Comparison of Endometrial Receptivity Markers Between Women with Polycystic Ovary Syndrome, Endometrioma and Unexplained Subfertility: A Cross-Sectional Study

Endometrial receptivity; endometrioma; insulin-like growth factor binding protein 1; osteopontin; prostaglandin E2 DOI: 10.5336/jcog.2022-89394 Correspondence: Emine DEMİR Deparment of Gynecology and Obstetrics, İzmir Katip Çelebi University, Atatürk Training and Research Hospital, İzmir, Türkiye E-mail: [email protected] Peer review under responsibility of Journal of Clinical Obstetrics & Gynecology. Re ce i ved: 02 Mar 2022 Received in revised form: 10 May 2022 Ac cep ted: 26 Jun 2022 Available online: 05 Jul 2022 2619-9467 / Copyright © 2022 by Türkiye Klinikleri. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Turkiye Klinikleri Journal of Internal Medicine Journal of Clinical Obstetrics & Gynecology ORIGINAL RESEARCH 78 increase in IGFBP-1 expression in endometrial stro- mal cells throughout decidualization may increase pregnancy rates in both spontaneous and assisted re- productive techniques. 9-11 Osteopontin (OPN) is located in the en- dometrium of fertile women with normal menstrual cycles, with maximum release throughout the im- plantation window. 12,13 Hence, it has been claimed that OPN is an effective marker of endometrial im- plantation and, together with its receptor αvβ3 inte- grin, promotes embryonic adhesion to the uterine epithelium. 13-18 Also, Wang, et al. showed that the OPN level was significantly suppressed in the group that failed in vitro fertilization (IVF) cycles and so established that OPN plays a role in success of IVF techniques. 19 Prostaglandins are lipid compounds that are also contained in the endometrium, of which the impor- tance in female fertility has been emphasized in the evidence up to now. Among the prostaglandins, prostaglandin E2 (PGE2) is thought to have a signif- icant role especially for decidualization and implan- tation of pregnancy. The levels of PGE2 in endometrial fluid have been implicated as a marker for endometrial receptivity. 20 In addition, elevated amounts of PGE2 were detected in polycystic ovarian cells compared to women with normal ovulation. 21,22 The purpose of the present study was to investi- gate the concentrations of IGFBP-1, OPN and PGE2 in endometrial flushing fluids of patients with ovula- tory polycystic ovarian syndrome (PCOS), en- dometrioma, unexplained subfertility and healthy fertile women.

This cross-sectional controlled study was carried out between January and June 2013 in Subfertility Unit of the Department of Obstetrics and Gynecology, İzmir Katip Çelebi University, Atatürk Training and Re- search Hospital İzmir, Türkiye. The unit is a tertiary center that treats referral patients from the region. The study design was in accordance with the ethical stan- dards of the Helsinki Declaration and good clinical practice, and was approved by the Institutional Review Board of İzmir Katip Çelebi University, School of Medicine, Atatürk Training and Research Hospital (date: 17.05.2012, no: 25). Detailed information was conveyed to all volunteers regarding the research and both verbal and written informed consent were taken. A total of 112 patients ranging between the ages 20 to 40 who referred to the subfertility outpatient clinic were included in the study. Voluntary patients diagnosed with PCOS (n=38), endometrioma (n=19) and unexplained subfertility (n=27) constituted the study groups, while the control group consisted of fertile women (n=28). In these 4 groups, 14 patients with PCOS, 2 patients with endometrioma, 2 patients diagnosed with unexplained subfertility and 10 pa- tients in the control group who were detected as hav- ing anovulation with blood progesterone levels on the 21 st day of menstruation were excluded from the study. The control group comprised of healthy women with no gynecologic disorder, not using an intrauterine device or hormonal contraception, or not receiving any medication that may affect en- dometrium and who had the intellectual capacity to give written informed consent and to understand the information concerning the study. Exclusion criteria for the volunteers were having a pregnancy, smok- ing, pelvic infection, a serum progesterone level of <3 ng/dL in the luteal phase or endometrial pathol- ogy (submucosal myoma, endometrial polyp etc.) during the endometrial fluid sampling or a patient’s reluctance to be included. A total of 24 patients who showed ovulatory phenotype and were diagnosed with PCOS accord- ing to the ESHRE Rotterdam 2003 criteria; a total of 17 patients who were diagnosed with endometrioma by clinical history, physical examination and transvaginal ultrasonography (Medison Sono Ace X8 Seoul, South Korea); and a total of 25 patients diag- nosed with unexplained subfertility after undergoing basic infertility evaluation which were performed ac- cording to American College of Obstetricians and Gynecologists diagnostic criteria were included. The control group consisted of a total of 18 healthy ovu- latory, parous women who had no history of subfer- tility and who were using the barrier contraception method. Emine DEMİR et al. JCOG. 2022;32(3):77-84 78 797979 In the study and control groups, after confirma- tion of ovulation with the blood progesterone levels on the 21 st day of menstruation, 0.154 mol/L sodium chloride was administered via a thin cannula (2 mL per administration into the uterine cavity, a total of 10 mL following fluid collection sampling performed 5 times in total) resembling the saline infusion sonog- raphy administration technique, 1 mL of uterine as- pirate was transferred to a standard micro test tube (Eppendorf, Hamburg, Germany), was frozen at - 20°C and finally stored at -80°C until biochemical analysis. After the endometrial fluid samples were ac- cumulated from study and control groups, IGFBP-1, OPN and PGE2 levels were analyzed by using East biopharm branded (Hangzhou East biopharm Co., Ltd./China) Elisa kits (PGE2 LOT: 20130924, OPN LOT: 20130924, IGFBP-1 LOT: 20130924, PGE2 Cat. No: CK-E10702, OPN Cat. No: CK-E10857, IGFBP-1 Cat. No: CK-E10159) with the Biotec branded Elisa device. The patients were warned about not having sexual intercourse until the endometrial fluid is obtained in that menstrual cycle. Patients were kept under observation for half an hour after the en- dometrial fluid sampling. The statistical analysis was performed using SPSS (version15.0, 2006; SPSS Inc., Chicago, IL, USA) program. Shapiro-Wilks and Levene tests were carried out to control the distribution of the data. Be- cause of the fundamental hypothesis of parametric statistics was not met, use of non-parametric tests was considered appropriate instead of parametric MANOVA. Therefore, Kruskal-Wallis tests were conducted for 2 variables, 4 groups were tested in the same hypothesis and paired comparisons were per- formed via Mann-Whitney U tests as follow-up tests between the groups in the event of statistically sig- nificant differences. Data analysis was considered significant when p values were less than 0.05.

Twenty eight (25%) of 112 women included in the study were excluded from the study in both study and control groups due to anovulation. This cross-sec- tional, controlled study consisted of 24 patients with ovulatory PCOS, 25 patients with unexplained sub- fertility, 17 patients with ovulatory endometrioma and 18 healthy fertile ovulatory women out of 84 vol- unteers. The demographic data and serum progesterone levels of the patients were shown in Table 1. While there was no statistically significant difference be- tween the endometrioma and control groups, there was a statistically significant difference in terms of age only between unexplained subfertility and en- dometrioma groups in all other pairwise comparisons. The mean body mass index (BMI) was highest in the PCOS group (28.67) and lowest in the endometrioma group (24.11), while it was similar in the unexplained infertility (24.25) and control (25.16) groups. In ad- dition, there was no statistically significant difference in terms of BMI when the 3 groups were compared with the control group and all 2-group comparisons. All groups were similar in terms of demographically, except for gravidity and parity. Statistically signifi- cant differences were detected between fertile group and each one of study groups as expected. The mean serum progesterone level at mid-luteal phase was highest in the unexplained subfertility group (10.38) and was similar in PCOS (9.98) and control groups (8.19), while it was statistically significantly lower in the endometrioma group (5.95) when compared to these other three groups. Emine DEMİR et al. JCOG. 2022;32(3):77-84 79 PCOS (n=24) US (n=25) End (n=17) Control (n=18) p-value* Age (year) 29.87±5.61 29.05±4.81 33.05±6.68 33.55±5.90 0.013 BMI (kg/m 2) 28.67±7.93 24.25±3.61 24.11±3.56 25.16±2.68 0.209 Gravida (n) 0.95± 1.04 0.48±0.96 1.02±1.28 3.66±2.08 0.000 Parite (n) 0.70±0.95 0.20±0.50 0.88±1.05 2.77±1.59 0.000 Progesteron (ng/mL) 9.98±4.61 10.38±5.51 5.95 ±3.32 8.19±3.92 0.006 TABLE 1: Evaluation of demographic and baseline data of the groups. Data are presented as mean±standard deviation, *Kruskal Wallis test, BMI: Body Mass Index; PCOS: Polycystic ovary syndrome; US: Unexplained subfertility; End: Endometrioma. Mean levels of IGFBP-1 (ng/mL) of endome- trial flushing fluid were 396.5, 310.2, 391.1 and 377.3 for the unexplained subfertility, PCOS, en- dometrioma and control groups, respectively ( Table 2). In addition, as seen in Table 3, there was no sta- tistically significant difference in all pairwise com- parisons when IGFBP-1 levels were compared between paired groups. Mean PGE2 (ng/mL) levels in the endometrial fluid were similar for endometri- oma (259.16), unexplained subfertility (292.6) and control groups (239.2). But, this marker was notably higher in the PCOS (367.7) patients relative to the endometrioma (p<0.05) and control groups (p<0.001). On the contrary, mean OPN levels (ng/mL) in endometrioma (16.67), ovulatory PCOS (12.09), unexplained subfertility (13.03) and control groups (10.04) were similar. No statistically signifi- cant difference was found in pairwise comparisons between all groups either.

This prospective study was conducted to evaluate the amounts of IGFBP-1, OPN and PGE2 in the uterine washing fluids of patients with ovulatory PCOS, en- dometrioma, unexplained subfertility and fertile women during implantation window. According to the findings of the present study, the levels of PGE2 were greater in ovulatory PCOS patients compared to the control group. Midluteal PGE2 expression was also found to be higher in patients with endometri- oma and unexplained infertility group compared to the control group, but the difference was not statisti- cally significant. It was determined that there was a similarity in IGFBP-1 and OPN values in both the pa- tient and control groups. The dysregulated expression of uterine receptiv- ity markers in women with PCOS has been addressed in most of the available evidence. In our study, mid- luteal PGE2 amount was greater in the ovulatory PCOS group than in the control group. Even though ovulatory dysfunction in PCOS appears to be the main reason for subfertility, following the ovulation induction, the weak link between ovulation and preg- nancy and low pregnancy rates despite providing ovulation are important indicators with regard to en- dometrial dysfunction. Recently, in PCOS as well as in other gynecological diseases which may influence fertility, endometrial receptivity studies concentrate on endometrial receptivity markers. Meaningful ele- vation of PGE2 levels has been noted in polycystic ovaries and this was parallel to our finding. 21,22 Fur- thermore, it has been pointed out that amount of PGE2 in the uterine endometrial fluid may be a po- tential endometrial receptivity marker. 20 Data ob- Emine DEMİR et al. JCOG. 2022;32(3):77-84 80 PCOS (n=24) US (n=25) End (n=17) Control (n=18) p-value* IGFBP-1, (ng/mL) 310.22±70.76 396.51±130.55 391.18±118.86 377.36±123.10 0.028 PGE2,(ng/mL) 367.75±96.37 292.68±123.42 259.16±117.80 239.25±106.97 0.003 OPN, (ng/mL) 12.09±7.72 13.03±9.61 16.67±6.27 10.04±4.74 0.029 TABLE 2: The distribution of IGFBP-1, PGE2 and OPN value according to the group. Data are presented as mean±standard deviation; *Kruskal Wallis test; IGFBP-1: Insulin-like growth factor binding protein 1; PGE2: Prostoglandin E2; OPN: Osteopontin; PCOS: Polycystic ovary syndrome; US: Unexplained subfertility; End: Endometrioma. PCOS vs control US vs control End. vs control PCOS vs US PCOS vs end US vs end IGFBP-1 (ng/mL) 0.349 1.000 1.000 0.055 0.152 1.000 PGE2 (ng/mL) 0.002 0.769 1.000 0.133 0.017 1.000 OPN (ng/mL) 1.000 1.000 0.068 1.000 0.356 0.794 TABLE 3: The comparison of IGFBP-1, PGE2 and OPN values between the 2 groups. *Mann-Whitney U test; IGFBP-1: Insulin-like growth factor binding protein 1; PGE2: Prostoglandin E2; OPN: Osteopontin; PCOS: Polycystic ovary syndrome; US: Unexplained sub- fertility; End: Endometrioma. Polycystic ovary syndrome; US: Unexplained subfertility; End: Endometrioma. tained in the present study showed similar findings. Increased PGE2 in PCOS is coupled with suppressed a propensity to apoptosis which plays a vital role with a delicate cell balance between proliferation and dif- ferentiation. It has been shown in both genital system cancers and the endometrial cells also. 23,24 It is known that cells overexpressing cyclooxygenase-2 have in- ability to increase proliferation and the ability to downregulate apoptotic processes. Besides the un- derlying of physiopathology that appears to be the re- sistance of endometrial cells to undergo programmed cell death, PGE2 may also contribute to endometrial dysfunction through its effects on cell proliferation, angiogenesis and immunosuppression by affecting the estrogen levels at the receptor level. Although the distribution of IGFBP-1 in our PCOS patients with ovulatory phenotype was lower than in the control group, the difference was statisti- cally insignificant. Low levels of IGFBP-1 have been found in PCOS and obesity, but the results of studies on this subject in the literature are contradictory. It has been mentioned that decreased amount of IGFBP-1 in PCOS patients may be related to BMI rather than ovarian hyperandrogenism. 25 Taking into account that the BMI of the ovulatory PCOS group was 28.6 kg/m 2 in our study, it may clarify the low IGFBP-1 levels although it is not statistically signif- icant. In addition, considering the trend of the data in the current study, the low expression of IGFBP-1 may gain statistical significance by increasing the number of cases. OPN levels were reported to be sim- ilar in patients with the ovulatory PCOS phenotype compared to the controls; in a recent study. 26 Impor- tant reduction of OPN levels was observed in infertile women with isolated PCO. But, ovulatory dysfunc- tion was the main factor for subfertility in this study. Of course, conflicting results affect the comparability of these data with our study. In the current study, the amounts of 3 markers evaluated in the midluteal phase of patients with en- dometriosis were found to be similar to the normal control group. Genetic factors are known to be asso- ciated with the development and progression of en- dometriosis, but endometriosis-associated genes have not been described. IGFBPs are thought to have major effects in cell apoptosis, proliferation and pathophysiology of endometriosis. It has been stated that IGFBP-1 is not associated with endometriosis, but IGFBP-3 has a significant relationship with en- dometriosis. 27 αvβ3 integrin and its extracellular ma- trix ligand OPN are involved in the regulation of endometrial receptivity. While OPN expression was unaffected in patients with endometriosis, αvβ3 inte- grin expression was shown to be decreased. On the other hand, OPN binding to the surface epithelium is so limited when αvβ3 expression is missing. This in- formation indicates that the endometrium of some women with endometriosis is dysfunctional and is re- sponsible for decreased fertilization. 28 In the current study, making the diagnosis of the endometrioma group by history, physical examination and transvagi- nal sonography imaging may be the reason for par- tially inconsistent data. In addition, the relative increase in OPN and IGFBP-1 levels and the relative low expression of PGE2 may negatively affect em- bryo implantation in endometriosis by causing both apoptosis inhibition and immune compromise. Diagnosis of unexplained subfertility made by exclusion in many guidelines, ovulation is diagnosed by excluding the male and tuboperitoneal factors. However, high prevalence of this diagnosis in all in- fertile couples and the perception that there is no treatment terminologically lead to serious perceptual problems. Nevertheless, many associated issues for infertility may go unnoticed with basic infertility re- search. Endometrial dysfunction in unexplained sub- fertility has been ignored until recently. In this study, the levels of all 3 markers in the midluteal phase in the unexplained subfertility group were found to be similar to the control group. In the literature, studies investigating endometrial dysfunction during the im- plantation window in patients with unexplained in- fertility are very limited. OPN and its receptor αvβ3 integrin, recently proposed as an important complex in embryo implantation, may be useful as endome- trial receptivity markers in a variety of infertility states. 13 In the last 20 years, an extremely consider- able rise was observed in genomic studies and an un- predictable amount of data was collected. A total of 1,453 gene pairs that have been identified are kept re- sponsible for implantation and nearly 200 of them Emine DEMİR et al. JCOG. 2022;32(3):77-84 81 have quite important functions. Whereas, this is not sufficient to explain the mechanism of implantation of a single marker expressed by each gene, because implantation has a complex physiopathology, and a decrease in a protein that a gene expresses is com- pensated by an increase in a protein that another gene expresses. 29 The strengths of our study were that diseases scarcely included in the literature were selected, the number of subjects were sufficient, the biomarkers studied were diverse and the subgroups were in- cluded in the analysis. But, the limitations of the pre- sent study were that the diagnosis of endometrioma was made by imaging methods, the control group consisted of random advanced age fertile women due to sequential collection, and fewer biomarkers were included in the study due to the limitation in gene ex- pression. Another limitation of this study was the lack of the power analysis. Therefore; although PGE2 lev- els were higher in patients with endometrioma as well as unexplained subfertility compared to the normal control group, the difference was not statistically sig- nificant.

Consequently, in the literature, endometriosis, un- explained subfertility and PCOS may be associated to a decreased fertility cycle and impaired en- dometrium receptivity. According to our results, PGE2 may be an indicator of poor endometrial re- ceptivity, which may be responsible for low preg- nancy rates in patients with ovulatory PCOS. A single marker is not satisfactory to explain the mechanism of implantation as well as many markers play role in endometrial receptivity. For this reason, there is a need for more comprehensive studies with a large number of markers in more different female infertility issues. Acknowledgments The authors thank all the women whose participation made this study possible. Source of Finance During this study, no financial or spiritual support was received neither from any pharmaceutical company that has a direct con- nection with the research subject, nor from a company that pro- vides or produces medical instruments and materials which may negatively affect the evaluation process of this study. Conflict of Interest No conflicts of interest between the authors and / or family mem- bers of the scientific and medical committee members or mem- bers of the potential conflicts of interest, counseling, expertise, working conditions, share holding and similar situations in any firm. Authorship Contributions Idea/Concept: Emine Demir, Fulya Oğuz Türkyılmaz, Sefa Kelekçi; Design: Emine Demir, Fulya Oğuz Türkyılmaz, Sefa Kelekçi; Con- trol/Supervision: Emine Demir, Fulya Oğuz Türkyılmaz, Mustafa Şengül, Sefa Kelekçi; Data Collection and/or Processing: Emine Demir, Fulya Oğuz Türkyılmaz, Sefa Kelekçi; Analysis and/or In- terpretation:Emine Demir, Fulya Oğuz Türkyılmaz, Mustafa Şengül, Sefa Kelekçi; Literature Review: Emine Demir, Fulya O ğuz Türkyılmaz, Mustafa Şengül, Sefa Kelekçi; Writing the Article: Emine Demir, Fulya Oğuz Türkyılmaz, Mustafa Şengül, Sefa Kelekçi; Critical Review: Emine Demir, Fulya O ğuz Türky ılmaz, Sefa Kelekçi; References and Fundings: İzmir Katip Çelebi University;

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