Semen analysis

Parameters of Semen Analysis

Parameter Normal value Abnormality
PH 7.2 or more
2- 6 ml <2 hypospermia
Volume >6 hyperspermia
No aspermia
Spermconcentration : more than 20 millions sperms/ml <20 oligozoospermia
Count Total amount of sperms : more than 40 millions sperms/ml >20 polyzoospermia
No azoospermia
Grade A: rapid progressive motility
Grade B: sluggish motility If A+B < 50 % asthenozoospermia
Grade C: abnormal rotatory ( move around himself )
Grade D: immotile sperms
Motility Normally:
A + B ( progressive mo lity ) > 50%
A only > 25%
Within 60 minute of ejacula on
Morphology If abnormal forms > %
15% normal morphology by strict Krugers criteria
Teratozoospermia
Vitality 75% or more live
WBCs Less than 10/HPF >10 pyospermia/leukosspermia
pyospermia
RBCs Less than 10/HPF >10 Haemospermia
H
Immuno-bead test
> 50% mo le sperms with beads bound

MAR test > 50% mo le sperms with adherent par cles

Normozoospermia
Oligoasthenozoospermia
Oligoteratozoospermia
Total number or concentration* of, and percentages of progressively motile and morphologically
normal, spermatozoa equal to or above the lower reference limits
Total number or concentration* of, and percentage of progressively motile, spermatozoa
below the lower reference limits
Total number or concentration* of, and percentage of morphologically normal, spermatozoa
below the lower reference limits

Asthenoteratozoospermia Percentages of both progressively motile and morphologically normal spermatozoa below
the lower reference limits

Oligoasthenoteratozoospermia Total number or concentration* of, and percentages of both progressively motile and
morphologically normal, spermatozoa below the lower reference limits.
limits

Cryptozoospermia Spermatozoa absent from fresh prepara ons but observed in a centrifuged pellet (3,000 g for 15 min)

Necrozoospermia Low percentage of live, and high percentage of immotile, spermatozoa in the ejaculate
 Physical Examination
Normally, a8er 3-5 days of sexual abs nence semen volume is 2-5 ml. The seminal vesicles are responsible for about two
thirds, while the prostate contributes to one third of the seminal plasma volume.

1- Semen Volume
1- Low Semen Volume Oligospermia :
Definition:
Low semen volume is the persistent ejacula on of semen of less than 1.5 cc despite the 35 days of preliminary sexual
abstinence.
Due to its etiological factors, low semen volume is usually associated with disorders in sperm count. However, even in
presence of normal sperm count and motility, the low semen volume in itself can be related to infertility due to wasting
of semen samples in vagina secretions and thus spermatozoa cannot reach the endocervix.

Etiology :
1- Bilateral and unilateral congenital absence of the vas.
2- Partial or complete obstruction of ejaculatory ducts.
3- Partial retrograde ejaculation.
4- Secretory defect: The secretory capacity of hypoplastic seminal vesicles and prostate is low in hypogonadal males.
The condition can also occur after severe prostato-vesiculitis due to post-inflammatory fibrosis.
5- Stress and neurological disorders of ejaculation.

Investigations:
1- Transrectal ultrasonography and CT scan to evaluate the size of the prostate and seminal vesicles and
to assess for the presence of Mullerian cyst.
2- Vasography gives an idea about the size of the seminal vesicles and if there is an obstructed
ejaculatory duct.
3- Urine examination after masturbation helps to diagnose cases of retrograde ejaculation.
4- Biochemical assay:
- Absence of fructose from semen occurs in 1- bilateral congenital absence of the vas as well as
2- in ejaculatory duct obstruction.
Fructose cannot be assayed in low volume specimens of 1 cc while zinc can be measured in
samples less than 0.1 cc

Treatment:
- Treatment is directed to the cause e.g. HCG and oral androgen therapy help to stimulate accessory gland secretions in
hypogonadal cases.
- Intrauterine insemination and ART may be needed if the etiological factor is difficult to correct.

2- High Semen Volume :

Definition:
- This is the persistent produc on of an ejaculate that is more than 5 cc. It is related to infer lity via:
Dilution oligozoospermia (low sperm concentration in a patient with average sperm count).
High semen volume is attributed to secretory dysfunction of the seminal vesicles.
Toxic factors from excess vesicular fluid may cause asthenozoospermia.

Management
- Artificial Insemination (AIH): using the first portion of split ejaculate, to eliminate the vesicular fraction.
- Coitus Interruptus: withdrawal of the penis after deposition of the first portion into the vagina.
 2- Semen Color
Normally, semen is a grayish white or grayish yellow gelatinous mixture, which is opaque to translucent according to its
sperm density, cellular content and debris. Abnormal semen color may be due to certain abnormal constituents e.g.

Urine:
In bladder neck disorders contamination of semen with urine gives it a faint yellow color. Diagnosis is easy by the
presence of the uriniferous odor, urea content and dead sperms due to the acidic pH of urine.

Blood
hemospermia: Pink discoloration of semen may be due to small traces of fresh blood. Bright red color is due to larger
amounts of fresh blood. And Brown semen signifies old blood.

Causes of hemospermia:
Bilharzial seminal vesiculitis.
Severe non-specific prostato-vesiculitis.
Tuberculosis of the prostate.
Prostatic calculi
Neoplastic conditions of male genital system
Trauma (e.g. fracture pelvis or after cystoscopy).
Hematological and vascular causes of bleeding e.g. coagulation disorders and hypertension.

Diagnosis:
The source of blood can be traced by split ejaculate technique
Prostatic smear examination may show evidence of prostato-vesiculitis.
Serological and endoscopic diagnosis of urogenital bilharziasis.
Imaging of accessory sex glands e.g. TRUS, CT scan and MRI.
Testing for bleeding tendency and coagulation defect.

Jaundice: Very bright yellow color is due to bilirubin. It has a little direct effect on semen but the observed semen
parameter disorder in such cases is due to liver damage.

3- Semen Liquefaction
Physiology:
Normally, semen is ejaculated in a liquid state and coagulates almost immediately as it touches the air due to the
presence of proteinaceous substances, produced by seminal vesicles, acted upon by vesiculase enzyme of the prostate.
Over the next 5-30 minutes it reliquies by prosta c enzymes, brinolysin and seminine.

Pathology:
- Complete failure of coagulation is indicative for absence of seminal vesicles as in cases of bilateral congenital absence
of vas deferens.
- Absence of semen liquefaction may cause infertility since spermatozoa entangled in the coagulum cannot travel up to
the cervical mucous after ejaculation. Non-Liquefaction or persistent coagulation results from decreased production
or absence of liquefying enzymes of the prostate, which may occur in chronic prostatitis and may benefit from
antibiotic and zinc sulphate therapy.

Assessment of liquefaction:
- Visual assessment:
If semen is totally unliquified, it appears as a gel-like coagulum
If it is partially liquefied, small gellike clots are formed.
Fully liquefied samples appear like a fluid.
- Comparison to 3rd frac on of split ejaculate: The 3rd fraction of split ejaculate from normal semen liquefies very slowly
and is not complete for up to 1-2 hours a8er ejacula on. This is placed in a nylon mesh net suspended in 2-3 ml
graduated tube and the filtered fluid is compared to the patients semen.
Treatment:
- Split ejaculation: Trying coital withdrawal after first portion ejaculation or using the first portion of split ejaculate for AIH.
- ART: If other semen parameters are normal, intrauterine insemination (IUI) can be done after induction of semen
liquefaction by:
Chemical mucolysis: Alevaire (mucolytic agent) and - amylase, - chemotrypsin or lysozyme (proteolytic
enzymes) can be used to liquefy semen either:
- In vitro: 0.1cc -amylase solu on is added to semen every 3-4 minutes ll liquefac on occurs and IUI is done.
- In vivo: In the ovulatory date of the wife -amylase vaginal suppository in cocoa butter base is inserted after
intercourse with eleva on of hips and knees over a pillow for 30-45 min.
Side effects are rare in low concentration of -amylase and include irritation, itching and some reduction in
sperm motility.

Mechanical mucolysis: Transferring semen into a syringe, then forcibly ejecting it back into a glass container.
This is repeated 5 mes before doing AIH.

Semen Viscosity
1- Normal Viscosity:
Semen viscosity is graded from (0) to (+4), where grade (0) denotes a sample that is capable of being poured into mul ple
small droplets while grade (+4) denotes thick semisolid mass that cannot be frac onated on pouring.
Viscosity can also be assessed by the degree of difficulty in aspiration of semen by Pasteur pipette.
Semen viscosity may be measured by the length of threads formed (in millimeters). Up to 40 mm is considered
normal. A thread length greater than 60 mm or the forma on of no thread at all is considered abnormal.

2- Seminal hyperviscosity:
Abnormally viscous semen is indirectly related to infertility via:
As sperms move in the viscid seminal plasma the grade of their motility is reduced.
High viscosity samples show uneven distribution of spermatozoa.
In a viscid seminal plasma sperms fail to catch the cervical mucous and fall rapidly from vagina.

Etiology: Highly viscid semen is attributed to accessory sex gland dysfunction (prostato-vesicular).
Diagnosis
of both increased viscosity and non-liquefaction should be supplemented with a sperm function test e.g.
postcoital test (PCT) to see if such problems would interfere with cervical mucous penetration or not.
Treatment:
Assisted Reproductive Techniques:
Helping males with hyperviscid semen by AIH, IVF, ICSI or other ART techniques can be done utilizing one of
the following preliminary measures:
- Spilt ejaculate:
- Mechanical mucolysis:
- Chemical mucolysis: Mucolytic compounds can better be used to reduce semen viscosity in-vitro, followed by AIH or ART

Reaction of Semen
According to the WHO Laboratory Manual (1999), a reference value for semen pH is 7.2 or higher.
For clinical purposes, a semen pH below 7.6 or above 8.6 is considered abnormal.
Chronic prosta s o8en displays a low pH (below 7.2),
Acute prosta s reveals a high pH (above 8.6).
Low semen volume, accompanied by low pH (below 7.2), is o8en due to a deciency in seminal vesicle uid
caused by obstruction of the ejaculatory ducts or bilateral congenital absence of the vasa.
 Microscopic Examination
1- Sperm Count
Sperm concentra on is the number of spermatozoa per ml, which normally ranges between 20-250 x106/ml. A better
evaluation is by calculating the sperm count, which is the multiplication of sperm concentration by semen volume.
Measuring Techniques:
1) Visual Assessment Eye-Balling
2) Neubauer coun ng chamber Hemocytometer
3) Makler Coun ng Chamber
4) Horwell Fer lity Coun ng Chamber
5) Laser Beam Sperm Count
Sperm Count Disorders :

(1) Azoospermia :
Definition:
Azoospermia means absence of spermatozoa from semen. Its only diagnosed after, at least, at least, two semen samples
showing no sperms even after centrifugation and examination of the sediment.
Etiology :
1- Functional (Non-obstructive) azoospermia:
This signifies the absence of sperm production from the testes is due to one of the following causative factors:
Congenital e.g. KF syndrome, SCO syndrome cryptorchidism, etc
Inflammatory e.g. Bilateral mumps orchitis
Toxic e.g. Post-irradiation, drugs
Endocrinal disturbances e.g. hypogonadism, hyperprolactinemia etc
Traumatic
2- Obstructive azoospermia:
This denotes normal spermatogenesis but the passage from the level of the vas efferentia to the ejaculatory ducts is
bilaterally obstructed. Various causes of genital duct obstruction are discussed before but the main general causes are:
Congenital e.g. Congenital bilateral absent vas, ejaculatory ducts atresia.
Post-inflammatory: Non-specific epididymitis, T.B epididymitis etc.
Traumatic: Vasectomy, inguinal and scrotal operations (e.g. hernia).

Classification of OA on the basis of ductal obstruction due to congenital and acquired causes
Conditions Congenital Acquired
Epididymal obstruction Idiopathic epididymal obstruction Post-infective (epididymitis)
Post-surgical (epididymal cysts)
Vas deferens obstruction Congenital absence of vas deferens Post-vasectomy
Post-surgical (hernia, scrotal surgery)
Ejaculatory duct obstruction Prostatic cysts (Mullerian cysts) Post-surgical (bladder neck surgery)
Post-infective

Classification
Intratesticular obstruction
Intratesticular obstruc on occurs in 15% of OA .
- Congenital forms (dysjunction between rete testis and efferent ductules) are less common than
- acquired forms, (i.e. post-inflammatory or post-traumatic obstructions).
Acquired forms are often associated with an obstruction of epididymis and vas deferens.
Epididymal obstruction
Congenital Post-infective Post-Surgical
Congenital epididymal obstruction usually manifests Acquired forms secondary to:
as CBAVD, which is associated with at least one acute (e.g. gonococcal) and subclinical after epididymal surgery,
muta on of the cys c brosis (CF) gene in 82% of (e.g. chlamydial) epididymitis are most such as cyst removal
cases (5). frequent.

This form is often accompanied by: Acute or chronic traumas can result in
- absence of the distal part of the epididymis and epididymal damage
- seminal vesicle agenesis
 Vas deferens obstruction
Congenital Post surgical
The most common congenital vasal Post-vasectomy Post-surgical (hernia, scrotal surgery)
obstruction is CBAVD, often accompanied acquired obstruction following vasectomy for Vasal obstruction may also occur after
by CF. sterilisation, with possible subsequent germ cell herniotomy (13). Polypropylene mesh
impairment and brosis (11,12). Approximately herniorrhaphy seems to induce a fibroblastic
2-6% of these men request vasectomy reversal. response able to entrap, or obliterate, the vas
Of those undergoing vaso-vasostomy, 5-10% deferens
have epididymal blockage as a result of tubule
rupture, making epididymo-vasostomy
mandatory
Ejaculatory duct obstruction
These obstructions can be classified as cystic or post-inflammatory.
Congenital Post-infective Post-surgical
In urogenital sinus abnormalities: one or Post-inflammatory obstructions of the
both ejaculatory ducts empty into the cyst ejaculatory duct are usually secondary to : Bladder neck surgery
,, while in Mullerian duct anomalies : - acute,
ejaculatory ducts are laterally displaced and - non-acute or
compressed by the cyst - chronic urethro-prostatitis
Congenital or acquired complete obstructions of the ejaculatory ducts are commonly associated with :
1- Low semen volume,
2- Acid pH.
3- Decreased or absent seminal fructose
4- The seminal vesicles are usually dilated (anterior-posterior diameter > 15 mm)
Diagnosis :
1) History data:
History suggestive of functional etiology (Postpubertal mumps orchitis,Chemo or radiotherapy, Delayed or failed
puberty and Testicular torsion or trauma)
History suggestive of obstructive etiology (Repeated UTI , Gonorrhea or NGU and Herniotomy during childhood)
ask about:
haematospermia;
post-ejaculatory pain;
previous or present urethritis or prostatitis;
obstructive or irritative urinary symptoms;
previous scrotal enlargement or pain or surgery;
previous inguinal herniorrhaphy or traumas;
chronic sino-pulmonary infections.

2) ExaminaAon:
The following findings indicate OA :
At least one tes s > 15 mL volume (although a smaller tes cular volume may be found in some patients with OA and concomitant
partial testicular failure).
Enlarged and hardened epididymis.
Nodules in the epididymis or vas deferens.
Absence or partial atresia of the vas.
Signs of urethritis.
Prostatic abnormalities.

3) Semen analysis data:

least two examinations must be carried out at an interval of 2-3 months, according to the WHO.
Azoospermia associated with low semen volume (< 1.5 mL) with an acid pH and low fructose level, suggests:
1- congenital absence of the vas (CBAVD) or
2- ejaculatory duct obstruction.
When semen volume is low, a search must be made for spermatozoa in urine after ejaculation, as their presence confirms an
ejaculatory disorder.

However the volume is also low in hypogonadal azoospermic males BUT showing normal pH and coagulation-
liquefaction phenomenon.
Azoospermia in presence of leukocytospermia arouses suspicion about an inflammatory obstructive etiology.

4) Hormonal assay:
- Seum FSH levels may be normal but do not exclude a testicular cause of azoospermia (e.g. spermatogenic arrest).
- FSH is normal in 40% of men with primary spermatogenic failure.
- Inhibin B appears has a higher predictive value for normal spermatogenesis
5) Imaging techniques:
For patients with a low seminal volume and in whom distal obstruction is suspected:
1- transurethral ultrasound (TRUS) is essential
2- Vasography
Scrotal ultrasound is mandatory and helps to :
1- find signs of obstruction (e.g dilatation of rete testis, enlarged epididymis with cystic lesions
and absence of vas deferens) and to
2- exclude signs of testicular dysgenesis (e.g. non-homogenous testicular architecture and
microcalcifications).
Transrectal ultrasound may also be used to aspirate seminal vesicle fluid
6) Invasive diagnosis : includes
1- testicular biopsy, (testicular biopsy may be indicated to exclude spermatogenic failure)
2- scrotal exploration and
3- distal seminal duct evaluation,
are indicated in patients with OA in whom an acquired obstruction of the seminal ducts is suspected.
Explorative and recanalisation surgery should be carried out at the same time.
7) Semen cytology:
Giemsa stain, MGG stain or Papanicolaou stain may be employed to study germ cells.
If spermatogenic cells are present in the seminal fluid of azoospermic fluid, this excludes obstructive pathology.
8) Semen markers
9) Cytogenetic studies:
In azoospermia, karyotyping and other cytogenetic studies allow identification of both numerical chromosomal
anomalies (e.g. KF syndrome) and structural anomalies (e.g. microdeletions of the Y-chromosome).
Management of azoospermia

Treatment
Intratesticular obstruction
- At this level seminal duct recanalisation is impossible;
- TESE or fine-needle aspiration is therefore recommended.
- The spermatozoa retrieved may be used immediately for ICSI or may be cryopreserved.
Both TESE and fine-needle aspiration allow sperm retrieval in nearly all OA patients.

Epididymal obstruction
- Microsurgical epididymal sperm aspiration (MESA) is indicated in men with CBAVD.
- Retrieved spermatozoa are usually used for ICSI.
- Usually, one MESA procedure provides sufficient material for several ICSI cycles and it produces high pregnancy and fertilisation rates.
- In patients with azoospermia due to acquired epididymal obstruction:end-to-end or end-to-side microsurgical epididymo-vasostomy is
recommended
- Reconstruction may be carried out unilaterally or bilaterally .
- Before microsurgery, it is important to check for full patency downstream of the epididymis.
- Anatomical recanalisa on following surgery may require 3-18 months.
- Before microsurgery (and in all cases where recanalisation is impossible), epididymal spermatozoa should be aspirated and
cryopreserved for use in ICSI in case of surgical failure.
 Proximal vas obstruction
- Proximal vas obstruction after vasectomy requires microsurgical vasectomy reversal .
- Vaso-vasostomy is also required in the rare cases of proximal vasal obstructions (iatrogenic, post-traumatic, post-inflammatory).
- When spermatozoa are absent in the intraoperative vas fluid, a secondary epididymal obstruction may be present, especially if the
seminal fluid of the proximal vas has a thick toothpaste appearance. Then microsurgical vaso-epididymostomy is indicated.

Distal vas deferens obstruction

- It is usually impossible to correct, large bilateral vas defects resulting from involuntary vas excision during hernia surgery in early
childhood or previous orchidopexy .
- In these cases, proximal vas deferens sperm aspiration or TESE/MESA can be used for cryopreservation for future ICSI.
- In large mono-lateral vas defects associated with contralateral testicular atrophy, the vas of the atrophic testis can be used for a
crossover vaso-vasostomy or vaso-epididymostomy.

Ejaculatory duct obstruction

Treatment of ejaculatory duct obstruction depends on the aetiology.
- In large post-inflammatory obstruction and when one or both ejaculatory ducts empty into an intraprostatic midline cyst :
transurethral resection of the ejaculatory ducts (TURED) can be used. Resection ma remove part of the verumontanum.
- In cases of obstruction due to a midline intraprostatic cyst : incision or unroofing of the cyst is required .
Intraoperative TRUS makes this procedure safer.
- If distal seminal tract evaluation is carried out at the time of the procedure, installation of methylene blue dye into the vas can help to
document opening of the ducts.
- The limited sucees rate of surgical treatment of ejaculatory duct obstruction in terms of spontaneous pregnancies should be weighed
against sperm aspiration and ICSI.
- Complications following TURED include :
retrograde ejaculation due to bladder neck injury,
and reflux of urine into ducts,
seminal vesicles and vasa (causing poor sperm motility, acid semen pH and epididymitis).

- Alternatives to TURED are :

MESA,
TESE,
proximal vas deferens sperm aspiration,
seminal vesicle ultrasonically guided aspiration and direct cyst aspiration.

- In cases of functional obstruction of the distal seminal ducts, TURED often fails to improve sperm output.
Spermatozoa can then be retrieved by antegrade seminal tract washout.
Spermatozoa retrieved by any of the aforementioned surgical techniques should always be cryopreserved for assisted reproductive
procedures.

(2)Oligozoospermia
Definition: Oligozoospermia refers to a sperm concentra on below 20 x106/cc.

Etiology: Oligozoospermia may be caused by several factors. According to their prevalence:

- Idiopathic: In the majority of cases no detectable cause is found.
- Varicocele:
The classic varicocele semen picture is a combination of oligo, astheno and teratozoospermia (mainly tapered forms).
However, one or two defects may be present in a high percentage of varicocele patients.
- Partial obstruction:
In unilateral obstruction, the presence of oligozoospermia is justifiable.
However, some controversy is raised about bilateral partial obstruction at the level of vasa
efferentia in varicocele patients or at the epididymal level as a post-inflammatory complication.
Bilharzial perivasitis has been claimed to cause functional obstruction, leading to oligozoospermia
- Partial retrograde ejaculation.
- Other causes:
Increased intrascrotal temperature.
Bilateral hydrocele.
Unilateral cryptorchidism.
Radiation in moderate doses.
Gonadotoxic drugs and chemical in small doses.
Chronic malnutrition.
Unilateral postpubertal mumps orchitis.
Systemic diseases e.g. renal failure, feversetc
(3)Polyzoospermia with Poor MoAlity :
Definition:
Polyzoospermia denotes sperm concentra on greater than 250 x106 /ml. It may be involved in couple infertility if only
mo lity of spermatozoa is poor (30%, grade II or less).
The high concentration of spermatozoa affects the sperm capacity to utilize oxygen, fructose one other nutrients.
The increased viscosity may also affect motility.
Diagnosis :
The period of abstinence before analysis is very important.
In normal fer le males we can get a picture of polyzoospermia with poor mo lity a8er 13 months abs nence.
It is advisable in cases of polyzoospermia with poor mo lity to obtain a semen sample a8er 24-72 hours of abs nence
following multiple ejaculations.

Management:
- Increase ejaculatory frequency:
Instructions for multiple daily ejaculations so as to reduce sperm count particularly around ovulatory period of wife.
- Artificial insemination after dilution:
Ar cial insemina on is done using a 1: 1 dila on of husbands semen with 5% dextrose in lactated Ringer solution
(D5 RL).
This is used for insemination utilizing a cervical cup.
A post insemina on cervical mucous examina on should be performed 4-6 hours a8er the ini al insemina on to
assess the efficiency of the procedure.
If improvement is noted, insemina on is con nued for at least 6 cycles or un l pregnancy occurs. If no improvement,
androgen suppressive therapy may be tried.
- Androgen suppressive therapy: Treatment with low-dose androgens has been claimed to improve sperm motility
without affecting sperm count.
- ART: ICSI can be tried particularly if no improvement occurs by other methods.

2- Sperm Motility Disorders

Asthenozoospermia
Asthenozoospermia usually occurs in association with oligozoospermia with almost the same etiological causes described
for that entity.

Causes of Isolated Asthenozoospermia in presence of normal sperm concentration include:

1- Artifacts during collection e.g. water, soap, detergents, condom collection, etc
2- Immotile cilia syndrome and Kartagner Syndrome.
3- Infection e.g. prostato-vesiculitis.
4- Fructose Deficiency:
Maldevelopment or secretory dysfunction of the seminal vesicles.
Polyzoospermia.
5- Immunological infertility (immobilizing antisperm antibody).

3- Sperm Viability
In specimens where spermatozoa are not moving in the field, its important to differentiate non-motile (absolute
asthenozoospermia) from dead spermatozoa (necrozoospermia). Dieren a on can be done by one of 2 methods:
Staining with Eosin-Negrosin stain: This stain cannot penetrate a living cell, so if spermatozoa get the red color they are dead.
Addition of sperm motility stimulants e.g. caffeine and Kallikrein, which cannot stimulate motility except in live sperms.

Normally, 50% or more of spermatozoa are alive. The percentage of alive sperms declines in association with genital tract
infections and disorders of sperm transport through the genital tract.
 4- Sperm Morphology
Sperm shape is an important indicator of sperm fertilizing capacity. Fertility declines as the percentage of normal shaped
sperm falls, par cularly in men with ejaculates with less than 5% normally-shaped sperm. Sperm are specially stained and
viewed under a microscope, with assessment of the head, middle and tail regions.

- Sperm Morphology Assessment:

Light Microscopy :
1-Wet film:
Many laboratories estimate abnormal forms by the unstained wet films. This method is unreliable, inaccurate
and does not study nuclear details.
2-Stained film:
After staining, examination under oil immersion is done to find out the percentage and type of abnormality in
at least 100 examined spermatozoa. The most popular stains used in assessment of sperm morphology are:
A- HxE Stain (simple and rapid)
B- Papanicolaou Stain (better but complex procedure)
C- Giemsa Stain (good)
D- Testsimplet: prestained slide

Electron Microscopy :
This is the best studying method for ultra structural sperm defects. However, it can not be used routinely as it
is time consuming and expensive.
1-Scanning Electron Microscopy :
SEM shows the outer sperm covering thus helping to show general sperm shape abnormalities.
2-Transmission Electron Microscopy :
TEM shows the internal structures and helps to detect axonemal disorders (absent dynein arms, nexin links).

- Sperm Morphological Anomalies :

WHO Criteria for normal sperm morphology defines :
The normal head to be oval and smooth. (Round, pyriform, pin, double and amorphous heads are all abnormal).
The midpiece should be straight and slightly thicker than the tail.
The tail should be single, unbroken, straight and without kinks or coils.
A minimum of 100 sperm must be counted that qualify the above criteria.
The use of Kruger criteria offers a more detailed analysis of sperm morphology. These criteria evaluate the shape and size
of the head, midpiece and tail much more stringently than WHO standards. Kruger morphology can assist the clinician in
determining the most appropriate reproductive technique for the patient.

A. Sperm head anomalies :

- Head numerical anomaly
- Head size anomaly
- Head shape anomaly
- Head structure anomaly
B. Head-midpiece connection defect
C. Midpiece abnormalities
D- Tail anomalies:
- Tail numerical anomalies
- Tail shape anomalies
- Tail size anomalies
- Tail ultrastructural anomalies
E. Axoneme defects
F. Outer fibres defects
G. Fibrous sheath defects
Teratozoospermia
Definition :
Teratozoospermia is dened as an increase in the percentage of abnormal sperm forms above 40%.

It has the main following causes:

- Idiopathic
- Varicocele
- Genetic factors
- Physical factors: heat, radiation, and pollution
- Psychic stress
- Toxic and chemical factors:
Arsenic, lead, mercury and Cadmium salts.
Pesticides and insecticides
Smoking
Chronic alcoholism

5- Sperm Agglutination
Definition:
Sperm agglutination means any type of random or organized attachment or clumping of spermatozoa to each other.

Etiology:
- Immunological (see before).
- Infection: Agglutination is associated with changes in semen volume, motility, viscosity and PNLs count. There is
controversy about change in seminal plasma zinc and the occurrence of infection or agglutination.
- High sperm count and polyzoospermia.
- Idiopathic (unexplained).

Types of agglutination:
- Organized agglutination (H-H, T-T, and H-T).
- Random clumping.

Treatment of agglutination:
1- Treatment of the cause: e.g.
Infection: antibiotics
Immunologic: corticosteroids.
Idiopathic: Vitamin C: 100-1000 mg /day. And Zinc therapy: if severe agglu na on due to infec on.
2- Semen Processing:
A) Descending sperm separation
B) Sperm Washing:
C) Ascending Sperm Separation

6- White Blood Cell Count

- A high number of white cells in semen may indicate infection or inflammation.
- White blood cells are considered significant if more than one million are found per milliliter of ejaculate.
- Finding high white cell count may contribute to sperm damage and should prompt investigation of infection and
antibiotic therapy.
- Germ cells and white blood cells appear identical on microscopic examination (round cells).
- Immunohistochemical stains are performed if such round cells are more than 5-10/HPF to dieren ate between the 2
cell types. An increased number of white blood cells may signify genital infection.
 Computer-Assisted Semen Analysis CASA
in the late 1980s, CASA employed Video Image Mo on Analysis which have the following advantages :
- Less time consuming.
- Assess lateral head displacement, which correlates well to the fertility potential.
- All aspects of sperm count, shape, size and morphology are assessed.

Parameters measured include:

Sperm concentration.
Percentage of sperm motility.
Mean velocity (a distribution histogram)
Mean linearity (straight line velocity)
Lateral head displacement.
Sperm morphology (size and shape).
Curvilinear velocity (average distance per unit time between successive sperm positions)
Straight-line velocity (speed of forward direction)
Linearity (straight-line velocity divided by the curvilinear velocity)
Average path velocity
Amplitude of lateral head displacement
Flagellar beat frequency

Disadvantages of CASA:
High initial cost
Inaccurate results when sperm concentrations are very high or very low. Computer systems of
sperm analysis need to incorporate a step of interactive object identification to work properly,
allowing the operator to confirm or correct possible computer misidentification. CASA does not
improve patient outcomes but is very helpful for research purposes.

Seminal Biochemical Assays

Tracing the chemical constituents of semen is not done during routine semen analysis. Upon clinical suspicion one may
request some biochemical markers that will help in establishing the diagnosis.
Testicular markers
- LDHX:
- Transferrin:
- Inhibin-B:
- Mullerian Inhibitory Substance MIS (Anti-Mullerian Hormone, AMH):
- Insulin Growth Factor-1 (IGF-1):
- Other Testicular Markers:
Many recently discovered markers are still under clinical trials for their clinical values e.g. Alpha 2-macroglobulin,
Laminin, Vitronectin, Hepatocyte growth factor, Nerve growth factor, Stem cell factor and Basic fibroblast
growth factor.
Epididymal markers
- Alpha Glucosidase:
- L-carnitine.
- Glyceryl-phosphoryl Choline (GPC).
- Alpha Mannosidase.
Seminal vesicle markers
- Fructose:
- Prostaglandin: PGF2 has also been used as a seminal vesicle marker.
Prostatic markers
- Acid Phosphatase, Citric Acid and Zinc:

Reactive oxygen species (ROS) testing

 Split Ejaculation
Examination of the two-fraction
fraction split ejaculate is a rather common procedure in clinical laboratory where semen is
fractionated :

First Portion
This is the prostatic and sperm rich portion that has the following criteria :
Higher concentration of spermatozoa than the whole ejaculate.
Motility of spermatozoa is better in the first portion than the second portion.
Morphology of sperms is beWer in the rst por on in 20% of cases.
The first portion being primarily from prostate contains liquefaction factors so coagulation does not
occur or if it occurs liquefaction takes place rapidly.
Viscosity is less in the first than second portion.
PH is lower.
IgG, transferrin and albumin concentration decreases from the first to the second portion.

Second Portion
This portion is mainly derived from the seminal vesicles and has the following characters:
Fructose content is higher in second portion.
Lactoferrin and prostaglandins are also present mainly in the second portion.
Rapid coagulation and slow liquefaction.
liquefact
Higher viscosity.
Motility and viability are lower in the second portion due to the presence of more than one factor
in seminal vesicle secretion which leads to rapid decline of sperm motility and viability. .

Value of Split ejaculation :

1- Value in research:
Split ejaculation technique permits a crude method to obtain prostatic and vesicular secretions. This
allows tracing the origin of various chemical compounds in seminal plasma.
2- Diagnostic value:
The source of hemospermia and leukocytospermia can be suspected using the 6-fraction
6 split ejaculate
technique.
3- Therapeutic value:
The first portion of split ejaculate can be used for AIH not only because of its better spermatozoal
parameters, but also because it is low in prostaglandins,
prostaglandins, which may lead to severe uterine contraction. It can
be used in the following indications: (High viscosity of semen., Large semen volume., Oligozoospermia.and
Asthenozoospermia.)