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J Gynecol Obstet Hum Reprod xxx (2018) xxx–xxx

Available online at

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Original Article

Estrogen receptor, progesterone receptor and CD8+ expression

in endometrium of women of unexplained infertility
Shilpi Gupta Dixit a,*, Surajit Ghatak a, Pratibha Singh b, Shilajit Bhattacharya c
a
Department of Anatomy, All India Institute of Medical Sciences, Jodhpur 342005, India
b
Department of Obstetrics & Gynecology, All India Institute of Medical Sciences, Jodhpur 342005, India
c
Department of Pathology, All India Institute of Medical Sciences, Jodhpur 342005, India

A R T I C L E I N F O A B S T R A C T

Article history: Objective. – The present study aimed to investigate the changes of endometrial progesterone and
Received 14 October 2017 estrogen receptors in luteal phase biopsy specimens of infertile women and find a correlation, if any,
Received in revised form 12 May 2018 between these and CD8+ receptors in the same.
Accepted 17 May 2018
Methods. – The study was conducted on luteal phase endometrial biopsy specimens of 30 women of
Available online xxx
unexplained infertility and 15 age matched controls. Paraffin sections were first H & E stained. A
standardized immunohistochemical protocol was then used to localize the estrogen, progesterone and
Keywords:
CD8+ receptors in these samples that were expressed as percentage positivity. Unpaired T test was
Infertility
Endometrial factor
applied between the controls and cases both for epithelial and stromal cells. The data was also analyzed
Immunohistochemistry for correlation in cases for the positivity of CD8+ Cells with that of ER and PR.
PR Results. – The positivity of estrogen receptors (ER) in stromal cells was significantly lower (p < 0.001) in
ER the infertile women when compared to controls and in both the epithelial and stromal cells for
CD8+ cells progesterone receptors (p < 0.001).
The results were non significant for CD8+ cells (p = 0.19) and also showed no significant correlation in
the positivity of CD8+ cells with that of ER and PR.
Conclusions. – The development of molecular probe like ER and PR positivity in endometrial epithelial
and stromal cells allows a new approach to be made to the characterization of normal and defective
endometrial function.
C 2018 Published by Elsevier Masson SAS.

1. Introduction range from very high to very low depending on demographic

Infertility is ‘‘a disease of the reproductive system defined by
the failure to achieve a clinical pregnancy after 12 months or more
of regular unprotected sexual intercourse (and there is no other
reason, such as breastfeeding or postpartum amenorrhea)
although work up is not delayed in women who are 35 years or
older’’ [1,2].
Demographers tend to define infertility as childlessness in a
population of women of reproductive age, whereas epidemiologi-
cal definition refers to ‘‘trying for’’ or ‘‘time to’’ a pregnancy
generally in a population exposed to a probability of conception.
There are many medical causes of infertility including some
that medical intervention can treat. The most common quoted
figure for prevalence of infertility is 10% of couples although it may
* Corresponding author at: All India Institute of Medical Sciences, 503/2, AIIMS
Residential Complex, Jodhpur 342005, Rajasthan, India.
E-mail address: shilpidr@gmail.com (S.G. Dixit).
regions out of which 1/3rd each is attributed to individual male
and female factors, 10% is a combination of both partners while
20% of cases are unexplained [3]. Various risk factors include
lifestyle habits, medicines, radiation and hormonal imbalances.
The hormone profiles of women with unexplained infertility have
been generally found to be normal on investigations.
Endometrium has been comparatively less explored factor of
infertility due to evolution of non-invasive, quantitative and more
sensitive endocrine assays. Endometrial morphology is no more
than a simple reflection of ovarian cycle.
Estrogen has always been responsible for uterine epithelial
proliferation and is obligatory for normal uterine epithelial
morphogenesis, cytodifferentiation, and secretory activity through
estrogen receptors (ER) which are expressed in both juvenile and
adult uterus [4]. Few researchers have used various methods for
detection of estrogen receptors [5–7].
Various studies have investigated the changes of progesterone
(PR) and estrogen (ER) receptors in endometrial glandular and

https://doi.org/10.1016/j.jogoh.2018.05.006
2468-7847/ C 2018 Published by Elsevier Masson SAS.

Please cite this article in press as: Dixit SG, et al. Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of
women of unexplained infertility. J Gynecol Obstet Hum Reprod (2018), https://doi.org/10.1016/j.jogoh.2018.05.006
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JOGOH-178; No. of Pages 5
2 S.G. Dixit et al. / J Gynecol Obstet Hum Reprod xxx (2018) xxx–xxx
stromal cells and found decreased PR content suggesting deficient
response of endometrium to proper stimulus of hormones in
patients of infertility with apparently normal hormonal assay [6–8].
Other studies have seen functional differences of endometrial
leukocytes between fertile and infertile groups.
Studies of endometrium even at ultrastructural level are still
not equipped to supply answers to unresolved questions of
reproductive failure. Therefore such studies related to histology of
endometrium will help to prepare the clinicians to adapt and
devise new assisted reproduction techniques based on discoveries
at receptor level and supplement their traditional methods of
assessment and treatment.
The present study aims to investigate the changes of
endometrial progesterone and estrogen receptors in luteal phase
biopsy specimens of infertile patients and find a correlation, if any,
between these and CD8+ receptors in the same.
2. Materials and methods
Ethical clearance was taken from Institutional Ethics Commit-
tee prior to the commencement of the study.
2.1. Cases
Thirty infertile women in reproductive age group (20–40 years,
mean age 34.5 years) who were unable to conceive for at least one
year of cohabitation without contraception and normal work up of
infertility did not reveal any cause identifiable for infertility and
showed normal results in hormonal assays and ultrasonography.
Routine work up included partner’s semen analysis, tubal patency
test, hormonal assays wherever indicated by history and exami-
nation (Thyroid function tests, LH, FSH, Serum Progesterone,
Menstrual blood for TB PCR, Bactec). Ultrasonography was done
both transabdominally and transvaginally to look for uterine
anatomy, ovarian size, number of follicles, antral follicle count and
to see for the dominant follicles at the time of ovulation.
2.2. Exclusion criteria
Various other factors related to infertility like in which male
partners did not show normal results on semen analysis like
anovulation, blocked tubes, and other pelvic pathologies like PID
and endometrioses were excluded in the study.
2.3. Controls
Fifteen age matched controls (mean age 37 years) who have
experienced at least one successful spontaneous pregnancy
without any infertility treatment or assisted reproductive tech-
nology and has undergone hysterectomy due to suspicious adnexal
mass or cervical dysplasia (CIN II, CIN III).
2.4. Exclusion criteria
Women with abnormal menstrual cycles or have received any
hormonal medications at least 3 months prior to the study or
inflammation related problems (ovarian cyst, benign tumors, etc.).
Table 1
Receptor positivity (in %) for estrogen (ER) and progesterone (PR) and CD8+ receptors in epithelial and stromal cells of endometrial biopsies.
ER positivity in %
Epithelial (mean  SD) Stromal (mean  SD)
* *
Cases 92  7.74 40  15.11
Controls 97.3  6.46 75  15.49
*
p < 0.001.
Please cite this article in press as: Dixit SG, et al. Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of
women of unexplained infertility. J Gynecol Obstet Hum Reprod (2018), https://doi.org/10.1016/j.jogoh.2018.05.006
Informed consent for participation was obtained from subjects
before enrolling them for the study.
2.5. Sample collection and preparation of sections
Samples were obtained from endometrial biopsy done in the
luteal phase of both the cases and controls. The biopsies were taken
as a day care procedure with Novak’s curette. Samples were
formalin fixed (10% buffered formalin pH buffering solution) for
10 days and then processed for paraffin embedding. Sections of 4–
5 mm of the embedded specimens were cut and slides were first
stained with Haemotoxylin and Eosin, then immunohistochemical
protocol was followed.
2.6. Immunohistochemistry
A standardized immunohistochemical protocol was used to
localize the estrogen, progesterone and CD8+ receptors in both
epithelial and stromal cells of the samples. To immunohisto-
chemically detect estrogen, progesterone, CD8 receptors, heat
epitope retrieval method was used for antigen retrieval. A
peroxidase blocking solution was used to inactivate the endoge-
nous peroxidase. This was followed by incubation with primary
and secondary antibodies (Dako, ISO8430-22-FLEX Monoclonal
Rabbit X-H-ER Alpha clone EP1, ISO6830-2-FLEX Monoclonal Mo
X-H-PR, Clone PgR 636, RTU, IS62330-2-FLEX Monoclonal Mo a Hu
CD8, Clone C8/144B, RTU and subsequent addition of chromogen
3,3Diaminobenzidene). Slides were counter stained with Haemo-
toxylin stain. Immunohistochemical results were evaluated and
scored according to the percentage of positively staining epithelial
and stromal cells of the sample of both the controls and cases.
2.7. Data analysis plan
The data obtained was analyzed using SPSS Version-23.
Unpaired T test was applied between the controls and cases both
for epithelial and stromal cells of endometrial specimens. The data
was also analyzed for correlation between ER, PR and CD8+ cells.
3. Results
Immunohistochemical results were evaluated in a semiquanti-
tative manner and scored according to the percentage of positively
staining cells.
1. The data when analyzed showed significantly (p < 0.001) low
positivity of estrogen receptors (ER) in stromal cells of cases as
compared to controls as shown in Table 1.
2. The positivity was also significantly low in both the epithelial
and stromal cells of infertility cases as compared to controls for
progesterone receptors as shown in Table 1.
3. The results were non significant for CD8+ cells.
4. The results also showed no significant correlation in the
positivity of CD8 cells with that of ER and PR.
5. The immunohistochemical images of ER, PR and CD8 positivity
of the cases of infertility and controls are shown in Fig. 1a,b,
PR positivity in % CD8+
Epithelial (mean  SD) Stromal (mean  SD) (mean  SD)
*
10  6.26 54  8.2 8.63  6.78
81.18  33.7 78.63  17.47 6.06  3.08
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Fig. 1. (a) Microphotograph showing ER positivity by IHC in cases (10) (300DPI  300 DPI); (b) Microphotograph showing ER in positivity by IHC in controls (20)
(300DPI  300 DPI).

Fig. 2a,b, and Fig. 3a,b respectively. Fig. 4a,b is showing multiple
stromal lymphoid aggregates.
4. Discussion
Prevalence of infertility ranges from 6 to almost 60% out of
which 1/3rd (30%) each is attributed to individual male and female
factors, 10% is a combination of both partners while 20% of cases
are unexplained [9,10]. Various risk factors include lifestyle habits,
medicines, radiation and hormonal imbalances. There are other
factors leading to DNA damage that can cause changes at receptor
level in an apparently healthy infertile couple with no hormonal
and anatomic abnormalities. The hormone profiles of women with
unexplained infertility have been generally found to be normal on
investigations.
The various factors that help in the initial interaction between
maternal and fetal epithelium causing temporal and spatial
expression of various endometrial peptides in response to estrogen
and progesterone hormones and making the endometrium ready
for receiving the blastocyst are still not clearly understood [11].
Identification of various causes of unexplained fertility is quite
complex with various overlapping etiologies.
Endometrium has been comparatively less explored factor of
infertility due to evolution of non-invasive, quantitative and more
sensitive endocrine assays as endometrial morphology was
considered no more than a simple reflection of ovarian cycle.
The cyclic changes taking place in human endometrium due to
estrogen and progesterone hormones is via their action through
specific nuclear receptors. Various pathophysiological states like
menstrual cycle (both normal and abnormal), pregnancy and
cancers etc. have different concentrations of estrogen and
progesterone receptors in endometrium.
There have been a wide variety of studies on male and female
infertility. Various studies have investigated the changes of
progesterone (PR) and estrogen (ER) receptors in endometrial
glandular and stromal cells and found decreased PR content
suggesting deficient response of endometrium to proper stimulus
of hormones in patients of infertility with apparently normal
hormonal assay [6–8]. Previously the work done was on hormonal
assays and anatomic abnormalities. Recently the work has shifted
to receptor levels.
Robertson in his review revaluated the importance of study on
endometrium for infertility [12]. Dia et al. investigated the
endometrial nuclear progesterone receptors (PgR) as well as its
relationship with retarded endometrial development (RED) in

Photo 2. (a) Microphotograph showing PR positivity by IHC in cases (10) (300DPI  300 DPI); (b) Microphotograph showing PR positivity by IHC in controls (20)
(300DPI  300 DPI).

Please cite this article in press as: Dixit SG, et al. Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of
women of unexplained infertility. J Gynecol Obstet Hum Reprod (2018), https://doi.org/10.1016/j.jogoh.2018.05.006
G Model
JOGOH-178; No. of Pages 5

4 S.G. Dixit et al. / J Gynecol Obstet Hum Reprod xxx (2018) xxx–xxx

Photo 3. (a) Microphotograph showing CD8+ positivity by IHC in cases (10); (b) Microphotograph showing CD8+positivity by IHC in controls (10).

infertile women immunohistochemically, and found significantly
lower levels of PRs in them that were similar to the findings of the
present study [8]. Godinjak and Bilalovic found significantly
reduced positive immunostaining for ER in stromal cells and for PR
in both stromal and epithelial cells in the endometrial biopsies of
patients of unexplained infertility. These findings have been
corroborated by the present study [13]. Law TM et al. suggested in
their study that there is a differential sensitivity of glandular and
stromal receptors to steroid regulation during normal menstrual
cycle. These may be affected in various gynecological disorders
[14]. Ohno et al. suggested that high PR expression in the pre-
ovulatory period is related to an adequate endometrial growth and
thereby increases the responsiveness of the endometrium to
progesterone stimulation after ovulation [15]. Lessey et al. in their
study came to a conclusion that the establishment of normal
endometrial receptivity appears to be tightly associated with the
down-regulation of epithelial PR [16]. Histologic delay, consistent
with luteal phase deficiency, is associated with a failure of PR
down-regulation and the lack of normal markers of endometrial
receptivity. Hirama and Ochiai suggested that level of cytosol ER
was significantly lower in out-of-phase endometrium regardless of
serum P level [17]. Bergqiuvst et al. compared the localization and
staining intensity of oestrogen and progesterone receptors in
endometrium and endometriotic tissue using monoclonal anti-
bodies and found a significant correlation between oestrogen
receptor score in endometriotic tissue and in endometrium but not
for progesterone receptor score [18]. Gracia et al. studied
variations in progesterone receptor staining and found that they
are potentially useful for determining the effect of progesterone on
endometrial maturation [19]. Kim et al. also evaluated relationship
between endometrial concentrations of estrogen and progesterone
receptors and sonographic endometrial findings of menstrual cycle
[20]. The positivity of ER in stromal cells which was found to be
significantly low in patients of infertility and not in epithelial cells
of the same patients in our study is probably due to the fact that the
mitogenic effects of estrogen on endometrium is mediated through
stromal ER. Cook et al. also stated the same in their study [4].
During the menstrual cycle leukocytes progressively infiltrate
the endometrium and they may constitute as many as 30% of
decidual cells in early pregnancy. Few authors observed a
difference in the immunological response of infertile women by
quantifying immunologically CD8+ and CD4+ cells that were
significantly reduced and increased respectively. Few others
suggested in their study that the endometrial lymphoid tissue of
women with endometriosis does not differ qualitatively or
quantitively from that of normal fertile controls which is similar
to the findings of our study [21,22].
The findings of various studies as well as the present study
suggest that changes in expression of ER and PR may lead to
impairment of endometrial proliferative processes. Therefore these

Photo 4. (a) Microphotograph showing multiple stromal lymphoid aggregates (shown in arrows) in ER positive slide; (b) Microphotograph showing the same multiple
stromal lymphoid aggregates in (a) which is showing positivity with CD8+ immunostaining (40).

Please cite this article in press as: Dixit SG, et al. Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of
women of unexplained infertility. J Gynecol Obstet Hum Reprod (2018), https://doi.org/10.1016/j.jogoh.2018.05.006
G Model
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S.G. Dixit et al. / J Gynecol Obstet Hum Reprod xxx (2018) xxx–xxx
may be sensitive indicators of receptive status of endometrium. The
decreased uterine receptivity for ER and PR exhibited by women of
unexplained infertility may be of assistance in the clinical arena in
choosing the schemes of assisted reproductive techniques.
5. Conclusion
The present study is a pilot study showing a significantly
decreased ER and PR positivity in patients of infertility thereby
reducing the receptivity of endometrium to steroid hormones and
thereby affecting the reproductive cycle. The CD8+ cells were
investigated with a hypothesis that underlying subclinical
inflammation could be a possible cause of unexplained infertility
wherein other investigations of the patients were normal. The
CD8+ cells were not significantly increased thereby indicating
absence of subclinical inflammation. The development of molecu-
lar probe to see ER and PR positivity in endometrial epithelial and
stromal cells allows a new approach to be made to the
characterization of normal and defective endometrial function.
However further studies with a larger sample size are needed to
substantiate the findings of this pilot study.
Ethical approval
All procedures performed in studies involving human partici-
pants were in accordance with the ethical standards of the
institutional research committee.
Funding
Intramural grant by AIIMS Jodhpur.
Informed consent
Written Informed consent was obtained from all individual
participants included in the study.
Disclosure of interest
The authors declare that they have no competing interest.
Please cite this article in press as: Dixit SG, et al. Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of
women of unexplained infertility. J Gynecol Obstet Hum Reprod (2018), https://doi.org/10.1016/j.jogoh.2018.05.006
5
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