D. and TEMPLETON A.: Early ovarian ageing: A hypothesis detection and clinical relevance, Hum Reprod., 18, No. 6 PP: 1137-9, 2003. 16- GOUGEON A., EOCOHARD R. and THALABARD: Age-related changes of the population of human ovarian follicles: Increase in the disaPP: Earance rate of nongrowing and growing follicles in aging. Biol. Reprod, 50, No. 4, PP: 653-63, 1994. 17- MONTSERRAT CREUS, JOANA PENARRUBIA F.F., ESTER V., FRANCISCO C., ROSER C.J., JUAN A. VANRELL and JUAN B.: Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum. Reprod. 15, No. 11, PP: 2341-6, 2000. 18- MAMAN E., BAUM M., MACHTINGER R., SEIDMAN D.S., DOR J. and HOURVITZ A.: IVF treatment should not be postponed for patients with high basal FSH concentrations. Reprod Biomed. Online. Nov.; 21 (5): 6315, 2010.
463
19- Practice committee of the American Society for Reproductive Medicine. Aging and infertility in women, Fertil Steril, 86, (S) 4, PP: S248-S51, 2006. 20- MUKHERJEE T., BENJAMIN S., ALAN B.C., MARIA B., ROBERT L. and LARRY GRUNFELD: An elevated day three follicle-stimulating hormone: Luteinizing hormone ratio (FSH: LH) in the presence of a normal day 3 FSH predicts a poor response to controlled ovarian Hyperstimulation. Fertil Steril, 65, No. 3, PP: 588-93, 1996. 21- SMOTRICH D.B., MICHAEL J., LEVY E.A.W., JERRY L.H., RAUL R.G. and ROBERT J.S.: Prognostic value of day 3 estradiol on in vitro fertilization outcome. Fertil Steril, 64, No. 6, PP: 1136-40, 1995. 22- EVERS Y.L., SLAATS P., LAND Y.A., DUMOULIN Y.C. and DUNSELMAN G.A.: Elevated level of basal estradial 17-B predict poor response in patients with normal basal levels of follicles stimulating hormone undergoing in vitro fertilization. Fertil Steril; 69, No. 6, PP: 1010-14, 1998. 23- FRATTARELLI J., PAUL A.B., MICHAEL R.D., FADY I.S. and RICHARD T.S. JR.: Evaluation of basal estradiol levels in assisted Reproductive technology cycles. Fertil Steril; No. 3, PP: 518-24, 2000. 24- SEIFER D.B., LAMBERT-MESSELIAN G., HOGAN Y.W., GARDINER A.C., BLAZAR A.S. and BERK C.A.: Day 3 serum Inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril. 67, No. 2, PP: 110-4, 1997. 25- SEIFER D.B., SCOTT R.T. and BERGH P.A.: Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin-B before a rise in day 3 follicle stimulating hormone. Fertil sterilit. 72, No. 5, PP: 63-5, 1999. 26- DANFORTH D.R., ARBOGAST L.K., MROUEH J., KIM M.H., KENNARD E.A., SEIFER D.B. and FRIEDMAN C.I.: Dimeric inhibin: A direct marker of ovarian aging. Fertil Steril.70 (1): 119-23, 1998. 27- HALL E., CORRINE K. and DANIEL W.C.: Inhibin A and inhibin B reflect ovarian function in assisted reproduction but are less useful at predicting outcome. Hum. Reprod, Vol. 10, No. 3, PP: 409-15, 1999. 28- SHANTHI M.K., HARRIS S., HUGH M.G. and MUTTUKRISHNA S.: Inhibin B and anti-Mullerian hormone: Markers of ovarian response in IVF/ICSI patients. Int. J. Obstet. Gynaecol., 111 (Suppl.): 1248-53, 2004. 29- RIGGS R.M., DURAN E.H., BAKER M.W., KIMBLE T.D., HOBEIKA E., YIN L., MATOS-BODDEN L., LEADER B. and STADTMAUER L.: Assessment of ovarian reserve with anti-Mllerian hormone: A comparison of the predictive value of anti-Mllerian hormone, follicle-stimulating hormone, inhibin B, and age. Am. J. Obstet. Gynecol. Aug., 199 (2): 202, 2008. 30- BROEKMANS F., KWEE J., HENDRIKS D. MOL B.W. and FAMBOLK C.B.: A systemic review of tests predicting ovarian reserve and IVF outcome. Hum. Reprod Update, 12, No. 6, PP: 685-718, 2006. 31- PAVLIK E., DEPRIEST P.D., GALLION H.H., UELAND F.R., REEDY M.B., KRYSCIO R.Y. and VAN NAGELL J.R.: Ovarian Volume related to age Gynecol. Oncol., 77, 410-2, 2002.
464

35- DURMUSOGLU F., ELTER K., YORUK P. and ERENUS M.: Combining cycle day 7 follicle count with the basal antral follicle count improves the prediction of ovarian response. Fertil Steril. 81 (4): 1073-8, 2004. 36- VERHAGEN T.E., HENDRIKS D.J., BANCSI L.F., MOL B.W. and BROEKMANS F.J.: The accuracy of multivariate models predicting ovarian reserve and pregnancy after in vitro fertilization: A meta-analysis. Hum. Reprod Update, 14: 95-100, 2008. 37- JAYAPRAKASAN K., DEB S., BATCHA M., HOPKISSON J., JOHNSON I., CAMPBELL B. and RAINEFENNING N.: The cohort of antral follicles measuring 2-6mm reflects the quantitative status of ovarian reserve as assessed by serum levels of anti-Mllerian hormone and response to controlled ovarian stimulation. Fertil Steril, 94 (5): 1775-81, 2010.
32- HAMISH W. WALLAE. and THOMAS W.K.: Ovarian Reserve and Reproductive age may be determined from measurement of ovarian Volume by transvaginal sonography. Hum. Reprod, 19, No. 7, PP: 1616-7, 2004. 33- FATIH DURMUSOGL, KORAY E., PINAR Y. and MITHAT E.: Combining cycle day 7 follicle count with the basal antral follicle count improves the prediction of ovarian response Fertil Steril. 8, No. 4, PP: 1073-8, 2004. 34- PAPOVIC-TODOROVIC B., LOFT A., LINDHARD A., BANGSBOLL S., ANDERSON A.M. and NYBOE A.: Andersen. A prospective study of predictive factors of ovarian response in standard IVF/ICSI patient treated with recombinant FSH. A suggestion for recombinant FSH dosage normogram. Hum. Reprod. Vol. 18, No. 4, PP: 781-7, 2003.
Expression of Apoptosis Related Proteins, P-Cadherin and Claudin-3 in Different Endometrial Lesions
AHLAM A. ABD EL-MAKSOUD, M.D.* and AHMED M. FAHMY, M.D.**
The Department of Pathology, Faculty of Medicine, Benha University* and National Cancer Institute (NCI), Cairo University**
Abstract
Objective: Endometrial adenocarcinomas are the eighth leading cause of cancer deaths in women in developed countries, with several well-established prognostic factors. This study aimed to evaluate the expression of apoptosis related proteins (survivin, PTEN and Fas) as well as P-cadherin and claudin-3 in normal, hyperplastic and endometrial carcinoma to investigate their role in endometrial carcinogenesis and to explore their correlations with clinico-pathological parameters of endometrial cancer. Material and Methods: Forty paraffin embedded tissues of endometrial carcinoma and 15 of endometrial hyperplasia (4 simple, 3 complex and 8 atypical) were studied using immunohistochemistry with antibodies against survivin, PTEN, Fas, P-cadherin and claudin-3. Results: The expression of apoptosis related proteins (survivin and Fas) as well as P-cadherin and claudin-3 increased progressively from normal proliferative endometrium to atypical hyperplasia to endometrioid endometrial carcinoma (EECA) (p<0.05). PTEN expression in cyclical endometrium and non atypical hyperplasia was higher than in atypical endometrial hyperplasia (ACH) and EECA (p<0.05). Survivin, Fas, claudin-3 and p-cadherin expression was significantly correlated with myometrial invasion (p<0.05). Fas and Pcadherin expression was significantly correlated with FIGO grade (p<0.01). Survivin, Fas and P-cadherin expression was significantly correlated with FIGO stage (p<0.05). Conclusions: Lack of PTEN expression may be the earliest event in endometrial carcinogenesis. Survivin may reflect an important mechanism in tumor progression of the endometrial mucosa. Fas-related apoptotic pathway is also involved in the regulation of apoptosis in the endometrial tissue and promotes the development and progression of endometrial neoplasia. Overexpression of P-cadherin is involved in endometrial carcinogenesis and is associated with aggressive subgroups. The expression data of claudin-3 in different premalignant and malignant endometrial alterations suggest that these proteins might serve as diagnostic and prognostic marker. Key Words: Endometrial lesions Survivin PTEN Fas P-cadherin Claudin-3. Correspondence to: Dr. Ahlam Abd El-Maksoud, Department of Pathology, Faculty of Medicine, Benha University, Email: dr_ahlampath@yahoo.com
Introduction ENDOMETRIAL hyperplasia is considered a precursor of endometrial carcinoma. Concurrent endometrial carcinoma in patients with endometrial hyperplasia is seen frequently. In developed countries, endometrial carcinoma is the fifth most common cancer of women; however in developing countries it is much less common than carcinoma of the cervix [1,2]. In Egypt, malignant tumors of the female genital tract represented 4.70% of total malignancies in females with endometrial carcinomas representing 14.27% [3]. The molecular mechanisms involved in the progression of endometrial cancer are unclear, which have hampered the development of an effective treatment. Abnormalities in the control of cell proliferation and apoptosis have been suggested to contribute to the development and progression of neoplasia. There are at least two pathways that activate apoptosis. The first is a mitochondriadependent route governed by bcl-2 family proteins. The second is a parallel mechanism which involves the activation of a group of tumor necrosis factor (TNF) receptors on the surface of cells, such as Fas (CD95, Apo-1) [4]. Fas is expressed at markedly elevated levels in subsets of human breast, ovarian, and prostate carcinomas that are associated with poor prognoses [5,6]. Survivin; a newly identified member of the inhibitor of apoptosis (IAP) family [7]; has been reported to be expressed in fetal and embryonic tissue and is transcriptionally silent in most normal differentiated adult tissues, but is highly overexpressed in virtually every human cancer and is associated with chemotherapy resistance, increased tumor recurrence, and shorter patient survival [8,9]. It is a key regulator in the antiapoptotic network, and its overexpression has been reported in endometrial hyperplasia and cancer [10].
465
466
Expression of Apoptosis Related Proteins, P-Cadherin & Claudin-3
Different molecular alterations have been described in endometrioid endometrial carcinoma (EECA) of which is loss of the PTEN protein [11,12] . PTEN is a tumor suppressor gene that is involved in the control of cell proliferation, differentiation, and apoptosis [13]. Mutation of PTEN is present in 83% of endometrial adenocarcinoma cases, making it the most frequent early molecular genetic alteration in well differentiated endometrial adenocarcinoma, which are generally associated with hyperplasia [6]. Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumors. Cadherins are multifunctional transmembrane proteins that sustain cell-tocell contacts, form connections with the actin cytoskeleton, and influence intracellular signaling. The expression of P-cadherin is only restricted to the basal or lower layers of stratified epithelia, including prostate and skin, and also to the breast myoepithelial cells. This unique distribution of Pcadherin suggests that in addition to maintaining cellular adhesion this molecule may also have other unknown functions which can be important in cell differentiation and proliferation [14]. Claudins are the main protein components of tight junctions which function as selective barriers by controlling paracellular diffusion, maintain cellular polarity and play a role in signal transduction. The expression pattern of the 24 known members of the claudin family proved to be organ and tissue specific. Claudins 3 and 4 were elevated in endometrial cancer. The expression data on claudins in different premalignant and malignant alterations suggest that these proteins might serve as diagnostic and prognostic markers and might be targets for future therapy [15,16]. The aim of this study was to examine the distribution and interrelation between the expression patterns of apoptosis-related proteins namely: Fas, survivin and PTEN in endometrial hyperplasia and adenocarcinoma as analyzed by immunohistochemistry. Also, to clarify the roles of claudins-3 and P-cadherin in endometrial tumorigenesis and to explore their correlations with clinicopathological parameters of endometrial cancer. Material and Methods Formalin fixed paraffin-embedded tissues of patients with endometrial lesions were retrieved from the archives of the Departments of Pathology, National Cancer Institute (NCI), Cairo University and Benha Faculty of Medicine, Benha University; received from 2005 to 2008. A total of 61 samples
were included and classified as: Proliferative endometrium (6 cases), simple hyperplasia (4 cases), complex hyperplasia (3 cases), hyperplasia with atypia (8 cases) and endometrial adenocarcinoma (40 cases). The age ranged from 34-65 years with a median of 49 year. H & E stained sections were examined and were graded according to the FIGO grading system [17]. Twelve cases were grade I, 14 cases were grade II, 10 cases were grade III and 4 cases were undifferentiated tumors. Staging of carcinoma followed FIGO staging system of endometrial cancer [18]. Nineteen cases were stage I, 8 were stage II, 8 were stage III and 6 cases were stage IV. All cancer patients were subjected to hysterectomy. Table (1) shows the clinicopathologic criteria of the cancerous patients. A significant correlation was found between different grades of endometrial adenocarcinoma and depth of invasion (p=.000), the presence of vascular invasion (p=.021) and their stages (p=.000). Immunostaining: From each paraffin block 5m thick sections were taken for immunohistochemical staining. The paraffin sections were processed after a standard procedure including blocking endogenous peroxidase activity in 1.0% hydrogen peroxide in PBS for 15min. and antigen retrieval performed by microwave heating in citrate buffer, pH 6. The monoclonal antibodies used, dilution and incubation times are listed in Table (2). Streptavidin-biotin immunoperoxidase method was used for each section (Dako, Universal LSAB-2 kit). 3, 3` diaminobenzidine tetrahydrochloride (DAB) solution was used as the final chromogen, and sections were counterstained with Mayers hematoxylin before mounting. Negative controls for non-specific binding; incubated with secondary antibodies only; were processed and revealed no signals. Positive controls recommended by manufacturer were used to confirm correct immunohistochemical staining. For P-cadherin (normal skin), for claudin-3 (colonic mucosa), for survivin (gastric mucosa), For Fas (small intestine epithelia) and for PTEN (normal prostatic tissue). One positive and one negative controls were used in each run. Immuostaining interpretation: Staining of P-cadherin was recorded according to Stefansson et al. [19] by considering the proportion of tumor cells showing a positive membranous reaction; the percentage of membranous staining area was recorded as 0 (no positive tumor cells),
Ahlam A. Abd El-Maksoud & Ahmed M. Fahmy
467
+1 (<10% positive tumor cells), +2 (10% to 50% positive tumor cells), and +3 (>50% positive tumor cells). The mean percentage of positive tumor cells for the expression of survivin was determined in at least five areas at 400-fold magnification, and cases with less than 10% positively stained cells were defined as negative. Cases with 10-29% positively stained cells were defined as +, 3059% as ++, and 60% or more as +++ [20] . Analysis of Fas was performed as survivin with fraction of positive cells evaluated as <10% = 1, 11-50% = 2, 51-80% = 3, >80 = 4). For purposes of discussion, scores of 3 or lower were considered negative [5,21]. For Caludin-3 the following values were given: Tumorous and non-tumorous epithelia were considered negative if less than 5% of the cells reacted, 1 (6-20% positivity), 2 (21-40% positivity), 3 (41-60% positivity), 4 (61-80% pos-
itivity), 5 (81-100% positivity) [16]. For purposes of discussion, score 1 and 2 were considered as low expression while score 3, 4 and 5 were considered as high expression. PTEN immunoreaction was scored as negative if <10%, +1 if 10%-50% and +2 if >50% of slide's area was stained positive [22]. All negative control sections were verified to be non reactive. Statistical analysis: Data analysis was performed using the SPSS program (0.8 for windows) software package according to Pearson's correlation coefficient. Correlation between the immunophenotypic variables and different clinicopathological parameters as histologic type, myometrial invasion, grade and stage were determined. p-value less than 0.05 (< 0.05) was considered significant and <0.01 is highly significant.
Table (1): Correlation between histopathological features and clinical data of endometrial adenocarcinoma. Histopathological parameters Mean age (years) Depth of invasion: Deep Superficial Vascular invasion: Positive Negative Staging: Stage I Stage II Stage III Stage IV Endometrial carcinoma Grade I (n 12) 52.6 2 (16.7%) 10 (83.3%) 4 (33.3%) 8 (66.7%) 10 (83.3%) 2 (16.7%) 0 0 Grade II (n 14) 58 8 (57.1%) 6 (42.9%) 6 (42.9%) 8 (57.1%) 6 (42.9%) 6 (42.9%) 2 (14.2%) 0 Grade III (n 10) 64.4 8 (80%) 2 (20%) 6 (60%) 4 (40%) 2 (20%) 0 4 (40%) 4 (40%) Undiff. Carcinoma (n 4) 51.7 4 (100%) 0 4 (100%) 0 0 0 2 (50%) 2 (50%)
Table (2): Characteristics of antibodies used in this study. Antibody P-cadherin Claudin 3 Survivin Fas (CD95) PTEN Dilution Ready to use 1:75 Ready to use 1:20 1:75 Incubation time 60 min 60 min Overnight 60 min 60 min Source Lab Vision Invitrogen Spring Bioscience Novocastra Invitrogen Staining pattern Membranous Membranous Nuclear and/or cytoplasmic Membranous cytoplasmic and/or nuclear
Results PTEN, survivin , Fas, claudin-3, and P-cadherin immunostaining: PTEN immunoreactivity was noted in all normal proliferative endometrium 6/6 (100%) and in 5/7 (71.4%) of non atypical endometrial hyperplasia.
In ACH and EECA immunoreactivity was positive in 62.5% and 47.5%; respectively (Table 3) and (Fig. 1), with significant inverse correlation (p= .040). PTEN immunoreactivity was heterogeneous, some cells within a gland or some glands were negative for PTEN staining in ACH and EECA; respectively. The normal proliferative endometrium
468
showed intense cytoplasm and/or nuclear staining in the glandular epithelial cells. The lowest PTEN immunoreactivity was detected in EECA and the differences were significant (p<0.05). PTEN expression in cyclical endometrium and non atypical hyperplasia was higher than in ACH and EECA. No significant association was detected between PTEN phenotype and tumor grade, depth of invasion or FIGO stage (p=.860, p=.302, and p=.370 respectively). Survivin was overexpressed in hyperplasia and endometrial adenocarcinoma more than proliferative endometrium. This was statistically highly significant (p=.000) (Fig. 1). Among carcinoma cases, survivin was strongly positive in 28/40 cases (70%) with abundant immunoreactivity in the nucleus and/or cytoplasm. Survivin expression was significantly correlated with classical prognostic factors of endometrial carcinoma such as myometrial invasion and FIGO stage (p=0.037 and p=.023 respectively). However, the correlation to the grad was insignificant (p=.106) (Table 4). Fas expression was significantly higher in the atypical hyperplasia and carcinoma group than in the proliferative and non atypical hyperplasia group
(p=.031) (Fig. 2). Fas expression was highly associated with high FIGO grade (p=.000), advanced stage (p=.000), and deep myometrial infiltration (p=0.007) (Table 5). Positive rate of claudin-3 was 16.7%, 28.6%, 62.5% and 90% in endometrium at proliferative phase, non atypical hyperplasia, atypical hyperplasia and endometrial cancer; respectively, with high significant difference (p=0.01) (Fig. 2), and it was statistically different between precancerous endometrium and endometrial cancer. Degree of claudin-3 expression was significantly correlated with the depth of myometrial invasion (p=0.027), however the correlation to the grade of endometrial carcinoma or the stage was insignificant (p=.896 and p=.315 respectively) (Table 6). High membranous expression of P-cadherin (+3) was found in 16.7%, 28.6%, 37.5% and 45% in endometrium at proliferative phase, non atypical hyperplasia, atypical hyperplasia and endometrial cancer; respectively with significant difference (p<0.05) (Fig. 1). High P-cadherin expression was highly associated with high FIGO grade (p=.000), advanced stage (p=.003), and deep myometrial infiltration (p=0.005) (Table 7).
Table (3): Correlation between PTEN expression and clinicopathological data in examined cases. Clinicopath variables ve Non-atypical hyperplasia: Simple Complex Atypical hyperplasia Adenocarcinoma Total Tumor grade: GI G II G III Undifferentiated Stage: I II III IV Depth of invasion: Superficial Deep Total 2 (28.6%) 1 (25%) 1 (33.3%) 3 (37.5%) 21 (52.5%) 26 7 (58.3%) 6 (42.9%) 4 (40%) 4 (50%) 8 (44.4%) 4 (50%) 5 (62.5%) 4 (66.7%) 8 (44.4%) 13 (59.1%) 21 PTEN expression +1 2 (28.6%) 2 (50%) 0 2 (25%) 16 (40%) 20 5 (41.7%) 8 (57.1%) 3 (30%) 0 9 (50%) 3 (37.5%) 2 (25%) 2 (33.3%) 8 (44.4%) 8 (36.4%) 16 +2 3 (42.8%) 1 (25%) 2 (66.7%) 3 (37.5%) 3 (7.5%) 9 0 0 3 (30%) 0 1 (5.6%) 1 (12.5%) 1 (12.5%) 0 2 (11.2%) 1 (4.5%) 3 7 4 3 8 40 55 12 14 10 4 18 8 8 6 18 22 40 Total p-value
p=.040
p=.860
p=.370
p=.302

Table (4): Correlation between survivin expression and clinicopathological data in examined cases. Survivin expression Clinicopath variables ve Non-atypical hyperplasia: Simple Complex Atypical hyperplasia Adenocarcinoma Total Tumor grade: GI G II G III Undifferentiated Stage: I II III IV Depth of invasion: Superficial Deep Total 4 (57.1%) 2 (50%) 2 (66.7%) 0 4 (10%) 8 2 (16.7%) 2 (14.3%) 0 0 4 (22.2%) 0 0 0 4 (22.2%) 0 4 +1 3 (42.9%) 2 (50%) 1 (33.3%) 2 (25%) 8 (20%) 13 2 (16.6%) 4 (28.6%) 2 (20%) 0 4 (22.2%) 2 (25%) 0 2 5 (27.8%) 3 (13.6%) 8 +2 0 0 0 5 (62.5%) 12 (30%) 17 6 (50%) 0 4 (40%) 2 (50%) 8 (44.4%) 0 2 (25%) 2 (50%) 4 (22.2%) 8 (36.4%) 12 +3 0 0 0 1 (12.5%) 16 (40%) 17 2 (16.6%) 8 (57.1%) 4 (40%) 2 (50%) 2 (11.1%) 6 (75%) 6 (75%) 2 (50%) 5 (27.8%) 11 (50%) 16 7 4 3 8 40 55 p=0.106 12 14 10 4 18 8 8 6 18 22 40 p=0.023 p=.000 Total p-value
469
p=0.037
Table (5): Correlation between Fas expression and clinicopathological data in examined cases. Clinicopath variables 1 Non-atypical hyperplasia: Simple Complex Atypical hyperplasia Adenocarcinoma Total Tumor grade: GI G II G III Undifferentiated Stage: I II III IV Depth of invasion: Superficial Deep Total 3 (42.9%) 2 (50%) 1 (33.3%) 2 (25%) 10 (25%) 15 6 (50%) 4 (28.6%) 0 0 6 (33.3%) 4 (50%) 0 0 8 (44.5%) 2 (9.1%) 4 Fas expression 2 4 (57.1%) 2 (50%) 2 (66.7%) 3 (37.5%) 8 (20%) 15 2 (16.7%) 4 (28.6%) 2 (20%) 0 8 (44.5%) 0 0 0 4 (22.2%) 4 (18.2%) 8 3 0 0 0 2 (25%) 14 (35%) 16 4 (33.3%) 6 (42.9%) 2 (20%) 2 (50%) 4 (22.2%) 4 (50%) 4 (50%) 2 (33.3%) 4 (22.2%) 10 (45.4%) 12 4 0 0 0 1 (12.5%) 8 (20%) 9 0 0 6 (60%) 2 (50%) 0 0 4 (50%) 4 (66.7%) 2 (11.1%) 6 (27.3%) 16 7 4 3 8 40 55 12 14 10 4 18 8 8 6 18 22 40 p=.000 Total p-value
p=.031
p=.000
p=.007
470

Table (6): Correlation between claudin-3 expression and clinicopathological data in examined cases. Clinicopath variables ve Non-atypical hyperplasia: Simple Complex Atypical hyperplasia Adenocarcinoma Total Tumor grade: GI G II G III Undifferentiated Stage: I II III IV Depth of invasion: Superficial Deep Total 5 (71.4%) 3 (75%) 2 (66.7%) 3 (37.5%) 4 (10%) 12 1 (8.3%) 1 (7.2%) 2 (20%) 0 0 2 (25%) 1 (12.5%) 1 (16.7%) 3 (16.7%) 1 (4.5%) 4 Claudin-3 expression Low expression 1 (14.3%) 1 (25%) 0 2 (25%) 15 (37.5%) 18 5 (41.7%) 5 (35.7%) 3 (30%) 2 (50%) 7 (38.9%) 3 (37.5%) 3 (37.5%) 2 (33.3%) 9 (50%) 6 (27.3%) 15 High expression 1 (14.3%) 0 1 (33.3%) 3 (37.5%) 21 (52.5%) 25 6 (50%) 8 (57.1%) 5 (50%) 2 (50%) 11 (61.1%) 3 (37.5%) 4 (50%) 3 (50%) 6 (33.3%) 15 (68.2%) 21 7 4 3 8 40 55 12 14 10 4 18 8 8 6 18 22 40 p=.896 Total p-value
p=0.01
p=.315
p=.027
Table (7): Correlation between P-cadherin expression and clinicopathological data in examined cases. Clinicopath variables 0 Non-atypical hyperplasia: Simple Complex Atypical hyperplasia Adenocarcinoma Total Tumor grade: GI G II G III Undifferentiated Stage: I II III IV Depth of invasion: Superficial Deep Total 2 (28.6%) 1 (25%) 1 (33.3%) 3 (37.5%) 10 (25%) 15 8 (66.7%) 2 (14.3%) 0 0 6 (33.3%0 3 (37.5%) 1 (12.5%) 0 8 (44.4%) 2 (9.1%) 10 P-cadherin expression +1 2 (28.6%) 1 (25%) 1 (33.3%) 2 (25%) 7 (17.5%) 11 3 (25%) 2 (14.3%) 2 (20%) 0 5 (27.8%) 2 (25%) 0 0 4 (22.2%) 3 (13.6%) 7 +2 1 (14.2%) 0 1 (33.3%) 0 5 (12.5%) 6 1 (8.3%) 2 (14.3%) 1 (10%) 1 (25%) 2 (11.1%) 0 2 (25%) 1 (16.7%) 1 (5.6%) 4 (18.2%) 5 +3 2 (28.6%) 2 (50%) 0 3 (37.5%) 18 (45%) 23 0 8 (57.1%) 7 (70%) 3 (75%) 5 (27.8%) 3 (37.5%) 5 (62.5%) 5 (83.3%) 5 (27.8%) 13 (59.1%) 18 7 4 3 8 40 55 12 14 10 4 18 8 8 6 18 22 40 p=.000 p<0.05 Total p-value
p=.003
p=.005

(A) (B)
471
(C)
(D)
Fig. (1): Immunohistochemical staining (Streptavidin-Biotin/DAB chromagen) results: Membranous staining for p-cadherin in grade II endometrial adenocarcinoma. Photomicrograph, x200 (a). Cytoplasmic staining for PTEN in adenomatous endometrial hyperplasia. Photomicrograph, X200 (b). Cytoplasmic staining for survivin in grade II (c) and grade III (d) endometrial adenocarcinoma. Photomicrograph, x200. (A) (B)
(C)
(D)
Fig. (2): Immunohistochemical staining (Streptavidin-Biotin/DAB chromagen) results: Cytoplasmic staining for FAS in adenomatous endometrial hyperplasia (a) and in grade II endometrial adenocarcinoma (b). Photomicrograph, x200. Dense cytoplasmic staining for claudin-3 in atypical adenomatous hyperplasia (c), photomicrograph, x200, and grade I endometrial adenocarcinoma (d), Photomicrograph, x400.
472
Discussion This is a retrospective case study that shows the interrelation and role of some apoptosis-related proteins, claudins-3 and P-cadherin in endometrial tumorigenesis. Several studies reported that PTEN inactivation is correlated with clonal growth patterns detected in endometrial hyperplasia and carcinoma [23,24]. In the present study, PTEN negative immunoreactivity was detected in the majority of EECA and ACH and in minority of non atypical hyperplasia, while positive immunoreactivity was noted in all normal proliferative endometrium and in 71.4% of non atypical endometrial hyperplasia. In ACH and EECA, immunoreactivity was positive in 62.5% and 47.5%; respectively with statistically significant difference. Such results are consistent with those of Tantbirojn et al. [1] and Samadi et al. [12] who reported a significant statistical difference of PTEN immunoreactivity among proliferative endometrium, endometrial hyperplasia and endometrial carcinoma group. Also, Allison et al., [25] found that the combination of loss of PTEN expression and particular histological features had most consistency to make a clear distinction between normal, endometrial hyperplasia and EECA. In the present study, no correlation was found between the grade of EECA, the stage or depth of myometrial invasion and PTEN expression. In consistent to these results, Erkanli et al., [22] found no correlation between PTEN gene and classical prognostic factors such as grade and myometrial invasion however, Kapucuoglu et al., [13] found a negative correlation between the grade and PTEN expression and speculated that decreased PTEN expression is associated with loss of differentiation. Data reported in this study showed higher levels of survivin protein expression in endometrial adenocarcinomas than in hyperplastic or normal endometrium. Survivin was overexpressed in 73.3% of hyperplasia and in 90% of endometrial adenocarcinoma compared to 16.7% positive immunoreactivity in proliferative endometrium. Going with this are reports of Takai et al., [7] who showed that survivin was weakly expressed in some normal endometria. Erkanli et al., [26] and Ai et al., [8] found that survivin expression increased from proliferative to hyperplasia to carcinoma cases. In the present work, survivin expression was significantly correlated with myometrial invasion and FIGO stage. Regarding FIGO grade of EECA, no significant correlation to survivin expression was found. With respect to this, Tarkowski et al., [26] noted that survivin expression was significantly
associated with clinical stage, histological grade, and myometrial invasion. Lehner et al. [27], Nancy et al. [9] and Erkanli et al. [22] reported that survivin overexpression in hyperplasia and endometrial adenocarcinoma cases were higher compared to proliferative endometrium and concluded that survivin not only inhibits apoptosis, but also accelerates cell proliferative activity. On the otherhand, Erkanli et al., [28] found that survivin was not correlated with grade and myometrial invasion. In the present study Fas expression was significantly higher in atypical hyperplasia and carcinoma than in the proliferativ and non atypical hyperplasia. In consistent with this, Atasoy et al., [5] reported that Fas score was significantly higher in carcinoma than in simple hyperplasia. In this study, elevated Fas expression was associated with higher tumor grades, advanced stage (p=.000), and deep myometrial infiltration. In accordance to these results, Pizer et al., [21] reported that the expression pattern of Fas supports a linkage between Fas expression, proliferation, tumor grade and poor prognosis. In the current study, absence or weak staining for claudin-3 was observed in normal and hyperplasitc endometrium while medium to intense staining was detected in atypical hyperplasia and carcinoma. The positive rate of claudin-3 was 16.7%, 28.6%, 62.5% and 90% in endometrium at proliferative phase, non atypical hyperplasia, atypical hyperplasia and endometrial cancer; respectively, with statisctically significant difference. Parallel to this, Pan et al., [29] demonstrated that claudin-3 and claudin-4 are strongly expressed in AEH and EECA, but less frequently in normal endometrium. In our study, degree of claudin-3 expression was significantly correlated with the depth of myometrial invasion, however the correlation to the grade or tumor stage was insignificant. The upregulation of claudins expression during endometrial carcinogenesis suggests their potential utility as diagnostic and prognostic biomarkers. In this study, increased membranous expression of P-cadherin was significantly higher in the carcinoma and atypical hyperplasia than in non atypical hyperplasia and proliferative endometrium. Also increased expression of P-cadherin was related to more aggressive endometrial cancers. This phenotype was highly associated with adverse prognostic factors, including a higher grade, advanced stage, and deep myometrial infiltration. The prognostic impact of this feature is similar to results found for breast cancer [30,31]. In a study carried out by Ingunn et al., [14] on subgroup of endometrioid carcinomas, strong staining of P-cadherin was
473
medical preventive measures. Eur. J. Cancer Prev., 17 (2): 133-8, 2008. 7- TAKAI N., MIYAZAKI T., NISHIDA M., NASU K. and MIYAKAWA I.: Survivin expression correlates with clinical stage, histological grade, invasive behavior and survival rate in endometrial carcinoma. Cancer Lett., 184 (1): 105-16, 2002. 8- AI Z., YIN L., ZHOU X., ZHU Y., ZHU D., YU Y. and FENG Y.: Inhibition of survivin reduces cell proliferation and induces apoptosis in human endometrial cancer. Cancer, 107 (4): 746-56, 2006. 9NANCY H. NABILSI, RUSSELL BROADDUS and DAVID S.: Loose Regulation of survivin expression in human endometrium.. Proc. Amer. Assoc. Cancer Res., 47: 55-9, 2006.
associated with high grade, deep myometrial infiltration, and pathologic expression of p53 protein, Conversely, Moreno-Bueno et al., [32] found that reduced P-cadherin expression was more frequent in carcinomas of more advanced stage. Conclusions: It was concluded that PTEN expression was significantly higher in cyclical endometrium than in atypical hyperplasia and endometrioid carcinoma. Lack of PTEN expression may be the earliest event in endometrial carcinogenesis. Survivin protein is assumed to be a diagnostic marker for endometrial carcinomas that may also yield prognostic information. The significant increase in Fas expression observed in carcinomas as compared with simple hyperplasias may suggest that the Fasrelated apoptotic pathway is also involved in the regulation of apoptosis in the endometrial tissue and promotes the development and progression of endometrial neoplasia. Because of the high Fas expression seen in high grade tumors, advanced stages and deeply invaded EECA, Fas detection has potential utility as a prognostic marker. The upregulation of claudins expression during endometrial carcinogenesis suggests their potential utility as diagnostic and prognostic biomarker and might be targets for future therapy. Also, strong P-cadherin staining may serve as a useful and novel marker of endometrial cancer progression, possibly by indicating a more invasive phenotype. References
1- TANTBIROJN P., TRIRATANACHAT S., TRIVIJITSILP P. and NIRUTHISARD S.: Detection of PTEN immunoreactivity in endometrial hyperplasia and adenocarcinoma. J. Med. Assoc. Thai., 91 (8): 1161-5, 2008. 2- CHEN X., ZHANG Z., FENG Y., FADARE O., WANG J., AI Z., JIN H., GU C. and ZHENG W.: Aberrant survivin expression in endometrial hyperplasia: Another mechanism of progestin resistance. Mod. Pathol., 22 (5): 699-708, 2009. 3- MOKHTAR N., GOUDA I. and ADEL I.: Malignant female genital system tumors. Chapter 8 in: Caner pathology registry 2003-2004 and time trend analysis, pp 77, 2007. 4- ABE H., SHIBATA M.A. and OTSUKI Y.: Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells. Mol. Hum. Reprod., 12 (9): 535-41, 2006. 5ATASOY P., BOZDOG AN O., EREKUL S., BOZDO GAN N. and BAYRAM M.: Fas-mediated pathway and apoptosis in normal, hyperplastic, and neoplastic endometrium, Gynecol. Oncol., 91 (2): 309-17, 2003.
10- ZHDANOV A.V., SUKHIKH G.T., DAVYDOVA M.P., SLUKINA T.V., CHERNUKHA G.E., SAMOILOVA T.E. and SMETNIK V.P.: Correlations in the cytokine system in endometrial hyperplasia. Bull Exp. Biol. Med., 136 (3): 270-2, 2003. 11- LACEY J.V. JR, MUTTER G.L., RONNETT B.M., IOFFE O.B., DUGGAN M.A., RUSH B.B., GLASS A.G., RICHESSON D.A., CHATTERJEE N., LANGHOLZ B. and SHERMAN M.E.: PTEN expression in endometrial biopsies as a marker of progression to endometrial carcinoma. Cancer Res., 68 (14): 6014-20, 2008. 12- SARMADI S., IZADI-MOOD N., SOTOUDEH K. and TAVANGAR S.M.: Altered PTEN expression; a diagnostic marker for differentiating normal, hyperplastic and neoplastic endometrium. Diagn. Pathol., 25: 4-41, 2009. 13- KAPUCUOGLU N., AKTEPE F., KAYA H., BIRCAN S., KARAHAN N. and CIRIS M.: Immunohistochemical expression of PTEN in normal, hyperplastic and malignant endometrium and its correlation with hormone receptors, bcl-2, bax, and apoptotic index. Pathol. Res. Pract., (3): 153-62, 2007. 14- INGUNN M. STEFANSSON, HELGA B. SALVESEN and LARS A. AKSLEN: Prognostic impact of alterations in P-Cadherin expression and related cell adhesion markers in endometrial cancer. Journal of Clinical Oncology, 22 (7): 1242-52, 2004. 15- DUAN ZHAO, ZHANG XIN and GAO YA: Expressions of claudin 4 and claudin 1 in endometrial cancer and their significance. Journal of Medical Colleges of PLA, 23 (3): 162-6, 2008. 16- SZAB I., KISS A., SCHAFF Z. and SOBEL G.: Claudins as diagnostic and prognostic markers in gynecological cancer. Histol. Histopathol., 24 (12): 1607-15, 2009. 17- KAPUCUOGLU N., BULBUL D., TULUNAY G. and TEMEL M.: Reproducibility of grading systems for endometrial endometrioid carcinoma and their relation with pathologic prognostic parameters., Int. J. of Gynecological Cancer, 18 (4): 790-6, 2008. 18- ZAINO R.J. FIGO staging of endometrial adenocarcinoma: A critical review and proposal. Int. J. Gynecol. Pathol., 28 (5): 477-8, 2009. 19- STEFANSSON I.M., SALVESEN H.B. and AKSLEN L.A.: Prognostic Impact of alterations in P-cadherin expression and related cell adhesion markers in endome-
6- BORUBAN M.C., ALTUNDAG K., KILIC G.S. and BLANKSTEIN J.: From endometrial hyperplasia to endometrial cancer: Insight into the biology and possible
474

trial cancer. Journal of Clinical Oncology, 22 (7): 124252, 2004. and hyperplastic endometrium. Ginekol. Pol., 71 (9): 1226-9, 2000. 27- LEHNER R., ENOMOTO T., MCGREGOR J.A., SHROYER L., HAUGEN B.R., PUGAZHENTHI U.S. and HROYER K.R.: Correlation of survivin mRNA detection with histologic diagnosis in normal endometrium and endometrial carcinoma. Acta. Obstet. Gynecol. Scand, 81 (2): 162-7, 2002. 28- ERKANLI S., KAYASELCUK F., KUSCU E., BAGIS T., BOLAT F., HABERAL A. and DEMIRHAN B.: Expression of survivin, PTEN and p27 in normal, hyperplastic, and carcinomatous endometrium. Int. J. Gynecol. Cancer, 16 (3): 1412-8, 2006. 29- PAN X.Y., WANG B., CHE Y.C., WENG Z.P., DAI H.Y. and PENG W.: Expression of claudin-3 and claudin-4 in normal, hyperplastic and malignant endometrial tissue. Int. J. Gynecol. Cancer, 17 (1): 233-41, 2007. 30- PERALTA SOLER A., KNUDSEN K.A. and SALAZAR H.: P-cadherin expression in breast carcinoma indicates poor survival. Cancer, 86: 1263-72, 1999. 31- GAMALLO C., MORENO-BUENO G. and SARRIO D.: The prognostic significance of P-cadherin in infiltrating ductal breast carcinoma. Mod. Pathol., 14: 650-4, 2001. 32- MORENO-BUENO G., HARDISSON D., SARRI D., SNCHEZ C., CASSIA R., PRAT J., HERMAN J.G., ESTELLER M., MATAS-GUIU X. and PALACIOS J.: Abnormalities of E- and P-cadherin and catenin (, catenin, and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia. The Journal of Pathology, 199 (4) 4: 471-8, 2003.
20- CHEN W.C., LIU Q., FU J.X. and KANG S.Y.: Expression of survivin and its significance in colorectal cancer. World J. Gastroenterol., 10 (19): 2886-9, 2004. 21- PIZER E.S., LAX S.F., KUHAJDA F.P., PASTERNACK G.R. and KURMAN R.J.: Fatty acid synthase expression in endometrial carcinoma. Cancer, 83: 528-37, 1998. 22- ERKANLI S., BOLAT F., KAYASELCUK F., DEMIRHAN B. and KUSCU E.: COX-2 and survivin are overexpressed and positively correlated in endometrial carcinoma. Gynecol. Oncol., 104 (2): 320-5, 2007. 23- ATHANASSIADOU P., ATHANASSIADES P., GRAPSA D., GONIDI M., ATHANASSIADOU A.M. and STAMATI P.N.: The prognostic value of PTEN, p53, and beta-catenin in endometrial carcinoma: A prospective immunocytochemical study. Int. J. Gynecol. Cancer, 17: 697-704, 2007. 24- HAYES M.P., WANG H., ESPINAL-WITTER R., DOUGLAS W., SOLOMON G.J. and BAKER S.J.: PIK3CA and PTEN mutations in uterine endometrioid carcinoma and complex atypical hyperplasia. Clin. Cancer Res., 12: 5932-5, 2006. 25- ALLISON K.H., TENPENNY E., REED S.D., SWISHER E.M. and GARICA R.L.: Immunohistochemical markers in endometrial hyperplasia: Is there a panel with promise? A review. Appl Immunohistochem. Mol. Morphol., 16: 329-43, 2008. 26- TARKOWSKI R., WOJCIEROWSKI J., POLAK G. and KOTARSKI J.: Expression of the survivin gene in normal
Ultrastructural Study of Hepatic Changes after Human Umbilical Cord Blood Stem Cell Transplantation in Chronic Murine Schistosomiasis
HALA N. HOSNI, M.D.*; HODA A. YEHIA, M.D.** and RANYA M. EL-SHEIKH, M.Sc.**
The Departments of Pathology*, Faculty of Medicine, Cairo University and Pathology & Electron Microscopy**, Theodor Bilharz Research Institute, Giza, Egypt
Abstract
Background: The contribution of hematopoietic stem cells to liver therapy in different forms of liver injury remains debatable. In the last decade, the number of transplantations of hematopoietic cells derived from cord blood has increased, where numerous literature reports documented the feasibility and effectiveness of the transplantation of cord blood for the treatment of a broad range of disorders. Aim of the Work: This xenogenic research is designed to highlight, by light and electron microscopic study, the possibility of engraftment of human umbilical cord blood derived stem cells in the livers of immune-competent mice infected with chronic schistosomiasis. Material and Methods: This study was conducted on 20 Swiss Albino immune-competent mice. The mice were subdivided into four groups (5 mice each). (Group 1) was infected with Schistosoma mansoni cercariae for 20 weeks duration, then intrahepatically transplanted with CD133+ human cord blood mononuclear cells, cultured on nutrient media, and isolated using the MACS Separation Unit from Miltenyi Biotec. (Group 2) was infected with S.mansoni, but not transplanted, (group 3) was normal and transplanted, and (group 4) was normal and non transplanted. All mice were sacrificed 3 weeks following the transplantation of groups 1 and 3. Engraftment of transplanted human cells was assessed by means of immunohistochemistry; using antibodies against human Hep Par 1 and -fetoprotein. Histological examination was performed using the Zeiss light microscope, and ultrastructural study was carried out by the Philips TEM 208 S electron microscope. Results: By light microscopic examination, livers of the infected transplanted group (group 1) and the infected non transplanted group (group 2) showed variable sized fibrocellular and fibrous schistosomal granulomas. (Group 1) exhibited as well more prominent bile duct proliferation than (group 2). Sections of (groups 1 & 3) showed small and large eosinophilic cells different from the surrounding murine hepatocytes. By immunohistochemistry, some cells in (group 1 & 3) sections showed positive cytoplasmic staining for the two anti human hepatocyte markers used; (Hep Par 1 and Correspondence to: Dr. Hala N. Hosni, The Department of Pathology, Faculty of Medicine, Cairo University.
fetoprotein). Electron microscopic examination of (group 1) grids distinguished variable immature cells in the form of small progenitor cells, intermediate hepatocyte-like oval cells and larger premature hepatocytic cells. The transplanted healthy group (group 3) showed similar cells. The previously noted cells were not seen in the remaining control groups, (groups 2 & 4). Conclusion: This research proved engraftment of the human umbilical cord blood hematopoietic stem cells after their intrahepatic transplantation into the livers of mice suffering from chronic hepatic schistosomiasis, and their attempt to give rise to premature forms of cells with hepatocytic lineage. Extensive studies are still needed to clarify the possible utility of these cells in resolving damaged organs and tissues. Key Words: Umbilical cord Blood stem cell transplantation Schistosomiasis Hepatic changes.
Introduction TREATMENT of liver disease has been greatly improved by the advent and evolution of liver transplantation. However as demand for donor organs continues to increase beyond their availability, the need for alternative liver therapies is clear [12]. Liver fibrosis occurs in the setting of chronic injury caused by different etiologies, constituting a serious worldwide public health problem. Schistosomiasis, hepatopathies due to viral hepatitis, drugs, alcohol, metabolic and autoimmune diseases, and congenital abnormalities are important causes of liver fibrosis [22]. In Egypt, viral hepatitis along with infection with Schistosoma mansoni (S.mansoni) is one of the major causes of chronic liver disease [9]. Chronic infection by Schistosoma mansoni is considered a good experimental model of chronic hepatic disease and fibrosis. New therapeutic strategies aiming to minimize damages caused by hepatic
475
476
Ultrastructural Study of Hepatic Changes after Human UCB Stem Cell Transplantation
fibrogenesis in chronic liver diseases are of great interest [22]. Several studies performed on animal models have highlighted the potential of hematopoietic stem cells (HSC) to differentiate into hematopoietic cell lineages, and to transdifferentiate into nonhematopoietic cells including hepatocytes [5,29]. Because of the lack of matched related donors and the risk of severe graft versus host disease (GVHD) that accompanies unrelated bone marrow or peripheral blood hematopoietic cell transplantation, alternative sources of hematopoietic stem cells have been considered [17]. Over the last few years, we have seen the growth in use of the common, often discarded stem cell source, umbilical cord blood. Cord blood stem cell transplantation successfully treated many diseases and debilitating conditions [3,19]. The frequency of umbilical cord blood (UCB) hematopoietic stem/progenitor cells equals or exceeds that of bone marrow (BM), and greatly surpasses that of adult peripheral blood [18,25] . Furthermore, UCB collection can be performed in a manner that entails less discomfort and morbidity for the donor than collection of bone marrow or peripheral blood stem cells. It is less expensive to store, and once banked, can be retrieved much more quickly than peripheral blood or bone marrow [17]. Efforts to understand the plasticity of the stem cells found in cord blood may lead to advances in regenerative medicine [25]. The aim of this work is designed to highlight, by light and electron microscopic study, the possibility of engraftment of human UCB derived stem cells in the livers of immune-competent mice with chronic schistosomiasis. Material and Methods Twenty female Swiss albino mice, 8 weeks old, weighing 20+/ 5gm were obtained, fed a standard commercial pelleted diet, and maintained under conventional conditions at the Schistosome Biological Supply Center (SBSC) of Theodor Bilharz Research Institute (TBRI). All animal protocols were conducted in accordance with the valid international guidelines for animal experimentation and were approved by the TBRIs animal research committee. Ten [10] Mice were inoculated with Schistosoma mansoni cercariae. Eighty cercariae, in a single dose, were administered subcutaneously to each mouse. The remaining 10 mice were not inoculated with the cercariae. All 20 mice used in the study
were subsequently kept at the SBSC for a further period of 20 weeks. After taking the informed consent of twelve pregnant mothers, and following delivery of the full term infant, the umbilical cord was clamped and cut in the usual manner. Prior to separation and delivery of the placenta, a four to eight inch of the cord was cleansed with an antiseptic solution. A gauge needle, connected to a sterile cord blood collection bag containing an anticoagulant solution (citrate-phosphate dextrose and adenine) was inserted into the umbilical vein at the cleansed site, and the blood was allowed to drain into the bag by gravity. A range of (60-120ml) of blood was collected from each umbilical cord. UCB samples were diluted 1:3 in phosphate buffer saline (PBS). Low-density mononuclear cells were separated over Ficoll-Hypaque (density 1.077g/dl; Pharmacia, Uppsala, Sweden). CD133+ve HSC were isolated from UCB mononuclear cell samples by the positive selection method using the magnetic activated cell sorting columns and microbead-conjugated antibodies (MACS and the CD 133 positive selection Kit, Miltenyi Biotec Gmbh, Bergisch Gladbach, Germany), as described by the manufacturer, in order to reach an optimum enrichment of the stem cells with depletion of the mature cells so as to escape the rejecting effect of the mature T cell according to [29] . Purity of sorted cells was assessed by fluorescence-activated cell sorting (FACS) analysis using anti-CD133 antibodies labeled with phycoerythrin (PE) from Miltenyi Biotec Gmbh. Cells were plated (5-6 x 106 cells/ml) in 9 well culture plates with Dulbecco's modified eagle's media, supplemented with 20% fetal calf serum (FCS), Penicillin/Streptomycin (100U/ml, 0.1mg/ ml), L-glutamine (2mM), all from Stem Cell Technologies Inc, Vancouver, Canada together with the following human recombinant growth factors and cytokines: Stem Cell Factor (SCF 100ng/ml), Flt3/Flk2-ligand (100ng/ml), Il 3 (20ng/ml) and Il 6 20ng/ml, all from R&D Systems, Inc. Cells were incubated at 37C in 5% CO 2 in a humidified atmosphere for one week. The positivity and concentration of CD 133+ve cells was confirmed in each step with flowcytometry using anti-CD133 antibodies labeled with phycoerythrin (PE). The cells were expanded in this culture media for a period of 3 weeks. In the 20th week post schistosomal infection, 10 non ablated mice were transplanted with 0.3 x106 umbilical cord blood stem cells each, through
Hala N. Hosni, et al.
477
an intrahepatic injection using an insulin needle. 5 of the transplanted mice were infected, and the other 5 were selected from the normal (non infected) group of mice. The studied immune-competent mice (n=20) were divided into four groups, consisting of 5 mice each. Group (1): Mice infected with Schistosoma mansoni and transplanted with human umbilical cord blood stem cells (HUCBSCs). Group (2): Mice infected with Schistosoma mansoni and not transplanted with HUCBSCs. Group (3): Normal mice not infected with Schistosoma mansoni and transplanted with HUCBSCs. Group (4): Normal mice not infected with Schistosoma mansoni and left out without stem cell transplantation. (Group 1 & 3) mice were sacrificed 3 weeks after their transplantation with stem cells and their livers were subsequently processed for further histological and immunohistochemical examination. (Group 2 & 4) mice were accordingly also sacrificed at the same time. Livers from all mouse groups were fixed in 10% phosphate-buffered formalin for 24 hours and processed to paraffin blocks. Four-micrometer thick paraffin sections were then rehydrated and stained with hematoxylin and eosin (H & E). 15 liver sections were prepared per mouse. The standard avidin-biotin immunoperoxidase technique was used [10] . From each mouse 10 paraffin sections were prepared and stained with the primary antibodies against human Hep Par1 (Dako, Denmark), and the other 5 sections were alternatively incubated with the primary antibodies against -fetoprotein (Santa Cruz Biotechnology, USA). Liver sections with voluntary omission of the primary antibody and its replacement by PBS served as negative control. Liver tissue specimens of the different groups of mice were minced into tiny pieces about 1mm 3 each, and immediately fixed in 4% glutaraldehyde solution buffered by 0.2M sodium cacodylate, for 1 hour at 4 C. The fixed specimens were then washed twice in equal volumes of sodium cacodylate 0.2M and sucrose 0.4M at 4C, post fixed in 2% osmium tetroxide for one hour and washed in distilled water. Dehydration in ascending grades of ethyl alcohol (50, 70, 90 and 100% ethanol) at 5min gap was then performed. The specimens were immersed in Epon for 3 successive days, and polymerized at 60C for 48 hours. Semithin sections
were cut, stained with methylene blue azur , and examined by light microscopy to choose the proper site for ultrathin sectioning. Then ultrathin sections were prepared using a Leica ultracut R ultramicrotome, and mounted on copper grids then double stained with uranyl acetate and lead citrate. Sections were examined under TEM (Philips EM 208S) electron microscopy. Results Liver sections of Schistosoma infected and umbilical cord blood stem cell transplanted mice (group 1), and infected non transplanted mice (group 2) showed partial apparent disturbance of the hepatic lobular architecture due to the presence of many scattered diffent sizes rounded and ovoid schistosomal granulomas of mostly fibrocellular granulomas representing the characteristic picture of the chronic stage of schistosomal infection. Fibrocellular granulomas were formed of central schistosomal ova surrounded by concentrically arranged chronic inflammatory cells, mainly lymphocytes, macrophages, epithelioid cells and by bundles of fibrous tissue. The rest were fibrous granulomas formed of fibrous bundles and reticular fibers. The granulomas may showed central calcified ova, or degenerated or absent ova. The portal tracts were enlarged by the presence of granulomas, increased deposition of collagen fibers around the portal blood vessels and the bile ductules, and the proliferative reaction of bile ductules. Besides the characteristic picture of schistosomal granulomas, sections of the infected transplanted livers of (group 1) mice revealed small and large cells with an intensely stained eosinophilic cytoplasm inside the hepatic lobule, which could be easily differentiated from the surrounding murine hepatocytic cells. Larger cells were more frequently seen than the smaller ones. These cells showed either central or eccentric basophilic nuclei, and were present either singly scattered throughout the hepatic lobule, or in groups. They were also present at the periphery of the granulomas, in periportal areas, and inside and around central veins. This group exhibited as well more prominent bile duct proliferation as compared to the infected only group (group 2), where bile duct proliferation in both groups was represented by groups of primitive bile ductules lined by cubical and some flattened epithelial cells with centrally located basophilic nuclei occupying most of the cytoplasm. In this infected transplanted group, some of the previously mentioned eosinophilic cells could be identified in the vicinity of and inside some of the bile ductules and vessels. By examining the liver sec-
478
tions of the normal transplanted mice of (group 3), they equally revealed the presence of similar eosinophilic cells between the normal murine hepatocytes and encircling the central veins. The smaller-sized cells was the dominant type in this group, some of which were ovoid in shape and some had angulated borders. The normal histology of murine liver was illustrated in Hematoxylin and Eosin stained liver sections of the normal non transplanted group (group 4). Immunohistochemically, by applying the two anti-human hepatocyte markers; Hep Par 1 and fetoprotein to the four groups of mice, by light microscopic examination revealed that:
Liver sections of the infected transplanted (group 1) mice showed positive cytoplasmic staining of some cells intercalated between mouse hepatocytes, adjacent to blood vessels, around central veins, at the periphery of portal tracts and around granulomas, either present singly or in clusters. Liver sections of the normal transplanted (group 3) mice showed positive staining of a number of cells for both markers. Negative staining for both markers was clearly demonstrated in the infected non transplanted group of mice, (group 2) and the normal non transplanted group of mice (group 4).
Fig. (1-A): A liver section from infected transplanted mouse showing cells with positive cytoplasmic staining of anti-hyman (Hep par1) seen nearby a granuloma (thin arrow) and interspersed between the rest of negatively stained hepatocytes (thick arrow) x200.
Fig. (1-B): A liver section from infected transplanted mouse showing cells with positive cytoplasmic staining for anti human (alpha fetoprotein). These cells are present around a central vein (thin arrow) and interspersed in between the rest of negatively stained hepatocytes (thick arrow) x200.
Electron microscopic examination of the ultrathin sections of livers of the infected transplanted (group 1) mice revealed the presence of fibrocellular and fibrotic granulomas, which also represented the main constituent of ultrathin sections of the infected non transplanted mice of (group 2). Some damage attributed to schistosomal infection was seen in the bile canaliculi of the infected transplanted and infected non transplanted groups with partial loss of their microvilli. Moreover, the infected groups of mice, (groups 1 & 2) showed congested sinusoidal spaces impacted with RBCs, and hypertrophied kupffer cells usually located at the junction of two sinusoids, having plumpy irregular nuclei, loaded with schistosomal pigment, and having primary and secondary lysosomes. Signs of hepatocyte regeneration were more clearly seen in (group 1) with proliferated rough endoplasmic reticulum encircling the mitochondria, binucleation, and irregularity of the nuclear membrane.
Besides the previously detected pathological changes, while screening the grids obtained from the infected transplanted mice three types of immature cells were identified; small oval-shaped cells of average diameter (5), (progenitor cells), oval-shaped cells with an average diameter of (7) showing large oval nuclei, a high nuclear cytoplasmic ratio and marginated chromatin, (intermediate hepatocyte-like cells), and lastly cells with an average diameter of (14) having few mitochondria and endoplasmic reticulum, looking larger and more mature than the former cells and resembling mature hepatocytes, but at the same time being smaller and less mature than the normal hepatocytes, (premature hepatocytes). The intermediate hepatocyte-like cells and the larger premature hepatocytes were more commonly seen in grids screened from this group. Such cells were seen in between inflammatory cells at the periphery of granulomas, at the sinusoidal poles of hepatocytes, and also in periportal areas. Similar cells sharing
479
the same morphological criteria of the previously mentioned immature cells were also seen in liver sections from (group 3), the normal transplanted group of mice. In this group however, they were mainly composed of the first category of small oval-shaped cells (the progenitor cells). Apart from the above noted pathological changes, most of the surrounding hepatocytes of the hepatic lobule in (groups 1, 2 & 3) revealed the normal ultrastructure of murine liver as presented in the normal non transplanted control group of mice (group 4). Hepatocytes measured (20-30) across with their
prominent nuclei having an average diameter of (10), showing finely dispersed chromatin condensed slightly around the nuclear envelope, and containing a large nucleolus. Binucleation was seen in some of the hepatocytes. The cytoplasm of hepatocytes was packed with organelles, including lots of mitochondria, rough and smooth endoplasmic reticulum, and Golgi complexes, which implies the active indulgement of the hepatocyte in protein synthesis. The sinusoids were clearly identified as wide vascular channels lined by endothelial cells.
Fig. (2-A)
Fig. (2-B)
Fig. (2-C)
Fig. (2-D)
480
Fig. (2-E)
Fig. (2-F)
Fig. (2): (A): Electron micrograph of a schistosomal egg shell (Sh) along with an eosinophil (E) showing the characteristic coffee-bean granules (arrows) and an adjacent fibroblast (F) as a part of fibrocellular granuloma. X6000. (B): Electron micrograph showing part of fibrocellular granuloma. A plasma cell is identified by the characteristic cart-wheel appearance (arrow) of its nucleus (N) and Rer arranged in a circular configurations. x7500. (C): Electron micrograph displaying schistosoma infected transplanted mouse liver with damage to a bile canaliculus in the form of partial loss of its microvilli (arrow). There are adjacent hepatocytes showing cytoplasm full of mitochondria (Mt) x10000. (D): Electron micrograph showing small progenitor cell (Pr) allocated at the sinusoidal surface of a hepatocyte, and an intermediate oval hepatocyte like cells (Int) with an oval shaped nucleus and more condensed heterochromatin than euchromatin, along with inflammatory cells, Neutrophil (N) with trilobed nucleus (arrows) and macrophage (M). X2000. (E): Electron micrograph from infected transplanted group showing an intermediate hepatocyte like oval shaped cell (Int). The N/C ratio is high with the nucleus (N). Few mitochondria (mt) can be spotted in its cytoplasm. X4500. (F): Electron micrograph showing premature hepatocytes (P) smaller in size than another adjacent large mature hepatocyte (M). The larger hepatocyte revelae signs of regeneration in the form of (irregularly nuclear membrane (thin arrow). Multiple nucleoli (n), and increased number of increased number of organelles e.g. mitochondria (thick arrow) x5000.
Discussion Hepatic fibrosis occurs in the setting of chronic liver diseases of different etiologies. Spontaneous regression of fibrosis occurs when the stimulus for hepatic liver damage is removed, and in some diseases the stimulus cannot be completely eradicated, in addition to being a slow process [22] . Thus, it is of great interest to explore new effective therapeutic techniques in an attempt to ameliorate the damages caused by fibrogenesis in chronic liver diseases. Lorenzini et al. [16] stated that in this scenario, stem cell therapy sounds particularly attractive for its potential to support tissue regeneration, requiring minimally invasive procedures with few complications. BM hematopoietic stem cells for long have been clinically introduced in regenerating many diseased organs including the liver, but UCB hematopoietic and mesenchymal stem cells are now newly proposed in the field of regenerative medi-
cine as a better and less invasive way to be used as a substitute for other types of stem cells [15]. This research intended to demonstrate the fate of human UCBHSCs after inoculation into murine diseased liver, in an attempt to set an experimental model of in vivo hepatocyte differentiation from stem cells. In this case, chronic schistosomiasis was selected as an example of liver disease. Schistosomiasis has been studied extensively since its discovery by Theodore Bilharz in 1851 [1]. HCV infection along with schistosomiasis are two major prevalent chronic debilitating diseases that threaten the Egyptian population [7,9]. In the present study, histopathological examination of the livers of Schistosoma infected and UCB transplanted mice, and the infected non transplanted mice revealed the typical picture of chronic hepatic schistosomiasis, in the form of fibrocellular and fibrous granulomas of variable shapes and sizes. Bile duct proliferation was noticed in the
481
liver sections as a consequence of schistosomal infection. It was though more prominent in the infected transplanted group, most probably due to the regenerative response induced by the inoculated stem cells. In addition, light microscopic examination of the H & E stained liver sections of the infected transplanted group showed identifiable eosinophilic cells in between the murine hepatocytic cells, present singly and in groups. These cells were also allocated at the periphery of the granulomas, in periportal areas, and around central veins. Similar cells were present in livers of the normal transplanted mice as well. Such results demonstrate the presence of distinctive non murine hepatocytes in the hepatic lobule of the stem cell transplanted mice. Using the anti human hepatocyte markers (Hep Par 1 and -fetoprotein), the livers of the infected transplanted group and the normal transplanted group showed cytoplasmic positivity of a number of cells. Negative staining for both used markers was clearly noticed in the liver sections of the infected non transplanted group (group 2), and the normal non transplanted group (group 4). This provides evidence of successful transplantation of the injected UCBHSCs in the livers of the experimented mice of (groups 1 & 3), and enhances the fact that these detected cells are of human origin. Though HepPar1 is considered an excellent marker of human hepatocyte lineage, it is known to be present, not only in fully differentiated human hepatocytes, but also in very early stages of liver development such as in human hepatoblasts [28]. Alpha fetoprotein is also considered to be an early hepatocytic marker [26], thus expression of such markers in our experiment might help in the allocation of premature hepatocytic cells that appear in liver tissue of mice after their intrahepatic transplantation with stem cells. In accordance with the above results, Newsome et al. [20] demonstrated the expression of the HepPar1 human hepatocyte-specific antigen in livers of NOD/SCID mice after inoculation with HUCBSCs, and concluded that cells from human cord blood become mature hepatocytes in livers of such mice. Likewise, Kogler et al. [14] transplanted adherently proliferating cells isolated from human cord blood into livers of fetal sheep. They observed the expression of albumin and human hepatocyte-specific antigen after transplantation, and concluded that the human cord blood cells differentiated into human parenchymal hepatic cells.
Ishikawa et al. [11] detected human albumin and the HepPar1 antigen in livers of immunodeficient mice, postulating that the engrafted cells from human cord blood functioned as hepatocytes. However, other scientists remained skeptical because they observed the expression of some, but not all hepatocyte markers that should be expressed by a real human hepatocyte [4]. In a study done by Akl et al. [2], transplantation with HUCBSCs in mice following their injury with CCL4, the inoculated undifferentiated cord blood cells proved homing into the livers with subsequent development of hepatocyte-like cells. In addition, a reduction in collagen deposition in livers of these mice was noticed. By electron microscopy, ultrathin sections of the livers of mice of both the infected transplanted and the infected non transplanted groups in the current experiment showed many fibrocellular and fibrous schistosomal granulomas. Sinusoidal spaces were seen congested with RBCs, and showed scattered hypertrophied kupffer cells with schistosomal pigment and primary and secondary lysosomes. This was perceived as a reaction to the Schistosomal infection. Regenerating hepatocytes were also present, having proliferated rER encircling the mitochondria, irregular nuclear membranes, and showing binucleation. Regenerative signs were otherwise more prominent in the infected transplanted group than in the infected non transplanted group of mice. Electron microscopic examination of the grids prepared from the transplanted groups of mice helped distinguish immature relatively small ovoid progenitor cells of average diameter (5), medium sized intermediate hepatocyte-like oval cells of average diameter (7) showing large oval nuclei, a high nuclear cytoplasmic ratio and marginated chromatin, and larger premature hepatocytic cells with an average diameter of (14). These large cells had few cytoplasmic organelles in the form of mitochondria and endoplasmic reticulum, approaching the morphological characteristics of mature hepatocytes. The above mentioned different forms of cells were situated mostly at the sinusoidal surface of hepatocytes. In accordance with the present results, Sell [23] previously described proliferation of small null intraportal cells that sequentially acquired markers of differentiation to hepatocytes after the acute form of allyl-alcohol liver injury. He noted that the progeny of these cells then extended across the necrotic zone, and acquired differentiation markers that reflected markers seen during maturation of
482
the fetal liver. His examination of these cells by electron microscopy revealed three cell types similar to those found in chronic ductular reactions and focal nodular hyperplasia of human liver which he named: Type I, small immature or progenitor cells; type II, bile duct-like progenitor cells; and type III, hepatocyte-like progenitor cells. Sell concluded that liver stem cells differentiate through stages of restitutive and transitional hepatocytes before developing the appearance of mature hepatocytes. Sell [24] stated that the terms oval cell, transitional hepatocyte, atypical ductular cell, neocholangiole, etc., are used to describe the previous cells. He also reported the presence of redundant proliferating bile ducts in zones of liver injury which we previously mentioned in our study by light microscopic examination of the specimens of the infected transplanted group of mice. The results of the present work suggest that the transplantation of cord blood stem cells in the livers of experimented mice has contributed in some way to the generation of premature forms of hepatocytic cells on their way to differentiate into mature hepatocytes. This proves that HUCBSCs can give rise to cells of hepatic lineage. In agreement with the results obtained in this work, Sobaniec-Lotowska et al. [27] detected two types of oval cells in liver biopsies from children with chronic hepatitis B; hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis, and in the vicinity of the limiting plate of the lobule. There was a distinct relationship between the prevalence of these oval cells and fibrosis stage. Hepatic progenitor cells were small (usually not exceeding 5 microns) and oval or nearly oval in shape. Intermediate hepatocyte-like cells varied in size, and were twice as large as the hepatic progenitor cells, whereas their diameter did not exceed one-half of the diameter of the mature hepatocytes. The nuclei were less abundant in heterochromatin compared to the progenitor cell nuclei, and occasionally contained nucleoli. Frequently, these nuclei resembled the nuclei of mature hepatocytes. The intermediate hepatocyte-like cell cytoplasm contained better developed cell organelles, mainly mitochondria and elements of the endoplasmic reticulum. In a study done by Xiong et al. [31], investigating cases of chronic viral hepatitis, similar hepatic oval cells were allocated predominantly in the periportal region and fibrosis septa, characterized by an ovoid nucleus, small size, and scant cytoplasm.
These results showed that UCB and BM cells cultured with the combination of FGF-1, FGF-2, SCF, and HGF were capable of producing hepatocyte lineage cells in vitro. Thus human UCB as well as BM cells proved their ability to generate hepatoblasts in an in vitro culture system in a proportion noted to be about (13%) in UCB and (23%) in BM according to the CK-18 expression. They concluded that UCB cells as well as BM cells differentiated into mature hepatocytes via the hepatic progenitor cells in the primary culture system. For clinical application, it is necessary to refine the methods to expand and isolate the generated hepatoblasts as well as to determine the specific hepatoblast markers [13]. The results of the current research showed that the intermediate-sized oval cells and premature hepatocytes constituted the dominant forms of newly formed cells observed in the grids of the infected transplanted group. It is explainable that the immature cells allocated here are mostly of the more differentiated type, since the mice in this experiment were sacrificed 3 weeks after transplantation. Thus, the stem cells here had plenty of time to develop and differentiate, in contrary to other reported studies where the sacrifice of mice was performed hours after the acute liver injury [23,24]. In the normal transplanted group however, light and electron microscopic examination highlighted that the relatively small ovoid progenitor hepatocytic cells were present more obviously than the medium sized intermediate oval cells and the larger premature hepatocytes. An explanation could be that the inoculated stem cells have successfully engrafted into the murine hepatic lobule, but there was no triggering infectious stimulus to motivate these cells to differentiate into the larger more mature forms, leaving the small progenitor cells as the mainly recognized type of cells. Transplantation of UCBHSCs into the livers of non irradiated mice infected with S.mansoni cercariae was carried out in this study, without prior immunosuppression therapy. Non-ablated immunecompetent Swiss Albino mice were sacrificed 3 weeks after their intrahepatic infusion with the stem cells, and demonstration of the actual transplantation of UCBHSCs followed by the study of their influence on the liver was carried out by utilizing both light and electron microscopy. This study demonstrated that the human-derived stem cells successfully engrafted into the mouse livers that were not previously subjected to immunosuppression.
483
[21]
It was previously reported by Nilsson et al. as well as Weiss et al. [30] that stem cells could survive till 6 weeks after transplantation into either the brain or the peripheral blood without the need for immunosuppression of the host. Similarly, Weiss et al. [30] transplanted porcine stem cells derived from umbilical cord mucous connective tissue into rat brains without immunosuppression [8]. This fact that hematopoietic stem cells were able to overcome host immunosurveillance, thus avoiding problems of immune rejection, could be an important advantage of the stem cell based therapy in comparison with whole organ transplantation [8]. Induction of tolerance, as another alternative to immunosuppression, has been suggested and explored by other authors. It is generally well established that auto-reactive T cell clones are completely deleted from the immune repertoire during fetal development. Indeed rats, intrafetally tolerated against human liver antigens, accept xenotransplantation with hepatocytes coming from the same donor [28]. In conclusion, the transplanted UCBHSCs in this current study proved engraftment into the livers of mice. However, to what extent can these cells repopulate a damaged liver is still under investigation. The stage of schistosomiasis, and the time interval between their infection, their transplantation with stem cells, and their sacrifice might be important variables affecting different experimental models used by different researchers. Establishment of a suitable model to study the extent of adult extrahepatic stem cell contribution to hepatic regeneration was felt mandatory. Mouse infected with schistosomiasis was chosen and was considered a suitable option for this study. Schistosomiasis was selected, being a disease that causes less complex architectural changes compared to known forms of liver cirrhosis [6]. There are several potential advantages of using adult rather than embryonic stem cells to regenerate tissues including fewer ethical concerns, better known biological behaviour, easier accessibility and, therefore, lower costs. Cord blood contains multiple populations of embryonic-like and other pluripotent stem cells capable of originating hematopoietic, epithelial, endothelial, and neural tissues both in vitro and in vivo. The isolation of HSCs and MSCs from cord blood is a relatively new procedure and only few studies have been published [16].
It would be helpful to use immuno-electron microscopy techniques in such studies. This helps to obtain a more precise evaluation of the source of the identified immature hepatocytic cells. Such technique might clarify whether the immature hepatocyte-like cells that appeared on electron microscopic examination were the product of the transplanted human umbilical cord blood hematopoietic stem cells, or the mobilized bone marrow hematopoietic stem cells from the mouse itself. Although anti human hepatocyte (Hep Par 1 and -fetoprotein) marker positivity claims the actual transplantation of human cord blood stem cell in this study, this positivity could be quantitatively and more objectively traced more accurately by immuno-electron microscopy. A broader understanding of the biological properties and use of extra-hepatic adult stem cells is required before proceeding into clinical applications. The possible carcinogenic potential of former types of stem cells should be investigated. We concluded that the engraftment of the human umbilical cord blood hematopoietic stem cells after their intrahepatic transplantation into the livers of mice suffering from chronic hepatic schistosomiasis, and their attempt to give rise to premature forms of cells with hepatocytic lineage. Extensive studies are still needed to clarify the possible utility of these cells in resolving damaged organs and tissues. References
1- ABDUL-GHANI R.A. and HASSAN A.A.: Murine schistosomiasis as a model for human schistosomiasis mansoni: Similarities and discrepancies. Parasitology Research Journal, 1432-1955, 2010. 2- AKL M., HAMMAM O., MAHMOUD S. and EL FANDY G.: Pathology of Hepatocytic differentiation from umbilical cord blood in injured livers by CCL4 mice, in 2009 World Stem Cell Summit-Science and Medicine, Poster #68, 2009. 3- BALLEN K.K.: New trends in umbilical cord blood transplantation. Blood, 105 (10): 3786-3792, 2005. 4- BRULPORT M., SCHORMANN W., BAUER A., HERMES M., ELSNER C., et al.: Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers. Hepatology, 46: 861-870, 2007. 5- BUNTING K.D. and HAWELY R.G.: The hematopoietic stem cells: Toward a defined theory of tissue regeneration. Scientific World Journal, 10, 2 (4): 983-995, 2002. MULO 6- COELHO P.M. A,B, ANDRADE Z.A. A,B, RO TEIXEIRA DE MELLOC, COSTA G.D., FlVIA SLVIA ES GUIMARA DIASE, et al.: Post-hepatectomy regeneration of the murine liver: I. Effect upon Schistosoma mansoni lesions, before and after chemotherapy. Acta. Tropica, 108: 104-108, 2008. 7- EL-AWADY M.K., YOUSSEF S.S. and OMRAN M.H.: Soluble egg antigen of Schistosoma Haematobium induces
484
HCV replication in PBMC from patients with chronic HCV infection. BMC Infectious Diseases, 6: 91, 2006. derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology, 124 (7): 1891-1900, 2003. 21- NILSSON S.K., DOONER M.S., WEIER H.U., FRENKEL B., LIAN J.B., et al.: Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice. Journal of Experimental Medicine, 189: 729-734, 1999. 22- OLIVEIRA S.A., SOUZA B.S.F., GUIMARESFERREIRA C.A., BARRETO E.S., SOUZA S.C., et al.: Therapy with bone marrow cells reduces liver alterations in mice chronically infected by Schistosoma mansoni. The World Journal of Gastroenterology, 14 (38): 58425850, 2008. 23- SELL S.: Electron microscopic identification of putative liver stem cells and intermediate hepatocytes following periportal necrosis induced in rats by allyl alcohol. Stem Cells, 15: 378-385, 1997. 24- SELL S.: Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology, 33: 738-750, 2001. 25- SIEFF C.A.: Overview of hematopoiesis and stem cell function. Up To Date for Patients, 16: 3, 2008. 26- SNYKERS S., VANHAECKE T., PAPELEU P., LUTTUN A., JIANG Y., et al.: Sequential exposure to cytokines reflecting embryogenesis: The Key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells, highlighted article, Toxicological Sciences, 94 (2): 330-341, 2006. 27- SOBANIEC-LOTOWSKA M.E., LOTOWSKA J.M. and LEBENSZTEJN D.M.: Ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on the stage of liver fibrosis: The first pediatric study. The World Journal of Gastroenterology, 13 (21): 29182922, 2007. 28- TURRINI P., MONEGO G., GONZALEZ J., CICUZZA S., BONANNO G., et al.: Human hepatocytes in mice receiving pre-immune injection with human cord blood cells. Biochemical and Biophysical Research Communications, 326 (1): 66-73, 2004. 29- WANG X., CROOKS G.M. and NOLTA J.A.: Albumin expressing hepatocyte like cells develop in the livers of immune deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood, 101 (10): 4201-4208, 2003. 30- WEISS M.L., MITCHELL K.E., HIX J.E., MEDICETTY S., EL-ZARKOUN S.Z., et al.: Transplantation of porcine umbilical cord matrix cells into the rat brain. Experimental Neurology, 182 (2): 288-299, 2003. 31- XIONG MA, DE KAI QIU and YAN SHEN PENG: Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis. The World Journal of Gastroenterology, 7 (2): 238-242, 2001.
8- ELKHAFIF N., YEHIA H., HAMMAM O., MAHMOUD S., HELMY A., et al.: Ultrastructural hepatic changes after bone marrow transplantation in murine schistosomiasis. Kasr El Ainy Medical Journal, 12 (1): 119-124, 2006. 9- HALIM A.B., GARRY R.F., DASH S. and GERBER M.A.: Effect of schistosomiasis and hepatitis on liver disease. American Journal of Tropical Medicine and Hygiene, 60 (6): 915-920, 1999. 10- HSU S.M., RAINE L. and FANGER H.: Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry, 29 (4): 577-580, 1981. 11- ISHIKAWA F., DRAKE C.J., YANG S., FLEMING P., MINAMIGUCHI H., et al.: Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Annals of the New York Academy of Sciences, 996: 174185, 2003. 12- JARED W.A. and SANGEETA N.B.: Engineering liver therapies for the future. Tissue Engineering, 8 (5): 725737, 2008. 13- JUNG Y., CHO K., WOO S. and SEOH J.: In vitro hepatic differentiation of human umbilical cord blood and bone marrow cells. Pediatric Hematology and Oncology, 25: 481-491, 2008. 14- KOGLER G., SENSKEN S., AIREY J.A., TRAPP T., MUSCHEN M., et al.: A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. The Journal of Experimental Medicine, 2: 123-135, 2004. 15- LEE O.K., KUO T.K., CHEN W., LEE K., HSIEH S., et al.: Hematopoiesis: Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood, 103 (5): 1669-1675, 2004. 16- LORENZINI S., GITTO S., GRANDINI E., ANDREONE P. and BERNARDI M.: Stem cells for end stage liver disease: How far have we got? The World Journal of Gastroenterology, 14 (29): 4593-4599, 2008. 17- LUBIN B.H. and GREENE M.F.: Collection and storage of umbilical cord blood for hematopoietic cell transplantation. Up to date, Version, 3 (16), 2008. 18- MAYANI H. and LANSDORP P.M.: Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cell, 16 (3): 153-165, 1998. 19- NEWCOMB J.D., SANBERG P.R., KLASKO S.K. and WILLING A.E.: Umbilical cord blood research: Current and future perspectives. Cell Transplant, 16: 151-158, 2007. 20- NEWSOME P.N., JOHANNESSEN I., BOYLE S., DALAKAS E., MCAULAY K.A., et al.: Human cord blood-
Impact of Intrauterine Growth Restriction on Neonatal Frontal Lobe Dimensions (Transcranial Sonographic Measurments)
EMAN A. AHMED, M.D.* and NAFISA H.R. ABD EL-AZIZ, M.D.**
The Departments of Diagnostic Imaging* and Pediatrics**, Assuit University Hospital
Abstract
Assessing the impact of restricted intrauterine growth on neonatal frontal lobe (FL) dimensions is important. Children with intrauterine growth restriction were found to have lower intelligence quotient, more frequent neuropsychological difficulties, difficulties in executive functioning, inflexible creativity, and language, indicative of frontal lobe dysfunction. This study aims to evaluate the impact of intrauterine growth restriction and small head circumference (HC) on neonatal frontal lobe dimensions. We measured frontal lobe dimensions at birth by using a recently published ultrasonic technique. We included 92 newborn infants born at GA of 37-41 WKS: (42) appropriate for GA and normal HC, and [50] small for GA and small HC. Infants with a 5-min Apgar score <7, severe congenital malformations, or chromosomal abnormalities were excluded. Through a coronal ultrasound scan via the anterior fontanel at the level where the most lateral point of the Sylvian fissure was best demonstrated, we drew a triangle connecting the most lateral point of the Sylvian fissure, the corpus callosum, and the subcalvarian point of the interhemispheric fissure. We measured the three sides of the triangle, Sylvian-fontanellar distance (SFD), Sylvian-callosal distance (SCD), and fontanellar-callosal distance (FCD), and calculated the frontal triangular area (FTA). The SGA neonates in our study had significantly smaller FL dimensions (SFD, SCD, FCD, and FTA) than their AGA mates. All four FL dimensions were strongly correlated with HC and birth weight in both AGA and SGA groups. It is concluded that IUGR has a great impact on FL dimensions which could have a considerable Abbreviations: AGA : Appropriate for gestational age. SGA : Small for gestational age. BW : Birth weight. FCD : Fontanellar-callosal distance. FL : Frontal lobe. FTA : Frontal triangular area. GA : Gestational age. HC : Head circumference. IUGR: Intrauterine growth restriction. MRI : Magnetic resonance imaging. SCD : Sylvian-callosal distance. SFD : Sylvian-fontaneller distance. US : Ultrasonography. Correspondence to: Dr. Eman A. Ahmed, The Department of Diagnostic Imaging, Assuit University Hospital.
effect on neurodevelopment of those neonates. US assessment proved to be simple, fast and reproducible in this situation. Key Words: Transcranial sonographic measurments Frontal lobe Intrauterine growth restriction.
Introduction STUDIES have shown that fetuses whose growth in utero is restricted have developed in an adverse environment, deficient in essential nutrients. Compensatory processes trigger circulatory changes and neural adaptive modifications aimed at conserving the developing brain [1]. However, these sparing processes may not suffice to fully compensate for the aberrant cellular successions and the altered neurodevelopmental route associated with it. Brodsky and Christon [2] have reported that children with intrauterine growth restriction had lower intelligence quotient and more frequent neuropsychological difficulties. Difficulties in executive functioning, inflexible creativity, and language, indicative of frontal lobe dysfunction, were typically affected by intrauterine growth restriction [2]. The FL constitutes approximately one third of the cerebral hemisphere [3]. Previous US studies have shown that FL hypoplasia and microcephaly in the fetus are very much interrelated [4,5], and that FL hypoplasia in the fetus might cause delayed growth of the fetal head and could result in microcephaly and mental retardation [6]. Recent study by Serralde et al., [7] showed that IUGR fetuses show difference in the volume of intracranial structures compared with AGA fetuses, with the largest difference found in frontal region. These differences might be explained by in-utero processes of neural reorganization induced by chronic hypoxia [7] . Furthermore a small FL on fetal US has been suggested as a marker for fetal trisomy 21 [8] .
485
486
Impact of Intrauterine Growth Restriction on Neonatal Frontal Lobe Dimensions
Ultrasonographic studies of the fetal head (axial scans) were based on three different measurements for the estimation of the fetal FL size: 1- From the inner calvaria to posterior cavum septum pellucidum [9]; 2- Frontothalamic distance, i.e. from the frontal bone to posterior edge of thalamus [4,8]; and 3- FL distance, i.e. from the anterior edge of the frontal horns of the lateral ventricles to the frontal bone [10]. More recently, Makhoul et al. [11]; developed a novel model for US biometry of the frontal lobe and established a nomogram of neonatal FL dimensions measurment. Aim of the study: Is to assess the frontal lobe dimensions for the normal and growth restricted neonates and to evaluate the impact of intrauterine growth restriction on neonatal frontal lobe dimensions. Patients and Methods A total of 92 neonates who were hospitalized in the neonatal intensive care unit for more than 24 hours were enrolled in this prospective study. They were born between 37 and 41 wk of gestation
at Assuit University Children's Hospital. Infants with a 5min Apgar score <7, severe congenital malformations, or chromosomal abnormalities were excluded. GA was calculated based on the date of the last menstrual period, early first trimester US data when available. Serial US scans of the neonatal brain for ruling out hemorrhage are routine procedures for all low birth weight infants hospitalized in our neonatal intensive care unit, therefore obtaining parental informed consent was not necessary. Out of the studied neonates, 42 were AGA and 50 were SGA. AGA status was defined as birth weight between the 10th and 97th percentiles [12]. SGA was defined as birth weight less than 10 th percentile or greater than 2 standard deviation below mean for gestational age [12]. HC was measured after the first 24h of life to minimize the impact of cranial subcutaneous edema, often seen after birth on HC measurement. HC and length were measured with non stretchable tape, weight was measured with electronic scale. Table (1) demonstrates the demographic and perinatal characteristics of the study population.
Table (1): Demographic data for both SGA and AGA groups. Item 1- Gestational age: (WKS mean SD) Range 2- Birth weight (KG): Mean SD Range 3- Length (cm): MeanSD Range 4- BMI (Kg/m): Mean SD Range 5- Head circumference (cm): Mean SD Range 6- Sex: Male Female 7- Age at examination: Mean SD Range Group1 SGA (n=50) 38.901.129 (37-41) 1.780.399 (0.994-2.37) 41.303.29 (35.o-49.00) 10.351.53 (7.09-13.93) 31.072.18 (24.0-34.0) 27(54%) 23(46%) 2.900.614 (2.0-4.0) Group2 AGA (n=42) 39.291.019 (37-41) 3.060.34 (2.58-3.95) 48.732.71 (44.0-54.0) 12.971.78 (9.50-17.48) 35.251.255 (32.0-38.0) 27(64.3%) 15(35.7%) 2,960.68 (1.00-4.00)
FL measurements: Transcranial US examinations were performed by each auther alone and the mean value of the two readings were recorded. We used a Digital Sonoace 5500 (Medison) equipped with a 5-to 7-MHz linear transducer. Between one
and 4 days of life, we scanned the FL in each neonate. There was no need for special preparation of the newborn, the examination was carried with neonate in the supine position and his head facing the examiner's head. A coronal scan was obtained
Eman A. Ahmed & Nafisa H.R. Abd El-Aziz
487
via the anterior fontanelle at the level where the most lateral point of the Sylvian fissure was best demonstrated. Using electronic calipers we drew the same triangle described by Makhoul et al., [11] in their US model, connecting the most lateral point of the Sylvian fissure, the corpus callosum where it cross-
es the interhemispheric fissure, and the subcalvarian point of the interhemispheric fissure (hereafter, fontanelle) and measured its three sides: SFD (a), SCD (b), and FCD (c), and then calculated the FTA: FTA = 0.25 (2a2b2+2b2c2+2a2c2+a4-b4-c4) Where a, b, and c represent the sides of the triangle (Figs. 1,2).
Fig. (1): Transcranial US, coronal scan measurement of frontal lobe dimensions in a newborn, AGA: SFD=4.2cm, SCD=5.7cm and FCD=3.2cm.
Fig. (2): Transcranial US, coronal scan measurement of frontal lobe dimensions in a newborn, SGA: SFD=3.4cm, SCD=4.6cm and FCD=2.1cm.
Statistical analysis: Data collected and analyzed using the computer program SPSS version 12. Data described as mean SD, number and percentage. Student t-test was used to determine significance for numeric variables. Pearson correlation was used to determine significance between variables in each group. N.S: p>0.05 is non significant, p<0.05 is significant, p<0.01 is moderately significant, p<0.001 is highly significant. Results The SGA neonates in our study had significantly smaller SFD, SCD, FCD, and FTA than their AGA mates (Table 2). Among SGA newborns, significant positive correlations were observed between BW and each of SFD, SCD and FCD (Fig. 3), and FTA (Fig. 4). Similarly significant positive correlations were detected between HC and each of SFD, SCD and FCD (Fig. 5) and FTA (Fig. 6). Similar observations were found among AGA newborns where significant positive correlations were observed between birth weight and each of SFD, SCD and FCD (Fig. 7), and FTA (Fig. 8). Similarly significant positive correlations were detected between HC and each of SFD, SCD and FCD (Fig. 9) and FTA (Fig. 10).
6 5 4 (cm) 3 2 1 0 0
SFD
SCD * FCD
* * ** **** * * * ** * ** * *** * *** ***** * ** ****** * * * * * * * *
0.5
1 1.5 Weight (kg)
2.5
SFD: r=0.634, p<0.05* SCD: r=0.636, p<0.000***
FCD: r=0.467, p<0.001** FTA: r=0.347, p<0.05*
Fig. (3): Correlation between weight and SFD, SCD, FCD and in SGA group. 20 15 FTA (cm2) 10 5 0 0 0.5 1 1.5 Weight (kg) r=0.347 p<0.05*
2.5
Fig. (4): Correlation between weight and FTA in SGA group.
488
6 5 SCD (cm) 4 3 2 1

20 15 10
SFD
SCD * FCD
r=0.662 p<0.000***
** * **** ** ******** *** *** * ** *

0 10
5 0 5
20 30 40 Hc (cm) Fig. (5): Correlation between head circumference and SFD, SCD, FCD and in SGA group. 6 5
10 15 20 25 30 35 Head circumference (cm) Fig. (6): Correlation between head circumference and FTA in SGA group. 7 r=0.195 6 p<0.05* 5 FTA (cm2)
SFD
SCD * FCD
4 3 2 1 2 3 4 Weight (kg) Fig. (7): Correlation between weight and SFD, SCD, FCD in AGA group. 6 5 0 0 1
* * ** * * * **** *** * * * ** * ** * * **** **
(cm)
4 3 2 1 0
2 3 4 Weight (kg) Fig. (8): Correlation between weight and FTA in AGA group.
SFD
SCD * FCD
7 6 5 FTA (cm2)
r=0.0108 p<0.05*
4 (cm) 3 2 1 10 20 30 40 Head circumference (cm) Fig. (9): Correlation between head circumference and SFD, SCD, FCD in AGA group. 0 0
**** ** * *** * * * * * * * ** ** * **
4 3 2 1 0
10 20 30 40 Head circumference (cm) Fig. (10): Correlation between head circumference and FTA in AGA group.
Table (2): The mean values of frontal lobe US dimensions (SFD, SCD, FCD, FTA) among both the SGA and the AGA groups. Item SFD SCD FCD FTA Group1 SGA (n=50) 4.480.29 3.950.27 2.090.211 4.212.72 Group2 AGA (n=42) 5.160.334 5.020.261 2.500.27 4.750.78 p-value p<0.001*** p<0.001*** p<0.001*** p<0.05
Eman A. Ahmed & Nafisa H.R. Abd El-Aziz
489
Discussion Results of the present study revealed that the dimensions of the FL (SFD, SCD, FCD, and FTA), as measured by US after birth, strongly correlated with BW and HC in both AGA and SGA neonates. The SGA neonates had significantly smaller SFD, SCD, FCD, and FTA than their AGA mates (Table 2). These findings are consistent with Makhoul, et al., [11] who found that the frontal lobe measures in the AGA group differed significantly from those of SGA group and they speculated that a sonographically small fetal HC implies growth restriction of the fetal frontal lobe. The specificity of the IUGR process to frontal lobe may be validated in view of Makhoul, et al. [13] reports that showed that frontal lobe measures increased significantly between 24 and 43 weeks of gestation (weeks at which IUGR can peak) and that fetuses who experienced severe IUGR commencing before 32wk gestation already had growth-restricted FL. On the other hands, Makhoul, et al. [13] reported that 60.7% of the SGA-small HC neonates had a preserved cerebellar width at birth, So contrary to the cerebellum, FL appears to be more affected by slowed head growth in the fetus. Frisk et al. [14] evaluated the effect of head growth compromise beginning in utero, the authors reported that SGA children with poor prenatal and postnatal head growth had the worst outcomes, followed by those with prenatal brain compromise, but good postnatal head growth. SGA children with preserved head growth in utero as well as good head growth after birth demonstrated the best outcomes although spelling skills were deficient relative to full term peers. They concluded that IUGR that slows brain growth in utero impairs the acquisition of some cognitive and academic abilities, even when followed by good catch up head growth after birth where as poor brain growth in utero followed by little or no catch up head growth results in wide spread impairments. These findings highlight the limits to brain plasticity and emphasize the importance of optimal prenatal and postnatal brain growth. Ronny, et al. [15] , showed that the prenatal growth restriction resulted in specific behavioral symptoms that correspond with malfunctioning of frontal structures. The specific neuropsychological profiles found at 9 years of age indicated that late onset IUGR compromises frontal network functioning. Findings reaffirm that functional coherence depends on preestablished structural growth and recognization of the CNS. Similar data were reported by Jellife, et al. [16] who followed-up infants
born full term, SGA at 8 months and 4 years and found them to have significantly increased risk for neurodevelopmental difficulties. On a longer study (26 years follow-up), Strauss, that adults who were born SGA had significant differences in academic achievement and professional attainment compared with adults who were AGA. However, there were no long term social or emotional consequences of being SGA.
[17] concluded
Conforming with the fact that the FL comprises about one third of the cerebral hemisphere, SGAneonates have smaller FL than their AGA mates, and a decrease in FL size will lead to a reduction of HC. Imaging options that can be used for FL measurements includes US [11,18], three dimentional US [7], quantitative MRI [3,19], three-dimensional MRI [20], and computed tomography of the brain that measures the frontal force index for evaluation of the development of the frontal lobe [6]. Although MRI of the brain might offer some advantages over US in terms of better assessment of FL volume and white matter thickness, MRI is impractical as a bedside screening modality in neonatal units and has limiting factors including economic aspects and availability. US is rapid, inexpensive, convenient and readily available for monitoring at bed side with easily movable equipment. Our observations regarding the smaller FL dimensions of the SGA neonates are in agreement with Serralde, et al. [7] reports on delayed growth of the FL in fetuses with severe IUGR and microcephaly. Other conditions associated with a small or slow-growing FL in infants and children include trisomy 21 [4,8], epilepsy [3], and malnutrition [20]. The FL dimensions correlated strongly with HC, and it appears that measurement of HC after birth reliably reflects the size of the FL in neonates, independent whether the third or the 10th percentile of HC was used as a cutoff line between normal HC and small HC. Our method for US assessment of the neonatal FL proved to be simple, fast, and reproducible, although it is practical only as long as the anterior fontanel is sufficiently open. When the US coronal plane is scanned through the anterior fontanel, the Sylvian, callosal, and frontal landmarks are easily identified. Our data may be of help for perinatologists and neonatologists when they are confronted with severe fetal IUGR and small head, or with an SGAsmall HC neonate. In both conditions, parents counseling should include information regarding small HC, small FL, and the possibility of an increased risk for future neuro-intellectual sequelae.
490

intracranial structures in growth restricted and appropriate -for- gestational age fetuses. Ultrasound in Obstetrics and Gynecology, 33: 530-7, 2009. 8- WINTER T.C., REICHMAN J.A., LUNA J.A., CHENG E.Y., DOLL A.M., KOMARNISKI C.A., NGHIEM H.V., SCHMIEDL U.P., SHIELDS L.E. and UHRICH S.B.: Frontal lobe shortening in second-trimester fetuses with trisomy 21: Usefulness as a US marker. Radiology, 207: 215-22, 1998. 9- PERSUTTE W.H., COURY A. and HOBBINS J.C.: Correlation of fetal frontal lobe and transcerebellar distance measurements: The utility of a new prenatal sonographic technique. Ultrasound Obstet. Gynecol., 10: 94-7, 1997. 10- GOLDSTEIN I., REECE E.A., PILU G., O'CONNOR T.Z., LOCKWOOD C.J. and HOBBINS J.C.: Sonographic assessment of the fetal frontal lobe: A potential tool for prenatal diagnosis of microcephaly. Am. J. Obstet. Gynecol., 158: 1057-62, 1988. 11- MAKHOUL I.R., SOUDAK M., GOLDSTEIN I., SMOLKEN T., TAMIR A. and SOUJOV P.: Sonographic biometry of the frontal lobe in normal and growth-restricted neanates. Pediatr. Res., 55: 877-83, 2004. 12- GOMELLA T.L., CUNNINGHAM M.D., EYAL F.G. and TUTTLE D.: Neonatology, Management, Prcedures, On-call problems, Diseases, and Drugs, Sixth edition, 97: 558-68, 2009. 13- MAKHOUL I.R., GOLDSTEIN I., TAMIR A., EPELMAN M., REECE A.E. and SUJOV P.: Transverse cerebellar distance in normal and growth restricted neonates. J. Matern. Fetal Med., 9: 155-60, 2000. 14- FRISK V., AMSEL R. and WHYTE H.E.: The importance of head growth patterns in predicting the cognitive abilities and literacy skills of small-for-gestational-age children. Dev. Neuropsychol., 22: 565-93, 2002. 15- RONNY GEFA P.H.D., RINA ESHHEL P.H.D., YAEL LEITNER M.D., AVIVA FATTAL VALEVSKI M.D., and SHAUL HARE: Neuropsychological Outcome of Children with Intrauterine Growth Restriction: A9-Year Prospective study Pediatrics, 118: 91-100, 2006. 16- JELLIFFE-PAWLOWSKI L.L. and HANSEN R.L.: Neurodevelopmental outcome at 8 months and 4 years infants born full-term small for gestational age J. Perinatology, 24: 505-14, 2004. 17- STRAUSS R.S.: Adult functional outcome of those born small for gestational age: Twenty-six-year follow-up of the British Birth Cohort. JAMA. 285 (5): 625-32, 2000. 18- GOMES N.F.P., ENRIQUES G., ROJAS R.A., PERAPOCH J., ANDRADE E.H. and GRATACOS E.: Prevelance of neonatal ultrasound brain lesions in premature infants with and without intrauterine growth restriction. Acta. Pediatrica, 96: 1582-7, 2007. 19- HARVEY I., RON M.A., DU BOULAY G., WICKS D., LEWIS S.W., MURRAY R.M.: Reduction of cortical volume in schizophrenia on magnetic resonance imaging. Psychol. Med., 23: 591-604, 1993. 20- KANEMURA H., AIHARA M. and NAKAZAWA S.: Measurements of the frontal and prefrontal lobe volumes by three dimensional magnetic resonance imaging scanIII. Analysis of sex differences with advanced age. No to Hattatsu, 34: 404-8, 2002.
Moreover, a small head, i.e. small FL, in a thirdtrimester fetus with severe IUGR poses a dilemma whether lengthening the pregnancy benefit the fetus or not. Conclusions and Recommendations: US assessment of neonatal FL dimensions may prove to be a useful tool in assessing the relative effect of IUGR on structural neuroanatomy. This US assessment proved to be simple, fast and reproducible, although it is practical only as long as the anterior fontanel is sufficiently open. These FL measurements correlated strongly with birth weight and HC. Quantitative US assessment of the frontal lobe could be used as a useful adjunctive first line screening tool for those with IUGR, SGA, small HC and those with malnutrition in early life, aiding in devising a tailored intervention of this developmentally at risk population. Furthermore follow up of such newborns to assess the effect of small FL dimensions on future neurodevelopmental status. Early postnatal high energy nutrient intake for SGA is needed to promote HC catch up growth and prevent negative consequences of under-nutrition. US would be helpful in follow-up of the established cases of IUGR to assess the FL development. Further studies are needed to assess the vulnerability of other cerebral lobes to severe IUGR. References
1- BONNIN P., FOURON J.C., TEYSSIER G., SONESSON S.E. and SKOLL A.: Quantitative assessment of circulatory changes in the fetal aortic isthmus during progressive increase of resistance to umbilical blood flow. Circulation, 88: 216-22, 1993. 2- BRODSKY D. and CHRISTON H.: Current concepts in intrauterine growth restriction. J. Intens. Care Med., 19: 307-19, 2004. 3- LAWSON J.A., VOGRIN S., BLEASEL A.F., COOK M.J. and BYE A.M.: Cerebral and cerebellar volume reduction in children with intractable epilepsy. Epilepsia, 41: 1456-62, 2000. 4- WINTER T.C., OSTROVSKY A.A., KOMARNISKI C.A. and UHRICH S.B: Cerebellar and frontal lobe hypoplasia in fetuses with trisomy 21: Usefulness as combined US markers. Radiology, 214: 533-8, 2000. 5- PERSUTTE W.H.: Microcephaly-no small deal. Ultrasound Obstet. Gynecol., 11: 317-8, 1998. 6- HIRAIWA M., NONAKA C., SEKIYAMA M., MISHIMA M., KOBAYASHI M., ABE T., FUJII R. and YASUKOCHI H.: Changes in frontal lobe size with age. A CT study in children. Acta. Radiol. Suppl., 369: 680-2, 1986. 7- SERRALDE A.B., ANDRADE E.H., DELGADO J.F., PLASENCIA W., SCHEIIER M., CRSPI F., FIGUERAS F., NICOLAIDES K.H. and GRATACCOS E.: Three dimensional sonographic calculation of the volume of
Carotid Intima Media Thickness (CIMT) in Nondiabetic Patients with non Alcoholic Fatty Liver Disease (NAFLD)
NOUMAN H. ALGAREM, M.D.; MONA A. AMIN, M.D.; MAY M. FAWZI, M.D. and ASHRAF H. AIAD, M.Sc.
The Department of Internal Medicine, Faculty of Medicine, Cairo University
Abstract
Introduction: Nonalcoholic Fatty liver disease (NAFLD) is currently the most common cause of abnormal liver function tests observed in the hepatology practice. Given the strong association between NAFLD and the Metabolic syndrome risk factors, patients with NAFLD would be expected to have an increased risk of cardiovascular disease. Aim of Work: The aim of the present work was to compare carotid intima-media thickness measured by arterial carotid duplex in patients with NAFLD diagnosed by transabdominal ultrasound and normal control persons and to correlate it with the different risk factors of atherosclerosis namely the lipid pattern, blood glucose levels, C-reactive protein, body mass index and hypertension. Results: Our study revealed a significant increase in carotid IMT in patients with NAFLD (0.730.1mm) than normal control persons (0.500.08mm) which was highly significant (p value <0.001). There was an association between the increase in the liver enzymes levels and the increase in carotid IMT in patients with NAFLD which was highly significant (p value <0.001). Moreover NAFLD patients had higher cholesterol, LDL and CRP levels as compared to control with a statistically highly significant p value (p value <0.001). Conclusion: Our findings support the hypothesis that the presence of NAFLD is strongly associated with early carotid atherosclerosis. Key Words: NAFLD CIMT.
media thickness (IMT) which is a reliable index of early atherosclerosis [2-4]. As a result of these studies, researchers have focused their studies on the relationship between hepatic steatosis and atherosclerosis. Clarification of this aspect may help to explain the underlying mechanism and may be of clinical importance in planning prevention and therapeutic strategies [2]. But, this aspect has been thoroughly investigated in diabetics with hepatic steatosis. So, the aim of the present work was to compare the carotid intima-media thickness in nondiabetic patients with NAFLD to normal controls and to correlate it with the different risk factors of atherosclerosis namely lipid pattern, blood glucose levels, C-reactive protein, body mass index and hypertension. Patients and Methods The study was conducted on 60 persons (45 patients and 15 controls). Patients were categorized into two groups: Group I (study group): It comprised 45 nondiabetic patients who were found to have hepatic steatosis detected by transabdominal ultrasound, elevated liver enzymes, negative serology for hepatitis B. & C. and no history of alcohol or drug intake. They were 9 males and 36 females with mean age of 503 years. Group II (control group): It comprised 15 patients serving as a control group, they were selected from those patients' relatives. They were apparently healthy volunteers; they had normal hepatic echogenicity on abdominal ultrasound and normal liver function tests. They
Introduction NON-ALCOHOLIC fatty liver disease is a clinicpathological syndrome that is closely associated with visceral obesity, dyslipidemia, insulin resistance and type II diabetes, thus suggesting that NAFLD is another feature of the metabolic syndrome [1]. Recent cross-sectional studies have shown that NAFLD is associated with increased carotid intimaCorrespondence to: Dr. May M. Fawzi, The Department of Internal Medicine, Faculty of Medicine, Cairo University.
491
492
Carotid Intima Media Thickness (CIMT) in Nondiabetic Patients
were matched for age, sex and BMI with the study group. They were 3 males and 12 females with mean age of 514 years. All persons were subjected to the following: 1- Clinical evaluation by history taking and clinical examination. 2- BMI was calculated by dividing weight in kilograms by the square of height in meters. 3- Abdominal ultrasound: Real time Gray Scale abdominal ultrasonography was done with a Phillips HDI machine using a convex-sector probe (3.5Mhz). Patients were examined after at least 8 hours of fasting. Scanning of the liver was concerned with its echogenicity, size, attenuation of the ultrasound wave, loss of definition of the diaphragm, and delineation of the intrahepatic architecture. The sonographer was unaware of the lab results or the carotid intima media (CIM) thickness of the patients studied. 4- Arterial Carotid Duplex: Carotid intima-media thickness was measured bilaterally in the upper common carotid artery far wall in the 1cm segment proximal to the dilatation of the carotid bulb where the vessel is usually at right angle to the ultrasound beam and always in plaquefree segments using a high frequency transducer (7 MHz). It is considered a reliable estimation of subclinical atherosclerosis [5]. 5- Laboratory investigations: Venous blood sample was drawn in the morning after an overnight fast. - Liver function tests: Aspartate Amino Transferase (AST), Alanine Amino Transferase (ALT), Alkaline Phosphatase (ALP), Prothrombin time and Albumin. - Serology for viral hepatitis B. and C.: HBsAg and HCVAb. - Lipid profile: Total cholesterol, HDL, LDL and Triglycerides. - CRP (quantitive). - Fasting blood sugar. Statistical methodology: Analysis of data was done by IBM computer using SPSS (statistical program for social science version 12) as follows: Description of quantitative variables as mean, SD and range. Description of qualitative variables as number and percentage.
Chi-square test was used to compare qualitative variables between groups. Unpaired t-test was used to compare quantitative variable between both groups. Correlation co-efficient test was used to rank different variables against each other positively or inversely. Results There was no statistically significant difference detected between cases and controls regarding the demographic data (p>value 0.05) (Table 1). There was no statistically significant difference detected between cases and controls as regards blood pressure values (p value >0.05). There was a highly statistically significant difference between cases and controls as regards liver function tests (p value <0.01) cholesterol, LDL, and CRP (p value <0.01) (Table 2). Group I patients had a larger liver size (Table 3) and a higher intima media thickness as compared to group II with statistically highly significant difference between them (p value <0.01) (Fig. 1). There was a highly statistically significant positive correlation between intima media thicknesses in group I patients and the general variables (age, weight, BMI, systolic and diastolic blood pressure) (p value <0.01, r=0.93, 0.95, 0.96, 0.89 and 0.88 respectively) as well as a highly statistically significant positive correlation between intima media thickness and all laboratory variables (Total cholesterol, LDL, TG, CRP, FBS, AST, ALT, ALP & PT) (p value <0.01, r=0.89, 0.83, 0.86, 0.92, 0.90, 0.82, 0.81, 0.82 & 0.94 respectively) except albumin and HDL where there was an inverse correlation (p value <0.01, r=0.92, 0.87 respectively) (Table 4). There was a highly statistically significant positive correlation between intima media thicknesses in the control group and the general variables (age, weight, BMI, systolic and diastolic blood pressure) (p value <0.01, r=0.80, 0.85, 0.86, 0.84 & 0.86 respectively) as well as a highly statistically significant positive correlation between intima media thickness and all laboratory variables (Total cholesterol, LDL, TG, CRP, FBS, AST, ALT, ALP & PT) (p value <0.01, r=0.83, 0.73, 0.66, 0.72, 0.70, 0.72, 0.81, 0.89 & 0.90 respectively) except albumin and HDL were there was inverse correlation (p value <0.01, r= 0.91, 0.80 respectively) (Table 5). There was a highly statistically significant positive correlation between intima media thicknesses and liver size in group I patients (p value <0.01, r=0.90). On the other hand there was no significant correlation among controls (p value >0.05, r=0.12).
Nouman H. Algarem, et al.

Table (1): Comparison between both groups as regard demographic data. Variables Age (yr.) Weight (kg) BMI (Kg/m2) Gender Female Male Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Cases N=45 503 752 27.52.9 36 (80%) 9 (20%) 1199 799 Controls N=15 514 744 274.2 12 (80%) 3 (20%) 12110 8110 0.6 0.4 >0.05 >0.05 t 0.7 1.2 1.4 0# p >0.05 >0.05 >0.05 >0.05
493
Table (2): Comparison between group I cases and controls as regard the laboratory variables. Variables AST (U/L) ALT (U/L) ALP (U/L) PT (sec) Albumin (g/dl) Total cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) CRP (mg/L) FBS (mg/dl) Cases N=45 68.45.1 674.7 1535.1 11.90.8 3.50.3 1642.9 40.73 845 1084.3 10.30.9 10422 Controls N=15 284 26.44.1 63.83.9 120.8 4.60.5 1623.3 422 793 105.43.5 6.10.7 10227 t 11 29 61 0.4 27 2.9 1.9 3 2.1 16 0.2 p <0.001 <0.001 <0.001 >0.05 <0.001 <0.05 >0.05 <0.01 <0.05 <0.001 >0.05
Table (3): Correlation between carotid intima media thickness and laboratory parameters among group I patients. Variables Total cholesterol HDL LDL TG CRP FBS AST ALT ALP PT Albumin Intimal thickness r 0.89 0.87 0.83 0.86 0.92 0.90 0.82 0.81 0.82 0.94 0.92 p <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Table (4): Liver size and CIMT values in groups I and II. Variables Liver size: Mean SD Range Intima media thickness IMT: Mean SD Range Cases N=45 18.92 16-23 0.730.1 0.6-0.9 Controls N=15 130.8 12-14 0.500.08 0.4-0.6 t p
10
<0.001
<0.001
494
Table (5): Correlation between intima media thickness and laboratory data among the studied controls. Variables Total cholesterol HDL LDL TG CRP FBS AST ALT ALP PT Albumin Intimal thickness r 0.83 0.80 0.73 0.66 0.72 0.70 0.72 0.81 0.89 0.90 0.91 p <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
0.8 0.6 0.4 0.2 0 Cases Controls Fig. (1): Comparison between cases and controls as regard intima media thickness.
172 170 168 Cholesterol HDL .5 94 92 90 88 LDL TG 86 84 82 80 78 76 .5 .6 .7 .8 Intimal thcickness .9 1.0 .6 .7 .8 Intimal thcickness .9 1.0 166 164 162 160 158
46 44 42 40 38 36 34 .5 116 114 112 110 108 106 104 102 100 .5 .6 .7 .8 Intimal thcickness .9 1.0 .6 .7 .8 Intimal thcickness .9 1.0
Fig. (2): Correlation between intima media thickness and lipid profile among cases.
Discussion Non-alcoholic Fatty Liver disease (NAFLD) is currently the most common cause of abnormal liver function tests observed in the clinical hepatology practice [6]. The importance of NAFLD and its relationship with the metabolic syndrome is now increasingly recognized and this has stimulated an interest in the possible role of NAFLD in the
development of cardiovascular disease (CVD) [7]. But, it is important to speculate whether NAFLD in itself confers a cardiovascular risk. Indeed, the possible impact of NAFLD on CVD risk deserves particular attention in view of the implications for screening/surveillance strategies in the growing number of patients with NAFLD [8].
Nouman H. Algarem, et al.
495
Our study, which was performed on nondiabetic patients with hepatic steatosis and raised liver enzymes indicating clear evidence of NAFLD, revealed a significant increase in carotid IMT (0.730.1mm) than in normal control persons (0.500.08mm) which was statistically highly significant (p<0.001) (Fig. 1). To ensure the absence of confounding factors our patients were age, sex and BMI matched to the controls. Thus our findings cannot be attributed to these factors that also affect the development of atherosclerosis. Moreover our patients were nondiabetics and even though their lipid profile was significantly raised than the normal controls (p value <0.001), it was within normal laboratory values. This has led us to speculate on the possible role of NAFLD in the presence of higher circulating serum cholesterol and LDL than in the normal population and the effect this could have on the assessment of such patients for cardiovascular morbidity. There was also a significantly positive correlation between the carotid intima media thickness and all our laboratory parameters except albumin and HDL where it is significantly negatively correlated. These results highlight the altered metabolism that accompanies hepatic steatosis and has recently intrigued researchers. We also found a highly statistically significant rise in the serum CRP between our patients and controls. Several studies have found CRP to be associated with an increased rate of atherosclerosis progression in the carotid vessels, and is recommended as a risk marker for cardiovascular disease both in diabetics and the normal population [9,10]. This also supports the fact that NAFLD patients may be at risk of early generalized atherosclerosis. There was a strong association between the increase of liver size and also the rise in liver enzymes and the increase in carotid IMT in patients with NAFLD which was highly significant (p value <0.001). This association highlights the significance of not only assessing the presence and degree of fatty infiltration of the liver but also the importance of the level of liver enzymes as a marker of altered physiology. These results were supported by pervious prospective studies reporting strong associations between elevated liver enzymes as surrogate markers of NAFLD and the incidence of cardiovascular disease (CVD) [11,12]. The pathogenetic mechanisms by which NAFLD could contribute to accelerated atherosclerosis are still poorly understood. But the possibility that the putative elevated CVD risk associated with NAFLD most likely reflects the overall atherogenic impact
of the metabolic syndrome did not neglect the effect of NAFLD as an isolated risk factor that may have its own special mechanisms by which it may cause or accelerate the incidence of early atherosclerosis. A possible atherogenic mechanism linking NAFLD and carotid IMT could be represented by increased oxidative stress and subclinical inflammation, which are thought to be causal factors in the progression of atherosclerosis, and simple steatosis to more advanced forms of NAFLD [13]. Another possible pathophysiologic mechanism is that fat overload may make the liver less prone to clear plasma free fatty acids, contributing to their accumulation in the blood, and to the augmentation of liver lipogenesis [14]. Our study has some limitations that should be kept in mind, the most important of which is that the diagnosis of NAFLD was exclusively based on ultrasound imaging and exclusion of other causes of elevated liver enzymes but was not confirmed by liver biopsy, which is an important diagnostic tool for confirming NAFLD. But, for the sake of the safety and in compliance with the wishes of the patients we chose the less accurate but easy, safe and non invasive method for diagnosis of NAFLD rather than the invasive liver biopsy due to its hazards and complications. Other studies have employed the same protocol [15]. From our point of view, our findings might have important clinical and public health implications. Our data emphasizes the importance of evaluating the CVD risk in patients diagnosed with NAFLD. Patients with NAFLD who are found to have increased carotid IMT should be selected for further laboratory investigations to detect elevations in their lipid profile and treated not only for their liver disease, but also by aggressive lifestyle modification and lipid modifying drugs if necessary, as well as addressing any other CVD risks that may be present. This may help to modify and potentially decrease the global CVD risk of these patients. Currently, it is not known whether improving NAFLD will ultimately prevent the development of CVD. However, it is worth noting that interventions known to be effective in preventing CVD, including weight reduction and treatment with insulin-sensitizing oral agents (thiazolidinediones or metformin), also improve NAFLD [1].
496

7- ADAMS L.A., SANDERSON S., LINDOR K.D. and ANGULO P.: The histological course of nonalcoholic fatty liver disease: A longitudinal study of 103 patients with sequential liver biopsies. J. Hepatol., 42: 132-8, 2005. 8- McCULLOUGH A.J.: The clinical features, diagnosis and natural history of non-alcoholic fatty liver disease. Clin. Liver Dis., 8: 521-33, 2004. 9- PFUTZNER A. and FORST T.: High sensitivity C-reactive protein as cardiovascular risk marker in patients with diabetes mellitus. Diabetes Technol. Ther., 8 (1): 28-36, 2006. 10- RIDKER P.M. and SILVERTOWN J.D.: Imflammation, C-reactive protein, and Atherothrombosis. J. Periodontol., 9 (suppl. 8): 1544-51, 2008. 11- IOANNOU G.N., WEISS N.S., BOYOKO E.J., MOZAFFARIAN D. and LEE S.P.: Elevated serum alanine aminotransferase activity and calculated risk of coronary heart disease in the United States. Hepatology, 43: 114551, 2006. 12- SCHINDHELM R.J., DEKKER J.M., NIJPELS G., BOUTER L.M., STEHOUWER C.D., HEINE R.J., et al.: Alanine aminotransferase predicts coronary heart disease events:a 10-year follow-up of the Hoorn study. Atherosclerosis, 191: 391-6, 2007. 13- CHALASANI N., DEEG M.A. and CRABB D.W.: Systemic levels of lipid peroxidation and its metabolic and dietary correlates in patients with non-alcoholic steatohepatitis. Am. J. Gastroenterol., 99: 1497-502, 2004. 14- TAMURA S. and SHIMOMURA I.: Contribution of adipose tissue and denovo lipogenesis to nonalcoholic fatty liver disease. J. Clin. Invest., 115: 1139-42, 2005. 15- CARALLO C., MANCUSO G., MAURO G., LAGHI F., MADAFFERI B., IRACE C., GNASSO A., SCAVELLI F., et al.: Hepatic steatosis, carotid atheroclerosis and metabolic syndrome: The STEATO Study. J. Gastroentrol., 44: 1156-61, 2009.
In conclusion, our findings support the hypothesis that the presence of NAFLD is strongly associated with early carotid atherosclerosis. Future follow-up studies are necessary to validate these findings, better estimate the risk of incident CVD among patients with biopsy proven NAFLD and to detect the exact mechanisms by which NAFLD increases the risk of early atherosclerosis and lastly, new lines in prevention and treatment of NAFLD. References
1- ANGULO P.: Nonalcoholic fatty liver disease. N. Engl. J. Med., 346: 1221-31, 2002. 2- VOLZKE H., ROBINSON D.M., KLEINE V., DEUTSCHER R., HOFFMANN W. and LUDEMANN J.: Hepatic steatosis is associated with an increased risk of carotid atherosclerosis. World J. Gastronterol., 11: 184853, 2005. 3- TARGHER G., BERTOLINI L., RODELLA S., TESSARI R., ZENARI L., LIPPI G., et al.: Nonalcoholic fatty liver disease is independently associated with an increased incidence of cardiovascular events in type 2 diabetic patients. Diabetes Care., 30: 2119-21, 2007. 4- O'LEARY D.H. and POLAK J.F.: Intima-media thickness: A tool for atherosclerosis imaging and event prediction. Am. J. Cardiol, 90: 18-21, 2002. 5- SIMON A., GARIEPY J., CHIRONI G., MEGNIEN J.L. and LEVENSON J.: Intima media thickness: A new tool for diagnosis and treatment of cardiovascular risk. J. Hypertension., 20 (2): 159-69, 2002. 6- MEDINA J., FERNANDEZ-SALAZAR L.I., GARCIABUEY L. and MORENO-OTERO R.: Approach to the pathogenesis and treatment of nonalcoholic steatohepatitis. Diabetes Care., 27: 2057-66, 2004.
The Diagnostic Utility of Third Generation TSH Electro-Chemilumenescence Immunoassay in Detecting the Incidence of Thyroid Dysfunctions in Saudi Arabia
ABDULAZIZ A.A. ALGHAITHY, Ph.D.* and ASMAA A. EL-REWENY, M.D.*,**
The Departments of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, Almadinah Almunawarah, Kingdom of Saudai Arabia* and Clinical & Chemical Pathology , Faculty of Medicine, Cairo University**
Abstract
Objective: To demonstrate how to get use of the highly sensitive third generation electrochemiluminescence immunoassay (ECLIA) in determining the incidence of various thyroid dysfunctions and making proper differentiation for these different thyroid disorders in KSA. Setting: The Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Taibah University, Outpatient Clinics and the Laboratory Section in Ohud Hospital at Almadinah Almunawarah, KSA. Subjects: Two groups were involved: Group I included 117 subjects of young volunteers (20-30 years) from Taibah University (TUV) as an apparently healthy group with no specific thyroid symptoms and group II represented by all attendants to outpatient clinics in Ohud Hospital at the same collection period (569 Subjects). Methods: All subjects were subjected to measurement of serum TSH and free thyroid hormones by ECLIA. The main 2 groups were subdivided on biochemical basis according to their TSH and Thyroid Hormones (TH) levels. Results: By the use of this immunoassay a percentage of 5.1% for subclinical hypothyroidism among TUV (group I) was reported. A higher percentage of 21.1% for subclinical hypothyroidism in group II was reported (group IIc). The difference between these two percentages was highly significant (p<0.0001). Percentages of 4% for overt hypothyroidism (group IId) and 7.2% for those with high TSH and border line thyroid hormones (group IIb) were also recorded among group II. The sum of percentages of cases with high TSH in group II (32.3%) was highly significantly increased when compared with TUV group (p<0.0001). Percentages of 2% for subclinical hyperthyroidism and 0.7% for overt hyperthyroidism in group II were reported, but no cases of hyperthyroidism (subclinicalor overt) were recorded in TUV group. Also, hypothyroidism was found to be the main thyroid dysfunction when compared to hyperthyroidism in group II (32.3% versus 2.7%) and also in group I (5.1% versus 0%). The mean values for TSH in all subgroups included in group II showed highly significant Correspondence to: Prof. Asmaa A. El-Reweny, E-mail: asmaarew@hotmail.com.
differences when compared with the normal control group (group Ia). The mean values for thyroid hormones (FT4 & FT3) showed significant differences in some but not all subgroups in group II when compared with normal control. Conclusion: The third generation TSH-ECLIA immunoassay is the single sensitive first-line test for accurate screening and early diagnosis of thyroid dysfunctions in clinical laboratory. From the 2 studied Saudian population samples, the test proves that increasing the age increases the susceptibility of having a thyroid diseases especially hypothyroidism. The age period of 20 to 30 years could be the period that regular screening for subclinical hypothyroidism should be done in order to face a warning silent pandemic of hypothyroidism and prevent its major complications. Key Words: TSH Electrochemiluminescence Immunoassay Third generation immunoassay Subclinical thyroid diseases.
Introduction THE whole system of thyroid pituitary hypothalamic axis is like a thermostat. When the body senses that there is no enough thyroid hormone, the hypothalamus releases Thyrotropin Releasing Hormone that causes the pituitary to secrete Thyroid Stimulating Hormone (TSH) that stimulate the thyroid gland to make and release thyroid hormones. When there is too much thyroid hormone the releasing hormones are turned off and the thyroid stops producing thyroid hormones [1]. The major function of thyroid hormones is their control of the basal metabolic rate and calorigenesis. Thyroid hormones are known to (a) Stimulate neural development and normal growth, (b) Promote sexual maturation, (c) Stimulate adrenergic activity with increased heart rate and myocardial contractility, (d) Stimulate protein synthesis and carbohydrate metabolism, (e) Increase the synthesis and degradation of cholesterol and triglycerides, (f)
497
498
The Diagnostic Utility of Third Generation TSH Electro-Chemilumenescence Immunoaasay
Increase the requirement for vitamins, (g) Increase the calcium and phosphorus metabolism, and (h) Enhance the sensitivity of adrenergic receptors to catecholamines. These effects are typically magnified in patients with either an overactive thyroid gland, such as in hyperthyroidism or reduced in patients with a sluggish thyroid function such as in hypothyroidism [2,3]. Thyroid-stimulating hormone (TSH, thyrotropin) is a glycoprotein having a molecular weight of approx. 30000 Daltons and consisting of two subunits. The -subunit carries the TSH-specific immunological and biological information, whereas -chain carries species-specific information and has an identical amino acid sequence to -chains of LH, FSH and hCG. TSH is formed in specific basophile cells of the anterior pituitary. The hypophyseal release of TSH is the central regulating mechanism for the biological action of thyroid hormones. TSH has a stimulating action in all stages of thyroid hormone formation and secretion; it also has a proliferative effect [4]. The thyroid hormone thyroxin (T4) is physiologically a part of the regulating circuit of the thyroid gland and has an effect on general metabolism. The major fraction of the total thyroxin is bound to transport proteins (TBG, prealbumin, and albumin). The free thyroxin (FT4) is the physiologically active thyroxin component [4-6]. Diseases of the thyroid gland are among the most abundant disorders worldwide second to diabetes. About 95% of the time hypothyroidism is the result of malfunction of the thyroid gland itself (primary hypothyroidism). Causes of primary hypothyroidism can be either, congenital or acquired. The most common occurs in iodine deficient countries, a daily iodine intake below 25 g particularly in preterm infants, is a more frequent cause of hypothyroidism accounting for many cases in Europe, Asia and Africa. Hypothyroidism is usually due to autoimmune thyroiditis and thyroid auto-antibodies are classified into three main categories, antibodies against microsomal components including; anti-microsomal antibodies (the thyroid peroxidase TPO), related to the production of thyroid hormones and other group of antibodies against TSH receptors. They included TSH stimulating antibodies and TSH binding inhibitory antibodies. The third group includes antithyroglobulin antibodies [7]. There are several causes of hyperthyroidism, The most common include immunologic conditions (Graves' disease and thyroiditis), toxic thyroid nodules (adenomas), and toxic multinodular goiter [1].
The term subclinical hypothyroidism is used for patients who have mildly increased levels of serum TSH but normal thyroid hormones levels [8]. The term Subclinical hyperthyroidism is defined as a persistently suppressed serum TSH with normal thyroxin and triiodothyronine in patients who do not have symptoms. Subclinical hyperthyroidism can be caused by the same thyroid disorders that result in clinical hyperthyroidism [9]. While signs and symptoms of overt hyper and hypothyroidism are well known, sub clinical thyroid conditions have subtle clinical manifestations and may mimic other diseases. Hence it is important to develop rational laboratory strategies to differentiate the various conditions to guide the physician towards correct diagnosis and treatment [10]. The determination of TSH serves as the initial test in thyroid diagnostics. Even very slight changes in the concentrations of the free thyroid hormones bring about much greater opposite changes in the TSH level. Accordingly, TSH is a very sensitive and specific parameter for assessing thyroid function and is particularly suitable for early detection or exclusion of disorders in the central regulating circuit between the hypothalamus, pituitary and thyroid [11-14]. The determination of free thyroxin is an important element in clinical routine diagnostics. Free T4 is measured together with TSH when thyroid function disorders are suspected. The determination of FT4 is also suitable for monitoring thyro-suppressive therapy. The determination of FT4 has the advantage of being independent of changes in the concentrations and binding properties of the binding proteins. Thus, additional determination of a binding parameter (T-uptake, TBG) is therefore unnecessary. A variety of methods are available for estimating the free thyroid hormone levels. The direct measurement of FT4 and FT3 via equilibrium dialysis or ultra filtration is mainly used as a reference method for standardizing the immunological procedures generally used for routine diagnostic purposes, but those methods are tedious and could not be used as routine tests [46]. Determination of TSH by sensitive electrochemiluminescence immunoassay "ECLIA" is currently judged as the most sensitive and also most costeffective first-line approach to thyroid function testing. Measuring TSH concentrations by third generation assay turned out to be advantageous in the follow-up of many clinical situations, (a) In patients with mildly suppressed but well detectable TSH concentrations due to functional thyroid autonomy (0.03-0.3mU/1), also, overt hyperthyroidism can be excluded by third generation TSH
Abdulaziz A.A. AlGhaithy & Asmaa A. El-Reweny
499
measurement alone without the need of additional thyroid hormone measurements; (b) In patients receiving long term suppressive T4 treatment after thyroidectomy for differentiated thyroid cancer, measurement of basal TSH by third generation assays allows accurate monitoring of hormone therapy without the need for TRH testing; (c) In most patients with severe non-thyroidal illnesses and decreased TSH levels, TSH concentrations measured by third generation assays are only moderately suppressed and could be clearly discriminated from undetectable levels in overt hyperthyroidism. Thus, the use of third generation TSH immunoassays is recommended in specialized clinical laboratories frequently analyzing samples taken in one of those clinical situations [15]. Electrochemiluminescence is one of the best of these immunoassays and it differs from chemiluminescence and bio-luminescence in that the reactive species that produce the chemiluminescenct reaction are electrochemically generated from stable precursors that are at the surface of an electrode. Electrochemiluminescence process has been demonstrated for many different molecules by several different mechanisms, including an oxidation-reduction reaction with tris ruthenium and tripropylamine. The chemiluminescence precursors are stable and relatively small and can be used to label haptens or large molecules like TSH. Multiple labels can be coupled to proteins or oligonucleotides. Their advantages include improved regent stability, simple regent preparation, and enhanced sensitivity. With its use, detection limits of 200 f mol/L could be achieved. It could be used as either in competitive or sandwich immunoassays. With the ruthenium label, various assays have been developed in a flow cell, with magnetic beads as the solid phase. Beads are captured at the electrode surface, and unbound label is wasted from the cell by a wash buffer. Label bound to the bead undergoes electrochemiluminescent reaction, and the light emission is measured by an adjacent photomultiplier tube [16]. The aim of this work was to demonstrate how to get use of this highly sensitive third generation electrochemiluminescence immunoassay in determining the incidence of various thyroid dysfunctions and making early proper differentiation for these different thyroid disorders in Almadinah Almounawarah, KSA. Subjects and Methods The current study included two groups of Saudian subjects, group (I) Included 117 volunteer
female subjects from Taibah University at Almadinah Almunawarh, KSA, they were having no specific symptoms of any thyroid disorders (their ages ranged from 20 to 30 years {an important inclusion criteria}), and group (II) Included 569 untreated subjects from all attendants at the outpatient clinics of Ohud hospital at Almadinah Almunawarah, KSA in the same period of collection (month 3 and 4 -1432), their ages ranged from 18 to 80 years and females represented 83% of this group (males were 17%). The two groups were subjected to laboratory tests that included highly sensitive serum TSH, FT4 and FT3 measurements. Five ml of venous blood was extracted from every subject and blood samples were allowed to clot at room temperature in vacutainer tubes with serum separator. The tubes were centrifuged and serum was separated and freezed at - 20C till assayed. The TSH and thyroid hormones were estimated using a highly sensitive electrochemiluminescence immunoassay method (ECLIA) on ELECSYS 2010 immunoassay analyzer from Roche. The reference ranges for the three hormones were primarily established as in the literature. These are 0.27-4.2 IU/ml (m IU/L), 12-22 p mol/L and 3.1-6.8 p mol/L for TSH, FT4 and FT3 respectively [16]. The two groups were subdivided biochemically according to TSH and thyroid hormones (TH) levels into subgroups. Group I was divided into group Ia that included 111 subjects with completely normal TSH and TH, this group was considered as the normal control for the whole studied groups and group Ib that included 6 subjects with elevated serum TSH level and normal TH. Group II was also subdivided biochemically into 6 subgroups, group IIa included those with normal TSH and normal TH, group IIb with high TSH and border line TH, group IIc with elevated serum TSH level and normal TH, group IId with elevated serum TSH level and low TH, group IIe with low serum TSH level and normal TH and group IIf with low TSH level and high TH. Test principle and steps of serum TSH automated electrochemiluminescence immunoassay method: This electrochemiluminescence TSH immuneassay employs monoclonal antibodies specifically directed against human TSH. The antibodies labeled with ruthenium complex consist of a chimeric construct from human and mouse-specific components. As a result, interfering effect due to human anti-mouse antibodies are largely eliminated. It is an immunoassay with sandwich principle. The total
500
duration of assay is 18 minutes. In the first incubation; 50 l of sample, a biotinylated monoclonal TSH-specific antibody and a monoclonal TSHspecific antibody labeled with a ruthenium complex react to form a sandwich complex. In the second incubation; after addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substance is then removed. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined via a calibration curve which is instrument-specifically generated by 2point calibration and a master curve provided via the reagent barcode [16]. Test principle and steps of serum FT4 & FT3 automated electrochemiluminescence immunoassay method: In this electrochemiluminescence assay, the determination of free hormone is made with the aid of a specific anti-T4 (or T3) antibody labeled with a ruthenium complex. The quantity of antibody used is so small that the equilibrium between bound and free T4 (or T3) remains virtually unaffected. It is an immunoassay with competition principle. Total duration of assay is 18 minutes. In the first incubation, 15 l of sample and T4 (or T3) specific antibody labeled with a ruthenium complex are mixed. In the second incubation, after addition of biotinylated T4 (or T3) and streptavidin-coated microparticles, the still-free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten complex. The entire complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined via a calibration curve which is instrumentspecifically generated by 2-point calibration and a master curve provided via the reagent barcode [16]. Statistical methods: The SPSS (Statistical Package for Social Sciences version 11) was used for data management and analysis and the Microsoft Excel for charts construction. Quantitative data were presented as meanSD. For comparison of the means, the Stu-
dent's t-test was used. The Chi-square test was used for comparison of percentages. All tests were two tailed and considered significant when p<0.01 and highly significant when p<0.0001. Results All the results of the current study are shown in the following figures and tables. The pie charts (Figs. 1-3) represent the percentage of various thyroid disorders diagnosed on biochemical basis in both groups; Fig. (1) for group I (TUV) and fig. (2) for percentages of different TSH levels (normal, high & low) in group II which represents hospital outpatient clinics attendants. Fig. (3) for all subgroups in group II. Table (1) shows the comparison of mean values for TSH and thyroid hormones in subclinical hypothyroid TUV versus normal TUV subjects. Table (2) shows the comparison of mean values for TSH and thyroid hormones in attendants to hospital laboratory in relation to normal TUV group. Table (3) shows the comparison of percentages of cases of subclinical hypothyroidism in TUV group with other subgroups in group II that also has either the same subclincal hypothyroid disorder, all hypothyroidism (subclinical- or overt) or all subjects with increased TSH level regardless the level of thyroid hormones. Table (4) shows comparison of percentages of cases with high TSH versus cases with low TSH in the main 2 groups. Figs. (4&5) show the comparison of mean values of serum TSH, FT4 and FT3 in all studied groups in relation to normal control respectively.
Normal TUV 94.87% Subclinicl Hypothyroidism 5.13%
*TUV: Taibah University Volunteers Normal TUV = Ia Subclinical Hypothyroidism = Ib
Fig. (1): Pie chart for thyroid disorders in goup 1 (TUV)*.

Normal TSH 65% Low TSH 2.70% High TSH 32.30%
Subjects with Normal TSH Subjects with High TSH
Subjects with Low TSH
Fig. (2): Pie chart for different TSH levels in group II.

Group IIf 0.70% Group IIa 65% Group IIe 2% Group IId 4% Group IIc 21.10%
501
Table (1): Comparison of mean values for TSH and thyroid hormones in subclinical hypothyroid TUV & normal TUV Group Ia Group Ib
Normal TUV TUV with Subclinical p value Hypothyroidism N = 111 N=6 TSH XSD (m IU/L) FT4 XSD (p mol/L) FT3 XSD (p mol/L)
** p<0.0001
Group IIb 7.20%
1.530.77 14.181.44 5.30.43
5.591.51** 13.512.24 4.951.38
<0.0001 0.67 0.34
Group IIa Group IIb Group IIc Group IId Group IIe Group IIf
: Normal TSH + Normal Thyroid Hormones (TH) : High TSH + border line TH : High TSH + Normal TH : High TSH + Low TH : Low TSH + Normal TH : Low TSH + High TH
Fig. (3): Pie chart for thyroid disorders in group II.
Table (2): Comparison of Mean Values for TSH and Thyroid Hormones in Attendants to Hospital Laboratory in Relation to Normal TUV. IIa Group Normal TSH+ Normal TH N= 370 (65%) TSH XSD (m IU/L) FT4 XSD (p mol/L) FT3 XSD (p mol/L)
* p=0.008 ** p<0.0001
IIb High TSH+ border line TH N= 41 (7.2%) 8.096.53** N=41 10.920.62** N=41 4.390.64** N=32
IIc High TSH+ Normal TH (Subclinical Hypothyroidism) N= 120 (21.1%) 8.676.67** N=120 14.112.02 N=120 4.770.86** N=102
IId High TSH+ Low TH (Hypo-thyroidism) N= 23 (4%) 44.4330.02** N=23 8.763.36** N=23 3.901.45** N=20
IIe Low TSH+ Normal TH (Subclinical Hyperthyroidism) N = 11 (2%) 0.0620.046** N=11 17.202.7** N=11 4.840.95* N=9
IIf Low TSH+ High TH (Hyperthyroidism) N = 4 (0.7%) 0.060.04** N=4 24.441.33** N=4 5.20.92 N=3
2.341.06** N=370 14.12.36 N=370 4.810.85** N=300
Table (3): Comparison of Percentages of Cases of Subclinical Hypothyroidism in TUV group with other related groups in group II. % Of cases in TUV (group Ib) Subclinical- Hypo -thyroidism within group I 5.1%
** p<0.0001
% Of cases in TUV (group Ib) Subclinical- Hypothyroidism within group I 21.1%
% Of cases in Groups IIc & IId Total Hypothyroidism (subclinical & overt) within group II 25.1%**
% Of cases in groups IIb, IIc & IId Total cases with high TSH within group II 32.3%** p-value <0.0001
Table (4): Comparison of percentages of cases with high TSH with cases with low TSH in the main 2 groups. % of cases with high TSH in group I 5.1%* % of cases with low TSH in group I 0% p value *<0.05 % of cases with high TSH in group II 32.3%** % of cases with low TSH in group II 2.7% p-value **<0.0001
502
TSH (m lU/L) 50 45 40 35 30 25 20 15 10 5 0 -5
44.43+30.02**
1.53+ 0.77
5.59+ 1.51**
8.09+ 8.67+ 6.53** 6.67** 2.34+ 1.06** 0.062+ 0.06+ 0.046** 0.04**
Ia
Ib
IIa
IIc IIb Groups
IId
IIe
IIf
Fig. (4): Comparison of mean values of serum TSH in all studied groups in relation to normal control.
FT4 (p mol/L) 30 25 20 15 10 5 0 Ia Ib IIa IIc IIb Groups IId IIe IIf
14.18+ 13.51+ 1.44 2.24 14.1+ 2.36 10.92+ 0.62** 14.11+ 2.02 8.76+ 3.36**
24.44+1.33** 17.20+ 2.7**
Fig. (5): Comparison of mean values of serum FT4 in all studied groups in relation to normal control.
FT3 (p mol/L) 6 5 4 3 2 1 0
5.3+ 0.43 4.95+ 1.38 4.81+ 4.77+ 0.85** 4.39+ 0.86** 0.64** 4.84+ 0.95** 3.90+ 1.45** 5.2+ 0.92
diagnosis of thyroid dysfunction. Immunoassay is the method of choice for the measurement of serum TSH in the clinical laboratory. Highly sensitive assays for TSH have become available that employ various detection signals, including chemiluminescece and assays with low end detection limits in the 0.01 to 0.05 m IU/L range [17]. Clinically, these assays are capable of measuring TSH at concentration required to accurately differentiate the low concentration of serum TSH found in patient with hyperthyroidism as represented in our study by 2% with subclinical hyperthyroidism; group IIe in group II (11 out of 569) in addition to 0.7% with overt hyperthyroidism in group IIf (4 out of 569) from other causes of low TSH. This means that low concentrations of TSH due to hyperthyroidism could be easily differentiated from the suppressed concentration found in patient with non-thyroidal illnesses that can develop because of other conditions not related to primary thyroid disease like hospitalization, some acute illnesses. etc. Group I in our study as we mentioned before is a group of volunteer people from Taibah University with no history of any thyroid disorders or non thyroidal illnesses and with no symptoms or signs of any of them. Their ages were ranging from 20 to 30 years which means with low susceptibility of developing any thyroid dysfunction especially hyperthyroid states which usually manifest in older ages. In spite of this young age and small sample volume (117), six cases with subclinical hypothyroidism were discovered (5.1%). We also reported percentage of 21.1% of subclinical hypothyroidism among group II subjects. The prevalence of subclinical hyperthyroidism varied amongst other reports. Prevalence of subclinical hyperthyroidism was reported to be 10% in Whickham Survey [9] and 12% in the Framingham study [18]. The colorado thyroid disease prevalence study involving 25,862 subjects showed a prevalence of 2.1% [19]. This is in agreement of our results for group IIe that showed percentage of 2%. Other investigators studied a representative population of 17,353 people aged 12 and above and found that the prevalence of subclinical hyperthyroidism was only 0.7% [7] . This means that increasing the age, increases the susceptibility of having thyroid diseases. The prevalence of overt hyperthyroidism in women is between 0.5 and 2%, and is ten times more common in women than in men in other reports. In the Whickham survey, the prevalence of undiagnosed hyperthyroidism was 4.7 per 1000 women (0.47%) [20]. The prevalence data in elderly persons show a wide range between 0.4% to 2.0%
Ia
Ib
IIa
IIc IIb Groups
IId
IIe
IIf
Fig. (6): Comparison of mean values of serum FT3 in all studied groups in relation to normal control.
Discussion It is well known that the most useful test for assessing thyroid function is the measurement of serum TSH. Measuring TSH with sufficient sensitivity to distinguish low levels from normal levels has become the preferred first-line test for accurate
503
Our results showed a close percentage (0.7%) in group IIf. A cross-sectional study of 2799 healthy adults aged 70 to 79 years in the US found evidence of hyperthyroidism (defined biochemically as a serum TSH concentration less than 0.1m U/L and a serum free T4 concentration greater than 23 p mol/L) in only five subjects. In Leiden study, only 2 of 558 subjects aged between 85 and 89 years had newly diagnosed overt hyperthyroidism [22,23]. The prevalence of a further 1% of adults was having a history of toxic nodular goiter [24]. In a population sample of 2656 from Copenhagen, newly diagnosed thyrotoxicosis was found in 1.2% of women and no men, and the prevalence of known thyrotoxicosis was 1.4% [25]. Thus our current highly sensitive TSH assay appeared to be very useful in differentiating mild, subclinical hyperthyroidism from overt Graves disease, and because of this reason, it could also be used in monitoring thyroid cancer patients on thyroxin, and in monitoring the adequacy of thyroid hormone replacement in hypothyroid patients. Also, if the basal serum TSH levels could be detected by ultrasensitive immuno-assay as the electrochemiluminescese immunoassay used in our study, then no diagnostic benefit is gained by performing a TRH stimulation test that is a dynamic test that requires the availability of TRH (which is not commercially available in a lot of countries) and the acceptance of patient to do this dynamic tedious test. This means that the use of this current sensitive TSH immuno-assay will save a lot of time and money and also efforts needed to reach the proper and correct diagnosis and only by very cheap easy fully automated accurate and precise immunoassay. Also, by this sensitive immunoassay we could detect an incidence of subclinical hypothyroidism (group Ib) that was 5.1% in group I subjects belonging to the apparently normal subjects that were taken from Taibah University volunteers. In addition to greater percentages that were shown among group II that included all attendants to hospital outpatient clinics, those subjects included mainly people with some non specific symptoms or signs of thyroid disorder. Those with high serum TSH and low FT4, group IId, (hypothyroid patients) represented 4% of group II in this study and those with high serum TSH and normal FT4, group IIc, (subclinical-hypothyroid patients) represented 21.1% of group II. Also we have an additional group, group IIb that contained those subjects with high serum TSH and border line FT4 in which we recorded a percentage of 7.2%. This means that the total percentage of subjects with high serum TSH levels represented 32.3% in group II which
[21].
is significantly higher than that recorded in group I (5.1% in TUV in our study), (see Table 3). The greater percentages of those with altered TSH levels among group II in the current study could be attributed to the difference in ages in the two included groups (age range for group I is 20-30 years & for group II is 18-80 years). This means that the reported percentages for subclinical hypothroidism ranging from 5.1% to 21.1% that varies according to age, sex and clinical presentation. The comparison between these two percentages showed highly significant difference (p<0.0001). This means that the incidence of subclinical hypothyroidism increases with increasing age and if this condition could be detected early in life, we can minimize the development of overt hypoyhyroidism with its complications. Also, all subjects with high TSH levels (32.3%) i.e. those with hypofunction represented majority in thyroid dysfunctions found in group II when compared with those with low TSH levels (hyperfunction) and we recorded a percentage of 2.7% for them and the difference was highly significant (p<0.0001). Other investigators reported close figures. The percentage of subjects with a high serum TSH concentration was higher for women than men in each decade of age, and ranged from 4 to 21% in women and 3 to 16% in men. An increase in serum TSH concentrations was also found in men in the NHANES III study. In the same study serum TSH concentrations increased with age in both men and women and were higher in whites than blacks, independent of serum antithyroid antibody concentrations [7]. Whickham survey showed 8% of women (10% of women over 55 years of age) and 3% of men had subclinical hypothyroidism [20]. In the Colorado USA study, 9.4% of the subjects had a high serum TSH concentration, of which 9.0% had subclinical hypothyroidism [19]. Others reported that approximately 10% of subjects over 60 years were having serum TSH values above the normal range [26,27]. Little number of researches were done in KSA, but a percentage of subclinical hypothyroidism (35%) was reported by authors in Jeddah with 61% of them having thyroid antibodies [28]. In Makkah, other investigator reported 50.1% of cases with hypothyroidism and 49.9% of cases with hyperthyroidism out of all studied cases having various thyroid diseases but not among general population [29]. Overt and subclinical hypothyroidism were reported in 9.3% and 14.9%, respectively, for pregnant women with significantly higher maternal age and with increased risk of pregnancy-induced hypertension, gestational diabetes mellitus and
504
delivery by cesarean section in a research that was done at Almadinah Almounawarah [30]. The clinical significance of identifying those with subclinical hypothyroidism and adjusting thresholds for treatment remain some -what unclear and are a continued topic of investigation. The great value of early discovery of those cases is for its potential benefits and risks of therapy for those with subclinical hypothyroidism that have been debated for two decades. The possible advantages of treating subclinical hypothyroidism generally include: Firstly, preventing the progression to overt hypothyroidism. Secondly, thyroxin therapy may improve the serum lipid profile and thereby potentially decrease the risk of death from cardiovascular causes. Finally, treatment may reverse the symptoms of mild hypothyroidism, including psychiatric and cognitive abnormalities [31]. In group II in our study we reported 23% of subjects with overt hypothyroidism with high TSH and low thyroid hormones (group IId), but we did not find any case among the small sampled group I (TUV). This may be attributed to the young age of this group and also the small size of the sample. This incidence reported by our work (23% in group II) is higher than those reported by other investigators that we will discuss later and further studies for larger population size should be done to confirm this figures. Also, other following studies that include tests determining the etiology of this observation like iodine levels and thyroid auto antibodies should urgently be done in different areas in KSA. In iodine-replete communities, the prevalence of spontaneous hypothyroidism is between 1% and 2%, and it is more common in older women and ten times more common in women than in men. In the Whickham survey, the prevalence of newly diagnosed overt hypothyroidism was 3 per 1000 women [20,21]. The prevalence of previously diagnosed and treated hypothyroidism was 14 per 1000 women, increasing to 19 per 1000 women when follow up was possible. The overall prevalence in men was less than 1 case per 1000. One third had been previously treated by surgery or radioiodine for thyrotoxicosis. Excluding iatrogenic causes, the prevalence of hypothyroidism was 10 per 1000 women, increasing to 15 per 1000. The mean age at diagnosis was 57 years. Other studies in Northern Europe, Japan and the USA have found the prevalence to range between 0.6 and 12 per 1000 women and between 1.3 and 4.0 per 1000 in men investigated [21] . In the Colorado and NHANES III studies, the prevalence of newly diagnosed hypothyroidism was 4 per 1000 and 3 per 1000 respectively [7,19]. In Pescopagano, Italy,
an area of mild iodine deficiency, the prevalence of newly diagnosed overt hypothyroidism was 0.3% of 573 women (autoimmune thyroiditis confirmed as etiology). There were no cases among 419 men, and no subject had been diagnosed and treated for hypothyroidism [24]. In borderline iodine-deficient Copenhagen, Denmark, 6 per 1000 of the women and 2 per 1000 men had overt but undiagnosed hypothyroidism, and 1% of all subjects were taking thyroxin [25] . The prevalence was higher in surveys of the elderly in the community in other researches [21]. The overall prevalence of hypothyroidism, including those already taking T4, in Birmingham, UK, of 1210 subjects aged 60 and over was 4% of women and 0.8% of men aged over 60 years. In subjects aged 60 years or more in Framingham, 4% had serum TSH concentration greater than 10 m U/L, of whom one-third had low serum T4 concentrations [32]. Overt hypothyroidism was found in 7% of 558 subjects aged between 85 and 89 years in Leiden, Netherlands [23]. From the current results and results reported by other authors, we can say that there is a silent pandemic of hypothyroidism that needs certain great attention for early diagnosis of subclinical hypothyroid indviduals. Follow up of levels of TSH followed by thyroxin supplements if needed could prevent or at least minimize the appearance of this pandemic. In addition, to the previously mentioned great sensitivity of the current TSH immunoassay, the calibration of the TSH by ECLIA (Elecsys 2010) is usually done once per reagent lot and also this is a great advantage as it reduces analysis time and also the analyzer automatically calculates results in m IU/L. The assay is also of good specificity as it is not affected by jaundiced, hemolysed or lipemic samples. The measuring range of the current assay is 0.005-100.0 IU/ml (m IU/L); the lower detection limit is 0.005 IU/ml which is the lowest TSH level that can be distinguished from zero. The functional sensitivity of this assay is 0.014 IU/ml [11]. Three generations of TSH immunoassays have been developed over the previous years. These assays are capable of diagnosing primary hypothyroidism with low thyroid hormones production and with elevated levels of TSH. Second generation TSH immuno assays, with detection limits of 0.1m U/L, can effectively screen for hyperthyroidism (better than first generation with detection limit of 1m U/L). However, the third generation TSH chemiluminometric assays, with detection limits of 0.01m U/L, are less likely to give false-negative
505
results and can more accurately distinguish between euthyroidism and hyperthyroidism. The assay used in our study detected very low TSH levels in group II down to 0.005 and 0.007m IU/L (represented by 0.18% for each), this is in addition to values like 0.01m IU/L (3 samples out of 569 samples represented by 0.53%), 0.033m IU/L (1 sample represented by 0.18%) and others such as 0.059, 0.076, 0.087, 0.088 and 0.09m IU/L. Thus, the third generation TSH assays should be routinely used to monitor and adjust thyroid hormone replacement therapy as well as screen for both hyperthyroidism and hypothyroidism [33]. The sensitivity of the third generation TSH assays has lead to the ability to detect subclinical thyroid disease - or a mild degree of thyroid dysfunction due to the large reciprocal change in TSH levels seen for even small change in free T4 [34]. In the group II which represents subjects coming from outpatient clinics with potential probability of having thyroid disease but not discovered yet, and they are supposed to have some specific or important symptoms or signs of thyroid disease, we have found great percentages of most of thyroid disorders. We recorded 120 cases with subclinical hypothyroidism out of 569 subjects (21.1%), 23 cases with overt hypothyroidism (4%), 11 cases with subclinical hyperthyroidism (2%), and 4 cases with overt hyperthyroidism (0.7%). In subclinical hypothyroidism, the mean TSH (m IU/L) was highly significantly increased in group Ib (5.591.51) when compared to normal TUV, group Ia (1.530.77), p value was <0.0001. Also there was no significant difference between the group of subclinical hypothyroidism in TUV (group Ib) and normal TUV group (Ia) as regards the levels of FT4 & FT3 (p mol/L). The mean values were 14.181.44 & 13.52.24 for FT4 in groups Ia & Ib respectively. The mean values were 5.30.43 & 4.951.38 for FT3 in groups Ia & Ib respectively. When we compared mean values for TSH in TUV normal group (1.530.77) and subgroups of hospital attendants (groups IIa, IIb, IIc, IId, IIe, IIf), there were highly significant differences between the normal TUV group and hospital groups (values were; 2.341.06, 8.096.53, 8.676.67, 44.4330.02, 0.0620.046, and 0.06 0.04 respectively) (p<0.0001). In the same way when we compared mean values for FT4 in TUV normal group (14.181.44) and groups including hospital attendants, there were highly significant differences between the normal TUV group and hospital groups except IIa and IIc (values were; 14.12.36, 10.920.62, 14.112.02, 8.763.36, 17.202.7 & 24.441.33 respectively, p<0.0001).
Also, when we compared mean values for FT3 in TUV normal group (5.30.43) and groups including hospital attendants, there were highly significant differences between the normal TUV group and all hospital subgroups except IIf (values were; 4.810.85, 4.390.64, 4.770.86, 3.901.45, 4.84 0.95 & 5.20.92 respectively p<0.0001 for all groups & =0.008 for group IIe). In conclusion, the use of highly sensitive third generation immunoassay like ECLIA was greatly useful in reporting a percentage of 5.1% for subclinical hypothyroidism among TUV which is a homogenous group of young female volunteers without any specific thyroid symptoms (group Ib). Also a percentage of 21.1% for subclinical hypothyroidism in larger heterogeneous group (group IIc in group II). The difference between these two percentages in the 2 groups was highly significant (p<0.0001). We also reported percentages of 4% for overt hypothyroidism (group IId) and 7.2% for those with high TSH and border line thyroid hormones (group IIb). A total percentage of hypothyroidism (subclinical and overt) was 25.1% which was also highly significant different from cases reported in TUV. Also, the sum of percentages of cases with high TSH in group II (32.3%) was highly significant from cases with high TSH in TUV group. Thus, we can suggest that the age period of 20 to 30 years could be the period that we should screen people regularly for subclinical hypothyroidism or at least at ending the stage of secondary school education in order to face this silent pandemic and prevent its major complications. We also reported percentage of 2% for subclinical hyperthyroidism and 0.7% for overt hyperthyroidism in group II, but no cases of hyperthyroidism (subclinical- or overt) in TUV group. Also, hypothyroidism was found to be the main thyroid dysfunction when compared to hyperthyroidism in group II (32.3% versus 2.7%) and also in group I (5.1% versus 0%). The mean values for TSH in all subgroups included in group II showed highly significant differences when compared with the normal control group (group Ia). The mean values for thyroid hormones (FT4 & FT3) showed significant differences in some but not all subgroups in group II when compared with normal control. Thus, this TSH assay is the single sensitive first-line test for accurate early diagnosis and differentiation of thyroid dysfunctions in clinical laboratory. In addition to its high sensitivity, it is also of good specificity and wide measuring rang. It is quit rapid, simple, fully automated method and needs no frequent calibration.
506

11- NICOLOFF J.T. and SPENCER C.A.: The use and misuse of the sensitive thyrotropin assays. J. Clin. Endocr. Metab., 71: 553-8, 1990. 12- SURKS M.I., CHOPRA I.J., MARIASH C.N., NICOLOFF J.T. and SOLON D.H.: American Thyroid Association Guidelines for the use of laboratory tests in thyroid disorders. JAMA, 263: 1529-32, 1990. 13- KEFFER J.H.: Preanalytical Considerations in Testing Thyroid Function. Clinical Chemistry, 42 (1): 125-35, 1996. 14- LADENSON P.W.: Optimal laboratory testing for diagnosis and monitoring of thyroid nodules, goier and thyroid cancer. Clin. Chem., 42: 183-7, 1996. 15- SALLER B., BRODA N., HEYDARIAN R., GORGES R. and MANN K.: Utility of third generation thyrotropin assays in thyroid function testing. Theme E. Journals, 106: s29-s33, 1998. 16- BLACKBURN G.F., SHAH H.P., KENTEN J.H., LELAND J., KAMIN R.A., LINK J., et al.: Electrochemiluminescence development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem., 37: 1534-9, 1991. 17- RAWLINS M.L. and ROBERTS W.L.: Performance characteristics of six third generation assays for thyroidstimulating hormone. Clin. Chem., 50: 2338-44. Epub 2004 Oct. 18- UTIGER R.D.: Subclinical hyperthyroidism - just as low serum thyrotropin concentration, or something more? N. Engl. J. Med., 331: 1302-3, 1994. 19- CANARIS G.J., MANOWITZ N.R., MAYOR G. and RIDGWAY C.: The Colorado Thyroid Disease Prevalence Study. Arch. Intern. Med., 160: 526-34, 2000. 20- TUNBRIDGE W.M.G., EVERED D.C., HALL R., APPLETON D., BREWISM, CLARK F., EVANS J.G., YOUNG E., BIRD T. and SMITH P.A.: The spectrum of thyroid disease in the community: The Whickham survey. Clin. Endocrinol., 7: 481-93, 1977. 21- VANDERPUMP M.P.J.: The epidemiology of thyroid diseases in: Braverman L.E., Utiger R.D., eds. Werner and Ingbar'sThe Thyroid: A. Fundamental and Clinical Text. 2005, 9th edn, pp 398-406. J.B. Lippincott-Raven, Philadelphia. 22- KANAYA A.M., HARRIS F., VOLPATO S., PREZSTABLE E.J., HARRIS T. and BAUER D.: Association between thyroid dysfunction and total cholesterol level in an older biracial population. The Health, Aging and Body Composition Study. Arch. Intern. Med., 162: 7739, 2002. 23- GUSSEKLOO J., VAN EXEL E., D.E. CRAEN A.J.M., FRLICH M. and WESTENDORP R.G.J.: Thyroid status, disability and cognitive function, and survival in old age. JAMA., 292: 2591-9, 2004. 24- AGHINI-LOMBARDI F., ANTONANGELI L., MARTINO E., VITTI P., MACHERINI D., LEOLI F., RAGO T., GRASSE L., VALERIANO R., BALESTRIERI A. and PINCHERA A.: The spectrum of thyroid disorders in an iodine-deficient community: The Pescopagano Survey. J. Clin. Endocrinol Metab., 84: 561-6, 1999.
Recommendations: We recommend performing further studies screening larger population samples in KSA that can reveal differences in incidence of thyroid dysfunction among different age groups and also measuring serum thyroid antibody concentrations in those different groups. Also, the influence of dietary iodine intake on the epidemiology of thyroid dysfunction should be studied in more detailed further studies in order to prevent this silent pandemic by treatment that potentially could decrease the risk of deaths from cardiovascular diseases in severe cases or at least reverse the mild abnormalities in mild cases. Also, we highly recommend a large study for establishing own reference ranges for thyroid hormones in Saudian population. References
1- Cecil Textbook of Medicine, 19th Edition, W.B. Saunders Company, 1248-71, 1992. 2- BALOCH Z., CARAGON P., CONTE DEVOLX B., et al.: Laboratory medicine practice guidelines: Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid., 13: 57-67, 2003. 3- LARSEN P.R., DVIES T.R., SCHLUMBERGER M. and HAY I.D.: The thyroid gland. in: Larsen P.R., Kronenberg H.M., Melmed S., Polonsky K.S., eds. Williams textbook of endocrinology, 10th ed. Philadelphia: WB Saunders, 331-491, 2003. 4- WHEELER M.H. and LAZARUS J.H.: Diseases of Thyroid. London, Glascow, Weinhem, New York, Tokyo, Melbourne, Madras: Chapman and Hall Medical, 10915, 1994. 5- EKINS R.P.: Measurement of free hormones in blood. Endocr. Rev., 11: 5-46, 1990. 6- EKINS R.P. and ELLIS S.M.: The radioimmunoassay of free thyroid hormones in serum. In Robbins J., Braverman L.E. (eds). Thyroid research, Proceedings of the Seventh international Thyroid Conference, Boston. Amsterdam, Excerpta Medica., 597, 1975. 7- HOLLOWELL J.G., STAEHING N.W., FLANDERS W.D., HANNON W.H., GUNTER E.W., SPENCER C.A. and BRAVERMAN L.E.: Serum TSH, T4, and thyroid antibodies in the United States Population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J. Clin. Endocrinol. Metab., 87 (2): 48999, 2002. 8- FATOURECHI V.: Subclinical Hypothyroidism: How should it be managed? Treat. Endocrinol., 1: 211-6, 2002. 9- WANG C. and CRAPO L.M.: The epidemiology of thyroid disease and implications for screening. Endocrinology and Metabolism Clinics, 26 (1): 189-218, 1997. 10- MOHANTY S., AMRUTHLAL W., REDDY G.C., et al.: Diagnostic strategies for subclinical hypothyroidism. Indian Journal of Clinical Biochemistry, 23 (3): 279-82, 2008.

25- KNUDSEN N., JRGENSEN T., RASMUSSEN S., CHRISTIANSEN and PERRILD H.: The prevalence of thyroid dysfunction in a population with borderline iodine deficiency. Clin. Endocrinol., 51: 361-7, 1999. 26- VANDERPUMP M.P.J. and TUNBRIDGE W.M.G.: Epidemiology and Prevention of clinical and subclinical hypothyroidism. Thyroid, 12: 839-47, 2002. 27- BIONDI B. and COOPER D.C.: The clinical significance of subclinical thyroid dysfunction. Endocr. Rev., 29: 7613, 2008. 28- AKBAR D.H., AHMED M.M. and HIJAZI N.A.: Subclinical hypothyroidism in elderly women attending an outpatient clinic. Med. Sci. Monit., 10 (5): C.R.229-32, 2004. 29- LAMFON H.A.: Thyroid Disorders in Makkah, Saudi Arabia. Ozean Jounal of Applied Sciences, 1 (1): 55-8, 2008. 30- TAHA I. and AL-HAZMI J.: Prevalence of overt and
507
subclinical hypothyroidism and associated maternal and fetal complications among pregnant women attending two referral hospitals in Saudi Arabia. (Accepted and presented at AACE 21st congress 12-16th April Sandiego USA), 2011. 31- COOPER D.S.: Subclinical hypothyoidism. N. Engl. J. Med., 345: 260-5, 2001. 32- MCGROGAN A., SEAMAN H.E., WRIGHT J.W. and DE VRIES C.S.: The incidence of autoimmune thyroid disease: A systematic review of the literature. Clin. Endocrinol, 69: 687-96, 2008. 33- ROSS D.S.: Laboratory assessment of thyroid function www.uptodate.-com, online 11.1, 2002. 34- SPENCER C.A., LOPRESITI J.S., PATEL A., et al.: Applications of a new chemiluminometric thyrotropin assay to subnormal measurement. J. Clin. Endocrinol Metab., 70: 453-60, 1990.
Comparison between Body Image and Self-Esteem Among Female Nursing Students in Three Different Arab Countries
NABILA TAHA, D.N.Sc.*; ESSMAT MANSOUR, D.N.Sc.**; AMAL SOBHY, D.N.Sc.*** and ENTISAR M. YOUNESS, D.N.Sc.*
The Departments of Obstetrics & Gynecological Nursing*, Faculty of Nursing, Assiut University; Medical Surgical Nursing**, Tanta University and Psychiatric & Mental Health Nursing***, Port-Said University.
Abstract
Current western culture promotes standards of beauty and success which focus on physical attractiveness. These standards can create feeling of inadequacy and body dissatisfaction. Traditionally, women have responded most strongly to cultural messages of bodily attractiveness, there by experiencing greater body dissatisfaction than men. Failure to achieve the ideal has been shown to lead to decreased self-esteem which compounds body dissatisfaction and body image disturbance. Therefore; this study aimed at assessing and comparing the level of body image and self-esteem among three communities based sample from Riyadh- Kindom of Saudia Arabia (KSA), Hadhramout-Yemen, and Assiut-Egypt, 300 female students were recruited, 100 female students from each country. The study was carried out in three different settings; Riyadh Female Health Science Collage-KSA, and Faculty of Nursing, Hadhramout University of Science and Technology-Yamen, Faculty of Nursing, Assiut University-Egypt. Tools for data collection included; body image scale, which developed by El-Desouki. This scale consisted of 20 statements about positive and negative sensation of body image, Rosenberg self-esteem scale, this scale consisted of 10 statements about the positive and negative sensation of self-esteem and interview Questionnaire which developed by the investigators, it includes information about student's age, residence, marital condition, economic condition, parent's educational level, number of brothers and sisters and their orders among them. Results of this study revealed that positive body image constitutes 86% in KSA, 84% from Egypt while 73% from Yemen. The majority of the students in the three countries had high self esteem (78% in KSA, 96% in Egypt and 84% in Yemen). The study also found that positive perception of body image is associated with high self esteem (85.9%, 84.8% and 69.0%) among the studied students in KSA, Egypt and Yemen respectively), fortunately; negative perception of body image is associated with low self esteem in (13.6%, 15.2% and 6.2%) among students of KSA, Egypt and Yemen respectively. In conclusion high level of self esteem positively correlated with body image. Age, parents education, marital status, numbers of brothers and sisters are significantly correlated to body image and self esteem. This study recommended another informative Correspondence to: Dr. Entisar Mohammad Youness Lecturer Obstetrics and Gynecological Nursing, Faculty of Nursing, Assuit University, E. mail: entisarmohamedyones@yahoo.com
study to investigate other dimensions of body image and self esteem in multi dimensional settings and studying the healthy lifestyle choices which are also a key to improve body image. Key Words: Body image Self esteem Body image scale Self esteem scale Female nursing students.
Introduction IN the light of the sudden and rapid changes in physical growth and psychological development among adolescents it was reported that body image related problems have become a critical determinant of nutritional status that place adolescents, particularly girls, as one of the nutritionally vulnerable groups. Evidences have shown that negative body image is significantly linked to various health issues, including a spectrum of disordered eating, low self esteem, depression and unhealthy weightloss practices [1,2]. Body image is the person's perception of his or her physical self. This perception includes conscious and unconscious perceptions of physical attributes and functioning, emotions, and sensation, it extends beyond the body to include objects such as clothing or items used in work [3]. A person's body image forms and changes as he or she develops, and is closely tied to self esteem and identity, both of which are threatened by a change in body image. Body image also is influenced by cultural beliefs and values and massages one receives from others [4]. Body image is changed as one progress through the different developmental stages of life. Persistent preoccupation with one's body image can impair emotional and cognitive development, interfere with interpersonal relationships, and place an individual at risk for the development of an eating disorder [5]. Any change in body image has destroyed all of his or her strengths. The client may
509
510
Comparison between Body Image & Self-Esteem Among Female Nursing Students
feel incapable of succeeding at anything and may need encouragement to attempt activities. Activities within the client's abilities provide opportunities for success. Positive feedback can enhance self esteem. Clients with low self esteem dont benefit from flattery or undue praise [3]. Negative body image is a serious issue during adolescence and should be duly addressed. Although body image has been increasingly studied over the last half century, no consensus was found in the definition of the body image concept. However, body image scholars [6] had agreed that body image comprises a multi-dimensional construct with various dimensions. One of the most common dimensions that have been described is body dissatisfaction, which is used interchangeably with negative body image or body image disturbance. Overlooking in the body image concept and failure to distinguish the various dimension of body image may hinder the important role of body image in population health and well-being. So, a thorough understanding of the body image concept is crucial in determining the etiology, prevention and treatment of body image and its related problems, particularly eating disorders and obesity [7]. As body image encompasses a complex and multi-dimensional construct, [8] recommended that multi scales should be used to assess body image. However, this may raise the issue of whether the items of the scales are overlapping to the point of redundancy. With a positive or healthy body image, a woman has a real perception of her size and shape. She also feels comfortable with her body. With a negative body image, a woman has a distorted perception of her shape and size, compares her body to others, and feels shame and anxiety about her body. Being unhappy with your body can affect how you think and feel about yourself as a person. A poor body image can lead to emotional distress, low self-esteem, unhealthy dieting habits, anxiety, depression, and eating disorders. Developing a positive body image and a healthy mental attitude is crucial to a woman's happiness and wellness [3]. Aim of the study: The aim of this study was to assess and compare the level of body image and self esteem of the female nursing students in three different Arabic countries (Saudi Arabia, Egypt and Yemen). Research question: What are the differences between the level of body image and self-esteem among female nursing students in the three different Arab Countries?
Subjects and Methods Research design: Comparative research design was utilized in this study. Settings: The study was carried out in three different setting; Faculty of Nursing, Assiut University, Egypt, Riyadh Female Health Science Collage, Kingdom of Saudia Arabia, and Faculty of Nursing, Hadhramout University of Science and Technology, Yemen. These faculties are governmental faculties (Ministry of Higher Education), and encompass multi-residential studies. Subjects: Subjects consisted of 300 female nursing students from three different Arab Countries, 100 students from each one. Their age ranges between 18 and 30 years. As the study involved only adolescent girls, the sample is limited by sampling location whereby these girls had became physically and psychologically mature, so all subjects were from the second year students in the three faculties which involved on the average of 100 female students. Tools of data collection: Data were collected using a self-administered questionnaire which has been translated into Arabic language. Three tools were utilized in this study: 1- Interview questionnaire: It includes information about sociodemographic characteristics such as student's age, residence, marital status, economic status, parents educational level, number and orders of brothers & sisters. 2- Body image scale: Body image scale which was developed by ElDesouki [9] were used to assess students body image. This is a factorial derived measure of male and female body esteem. This scale consisted of 20 statements about positive and negative sensation of body image. For example I accept my shape as it is. Items were rated on a five-point Likert scale, from strongly negative feelings to strongly positive feelings. Each scoring response is classified to 1-5 degrees: 1 for never, 2 for rare, 3 for sometimes, 4 for mostly, 5 for always. In total score for each student the high score indicates positive body image and the low score indicates negative body image.
Nabila Taha, et al.
511
3- Self-esteem scale: Self-esteem was assessed through using a Rosenberg self-esteem scale (1965) [10]; this scale consisted of 10 statements about the positive and negative sensation of self-worth, an example of which is I feel that I have a number of good qualities. There were five positive statements and five negative statements, with four-point Likert scale ranging from strongly agree to strongly disagree Each scoring response is classified to 14 degrees. 1 degree is assigned for strongly agree, 2 degrees for agree, 3 degrees for disagree, and 4 degrees for strongly disagree. In total score for each student the high score indicates high selfesteem and the low score indicates low self-esteem. Content validity: It was established by panel of 5 experts who reviewed the instruments for clarity, relevance, comprehensiveness, understanding, applicability and easiness. Pilot study: A pilot study was conducted on 10 students from each country, to test feasibility of tools and time required to be applied. Simple modification was done of some items of the interview questionnaire sheet that they were not consistent with this study. Administrative design: Before the conduction of the pilot study as well as the actual study, an official permission was obtained from the dean of each collage in each country. Procedure: The official permission was obtained from the research ethical committee in the three nursing faculties in the three countries to approve this study. The students were gathered in the faculty class for each session of data collection. An information sheet explaining the study purpose and its protocol was disseminated and explained before informed consent was obtained from the students. Data were collected using a self-administered questionnaire and the tools utilized to collect the desired data were explained sufficiently. The students were interviewed in groups, 25 students in each session in their classes for about 20 minutes. The duration for data collection lasts for 3 months precisely from April to June, 2010. Ethical consideration: The study protocol was approved by pertinent research and ethical committees. Informed consent was taken from every girl before inclusion in the
study. No health hazards were present. Participants were assured that all their data are highly confidential, anonymity was also assured through assigning a number for each student instead of names to protect their privacy. Data was only available to the researchers and the participants. Statistical analysis: Collected data were coded and analyzed. Descriptive statistics for the variables were calculated. Variables were compared using chi-square test. The variables were significant at p-value <0.05. All the statistical analysis was performed using SPSS package version 11. Results Table (1) Describes the socio-demographic characteristics of the students. It shows that 91%, 68% and 88% of the students were in the age group from 20 to less than 25 years in KSA, Egypt and Yemen respectively. Most of mothers and fathers education in three countries had basic education. There is a highly statistical significant difference between the three countries regarding age (X 2= 41.774, p=0.000) and mothers education (X 2 = 18.78, p=0.005) while there are no statistically significant differences between them as regards fathers education (X2=9.006, p=.170). Regarding mothers job, the majority of mothers in the three countries were housewives (73%, 77% and 89%) in KSA, Egypt and Yemen respectively. In relation to place of residency, it was found that 93% of students in KSA, 35% of students in Egypt and 87% of students in Yemen are coming from urban areas and the majority of students had intermediate family income in the three countries (79% from KSA, 92% from Egypt and 61% from Yemen). There is a statistical significant difference between the three countries regarding mothers job (X2= 8.560, p=0.014), place of residency (X2=100.202, p=0.000) and family income/month (X2=56.434, p=.000). Also Table (1) represented that about half of the females had from 1-3 brothers and sisters (51%, 52% and 46%) and more than half of them in the medium of birth order (57%, 55% and 56%) in KSA, Egypt and Yemen respectively. Regarding marital status, about one quarter of the students in KSA (24%), only 3% of students of Egypt, while 86% of Yemen were married. There is a highly statistical significant differences between the three countries regarding number of brothers and sisters (X2=26.775, p=0.000), marital status (X2=161.041, p=0.000) while there is no statistically significant
512
differences between them regarding birth order (X2=.648, p=.958). Distribution of the study sample according to their self esteem represented in Table (3), the subjects were grouped according to their scores of the level of self esteem into two groups (low and
high self esteem). The majority of the students in the three countries had high self esteem (78% in KSA, 96% in Egypt and 84% in Yemen) while the rest of subjects had low self esteem (22% of KSA students, 4% of Egypt students and 16% of Yemen students) with a highly statistically significant difference (X2=13.953, p=.001).
Table (1): Distribution of the subjects according to their socio demographic characteristics. Country Socio-demographic characteristics Age (years): <20 2025-30 Mothers education: Illiterate Basic Secondary University Fathers education: Illiterate Basic Secondary University Mothers job: Housewife Employed Place of Residency: Urban Rural Family income/month: High Intermediate Low Number of brothers & sisters: Null 1-3 4-6 More than 7 Birth Order: First Medium The last Marital status: Single Married Divorced and widow (ed) KSA (100) N 4 91 5 % 4 91 5 Egypt (100) N 32 68 0 % 32 68 0 Yemen (100) N 7 88 5 % 7 88 5 Total (300) N 43 247 10 % 14.3 82.4 3.3 Chi-Square Test p-value
41.774
.000 S
20 36 20 24
20 36 20 24
28 32 28 12
28 32 28 12
21 53 13 13
21 53 13 13
69 121 61 49
23.0 40.4 20.3 16.3
18.787
.005 S
11 34 28 27
11 34 28 27
13 31 30 26
13 31 30 26
6 38 40 16
6 38 40 16
30 103 98 69
10.0 34.3 32.7 23.0
9.006
.170 N.S
73 27
73 27
77 23
77 23
89 11
89 11
239 61
79.7 20.3
8.560
.014 S
93 7
93 7
35 65
35 65
87 13
87 13
215 85
71.7 28.3
100.202
.000 S
5 79 16
5 79 16
0 92 8
0 92 8
31 61 8
31 61 8
36 232 32
12.0 77.3 10.7
56.434
.000 S
3 3 51 43
3 3 51 43
0 23 52 25
0 23 52 25
2 25 46 27
2 25 46 27
5 51 149 95
1.7 17.0 49.6 31.7
26.775
.000 S
24 57 19
24 57 19
23 55 22
23 55 22
21 56 23
21 56 23
68 168 64
22.7 56.0 21.3
.648
.958 N.S
73 24 3
73 24 3
96 3 1
96 3 1
14 86 0
14 86 0
183 113 4
61.0 37.7 1.3
161.041
.000 S
Nabila Taha, et al.

Table (2): Distribution of the students according to their body image perception. Country Body image KSA (100) 86 (86%) 14 (14%) Egypt (100) 84 (84%) 16 (16%) Yemen (100) 73 (73%) 27 (27%) Total (300) 243 (81%) 57 (19%) Chi-Square Test 6.368 p.041 S
513
Positive perception Negative perception
Table (3): Distribution of the subjects according to self esteem. Country Self esteem KSA (100) Low self esteem High self esteem 22 22% 78 78% Egypt (100) 4 4% 96 96% Yemen (100) 16 16% 84 84% Total (300) 42 14% 258 86% Chi-Square Test
13.953 p.001 S
The relation between self esteem and body image among the studied students is clarified in Table (4), it was found that 85.9%, 84.4% and 69.0% of the subjects with a high self esteem had positive body image among the studied students in KSA, Egypt and Yemen respectively, with a statistically significant difference (X 2 =13.58, p=.001) between groups, while 75.0%, 84.8% and 93.8% of the subjects with a low self esteem had positive body image among the studied students in Saudi, Egypt and Yemen respectively, with no statistically significant differences. Comparison between sociodemographic characteristics in relation to body image in the three countries was presented in Table (5). It was found that students' age from 20-25 years is correlated positively with positive perception of the body image among students of KSA, Egypt and Yemen (94.2%, 65.5% and 86.3%) respectively with a highly statistical significant differences between groups (X2=34.75, p=.000). Fortunately mother's
Table (4): Relation between self esteem and body image.
education, urban areas, mediated family income, birth order among sisters and brothers and marital status all are associated with positive perception of body image with a highly statistical significant differences between groups owing this to the communities differences. Concerning the comparison between sociodemographic characteristics in relation to selfesteem in the three countries Table (6) presented that, students' age from 20-25 years is correlated positively with high selfesteem among students of the thre countries KSA, Egypt and Yemen (89.7%, 69.8% and 85.7%) respectively with a highly statistical significant differences between groups (X 2 =31.98, p=.000). Fortunately mother's job, urban areas, mediated family income, number of sisters and brothers and marital status all are associated with high self esteem with a highly statistical significant differences between groups.
Student self esteem High self esteem Student body image Saudia (78) N Positive perception Negative perception 67 11 % 85.9 14.1 Egypt (96) N 81 15 % 84.4 15.6 Yemen (84) N 58 26 % 69.0 31.0 13.58 p=.001 S Test 2 Saudia (22) N 19 3 % 75.0 25.0 Low self esteem Egypt (4) N 3 1 % 84.8 51.6 Yemen (16) N 15 1 % 93.8 6.2
514
Table (5): Comparison between students sociodemographic characteristics in relation to the three Arab Countries. Student body image Socio-demographic Characteristics Positive perception Al-Riyadh (86) N Age (years): <20 2025-30 Mothers education: Illiterate Basic Secondary University Fathers education: Illiterate Basic Secondary University Mothers job: Housewife Employed Place of Residency: Urban Rural Family income/month: Sufficient Mediate Insufficient Number of daughters and sons: Null 1-3 4-6 More than 7 Birth order: First Medium The last Marital status: Single Married Divorced and widow (ed) % N Assiut (84) % Yemen (73) N % Test 2 Negative perception Al-Riyadh (14) N % N Assiut (16) % N Yemen (17) %
4 81 1
4.7 94.2 1.2
29 55 0
34.5 65.5 0
7 63 3
9.6 86.3 4.1
34.75 p=.000 S
0 10 4
0 71.4 28.6
3 13 0
18.8 81.3 0
0 25 2
0 92.6 7.4
15 30 19 22
17.4 34.9 22.1 25.6
23 28 23 10
27.4 33.3 27.4 11.9
15 41 10 7
20.5 56.2 13.7 9.6
19.23 p=.004 S
5 6 1 2
35.7 42.9 7.1 14.3
5 4 5 2
31.3 25.0 31.3 12.4
6 12 3 6
22.2 44.4 11.1 22.2
7 32 22 25
8.1 37.2 25.6 29.1
11 26 26 21
13.0 31.0 31.0 25.0
4 32 28 9
5.5 43.8 38.4 12.3
11.65 p=.070 N.S
4 2 6 2
28.6 14.3 42.9 14.3
2 5 4 5
12.4 31.3 25.0 31.3
2 6 12 7
7.5 22.2 44.4 25.9
61 25
70.9 29.1
65 19
77.4 22.6
65 8
89.0 11.0
7.81 p=.020S
12 2
85.7 14.3
12 4
75.0 25.0.
24 3
88.9 11.1
84 2
97.7 2.3
30 54
35.7 64.3
61 12
83.6 16.4
87.85 p=.000S
9 5
64.3 35.7
5 11
31.3 68.7
26 1
96.3 3.7
2 68 16
2.3 79.1 18.6
0 76 8
0 90.5 9.5
24 44 5
32.9 60.3 6.8
58.03 p=.000 S
3 11 0
21.4 78.6 0
0 16 0
0 100 0
7 17 3
25.9 63.0 11.1
3 2 44 37
3.5 2.3 51.2 43.0
0 21 39 24
0 25.0 46.4 28.6
2 22 30 19
2.7 30.1 41.1 26.0
27.22 p=.000 S
0 1 7 6
0 7.1 50.0 42.9
0 2 13 1
0 12.5 81.3 6.3
0 3 16 8
0 11.1 59.3 29.6
19 51 16
22.1 59.3 18.6
20 46 18
23.8 54.8 21.4
17 38 18
23.3 52.1 24.7
1.12 p=.891 N.S
5 6 3
35.7 42.9 21.4
3 9 4
18.8 56.2 25.0
4 18 5
14.8 66.7 18.5
65 19 2
75.6 22.1 2.3
81 2 1
96.4 2.4 1.2
12 61 0
16.4 83.6 0
124.08 p=.000 S
8 5 1
57.1 35.7 7.1
15 1 0
93.7 6.3 0
2 25 0
7.4 92.6 0
Nabila Taha, et al.
515
Table (6): Comparison between students sociodemographic characteristics in relation to selfesteem in the three different Arab Countries. Student self esteem Socio-demographic Characteristics Low self esteem Al-Riyadh (22) N Age (years): <20 2025-30 Mothers education: Illiterate Basic Secondary University Fathers education: Illiterate Basic Secondary University Mothers job: Housewife Employed Place of Residency: Urban Rural Family income/month: Sufficient Mediate Insufficient Number of sisters and brothers: Null 1-3 4-6 More than 7 Birth order: First Medium The last Marital status: Single Married Divorced and widow (ed) 1 21 0 2 11 3 6 1 9 9 3 14 8 20 2 2 18 2 % 4.5 95.5 0 9.1 50.0 13.6 27.3 4.5 40.9 40.9 13.7 63.6 36.4 90.9 9.1 9.1 81.8 9.1 N 3 1 0 0 1 2 1 1 0 2 1 2 2 1 3 0 4 0 Assiut (4) % 75.0 25.0 0 0 25.0 75.0 25.0 25.0 0 50.0 25.0 50.0 50.0 25.0 75.0 0 100 0 Yemen (16) N 0 16 0 0 15 0 1 0 13 2 1 14 2 10 6 0 16 0 % 0 100 0 0 93.7 0 6.3 0 81.3 12.5 6.3 87.5 12.5 62.5 37.5 0 100 0 22.22 p=.000 S 14.71 p=.023 S Test 2 Al-Riyadh (78) N 3 70 5 18 25 17 18 10 25 19 24 59 19 73 5 3 61 14 % 3.8 89.7 6.5 23.1 32.1 21.8 23.1 12.7 32.1 24.4 30.8 75.6 24.4 93.6 6.4 3.8 78.2 17.9 N 29 67 0 28 31 26 11 12 31 28 25 75 21 34 62 0 88 8 High self esteem Assiut (96) % 30.2 69.8 0 29.2 32.3 27.1 11.5 12.5 32.3 29.2 26.0 78.1 21.9 35.4 64.6 0 91.7 8.3 Yemen (84) N 7 52 5 21 38 13 12 6 25 38 15 75 9 77 7 31 45 8 % 8.3 85.7 6.0 25.0 45.2 15.5 14.3 7.1 29.8 45.2 17.9 89.3 10.7 91.7 8.3 36.9 53.6 9.5
13.39 p=.037 S
3.58 p=.167 N.S 9.32 p=.009 S 4.02 p=.403 N.S
3 0 11 8 3 13 6 17 5 0
13.6 0 50.0 36.4 13.6 59.1 27.3 77.3 22.7 0
0 0 2 2 1 3 0 4 0 0
0 0 50.0 50.0 25.0 75.0 0 100 0 0
0 10 3 3 1 9 6 0 5 11
0 62.4 18.8 18.8 6.3 56.2 37.5 0 31.3 68.7
22.69 p=.001 S
0 3 40 35 21 44 13 56 19 3
0 3.8 51.3 44.9 26.9 56.4 16.7 71.8 24.4 3.8
0 23 50 23 22 52 22 92 3 1
0 24.0 52.0 24.0 22.9 54.2 22.9 95.8 3.1 1.0
2 15 43 24 20 47 17 0 9 75
2.4 17.8 51.2 28.6 23.8 56.0 20.0 0 10.7 89.3
2.85 p=.583 N.S 11.04 p=.004 S
Discussion Over the past ten to twelve years, there has been an increase of study in the area of self esteem; self-esteem is the judgment of worth an individual assigns to his or her self [11]. In the areas of self concept and self esteem there has been a particular emphasis on college students in their second years
of studies. In this population self esteem appears to correlate with body image, the overall perception on individual has about their physical body [12]. The aim of this study was to assess and compare the level of body image and self esteem of the females nursing students in three different Arabic countries (KSA, Egypt and Yemen).
516
The study results have shown that 79.8% of the female with positive body image had high self esteem; this finding expresses a strong relationship between body image and self esteem. This is in agreement with Friedman and Haaga [13] who found in their research that, the participants who endorsed more depressive symptoms and reported lower levels of self esteem used proportionately more negative terms when describing themselves. Moreover, Dimitra [14] says that, for many women self esteem is based executively on their body image. On the other hand Trampe [15], supported the negative effects these perfect images have ones self esteem.She found in her study that, the more body dissatisfied women were the more they indicated that they compare their body with that of other women. It follows that if in fact these images can change the way women feel about themselves over time, they can be even more damaging to anyone who is already dissatisfied with his/her body. Mean while Killi [16] found a weaker relation between body image dissatisfaction and self esteem. The current study revealed that, the majority of the subjects were with positive body image, there is a contribution to this finding, in which most of Arabic females usually tend to reject external standards of beauty, essentially in an effort to protect their self esteem, by the virtue of satisfaction which is acquired from the religion, the Arabic females have been described as working with they have instead of trying to obtain the ideal beauty presented by the media, So they tend to positively accept their body as it is. In relation to the level of self esteem, high level of self esteem 96% was observed in the study sample of Egypt followed by Yemen 84% then KSA 78%, It may contributed to the nature of society in both Riyadh (KSA) and Yemen which is a male dominant society, and the female considered as dependant and follower to the male and in most occasions she losses her identity which will negatively affect her self esteem. This comes in line with Lightstone [17], who said that self esteem can be ever change and is not created based on fact but influenced by environmental, physical and physiological experiences; it is not inborn but learnt. The learning comes from family and peers but only reinforces what is learnt and experience culturally. Concerning the socio-demographic characteristics of the study sample, the findings illustrated that the percentage of the students with a positive body image is more higher than the percentage of
the students with a negative body image and were found between 20 to less than 25 years old. This result isn't in accordance with Yuerk et al. [18] who found no effect of age on the level of body image, but it is contradictory with other studies where they find a relation between age and body image [19] , and Arnet [20] , suggested that individuals experience some identity confusion during the middle adulthood (20-30 years), during this time females between the ages of 20-24 years old may feel negative body image about themselves. As regards to the fathers and mothers education, the study showed that, the percentage of fathers and mothers who had basic education were higher between subjects with a low self esteem, it may be due to the lack of satisfactory level of education which may affect the personality of the parents and the way of communications they used to relate with their daughters as a consequence it will affect the subjects way of thinking about the self concept and self esteem. Regarding the marital status in relation to body image, the present study found that, the single students constituted (65%,96.4% and 16.4%) of students who have positive body image of KSA, Egypt and Yemen students respectively with a highly statistical significant difference between them, (X 2 =124.08, p=.000) compared to (57.1, 93.7% and 7.4% of the married females who had negative body image. This finding is contradicting with the Dimitra [14] , findings which indicated that, marital status was related significantly with body image, married women having a more positive body image than women that were single, divorced or widowed. On the same line Spencer [21] found that married women accept more and view their bodies better than the women who are not married. Concerning the number of brothers and sisters in the family, the findings of the present revealed that most of the students who had 4-6 brothers and sisters were with a high self esteem. This comes in line with Dimitra [14] , who stressed that the different sources of social support (family members, parents, and friends) they all seemed important in developing healthy body image and self esteem. Conclusion: In conclusion high level of self esteem positively correlated with body image. Hence, female students with lower self-esteem were more likely to be at risk of negative body image. Age, parents education, marital status, numbers of brothers and sisters, all are significantly correlated to body image and self esteem.
Nabila Taha, et al.
517
9- EL-DESOUKI M.M.: Body Image Questionnaire, ElAnglo Egyptian Library, 2002. 10- ROSENBERG M.: Society and the adolescent self-image. Princeton University Press, Princeton, New York. USA, 1965. 11- LAURA S., and STORMER C.: Socio cultural variation the body image perceptions of urban adolescent females: Journal of Youth and Adolescent, 31 (6): 443-450, 2002. 12- Vallerand R.: On obsessive and harmonious Passion: Journal of Personality and Social Psychology, 85: 765767, 2003. 13- FRIEDMAN E. and HAAGA D.: Using hierarchical classes to analyze organization of the self concept. European Journal of Psychological Assessment, 23: 9-14, 2007. 14- DIMITRA M.: Body image in relation to self-esteem in a sample of Spanish women with early stage breast cancer. Psicooncologia, 2 (1): 103-116, 2005. 15- TRAMPE D., STAPEL D. and SIERO F.: On models and vases: Body dissatisfaction and proneness to social psychology, 92: 106-118, 2007. 16- KILLI E.: Body image partially mediates the relationship between obesity and psychological distress. Obesity Research, 10: 33-41, 2002. 17- LIGHTSTONE J.: Improving body image, 2005. http://www.edreferral.com /body image. htm. 18- YUREK D., FARRAR W. and ANDERSEN B.: Breast cancer surgery: Ring surgical groups and determining individual differences in postoperative sexuality and body change stress. J. Consult Clin. Psycho., 68: 69770, 2000. 19- MAYER A.: Psychosocial outcomes of breast-conserving surgery versus mastectomy; a meta- analytic review health Psycho., 16: 284-98, 1997. 20- ARNET J.: Emerging adulthood the winding road from teens through the twenties, 2004. http//www.joffereyamett. com/winding road.htm. 21- SPENCER S.: Concerns about breast cancer and relations to psychosocial well-being in a multiethnic sample. Health Psycho, 18: 159-68, 1999.
Recommendations: This study recommended: Another informative study is advised to investigate other dimensions of body image and self esteem in multi-dimensional setting. Studying the healthy lifestyle choices which are also a key to improve body image. Acknowledgement: We would like to acknowledge the help of our nursing students of the three Arab countries for their valuable participation. References
1- World Health Organization: Physical status. The use and interpretation of anthropometry, WHO Technical Series Report No. 854, Geneva, Switzerland, pp. 263-311, 1995. 2NEUMARK-SZTAINE D., PAXTON S.J., HANNAN P.J., HAINES J. and STORY M.: Does body dissatisfaction matter? Five year longitudinal associations between body satisfaction and health behaviors in adolescent females and males. J. Adolesec Health, 39: 244-251, 2006.
3- JUDITH M., SCHULTZ I. and SHEILA L. VIDE BECK: Disturbed Body Image: Lippincot's Manual of Psychiatric Nursing Care plans. Eighth ed. Lippincott Williams & Wilkins, pp. 359-360, 2009. 4- MELAND E., HAUUGLAND S. and BRIIDABLIK H.J.: Body image and perceived health in adolescence. Health Education Research, 223 (3): 342-350, 2007. 5- BENSING K.: Unveiling the mystery of body image. Advance for Nurses, 4 (7): 13-17, 2003. 6- CASH T.F.: Body image: Past, present and future. Body Image, 1: 1-5, 2004. 7- CHIN Y.S., TAIB M.N., SHARIFF Z.M. and LINKHOR G.: Development of multi-dimensional body image scale for Malaysian female adolescents Nutr. Res. Pract., 2 (22): 85-92, 2008. 8THOMPSON J.K.: The (mis) measurements of body image: Ten strategies to improve assessment for applied and research purposes. Body Image, 1: 7-14, 2004.
Effect of Cinnamon Beverage on Some Biochemical Aspects of Diabetic Male Albino Rats
MAGDA M. KHALIL, Ph.D.; SHADIA A. FREIG, Ph.D. and EMILY T. HANNA, Ph.D.
The Department of Nutritional Biochemistry and Metabolism, National Nutrition Institute, Cairo, Egypt
Abstract
Introduction: Cinnamon has been shown to improve the insulin receptor function in rats, which leads to enhanced cellular glucose uptake. Aim of the Study: This study was conducted to investigate the changes of blood glucose, hemoglobin [HB] and hematocrit [HCT], serum total lipids, triglycerides [TG], total cholesterol [TC], High density lipoprotein-cholesterol [HDL-C] and low density lipoprotein-cholesterol [LDL-C] level, serum urea nitrogen, serum uric acid, and serum creatinine in diabetic rats received cinnamon beverage orally. Experimental Design: Thirty adult male albino rats Sprague-Dawley strain weighing 160-180g 12 weeks old were divided into five groups each of six rats. The first group was negative control. The other groups were induced diabetic by alloxan injection. One of them was positive control fed standard diet only. The three other groups were fed standard diet and received cinnamon beverage orally 2.5, 5 and 7.5ml/day respectively during 4 weeks. At the end of the experiment blood samples were taken in centrifuge tubes then a part of blood was subjected to glucose, hemoglobin [HB] and hematocrit [HCT] determination. Serum was separated and subjected to biochemical analysis. Results: The results indicated that the lowest feed intake and body weight gain were found in group of rats received 7.5ml/day of cinnamon beverage orally. This group of rats recorded a reduction in blood glucose level, blood hemoglobin [HB] and hematocrit [HCT] values compared with positive control group but significantly higher in serum uric acid. Total cholesterol value showed a significant decrease and HDL-C significant increase by increasing the level of cinnamon beverage 2.5, 5 and 7.5ml/day. Conclusion: Cinnamon is commonly used in Egypt as a hot beverage in winter. It can be added to the foods to give a palatable taste and smell. The study showed a decrease of blood glucose, total cholesterol, LDL-C, improved the HDLC but lowered the HB and HCT values and increased serum uric acid. Key Words: Cinnamon Diabetic Hemoglobin Total lipid Urea, uric acid and creatinine. Correspondence to: Dr. Magda M. Khalil, The Department of Nutritional Biochemistry and Metabolism, National Nutrition Institute, Cairo, Egypt.
Introduction VARIOUS herbs and medicinal plants have been investigated. Cinnamon has been shown to be one of the most effective regulating blood glucose. Cinnamon extract has a moderate effect in reducing fasting blood glucose concentration in both diabetic patients and animals [1-5]. Cinnamon oil has been widely used as a flavoring agent and additive for centuries in the food industries. It has been used in seasoning chili sauces baked goods, and non alcoholic beverages. In addition, it is safe for human body and has a delicate aroma along with a sweet pungent taste [6]. The active compound of cinnamon have been reported as water soluble poly phenol type-A polymers [7,8,9], Cinnamaldehyde [10] and cinnamic acid [11]. Changes in life style; such as increase in energy intake and decrease in physical activity, are causing over weight and obesity and leading to an epidemic increase in type 2 diabetes [12]. Diabetes mellitus is one of the most significant chronic disease and a growing health problem in most countries [13,14,15]. It is metabolic disorder caused by an absolute or relative lack of insulin resistance of peripheral tissues in case of type 2 diabetes [16]. Cinnamon has been shown to improve the insulin receptor function in rats, which leads to enhanced cellular glucose uptake [3,13,14, 15,17]. Aim of the study: This study aimed to investigate the changes of some biochemical aspects of diabetic rats receiving cinnamon beverage orally. Material and Methods Cinnamon was obtained from local market at Cairo governorate. Alloxan pure chemical fine (BDH), was obtained from Sigma and used for inducing diabetes in this study. Sprague-Dawley strain male albino rats were derived from Vaccine
519
520
Effect of Cinnamon Beverage on Some Biochemical Aspects
and Immunity Organization, Hellwan farm, Cairo, Egypt. Diet: Standard diet was prepared to give the efficiency for maintaining and growth of experimental animals. Preparation of beverage: Beverage was prepared as follows: 10gm of cinnamon powder was dissolved in one liter of water, boiled for 10 minutes in conical flask with air condenser then cooled at room temperature. Experimental diet: Standard diet was prepared from the following ingredients per 100g as follows: Sun flower oil 10%, salt mixture 4% [18], vitamin mixture 1% [19], DL methionine 0.3%, cellulose 5% and cholin chloride 0.2%. As to represent 14% protein according to [20], the protein was added at the expense of starch. Experimental design: Thirty adult male albino rats Sprague-Dawley strain weighing 160-180g,12 weeks old were divided into five groups each of six rats. All groups were similar in total body weight, and housed individually in wire cages under healthy environmental conditions. Diabetes inducing: Diabetes was induced in normal healthy male albino rats by subcutaneous injection of alloxan 150mg/kg body weight according to [21] after that, blood samples were taken from optic vein to estimate fasting blood glucose. Rats having glucose level more than 170mg/dl were considered diabetic according to [22]. The first group (negative control) was fed standard diet only. The other four groups were induced diabetic by alloxan injection. The second group (positive control) received standard diet only. The third group was fed standard diet and received 2.5ml/day of cinnamon beverage by oral administration once per day by stomach tube for three weeks. The fourth group was fed standard diet and received 5ml/day of cinnamon beverage by oral administration once per day by stomach tube. The fifth group was fed standard diet and received 7.5ml/day of cinnamon beverage by oral administration once per day by stomach tube. At the end of the experimental period (3 weeks), all rats were sacrificed under ether anesthesia and blood samples were taken from hepatic portal vein after overnight fasting then a part of blood was subjected to glucose, hemoglobin (HB) and hema-
tocrit (HCT) determination. The rest of blood was left in centrifuge tubes at room temperature for 10 minutes and centrifuged at 4000 r.p.m. for 15 minutes. The serum was separated and subjected to biochemical analysis. Biochemical analysis: Determination of blood glucose: Enzymatic colorimetric method for glucose was determined according to [23]. Determination of hemoglobin [HB] and hematocrit [HCT]: Hemoglobin was determined by the cyanomethoglobin method according to [24] . Packed cell volume [P.C.V] and Hematocrit was measured according to [25]. Determination of total lipids: Total lipids were determined by colorimetric method using kits purchased from Egyptian American Co. for Laboratory services according to [26]. Determination of triglycerides: Quantitative enzymatic colorimetric determination of triglycerides in serum using kits Stanbio laboratory according to [27]. Determination of total cholesterol: Quantitative enzymatic colorimetric determination of total cholesterol in serum using kits Stanbio laboratory according to [28]. Determination of HDL-C: HDL-C level was determined in serum using kits Stanbio laboratory according to [29]. Determination of LDL-C: LDL-C was calculated according to [29] with a few restrictions, as follows:
LDL-C (mg/dl) = TC [HDL-C + VLDL-C]
Calculation of very low density lipoprotein cholesterol: VLDL-C was calculated as mg/dl according to the equation given by [29].
VLDL-C = (Triglycerides/5)
Determination of serum urea nitrogen: Serum urea was determined as described by [30]. Determination of serum uric acid: Serum uric acid was determined according to [31]. Determination of serum creatinine: Serum creatinine was determined according to the method described by [32]. Statistical analysis: The collected data were processed using the software SPSS (Statistical Package for Social Science, version 10), 2002. Independent F-tests one-way analysis of variance (ANOVA) and the least significant difference (LSD) were conducted according to [33].
Magda M. Khalil, et al.
521
Results and Discussion Table (1) showed that the highest feed intake was found in the negative control group (500g) than the other groups of rats received cinnamon beverage orally. The positive control group feed intake was (300g) while the lowest feed intake was found in group of rats received 7.5ml/day of cinnamon beverage orally (270g). These variations may be due to the palatability of feed and metabolic effect. The same trend was found in body weight gain BWG. It could be seen that groups of rats received 5 and 7.5ml/day of cinnamon beverage orally recorded significant reduction (p<0.01) in body weight gain (16.81.9g and 14.61.3g) respectively compared with negative control group (40.12.8g) and lower than positive control group [18.22.6g]. These variations may be due to the diabetic effect in which catabolic process was more than anabolic. Rats group received 2.5ml/day of cinnamon beverage orally ml/day showed improvement in body weight gain (20.52.3g) compared with positive control (18.22.6g) and other groups. This phenomenon may be due to the decrease in
feed intake. Also the beverage may be caused anorexia. Table (2) indicated that all groups of rats received different levels of cinnamon beverage orally ml/day recorded significant reduction (p<0.01) in blood glucose level compared to positive control group (1703.2mg/dl), while induced significant increase (p<0.01) compared to negative control group (80.43. 3mg/dl). Rats group received 2.5ml/ day of cinnamon beverage orally showed the highest value (143.41.9 mg/dl) in blood glucose level while the lowest value was found in group of rats received 7.5ml/day of cinnamon beverage orally (110.83.4mg/dl). This phenomenon may be due to cinnamon function which has been shown to improve the insulin receptor in rats, which lead to enhanced cellular glucose uptake [3,17]. Also may be due to the cinnamaldehyde, a main composition of cinnamon which has been shown to reduce the blood glucose level [10]. Also cinnamon may delay gastric emptying leading to a lower postprandial blood glucose concentration and feeling of hunger [34,35].
Table (1): Effect of receiving cinnamon beverage with different levels orally ml/day to diabetic rats on feed intake and body weight gain g (MeanS.D). Groups Parameters Feed intake g BWG g
* (p<0.05).
Control Negative 500 40.12.8

** (p<0.01).
Cinnamon orally ml/day p< 2.5 290 20.52.3 5 285 16.81.9 7.5 270 14.61.3 ** **
Positive 300 18.22.6
Table (2): Effect of receiving cinnamon beverage with different levels orally ml/day to diabetic rats on blood glucose mg/dl (MeanS.D). Groups Parameter Glucose mg/dl
* (p<0.05).
Control Negative 80.43.26 Positive 1703.2
Cinnamon orally ml/day p< 2.5 143.41.9 5 115.23.4 7.5 110.83.4 **
** (p<0.01).
Table (3) illustrated that Diabetic rats which received 2.5, 5 and 7.5ml/day of cinnamon beverage orally showed significant reduction (p< 0.01) in hemoglobin value (11.72.3, 9.62.3 and 9.11.8 ml/day) respectively compared with negative control group (16.51.8mg/dl) while rats group received 2.5ml/day of cinnamon beverage orally showed the highest hemoglobin value (11.72.3ml/ day) in treated rats and showed insignificantly lower value compared with positive control group (12.82.1mg/dl) in this parameters. Increasing the level of cinnamon beverage caused a decrease in
hemoglobin and hematocrit values. This observation may be due to that cinnamon contain phenol and poly phenol compounds [9] such as tannin which prevents iron absorption causing iron deficiency anemia. It could be noticed from Table (4) that serum urea nitrogen for the groups of rats received 5 and 7.5ml/day of cinnamon beverage orally showed significantly higher values in serum urea nitrogen (50.10.8 and 53.20.063mg/dl) respectively compared with both negative control group (34.1 3.1mg/dl) and rats group received 2.5ml/day of
522
cinnamon beverage orally (33.82.8mg/dl) while showed significantly lower value than positive control group (64.23.5mg/dl) at (p<0.01). This useful effect of cinnamon was due to its ability in improving renal function and protecting renal glomurlar against oxidative damage via increasing both levels and activities of antioxidants status [1]. The same trend was found in serum uric acid value. It could be seen that rats groups received 5 and 7.5ml /day of cinnamon beverage orally showed significantly higher value (3.10.063 and 3.1 0.063ml/dl) at (p<0.01) in serum uric acid compared with both negative control group (2.30.025mg/dl) and rats group received 2.5ml/day of cinnamon
beverage (2.40.090ml/dl) while showed insignificant difference than positive control group (3.2 0.102mg/dl]. This may be due to inhibiting liver in vivo activities of xanthine Dehydrogenase (XDH) and xanthine oxidase (XOD) [3]. It could be concluded from Table (5) that there is a significant decrease (p<0.01) in total cholesterol value in rats treated with 2.5, 5 and 7.5ml/day of cinnamon beverage orally (130.13.5, 113.10 .74 and 110.81.8mg/dl) respectively compared with positive control group (152.62.9mg/dl) however these values recorded a significant increase (p<0.01) as compared to negative control group (95.13.4mg/dl).
Table (3): Effect of receiving cinnamon beverage with different levels orally ml/day to diabetic rats on hemoglobin and hematocrit values (MeanS.D). Groups Parameters Hemoglobin g/dl Hematocrit g/dl
* (p<0.05).
Control Negative 16.51.8 49.53.8 Positive 12.82.1 38.44.1
Cinnamon orally ml/day p< 2.5 11.72.3 35.14.3 5 9.62.3 28.84.3 7.5 9.11.8 27.33.8 ** **
** (p<0.01).
Table (4): Effect of receiving cinnamon beverage with different levels orally ml/day to diabetic rats on urea uric acid and creatinine mg/dl (MeanS.D). Groups Parameters Control Negative Positive Cinnamon orally ml/day p< 2.5 5 7.5 Urea mg/dl 34.13.1 64.23.5 33.82.8 50.10.8 35.20.063 ** Uric acid mg/dl 2.30.025 3.20.102 2.40.090 33.10.063 3.10.063 ** Creatinine mg/dl 0.60.025 1.80.102 1.70.090 1.50.063 1.50.03 **
* (p<0.05). ** (p<0.01).
Table (5): Effect of receiving different levels of cinnamon beverage orally ml/day to diabetic rats on lipid pattern mg/dl (MeanS.D). Groups Parameters TC mg/dl TG mg/dl HDL-C mg/dl LDL-C mg/dl VLDL-C mg/dl
* (p<0.05).
Control Negative 95.13.4 58.82.8 42.52.2 32.30.82 11.76 Positive 152.62.9 85.52.5 29.42.8 99.20.83 17.1
Cinnamon orally ml/day p< 2.5 130.13.5 72.31.9 330.8 74.72.5 14.46 5 113.10.74 68.12.6 39.290.5 57.60.26 13.62 7.5 110.81.8 64.63.4 39.220.5 57.60.26 12.92 ** ** ** ** **
** (p<0.01).
Triglycerides; total cholesterol and LDL-C values showed the same trend with an increase in HDL-C level, it could be seen that significantly higher value (p<0.01) of HDL-C was found in treated rats compared with positive control group (29.482.8mg/dl) these values recorded significant decrease (p<0.01) in the mean values of HDL-C compared to negative control group (42.542.2mg/
dl). Group of rats received 2.5ml/day of cinnamon beverage orally has the lowest value of HDL-C (330.8mg/dl) of treated rats. This observation referred to cinnamon effect which contains antioxidant compounds such as water soluble poly phenol type-A polymers which have a positive role in enhancing insulin effectiveness and reducing its resistance, thus regulating the levels of serum lipids
Magda M. Khalil, et al.
523
12- HLEBOWICZ J., HLEBOWICZ A., LINDSTEDT S., BJORGELL O., HOGLUND P., HOLST J., DARWICHE G. and ALMER L.: Effect of 1 and 3g cinnamon on gastric empting, satiety, and postprandial blood glucose Insulin, glocose-dependant insulinotropic polypeptide, glucagonlike peptide 1, and ghrelin concentrations in healthy subjects. Am. J. Clin. Nutr., 89: 815-21, 2009. 13- GAVARD J.A., LUSTMAN P.J. and CLOSE R.E.: Prevalence of depression in adult with diabetes: An epidemiological evaluation. Diabetes Care, 16: 1167-1178, 1993. 14- BELL D.S.: Importance of postprandial glucose control. South. Med. J., 94: 804-809, 2001. 15- ANDERSON R.J., FREEDLAND K.E. and CLOUSE R.E. and LUSTMAN P.J.: The prevalence of comor bid depressin in adults with diabetes: Ameta-analysis Diabetes Care, 24: 1069-1078, 2001. 16- PING H., ZHANG G. and REN G.: Antidiabetic effect of cinnamon oil in diabetic KK-A mice. Food and chemical toxicology, 48: 2344-2349, 2010. 17- QIN B., NAGASAKI M., REN M., BAJOTTO G., OSHIDA Y. and SATO Y.: Cinnamon extract prevents the insulin resistance induced by a high-fructose diet. Horm. Metab. Res., 36: 119-25, 2004. 18- HEGSTED D.M., MILLS R.C., ELVEHJEN C.A. and HART E.B.: Cholin in chicks. J. Biol. Chem., 138: 459, 1941. 19- CAMBELL J.A.: Methodology of protein evaluation, PAG. Nutr. Document R.101, June, Meeting, New York, 1961. 20- Ain American Institute of Nutrition. Purifid diet for laboratory Rodents final report Hoc Writing Committee on the reformulation of the Ain 76A Rodent diet. J. of Nutr., 123: 1939-1951, 1993. 21- DESAI A. and BHIDE M.: Hypoglycemic effect of hanitonia suaveolens. Indian J. Med., 81: 86-91, 1985. 22- National Diabetes Data Group (NDDG): Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes, 28: 1039-1057, 1994. 23- TRINDER P.: Chemical kit method. Ann. Clin. Biochem., 6: 24, 1969. 24- DRABKIN: The standardization of hemoglobin measurement. Am. J. Med. Sci., 21: 710, 1949. 25- MC-INORY M.: A micro hematocrit for determination the packed cell and hemoglobin concentration on capillary blood. J. Clin. Biochem., 32, 1954. 26- THANNHAUSER J.S.: Lipidoses. 3 rd ed. Grune and Stration; New York and London. Review: Eggstein M. (1961) Med. U. Ernahr., 2: 1, 1958. 27- WAHLEFELD A.W.: In methods of enzymatic analysis, Vol. 5, H. Bergmeyer, ed. Academic, New York, p. 18311835, 1974. 28- STEIN E.A.: In textbook of clinical chemistry. W. Tiez, ed W.B. Sander, Philadelphia P. 879-886, 1818, 1829, 1986. 29- FRIEDEWALD W.T., LEVY R.I. and FREDTIEK-SON D.S.: Estimation of concentration of low-density lipoproteins separated by three different methods, Clin. Chem., 28: 2077, 1972.
and lipid fraction [37,38]. On the other hand, this observation may be an encouraging result that improves the insulin secretion by cinnamon beverage and the function of pancreas islet -cells [16]. Generally, the present study revealed that cinnamon beverage was found to have alleviative effect to reduce the overweight, blood glucose level and hyperlipedimia. It could be concluded that cinnamon beverage may be useful when consumed by hyperlipedimic person and diabetic patients to enjoy healthy life and as a source of minerals and antioxidants for healthy people. References
1- BAILEY C. J. and DAY C.: Traditional plant medicines as treatment for diabetes. Diabetes Care, 12: 553-564, 1989. 2- KHAN A., SAFDAR M., KHAN M.H.A., KHATTAK K.N. and ANDERSON R.A.: Cinnamon improves glucose and liquids of people with type 2 diabetes. Diabetes Care, 26: 3215-3218, 2003. 3- QIN B., NAGASAKI M., REN M., BAJOTTO G., OSHIDA Y. and SATO Y.: Cinnamon extract [traditional herb] potentates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats. Diabetes Res. Clin. Pract, 62: 139-48, 2003. 4- MANG B., WOLTERS M., SCHMITT KELB K., LICHTINGHAGEN R., STICHTENOTH D.O. and HAHN A.: Effect of cinnamon extract on plasma glucose, HbA and serum lipids in diabetes mellitus type 2. Eur. J. Clin. Investig., 36: 340-344, 2006. 5- KIM S.H., HYUN S.H. and CHOUNG S .Y.: Anti-diabetic effect of cinnamon extract on blood glucose in db/db mice. Ethnopharmacol., 104: 119-123, 2006. 6- HUA P., GUIJUN Z. and GUIXING R..: Antidiabetic effects of cinnamon oil in diabetic KK-A mice Food and Chemical Toxicology, 48: 2344-2349, 2010. 7- JARVILL-TAYLOR K.J., ANDERSON R.A. & GRAVES D.J.: A hydroxychalcone derived from cinnamon function as amimetic for insulin in 3T3-L1 Adipocytes. J. AM. Coll. Nutr., 20: 327-336, 2001. 8ANDERSON R.A., BROADHURST C.L., POLANSKY M.M., SCHMIDT W.F., KAHAN A., FLANAGAN V.P.S., CHOENE N.W. and GRAVES D.L.: Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity. J. Agric. Food Chem., 52 (1): 65-70, 2004.
9- CAO H., POLANSKY M.M. and ANDERSON R.A.: Cinnamon extract and polyphenols affect the expression of tristetraprolin insulin reseptor, and glucose transporter 4 in mouse 3t3 L1 adipocytes Arch. Biochem. Biophys., 459: 214-222, 2007. 10- BABU P.S., PRABUSEENIVASAN S. and LGNACIMUTHU S.: Cinnamaidehyde-Apotential antidiabetic agent. Phytomedicine, 14: 15-22, 2007. 11- XIANG X.: The use of cinnamic acid to prepare for antidiabetic medicine. Paten Number 99115661, 7, 1999.
524

35- NASLUND E., BARKELING B. and KING N.: Energy intake and appetite are suppressed by glucagons-like peptide-1 [GLP] in obese men. Int. J. Obes. Relat. Metab. Disord, 23: 304-11, 1999. 36- ZHAO X., ZHU J.X., MO S.F., PAN Y. and KONG L.D.: Effects of cassia oil on serum and hepatic uric acid levels in oxonate induced mice and Xanthine dehydrogenase and xanthine oxidase activities in mouse liver. J. Ethnopharmacol., 20 (103): 357-365, 2006. 37- KIM S.H., HYUN S.H. and CHOUNG S.Y.: Antioxidative effects of cinnamomi Cssiae and Rhodiola rosea extracts in liver of diabetic mice. Biofactors, 26 (3): 209-219, 2006. 38- HARRY G., PREUSS M.D., BOBBY-ECHARD M.T., MARILYN M., POLANSKY M.S. and RICHARD A.D.: Whole cinnamon and aqueous Extract ameliorate sucroseinduced blood pressure elevations in spontaneously hypertensive rats. Journal of the American College of Nutrition, 25 (2): 144-150, 2006.
30- YOUNG D.S., PESTANER L.C. and GIBBERMAN V.: Quantitative enzymatic Colorimetric determination of blood urea nitrogen [BUN] in serum Clin. Chem., 21: 1D, 1975. 31- FOSSATI P., TRINDER P. and BORKAM D.: Estimation of serum uric acid. Clin. Chem., 26: 227-234, 1980. 32- SPIERTO F.W., M.C. NEIL M.L. and BURTIES C.A.: Determination of creatinine in serum after deproteinization according to Jaffe' reaction. Clin. Biochem., 12: 18-21, 1975. 33- ARMITAG P., BERRY G.: Statistical methods in medical research, 2 ed. Oxford. Blackwell scientific publications, 1987. 34- NASLUND E.,GUTNIAK M., SKOGAR S., ROSSNER S. and HELLSTROM P.M.: Glocagon-Like peptide 1 increases the period of postprandial satiety and slows gastric emptying in obese men. Am. J. Clin. Nutr., 68: 525-30, 1998.
Patterns of Seizures Among Children in Aseer Region, Saudi Arabia

MANSOUR Y. OTAIF, SSC-P (Ped. Neuro.)*; AYED A. SHATI, MBBS**; ALI M. ALSUHEEL, MBBS**; MOHAMED A. HUNEIF, MBBS** and MOHAMED E. ELAWAD, FRCPCH; FRCPI; FRCPE**
The Departments of Pediatrics, Aseer Central Hospital* and Child Health**, College of Medicine, King Khalid University, Abha, KSA
Abstract
Objective: To identify the pattern of seizures among children admitted to Aseer Central Hospital in Abha, Aseer Region, Kingdom of Saudi Arabia. Methodology: This research explored retrospectively all hospital records of the Pediatrics Department, with the diagnosis of seizure during the period from January to December, 2010. A total of 272 records were analyzed (144 were for males, 52.9% and 128 were for females 47.1%). Age ranged from one month to 18 years (MeanSD: 6.94.4 years). The study variables included age, sex, residence, etiology and type of seizure, family history of seizures, and received medications. Results: Idiopathic and cryptogenic seizures were the most common (42.6% and 42.3%, respectively). General seizures were the most common type (50.7%), while partial seizures affected 29.4% and in almost one fifth of cases, it was unclassified (19.9%). Family history of seizures was positive among 9.9% of cases. The most common prescribed medications for management of seizures were valproic acid (43.8%), phenobarbital (31.6%) and carbamazepine (22.8%). Conclusions: Seizures among children in Aseer Region affect more males than females, those who live in rural areas than in urban areas. Idiopathic and cryptogenic seizures are the most common etiologies for seizures. General seizures are the most common type. Most commonly used medications have their proven side effects on the cognitive and behavioral domains of affected children. Key Words: Seizures Children Aseer Kingdom of Saudi Arabia.
brain; it may cause physical or mental symptoms and may be convulsive or nonconvulsive [2]. The classification of seizure type is dependent on accuracy of history, availability and sophistication of diagnostic tests used, and age at which the patients seizure type was classified. When making a diagnosis, one of the following terms may be used: idiopathic, cryptogenic, symptomatic, generalized, focal, or partial [3]. When a person presents to the healthcare system with a first seizure, it is almost always a convulsive seizure, either generalized or focal. Other seizure types such as absence or complex partial seizures typically occur several times before the person or family become concerned [4]. Febrile seizures are the most common cause of convulsions in children and a frequent cause of emergency hospital admissions. Between 2 and 5% of children (more common in boys) in Europe and the United States experience at least one FS before the age of 5 years [5]. Initial seizure pattern appeared to be a predictor for seizure control in several studies of childhoodonset epilepsy [6,7] . Frequent seizures from the onset may be the first manifestation of various epileptic syndromes with a wide range of severity, particularly in childhood [8]. During childhood, the incidence of acute seizures was found to be highest during the neonatal period [9]. Among neonates, seizures are the most common manifestation of neurological conditions and a major risk factor for inpatient death and subsequent neurological disability [10-13]. They show marked differences from seizures in older age groups: Tonic-clonic seizures, common in children and adults, are rarely seen in the neonate [14].
Introduction THE terms convulsion, fit and seizure are used interchangeably to describe episodes associated with involuntary motor activity. Not everything that twitches is a seizure and therefore it is important to consider other conditions before diagnosing epilepsy [1]. Seizure is defined as an uncontrolled, paroxysmal neuronal discharge in any part of the
Correspondence to: Dr. Mansour Y. Otaif, The Department of Pediatrics, Aseer Central Hospital, College of Medicine, King Khalid University, Abha, KSA.
525
526
Seizures are the most common acute neurologic problem in the United States [2]. Scanty studies explored patterns of seizures in Saudi Arabia. AlRajeh et al. [15] reported that epilepsy and other convulsive disorders were common with a hospital frequency rate of 8 per 1,000. Males were more frequently affected than females and 60% of the patients were under 10 years old at the onset of their illness. In Aseer Region, KSA, Izuora and Anis [16] reported that convulsive disorders accounted for 28.9% of the neurological disorders affecting children at Al-Majardah General Hospital. None of these studies were exclusively describing seizures among children. The present study aims to identify the pattern of seizures among children admitted to Aseer Central Hospital in Abha, Aseer Region, Kingdom of Saudi Arabia. Material and Methods The present study was performed in Aseer Central Hospital. It is the highest tertiary care referral level hospital within Aseer Region, at the southwestern part of Kingdom of Saudi Arabia (KSA). This research explored retrospectively all hospital records of the Pediatrics Department, with the diagnosis of seizure during the period from January to December, 2010. A total of 272 records were analyzed (144 were for males, 52.9% and 128 were for females 47.1%). Age ranged from one month to 18 years (MeanSD: 6.94.4 years). The study variables included age, sex, residence, etiology and type of seizure, family history of seizures, and received medications. Results Table (1) shows a gradual decline in proportion of cases according to their age groups. The youngest age group (i.e., <5 years) comprised the highest proportion of cases (35.3%), while the oldest age groups (i.e., 10-15 years and >15 years) comprised the least proportions of cases (30.1% and 2.2%, respectively). About one third of cases aged <5 years, higher proportion of seizures among males than females (52.9% and 47.1%, respectively), and more rural residents than urban residents (58.8% and 41.2%, respectively). Table (2) shows that as regard etiology of seizures, idiopathic and cryptogenic seizures were the most common (42.6% and 42.3%, respectively). General seizures were the most common type
(50.7%), while partial seizures affected 29.4% and in almost one fifth of cases, it was unclassified (19.9%), as shown in Fig. (1). Family history of seizures was positive among 9.9% of cases. The most common prescribed medications for management of seizures were valproic acid (43.8%), phenobarbital (31.6%) and carbamazepine (22.8%). Table (3) shows that distribution of seizure types according to characteristics of patients (i.e., age, gender, residence and family history) was not statistically significant.
Table (1): Characteristic of study sample. Personal characteristics Age: <5 years 5-9 years 10-15 years >15 years Gender: Male Female Residence: Rural Urban No. 96 88 82 6 144 128 160 112 % 35.3 32.4 30.1 2.2 52.9 47.1 58.8 41.2
Table (2): Characteristic of seizures. Seizures characteristics Etiology of seizure: Idiopathic Cryptogenic Symptomatic Type of seizure: Generalized Partial Unclassified Family history of seizures: Positive Negative Received medications: Valproic acid Phenobarbital Carbamazepine Clonazepam Topiramate Lamotrigine Levetiracetam Phenytoin Clobazam Others No. %
116 115 41 138 80 54 27 245 119 86 62 26 23 18 13 9 5 27
42.6 42.3 15.1 50.7 29.4 19.9 9.9 90.1 43.8 31.6 22.8 9.6 8.5 6.6 4.8 3.3 1.8 9.9
Mansour Y. Otaif, et al.

Table (3): Distribution of seizure types according to characteristics of patients. Generalized (n=138) No. Age: <5 years 5-9 years 10-15 years >15 years Gender: Male Female Residence: Rural Urban Family history of seizures: Positive Negative 50 44 41 3 74 64 82 56 % 36.2 31.9 29.7 2.2 53.6 46.4 59.4 40.6 Partial (n=80) No. 29 24 25 2 43 37 47 33 % 36.3 30.0 31.3 2.5 53.8 46.2 58.8 41.2 Unclassified (n=54) No. 17 20 16 1 27 27 31 23 % p value
527
Characteristics
31.5 37.0 29.6 1.9 0.898 50.0 50.0 0.889 57.4 42.6 0.968
were more frequently affected than females and 60% of the patients were under 10 years old at the onset of their illness. The epilepsies were the commonest type (74%). Generalized seizures (71%) were more frequent than partial (29%). No specific etiology of the epilepsy was determined in the majority of the cases (63%). A family history of epilepsy in close relations was obtained in 23% of the cases. Al-Rajeh et al. [17] conducted a total population survey of Thugbah community in the Eastern Province of Saudi Arabia. There were marginally more females (50.2%) than males (49.8%). In Al-Khobar, KSA, Al-Sulaiman and Ismail [18] reported that the age of onset was within the first year of life in 128 (48.7%) of the patients. The main seizure types were generalized in 60.4% and partial in 32.7%. Al Rajeh et al. [19] studied the prevalence of epilepsy in Saudi Arabia. Twenty-eight percent of the patients had partial seizures, 21% generalized seizures. In Pakistan, Khatri et al. [20] reported a higher prevalence of seizures among rural population. Epilepsy was considered idiopathic in 21 to 76% cases. Generalized seizures were the most common seizure type noted.
13 9.4 125 90.6
8 72
10.0 90.0
6 48
11.1 88.9 0.940
Unclassified 19.9
Partial 29.4
Generalized 50.7
Fig. (1): Percentage distribution of seizure types.
Benamer and Grosset [21] studied and systematically reviewed the epidemiologic aspects of epilepsy in Arab countries. They noted higher prevalence in males. Idiopathic epilepsy represented 73.5-82.6% of cases. Primary generalized seizures are reported in 28-97% of cases, partial seizures in 3-43.8%, and unclassified seizures in 18-51%. In Turku, Finland, Sillanpaa and Schmidt [22] reported that male patients constituted 52% while females 48%. Patients aged less than 6 years constituted 63.7%, while those aged above 6 years 36.3%, localized cases were more than, generalized (52.9% and 36.3%, respectively). In the current study, 9.9% of cases gave positive family history of seizures. Abou-Khalila et al. [23] reported that patients with generalized epilepsy were more likely to have first and second degree relatives with epilepsy than those with partial epilepsy and also had a greater proportion of affected first degree relatives. The most common prescribed medications for management of seizures among pediatric cases in the present study were valproic acid, phenobarbital and carbamazepine. It is to be considered that antiepileptic drugs have the potential for affecting the cognitive and behavioral domains. Of the classical drugs, phenobarbitone and benzodiazepines are
Discussion This study revealed that the main characteristics of seizures among children in Aseer Region, KSA, are that the youngest age groups have the highest proportion of cases, slightly higher proportion of seizures among males than females, more rural residents than urban residents. Regarding etiology of seizures, the present study revealed that idiopathic and cryptogenic seizures were the most common. Generalized seizures were the most common type, while partial seizures affected 29.4%. Family history of seizures was positive among almost one-tenth of cases. Distribution of types of seizures was not statistically significant according to patients characteristics. Results of studying the patterns of seizures seem to differ widely according to different study areas. In Saudi Arabia, Al-Rajeh et al. [15] studied the pattern of epilepsy and other convulsive disorders in 1,000 consecutive Saudi nationals. Males
528

10- TEKGUL H., GAUVREAU K., SOUL J., MURPHY L., ROBERTSON R., STEWART J., VOLPE J., BOURGEOIS B. and DU PLESSIS A.J.: The current etiologic profile and neurodevelopmental outcome of seizures in term newborn infants. Pediatrics, 117 (4): 1270-1280, 2006. 11- RONEN G.M., BUCKLEY D., PENNEY S. and STREINER D.L.: Long-term prognosis in children with neonatal seizures: A population-based study. Neurology, 69 (19): 1816-1822, 2007. 12- PISANI F., SISTI L. and SERI S.: A scoring system for early prognostic Assessment after neonatal seizures. Pediatrics, 124 (4): e580-587, 2009. 13- MWANIKI M., MATHENGE A., GWER S., MTURI N., BAUNI E., NEWTON C.R.J.C., BERKLEY J. and IDRO R.: Neonatal seizures in a rural Kenyan District Hospital: aetiology, Incidence and outcome of hospitalization. BMC Medicine, 8: 16, 2010. 14- SCHMITT B., WOHLRAB G., SANDER T., STEINLEIN O.K. and HAJNAL B.L.: Neonatal seizures with tonic clonic sequences and poor developmental outcome. Epilepsy Res., 65 (3): 161-168, 2005. 15- AL-RAJEH S., ABOMELHA A., AWADA A., BADEMOSI O. and ISMAIL H.: Epilepsy and other convulsive disorders in Saudi Arabia: A prospective study of 1,000 consecutive cases. Acta. Neurol. Scand, 82 (5): 341-5, 1990. 16- IZUORA G.I. and ANIS A.S.: Neurological disorders in Saudi children at the Al-Majardah General Hospital, Asir region. Ann. Saudi Med., 12 (2): 191-5, 1992. 17- AL RAJEH S., BADEMOSI O., ISMAIL H., AWADA A., DAWODU A., AL-FREIHI H., ASSUHAIMI S., BOROLLOSI M. and AL-SHAMMASI S.: A community survey of neurological disorders in Saudi Arabia: The Thugbah study. Neuroepidemiology, 12 (3): 164-78, 1993. 18- AL-SULAIMAN A.A. and ISMAIL H.M.: Clinical pattern of newly-diagnosed seizures in Saudi Arabia: A prospective study of 263 children. Childs Nerv. Syst., 15 (9): 468-71, 1999. 19- AL RAJEH S., AWADA A., BADEMOSI O. and OGUNNIYI A.: The prevalence of epilepsy and other seizure disorders in an Arab population: A community-based study. Seizure, 10 (6): 410-4, 2001. 20- KHATRI I.A., IANNACCONE S.T., ILYAS M.S., ABDULLAH M. and SALEEM S.: Epidemiology of epilepsy in Pakistan: Review of literature. J. Pak. Med. Assoc., 53 (12): 594-7, 2003. 21- BENAMER H.T. and GROSSET D.G.: A systematic review of the epidemiology of epilepsy in Arab countries. Epilepsia, 50 (10): 2301-4, 2009. 22- SILLANPAA M. and SCHMIDT D.: Early seizure frequency and aetiology predict long-term medical outcome in childhood-onset epilepsy. Brain, 132: 989-998, 2009. 23- ABOU-KHALILA B., KREIA L., LAZENBYA B., HARRISBC P.A., HAINESD J.L. and HEDERAA P.: Familial genetic predisposition, epilepsy localization and antecedent febrile seizures. Epilepsy Researcher, 73 (1): 104-10, 2007. 24- VINAYAN K.P.: Epilepsy, antiepileptic drugs and educational problems. Indian Pediatr., 43 (9): 786-94, 2006.
the major ones reported to produce learning disorders. Phenytoin has also been found to cause impairment in memory and psychomotor speed. Cognitive and behavior problems on carbamazepine are relatively rare. However it can occasionally cause an increase in subtle seizures leading to learning problems. Valproate can also produce learning problems in higher doses. So, these patients may need concurrent cognitive and educational rehabilitation beside their medical treatment [24]. In Conclusion: Seizures among children in Aseer Region, KSA affect more males than females, those who live in rural areas than in urban areas. Idiopathic and cryptogenic seizures are the most common etiologies for seizures. General seizures are the most common type. Family history of seizures is positive among one-tenth of cases. Most commonly used medications have their proven side effects on the cognitive and behavioral domains of affected children. References
1- MACKAY M.: Fits, faints and funny turns in children. Australian Family Physician, 34 (12): 994-1061, 2009. 2- KAMMERMAN S. and WASSERMAN L.: Seizure disorders: Part 1. Classification and diagnosis. West J. Med., 175 (2): 99-103, 2001. 3- ROCCA W.A., SAVETTIERI G., ANDERSON D.W., MENEGHINI F., GRIGOLETTO F., MORGANTE L., REGGIO A., SALEMI G., PATTI F. and DI PERRI R.: Door-to-door prevalence survey of epilepsy in three Sicilian municipalities. Neuroepidemiology, 20 (4): 237241, 2001. 4- BANERJEE P.N., FILIPPI D. and HAUSER W.A.: The descriptive epidemiology of epilepsy-a review. Epilepsy Res., 85 (1): 31-45, 2009. 5- KARANDE S.: Febrile seizures: A review for family physicians. Indian Journal of Medical Sciences, 61 (3): 161-72, 2007. 6- BERG A.T., SHINNAR D., LEVY S.R., et al.: Early development of intractable epilepsy in children: A prospective study. Neurology, 56: 1445-52, 2001. 7- SPOONER C.G., BERKOVIC S.F., MITCHELL L.A., WRENNALL J.A. and HARVEY A.S.: New onset temporal lobe epilepsy in children: Lesion on MRI predicts poor seizure outcome. Neurology, 67: 2117-8, 2006. 8- DULAC O., LEPPIK I.E., CHADWICK D.W. and SPECCHIO L.: Starting and stopping treatment. In: Engel J. Jr, Pedley T.A. (eds). Epilepsy. A comprehensive textbook. Philadelphia: Wolters-Kluver-Lippincott Williams & Wilkins, p. 1301-9, 2007. 9- IDRO R., GWER S., KAHINDI M., GATAKAA H., KAZUNGU T., NDIRITU M., MAITLAND K., NEVILLE B.G., KAGER P.A. and NEWTON C.R.: The incidence, aetiology and outcome of acute seizures in children admitted to a rural Kenyan district hospital. BMC Pediatr., 8: 5, 2008.
Pattern of Infertility Among Couples in Gezira Area, Sudan

NEZAR M. ABDALLA, M.D.
The Department of Medical Microbiology, College of Medicine, King Khalid University, Abha
Abstract
Objectives: To screen couples in Gezira area, central of Sudan, to identify prevalence, types and main causes of infertility. Patients and Methods: This study was performed in Gezira State (Wad Medani City), Central Sudan. Four primary health care (PHC) centers were randomly chosen. A consecutive sample was followed to include 2000 couples (i.e., 2000 husbands and 2000 wives) attending the selected 4 PHC centers for any complaint. A questionnaire was constructed by the researcher for collection of variables related to full history of both the wife and the husband, present complaint, past complaint, surgical history, family history of chronic diseases and infertility, sexual intercourse history, results of all necessary physical examination and conducted investigations. Results: Almost two-thirds of participant wives aged 3039 years (62.5%). Only 3.7% of wives had university education, compared with 9.5%, of husbands. Prevalence of infertility was 10.4%, mainly primary infertility (77.4%), with a duration of 5-9 years among 46.6% of the couples and >10 years among 26.4% of the couples. In about one third of the couples, infertility was due to female factors (33.7%), while in about one fourth of the couples, infertility was due to male factors (25.5%). However, cause of infertility was unknown in 38% of couples. Sexually transmitted infections were identified in only 4 infertile couples (1.9%), of whom two couples had N. gonorrheae and the other two had C. trachomatis. The great majority of wives (98.6%) underwent female genital mutilation. A minority of husbands received previous treatment for infertility (5.3%) compared with the majority of wives (90.4%). Conclusions: Infertility is a public health problem in Central Sudan with a prevalence rate of about 10%. The main infertility pattern is primary rather than secondary infertility. There is delay in conducting investigations for infertility, especially among husbands. Sexually transmitted diseases are not the main causative cause for infertility. Almost all wives underwent extensive female genital mutilation during their childhood. Key Words: Reproductive health Infertility Sexually transmitted diseases Sudan Female genital mutilation. Correspondence to: Dr. Nezar M. Abdalla, The Department of Medical Microbiology, College of Medicine, King Khalid University, Abha.
Introduction REPRODUCTIVE health (RH) is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity, in all matters relating to the reproductive system, and to its functions and processes. RH includes the right of access to appropriate health-care services that will enable women to go safely through pregnancy and childbirth and provide couples with the best chance of having a healthy infant. In line with the above definition, RH care is defined as the constellation of methods, techniques and services that contribute to RH and well-being by preventing and solving RH problems [1]. Although the human population is growing rapidly and is predicted to reach 9 billion by 2050, 15% of couples worldwide are childless because of infertility [2]. A vast proportion of the world's population has no access to medical treatment for infertility, and even in developed and emerging economies there are great inequalities in access to proper diagnosis and treatment [3]. Infertility is the inability to conceive after one year of unprotected adequately time. Approximately 20% of couples consult their family physician because of difficulty conceiving, and half of those couples require specialist care intercourse [4]. The World Health Organization has repeatedly requested research on infertility in Africa. Larsen [5] documented that infections, either from a sexually transmitted disease, after childbirth, or an abortion, were the major cause of infertility in subSaharan Africa. In Sudan, there has been no actual study to assess the magnitude of infertility. However, to screen infertility, primary health care (PHC) physicians should consider simple investigations which will bring out most of treatable causes of infertility and to avoid expensive, time consuming and usually inconclusive procedures [6].
529
530
This study aimed to screen couples in Gezira area, central of Sudan, to identify prevalence, types and main causes of infertility. Material and Methods This study followed a cross-sectional analytical design. It was performed during July-August 2009 to screen couples in Gezira area, (Wad Medani City), Central Sudan for infertility. To have a representative sample, out of 17 PHC centers, four health care centers were randomly chosen (i.e., Police, Banat, Eldibagha, and Gezira health centers). A consecutive sample was followed to include 2000 couples (i.e., 2000 husbands and 2000 wives) attending the selected 4 PHC centers for any complaint. Before starting interviewing the couples, their consent was secured after clear explanation of this study to the couples. A questionnaire was constructed by the researcher for data collection of variables related to full history of both the wife and the husband, present complaint, past complaint, surgical (abdominal and/or pelvic) history, family history of chronic diseases and infertility, sexual intercourse history (the frequency and timing), results of physical examination and investigations. The following investigations were performed: Hemoglobin (%), total white blood count, erythrocyte sedimentation rate, urine analysis and seminal analysis. These investigations were performed at the laboratory of the PHC center. Moreover, to solve the expected problem of male refusal to perform seminal analysis, they were asked to perform the semen sample at their own home and to bring it immediately to the nearby PHC center laboratory. The WHO classification [7] was followed for results of seminal analysis, which is: The volume will be more than 2 mL. Sperm count is as follows: Normal sperm count >20 million/HPF, moderately oligospermia >5 million but <20 million/HPF, severe oligospermia <5 million/HPF, while azoospermia is no spermatozoa seen. Sperm motility more than 60% of the sperms will be considered as actively motile after production (within half an hour). More than 60% of spermatozoa should be normal in shape. Any husband with a sperm count <20 million/HPF was asked to repeat the test after 2-3 weeks with avoidance of intercourse for 3-7 days prior to the test. In Gezira University Medical Laboratory for Microbiology, high vaginal swab and vaginal se-
cretions for culture and sensitivity were done. Moreover, urine samples were collected for testing men using nucleic acid amplification tests for sexually transmitted infections (e.g., N. gonorrheae, C. trachomatis, etc), while for female screening, vaginal swab specimens were collected. Abdominal and pelvic ultrasonography was done and testicular biopsy was taken from azoospermic men at the Surgery Department in Wad Medani Teaching Hospital. Hysterosalpingography was performed at the Radiology Department of Wad Medani Teaching Hospital, with urographin being used as radioopaque contrast medium. This procedure was done from day 6 to day 9 from the first day of the cycle. Reproductive hormonal immune assay was performed at the Institute of Nuclear Medicine and Molecular Biology in Gezira University. Four reproductive hormones were estimated. Luteinizing hormone (LH), follicular stimulating hormone (FSH) and progesterone were assessed in day 21 from the first day of the cycle. A blood level of progesterone more than 20 nmol/L indicated ovulation. Prolactin hormone level was assessed on days 3-5 of the cycle. A blood level >500 nmol/L was considered as high level. Hormonal analysis was done by the method of Immuno-Radiometric Assay. Results Table (1) shows that almost two-thirds of participant wives aged 30-39 years (62.5%) while most husbands aged 30-39 years (38.9%) or 4049 years (39.6%). Almost half of wives had secondary school education (49.1%) and 3.7% had university level of education, compared with 43.6% of husbands who had secondary education and 9.5%, who had university education. The great majority of wives (98.6%) underwent extensive female genital mutilation (FGM) during their childhood, mainly of the Pharonic type. A minority of husbands received previous treatment for infertility (5.3%) compared with the majority of wives (90.4%). Table (2) shows that prevalence of infertility among the study group was 10.4%. Infertility among more than three-fourths of couples was primary infertility (77.4%), with a duration of <5 years among 26.9% of the couples, 5-9 years among 46.6% of the couples and >10 years among 26.4% of the couples. In about one third of the couples, infertility was due to a female factor (33.7%), e.g.,
Nezar M. Abdalla
531
ovulation problem, tubal blockage, hormonal problems, or uterine problem, while in about one fourth of the couples, infertility was due to a male factor (25.5%), i.e., low semen quality. However, cause of infertility was unknown in 38% of couples. Sexually transmitted infections were identified in
Table (1): Characteristics of study sample. Variables/characteristics Age of wives (in years): <20 20-29 30-39 40 Age of husbands (in years): <30 30-39 40-49 50-59 60 Educational level of wives: Illiterate/read & write Primary/preparatory Secondary University or higher education Educational level of husbands: Illiterate/read & write Primary/preparatory Secondary University or higher education Female circumcision (genital mutilation) among wives: No Yes No. 11 618 1249 122 80 777 792 261 90 245 699 981 75 300 638 872 190 % 0.6 30.9 62.5 6.1 4.0 38.9 39.6 13.1 4.5 12.3 35.0 49.1 3.7 15.0 31.9 43.6 9.5
only 4 infertile couples (1.9%), of whom two couples had N. gonorrheae and the other two had C. trachomatis. in only 1.9% of infertile couples. Almost all wives (90.4%) received previous management for infertility, compared with only 5.3% of husbands.
Table (2): Characteristics of infertility. Variables/characteristics Presence of infertility among couples: Absent Present Type of infertility among couples: Primary Secondary Duration of infertility (in years): <5 years 5-9 10 Causes of infertility: Male factors Female factor Both Idiopathic Sexually transmitted infections: No Yes - N. gonorrheae - C. trachomatis Receiving previous management for infertility: Husbands Wives No. 1792 208 161 47 56 97 55 53 70 6 79 204 4 2 2 % 89.6 10.4 77.4 22.6 26.9 46.6 26.4 25.5 33.7 2.9 38.0 98.1 1.9 0.96 0.96
3 205
1.4 98.6
11 188
5.3 90.4
Discussion Worldwide the overall incidence of infertility related to both male and female factors continue to rise despite the many achieved advances in reproductive technology [8]. In this study, almost two thirds of female couples with infertility were within the age group of 35-39 years old. Moreover, almost three fourths of couples had their infertility for five years or more. This documents a prolonged delay in seeking medical consultation regarding infertility among the study population, which would lower the probability of successful management. This delay can be explained by the relatively low educational status of study population, in which only 3.7% of wives and 9.5 of husbands had university education. So, raising awareness and promoting health education constitute a necessary primary strategy for success in managing infertility.
Moreover, in Sudan FGM is extensively practiced during childhood. This type of circumcision has been incriminated in several cases of primary infertility. Sakar et al. [9] reported that almost half of infertile women had adnexal pathology, indicative of previous inflammation due to undergoing the most extensive form of female genital mutilation, involving the labia majora (odds ratio 469, 95% CI 149-197). They concluded that the anatomical extent of FGM, rather than whether or not the vulva had been sutured or closed, was associated with primary infertility. Though the male factors for infertility were more prevalent in the present study than the female factors, only 5.3% of the husbands received previous treatment for infertility compared with 90.4% of the wives. This differential in compliance to treatment between male and female couples is a common finding that was confirmed by several
532
researchers. The female is usually more enthusiastic and energetic than her husband in undergoing all necessary investigations and complying with treatment [10-12]. After fully examining and conducting all investigations for all infertile couples, infertility was attributable to female factors in about one third of couples, while it was attributable to male factors in one about fourth of couples. However, the reason for infertility remained unknown in more than one third of the couples. This finding is similar to those reported in other studies [6]. Sexually transmitted infections were identified in only four couples (1.9%). This low prevalence is not in agreement with the higher prevalence rates reported in western countries. Devroey et al. [13] stated that the main causes of infertility include pelvic infections including sexually transmitted diseases (STDs). Garnett (14) added that infertility due to obstruction of the fallopian tubes is one of the main severe and lasting consequences of infection with the bacterial sexually transmitted infections due to gonorrhea and Chlamydia. Conclusions: Infertility is a public health problem in Central Sudan, with a prevalence rate of about 10%. The main pattern is primary rather than secondary infertility. There is delay in conducting investigations for infertility, especially among husbands. STDs are not the main causative cause for infertility. Almost all wives underwent extensive female genital mutilation during their childhood. Acknowledgements: The author would like to confer our gratitude to the UNFPA (United Nation, Khartoum, Sudan), The Head of The Population Centre, Gezira University (Prof. Ahmed H. Elnoory), The health centers staff (Police, Banat, Eldibagha, Gezira, Wad Medani, Sudan) and Wad Medani Teaching Hospital for their meticulous collaboration and efforts.
References
1- TEMPLETON A.: Infertility and the establishment of pregnancy-overview. Br. Med. Bull., 56 (3): 577-87, 2000. 2- Dey S.K.: How we are born. J. Clin. Invest, 120 (4): 9525, 2010. 3- CHAMBERLAIN G.: Gynaecology by ten teachers, 17thed. London, Arnolds, 2000. 4- GORKEMLI H., CICEK M.N., AKYUREK C. and CELIK C.: Approach to infertile patient. In Gynecology and obstetrics. Chapter 29. Ankara, Turkey: Gunes Kitabevi, p.1081, 2006. 5- LARSEN U.: Primary and secondary infertility in subSaharan Africa. International Journal of Epidemiology, 29: 285-91, 2000. 6- JEJEEBHOY S.J. and SATHAR Z.A.: Women's Autonomy in India and Pakistan. The Influence of Religion and Region. Population & Development Review, 27: 687712, 2001. 7- COOPER T.G., NOONAN E., VON ECKARDSTEIN S., AUGER J., BAKER H.W.G., BEHRE H.M., HAUGEN T.B., KRUGER T., WANG C., MBIZVO M.T. and VOGELSONG K.M.: World Health Organization reference values for human semen characteristics. Human Reproduction Update, pp. 1-15, 2009. 8- WILKINS K.M., WARNOCK J.K., SERRANO E., ZEGERS-HOCHSCHILD F., SCHWARZE J.E. and ALAM V.: Depressive Symptoms Related to Infertility and Infertility Treatments. Psychiatric Clinics of North America, 33 (2): 309-321, 2010. 9- SAKAR M.N., GUL T., ATAY A.E. and CELIK Y.: Comparison of hysterosalpingography and laparoscopy in the evaluation of infertile women. Saudi Medical Journal, 29 (9): 1315-18, 2008. 10- CLAYTON S.G., LEWIS L.T. and PINKER G.: Gynecology by Ten Teachers, 1985. 11- TRANTHAM P.: The infertile couple. American Family Physician, 54 (3): 1001-10, 1996. 12- PRASAD S.V. and DUNBAR B.S.: Human oocyte recognition and infertility Seminars in Reproductive Medicine, 18 (2): 141-9, 2000. 13- DEVROEY P., FAUSER B.C.J.M. and DIEDRICH K.: Approaches to improve the diagnosis and management of infertility. Human Reproduction Update, 15 (4): 391408, 2009. 14- GARNETT G.P.: How much infertility does chlamydia cause? Sex Transm. Infect., 84:157-158, 2008.
Leishmaniasis in Western Sudan: Prevalence and Clinical Picture

AMANI A. OSMAN, M.D.
The Department of Family and Community Medicine, College of Medicine, King Khalid University, Abha, Saudi Arabia
Abstract
Aim of Study: To identify prevalence of leishmania cases and the clinical picture among inhabitants of the Green Valley villages in Rashad Province. Material and Methods: The study included the total population of The Green Valley villages, Rashad Province, west of Sudan villages (N=332). Demographic data were collected using a special questionnaire. Clinical examination of all villagers was conducted. Screening for symptoms and signs related to leishmaniasis was performed. Leishmanin skin test (LST) was done to all villagers. The ball pin technique was applied after 48-72 hours. Indurations, redness and swelling more than 5mm were considered positive. Finger prick blood spotted filter papers were collected from all the villagers. All the samples were tested for leishmania parasite detection using specific donovani primers. Results: LST results showed that 170 cases were positive (51.2%). Distribution of LST results by clinical grades according to gender showed no significant difference. Clinically, only 3% complained of diarrhea. Fever was the complaint of 34% of children and 16% adults, while abdominal pain experienced by 6.7% of children and 13.5% of adults. Clinical assessment showed that 14 individuals had enlarged liver while 50 persons showed enlarged spleen and only 63 individuals showed enlarged lymph nodes localized to the inguinal region. Conclusions: The capability of leishmaniasis to exist in deserted areas can be explained by the disease capability to maintain internal circulation within the vectors and animal reservoirs and this can last as long as twenty years. Also there is a need to do further serological study to confirm sub clinical infection in this area. Key Words: Visceral Cutaneous Leishmaniasis Subclinical infection LST (Leishmanin skin test) Prevalence.
bites of female sandfly, mainly due to Phlebatomous spp. (papatasi, orientalis). L. donovani complex (donovani, infantum and chagasi) cause visceral leishmaniasis while L. tropica (arthroponotic) and L. major (zoonotic) are responsible for cutaneous leishmaniasis. Leishmania donovani is the main causative species for visceral leishmaniasis while Leishmania major needs an animal reservoir and it is the main causative parasite for cutaneous leishmaniasis in Sudan [1,2]. In Sudan the Leishmania Belt extends from Gadarif Region from the east, where visceral leishmania is the predominant form to Darfor Region to the west, where the cutaneous form has been reported [3] . The Nuba Mountain is situated in Kordofan Region which lies in the midway in the leishmania belt. This area has rarely been studied [4,5], while several studies in the field of leishmaniasis have been conducted, mainly in the eastern and southern Sudan [3,6]. Many mobile, nomadic tribes are settled in this area from various ethnicities, such as Massaleet, Arabs and Nuba. They work as shepherds and farmers. Variable types of domestic and wild animals are found such as camels, cows, sheep, dogs and rodents. The ecology is suitable for the vector (sandfly) existence and breeding. This is shown by the presence of the sub-savanna climate and the plants forests belonging to Acasia balanitis. The cracked dry muddy sand offer sandflies the optimal breeding sites [7]. Investigations are available for detection of leishmania cases, but still the most reliable and easies test used in screening and epidemiological tool to be used in field studies is the leishmania skin test (LST, or Montonegro test) [8]. This study aimed to identify prevalence and clinical picture of leishmania cases among inhabitants of the Green Valley villages in Rashad Province.
Introduction LEISHMANIASIS is an important parasitic disease that constitutes a great public health problem to many countries. Transmission to human is via
Correspondence to: Dr. Amani A. Osman, The Department of Family and Community Medicine, College of Medicine, King Khalid University, Abha, Saudi Arabia.
533
534
Leishmaniasis in Western Sudan: Prevalence & Clinical Picture
Material and Methods This study followed a cross-sectional descriptive design. It was applied in the Green Valley villages, which are small villages in Rashad Province, west of Sudan. They consist of three adjacent small villages, each consists of around 24 cottages, each cottage represents a family. These villages were deserted for twenty years due to a killing disease that resembles leishmaniasis clinically, as described by the villagers. Resettling in these villages has lead to re-appearance of similar illness. The total population of these villages is 332. All inhabitants of this village were included. The field study was performed during the months of July and August 2009. Demographic data were collected using a special questionnaire. Clinical examination of all villagers was conducted. Screening for symptoms and signs related to leishmaniasis (i.e., fever, epistaxis, abdominal pain, anaemia, enlarged liver, spleen and lymph nodes for visceral and skin ulcers, scars and mucocutaneous lesions for cutaneous) was performed. Leishmanin skin test (LST) was done to all villagers, 0.1mL leishmanin was injected subcutaneously in the upper extensor part of the left arm. The ball pin technique was applied after 48-72 hours. Indurations, redness and swelling more than 5mm were considered positive. The LST grades were applied to plot the test results [9]. Finger prick blood spotted filter papers were collected from all the villagers. All the samples were tested for leishmania parasite detection using specific donovani primers [10] . All cases received specific antileishmaniasis treatment (e.g., amphotericin B), while all participants received health education on how to avoid the vectors bite. Results Table (1) shows that the total study population was 332. Females were more than males (56.1% and 43.9%, respectively). LST results showed that 170 cases were positive (51.2%). Distribution of LST results by clinical grades according to gender showed no significant difference (Table 2). Clinically, only 3% complained of diarrhea. Fever was the complaint of 34% of children and 16% adults, while abdominal pain experienced by 6.7% of children and 13.5% of adults. Clinical assessment of liver and spleen enlargement among the study group showed that 14 individuals had enlarged liver while 50 persons showed enlarged spleen and only 63 individuals showed enlarged lymph nodes that were localized to the inguinal region (Table 3).
Table (1): Distribution of the study population by gender and age groups. Age groups (in years) 0-5 6-10 11-20 > 20 Total Males No. 26 22 27 71 146 % 46.4 37.9 35.1 50.4 43.9 Females No. 30 36 50 70 186 % 53.6 62.1 64.9 49.6 56.1 No. 56 58 77 141 332 Total % 16.9 17.5 23.1 42.5 100.0
Table (2): Distribution of LST results clinical grades in relation to gender. Clinical Grades 0-I II III IV
2=4.71.
Size (in cm) <5 5-8 >8 >8 with skin bolus
p=0.196
Males (n=146) No. 65 6 70 5 % 44.5 4.1 47.9 3.4
Females (n=186) No. 97 13 73 3 % 52.2 7.0 39.2 1.6
Total No. 162 19 143 8 % 48.8 5.7 43.1 2.4
Table (3): Prevalence rates of leishmaniasis according to age groups. Age group (years) 0-5 6-10 11-20 > 20 Total
2=21.798.
Population 56 58 77 141 332

p<0.001
No. of infected cases 42 35 36 57 170
Prevalence rate 75.0 60.3 46.8 40.4 51.2
Table (4): Clinical assessment of 170 cases with leishmaniasis. Clinical picture Symptoms: Fever Abdominal pain Diarrhea Signs: Enlarged lymph nodes Enlarged liver 1-2 cm 3-4 cm 5+ cm Enlarged spleen 1-2 cm 3-4 cm 5+ cm No. %
81 34 5 63 14 7 6 1 50 15 22 13
47.6 20.0 2.9 37.1 8.2 4.1 3.5 0.6 29.4 8.8 12.9 7.6
Amani A. Osman
535
Discussion This study revealed that prevalence rate for leishmaniasis among inhabitants of the Green Valley villages in Rashad Province is quite high, as more than half of the population were infected, i.e., 51.2%. Moreover, the distribution of LST results according to clinical grades among the study population showed that 43.1% had grade III, reflecting the long duration of their disease. These findings indicate the pressing necessity for efforts to control leishmaniasis at that endemic area. Younger age groups had significantly higher prevalence rates than older ones (p<0.001). Children aged up to 5 years had the highest prevalence rate, i.e., 75%. Epidemiological studies from endemic regions worldwide showed the predominance of leishmania cases among children in indoors infections. This has been explained by the low immunity in children in comparison to adults [1113]. In this study there were no significant gender differences as regard prevalence of leishmaniasis. This finding is not in agreement with that reported by Zhang et al. [14], who stated that sex-associated differences are known to exist in numerous diseases. Males are, in general, more susceptible, while females are more resistant to diseases. Such sexdependence has been described for visceral leishmaniasis, which is more common in males than females. However, the nature of the studied area in Sudan, where women usually work outside homes and do all field works, similar to men may explain the gender differences based on exposure differences rather than sex-related immunity differences, as stated by Wilson et al. [12]. Clinical examination for our population revealed that fever, abdominal pain, enlargement of lymph nodes and spleen were the major symptoms and signs in our patients. Stanley and Engwerda [15] explained the clinical picture of leishmaniasis by stating that that leishmaniasis is characterized by long term fever, spleen enlargement, immunosuppersion and weight loss. The parasites inhabit the macrophages of the spleen, liver and bone marrow in the aflagellated amastigotes. During the course of the disease, there is a marked depression of cellular immunity to leishmania antigens. This study have screened these villages and showed that the capability of leishmaniasis to exist in deserted areas can be explained by the disease
capability to maintain internal circulation within the vectors and animal reservoirs and this can last as long as twenty years. Also there is a need to do further serological study to confirm sub clinical infection in this area. Acknowledgements: The researcher confers her gratitude to the Institute of Nuclear Medicine, Molecular Biology and Oncology (University of Gezira, Sudan), Institute of Endemic Diseases (University of Khartoum, Sudan), and to Prof. El-Hassan, LRG (Sudanese Leishmania Research Group, Khartoum), who kindly provided the leishmanin skin test (LST). References
1- GORSKI S., COLLIN S.M., RITMEIJER K., KEUS K., GATLUAK F., MUELLER M. and DAVIDSON R.N.: Visceral leishmaniasis relapse in Southern Sudan (19992007): A retrospective study of risk factors and trends. PLoS Negl. Trop. Dis., 4 (6): e705, 2010. 2- KOLACZINSKI J.H., HOPE A., RUIZ J.A., RUMUNU J., RICHER M. and SEAMAN J.: Kala-azar epidemiology and control, southern Sudan. Emerg. Infect. Dis., 14 (4): 664-6, 2008. 3- EL-HASSAN A.M. and ZIJLSTRA E.E.: Leishmaniasis in Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene, 95: (Suppl. 1), 2001. 4- ABDALLA N.M., IBRAHIM M.E., EL-HASSAN A.M., OSMAN O.F., DAIFALLA N.S., BARKER D.C., LAMBSON B., MILES M., MAURICIO I. and MAGZOUB M.M.: Clinico-epidemiological Study On Leishmaniasis In The Nuba Mountain-Sudan. Acta. Parasitologica Turcica, 26 (1), 2002. 5- ABDALLA N.M., ELDOSH A.A., ABDULGANI A.M., YUSIF B.E. and MAGZOUB M.M.: Typing and characterization of leishmania sub-clinical isolates from Nuba Mountain, west of Sudan. Infection, Genetic and Evolution J., 2: 4. 6- SIDDIG A.M., GHALIB H., SHILINGTON D.C., PETERSON E.A. and KHIDIR S.: Visceral leishmaniasis in Sudan. Trop. Geogr. Med., 42: 107-112, 1990. 7- KIRK R. and LEWIS D.J.: Studies on Leishmaniasis in the Anglo-Eygptian Sudan. XI. Phlebotomus in relation to leishmaniasis in the Sudan. Trans. R Soc. Trop. Med. Hyg., 49: 229-40, 1955. 8- NOGUEIRA M.F., GOTO H., SOTTO M.N. and CUC L.C.: Cytokine Profile in Montenegro Skin Test of Patients with Localized Cutaneous and Mucocutaneous Leishmaniasis. Rev. Inst. Med. Trop. S. Paulo., 50 (6): 333-337, 2008. 9- CHAPPUIS F., SUNDAR S., HAILU A., GHALIB H., RIJAL S., PEELING R.W., ALVAR J. and BOELAERT M.: Visceral leishmaniasis: What are the needs for diagnosis, treatment and control? Nature Reviews Microbiology, S7-S16, 2007. 10- GIDWANI K., RAI M., CHAKRAVARTY J., BOELAERT M. and SUNDAR S.: Evaluation of leishmanin skin test
536
Leishmaniasis in Western Sudan: Prevalence & Clinical Picture

in Indian visceral leishmaniasis. Am. J. Trop. Med. Hyg., 80 (4): 566-7, 2009. 13- PALUMBO E.: Visceral leishmaniasis in children: A review. Minerva Pediatr., 62 (4): 389-95, 2010. 14- ZHANG H., ZHAO J., WANG P. and QIAO Z.: Effect of testosterone on Leishmania donovani infection of macrophages. Parasitology Research, 87 (8): 674-676, 2009. 15- STANLEY A.C. and ENGWERDA C.R.: Balancing immunity and pathology in visceral leishmaniasis immunity and pathology in VL. Immunology and Cell Biology, 85: 138-147, 2007.
11- JAHN A., LELMETT J.M. and DIESFELD H.J.: Seroepidemiological study on Kala-azar in Baringo District, Keya. J. Trop. Med. Hyg., 89: 91-104, 1986. 12- WILSON M.E., JERONIMO S.M.B. and PEARSON R.D.: Immunopathogenesis of infection with the visceralizing Leishmania species. Microbial Pathogenesis, 38: 147160, 2005.
Profile of Idiopathic Pulmonary Arterial Hypertension in High Altitude Aseer Region, Saudi Arabia
ABDULLAH S. ASSIRI, M.D. (Hon), ABIM, ABCVM, FRCP(C)
The Department of Internal Medicine, King Khalid University, Abha, Saudi Arabia
Abstract
Objectives: To characterize the demographic and clinical profiles of patients with Idiopathic Pulmonary Arterial Hypertension (IPAH) admitted to the tertiary care hospital in Aseer region, and to evaluate the altitude effect. Design: A retrospective cohort study. Setting: Aseer Central Hospital. Subjects: All patients admitted with the diagnosis of (IPAH) during the period from January 2001 to January 2009 were enrolled. Main Outcome Measures: Descriptive analysis of demographic and clinical profiles data, and comparative analysis of data among high and low altitude groups. Results: Overall, we found 29 patients with IPAH. The mean age was 42.125.2 years. 58.6% were females, 86.2% were Saudi nationals, 79.3% were living at high altitude, the most common symptom of presentation was dyspnea in 89.7%. Echocardiography showed evidence of right ventricular enlargement in 65.5%, and the average systolic pulmonary artery pressure was 63.823.6 mmHG equivalent to mean pulmonary artery pressure of 40.916.4 mmHG. The average number of admissions was 6.28.2. No statistically significant difference between the high and the low altitude groups was found regarding the demographic or clinical profiles. Conclusions: IPAH is a rare disease in Aseer region, affecting more female than male patients in the middle age group; the majority of these patients were living at high altitude, with no statistically significant difference between the high and the low altitude groups, regarding the demographic or the clinical profiles. Our study was not intended to give prevalence or survival data on IPAH in Aseer region and a national survey is needed to characterize the incidence and prevalence of IPAH in Saudi Arabia. Key Words: Hypertension Pulmonary High altitude.
hypertension, is a rare disorder characterized by progressive increase of pulmonary vascular resistance and pulmonary artery pressure leading to right ventricular overload and failure without apparent cause [1] . Dresdale and colleagues first reported a hemodynamic account of IPAH in 1951 [2]. The diagnosis of IPAH can only be made after exclusion of other secondary causes of pulmonary hypertension. The pathophysiology of IPAH is poorly understood, but it is believed that certain external and internal insults to the endothelium may occur to susceptible pulmonary vascular tree resulting in a cascade of events including endothelial dysfunction, vascular scarring and medial smooth muscle proliferation, leading to vasoconstriction and elevation of pulmonary arterial pressure [3,4]. A familial form is detected in 15-20% of cases of IPAH in which the genetic defect has been identified recently. The most common genetic defect is related to the BMPR-II gene mutation [5,6] . As a consequence of increased pulmonary vascular pressure and thrombosis, pulmonary arteriopathy may occur, characterized by in situ thrombosis of small pulmonary arteries and in late stages it can lead to pulmonary artery intimal fibrosis. IPAH is responsible for approximately 125-150 deaths per year in the USA and worldwide and the incidence rate is approximately 2-6 cases per million populations per year in France [7]. The Aseer region (population of 1,200,000) is located in the southwest of Saudi Arabia covering an area of more than 80,000km2. The region extends from the high mountains of Sarawat (with an altitude of 3200m above the sea level) to the Red Sea. Health services delivery in Aseer region is provided by a network of 244 primary health care centers, 16 referral hospitals and one tertiary hospital, Aseer Central Hospital (ACH). ACH, with 500 beds, is
Introduction IDIOPATHIC pulmonary arterial hypertension (IPAH), previously known as primary pulmonary
Correspondence to: Dr. Abdullah S. Assiri, The Department of Internal Medicine, King Khalid University, Abha, Saudi Arabia.
537
538
Profile of Idiopathic Pulmonary Arterial Hypertension
run by the Ministry of Health and the College of Medicine of King Khalid University in Abha. In Saudi Arabia, there have been no previous studies exploring any association between IPAH and high altitude. This study aimed to analyze the demographic and clinical profiles of patients admitted with the diagnosis of IPAH during the period from January 2001 and January 2009 to ACH, Aseer region, Saudi Arabia, and to analyze the effect, if any, of the altitude level. Subjects and Methods A retrospective review of all records for consecutive patients admitted to ACH with the diagnosis of IPAH or primary pulmonary hypertension during the period from January 2001 to January 2009 was conducted. Only records satisfying the definition of IPAH were included, defined as a mean pulmonary artery pressure (MPAP) greater than 25mmHg at rest causing right side heart failure without obvious cause [8] , and where other secondary causes of pulmonary hypertension were excluded (including: connective tissue diseases, congenital left-to-right shunt, portal hypertension, human immunodeficiency infection, appetite-suppressant drugs, pulmonary veno-occlusive disease, left-sided heart disease, chronic obstructive pulmonary disease, interstitial lung disease, and sleep-disordered breathing) by appropriate investigations. In our study the MPAP was estimated by Doppler echocardiography technique, where the transtricuspid pressure gradient was calculated using the modified Bernoulli equation (4 v2) where v is the maximum velocity of the tricuspid valve regurgitant jet. Then the right atrial pressure (RAP) was estimated by the respiratory variation in the diameter of the inferior vena cava and was categorized as 5, 10 or 15mmHg. The systolic pulmonary artery pressure (SPAP) was then calculated by adding the trans-tricuspid pressure gradient to the RAP estimate. The MPAP was then calculated using the equation (MPAP = 0.61* SPAP+2) [9]. Patients data were reviewed and tabulated. These data included demographic and clinical profile data: such as, age, gender, nationality, living altitude, symptoms and signs at presentation, electrocardiographic and echocardiographic features and number of admissions were coded for subsequent analysis. Approval of the local medical-ethical committee was obtained. Data were analyzed using SPSS software package. Frequency, percentage, mean, standard deviation and median were used to present
the data. Chi square and Student t-test were used to test significance at 5% level. Results A total of 29 cases, who satisfied the definition of IPAH, were thus included. Table (1) shows the demographic distribution of the cohort. More than half of patients were females (17, 58.6%) while 12 (41.4%) were males, with a female to male ratio of 1.4:1. The mean age of the cohort was 42.125.2 years. Most of patients were Saudis (25, 86.2%), 23 (79.3%) were living at high altitude (3200 m above the sea level) and the rest of the cohort were living at the sea level.
Table (1): Demographic profile of idiopathic pulmonary arterial hypertension cases. N = 29 Patients Age mean SD Gender: Female Male Nationality: Saudis Non-Saudis Altitude: High altitude Low altitude 42.125.2 years 17 (58.6%) 12 (41.4%) 25 (86.2%) 4 (13.8%) 23 (79.3%) 6 (20.7%)
Table (2) shows the clinical profile of the cohort. The most common symptom of presentation was dyspnea in 26 (89.7%) and the most common physical signs were hepatomegaly in 18 (62%), loud pulmonary second heart sound in 17 (58.6%) and raised jugular venous pressure in 16 (55%).
Table (2): Clinical profile of idiopathic pulmonary arterial hypertension cases. N = 29 Patients Symptoms: Dyspnea Chest pain Syncope Physical signs: Loud pulmonary sound Elevated jugular venous pressure Hepatomegaly Right ventricular heave Tricuspid regurgitation Electrocardiogram: Right bundle branch block Right ventricular hypertrophy Right atrial enlargement Echocardiography: Right ventricular dilatation Severe tricuspid regurgitation Mean PAP SD (mm Hg) Number of admissions 26 (89.7%) 4 (13.8%) 2 (6.9%) 17 (58.6%) 16 (55.2%) 18 (62.1%) 4 (13.8%) 1 (3.4%0 11 (37.9%) 13 (44.8%) 11 (37.9%) 19 (65.5%) 14 (48.3%) 40.916.4 mmHg 6.28.2
Abdullah S. Assiri
539
Electrocardiogram showed evidence of right ventricular hypertrophy in 13 (44.8%) of cases. Doppler echocardiography showed an average systolic pulmonary artery pressure of 63.823.6 (equivalent to MPAP of 40.916.4mmHg), evidence of right ventricular dilatation in 19 (65.5%) of cases and evidence of severe tricuspid regurgitation in 14 (48.3%) of cases. The average number of admissions was 6.28.2. No statistically significant difference was found between the high and the low altitude groups regarding the demographic or the clinical profiles (Table 3).
Table (3): Distribution of demographic and clinical variables among high and low altitude groups. High altitude group 23 (79.3%) Low altitude group 6 (20.7%)
Institutes of Health Registry on IPAH showed that it is a fatal disease and the majority of patients die within 2-3 years from the diagnosis and the 5-year survival rate is only 34% [1,10]. In our cohort, the mean age of patients was 42.1 years, which is older than the reported mean age in USA registry (36 years). Typically younger women at child-bearing age are affected by IPAH. However, it can also affect individuals at older age in the 5th and 6th decades of life or older [7]. In our cohort, the female to male ratio was 1.4:1, which is lower than the US reported ratio of 2:1 [10]. Typically, the IPAH is more common in females patients compared with males. However, the reasons for this female predilection remain unknown. Most patients within our cohort (79.3%) were living at high altitude areas (more than 3000 meters above sea level). The relation between the pulmonary hypertension in humans and the altitude had been investigated in previous reports and a positive correlation was reported, as chronic exposure to hypoxia is a well-recognized stimulus for pulmonary vascular remodeling and the development of IPAH, though animal studies on mice, however showed that chronic hypoxia elicited less severe IPAH, vascular modeling, and right ventricular hypertrophy [11]. In our series we did not find a statistically significant difference between the high and the low altitude groups regarding the demographic or the clinical profiles, this could be due to the overall small sample size or to the relatively small proportion of patients in the low altitude group (20.7%). Patients in our series presented with dyspnea in 89.7% of cases, which is higher than the reported rate of dyspnea in the US national registry (60%). Patients with IPAH usually present with symptoms 2 years later after the diagnosis and female patients are more symptomatic than males [1,10]. The electrocardiogram findings in our study showed evidence of right side cardiac chamber enlargement in 44.8% of cases, and the echocardiography showed that 65.5% of cases had evidence of right ventricular enlargement, and the average systolic pulmonary artery pressure was 63.8 23.6mmHg, which is equivalent to MPAP of 40.9 16.4mmHg. This cohort is considered to have a moderate to severe pulmonary hypertension since the average MPAP exceeds the normal value by more than 1.6 folds. Similar findings were reported in other series [12].
Variables
p value
Age mean SD Female gender Saudi nationals Symptoms: Dyspnea Chest pain Syncope Physical signs: Loud pulmonary sound Elevated jugular venous pressure Hepatomegaly Right ventricular heave Severe tricuspid regurgitation
42.0124.8 42.6728.9 0.957 13 (56.5%) 4 (66.7%) 0.653 19 (82.6%) 6 (100%) 0.553 20 (87%) 3 (13%) 1 (4.3%) 6 (100%) 1 (16.7%) 1 (16.7%) 0.351 0.819 0.337 0.669 0.183 0.058 0.180 0.924
14 (60.9%) 3 (50%) 11 (47.8%) 5 (83.3%) 12 (52.2%) 6 (100%) 2 (8.7%) 2 (33.3%) 11 (47.8%) 3 (50%)
Electrocardiogram: Right bundle branch 9 (39.1%) 2 (33.3%) block Right ventricular 11 (47.8%) 2 (33.3%) hypertrophy Right atrial enlargement 9 (39.1%) 2 (33.3%) Echocardiography: Right ventricular dilatation Severe tricuspid regurgitation Mean PAP SD (mm Hg) Number of admissions 17 (73.9%) 2 (33.3%) 11 (47.8%) 3 (50%) 42.316 6.578.7 36.618.7 4.86.4
0.794 0.663 0.794 0.143 0.924 0.510 0.353
Discussion The incidence of IPAH in Saudi Arabia is not known yet. This study was not intended to give the incidence of the disease in Aseer area or Saudi Arabia as it is a retrospective review for cases admitted to the tertiary hospital ACH. The estimated worldwide incidence is 1-2 cases per million population per year [10]. Data from the USA National
540
Profile of Idiopathic Pulmonary Arterial Hypertension

primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptorII gene. Am. J. Hum. Genet., 67: 737-44, 2000. 4- FISHMAN A.P.: Primary pulmonary arterial hypertension: A look back. J Am Coll Cardiol., 43: S2-4, 2004. 5- HUMBERT M., MORRELL N., ARCHER S., et al.: Cellular and molecular pathobiology of pulmonary arterial hypertension. J. Am. Coll. Cardiol., 43: S13-24, 2004. 6- BUDHIRAJA R., TUDER R.M. and HASSOUN P.M.: Endothelial dysfunction in pulmonary hypertension. Circulation, 109: 159-65, 2004. 7- HUMBERT M., SITBON O., CHAOUAT A., et al.: Pulmonary arterial hypertension in France: Results from a national registry. Am. J. Respir. Crit Care Med. May 1, 173 (9): 1023-30, 2006. 8- BADESCH D.B., CHAMPION H.C., SANCHEZ M.A., et al.: Diagnosis and assessment of pulmonary arterial hypertension. J. Am. Coll. Cardiol., 54: S55, 2009. 9- CHEMLA D., CASTELAIN V., HUMBERT M., et al.: New formula for predicting mean pulmonary artery pressure using systolic pulmonary artery pressure. Chest, 126: 1313-1317, 2004. 10- RICH S., DANTZKER D.R., AYRES S.M., et al.: Primary pulmonary hypertension: A national prospective study. Ann. Intern. Med., 107: 216-230, 1987. 11- EDDAHIBI S., HANOUN N., LANFUMEY L., et al.: Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J. Clin. Invest, 105: 1555-1562, 2000. 12- APPELBAUM L., YIGLA M., BENDAYAN D., et al.: Primary Pulmonary Hypertension in Israel A National Survey. Chest, 119: 1801-1806, 2001. 13- IDREES M.: Pulmonary hypertension: Where are we? Egyptian Journal of Bronchology, 2: 138-142, 2008. 14- IDREES M., AL-HAJJAJ M., KHAN J., et al.: Saudi guidelines on diagnosis and treatment of pulmonary arterial hypertension. Ann. Thorac. Med., 3: S1-57, 2008.
In Saudi Arabia, the Saudi Advisory Group for Pulmonary Hypertension (SAPH) was established in 2004 as a regional scientific body that deals with PAH patients, with the missions of health education and treatment of patients with IPAH. A national registry to study the prevalence of the disease in the Kingdom of Saudi Arabia was started [13]. In 2008, the Saudi guidelines on the management and treatment of pulmonary hypertension were published [14]. Conclusions: In conclusion, IPAH is a rare disease in Aseer region, predominantly affecting female patients in the middle age group, the majority of whom live in high altitude, but with no statistically significant difference between the high and the low altitude groups regarding the demographic or the clinical profiles. Our study was not intended to give prevalence or survival data on IPAH in Aseer region, but to shed some light on demographic and clinical patterns of this rare disease in Aseer area which is a high altitude region. A national survey is needed to evaluate the incidence and survival pattern of IPAH in Saudi Arabia as a whole. References
1- D'ALONZO G.E., BARST R.J., AYRES S.M., et al.: Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann. Intern. Med., 115: 343-9, 1991. 2- DRESDALE D.T., SCHULTZ M. and MICHTOM R.J.: Primary pulmonary hypertension. I. Clinical and hemodynamic study. Am. J. Med. Dec., 11 (6): 686-705, 1951. 3- DENG Z., MORSE J.H., SLAGER S.L., et al.: Familial
Med. J. Cairo Univ., Vol. 79, No. 1, September: 541-553 , 2011 www.medicaljournalofcairouniversity.com
Sources of Stress as Perceived by Nursing Students at King Saud University

MOFIDA YOUNIS AL-BARRAK, D.N.Sc.; MONA TALAT EL-NADY, D.N.Sc. and ELHAM ABDELKADER FAYAD, D.N.Sc.
The Department of Psychiatric Mental Health Nursing, College of Nursing, King Saud University
Abstract
Nursing students suffer higher levels of stress during their college years than college students in other disciplines. Nursing students and persons employed in the nursing profession have been identified as a population with an elevated stress level. Stressors for student nurses included adjusting to a rigorous program of theory, long hours of study and pressures of student clinical practice requiring emotional and personal maturity. So the aim of the present study was to identify sources of stress as perceived by undergraduate nursing students at King Saud University. A descriptive, correlation design was utilized in this study. The study was conducted at Female College of Nursing, King Saud University at Al-Riyadh City. The total proposed subjects included in this study were 300 students covering all levels of the nursing college, using convenience data. The tools included two parts: Part 1 included sociodemographic data for students such as age, marital status, and health related data, part 2 was the Student Stress Survey Sheet (SSSS) which used to study the major sources of stressors among college students; it included items addressed academic, intrapersonal, interpersonal, and environmental sources of stress. The present study results concluded that there were a variety of stressors placed on the studied subjects. The major sources of stress as perceived by King Saud University College of Nursing students were the academic followed by intrapersonal, then environmental and the last was interpersonal. The results of the current study recommended that it is important for the university to maintain well balanced academic environment conducive for better learning, with the focus on the students personal needs. The university should be affording adequate resources such as books, computers to do assignments; also nursing college should do an effort to narrow the gap between students expectations, goals, and values to be integrated with that of the university. Key Words: Identify Stress Stressors Undergraduate Nursing Students.
many years. Researchers have found that the perception of high stress levels in students can lead to poor academic performance, depression, attrition and serious health problems [1,2]. Many scholars in the field of behavioral science have carried out extensive research on stress and its outcomes on individuals then concluded that the topic needed more attention, especially in nursing students who face many stresses [3,4]. Beck, et al. [5] stated that nursing students suffer higher levels of stress during their college years than college students in other disciplines. Nursing students and persons employed in the nursing profession have been identified as a population with an elevated stress level. Stressors for student nurses included adjusting to a rigorous program of theory, long hours of study and pressures of student clinical practice requiring emotional and personal maturity. The practicum portion of nursing education was identified by nursing students as more stressful than didactic courses [6,7]. Sources of stress among nursing students can be viewed from four domains namely; academic, intrapersonal, interpersonal, and environment. As regard academic stressors which refer to stress associated with studying, including study load, performance, and conflict with lecturers or tutors. Such as: Midterms, final examinations, research papers and other assignments. In addition, nursing students experience a clinical component, which is highly stressful [8]. Nwadiani and Ofoegbu [9] said that students have a large amount of preparatory work before their clinical assignments. They often must travel long distances to clinical sites and use highly technical equipment. In addition, they must perform procedures that can cause serious harm to their
Introduction THE issue of stress among college or university students has been the subject of much research for
Correspondence to: Dr. Mofida Younis Al-Barrak, The Department of Psychiatric Mental Health Nursing, College of Nursing, King Saud University.
541
542
patients, thus enhancing their fear of making mistakes. Ross, et al. [10] concluded that academic stress among college students has been a topic of interest for many years. College students experience high stress at predictable times each semester due to academic commitments, financial pressures, and lack of time management skills. When stress is perceived negatively or becomes excessive, it can affect both health and academic performance [6,10]. Role conflict is a common part of the college experience. College students must learn to balance the competing demands of academics, developing new social contacts and being responsible for their own daily needs (e.g., nutrition and clean clothing). In addition, the academic workload requires that students face a series of peak periods such as midterms and finals; there is a relatively constant underlying pressure to complete an upcoming assignment [11]. In relation to intrapersonal stressors which include stress from physical health, financial situation, and mental health issues (e.g. depression, anxiety, Poor diet, living on caffeine, anxiety, worrying about money [5,12]. These stressors can include positive or negative events. Examples of positive events are marriage, graduation, starting a business, or the birth of a baby. In other hand negative changes include events like losing a job, divorce, or death in the family etc. Interpersonal stressors include stress that results from relationships with roommates, problems with friend's also romantic partners, and family members. Marital disagreements, dysfunctional relationships, or caring for a chronically-ill family member or a child with special needs can all increase stress levels [12,13]. Many authors concluded that peers may also influence the perception of and reaction to stress. College norms that define certain types of behavior as "appropriate" under certain conditions, e.g., staying up all night to study for an exam, may be stress inducing and may lead to less healthy practices. Nursing students are now older and are more culturally diverse. They are more likely to be married and have children. Many are employed. These changes have major implications for nursing education programs, as well as colleges and universities in general [14]. Environmental stressors refer to physical surroundings in the college which can set off the stress response including new and unfamiliar situations or conflict between people live in campus. Examples of environmental stressors an unsafe neigh-
borhood, pollution, noise, change in sleeping habits, vacations, breaks, and change in eating habits, and new responsibilities. Furthermore, stress may result from being separated from home for the first time, the transition from a personal to an impersonal academic environment, and the structure of the academic experience at the college level [15,13]. The environment in which college students live is quite different. The pressure to earn good grades and to earn a degree is very high [16]. Earning high grades is not the only source of stress for college students. Other potential sources of stress include excessive homework, unclear assignments, and uncomfortable classrooms [17]. Although it is normal for students to experience stress, especially as they adjust to college life at a highly competitive institution. However, if stress is severe and/or prolonged, it can reduce academic performance and interfere with a student's ability to participate in and contribute to campus life [1]. Significance: College students, especially nursing are particularly prone to stress due to the transitional nature of college life. For example, many college students move away from home for the first time, which can necessitate leaving all previously learned support systems such as parents, siblings and high school friends. Students may need to develop entirely new social contacts and are expected to take responsibility for their own needs. They may have difficulty adjusting to more rigorous academic expectations and the need to learn to deal with individuals of differing cultures and beliefs [1]. At King Saud University, College of Nursing, no research studies were conducted in this respect, so an in depth research is needed to clarify the magnitude of the problem and to emphasize whether these stressors are mostly related to daily hassles, major live events or academic and teaching style problems. It is concluded that stress in academic institutions can have both positive and negative consequences if not well managed. It is important to investigate the stress that undergraduate students experience because of being nursing student can make it even more difficult to handle stress as an undergraduate. High levels of stress are believed to affect students health and academic functions. If the stress is not dealt with effectively, feelings of loneliness, nervousness, sleeplessness and worrying may result. It is important to look at the different factors of stress to help them cope effectively [18,19] . So the aim of the present study was to identify sources of stress as perceived by undergraduate nursing students at
Mofida Y. Al-Barrak, et al.
543
King Saud University, will help to identify the major area of stress and dealing accordingly. Subjects and Methods Research design: A descriptive, correlation design was utilized in this study. Setting: The study was conducted at Female College of Nursing, King Saud University at Al-Riyadh City. The college of nursing is composed of two floors at college of community service. It includes four organizational departments namely: Community and Mental Health Nursing Department, Maternity and Pediatric Nursing Department, Medical Surgical Nursing Department and Department of nursing administration. Subjects: The college has eight academic levels; total number of students enrolled at all levels is 430 at the time of data collection for this research. Total proposed subjects were 298 students covering all levels of the nursing college, using convenience data collected from 32 students from level one, 66 selected from level two, 35 students from level three, 30 students from level four, while 43 students shared from level five, 34 students participated from level six, 41 from level seven, and 37 students from level eight. Ethical consideration: Ethical rules of research guaranteed for each participant to regret from sharing in the study at any stage. Student was assured that the data is confidential and used only for research purposes. Tools for the data collection: A structured interview questionnaire was developed by the researchers based on literature review to collect needed information about the study subjects. The stress factors items were scored as Likert scale ranged from 0 to 5. For each domain of stressor factors, the scores of the items were summed-up and the total divided by the number of the items, giving a mean score for the part. These scores were converted into a percent score. The stressor domain was considered to be high if the percent score was 60% or more, and low if less than 60%. The tools included two parts presented as following: Part 1: Socio demographic data for students included: Age, marital status, and health related data.
Part 2: After reviewing related literature, the Student Stress Survey Sheet (SSSS) was used to study the major sources of stress among college students; it included items of the survey sheet addressed to academic, intrapersonal, interpersonal, and environmental sources of stress [6,10,13]. Procedure: Permission was obtained through an official letter to the director of nursing college at king Saud University to carry out the study, after explanation of the aim and methods of the study and process for data collection. Oral consent was taken from each student. The purpose of the study was explained before starting the interview to gain student confidence, cooperation and to alleviate her fear and anxiety. Data was collected during 1430 on duration of three months (Rabi' II to Rajab), around 10 questionnaire sheets per day. Every sheet took an average time for 25-30 minutes from the students during their studying day, from 8am-3pm. Pilot study: Pilot study was carried out on 10% of nursing students who attended the classes to test the clarity and relevance of the tool. According to the results of pilot study, necessary modifications in the sheet were done. Based on the pilot; the meaning of some questions was vague for the students, so the researchers omitted it, also some of questions were difficult in language modified to be easy for the students. Jury members: A jury of (13) faculty members: Two professors, four associate professor and seven assistant professors were chosen to establish content validity. Statistical analysis: Data entry and statistical analysis were done using SPSS 14.0 statistical software package. Quality control was done at the stages of coding and data entry. Data were presented using descriptive statistics in the form of frequencies and percentages for qualitative variables, and means and standard deviations for quantitative variables. Qualitative categorical variables were compared using chi-square test. Statistical significance was considered at p-value<0.05. Results The results of this study described the sociodemographic characteristics of the studied subjects and indicated that age ranged from 18-35 years
544

Table (1): Socio-demographic and health characteristics of studied subjects (n=300). Socio-demographic characteristics Age (years): <20 2022+ Range MeanSD Pre-college education: Public Private Marital status: Single Engaged Married Divorced Birth order: 1 2-4 5+ Have chronic disease: No Yes Types of chronic diseases (n=48):@ Allergies Bronchial asthma Sickle cell anemia Hypertension Migraine Heart disease Back problems Kidney disease Liver disease Diabetes Have impairment/disability: No Yes Have problems with menstruation that affect study
@ Responses are not mutually exclusive.
old, with mean standard deviation 20.61.8 years. Regarding age more than two-fifth 42.6% of the subjects were at the age group 20-21 years old. The majority of students 72.5% had public precollege education, and were single 82.5%, while about one-tenth 10.4% were married. The birth order of students was mostly second to fourth 41.9%. Concerning students health status and medical history, this table showed that 16.1% had a history of chronic disease. The most commonly medical problems reported were allergies, bronchial asthma, sickle cell anemia, hypertension, and migraine, 29.2, 18.8, 14.6, 10.4, and 10.4 percent, respectively. Meanwhile, none of them had diabetes. It was noticed that about two-thirds of the students 63.4% reported problems with menstruation that affect their study Table (1). Concerning stressors reported by students, Table (2) demonstrated that the most frequent academic sources of stress as perceived by students were increased workload 64.9%, and anticipation of graduation 54.7%. At the other extreme, the least reported factors were those of missing too many classes and serious argument with instructors, 13.3% and 8.9%, respectively. Regarding intrapersonal sources of stress, Table (3) demonstrated that the highest sources perceived by students were related to changes in sleeping habits 56.5%, eating habits 49.7%, in addition to new responsibilities 46.9%. On the other hand, the least intrapersonal factors reported as sources of stress were feeling helpless for not being able to improve patient's condition 12.7%, and inability to answer question of patients or their families 10.6%.
Frequency 89 127 82 18-35 20.61.8 198 75 245 21 31 1 68 125 105 250 48 14 9 7 5 5 3 3 1 1 0 189 63 189
Percent 29.9 42.6 27.5
72.5 27.5 82.2 7.0 10.4 0.3 22.8 41.9 35.2 83.9 16.1 29.2 18.8 14.6 10.4 10.4 6.3 6.3 2.1 2.1 0.0 63.4 21.1 63.4
Table (2): Academic sources of stress as perceived by the studied subjects (n=300). Academic stressors Increased study workload Anticipation of graduation Lower grade than anticipated Change of Major (Career) Vast differences between lectures at school and actual practice Fear of having to read too much information and English books Trouble dealing with particular or complicated conditions Unfairly given more work and difficult case studies than other classmates Teaching contents are far from what I expected Do not know how to do a case report Missing too many classes Serious argument with instructor Never/rare No. 0 1 0 0 2 0 3 2 5 7 0 4 % 0.0 0.3 0.0 0.0 0.7 0.0 1.0 0.7 1.7 2.4 0.0 1.4 Sometimes No. 102 130 151 174 181 212 217 221 230 243 254 261 % 35.1 45.0 51.5 59.8 62.0 71.9 73.8 75.7 79.0 83.2 86.7 89.7 Often/very often No. 189 158 142 117 109 83 74 69 56 42 39 26 % 64.9 54.7 48.5 40.2 37.3 28.1 25.2 23.6 19.2 14.4 13.3 8.9

Table (3): Intrapersonal sources of stress as perceived by the studied subjects (n=300). Intrapersonal stressors Change in sleeping habits Change in eating habits New responsibilities Responsibility violation (abuse) Financial difficulties Decline in personal health Embarrassment of speaking in public Fear of contagious diseases at the hospital Conflict with customs/beliefs Major family event (death/illness) Being a student, not trusted by patients or their families Major personal event (engagement/ marriage/divorce/delivery) Unable to handle patients' psychological, financial or other situations Feeling helpless for not being able to improve a patient's condition Can't answer questions of patients or their families Never/rare No. 0 0 0 0 0 0 0 16 0 0 19 2 19 17 20 % 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.5 0.0 0.0 6.5 0.7 6.5 5.8 6.8 Sometimes No. 128 148 154 200 201 204 207 211 234 235 218 238 222 238 242 % 43.5 50.3 53.1 68.3 68.6 70.1 71.4 72.5 79.3 80.2 74.7 81.5 76.3 81.5 82.6
545
Often/very often No. 166 146 136 93 92 87 83 64 61 58 55 52 50 37 31 % 56.5 49.7 46.9 31.7 31.4 29.9 28.6 22.0 20.7 19.8 18.8 17.8 17.2 12.7 10.6
As regard interpersonal sources of stress as perceived by students, results indicated that all of them reported change in social activities and working with people not known to them, fight with family members, and with new colleagues were often or sometimes sources of interpersonal stress. At the other end, trouble with instructors was the least factor "often" perceived by students 8.8%. Additionally, Roommate conflict 86.2% sometimes experience interpersonal stress, while 17.5% not knowing how to communicate with other medical staff about patients' condition and it was the factor with the highest percentage of "never/rare" response Table (4). Table (5) described the environmental sources of stress as perceived by students. It indicated almost all the factors were mentioned by all students as occurring "sometimes, often, or very often." The highest environmental factor was college
discipline 43.6%. On the other hand, only three factors were mentioned to never or rarely be sources of stress; these were negative attitude from hospital staff, lack of awareness of hospital resources, and lack of hospital resources, 5.5%, 5.6%, and 5.6%, respectively. The relationship between total sources of stress as perceived by students and their sociodemographic characteristics are illustrated in this table. Results indicated statistically significant relations with student's academic level (p=0.003), and age (p=0.002). As evident from this table, the highest stress was among sixth level students 70.0%, while the lowest was in the first 20.0% and second 22.0% levels. Also, stress was lowest among students younger than 20 years age 18.9%. Meanwhile, none of the other students characteristics had statistically significant relations with total stressors Table (6).
Table (4): Interpersonal sources of stress as perceived by the studied subjects (n=300). Interpersonal stressors Change in social activities Work with people you don't know Fear of my clinical performance being of lower grade than other classmates Nurse's job was not being respected by patients and other hospital staff Not knowing how to communicate to other medical staff about patients' condition Fight with family member Roommate conflict Lack of interpersonal skills to communicate with patients and their families Fight with new colleagues Trouble with instructors
@ Responses are not mutually exclusive
Never/rare No. 0 0 10 16 19 0 3 16 0 6 % 0.0 0.0 3.4 5.5 6.5 0.0 1.0 5.5 0.0 2.1
Sometimes No. 153 207 222 220 222 245 250 238 263 253 % 52.0 70.9 76.3 75.3 76.0 83.3 86.2 81.8 90.7 89.1
Often/very often No. 141 85 59 56 51 49 37 37 27 25 % 48.0 29.1 20.3 19.2 17.5 16.7 12.8 12.7 9.3 8.8
546

Table (5): Environmental sources of stress as perceived by the studied subjects (n=300). Environmental stressors College discipline Non-flexible and long working hours that affect normal life Change in living conditions Computer problems Transportation problems Negative attitude from hospital staff Placed in unfamiliar situation Lack of awareness about hospital resources Waiting long time between lectures Lack of hospital resources Never/rare No. 0 0 0 0 0 16 0 16 0 16 % 0.0 0.0 0.0 0.0 0.0 5.5 0.0 5.6 0.0 5.6 Sometimes No. 164 178 201 210 214 200 227 217 246 230 % 56.4 60.5 69.3 71.4 73.3 69.0 77.7 75.6 84.0 79.9 Often/very often No. 127 116 89 84 78 74 65 54 47 42 % 43.6 39.5 30.7 28.6 26.7 25.5 22.3 18.8 16.0 14.6
Table (6): Relationship between total sources of stress as perceived by the studied subjects and their sociodemographic characteristics (n=300). Total stressors Variables No. Academic level: 1st 2nd 3rd 4th 5th 6th 7th 8th Age (years): <20 2022+ Marital status: Single Engaged Married/divorced Have chronic disease: No Yes Have menstruation problems that affect study: No Yes Have impairment/disability: No Yes
* Statistically significant at p<0.05
High. % 20.0 22.0 38.1 28.6 55.0 70.0 36.4 37.5 18.9 41.8 43.5 34.2 33.3 43.5 33.2 44.7 39.3 32.4 36.7 28.3 No. 24 39 13 20 9 6 21 20 60 57 35 129 10 13 131 21 54 98 119 33
Low % 80.0 78.0 61.9 71.4 45.0 30.0 63.6 62.5 81.1 58.2 56.5 65.8 66.7 56.5 66.8 55.3 60.7 67.6 63.3 71.7
X2 Test
p-value
6 11 8 8 11 14 12 12 14 41 27 67 5 10 65 17 35 47 69 13
21.67
0.003*
12.41
0.002*
0.80
0.67
1.87
0.17
1.16
0.28
1.16
0.28
The relationship between academic sources of stress as perceived by students and their sociodemographic characteristics are presented in this table. It pointed to statistically significant relations with student's academic level (p=0.009), and age (p=0.04). It was noticed that the highest stress was among fifth and sixth-level students, 80.0% and 90.0%, respectively. Meanwhile, the lowest was
in the first levels 43.3%. Regarding age, stress was highest among students in the age group 20-21 years 64.3%. No statistically significant relations were revealed between any of the other characteristics and academic stressors Table (7). Table (8) displayed the relationship between intrapersonal sources of stress as perceived by
547
students and their socio-demographic characteristics. It showed statistically significant relations with student's academic level (p=0.03), age (p=0.03), and chronic diseases (p=0.006). It was noticed that the highest stress was among third, fifth, and sixth-level students 66.7%, 60.0%, and 60.0%, respectively, while the lowest was in the first and second levels 33.3% and 32.0, respectively. Moreover, stress was lowest among students younger than 20 years age 32.4%. As for the presence of chronic diseases, about two thirds of students with chronic diseases 65.8% had high stress, compared to 41.3% of those with no chronic diseases. The other students characteristics had no statistically significant relations with intrapersonal stressors. Table (9) described the relation between interpersonal sources of stress as perceived by students
and their socio-demographic characteristics. It indicated statistically significant relationship with student's academic level (p=0.001), and age (p=0.001). It was evident that the highest stress was among fifht level students 70.0%, while the lowest was in the first and second levels 10.0%. Also, stress was lowest among students younger than 20 years age 9.5%. Meanwhile, neither marital status nor birth order had statistically significant relations with interpersonal stressors. The relationship between environmental sources of stress as perceived by students and their sociodemographic characteristics, no statistically significant relationship was found with any of the students socio-demographic characteristics and environmental sources Table (10).
Table (7): Relationship between academic sources of stress as perceived by the studied subjects and their socio-demographic characteristics (n=300). Academic stressors Variables No. Academic level: 1st 2nd 3rd 4th 5th 6th 7th 8th Age (years): <20 2022+ Marital status: Single Engaged Married/divorced Have chronic disease: No Yes Have menstruation problems that affect study: No Yes Have impairment/disability: No Yes
High. % No.
Low %
X2 Test
p-value
13 23 12 15 16 18 17 16 33 63 34 110 6 14 105 25 47 83 104 26
43.3 46.0 57.1 53.6 80.0 90.0 51.5 50.0 44.6 64.3 54.8 56.1 40.0 60.9 53.6 65.8 52.8 57.2 55.3 56.5
17 27 9 13 4 2 16 16 41 35 28 86 9 9 91 13 42 62 84 20
56.7 54.0 42.9 46.4 20.0 10.0 48.5 50.0 55.4 35.7 45.2 43.9 60.0 39.1 46.4 34.2 47.2 42.8 44.7 43.5
18.80
0.009*
6.64
0.04*
1.76
0.42
1.92
0.17
0.44
0.51
0.02
0.88
548

Table (8): Relationship between intrapersonal sources of stress as perceived by the studied subjects and their socio-demographic characteristics (n=300). Intrapersonal stressors X2 Variables p-value High. Low Test No. % No. % Academic level: 1st 10 33.3 20 66.7 16 32.0 34 68.0 2nd 14 66.7 7 33.3 3rd 11 39.3 17 60.7 4th 60.0 8 40.0 15.86 0.03* 5th 12 60.0 8 40.0 6th 12 57.6 14 42.4 7th 19 37.5 20 62.5 8th 12 Age (years): 32.4 50 67.6 24 <20 51.0 48 49.0 50 7.24 0.03* 2051.6 30 48.4 32 22+ Marital status: 44.4 109 55.6 87 Single 53.3 7 46.7 8 0.52 0.77 Engaged 47.8 12 52.2 11 Married/divorced Birth order: 46.2 28 53.8 24 1 44.9 54 55.1 44 0.02 0.99 2-4 45.2 46 54.8 38 5+ Have chronic disease: 41.3 115 58.7 81 No 65.8 34.2 25 13 7.69 0.006* Yes Have menstruation problems that affect study: 46.1 53.9 41 48 No 44.8 55.2 65 80 0.03 0.85 Yes Have impairment/disability: 44.7 55.3 84 104 No 47.8 52.2 22 24 0.15 0.70 Yes
Table (9): Relationship between interpersonal sources of stress as perceived by the studied subjects and their socio-demographic characteristics (n=300). Interpersonal stressors X2 High. Low Variables p-value Test No. % No. % Academic level: 1st 10.0 27 90.0 3 10.0 45 90.0 2nd 5 33.3 14 66.7 3rd 7 25.0 21 75.0 4th 7 31.97 <0.001* 30.0 14 70.0 5th 6 70.0 6 30.0 6th 14 27.3 24 72.7 7th 9 31.3 22 68.8 8th 10 Age (years): 9.5 67 90.5 <20 7 16.00 <0.001* 35.7 63 64.3 2035 30.6 43 69.4 22+ 19 Marital status: 25.5 146 74.5 50 Single 0.26 0.88 26.7 11 73.3 4 Engaged 30.4 69.6 16 7 Married/divorced Birth order: 34.6 65.4 34 18 1 3.68 0.16 20.4 79.6 78 20 2-4 27.4 72.6 61 23 5+

Table (10): Relationship between environmental sources of stress as perceived by the studied subjects and their socio-demographic characteristics (n=300). Environmental stressors Variables No. Academic level: 1st 2nd 3rd 4th 5th 6th 7th 8th Age (years): <20 2022+ Marital status: Single Engaged Married/divorced Birth order: 1 2-4 5+
549
High. % 33.3 44.0 28.6 35.7 65.0 50.0 36.4 40.6 36.5 43.9 41.9 39.3 46.7 52.2 44.2 41.8 38.1 No. 20 28 15 18 7 10 21 19 47 55 36 119 8 11 29 57 52
Low % 66.7 56.0 71.4 64.3 35.0 50.0 63.6 59.4 63.5 56.1 58.1 60.7 53.3 47.8 55.8 58.2 61.9
X2 Test
p-value
10 22 6 10 13 10 12 13 27 43 26 77 7 12 23 41 32
8.30
0.31
0.98
0.61
1.62
0.44
0.55
0.76
Discussion University students, however, often experience an undue amount of stress, which can have negative academic, emotional, or health outcomes [5]. This can occur at different time periods during a semester or years in college, during the transition from undergraduate to professional or graduate programs, or upon graduation. Stress in university students has many sources, including academics, personal situations, environment, time, and economic circumstances [20] . Therefore the aim of this study was to identify sources of stress as perceived by undergraduate nursing students at College of Nursing, King Saud University. This study included students with mean age 20.61.8 years, 82.5% were single. Studies on major sources of stress among academic baccalaureate students proved that the same characteristics are congruent with this present study [16]. Stress may be perceived negative or positive, intense or not intense in relation to several factors that calculate the effect of stress outcome and impact of stress on the individual in various situations that he is confronted with through life experience. Among this factors that influence stress perception are: Previous personal experience, intensity of stress, maturation and duration of stress experience.
According to the current study findings, more than half of students 54.7% said that anticipation of graduation was very often an academic source of stress. Result of this study proved that 37.3% of students mentioned that there are vast difference between lectures in school and actual practice and 89.7% of study subjects perceived that serious argument with an instructor sometimes is an academic source of stress. More than three quarters 79% mention that sometimes teaching content is far from there expectation. Integration of theory to practice, insure the theoretical relation behind clinical action and clear communication challenge during clinical training of students are big issues and dilemma to be resolved for academic education. Evan and Kelly [21] concluded that nursing students have the same academic stressors as other college students, such as midterm and final examinations, research papers and other assignments. In addition, nursing students experience a clinical component, which is highly stressful. Students have a large amount of preparatory work before their clinical assignments. It was noticed in this study that the highest sources of stress among King Saud University College of nursing students was presented by the fifth and sixth-level students, 80.0% and 90.0%,
550
respectively. Meanwhile, the lowest was in the first levels 43.3%, as regard age, stress was highest among students in the age group 20-21 years 64.3%. Beck and Srivastava [22] investigated the perception of level and sources of stress in nursing students in the various years of the baccalaureate nursing program which conducted at School of Nursing concluded that the second, third and fourth year nursing students reported that clinical experience was the most stressful part of the nursing program. However Beck et al. [5] in their study about sources of stress reported that a highest level of stress was in second year of nursing students in a medical college. Larson [23] concluded that stress among nursing students is concerned with the recent move to third level education in nursing education programmers. Also Singh and Upadhyay [24] members in the Indian Academy of Applied Psychology demonstrated that first year students experienced higher degree of academic stress in comparison of third year students. Intrapersonal sources of stressors as perceived by nursing students at King Saud University the highest stressors perceived by students were related to changes in sleeping habits 56.5%, eating habits 49.7%, in addition to new responsibilities 46.9%. On the other hand, the least intrapersonal factors reported as sources of stress were feeling helpless for not being able to improve patient's condition 12.7%, and inability to answer question of patients or their families 10.6%. These pervious results for intrapersonal categories come among students' mostly were live in dormitories 26.5% this may cause more stress than for students who live inside Riyadh. In addition the vast difference between school and university was another source of stress, which leads them to residence in college campus or renting special home other than their own home often, they experience ambivalent feelings resulting from the need for parental/familial support and the drive for independence. Rocha-Singh, [25] concluded from his study on graduated Pre-Diploma Science students of University Technology MARA (UITM)" is a Malaysian public university" stated that the top five intrapersonal stressors were, in order: Change in sleeping habits 89%, vacations/breaks 82%, and change in eating habits 74%, new responsibilities 73%, and increased class workload 73%. Financial difficulties 71% and change in social activities 71% were also frequently reported stressors. The five least frequently reported stressors were; death of a friend 6%, severe injury 5%,
transferred schools 3%, engagement/marriage 2%, and divorce between parents 1%. Quitting one's job 8% was also infrequently reported stressor. Another interesting result was that, in a college setting, events such as missing too many classes 21% and arguing with an instructor 11% only comprised 15% of the total responses. Seyedfatemi [13] went in the same line with this study and concluded that the most common intrapersonal sources of stress were "new responsibilities" 72.1% and change in sleeping habits" were significantly greater stressors in first year students than in students of other years. Another study that examined sources of stress among nursing student this study agreed that the stress factors such, not getting enough sleep or sleeping too much, social activities, finance, course loads and problems with boyfriends/girlfriends (partners) pose major problems to the students even to the end of the semester which affect their academic performance [26]. On the contrary Ross, et al. [10] concluded that daily hassles were responsible for 77.3% of the intrapersonal stressors. The relationship between total factors perceived by students as sources of stress and their socio demographic characteristics in this research indicated statistical significant relations with student academic level and age, the highest stress was among students at level sixth and lowers was among students more than 20 years old. These results went in the same line with [27] who concluded that there is consistent evidence to suggest that certain demographic characteristics may be indicators of a greater or lesser susceptibility to stress as age, gender, and relationship status. The relationship between intrapersonal factors perceived by students as sources of stress and their socio-demographic characteristics among nursing college King Saud University in this present study. Results revealed that stress was lowest among students younger than 20 years age 32.4% this could be interpreted by that majority of age group in the current study was in the age group 20-22 years and above. It was noticed that the highest stress was among third, fifth, and sixth-level students 66.7%, 60.0%, and 60.0% respectively, while the lowest was in the first and second levels 33.3% and 32.0 respectively. From researchers point of view this may be due to at this level the students begin clinical training and this intrapersonal stress may lead to negative impact on students academic and interpersonal stress. This opinion went in the same line
551
with [28] who asked college students about their most stressful daily hassles. They observed that the most irritating daily hassles were usually schoolrelated stressors such as constant pressure of studying, too little time, writing term papers, taking tests, plans, and boring instructors. Al-Omar [29] agreed with this study, he studied the knowledge, attitudes, and intention among the Saudi high school students towards the nursing profession and identify students perception of causes preventing them to become nurses. He concluded that the major stressors were in order; long working-hours 59.3%. Community does not appreciate nursing profession 58.9%, night shifts 56.2%, requires working with opposite sex 53.9%, majority of people down grade, nurses 52.6%, Ignored-job by decision makers 48.3%, unpleasant environment to work 38.9%. People do not respect nursing profession 42.2%, afraid of getting diseases 39.6%, hating the sight of blood 37.2%, High workload compared to other jobs 41.2%. These results coincide with the current study, may be because of the study done in the same culture for example as in the study results 79.3% were mentioned that the conflict with customs/beliefs are sometimes stressful. As regard interpersonal factors perceived by students as sources of stress, results indicated that the entire subjects reported change in social activities and working with people not known to them, fight with family member, and with new colleagues were often or sometimes sources of interpersonal stress. On the other end, trouble with instructors was the least factor "often" perceived by students 8.8%. Additionally, Roommate conflict 86.2% Sometimes experience interpersonal stress, while 17.5% not knowing how to communicate to other medical staff about patients condition was the factor with the highest percentage of "never/rare" response. This result may be due to female students in this culture spend more time in their residential environment and share more involvement with their roommates, or family members so they are more susceptible to interpersonal stresses related to miss communication. Ahern [30] agreed with the results of current study and stated that social circumstances and low complexity activities in which stress was experienced. While Frazier, and Schauben [31] added other stressors to be among the major sources of stress and play a role enhancing or hindering academic achievement with the results of current study and concluded that the five most frequently named stressors were test pressure, financial problems,
being rejected by someone, relationship breakups, and failing a test, had experienced the death of a significant other (e.g., parent, sibling, or friend). Higher amounts of stress were associated with more psychological symptoms and more disrupted beliefs. Naidu, et al. [32] concluded that the problems encountered by dental students in the United States and abroad are the difficult interactions with faculty. Also the major stressors were fear of failing or poor performance as stressful events, continuous poor academic performance and expectation from family or friends. Giacobbi, et al. [33] assessed stress and coping during the transition to university for first-year female athletes and revealed that the major sources of stress were high performance expectations, interpersonal relationships, and being away from home. From the researchers point of view all results agreed that putting more expectation on students leads to more stress especially in Arab countries that put more stress on their students and obligate them to obtain high scores regardless their capabilities. So this pressure to achieve good grades can result in a battle for self-esteem of students. Being in different environment can cause a great deal with stress, hence environmental factors may be perceived by students as major sources of stress. The results of this study indicated that the highest environmental factor was college discipline 43.6% very often. On the other hand, only three factors were mentioned to never or rarely be sources of stress; these were negative attitude from hospital staff, lack of awareness of hospital resources, and lack of hospital resources, 5.5%, 5.6%, and 5.6%, respectively. This means that student experience on lab are helpful for orienting the student about hospital situation resources and facilities, hence lab experience on the college may be very beneficial in relation to those two aspect. Daily hassles accounted for 88.2% of the environmental stressors. Daily hassles were responsible for 77.3% of the intrapersonal stressors, and 67.2% of the academic sources of stress. Overall, 81.1% of the identified stress sources could be classified as daily hassles. Studies on the lived experience of new graduate baccalaureate-prepared registered nurse and found that the majority of the participants expected the graduate program mention that learning environment to be challenging, friendly and supportive. They are therefore placed in unfamiliar surroundings, with crowded streets and other stressors
552
endemic to a capital city. Thus when students doing their clinical learning in hospital, higher stress level was reported than just studying in college or inside the university [34]. Sayedfatimi [13] concluded that among environmental sources of stress, most students reported that "being placed in unfamiliar situations" 64.2% and "waiting in long lines" 60.4% were the most frequent stressors. The binomial test showed that "being placed in unfamiliar situations" was a significant stressor in all 4 years, whereas "waiting in long lines" and "change in living environment" were significantly greater in first year students than in students of other years. A study on college nursing students proved that Nigerian nursing students have high levels of stress, with the most common stressors including excessive schoolwork, financial problems, inadequate recreational facilities, and overcrowded accommodations. These findings indicated a need for counseling and other support services among nursing students [35]. Schaufeli, et al. [36]. Emphasized that environmental role as sources of stress, focused on different conditions in the job environment and have found that role stressors, such as long hours, are associated with burnout and high levels of turnover. Another study for environment as sources of stress concluded that the major stress factors in male house officers were lack of resources, work overload and lack of communication and comfort with supervisors and colleagues. Lack of resources was the factor observed in all the departments. Female house officers do not experience much stress compare to males as they have support from their supervisors and colleagues [37]. Hamill [38] found that nursing students often have difficulty adjusting to the academic environment of higher education, as well as difficulty adjusting to the environment of a nursing unit. The most likely explanation is that causes of burnout are found in both the individual and the environment. College stress cannot be prevented, but educators and administrators can do better job to guide students on how to pre-departure preparation and stress management strategies. Conclusively, stress may be exacerbated among health allied science students with their start application performance on actual clinical setup especially at hospital. Therefore simulation labs are conducted for students and should be designed accurately to resemble the actual clinical situation and should be equipped
with advanced technology that help student to handle simulators as if it is a real patient, this may decreased the shock of the direct exposure to actual clinical setup and actual patient and show decrease the level of stress on advanced academic year that the student need to be in actual clinical setup [39]. The present study results concluded that their where a variety of stressors placed on the studied subjects. The major sources of stress as perceived by King Saud University College of Nursing students were the academic followed by intrapersonal, then environmental and the last sources of stress for these students was interpersonal. The results of the current study emphasized the following aspects as recommendations: - It is important for the university to maintain well balanced academic environment conducive for better learning, with the focus on the students personal needs. The university should be affording adequate resources such as books, computers to do assignments. - Nursing college should do an effort to narrow the gap between Students expectations, goals, and values to be integrated with that of the university. - Stress management workshops are important to train student's different ways to deal with unexpected stressors. - Faculty members should train students to cope with stress by using stress management techniques which is either physical or mental. References
1- REES C. and REDFERN D.: Recognizing the perceived causes of Stress-training and development perspective. Industrial and Commercial Training Journal, 4 (32): 1207, 2000. 2- LEE H. A.: Study on stress and coping method of nursing students. Journal of Korean Academy of Psychiatric and Mental Health Nursing, 12: 586-93, 2003. 3- TULLY A.: Stress, sources of stress and ways of coping among psychiatric nursing students. Journal of Psychiatric and Mental Health Nursing, 11: 43-7, 2004. 4- AWINO J. and AGOLLA J.: A quest for sustainable quality assurance measurement for universities. Case of study of the University of Botswana. Educational Research and Review Journal, 2 (4): 63-70, 2009. 5- BECK D.L., HACKET, M.B., SRIVASTAVA R., MCKIM E. and ROCKWELL B.: Perceived level and sources of stress in university professional schools. J. Nurs Educ., (36) 4: 180-6, 1997. 6- MISRA R., and MCKEAN M.: College students academic stress and its relation to their anxiety, time management,

and leisure satisfaction. American Journal of Health Studies, 1 (16): 41-51, 2000. 7- PRATER L. and MCEWEN M.: Perceptions of student nurses. Journal of Holistic Nursing, 1 (24): 63-9, 2006. 8- ROBIN L.: A Comparison of perceived stress levels and coping styles of junior and senior students in nursing and social work programs. Doctorate of Education Dissertation College of Graduate Studies, Marshall University, 2002. 9- NWADIANI M. and OFOEGBU F.: Perceived Level of Stress among First-times in Nigerian Universities. College Student Journal, 1 (35): 1, 2001. 10- ROSS S., NIEBLING B. and HECKERT T.: Sources of stress among college students. College Student Journal, (33): 312-7, 1999. 11- BAYKAL U., SOKMEN S., KORKMAZ S. and AKGUN E.: Determining student satisfaction in a nursing college. Nurse Educ Today, 4 (25): 255-6, 2005. 12- NICHOLL H. and TIMMINS F.: Program-related stressors among part-time undergraduate nursing students. Journal of Advanced Nursing, 1 (50): 93-100, 2005. 13- SEYED FATEMI N., TAFRESHI M. and HAGANI H.: Experienced stressors and coping strategies among Iranian nursing students. BMC Nursing, 11 (6): 1-10, 2007. 14- YOKOZUKA N.: Stress at college: Effects on health habits, health status and self-esteem. College Student Journal, 4 (3): 217-22, 2000. 15- OLPIN M.: Perceived stress levels and sources of stress among college students: Methods, frequency, and effectiveness of International Section A: Humanities and Social Sciences, (57): 4279-88, 1997. 16- NELSON N., DELL' OLIVER C., KOCH C. and BUCKLER R.: Stress, coping, and success among graduate students in clinical psychology. Psychological Reports, 3 (88): 759-67, 2001. 17- TOPPER E.: Stress in the library workplace. New Library World, 108 (11/12): 561-4, 2007. 18- TIMMINS F. and KALISZER M.: Aspects of nurse education programmers that frequently cause stress to nursing students - fact-finding sample survey. Nurse Education Today, 3 (22): 203-11, 2002. 19- DYSON R. and RENK K.: Freshmen adaptation to university life: Depressive symptoms, stress, and coping. Journal of Clinical Psychology, 10 (62): 1231-44, 2006. 20- HEINS M., FAHEY S.N. and LEIDEN L.: Perceived stress in medical, law, and graduate students. J. Med. Educ., 3 (59): 169-79, 1984. 21- EVANS W. and KELLY B.: Pre-registration diploma student nurse stress and coping measures. Nurse Education Today, 6 (24): 473-82, 2004. 22- BECK D. and SRIVASTAVA R.: Perceived level and sources of stress in baccalaureate nursing students. Journal of Nursing Education, (30): 127-33, 1991. 23- LARSON E.: Stress in the lives of college women: Lots to do and not much time. Journal of Adolescent Research, 6 (21): 579-606, 2006. 24- SINGH A. and UPADHYAY A.: Age and sex differences in academic stress among college students. Social Science International, 1 (24): 78-88, 2008.
553
25- ROCHA-SINGH I.: Perceived stress among graduate students: Development and validation of the Graduate Stress Inventory. Educational and Psychological Measurement, 3 (54): 714-27, 1994. 26- GOLDSTEIN M.: Sources of stress and interpersonal support among first-year dental students. J. Dent. Educ., 1 (53): 718-21, 1989. 27- CHEUNARROM C. and BENJAKUL P.: Student adjustment problems in two dental schools in Thailand. J. Dent. Educ., 5 (64): 365-9, 2000. 28- JENKINS R. and ELLTOTT P.: Stressors, burnout and social support: Nurses in acute mental health settings. Journal of Advanced Nursing, 6 (48): 622-31, 2004. 29- AL-OMAR B.A.: Knowledge, attitudes and intention of high school students towards the nursing profession in Riyadh city, Saudi Arabia. Saudi Med. J., 2 (25): 150-5, 2004. 30- AHERN N.R.: Risky behaviors of adolescent college students. Journal of Psychosocial Nursing and Mental Health Services, 4 (47): 21- 55, 2009. 31- FRAZIER P., and SCHAUBEN L.: Stressful life events and psychological adjustment among female college students. Measurement and Evaluation in Counseling and Development, 1 (27): 280-92, 1994. 32- NAIDU R.S., ADAMS J.S., SIMEON D. and PERSAD S.: Sources of stress and psychological disturbance among dental students in the West Indies. J. Dent. Educ. 1 (66): 1021-3021, 2002. 33- GIACOBBI P., LYNN T., WETHERINGTON J., JENKINS J., BODENDORF M. and LANGLEY B.: Stress and coping during the transition to university for first-year female athletes. The Sport Psychologist, 1 (18): 1-20, 2004. 34- HARPER J. R.: The lived experience of new graduate baccalaureate-prepared registered nurses working in an acute care hospital setting. Doctor of Philosophy, Louisiana State University and agricultural and Mechanical College, 2005. 35- OMIGBODUN O., ONIBOKUN A., YUSUF B., ODUKOGBE A. and OMIGBODUN A.: Stressors and counseling needs of undergraduate nursing students in Ibadan, Nigeria. Journal of Nursing Education, 9 (43): 412-5, 2004. 36- SCHAUFELI W., BAKKER B., HOOGDUIN K., SCHAAP C. and KLADLER A.: On the clinical validity of the Maslach Burnout Inventory and the burnout measure. Psychology and Health, 5 (16): 565-82, 2001. 37- KAZMI R., SHEHLA A. and KHAN D.: Occupational stress and its effect on job performance a case study of medical house officers of district Abbottabad. J. Ayub Med. Coll Abbottabad, 3 (20): 135-9, 2008. 38- HAMILL C.: The phenomena of stress as perceived by Project 2000 student nurses: A case study. Journal of Advanced Nursing, 3 (21): 528-36, 1995. 39- SMITH T. and RENK K.: Predictors of academic-related stress in college students: An examination of coping, social support, parenting, and anxiety. NASPA Journal, 3 (44): 78-90, 2007.

Acropat

Transféré par

Informations du document

Copyright

Formats disponibles

Partager ce document

Partager ou intégrer le document

Options de partage

Avez-vous trouvé ce document utile ?

Ce contenu est-il inapproprié ?

Droits d'auteur :

Formats disponibles

Acropat

Transféré par

Droits d'auteur :

Formats disponibles

FACULTY OF ME D IC N E UA ISSN 0045 - 3803 NLM (PubMed) Id: 0362256 Since 1967.

Gr Id: 28312 Input of the IMEMR (WHO) Since 1986.

Vol. 79, No. 1, September 2011

369 377 383

389 407 415 421

441 449 457 465

Overexpression of C-KIT in Schistosomal Urinary Bladder Carcinoma, TAHANY SHAMS and

509 519 525 529 533 537 541

Topiramate for Prophylaxis of Basilar Migraine in Children and Adolescents

Topiramate for Prophylaxis of Basilar Migraine in Children & Adolescents

Sayed A. Amin & Waleed R. Abou Seree

Fisher's exact test, p=.13

Fisher's exact test, p=1.0

Fisher's exact test, p=1.0

Topiramate for Prophylaxis of Basilar Migraine in Children & Adolescents

Sayed A. Amin & Waleed R. Abou Seree

Topiramate for Prophylaxis of Basilar Migraine in Children & Adolescents

Ossama A. Most afa, et al.

Ossama A. Most afa, et al.

11.7 181 88.3 45.6 106 54.4

Ossama A. Most afa, et al.

Response to Combined Pegylated Interferon & Ribavirin among Patients

Saad Zaky, et al.

Response to Combined Pegylated Interferon & Ribavirin among Patients

318 67 15 23 194 126 57 190 149 61

At 24 weeks Responders 289 (72.2%) No. 41.69.3 %

At 48 weeks Responders 257 (64.2%) No. 41.79.3 %

SVR Yes 96 (41.2%) No. 41.98.9 %

90.6 9.4 75.613.5 165.77.0 26.64.1

72.612.5* 166.45.6 26.44.3* 84 122 86 28.8 41.8 29.5 81 119 89

76.913.0 166.25.8 26.73.3 28.0 41.2 30.8 74

76.913.1 166.15.9 27.84.6 28.7 41.5 29.8 32 39 25 107 77

33.3 40.6 26.0

Saad Zaky, et al.

At 48 weeks Responders 257 (64.2%) No. % 14.19.8 54.3 45.7

90 (67.2%) 44 (32.8%) 134

6 (6.1%) 0.000* 93 (93.9%) 99

Response to Combined Pegylated Interferon & Ribavirin among Patients

Saad Zaky, et al.

Response to Combined Pegylated Interferon & Ribavirin among Patients

Maternal Weight & the Success of Trial of Vaginal Delivery after CS

Waleed A.S. Ahmed & Ayman Morsy

Maternal Weight & the Success of Trial of Vaginal Delivery after CS

Waleed A.S. Ahmed & Ayman Morsy

Impact of Adjuvant Chemotherapy Delay on Outcome in Cancer Colon Patients

Impact of Adjuvant Chemotherapy Delay on Outcome in Cancer Colon Patients

Zeinab M. Abd El-Hafeez, et al.

CT: Chemotherapy. FOLFOX: 5FU/leucovorin/oxaloplatin. LN: Lymph node metastases.

Impact of Adjuvant Chemotherapy Delay on Outcome in Cancer Colon Patients

Zeinab M. Abd El-Hafeez, et al.

Detection of Coronary Artery Stenoses

Sherif M. Maher, et al.

Detection of Coronary Artery Stenoses

Sherif M. Maher, et al.

Detection of Coronary Artery Stenoses

Nonsig. stenosis 1 1 2 0 1 4 0 0 0 Occlusion 1 0 0 0 0 1 0 0 0

NSS = Nonsignificant stenosis. SS = Significant stenosis.

Sherif M. Maher, et al.

Detection of Coronary Artery Stenoses

Sherif M. Maher, et al.

Detection of Coronary Artery Stenoses

Sherif M. Maher, et al.

Detection of Coronary Artery Stenoses

Sherif M. Maher, et al.

Combination 43.81.7#$@ 1.70.9#@ 0.590.02#$@ 277.59.1#$@