Evaluation of Endometrial Receptivity in Patients with Recurrent Pregnancy Loss and Recurrent Implantation Failure

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Evaluation of Endometrial Receptivity in Patients with Recurrent Pregnancy Loss and Recurrent Implantation Failure

https://doi.org/10.21203/rs.3.rs-3246580/v1

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Background: The cause of implantation defects in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) has not been clearly established. We aimed to evaluate the immunohistochemical changes of HOXA-11, β1 integrin, Focal Adhesion Kinase (FAK), CD44, and Extracellular Matrix Protein 1 (ECM1) in the endometrium of patients with defective implantation -RIF and RPL- in the receptive period.

Methods: This retrospective case-control study was conducted in a university hospital. After the exclusion of all cases with pathology that may cause a change in the level of receptors in the endometrium, biopsies taken during the receptive period were selected, and the patients were divided into RPL (n=15), RIF (n=16), and control (n=16) groups. All preparations were immunohistochemically stained for HOXA11, β1 integrin, FAK, CD44, and ECM1. The presence or absence of staining and staining intensity were compared between groups.

Results: When the RIF group was compared with the control group, there was no significant difference in HOXA-11 and β1 Integrin expression (p>0.05), FAK expression was significantly increased compared to the control group (p<0.01); ECM1 and CD44 expressions were found to be significantly decreased (p<0.01). When the RPL group was compared with the control group, there was no significant difference in the endometrial staining of HOXA-11, FAK, and ECM1 in women with a history of RPL (p>0.05); β1 Integrin and CD44 levels were significantly decreased (p<0.05).

Conclusion: CD44 reduction may play a role in pathophysiology in both patient groups. In addition, decreased β1 integrin may also play a role in RPL. In RIF, the increase in FAK and decrease in ECM1 seem to be involved in the mechanisms of the disease.

Health sciences/Diseases/Reproductive disorders/Infertility

Health sciences/Molecular medicine

Health sciences/Biomarkers/Predictive markers

Health sciences/Biomarkers/Diagnostic markers

adhesion molecules

defective implantation

endometrial receptivity

recurrent implantation failure

recurrent pregnancy loss

The human endometrium has various changes in the secretory and proliferative phases of the menstrual cycle. It becomes available for embryo implantation in a short period, known as the "implantation window" [1]. A live blastocyst must reach the receptive endometrium at this appropriate time for successful implantation [2]. Although this information is well known, pregnancy losses are estimated at approximately 50–75% after in vitro fertilization (IVF) due to implantation failure [3]. While 50% of early pregnancy losses are associated with abnormal embryo karyotype, the remaining 50% is related to the poor relationship between embryo and endometrium [4].

After organic endometrial pathologies such as chronic endometritis and endometrial polyps have been treated, endometrial receptivity studies have gained momentum with the persistence of recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) in some patients to understand immunological mechanisms better for implantation and implantation window concepts [5]. Many immunohistochemical markers may affect endometrial receptivity and be expressed at different rates in various diseases of the uterus [6]. However, all markers still need to be clearly understood, and there are still markers whose effects cannot be demonstrated.

Interactions between various growth factors, cell adhesion molecules, extracellular matrix proteins, and cytokines have been defined in the implantation process. Expression of most of these factors in the receptive endometrium of the implantation window has previously been identified. Among these markers, data on HOXA11, Focal Adhesion Kinase (FAK), CD44, β1 integrin, and Extracellular Matrix Protein 1 (ECM1) are limited. Some real-time PCR studies found a change in the HOXA11- a transcription factor in the homeobox gen family- expression during the implantation window. Nevertheless, immunohistochemical studies are limited [7]. β1 integrin and FAK are molecules that communicate with each other in the cell adhesion process. Previously, these molecules have been implicated in the process of ectopic implantation of endometrium in endometriotic implants [8, 9]. Although the expression of these markers during the implantation process has been shown [10], their changes in RIF and RPL have yet to be defined. Similarly, studies on the expression of cell adhesion molecules of CD44 and ECM1- proposed to affect the implantation process in infertile patients- in RIF are limited [11]. Considering the complexity of endometrial receptivity and the lack of essential markers on implantation, immunohistochemical studies on implantation failure and pregnancy loss are intriguing and promising for clinicians.

This study aimed to evaluate the expression changes of the five adhesion proteins (HOXA-11, β1 Integrin, FAK, CD44, and ECM1) during the secretory period when the endometrium is receptive for implantation on the endometrial receptivity in patients with RIF and RPL.

This retrospective case-control study was conducted at the Department of Obstetrics and Gynecology of Gazi University Faculty of Medicine. Gazi University Ethics Committee of Clinical Studies approved the study (Approval no: 2019 − 129). All experiments were performed in accordance with relevant guidelines and regulations. In the study, patient preparations in the archive were used, and informed consent form was not obtained from the patients because it was stated that there was no need by the ethics committee.

Data Collection and Patient Selection

Patients who applied to the Obstetrics and Gynecology Clinics between January 2019 and January 2020 and underwent endometrial biopsy between the ages of 21 and 40 were examined from the hospital's medical records (n = 349). These groups were formed according to their obstetrical history: RIF group, RPL group, and control group. RIF was defined as the absence of pregnancy, although at least four high-quality embryos were transferred in at least three fresh or frozen-thawed embryo transfer cycles [12]. RPL was defined as three or more miscarriages with an unknown cause before 20 weeks of gestation or weighing less than 500 grams [13]. All patients with RIF or RPL who met mentioned criteria were included in the study. Patients with at least one child undergoing endometrial biopsy for reasons other than infertility were evaluated as the control group. No endometrial pathology was reported in all control group patients. Patients with endometrial pathology, including endometrial polyp, chronic endometritis, or submucous leiomyoma; endometrial biopsies were taken outside the receptive period of the endometrium (between 21-24th days were accepted as the receptive period); patients with systemic diseases, that may affect endometrial receptivity, such as diabetes mellitus, gynecological malignancy, or any malignancy associated with estrogen or progesterone receptors; patients whose endometrial biopsy results were compatible with ectopic pregnancy; infertile patients with endometriosis, hydrosalpinx were excluded. The flowchart of the study and patient selection is shown in Fig. 1.

Interpretation of the Morphology

Tissue specimens were pathology archive paraffin blocks. Hematoxylin&Eosin archive slides were reviewed by a dedicated pathologist and chosen for the research. Tissues were re-examined for endometrial dating before the immunohistochemical process.

Immunohistochemistry

Paraffin-embedded blocks were sectioned at a four µm thickness, deparaffinized in xylene, and inserted into the Ventana-XT (Roche, US) automated staining device.

The antibodies used were polyclonal rabbit anti-human against HOXA-11 (1:500 dilutions, Thermo Fischer Scientific, US), monoclonal rabbit anti-human against β1 integrin (clone: EPR16895, 1:1000 dilutions, Abcam, US), monoclonal rabbit anti-human against FAK (clone: EP69Y, 1:250 dilutions, Abcam, US), monoclonal rabbit anti-human against CD44 (clone: EPR1013Y, 1:100 dilutions, Abcam, US) and monoclonal rabbit anti-human against Extracellular Matrix Protein-1 (clone: EPR6701, 1:250 dilutions, Abcam, US). Positive controls included endometrium biopsies, colon, spleen, hepatocellular carcinoma, tonsil, and kidney.

Scoring of Markers

The immunohistochemical staining was evaluated independently by two authors without knowledge of the clinicopathological information. The immunoreactive scores of the markers were independently noted at the endometrial stroma and epithelium. The multiplication of extension and intensity determined the immunoreactivity score. The staining intensity was scored using the following scale: Negative, no staining, mild staining (+), and marked staining (++). The staining area was evaluated and recorded as a percentage: negative, less than 10% (+), and 11–100% (++). The first statistical analysis was made between the positive and negative cases. The insignificant cases statistically were re-evaluated and compared thru their intensity.

Outcome Measures

The primary outcome measure was that the positive and negative staining differed significantly from the control group. The secondary outcome measure was the difference between staining intensities.

Statistical analysis

Statistical analyses were performed using Statistical Package for Social Sciences (SPSS, version 21.0, Statistics, 2013, Chicago, IBM, USA). The variables were evaluated using visual (histograms, probability plots) and analytical methods (Kolmogorov-Smirnov test) to determine whether they are normally distributed or not. The One-way ANOVA test analyzed demographic characteristics, and Bonferroni was used as a post hoc test. Chi-square or Fisher's exact test was used to evaluate categorical data. Data were presented as mean ± standard deviation (SD). Statistical significance was defined as p < 0.05.

The demographic characteristics and history of the women included in the study are summarized in Table 1.

When the endometrial glandular β1 Integrin, FAK, CD44, and ECM1 stainings were compared to the control group, it was detected that ECM1 staining was decreased and FAK staining increased in patients with RIF. In patients with RPL, there was a significant decrease in the endometrial glandular staining of CD44. No significant difference was found in the patient groups regarding other molecules examined. The results are shown in Table 2.

When the endometrial stromal stainings for all studied markers of RIF and RPL groups were compared to the control group, it was observed that there was a decrease in CD44 staining in both RIF and RPL groups. A decrease in endometrial stromal β1 integrin levels was observed in RPL patients; no such change was detected in RPL patients. HOXA11 had nearly complete stromal staining in all groups. No statistically significant difference was found in the endometrial stromal staining in both patient groups for the other molecules in the study (Table 2).

A staining density method was used to compare further groups in which there was no difference in the staining properties for molecules studied. The intensity of staining in the stroma of ECM1 was higher in the RPL and RIF groups compared to the control group. No difference was detected for other molecules.

Stainings in the endometrium are seen in Fig. 2.

Endometrial receptivity has been studied since the Noyes criteria were established in the 1950s [14]. Unfortunately, little is known about the molecular mechanisms of embryo implantation, although much research has been done for at least 50 years [15]. With the increasing use of IVF technologies, endometrial receptivity studies have gained more importance due to the RIF in some patients. Studies that provide insight into the molecular mechanisms of endometrial receptivity in RIF and RPL patients will allow an understanding of the etiology and increase treatment options in both patient groups. It was emphasized that endometrial receptivity is a complex process involving hormonal, biochemical, and molecular mechanisms, and molecular studies play a role in understanding receptivity and achieving successful implantation [16].

Integrins are large transmembrane proteins composed of α and β subunits that mediate cell-cell and cell-extracellular matrix adhesion. Integrins bind to cytoskeletal elements, allowing the uterine epithelial cells to bind more tightly. After the binding of integrins to cytoskeletal proteins, FAK, a tyrosine kinase located at focal adhesion sites, is activated.

The results of our study on integrin and related FAK immunostaining in the RIF group showed a statistically insignificant mild increase compared to controls (100% in the RIF group and 94% in the control group). In addition, the FAK level was higher in the RIF group. These findings suggest that there may be an increase in the barrier function of the endometrium in case of recurrent implantation failure, which supports the results of a former study on rats. In this study, it was shown that increased focal adhesions could act as a barrier to implantation by making uterine endometrial cells more compact, suggesting the decreased ability of uterine epithelial cells to adhere together during implantation is necessary to allow the blastocyst to enter the underlying stroma and subsequently form a placenta. [10].

RPL is hypothetically considered to occur due to decreased selectivity in the endometrium and/or superfertilization [17]. In our study, a statistically significant decrease in β1 Integrin levels in the endometrial stroma of patients with RPL supports this hypothesis. The decrease of β1 integrin in stromal cells may prevent endometrial cells from tightly bonding, thus reducing endometrial selection for the embryo. In this case, it may result in an increased rate of early pregnancy loss as the implantation of defective ova is increased. On the other hand, there was no difference in FAK levels questioning the role of the FAK system. This may be due to the small sample size in this study.

HOXA-11 is a member of the homeobox gene family. HOX transcription genes play an essential role in implantation, modulating cell to cell and cell to extracellular matrix adhesion [18]. In our study, the change in HOXA-11 expression could not be demonstrated immunohistochemically in the endometrium. Similarly, in a study conducted with unexplained infertile patients with endometrioma, although an increase in HOXA-11 expression in eutopic endometrium was detected by PCR, immunohistochemical staining for HOXA-11 protein level was not different [19].

CD44 is a transmembrane glycoprotein that might play a role in implantation. It has been revealed that its expression is physiologically increased in the secretory phase, including the implantation window period in the endometrium [20] [21]. It has recently been reported that mid-secretory expression is decreased in patients with RIF [22]. In our study, we similarly found decreased expression of CD44 in the endometrial stroma of patients with RIF. It has been previously hypothesized that increased CD44 expression is responsible for the excessive invasion of endometriotic implants[23]. In our study, on the contrary, CD44 reduction confirms the effect of CD44 on implantation in cases where implantation decreases. It has also been known to play a role in unexplained miscarriages [24]. CD44 expression was decreased significantly in the endometrium of our patients with RPL. This finding suggests a defect in vascular invasion and placental angiogenesis in RPL cases. This might be considered a possible pathophysiological mechanism for increased abortion.

In our study, endometrial biopsies of patients with RIF were performed during the receptive midsecretory phase of the endometrium. We observed decreased ECM1 staining in the endometrial glandular tissue in RIF patients. Our data is the first that ECM1 is studied in the receptive period of the endometrium in patients with RIF. ECM1 is an extracellular matrix member glycoprotein first identified in murine osteogenic stromal cells, which acts as a bridge between cells in the skin, and its expression changes in many dermatological diseases [25]. Research in 2008 by Hannan N.J. et al. investigated maternal-fetal surface ECM1 expression change in first-trimester curettage materials, and it was found that ECM1 expression is increased at the implantation site [26]. Fitzgerald HC et al. reported a decrease in ECM1 expression by PCR in the uterine lavage fluid of unexplained infertile women; however, immunohistochemical staining did not show this decrease [11]. Endometrial samples in this study were reported to be performed in the proliferative phase and did not coincide with the implantation window. In a recent study of endometrial transcriptome of metabolic and inflammatory pathways in the implantation window period patients with polycystic ovary syndrome, differential expression of ECM1 has been reported, and ECM1 has been related to obesity [27]. Our study is also the first ECM1 study in patients with RPL. Although not statistically significant, a decrease in endometrial glandular ECM1 level was detected in patients with RPL. This can be considered one of the possible mechanisms of very early pregnancy loss, and more studies are needed.

The data of our study strongly supports the association between adhesion formation and pregnancy failures; defective implantation in RIF and RPL might also be related to altered adhesion mechanisms involved in endometrial receptivity. Changes in the expression of adhesion-related CD44, ECM1, and FAK molecules - rarely studied before and whose effects have not been elucidated- in these two groups, which progressed with implantation defects during the implantation window period, are noteworthy.

RIF and RPL are critical clinical entities of defective implantation. The importance of implantation defects is increasing with the widespread use of IVF technologies. The pathophysiology of both has yet to be thoroughly elucidated. Endometrial receptivity studies have gained momentum and hold promise for understanding the pathophysiology. In this study, the changes in the expression of HOXA11, FAK, β1 integrin, CD44, and ECM1, which are involved in adhesion, were examined in the endometrial glands and stroma in the implantation window of patients with RIF and RPL. It was suggested that they may be altered in implantation failures.

CD44

Cluster of differentiation 44

ECM1

Extracellular matrix protein 1

FAK

Focal adhesion kinase

IBM

International Business Machines

IVF

In vitro fertilization

PCR

Protein chain reaction

RIF

Recurrent implantation failure

RPL

Recurrent pregnancy loss

Standard deviation

SPSS

Statistical Package for Social Sciences

United States

USA

United States of America

β1 integrin

beta1 integrin

Acknowledgements

Not applicable.

Funding

This study is supported by Gazi University Scientific Research Projects Coordination Unit. The funds were paid for purchasing antibodies.

Ethics approval and consent to participate

The study was approved by Gazi University Ethics Committee of Clinical Studies approved the study. Approval no: 2019-129.

Consent for publication

Not applicable.

Competing interests

The authors declare that there are no competing interests.

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Authors Information

Department of Obstetrics and Gynecology, Sakarya Training and Research Hospital, Sakarya, Turkey

Sultan Canan

Department of Pathology, Faculty of Medicine, Gazi University, Ankara, Turkey
Mehmet Arda İnan, Mualla İlknur Gündüz & Özlem Erdem

Department of Obstetrics and Gynaecology, Faculty of Medicine, Gazi University, Ankara, Turkey

Ahmet Erdem, Erhan Demirdağ & Mehmet Erdem

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Table 1: Demographic Characteristics

Variant	Control (n=16)	RPL (n=16)	RIF (n=15)	p-value
Variant	Control (n=16)	RPL (n=16)	RIF (n=15)	p-value
Age (Year)	36,7 ± 3,1	34,2 ± 3,6	35,1 ± 2,4	0,07
BMI (kg/m2)	26,3 ± 3,2	24,3 ± 3,7	23,9 ± 2,7	0,21
Previous live birth	2,13 ± 0,71	0,13 ± 0,34	0,13 ± 0,35	Sig. a
Previous abortion	0,13 ± 0,34	3,00 ± 0,81	0,07 ± 0,25	Sig. b

Data are noted as mean value ± Standard Deviation. RPL: Recurrent Pregnancy Loss RIF: Recurrent Implantation Failure

a: Control between RPL: p<0,01 Control between RIF: p<0,01 RIF between RPL: p=1,00

b: Control between RPL: p<0,01 Control between RIF: p=1,00 RPL between RIF: p<0,01

Table 2: Changes in the staining of endometrial β1 Integrin, FAK, HOXA-11, CD44, and ECM1 in the RPL and RIF groups compared to the control group.

Molecule	RPL	RIF
Stromal β1 Integrin	Decreased	No difference
	(p=0,03)	(p=0,43)
Epithelial β1 Integrin	No difference	No difference
Epithelial β1 Integrin	(p=0,50)	(p=0,52)
Stromal FAK	No difference (p=0,11)	No difference (p=0.52)
Epithelial FAK	No difference	Increased
Epithelial FAK	(p=0,11)	(p<0,01)
Stromal HOXA-11	(p=0,50)	(p*)
Stromal CD44	Decreased (p<0,01)	Decreased (p=0,02)
Epithelial CD44	Decreased	No difference
Epithelial CD44	(p=0,04)	(p=0,55)
Stromal ECM1	No difference	No difference
Stromal ECM1	(p=0,30)	(p=0,12)
Epithelial ECM1	No difference	Decreased
Epithelial ECM1	(p=0,14)	(p<0,01)

RPL: Recurrent Pregnancy Loss RIF: Recurrent Implantation Failure FAK: Focal Adhesion Kinase ECM1: Extracellular Matrix Protein 1 CD44: Cluster of Differentiation 44

*: Analysis was not possible as both groups showed 100% staining.

No competing interests reported.

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Evaluation of Endometrial Receptivity in Patients with Recurrent Pregnancy Loss and Recurrent Implantation Failure

Status:

Version 1

Abstract

Figures

Background

Material Method

Data Collection and Patient Selection

Interpretation of the Morphology

Immunohistochemistry

Scoring of Markers

Outcome Measures

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

Acknowledgements

Funding

Ethics approval and consent to participate

Consent for publication

Competing interests

Authors Information

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1