Despite one year of unprotected intercourse of a woman with normal ovulation and tubal functions and a man with normal semen analysis the absence of conception is called unexplained infertility [1]. In the light of this definition, we should consider that mechanisms other than tubal, ovulatory and semen parameters are responsible for the etiology of unexplained infertility (UEI) [2]. However, the range of data that we can ascribe to etiology is so wide that we have to include many disorders involving edocrinological, immunological, genetic and reproductive physiology [3]. This is one of the main factors that make it difficult to find a solution to the problem. Interestingly, the endometrium, which has a critical importance in embryo implantation, has not been adequately studied in UEI cases [2, 4]. One of the main reasons for this is that we do not have a non-invasive method other than ultrasonography (USG) to evaluate the endometrium [5]. On the other hand, endometrium evaluation with USG is a method with low sensitivity is about 30% [6]. We have very little data about what kind of changes occur in histo-morphological and molecular levels in the endometrium of UEI cases. The fact that endometrial sampling is an invasive method is the main reason for very few studies on this subject.

Failure of implantation despite healthy embryo transfer in UEI cases undergoing fertilization - embryo transfer (IVF-ET) generated the hypothesis that there may be a defect in the genes or growth factors that regulate endometrial receptivity. In addition to the presence of a decidua resulting from differentiation of endometrial stromal cells, adequate expression of steroid hormones and their receptors, cytokine and receptivity genes is required for successful implantation [7, 8]. The bone morphogenetic protein 2 (BMP-2) is one of the critical growth factors involved in embryo implantation. It belongs to the transforming growth factor-beta (TGF-$\beta$) superfamily and plays a role in endometrial proliferation and differentiation as well as embryo navigation [9, 10]. BMP-2 expression in endometrial cell is regulated by progesterone and critical for implantation and decidualization. Gradual expression of BMP-2 and its receptor in the endometrial stromal cells during early pregnancy indicates a possible link between BMP-2 expression and stromal cells differentiation [9, 10]. In experimental models with BMP-2 ablation (BMP-2${}^{d/d}$), both the implantation zone and decidua formation are almost completely blocked, and the animal becomes absolutely infertile [9]. In literature review, we could not find a study on endometrial BMP-2 levels of UEI patients who underwent IVF-ET. The aim of this study is to measure BMP-2 levels in endometrial samples of patients scheduled for IVF-ET with a diagnosis of UEI.

The demographic and laboratory findings of the patients are shown in Table 1. The patients in the UEI group and control group were found to be similar in terms of age and body mass index values. Serum total testosterone, LH (luteinizing hormone) and estradiol levels of patients in the control group were significantly higher than the UEI group. similarly, fasting insulin, glucose and HOMA-IR levels of the control group were found to be significantly higher than the UEI group. The number of total oocytes, MII oocytes and 2PN embryos in the control group were significantly higher than in the UEI group. The probable reason for the difference between the two groups in metabolic and hormonal markers may be that the patients in the control group were selected from PCOS patients.

The mean BMP-2 level was significantly lower in the endometrium of women with UEI compared to control cases. BMP-2 levels in the endometrial supernatants of UEI patients were found to be about 2 times lower than the patients in the control group (984.5 (350) pg/mg wet tissue vs. 1720 (318) pg/mg wet tissue, p 0.000, Z = –6.6). A strong and significant positive correlation was found between endometrial thickness, estradiol levels measured on hCG day and BMP-2 levels. A positive and significant correlation was found between BMP-2 levels and serum LH levels in the UEI group. A positive and significant correlation was found between endometrial BMP2 levels and fasting insulin, glucose, testosterone, HOMA-IR and mature oocyte counts. There was no significant correlation between other hormonal and reproductive parameters and BMP-2 levels in the UEI and control groups (Table 2).

The main result we found in our study is a significantly lower BMP-2 levels in the endometrium of UEI patients. Decreased endometrial BMP-2 levels in UEI cases without tubal, ovulatory and male factor infertility may be one of the underlying causes of subfertility due to UEI. In order for this idea to be better understood, we need to explain the role of BMP-2 in implantation in more detail. BMP-2 is an essential growth factor that is released from endometrial stromal cells (ESC) and contributes to decidualization by acting paracrine on the same cell [9, 10, 12]. ESC cells contain BMP membrane receptors (BMPRs) type I and type II. BMP-2 phosphorylates smad-1 in the cytoplasm by attaching to both receptors in the cell membrane [8, 13]. Smad-1p then interacts with smad-4 to form smad-4p. Smad-4p enters the nucleus of the ESC and initiates implantation by increasing the expression of homeobox A10 (HOXA 10) and leukemia inhibitory factor (LIF), the main genes involved in decidualization [7, 13]. When compared with the patients in the control group, we found 2-fold decreased endometrial bmp-2 levels in patients in the UEI group. This significant decrease in BMP-2 levels may lead to insufficient decidualization and negatively affect the implantation of the transferred embryos. Sinclair et al. [13] reported that in the case of decreased BMP-2 levels or resistance to BMP-2, HOXA 10 and LIF gene expression decreased. Although receptivity genes were not evaluated in our study, we can argue that the significant reduction in BMP-2 may contribute to UEI-related subfertility by disrupting receptivity gene expression and subsequent decidualization [12].

Another critical finding we obtained in our study is the positive correlation between endometrial BMP-2 levels and LH, estradiol and endometrial thickness. This finding is important in that it supports the close relationship of BMP-2 synthesis and release with estrogen synthesis. Estrogen is critical for establishment of blastocyst attachment, implantation, and proliferation [14]. The lack of difference between the UEI and the control group in terms of endometrial thickness and estradiol levels on the day of hCG suggests that the decrease in BMP-2 levels in UEI cases occurs by a different mechanism. An example of this idea is that endometrial stromal cells of patients with myoma secrete less BMP-2 than those without fibroids. Similarly, myomas secrete TGF-$\beta$3 and block BMP-2 from acting through ESC [13]. When these findings and our findings are evaluated together, significantly lower endometrial BMP-2 levels in UEI cases may be due to the presence of a neglected small myoma, intrauterine adhesions, endometrial polyp or peritoneal or ovarian endometriosis/endometrioma rather than hormonal factors [15, 16]. For this reason, we recommend that ovaries, fallopian tubes, endometrium and myometrium should be evaluated in detail, as well as HSG, semen analysis and ovulatory evaluation in patients with UEI with recurrent implantation failure. Lower BMP-2 levels in UEI cases may indicate the presence of a non-clinical pathology located in the intrauterine cavity or cervical canal. Similar to the decrease in BMP-2 secretion in the presence of fibroids [13], lesions located in the cavity or in the cervical canal may impair BMP-2 secretion or the interaction between BMP-2 and endometrial stromal cells. Hysteroscopic evaluation of UEI cases may be important in this respect. Chiofaloa et al. [17] reported that office hysteroscopy is a highly effective diagnostic and therapeutic intervention for the management of cervico-isthmic and intrauterine adhesions, as well as for small polyps. Further, surgical removal of these lesions detected in hysteroscopy may contribute positively to receptivity [15, 16]. Unlu et al. [16] reported that myomectomy has a favorable effect on endometrial home box gene expression. Celik et al. [15] reported that surgical removal of endometrioma improves endometrial receptivity suggesting that there is a functional link between reptoductive tractus lesions and endometirum receptivity. When our findings and literature data are evaluated together, we can conclude that decreased BMP-2 levels in UEI cases may coexist with other pathologies [13, 15, 16]. Surgical treatment of these pathologies may lead to improvement in endometrial receptivity in UEI cases, but this idea needs to be confirmed by further studies.

We do not have sufficient data on how metabolic parameters affect receptivity in UEI cases. Since our control group patients were selected among those with PCOS, we evaluated serum insulin, testosterone and HOMA-IR in both groups. High testosterone, insulin and HOMA-IR values we found in control group may cause subfertility directly or indirectly [18]. The positive correlation between BMP-2 and HOMA-IR, testosterone and fasting insulin levels suggests that metabolic parameters may affect endometrial receptivity in UEI cases. For this reason, we can say that the synthesis and release of BMP-2 from endometrial stromal cells can be affected by the metabolic parameters in women with UEI. This data is important in terms of providing clues that the subfertility-causing effects of metabolic diseases such as PCOS are through BMP-2 [19]. Similarly, disturbed metabolic parameters may have an effect on BMP-2 release and leading to subfertility in UEI patients. However, although there was no significant difference between estrogen and progesterone levels measured on hCG day between the two groups in our study, the lower BMP-2 levels in the UEI group suggests that BMP-2 was regulated differently in this patient group. This paradoxical situation needs to be clarified because it is a known fact that both estrogen and progesterone stimulate synthesis and release of receptivity molecules [18].

Expression levels of BMP-2, one of the most important regulators of the endometrium at the implantation stage, were significantly lower in UEI cases. Endometrial BMP-2 is associated with UEI and mechanistically it may be useful to study this further to determine is this is causal or merely a biomarker. Finding the underlying reason for the significanlty lower endometrial BMP-2 levels and arranging treatment accordingly may improve reproductive outcome in women with UEI.

AY designed the research study and wrote the manuscript. NDG and TG performed the research and analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Ethical approval was obtained from the local Ethics Committee of the Beykoz University (2020/4), informed consent was obtained from all participants at the time of enrollment.

We thank participants, anonymous reviewers for excellent criticism of the article.

This research received no external funding.

The authors declare no conflict of interest.