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J Mol Hist

DOI 10.1007/s10735-017-9722-7

ORIGINAL PAPER

Correlation betweenthree-dimensional power Doppler


andmorphometric measurement ofendometrial vascularity
atthetime ofembryo implantation inwomen withunexplained
recurrent miscarriage
XiaoyanChen1 SotiriosH.Saravelos1,2 YingyuLiu1 JinHuang1
ChiChiuWang1,3,4 TinChiuLi1

Received: 16 February 2017 / Accepted: 12 April 2017


Springer Science+Business Media Dordrecht 2017

Abstract Power Doppler in combination with three- and volume fraction measurement of vessel). This study
dimensional (3D-PD) ultrasonography has been used as indicates that endometrial vascularity assessed by 3D-PD
a noninvasive tool to evaluate the vascularity. However, could not be used to reflect changes in micro vessels of the
it is unclear whether 3D-PD can accurately reflect endo- endometrium at the time of embryo implantation in women
metrial vascularization and replace the invasive endome- with unexplained recurrent miscarriage.
trial biopsy. This study aims to investigate the correlation
between 3D-PD and micro vessel morphometric meas- Keywords Vascularization 3D power Doppler
urement of endometrial vascularity. Twenty-five women ultrasound Morphometric measurement Unexplained
with unexplained recurrent miscarriage were recruited for recurrent miscarriage
3D-PD and endometrial biopsy on precisely day LH+7.
Immunohistochemistry using vWF was employed to iden-
tify micro vessels in endometrial biopsy specimens fol- Introduction
lowed by the use of morphometric technique to measure the
mean vessel diameter and volume fractions. The vasculari- Recurrent miscarriage (RM) is defined as three or more
zation index (VI), flow index (FI) and vascularization flow consecutive spontaneous miscarriages before 24 weeks
index (VFI) assessed by 3D-PD were calculated for both gestation (RCOG 2011) and there are some recognized
the endometrial and sub-endometrial regions. There were causes, such as genetic disorders, uterine malformations,
no significant correlations between any of the ultrasono- hormonal abnormalities, hereditary thrombophilia, thy-
graphic measurements (endometrial thickness, endometrial roid disease and immunological disorders (Christiansen
volume, endometrial VI/FI/VFI, sub-endometrial volume, 2014). Currently, around 50% of women with recurrent
sub-endometrial VI/FI/VFI) and morphometric features miscarriage are of unknown etiology, and an altered endo-
(number of micro vessel, mean diameter of micro vessel metrial environment is thought to be associated with this
idiopathic condition (Christiansen 2014). Compared with
Xiaoyan Chen and Sotirios H Saravelos: similar in author order. control group, an alteration of impaired decidual vascu-
lar remodeling related to decidual natural killer cell was
* Tin Chiu Li observed in women with unexplained recurrent miscarriage
tinchiu.li@gmail.com
(Almasry etal. 2015). It is also well accepted that the vas-
1
Department ofObstetrics andGynaecology, The Chinese cularization of human endometrium plays an important role
University ofHong Kong, Shatin, HongKong in implantation, when the endometrium transforms into a
2
Department ofObstetrics andGynaecology, Imperial College well-vascularized and receptive tissue (Plaisier 2011). The
London, London, UK characteristic of vascularization observed at the time of
3
Li Ka Shing Institute ofHealth Science, The Chinese implantation has been considered as an indicator of endo-
University ofHong Kong, Shatin, HongKong metrial receptivity.
4
School ofBiomedical Science, The Chinese University Endometrial biopsy for histological examination of
ofHong Kong, Shatin, HongKong micro vessel is a traditional and informative method to

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evaluate endometrial vascularity. In our previous study The inclusion criteria for the subjects were: (i) more than
(Chen et al. 2016), a significantly increased number of three consecutive miscarriages before 24 gestational weeks
micro blood vessels was found in peri-implantation endo- and be fathered by the present partner; (ii) regular men-
metrial biopsies from women with recurrent miscarriage, strual cycle. According to our and others previous stud-
although the mean diameter of micro blood vessel did not ies (Li etal. 2002; Christiansen etal. 2008; Saravelos and
differ between recurrent miscarriage group and control Li 2012), the exclusion criteria for the whole study period
group. However, endometrial biopsy is an invasive method were: (i) age <18 or >40 years; (ii) uterine abnormalities;
to assess the rapid changes in endometrial vascularization (iii) abnormal hystersalpingogram result; (iv) parental chro-
during the process of implantation and it could not be used mosomal abnormalities; (v) Day 2 FSH >10IU/L or mid-
in conception cycles. luteal progesterone <30nmol/l; (vi) positively for the lupus
On the other hand, three-dimensional (3D) ultrasonogra- anticoagulant, anti-cardiolipin IgG and IgM antibodies at
phy in combination with power Doppler (3D-PD), a nonin- repeated tests; (vii) positive heritable thrombophilia tests;
vasive tool to evaluate the blood supply in the endometrium (viii) abnormal thyroid function; (ix) manifestations of
and the sub-endometrial regions has been used to indicate autoimmune or thrombotic diseases; and (x) chronic intake
endometrial receptivity and pregnancy outcome in a recent of drugs with anti-inflammatory or anticoagulative effect or
study (Mishra et al. 2016). Furthermore, 3D-PD has also had hormonal treatment during preceding 3 months.
been used to assess endometrial vascularization in women
with recurrent miscarriage (Chen et al. 2011; Tan et al. Timing ofmeasurement
2015). However, it is still unclear whether the non-invasive
3D-PD examination can accurately reflect vascularity and As described before (Chen et al. 2016), all subjects in
replace the invasive biopsy. In animal tumor studies, one this study had daily urine dipstick test from day 9 of the
earlier study found that neither 2D nor 3D power Doppler menstrual cycle onwards to identify LH surge (ovulation),
parameters correlate with micro vessel count (Fleischer which was used to precisely time the ultrasonographic and
etal. 1999), while another more recent study showed a sig- Doppler study, followed immediately by an endometrial
nificant correlation between these two methods for detect- biopsy.
ing tumor vascularization (Xuan et al. 2007). In humans,
a previous study did report on a lack of significant correla- Threedimensional power Doppler assessment
tion between 3D-PD parameters and micro vessel density
for detecting endometrial vascularization in women with All examinations were performed by a single operator
recurrent miscarriage (Chen etal. 2012). using standardized settings with a Voluson E8 Expert
The main aim of this study to re-examine the correla- series ultrasound machine and RIC5-9-D 3D transvaginal
tion between 3D-PD and histological assessment of endo- probe (Ge Medical Systems Kretztechnik GmbH & Co,
metrial vascularity by using improved methods based Austria) as described in our previous study (Saravelos
on quantitative histologically technique (morphometric and Li 2015). Typically, the setting included: dynamic
method), to measure the volume fraction of micro vessels contrast: 8, focal zones: 1, harmonic frequency: mid-
of precisely timed endometrial biopsy in women with unex- dle (2D settings); render mode: default 3D PD, volume
plained recurrent miscarriage. We also wish to investigate angle: 90%, quality: high 1, Compound Resolution Imag-
if 3D-PD, as a non-invasive alternative, can replace histo- ing 3 (3D settings). Power Doppler characteristics were
logical method to study endometrial vascularity clinically. set as followed: frequency: 39 MHz, quality of color:
normal, color gain: 3.4, color map: 5, wall motion filter:
low 1, pulse repetition frequency: 0.6 kHz. The power
Materials andmethods Doppler patients were scanned with an empty bladder,
and the depth was adjusted so the uterus could be seen in
Subjects its entirety on the sagittal plane. An automated acquisi-
tion sweep was then performed with the lying completely
The experimental design was a cohort observational study. still. Virtual Organ Computer Aided Analysis (VOCAL)
In this study, 25 women were recruited for 3D-PD assess- was then used in the coronal plane and 9 rotational steps
ment and endometrial biopsy from March 2015 to January of manual endometrial outlining to estimate acquire the
2016 from the Prince of Wales of Hospital, The Chinese endometrial volume and sub-endometrial volume (3 mm
University of Hong Kong. Approval from local ethical outer shell). The vascularization index (VI), flow index
committees was obtained for this study. Written, informed (FI) and vascularization flow index (VFI) were then
consent was obtained from subjects prior to undergoing 3D calculated for both the endometrial and sub-endome-
Power Doppler study and endometrial biopsy. trial regions, where VI represents the number of blood

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J Mol Hist

vessels; FI represents the blood flow strength sensed over Identification ofvessels
3D-scanning time; and VFI represents the sum of blood
flow and vascularization. Any brown-staining endothelial cell or endothelial cell
cluster that was clearly separate from adjacent vessels
was considered as a single, countable vessel. Red blood
Endometrial biopsy cells were not used to define a vessel lumen. Branching
structures were counted as a single vessel.
All biopsies were obtained using a Pipelle sampler
(Prodimed, France) or Pipet Curet (Cooper Surgical, USA),
Micro vessel density
which has been designed to obtain specimens from the
superficial layers and from a wider area of the uterine cav-
The mean number of micro vessels/field was determined
ity. The specimens were immediately placed into 10% neu-
by the use of Image J software, from which the ves-
tral buffered formalin for over-night fixation at room tem-
sel density (number of micro vessels per mm2) could be
perature and then embedded into paraffin wax.
calculated.

Immunohistochemistry staining forvWF Mean diameter ofmicro vessel

As described in our earlier study (Chen et al. 2016), par- The diameter of positive-stained vessels in five chosen
affin-embedded human endometrial tissue sections (3 m) fields was measured by Image J and the mean vessel
were dewaxed in xylene, rehydrated through descend- diameter was calculated.
ing ethanol to phosphate-buffered saline, and quenched
in 0.3% hydrogen peroxide in methanol for 20 min. Anti-
Volume fraction
gen retrieval was performed in a microwave oven with
10 mmol/l citrate buffer. The sections were blocked with
Volume fraction measurement was derived by point-
donkey serum and then incubated overnight at 4C in pri-
counting method with the use of square lattices with
mary rabbit polyclonal anti-human vWF antibody (DAKO,
121 intersection points superimposed onto the micro-
A0082) at a dilution of 1:800. After incubation, the slides
scopic field (Li et al. 1988). The number of points fall-
were washed in phosphate-buffered saline-Tween20 and
ing on a vessel (v), stroma (s) and gland (g) were deter-
incubated in secondary donkey anti-rabbit antibody at a
mined, from which the volume fraction of endometrium
dilution of 1:1000. The specific antibody binding was visu-
(v+s+g) occupied by vessel (v) may be determined and
alized by incubation with peroxidase substrate 3,3-diamin-
the volume fraction of stroma (v+s) occupied by vessel
obenzidene tetrahydrochloride (Dako) and counterstained
(v) may also be determined.
with hematoxylin. Rabbit IgG was substituted for primary
antibody in the negative controls.
Intra andinterobserver variability

Morphometric analysis ofmicro vessels To determine the intra-observer variability, the immunohis-
tochemistry and morphometric analysis were repeated by
Field selection one of the observers (X. C.) on ten blind slides on a differ-
ent occasion without knowledge of the results of the earlier
A strategy of systematic random field selection was adopted measurement. Inter-observer variability was evaluated by
to reduce observer bias in field selection. The first field to two observers (X.C. and Y. L.). Eight sections were ran-
be captured was randomly selected over the L upper corner domly selected, processed, stained and counted using the
of the microscopic field. Subsequent fields were obtained agreed protocol to determine the inter-observer variability.
by moving horizontally, one field width at a time to the
right of the first field; when it reaches the left side margin Statistical analysis
of the specimen, the field was moved vertically downwards
by a distance of one field; afterwards the field was moved The data distribution was checked by the ShapiroWilk test.
horizontally to the left and the it continued in this manner, All results were presented as median and range for skewed
sideways and downwards, each time moving by a prede- data. Spearman correlation tests were applied to nonpara-
fined distance until all five completed microscopic fields metric data. p<0.05 was considered statistically significant.
had been examined. All of the data were analyzed with SPSS 18.0 software.

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J Mol Hist

Results (range 0.0110.046) respectively for the endometrium


and 0.32 (range 00.48), 23.62 (range 040.96) and 0.083
From March 2015 to January 2016, a total of 34 women (range 00.056) for the subendometrium. An example of
with RM were assessed for eligibility. Only 25 women estimating endometrial volume and vascularization using
were recruited into the study for 3D-PD ultrasound VOCAL in the multiplanar display was shown in Fig.1.
examination and endometrial biopsy. The remaining nine
patients were excluded due to abnormal maternal kar- vWF intheendometrium andmicro vessel
yotyping (1 patient), abnormal paternal karyotyping (1 determination
patient), intrauterine adhesion (2 patients), endometrial
polys (1 patient), Day 2 FSH >10IU/L (1 patient), mid- Figure2a showed immunostaining of vWF in the endome-
luteal P4 <30nmol/l (1 patient), positive anti-cardiolipin trium from women with unexplained recurrent miscarriage.
IgM antibody (1 patient), and loss to follow-up (1 patient). Positive staining for the vWF representing endothelial cell
None of the enrolled women were current or previous or endothelial cell cluster in stromal compartment of endo-
tobacco or alcohol users. Only one of the 25 women was metrium was considered as a single, countable vessel. Mor-
secondary RM patient and the remaining 24 women were phometric measurement was calculated by point-counting
diagnosed as primary RM. None of the subjects had mani- method with 121 intersection points (Fig.2b).
festations of autoimmune or thrombotic diseases or used
any anti-inflammatory, anti-coagulative or hormonal treat- Correlation between3D power Doppler parameters
ment for at least 3 months prior to the 3D-PD examination andendometrial micro vessels
and endometrial biopsy. The demographics data, 3D-PD
vascularization parameters and endometrial micro blood Serial bivariate correlations were performed between all
vessel measurements are summarized in Table1. histological and power Doppler variables. There were no
significant correlations between any of the power Dop-
3D power Doppler assessment pler variables and each of the morphometric measure-
ments (Table2).
In terms of uterine characteristics, 17/25 of uteri were
anteverted, the median endometrial volume was 3.82 ml
(range 1.659.72ml) and the median endometrial thickness Intra andinterobserver variability
was 8.94mm (range 5.6514.30mm). In terms of 3D-PD
vascularization parameters, the median VI, FI and VFI For intra-observer variability, there was significant
were 0.15 (range 00.45), 20.30 (range 033.02) and 0.035 correlation between volume fraction of endometrium

Table1Demographics Parameter Median Range


data, 3D-PD vascularization
parameters and endometrial Age (years) 36 3040
micro blood vessel
Day 2 FSH (IU/L) 5.8 4.515.1
measurements on day LH+7
in women with unexplained Day 2 LH (IU/L) 3.4 2.75.4
recurrent miscarriage (n=25) Day 21 progesterone (nmol/l) 45.9 24.893.0
Number of miscarriage 3 35
Endometrial thickness (mm) 8.94 5.6514.30
Endometrial volume (ml) 3.82 1.659.72
Endometrial VI 0.15 0.000.45
Endometrial FI 20.30 0.0033.02
Endometrial VFI 0.035 0.0110.046
Subendometrial volume (ml) 5.18 2.9922.30
Subendometrial VI 0.32 0.000.48
Subendometrial FI 23.62 0.0040.96
Subendometrial VFI 0.083 0.0000.056
m2
Number of micro blood vessels per m 15 1119
Diameter of micro blood vessels (m) 19.8 15.422.1
Volume fraction of endometrium occupied by micro vessels (%) 14 1125
Volume fraction of stroma occupied by micro vessels (%) 20 1429

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J Mol Hist

Fig.1An example of assessing the endometrial volume and vascu- typically represent the longitudinal (x), transverse (y) and coronal (z)
larization indices using 3D power Doppler ultrasound in the multi- planes of the uterus respectively, where manual outlining of the endo-
planar display. The built-in VOCAL imaging program can be used in metrial cavity is performed. The final lower-right image 3D repre-
the analysis to measure the endometrial and sub-endometrial volumes sents the 3D volume reconstruction of the endometrium, from which
and indices of blood flow within the endometrium. Images a, b and c the volume and vascularization parameters can be deduced

occupied by micro vessel (r=0.917, p=0.001) and endometrium transforms into a well-vascularized receptive
volume fraction of stroma occupied by micro vessel tissue characterized by increased vascular permeability,
(r=0.924, p=0.001) measured on each occasion. oedema, vascular remodeling and angiogenesis (Plaisier
For inter-observer variability, there was significant 2011). The characteristic and profound vascular changes
correlation between measurements of volume fraction observed at the time of implantation has been used as a
of endometrium occupied by micro vessel (r=0.821, marker of endometrial receptivity.
p=0.003) and volume fraction of stroma occupied by Traditionally, the assessment of vascular changes in the
micro vessel (r=0.800, p=0.010) of two observers. endometrium is based on histological examination of endo-
Therefore, acceptable agreement was reached between metrium biopsy specimens, such as measurement of micro
the results obtained by the two different observers. vessel density. An earlier study found no significant differ-
ence in micro vessel density between women with recurrent
miscarriage and fertile controls (Chen et al. 2012). How-
Discussion ever, in our own recent study, the number of micro blood
vessels in women with unexplained recurrent miscarriage
In this study we were unable to detect any significant corre- was observed to be higher than those in normal fertile
lation between the two methods of measuring endometrial women (Chen et al. 2016). Although histological exami-
vascularity around the time of implantation. nation is informative, it suffers from two important disad-
Vascular modification and new blood vessels growth vantages. Firstly, it cannot be used to study changes in the
are essential components of endometrial physiology. Dra- conception cycle as an endometrial biopsy may disturb the
matic changes in the vascular component of the endome- implantation process. Secondly, it cannot be repeated seri-
trium occur around the time of implantation, when the ally in the same cycle to examine the rapid and dynamic

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J Mol Hist

changes occurring in the endometrium as an endometrial


biopsy will necessarily induce an inflammatory reaction
and bring about iatrogenic vascular changes.
On the other hand, Doppler investigation has a number
of potential advantages over histology in that it is non-inva-
sive, may be repeated serially in the same cycle without
affecting the physiological process and may be also be used
to study changes occurring in the conception cycle. For this
reason, a number of investigators have attempted to meas-
ure endometrial vascularity by using Doppler ultrasonog-
raphy instead, but the findings are rather controversial. An
earlier study demonstrated that the pulsatility index (PI)-
measured by 2D Doppler ultrasonography in the mid-luteal
phase in women with unexplained recurrent miscarriage
was significantly higher than that of the control women
(Habara et al. 2002). However, in other studies, a signifi-
cantly lower endometrial and sub-endometrial vascularity
(VI, FI, VFI) was observed in women with miscarriage (Ng
etal. 2007; Chen etal. 2012).
Fig.2Immunohistochemical staining of endometrial biopsy
The main aim of our study was to determine how well
obtained on day LH+7 from women with unexplained recurrent mis-
carriage. The glands remain dilated and secretion can be seen. The 3D-PD ultrasonography and histologically vascular-
stroma becomes looser and oedematous. The micro blood vessels ity assessment correlate with each other and to establish
were identified by anti-vWFstained sections. Representative image if 3D-PD can be used clinically to replace histology as a
of micro blood vessels (black arrow) localized in endometrium from
non-invasive alternative to study endometrial vascularity
women with recurrent miscarriage is shown (a). Morphometric meas-
urement of volume fraction was calculated by point-counting method around the time of embryo implantation. An earlier study
with the use of square lattices with 121 intersection points (b). The focusing on animal tumor neovascularization has shown
number of points falling on a vessel (v), stroma (s) and gland (g) that neither 2D nor 3D power Doppler parameter corre-
were determined, from which the volume fraction of endometrium
late with micro vessel count (Fleischer etal. 1999), while
(v+s+g) occupied by vessel (v) may be determined and the volume
fraction of stroma (v+s) occupied by vessel (v) may also be deter- a more recent study found a significant correlation between
mined. Magnification 200. Scale bar 100m these two methods for detecting animal tumor vasculariza-
tion (Xuan et al. 2007). In humans, a previous study did

Table2Correlation between 3D power Doppler parameters and endometrial micro vessels measurements in women with unexplained recurrent
miscarriage (n=25, p>0.3 in all cases)

Parameter Number of micro blood Mean diameter of micro Volume fraction of endometrium Volume fraction of stroma
m2
vessels per m blood vessels occupied by micro vessels occupied by micro vessels

Endometrial
Thickness r=0.21 r=0.04 r=0.17 r=0.09
Volume r=0.03 r=0.08 r=0.11 r=0.19
VI r=0.10 r=0.09 r=0.18 r=0.22
FI r=0.10 r=0.09 r=0.09 r=0.12
VFI r=0.11 r=0.08 r=0.14 r=0.10
Sub-endometrial
Volume r=0.15 r=0.01 r=0.07 r=0.10
VI r=0.16 r=0.07 r=0.12 r=0.16
FI r=0.09 r=0.03 r=0.11 r=0.09
VFI r=0.18 r=0.07 r=0.11 r=0.17

Spearman correlation tests were applied to analyze the correlations


There were no significant correlations between any of the power Doppler variables and each of the morphometric measurements

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J Mol Hist

report on a lack of correlation between 3D-PD parameters to diagnose unexplained recurrent miscarriage. More
and micro vessel density in peri-implantation endometrium extensive investigations, such as plasma IgA level and
from women with recurrent miscarriage and tested posi- maternal HLA-DRB1 alleles, as suggested by Christiansen
tive for antiphospholipid antibodies (Chen et al. 2012). In etal. (2008), would be considered in the future study.
that study, however, the authors only measured micro ves- To conclude, we did not find any correlation between
sel density, determined by the number of CD34-positive endometrial vascularity evaluated by 3D-PD ultrasonogra-
micro vessels in three randomly selected fields, which is phy and morphometric analysis of micro vessels. It appears
prone to various systematic biases and sampling error. In that the 3D-PD examination could not be used clinically
this study, we have employed morphometric techniques to to replace the histological morphometric measurement of
examine five microscopic fields and systemic random field micro vessel density in women with unexplained recurrent
selection to reduce systematic bias. In addition, we had miscarriage. The assessment of endometrial angiogenesis
measured volume fraction by point counting method, which status may rather rely on the direct histologically method,
is recognized to be more objective and reliable (Li et al. although it is invasive.
1988). Using this improved method, we have confirmed the
earlier, preliminary findings of a lack of significant corre- Funding This study was supported by Hong Kong Obstetrical and
Gynaecological Trust Fund in 2016, Hong Kong. Xiaoyan Chen is a
lation between 3D-PD and histological studies of vascular recipient of Hong Kong PhD Fellowship from Hong Kong Research
changes in the endometrium at the time of embryo implan- Grants Council.
tation. The main reason accounting for the lack of correla-
tion may be that 3D ultrasonography possibly reflects the Compliance with ethical standards
dynamic blood flow invivo rather than the static histologi-
cal angiogenesis indicator, which is assessed by micro ves- Conflict of interests The authors declare no conflict of interests.
sel density in tissue (Chen etal. 2012).
There are several additional strengths regarding our
study. Firstly, all the observations were made on a precise References
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