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Author's personal copy
Altered endometrial expression of endothelial
nitric oxide synthase in women with
unexplained recurrent miscarriage and infertility
Tohid Najafia, Marefat Ghaffari Novina,*, Reza Ghazib, Omid Khorramb
aInfertility and Reproductive Health Research Center, Shahid Beheshti Medical University, Tehran, Iran;bDepartment
of Obstetrics and Gynecology, Harbor-UCLA Medical Center and Los Angeles Biomedical Institute, Torrance, California, USA
*Corresponding author. E-mail address: firstname.lastname@example.org (MG Novin).
Tohid Najafi, MSc Embryology and Anatomical Science, was born in Iran and graduated from Shahid Beheshti
Medical University in 2011. His major research fields are reproductive biology, specifically expression of nitric
oxide synthase in normal and infertile endometrial tissues. He has also published articles about other aspects of
the reproductive tract, for example regarding IVF and ultrastructure of spermatozoa. He also worked as an
embryologist in infertility treatment centres in Iran. Tohid now started to pursue his PhD in Reproductive
Endocrinology at Wayne State University of Michigan, USA Iran.
implantation. Aberrant expression of eNOS could therefore be significant in the pathogenesis of disorders of implantation. In this
study, eNOS protein and mRNA levels in the endometrium of women with recurrent miscarriages, unexplained infertility and a con-
trol group were determined by compartmental quantitative immunohistochemistry and real-time reverse-transcription PCR. eNOS
was found to be immunolocalized to all layers of the endometrium and vascular endothelium. eNOS protein was higher in glandular
epithelium (P = 0.004) and luminal epithelium (P = 0.002), but not vascular endothelium and stroma, in women with recurrent mis-
carriage. Similarly, in women with unexplained infertility, eNOS was significantly higher (P < 0.03) in luminal epithelium but not in
any other compartments compared with the control group. The levels of mRNA confirmed the protein data, demonstrating higher
eNOS mRNA in the endometrium of women with recurrent miscarriage and unexplained infertility compared with controls. In con-
clusion, increased expression of eNOS in glandular and luminal epithelium of the endometrium in women with recurrent miscarriages
and unexplained infertility suggests a detrimental effect of excess nitric oxide in endometrial receptivity and implantation.
ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Endothelial nitric oxide synthase (eNOS) has diverse roles in the female reproductive system including a role in blastocyst
KEYWORDS: endometrium, eNOS, immunohistochemistry, real-time RT-PCR, recurrent miscarriage, unexplained infertility
Recurrent miscarriage is described as at least three consec-
utive spontaneous pregnancy losses in the first trimester and
compromises 3% of couples (Regan and Rai, 2000; Taylor,
2003). Genetic and developmental abnormalities are the
major causes of recurrent miscarriage; however, immune,
endocrine and endometrial anatomical or non-anatomical
factors as well as thrombophilias can lead to recurrent mis-
carriage. In about 50% of cases, the cause of miscarriage
remains unknown (Li et al., 2002a,b). Unexplained infertil-
ity affects 15% of infertile couples and is defined as when
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Reproductive BioMedicine Online (2012) 25, 408–414
Author's personal copy
all the tests of a basic infertility evaluation, including
semen analysis, hysterosalpingogram, ovarian reserve test-
ing, pelvic ultrasound and possibly laparoscopic evaluation
of the pelvis, are within normal limits (Hatasaka, 2011;
Smith et al., 2003). Recent data have shown that unex-
plained infertility and recurrent miscarriage are distinctly
different diagnoses and that unexplained infertility is not
due to recurrent preclinical pregnancy loss (Koot et al.,
2011), although these two conditions might share common
endometrial function defects.
Nitric oxide (NO) is a vasodilator synthesized from L-argi-
nine through the action of nitric oxide synthase (NOS).
Three isoforms of NOS are expressed in the human endome-
trium, although eNOS is the predominant form (Khorram
et al., 1999). The endometrial expression of eNOS is cyclic,
with peak expression during the window of implantation in
humans (Khorram et al., 1999; Ota et al., 1998), and rodents
(Purcell et al., 1999). Both oestrogen and progesterone (Han
et al., 2005; Khorram and Han, 2009; Zervou et al., 1999)
regulate the expression of eNOS in the human endometrium
(Khorram et al., 1999). NO, by virtue of its properties as a
potent vasodilator (Palmer et al., 1987) and a myometrial
smooth muscle relaxant (Buxton, 2004; Norman et al., 1997)
and its participation in signal transduction pathways
(Thomas et al., 2008), might play a significant role in estab-
lishment and maintenance of pregnancy. By virtue of these
properties of NO, this study postulated that aberrant endo-
metrial expression of eNOS as in endometriosis (Dong et al.,
2001; Khorram and Lessey, 2002; Ota et al., 1998; Wu et al.,
2003) and adenomyosis (Ota et al., 1998) could occur in
patients with unexplained infertility and recurrent miscar-
riage. Since oxidative stress plays an important role at least
in idiopathic recurrent pregnancy loss (Gupta et al., 2007),
and NO in high concentrations can induce nitrosative stress
(Agarwal et al., 2008), it was also postulated that increased
endometrial eNOS expression and thereby NO generation in
patients with unexplained infertility and recurrent miscar-
riage could impair endometrial function by either inducing
cellular apoptosis (Wang et al., 2010) or through nitrosyla-
tion of key endometrial proteins (Gu et al., 2010; Weiner
et al., 2009), impairing their physiological function.
Materials and methods
Sample collection and preparation of sections
The protocol for this study was approved by the Human Sub-
jects Committee at Shahid Beheshti Medical University (84th
Session, 5/2010). Endometrial biopsies were obtained from
three groups of women using a pipelle curette 7–9 days post
ovulation as determined by serial ultrasound scans. The
recurrent miscarriage group (n = 10) consisted of women
with a mean age of 32.8 years and a mean of 4.7 consecutive
pregnancy losses. Women with secondary miscarriages or
less than three miscarriages were excluded. Evaluation of
recurrent miscarriage group including karyotype analysis,
antiphospholipid antibody and thrombophilia testing were
all within normal range. Endometrial cavity as assessed by
hysterosalpingogram was normal in recurrent miscarriage
patients. Women with unexplained infertility (n = 10) con-
sisted of individuals with a mean age of 29.8 years who were
unable to conceive for more than 2 years with a normal
basic infertility evaluation. This evaluation consisted of
endocrine tests (TSH, cycle-day-3 FSH and oestradiol con-
centrations, prolactin and progesterone concentrations
greater than 10 ng/ml in mid-luteal phase), anatomical
tests (hysterosalpingography and pelvic ultrasonography)
and semen analysis (WHO criteria; WHO, 1999). The control
group (n = 10) consisted of women with a mean age of
36.1 years who presented for tubal sterilization. Women in
this group had normal menstrual cycles (26–33 days), had
a mean parity of 1.4 and had no prior history of pregnancy
losses and no prior use of assisted reproductive techniques
Endometrial biopsy specimens were divided into three
portions. One piece was placed in 4% paraformaldhyde tis-
sue fixative for 24 h and then switched to 70% ethanol for
later processing. Another piece was placed in RNA Later
preservative and stored at ?80?C and one section was fixed
for histological dating of the endometrium using the criteria
of Noyes et al. (1975). Human placental tissue was used as a
positive control (Bhuiyan et al., 2006).
Endometrial specimens were cut into 6 lm sections using a
Cryocut and placed on poly-L-lysin coated slides and stored
at ?70?C for immunostaining. A monoclonal mouse antihu-
man antibody (6H2, cat. no. 91205; Abcam, UK) was used
to detect the intensity and distribution of eNOS immuno-
staining using standard immunohistochemical protocol.
Briefly after rinsing the slides with buffer (0.1 mol/l phos-
phate-buffered saline (PBS), pH 7.4), endogenous peroxi-
dases were quenched by incubation in 0.3% H2O2 in
methanol for 15 min. Repeated rinses with 0.05% bovine
serum albumin in PBS were performed followed by antigen
retrieval using trypsin. Normal goat serum (1.5%; DAKO,
Denmark) was then added to the slides in humidified cham-
bers for 20 min at room temperature to prevent nonspecific
binding of antibody. The primary antibody against eNOS
(1:100) was added to slides and incubated at 37?C for 1 h.
Slides were then rinsed three times with wash buffer, fol-
lowed by incubation of sections with the secondary anti-
body, rabbit antimouse IgG, H and L (cat. no. ab6728;
Abcam) diluted 1:1000 in PBS. Incubation with the second-
ary antibody was performed for 1 h at 37?C in an incubator.
Slides were then exposed to 3,3-diaminobenzidine in H2O2
(DAKO) was for 15 min. Thereafter, the sections were coun-
terstained with haematoxylin and mounted. In case of neg-
ative controls, a similar method was used but PBS replaced
the primary antibody. Human full-term placental tissue
served as an external positive control. Staining intensity
of sections was determined by Image Pro Plus software by
a blinded reviewer (TN) using previously described methods
(Khorram et al., 2007). Six different areas of the sections
were analysed at a magnification of ·40 and the mean was
used for statistical analysis. The results were expressed as
percentage integrated optical density.
Real-time reverse-transcription PCR
RNA was isolated using the High Pure RNA Isolation kit
(Roche Applied science). Ribonucleic acid was DNase
Expression of eNOS in unexplained recurrent miscarriage and infertility409
Author's personal copy
treated and quantitated by measurement of absorbance in a
NanoDrop spectrophotometer. Total RNA (1 lg) was reverse
(cDNA) with use of the Omniscript Reverse Transcription
kit (GeneON, Germany) at 37?C for 60 min in a total volume
of 20 ll. The reaction mix consisted of 1 ml of 10-fold
diluted cDNA, qPCR MasterMix Plus for SYBR green I reagent
(GeneON) and optimized forward and reverse gene-specific
primers (300 nmol/l each). Reactions were run in triplicate
in 96-well plates with a Mx3000P real-time PCR system
(Stratagene, Santa Clara, CA, USA). The thermal cycling
program was 95?C for 10 min followed by 40 cycles of 95?C
for 15 s and 60?C for 1 min. The internal control used was
(de Kok et al., 2005). Data were analysed to select a thresh-
old level of fluorescence that was in the linear phase of the
PCR product accumulation (the threshold cycle, CT) for that
reaction. The CT value for the control was subtracted from
the CT value of eNOS gene to obtain a DCT value. The rela-
tive fold change for each gene was calculated with use of
the DDCT method (Livak and Schmittgen, 2001).
Results were analysed by ANOVA, comparing percentage
integrated optical density for the immunohistochemical
data and fold change in case of real-time reverse-transcrip-
tion PCR using Statistical Package for Social Sciences soft-
ware (SPSS, USA). Post-hoc analysis was performed using
the Student–Newman–Keuls test. P < 0.05 was considered
The demographics of the three groups of women studied are
shown in Table 1. Women in the unexplained infertility
(P = 0.036). There were no differences in body mass index
among the three groups. Four subjects in the control group
and two in the recurrent miscarriage and unexplained infer-
tility groups were light smokers (<3 cigarettes/day). None
of the subjects had chronic medical illnesses. Laparoscopic
evaluation in all three groups showed a normal pelvis.
As previously demonstrated, eNOS was expressed in all
layers of the endometrium (Figure 1). eNOS protein staining
in glandular epithelium (Figure 1A–C) was less intense than
in luminal epithelium (Figure 1D–F). Weak staining of eNOS
was detected in the vascular endothelium (Figure 1G) and
the stromal layer (Figure 1H). eNOS expression was higher
in glandular epithelium of patients with recurrent miscar-
riage and unexplained infertility compared with control
(Figure 1A–C). A similar pattern was found in the luminal
epithelium only in patients with Recurrent Miscarriage
A summary of image analysis for eNOS expression in dif-
ferent layers of the endometrium is given in Figure 2. In
women with recurrent miscarriage, glandular (P = 0.004)
and luminal epithelial (P = 0.002) expression of eNOS was
greater compared with controls, whereas stromal and
vascular endothelial expression of eNOS was not signifi-
cantly different for recurrent miscarriage and unexplained
infertility as compared with the control group. In women
with unexplained infertility, the expression of eNOS was
only higher in luminal epithelium compared with controls
(P < 0.021), with no significant differences in the other
The expression of eNOS mRNA in the endometrium of
women with recurrent miscarriage and unexplained infertil-
(P < 0.05; Figure 3), confirming the protein data.
The data confirm prior reports on endometrial eNOS immu-
nolocalization (Khorram et al., 1999) and demonstrate a dif-
ferential site-specific alteration in expression of eNOS
protein in women with unexplained infertility and recurrent
miscarriage compared with controls during the window of
implantation. The most prominent changes were found in
the luminal epithelium, with greater expression of eNOS
protein in both the recurrent miscarriage and unexplained
infertility groups in this area. The expression of eNOS mRNA
in the endometrium of women with unexplained infertility
and recurrent miscarriage was significantly higher compared
with controls, thus confirming the protein data. The luminal
eNOS protein changes in both recurrent miscarriage and
unexplained infertility support the importance of this
enzyme for implantation and its dysregulation as a possible
cause of implantation failures in these patients.
The significance of NO in the implantation process has
been demonstrated in animal studies in which pharmacolog-
ical blockers of NOS impair implantation. Novaro et al.
(1996) demonstrated that the expression of NOS and prosta-
glandins E and F increase 1 day before implantation and that
(L-NAME) decreased production of prostaglandins E and F2
on the day of implantation in rats. Biswas et al. (1998)
injected L-NAME into the rat uterine horn and demonstrated
Demographic characteristics of the study population.
Control (n = 10)Recurrent miscarriage
(n = 10)
infertility (n = 10)
Body mass index (kg/m2)
36.1 ± 1.1
26.1 ± 1.6
1.4 ± 0.2
32.8 ± 1.9
23.9 ± 1
4.7 ± 0.7
29.8 ± 0.8a
25 ± 1
Values are mean ± SEM.
aP = 0.036.
410T Najafi et al.
Author's personal copy
an inhibition of implantation, and similar results were
obtained by Duran-Reyes et al. (1999) using a different
NOS inhibitor. In contrast to these studies, knockout mice
for various isoforms of NOS do not show reduced litter size
(Huang et al., 1993, 1995; MacMicking et al., 1995), indicat-
ing the importance of multiple pathways and redundancy in
the implantation process. Since implantation failure sec-
ondary to endometrial factors may be common to unex-
plained infertility (Koot et al., 2011) and recurrent
miscarriage (Li et al., 2002b), the present study sought to
determine if aberrant endometrial expression of eNOS could
be associated with these two conditions. The common find-
ing of luminal increase in the expression of eNOS protein in
both recurrent miscarriage and unexplained infertility
patients suggest that luminal eNOS is important in the path-
ogenesis of these disorders. Excess NO could impair implan-
tation through several mechanisms. NO has been shown to
induce endometrial epithelial apoptosis (Castro et al., 2002;
Johnson et al., 2004; Li et al., 2001) and increased eNOS
expression, so therefore localized excess NO production at
the luminal surface could induce epithelial apoptosis and
implantation failure. Vatansever et al. (2005) also reported
increased eNOS immunoreactivity in the endometrium of
unexplained infertility patients, although this was associ-
ated with a lower number of apoptotic cells. A second
mechanism by which NO might impair implantation is
through localized nitrosative stress. NO by virtue of its
unpaired electron is a highly reactive free radical that, in
excess, can damage protein, carbohydrates, nucleotides
and lipids (Agarwal et al., 2008). Based on the present data,
it is proposed that excess eNOS expression in luminal epithe-
lium of patients with recurrent miscarriage and unexplained
infertility can create local oxidative stress which could
impair implantation, similar to other inflammatory gynaeco-
logical conditions such as endometriosis (Dong et al., 2001;
Khorram and Lessey, 2002; Ota et al., 1998; Wu et al.,
luminal epithelium (D–F) of a control subject (A, D), recurrent miscarriage patient (B, E) and unexplained infertility patient (C, F)
and vascular endothelium (G) and stroma (H) of a control subject. Negative (I) and positive (J) controls in a human placental section.
Arrows indicate areas with positive eNOS immunostaining. The magnification used was ·40; bar = 40 lm.
eNOS immunostaining in different endometrial compartments of the study population. Glandular epithelium (A–C) and
Expression of eNOS in unexplained recurrent miscarriage and infertility 411
Author's personal copy
2003), adenomyosis (Ota et al., 1998) and adhesions (Saed
and Diamond, 2004). Exogenous factors such as cigarette
smoking, which has been associated with recurrent miscar-
riages (Cramer and Wise, 2000), can also induce nitrosative
stress through direct endometrial cell stimulation of eNOS
expression, an effect which can be blocked by antioxidants
such as ascorbic acid (Khorram et al., 2010). Similarly,
ascorbate has recently been shown to activate eNOS activity
by rapid modulation of its phosphorylation status (Ladurner
et al., 2012).
Several studies have attempted to find a genetic link for
recurrent miscarriage, and in so doing have examined poly-
morphism of the eNOS gene in different ethnic groups A
recent meta-analysis of these studies showed a significant
association in eNOS Glu298Asp polymorphism and recurrent
miscarriage (Su et al., 2011). These genetic abnormalities
could lead to reduced NO production and impaired endome-
trial function. The present data did not show any differ-
ences in endothelial microvascular eNOS concentrations in
recurrent miscarriage and unexplained infertility patients,
suggesting that localized endometrial blood flow mediated
by the NO pathway during the implantation window is not
a significant factor in the pathogenesis of these disorders
and also that other blood-flow-regulating factors not exam-
ined in this study may be of greater importance. Although
weak expression of eNOS in vascular endothelium, as seen
in these endometrial samples, could be a factor which can
lead to infertility and miscarriage, these expression levels
were not significantly different compared with the control
group. This suggests that dysregulated expression of eNOS
in non-endothelial sites plays a more significant role in the
pathophysiology of recurrent miscarriages and unexplained
In conclusion, the present data demonstrate an overex-
pression of eNOS protein and mRNA in the endometrium of
recurrent miscarriage and unexplained infertility. This aber-
rant pattern of eNOS protein and mRNA expression is similar
to other inflammatory gynaecological conditions such as
endometriosis and adenomyosis. Although eNOS expression
in some amounts is essential for implantation, excess eNOS
expression and therefore excess generation of NO in the
endometrium of patients with recurrent miscarriage and
unexplained infertility is deleterious and could induce nitro-
sative stress. which could lead to implantation failures or
failure of early pregnancy maintenance. More information
about this can be obtained in the future, studying NO
expression during full-term pregnancies, and it would also
be helpful to compare the expression of eNOS in full and
This work was supported by the National Institutes of Health
(RO3 HD 41409-01, to OK) and the Infertility and Reproduc-
tive Health Research Centre to MGN. The authors thank the
secretarial assistance of Ms Jeannie Park.
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Declaration: The authors report no financial or commercial
conflicts of interest.
Received 18 March 2012; refereed 4 July 2012; accepted 9 July
414T Najafi et al.