Altered Expression of Interleukin-6, Glycodelin A and Heparin-Binding Epidermal Growth Factor, in The Endometrium of Women with Hydrosalpinx: A Case-Control Study.

Tubal infertility accounts for approximately 25 to 35% of female infertility cases. Hydrosalpinx is a severe form of tubal disease characterized by occlusion of the distal segment of the fallopian tube, leading to fluid aggregation ( 1 ). The occurrence of a hydrosalpinx correlated with a 50% reduction in embryo implantation and clinical pregnancy rate, as well as double rates of spontaneous abortion and miscarriage ( 2 , 3 ). Hydrosalpinx management through salpingectomy, tubal occlusion or Aspiration of hydrosalpinx improved pregnancy success rates during in vitro fertilization (IVF) ( 4 ). Research on hydrosalpinx fluid (HF) components reveals a mixture of protein and bicarbonate with concentrations lower than those in serum. Additionally, lymphocyte debris, cytokines, prostaglandins, leukotrienes, and catecholamines have been identified in HF ( 5 ). The precise mechanism of hydrosalpinx on endometrial receptivity (ER) and embryo implantation is still elucidate. It was suggested that HF may have a flushing effect on the embryo and disrupt the contact between the blastocyst and endometrial surface, hence prevented its attachment. Another theory proposed that HF may diminish sperm motility, have toxic effects on gametes/embryos, and alter the expression of several genes and proteins ( 6 - 8 ), such as cytokines, transcription factors, growth factors, cell adhesion molecules (CAMs), glycoproteins, pinopode structures, and other factors which is crucial for successful embryo implantation. It could affect the receptive endometrium's characteristics and avert embryo implantation ( 7 , 9 - 11 ). Some of these genes have been investigated in ER and embryo implantation. Interleukin-6 (IL-6) is a multifunctional cytokine that has both pro and anti-inflammatory roles in different tissues, such as the uterine endometrium ( 8 ). IL-6 is necessary for embryo implantation, placental growth, the immune system's adaptation for pregnancy tolerance, and the body’s inflammatory response ( 10 ). Heparin-binding epidermal growth factor (HB-EGF) is one of the steroid hormones depending on members of the EGF family, which participates in various activities such as pinopode formation, cell adhesion, decidualization, and embryo implantation ( 12 , 13 ). Glycodelin A (GdA) is one of the main progesteroneregulated glycoproteins secreted into the uterine luminal cavity by secretory/decasualized endometrial cells ( 14 ) GdA participates in pinopodes formation ( 13 ) and its concentration significantly decreased in women who were suffering from early pregnancy loss and growth retardation of the endometrium ( 15 , 16 ). Implantation is a physiological inflammatory process during a specific timeframe known as the window of implantation (WOI). Various inflammatory factors like cytokines and immune cells regulate this process. Any disruptions in the cytokine network and the balance between them can impact ER and embryo implantation ( 17 ). Therefore as a pathological inflammatory condition, hydrosalpinx, can alter the ER by affecting the inflammatory response in the endometrium ( 18 ). These changes may include IL-6 , GdA , and HB-EHG , that previously well-known in ER and embryo implantation. In this current investigation, we have examined the altered expression of the mentioned genes in the endometrial tissue of hydrosalpinx patients. We aim to compare these findings with a control group, to gain insight into the effect of HF on the ER and embryo implantation.

The demographic information is summarized in Table 2. There were no significant statistical differences between the hydrosalpinx group and fertile women in terms of age, BMI, and hormonal profile. All the hydrosalpinx patients were sono visible, and hydrosalpinx size was >10 mm. Demographic characteristics of the participants (n=30) BMI; Body mass index, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, AMH; Anti-Müllerian hormone, PRL; Prolactin, TSH; Thyroid-stimulating hormone, IQR; Interquartile range, a ; Mann-Whitney test applied for quantitative variables with non-normal distribution, and b ; t test was applied for the quantitative variable with normal distribution. P<0.05 is accepted as statistically significant. The mRNA expression level of IL-6 in the endometrium of the case group was significantly higher (a 3.52-fold increase) than the control group (P=0.042, Fig .2A ). As shown in Figure 2B, the mRNA expression of HB-EGF in the endometrium tissue of the case group was significantly lower (fold change=3.79) compared in the control group (P=0.007). In Figure 2C, the mRNA expression of GdA in the endometrium tissue of the case group was lower than in the control group (a fold decrease =2), but this reduction was not statistically significant (P=0.849). Comparison of endometrial mRNA expression levels of IL-6, HB-EGF , and GdA genes in patients with hydrosalpinx and fertile controls. A. IL-6 , B. HB-EGF , and C. Glycodelin A (GdA) . The values are provided as mean ± SEM. *; P<0.05, **; P<0.001, and ns; Not significant.

The current study evaluates the altered mRNA expression levels of IL-6, GdA , and HB-EGF genes observed in the endometrial tissue of the hydrosalpinx group compared to the fertile subjects. Previous research findings indicate an alteration in the expression of receptivity genes in endometrium when exposed to hydrosalpinx fluid, along with a rise in proinflammatory cytokines ( 8 , 18 - 23 ). Although the exact mechanism of HF remains unspecified, its effects on ER and embryo implantation after IVFembryo transfer were well-demonstrated ( 2 , 24 ). Our findings reveal that the mRNA expression level of the IL-6 gene was dramatically elevated in the hydrosalpinx group compared to the control group. This data is consistent with several previous studies ( 9 , 21 , 25 , 26 ). Incognito et al. ( 25 ) found a correlation between serum and peritoneal fluid IL-6 elevation in endometriosis-infertile patients. Similarly, Hadi et al. ( 26 ) reported a significant elevation in the concentration of IL-6 in ectopic pregnancy (EP) patients. In addition, several studies have revealed a significant increase in the expression pattern of IL-6 in PCOS women ( 27 - 29 ). The results of a more recent study showed that the JAK/STAT signaling pathway mediated by IL‐6 may contribute to the pathogenesis of hydrosalpinx ( 21 ), Deng et al. ( 21 ) proposed that the excessive activation of IL-6 cytokines results in the induction of STAT3 , which essential in causing an inflammatory condition in the fallopian tubes. Various inflammatory markers such as TNF-α, IL-1, IL-17, IL-7 , and NF-kappa B have been investigated in endometrium exposed to hydrosalpinx fluid, and the upregulation of these genes has been reported in these women ( 6 , 8 , 22 ). Despite we did not measure NF-kappa B gene expression in this study, prior research has demonstrated that NF-kappa B is the key element in activating of inflammatory pathways in hydrosalpinx and other inflammatory conditions such as endometriosis ( 6 , 30 ). NF-kB stimulates IL-6 production and transforms macrophages from the M0 to the M1 subtype, this process amplifies the secretion of pro-inflammatory cytokines and disrupts the balance of the cytokine network, leading to ER defects and implantation failure ( 6 ). In addition, we investigated HB-EGF mRNA levels in the endometrium of hydrosalpinx patients for the first time. Our findings demonstrated a significant decrease in the endometrial HB-EGF expression level in patients with communicating hydrosalpinx compared to controls. The results of two recent studies on the alteration of HB-EGF in patients with ectopic pregnancy (EP) revealed a significant upregulation in the expression level of HB-EGF in the implantation site of the embryo in the ampullary segment of the fallopian tube, in contrast, no statistical differences were observed in endometrial samples of these cases ( 31 , 32 ). These data highlight the significant role of HB-EGF in embryo implantation. In contrast, Broi et al. ( 33 ) found no remarkable changes in the endometrial expression of HB-EGF during the implantation window in patients diagnosed with endometriosis. Rarani et al. ( 11 ) noted that HB-EGF is associated with developing pinopodes and facilitates blastocyst attachment. The most prominent role of HB-EGF is communicating in the initial cross-talk between the uterine epithelium and the blastocyst, decidualization, and trophoblast invasion ( 34 ). HB-EGF is expressed in response to steroids in the uterine stroma and facilitates blastocyst attachment by the pinopode developments ( 11 ). In addition, HB-EGF could shield uterine decidualization against oxidative stress by increasing antioxidant capacity, restoring mitochondrial function preventing cell apoptosis ( 35 ), and participating in ER and embryo implantation. In the current study, the GdA gene expression decreased in the hydrosalpinx group compared to the control, but this reduction was not statistically significant. GdA exhibits immunosuppressive properties at the feto-maternal interface by inducing a tolerogenic phenotype in dendritic cells and macrophages. Additionally, it enhances immune tolerance through its modulatory impact on natural killer cells (NK cells) ( 11 , 36 ). Moreover, GdA is associated with the developing pinopodes and ameliorates blastocyst adhesion ( 11 ). GdA concentration and its changes were evaluated in several studies ( 37 , 38 ). Focarelli et al. ( 37 ) showed the GdA expression seems to be altered in the endometrial epithelium of individuals with endometriosis, explicitly demonstrating a notable rise in the late secretory phase. This suggests that irregularities in the endometrium could result in an unreceptive environment, potentially caused by factors like inflammation, immune dysregulation, and apoptosis activation. Demir et al. ( 38 ) reported Gd A level was significantly higher in patients with ovarian endometrioma rather than in the PCOS and myoma uteri, compared to the control group during the mid-luteal phase of the menstrual cycle. In line with our study, Farimani Sanoee et al. ( 39 ) found that mRNA expression level of GdA significantly decreased in the endometrium of women with myoma, and myomectomy led to significant elevation in GdA gene expression level in these women. Non-significant differences in GdA mRNA expression levels between the study’s hydrosalpinx and control groups can be attributed to the small sample size. It is possible that with a larger sample size, this difference could become statistically significant. However, it is essential to note that despite this result, GdA is still crucial for successful embryo implantation and plays a substantial role in initiating communication between the blastocyst and endometrium during the early stages. Although physiological inflammation is necessary for implantation and maintenance of pregnancy, any disturbances can lead to changes in the expression pattern of effective genes in the implantation process and create an unfavorable milieu in the uterus, and as mentioned earlier, decreases the rate of embryo implantation and increase spontaneous abortion ( 24 ). In light of our findings in the present study and other previous that have been conducted in the investigation of the altered expression of genes affecting ER and embryo implantation in hydrosalpinx patients, it can be concluded that Hydrosalpinx Fluid can disrupt the cytokine network balance and lead to move the inflammation state from the physiological level to the pathological state. Conducting more studies in this field will help better understand influential factors in embryo implantation, and their interactions and can lead to improved assisted reproductive techniques (ART). Although this study is groundbreaking, there were some limitations. With the limited number of cases volunteered within each study group, validating these findings with larger sample sizes is advisable. Additional research will be required to ascertain whether measurement of these genes can be beneficial to finding biomarkers for ER and embryo implantation. It is suggested that following up on these genes at the protein level can confirm the results of this research and lead to comprehension better of ER, the implantation process, and successful ART.

It is concluded that IL-6 gene expression in individuals with hydrosalpinx is increased compared to the control group due to inflammation conditions. Additionally, the gene expression of GdA and HB-EGF in patients with hydrosalpinx is lower than that of fertile women. In our study, the alteration in GdA expression level was not statistically significant due to the restricted number of subjects. Despite this, these genes are essential for successful implantation, and disturbances in their expression patterns may be linked to hydrosalpinx. Therefore, further research with a larger population will be necessary to better understand the hydrosalpinx disorder and its impact on ER and the implantation process.

In this case-control study, 15 infertile women with hydrosalpinx who underwent laparoscopic salpingectomy in Royan Institute were allocated. The controls were included 15 healthy fertile women who volunteered to donate eggs. The study was approved by the Ethics commitee of Shahid Beheshti University of Medical sciences, Iran (IR.SBMU. RETECH.REC.1397.1180) and Royan Institute (IR.ACECR.ROYAN.REC.1398.169). All participants signed written informed consent before the collection of tissue samples. In the case group, all subjects were aged between 20-37 years old, had body mass index (BMI) within the range of 18.5-30 kg/m 2 according to the World Health Organization (WHO) categories, had regular menstruation and not received any exogenous hormones in the three months leading up to the endometrial biopsy could participate in this study. Subjects with a history of endometriosis, polycystic ovarian syndrome (PCOS), uterine cancer and abnormalities, myomectomy, adenomiosis, fibroid surgery, tuberculosis, diabetes, and other hormonal disorders, as well as those with unexplained infertility, smoking, and repeated miscarriages, were not included in the study. Hydrosalpinx diagnosis was established by identifying obstruction, enlargement, and a fluid-filled ampullary portion of fallopian tubes without a free intraperitoneal spill of contrast on laparoscopy or hysterosalpingography. Cases with sono-visible hydrosalpinx (diameter>10 mm) were considered for inclusion. A control group, including 15 age-matched healthy fertile women, was recruited. These volunteers were 20-37 years old, with a BMI (18.5-30 kg/m 2 ) and regular menstruation (normal hormonal profile). They had not received any exogenous hormones in the past three months and had a history of at least one previous successful spontaneous pregnancy with live birth. Subjects with a history of endometriosis, PCOS, uterine cancer and abnormalities, myomectomy, adenomiosis, fibroid surgery, tuberculosis, diabetes, and other hormonal disorders, as well as those with unexplained infertility, smoking, and repeated miscarriages, were not included in the study. Endometrial biopsy was performed after measurement of mid-luteal progesterone, by the Pipple (Laboratoire CCD, Paris, France) during the 21 ± 2 day of the menstrual cycle, at the WOI period. The endometrial specimens were transferred to 2-ml cryovial tubes (Greiner BioOne, Germany) with RNA later (Ambion, UK) and quickly frozen in liquid nitrogen (196 °C) for 30 seconds. They were then promptly stored at -80°C to ensure RNA stability. The primers for the genes under study were designed using Perl Primer and Gen Runner software and synthesized (Metabion, Martinsried, Germany). All sequences of primers are provided in Table 1. Primer sequences used for real-time polymerase chain reaction (RT-PCR) The endometrial biopsy specimens were obtained during the mid-secretory phase and carefully timed according to the criteria for a regular menstrual cycle. Histological dating was verified through independent examinations by an experienced pathologist (M.L), and only specimens indicating the absence of other gynecological pathologies in clinical analysis were included. The collected endometrial samples underwent saline rinsing to eliminate blood and were promptly fixed in 10% neutral buffered formalin. They were then embedded in paraffin, sectioned with a microtome to produce 4-5 μm-thick paraffin sections, and stained with Hematoxylin and eosin (H&E). Histological dating was performed according to conventional dating criteria. Hematoxylin and Eosin staining of human endometrium. A. The light microscopic appearance of the mid-secretory phase of endometrium in a fertile woman who is considered control (400x). The presence of abundant pinopodes (indicated by black arrows) can be seen on the throughput of apical surfaces of the epithelial (Ep) cells, suggestive of the window of implantation time frame. Endometrial glands are tubular and can be observed extending into the depths of the stroma (S), blood vessels (Bv) and stromal cells. The lumen (Lu) is also visible. (H&E). B. Light microscopic examination reveals the patients endometrium with hydrosalpinx. The endometrium surface epithelium (Ep) shows thinning without pinopodes, and the endometrial glands within the stroma (S) are seen (scale bar: 100 μm). Total RNA was extracted using TRIzol (Thermo Fisher Scientific, USA). To remove genomic contamination, the extracted RNA was treated with DNase I (Thermo Fisher Scientific, USA) before cDNA synthesis to eliminate genomic DNA. cDNA synthesis was done utilizing the Takara Kit (Takara Bio, Japan, Cat # RR037A-100). The qRT-PCR reaction was done on the StepOnePlus Apparatus (Applied Biosystems SDS 7000 Foster, USA). All reactions were triplicated with the constructed cDNAs. The mixture prepared for reaction including 5 μL cyber green (Real Q Plus Master Mix Green high ROX Cat No: RR820L, Takara, China), 1 μL forward primer, 1 μL reverse primer, 2 μl cDNA at 1000 ng/μl and DEPC water up to 20 μL volume. RT-qPCR conditions were 1 cycle of reverse-transcription at 42°C for 5 minutes, 1 cycle of reverse-transcriptase inactivation at 95°C for 5 minutes, 40 cycles of amplification at 95°C for 15 seconds, 56°C for 30 seconds, then 72°C for 30 seconds. The relative quantity of the gene of interest was normalized to the relative quantity of β-actin , as a reference gene. It was reported as a fold change in gene expression. The melting curve was shaped after the final amplification cycle to calculate the specificity of the real-time PCR fragments. The relative abundance of each sample was calculated using the 2 -ΔΔCt method. The normality of the variables in the groups was evaluated using the Kolmogorov-Smirnov test. Since the gene expression variables were not normally distributed, nonparametric tests (the Mann-Whitney test) were used to compare the two groups. In light of the non-normality of the variables, the data was described using the median and interquartile range. Analyzing was performed using SPSS software (version 24 SPSS, IBM, USA), and Prism version 9.0.0. P<0.05 is considered to be statistically significant.