Laparoscopic Salpingectomy Regulates The Expression of Endometrial Homeobox Genes (HOXA9, HOXA10, HOXA11, HOXD10) and HOX Transcript Antisense Intergenic RNA in Women with Communicating Hydrosalpinx: A Prospective Study.

Tubal factor is the most common cause of female infertility, responsible for approximately 25 to 30% of all female infertility problems ( 1 ). The most severe form of tubal pathology is hydrosalpix, characterized by obstruction and accumulation of watery fluid in the distal part of one or both fallopian tubes. Hydrosalpinx accounts for 10-30% of tubal disorders, according to the diagnostic modality used ( 2 ). The most common cause for hydrosalpinx formation is pelvic inflammatory disease resulting from prior sexually transmitted diseases. Other causes include endometriosis, previous pelvic or abdominal surgery due to appendectomy, myomectomy, and ectopic tubal pregnancy ( 2 ). Several studies have demonstrated that the presence of a hydrosalpinx is associated with an adverse effect on fertility outcomes in patients undergoing in vitro fertilization (IVF) cycles, leading to a 50% reduction in implantation and pregnancy rates and a two-fold increase in spontaneous miscarriage ( 1 , 3 - 5 ). Evidence from prospective trials strongly suggests that laparoscopic salpingectomy before IVF in patients with ultrasound-visible hydrosalpinges significantly improves implantation and pregnancy rates ( 4 - 8 ). Various theories have been proposed to explain the underlying mechanisms by which hydrosalpinges fluid can have detrimental impacts on implantation and pregnancy, however, the exact mechanism is not well understood ( 8 - 11 ). A combination of mechanical and chemical factors is thought to interfere with embryo implantation ( 8 ). Some authors have suggested that fluid may potentially be embryotoxic ( 9 , 10 ), disturb sperm motility ( 9 ), and early embryo development ( 9 , 10 ). In contrast, others proposed that the reflux of fluid into the uterus could produce a flushing effect that mechanically inhibits implantation ( 11 ). The most accepted theory is poor endometrial receptivity caused by altered expression levels of growth factors, inflammatory cytokines, enzymes, peptides, steroid hormones, and significantly lower expression of key receptivity molecules such as HOXA10 ( 12 , 13 ), αVβ3 integrin ( 14 ), and leukemia inhibitory factor (LIF) ( 15 ) in the endometrium exposed to hydrosalpinges fluid at the time of implantation. HOX genes are a family of transcriptional regulators that play critical roles in embryonic development and adult functional tissue differentiation ( 16 ). The paralogs of Hox genes display similar functions and overlapping expression patterns during embryogenesis in mice and humans ( 17 ). Specifically, HOXA10 is a well-known endometrial marker for both the development and receptivity of the endometrium. It is expressed at the highest level in the midsecretory phase in response toHOXA10, several HOX sex hormones ( 16 ). In addition to HOXA10 , several HOX family member genes including HOXA9, HOXA11 , and HOXD10 show significant upregulation during the mid-secretory phase, suggesting a similar role in the process of decidualization and implantation ( 17 ). HOX gene expressions are regulated by long noncoding RNAs ( lncRNA ), a class of genes with longer than 200 nucleotides in length or little and no protein-coding potential ( 18 ). In the last twenty years, the role of lncRNA in the epigenetic regulation of wide biological processes such as differentiation, development, and multiple human diseases has been confirmed ( 18 , 19 ). Human HOX transcript antisense intergenic RNA ( HOTAIR ) is the first identified trans-acting repressor lncRNA located between HOXC11 and HOXC12 on chromosome 12 and acts in trans leading to epigenetic silencing of posterior HOXD genes located in chromosome 2 ( 20 ). Recent studies have shown that HOTAIR is involved in inflammatory cytokines production, and inflammation ( 20 , 21 ). Research shows that HOTAIR plays an important role in promoting endometriosis, endometrial fibrosis, and intrauterine adhesion ( 22 , 23 ). To the best of our knowledge, no studies have been performed to evaluate the impact of salpingectomy on the expression level of a panel of homeobox genes and lnc HOTAIR in the endometrium of infertile patients with hydrosalpinx. Therefore, in this prospective study, we first investigated the alteration of these genes in the endometrium of infertile patients exposed to hydrosalpinx fluid before and after salpingectomy. We next predicted the potential functions of HOTAIR by analyzing its cisand trans-regulating protein-coding genes.

The flow diagram clearly illustrates the enrollment, follow-up, and analysis. As the flow diagram shows, 17 patients with hydrosalpinx underwent salpingectomy and were allocated for the first endometrial biopsy in the mid-luteal phase before salpingectomy. In the control group, 15 healthy fertile women who were candidates for egg- donation were recruited for the first biopsy in the mid-luteal phase. In the hydrosalpinx group, three patients were excluded from the study due to incomplete data and 14 patients completed the study. In the control group, 1 woman was excluded from the study due to insufficient RNA isolation and 14 women completed the study ( Fig .1 ). Study flow chart illustrates the participants in hydrosalpinx and nonhydrosalpinx groups. Table 2 shows the demographic characteristics and the hormonal profile of hydrosalpinx and control group. There were statistically no significant differences between the hydrosalpinx and healthy fertile women in the mean of age, BMI, basal hormonal profiles, basal FSH, LH, AMH, prolactin (PRL), and mid-secretory progesterone levels (P>0.05). Clinical and hormonal characteristics of the hydrosalpinx and non-hydrosalpinx groups t test was applied for quantitative variables with non-normal distribution. P<0.05 is accepted as statistically significant. BMI; Body mass index, FSH; Follicular stimulating hormone, LH; Luteinizing hormone, AMH; Anti-mullerian hormone, PRL; Prolactin, a ; Mann-Whitney test was applied for quantitative variables with non-normal distribution, and IQR; Interquartile range. First, we investigated and compared endogenous HOXA9, HOXA10, HOXA11 , and HOXD10 mRNA expression levels in parallel with the expression of lnc RNA HOTAIR in the endometrium of infertile women with hydrosalpinx before and four cycles after salpingectomy during the mid-luteal phase ( 13 ). Furthermore, we predicted the potential functions of HOTAIR by analyzing its co-expressed protein-coding genes. Compared with fertile control subjects, the expression levels of HOXA9 (P<0.001), HOXA10 (P=0.001), HOXA11 (P=0.003), and HOXD10 (P=0.004) mRNA were significantly lower during the mid-secretory phase in the endometrium of patients with hydrosalpinges. To investigate the effect of surgical removal of hydrosalpinx on the expression of endometrial receptivity HOX genes in patients with hydrosalpinges pre- and postoperative mRNA expression levels of each gene in each subject were analyzed ( Fig .2A-D ). Surgical removal of hydrosalpinx restored impaired endometrial expression of HOXA9 and HOXA10 , in the endometrium of 78.57 % (11 of 14) and 71.4% (10 of 14) of infertile patients with hydrosalpingies respectively. After salpingectomy, we observed a 6-fold increase in the expression of HOXA9 and a 7.64-fold increase in the expression of HOXA10 mRNA levels in comparison with before salpingectomy samples (P=0.006 and P=0.023 respectively, Fig.2A, B). Similarly, HOXA11 and HOXD10 levels increased in 71.4% (10/14) of patients after surgery. Data obtained in the present study showed a significant raising in endometrial HOXA11 (6.78-fold increase, P=0.012) and HOXD10 mRNA expression levels (5-fold increase, P=0.013) in postoperative samples compared to preoperative tissue ( Fig .2C, D ). Moreover, there were no significant differences in endometrial expression levels of HOXA9, HOXA10, HOXA11 , and HOXD10 mRNA between patients undergoing salpingectomy and control groups (P=0.462, P=0.494, P=0.631 and P=0.767, respectively). Interestingly, lnc HOTAIR expression level was significantly higher in the endometrium of women with hydrosalpinges compared to control groups (P=0.020, Fig .2E ). After salpingectomy, we found a significant decrease in the mean relative expression level of HOTAIR (2.89-fold decrease) compared to before salpingectomy samples (P=0.010). HOTAIR expression decreased in the endometrium of 71.4% (10/14) of the endometrium exposed to hydrosalpinges fluid. Our findings showed no evidence for an inverse correlation between HOTAIR and HOXD10 expression due to limited sample size (r Pearson=-0.067, P=0.821). Relative gene expression levels of HOX-9/HOXA10/HOXA11/ HOX-D10 and lncRNA HOTAIR in infertile women with Hydrosalpinx before and after salpingectomy (n=14) compared to healthy fertile women (n=14). A-D. In the hydrosalpinx group, the surgery restored a 6-fold increase in HOXA9 , a 7.64-fold increase in HOXA10 , a 6.78-fold increase in HOXA11 , and a 5-fold increase in HOXD10 mRNA expression levels. E. The expression level of lnc HOTAIR was found to be significantly higher in the endometrium-induced hydrosalpinx fluid compared to controls (P=0.020), which had a 2.89-fold decrease following salpingectomy (P=0.010). The values are expressed as mean ± SEM. The data were analyzed by nonparametric Wilcoxon’s rank test. * ; P<0.05, ** ; P<0.01, *** ; P<0.001, B.S; Before salpingectomy, A.S; After salpingectomy, and FC; Fertile control.

The cross-talk between trophoblast and endometrium is highly mediated by the expression of specific receptivity genes and inflammatory cytokines. A complex network of signaling is required for implantation. Alteration of these signaling pathways often results in pathological conditions contributing to infertility ( 25 ). In this study, we represent the first report of remarkable disruption in the expression pattern of the lnc RNA HOTAIR and endometrial receptivity HOX genes mRNA ( HOXA9, HOXA11 , and HOXD10 mRNA, in addition to HOXAl0 ), during implantation window in infertile patients with hydrosalpinges compared to samples derived from fertile healthy women. Our findings revealed that cyclic upregulation of HOX genes involved in the receptivity of endometrium does not occur during the implantation window in infertile women with hydrosalpinges and salpingectomy can restore expression of these genes to physiological levels in two-thirds of patients. In line with our findings, several studies have shown a detrimental effect of hydrosalpinx fluid on endometrial receptivity markers, for instance, HOXA10, LIF, Integrin αʋβ3, MUC1, and pinopodes expression levels and the benefit of salpingectomy as a therapeutic option for management of hydrosalpinx prior IVF programs ( 13 - 15 , 26 , 27 ). In an in vitro study, Daftary and Taylor ( 12 ), reported significantly lower expression levels of HOXA10 mRNA using hydrosalpinx fluid in a concentrationdependent manner in Ishikawa cells. Subsequently, the authors in a prospective study examined the expression of endometrial HOXA10 mRNA levels in women with hydrosalpinges before and after salpingectomy and found that HOXA10 mRNA levels returned to normal physiological levels (15-fold increase) after salpingectomy ( 13 ), indicating surgical removal of hydrosalpinx restores HOXA10 expression to normal levels. Increased HOXA10 mRNA levels were detected in both glandular cells and endometrial stroma. Similarly, the result of two other studies demonstrated the detrimental effect of hydrosalpinx on endometrial expression of integrin ανβ3 and its restoration in 70% of patients following salpingectomy ( 14 , 26 ). Likewise, Li et al. ( 25 ) noted that the remarkable reduction of LIF, integrin b3, and MUC1 expression during the mid-secretory phase might be one of the reasons for the low pregnancy rate in women with hydrosalpinx. In women with normal cyclicity, expression of HOX genes ( HOXA9, HOXA10, HOXA11 , and HOXD10 ) involved in endometrial development, receptivity, decidualization, and implantation increase under the control of ovarian steroids, especially progesterone and reach to the maximal level in mid-secretory phase. In humans, the above-mentioned genes are important transcriptional regulators, which mediate (activate or repress) the multiple downstream target genes; including EMX2, IGFBP1, ITGB3, and are important for decidualization, and embryo implantation ( 17 ). HOXA10 dramatically regulates the expression of integrin αvβ3 and pinopode implantation efficiency ( 27 ). A defect in the endometrial expression of the HOXA10/ HOXA11 has previously been described in several pathological conditions related to endometrium such as recurrent implantation failure, endometriosis, submucosal leiomyomas, and polycystic ovarian syndrome ( 28 - 30 ). The hydrosalpingeal fluid in the damaged tubes is a result of pelvic inflammatory disease and consists of inflammatory cells and pro-inflammatory cytokines, which prevents the formation of a receptive endometrium ( 31 , 32 ). Although acute inflammation of endometrium is a part of the embryo-uterine “dialogue” and promotes successful trophoblast invasion ( 33 ), chronic inflammation is a pathological condition that may adversely affect uterine receptivity expression markers involving in endometrial apposition and embryo adhesion ( 31 , 32 ). The nuclear factor -Kappa B (NF-κB) pathway has shown that mainly regulates inflammatory processes in the endometrium-induced hydrosalpinx fluid. We did not measure the expression level of NF-KB in our samples; however, several studies have strongly suggested that the presence of hydrosalpinx or endometrioma is associated with considerably increased endometrial NF-κB ( 31 , 32 ). Moreover, the result of a more recent study demonstrated that overexpression of TNF-α, IL-7 , and NF-κB genes may induce potentiation of the proinflammatory environment in the endometrium, leading to the following downregulation of HOX genes involved in implantation ( 32 ). On the other hand, the research on HOX genes and NF-KB pathways in inflammatory conditions indicates a negative relationship between NF-kB and receptivity genes ( 34 - 36 ). Based on the literature, activation of the canonical NF-κB pathway can result in transcriptional repression of HOX genes and the cross-talk between these two pathways plays an important role in pathogenic inflammatory microenvironments such as breast cancer, atherosclerosis, and endometrioma ( 34 , 35 ). Trivedi et al. ( 34 ) reported that HOXA9 expression rapidly downregulates in response to TNF-α and NF-KB expressions in endothelial cells. Their findings indicated the presence of an NF-κB binding site in the HOXA9 promoter proximal to the transcription start site. Similar to these findings, Dokuzeylül Güngör et al. ( 36 ) found a significant inverse correlation between endometrial upregulated NF-kB and downregulation of HOXA10, HOXA11 , and LIF expression levels in the presence of type 0 or 1 fibroid that returned to normal values after hysteroscopic myomectomy. Aberrant DNA methylation is a possible epigenetic mechanism that is responsible for the aberrant expression of endometrial receptivity genes ( 37 ). A growth body of evidence supports NF-kB regulates the epigenetic changes associated with inflammation through DNA methylation or demethylation in the promoter regions of HOXA10/HOXA11 in endometriosis, so increased endometrial NF-kB expression may be a probable mechanism, which suppresses the expression of HOX genes through hypermethylation ( 37 , 38 ). Besides, the retrograde flow of fluid into the uterine cavity is accompanied by the aberrant expression of endometrial inflammatory microRNAs for example; miR-135a, miR-135b, and miR-145 ( 39 , 40 ). Specifically, miR-135a and miR-135b suppress HOXA10 expression leading to impaired endometrial receptivity in women with endometriosis ( 39 ). Likewise, overexpression of miR-145 in hydrosalpinx-induce defective endometrium is significantly associated with down-regulation of HOXA10 through facilitating the secretion of TGF- β1, TNF-α, IL-6, and IL-8 by endometrial cells ( 40 ). Additionally, we evaluated and compared HOTAIR expression levels before and after surgery. We observed that the endometrial HOTAIR expression level in patients with hydrosalpinx was significantly higher than in controls and salpingectomy returned pre-implantation endometrial HOTAIR expression to physiological level. In the current study, we selected HOTAIR because of its inflammatory nature and epigenetic modulatory function ( 20 ). The aberrant endometrial HOTAIR expression has been seen in severe endometriosis and polycystic ovarian syndrome, which leads to downregulation of HOXD10 and alterations of the microenvironment of endometrium and receptivity ( 22 ). However, in our study, the differences failed to show a statistically inverse correlation between HOTAIR and HOXD10 expression levels due to the limited sample size. Beyond its role in chromatin modification and gene repression, HOTAIR was found to promote an inflammatory response in different pathological situations such as diabetes, arthritis, acute myocardium infarction, intrauterine adhesion, endometriosis, cancer, and other inflammatory diseases ( 20 - 23 ). HOTAIR triggers the NF-KB pathway and immune response through the reduction of its inhibitor, IκBα. Upon activation, NF-κB translocates to the nucleus and binds to the promoters of pro-inflammatory cytokines, resulting in an upregulation of their expression. The overexpression of HOTAIR has been reported with an increased expression of IFN-γ, IL6, IL-17, TNF-α, IL-1β , and IL-6R in monocytes ( 20 ). The aberrant endometrial HOTAIR expression has been seen in severe endometriosis, which leads to the downregulation of HOXD10 and alterations of the microenvironment of endometrium and receptivity ( 22 ). The possible limitation of our study could be the small number of patients. Although our findings showed a significant increase in HOTAIR expression levels in preoperative samples compared to postoperative tissue, the results failed to show a statistically inverse correlation between HOTAIR and HOXD10 expression levels due to the limited sample size.

Our findings suggest that the presence of hydrosalpinx is associated with overexpression of the pro-inflammatory lncRNA HOTAIR, which may impair the regulation of HOX genes involved in endometrial receptivity. Nevertheless, our current knowledge about the role of lncRNAs in endometrium exposed to hydrosalpinx fluid is limited. Additional research with more sample sizes is required to explore the HOTAIR pro-inflammatory signaling pathway and its inverse co-expression with endometrial receptivity HOX genes in these subjects.

In this prospective study, 14 infertile women aged 18- 40 years- old with unilateral communicating hydrosalpinx detected by hysterosalpingography or laparoscopy who underwent laparoscopic salpingectomy from May 2022 to September 2023 were evaluated. Royan Institute Ethics Committee approved the study for Research on Human Subjects (IR.ACECR.ROYAN.REC.1401.015) and all participants signed written informed consent before the collection of tissue samples. The etiology for hydrosalpinx was a history of pelvic inflammatory disease, appendectomy, previous abdominal surgery, ectopic pregnancy, and peritubal adhesions. Exclusion criteria were the presence of endometriosis, intrauterine pathologies such as a polyp, submucous or intramural myoma, Asherman syndrome, history of previous uterine surgery, Mullerian anomaly, habitual miscarriage, and etiology other than hydrosalpinx. Male factors were excluded from the study (normal semen analysis according to World Health Organization criteria, 1992). Diagnosis of hydrosalpinx was made based on the presence of obstruction, enlargement, and a fluid-filled ampullary portion of fallopian tubes with no free intraperitoneal spill of contrast on hysterosalpingography or laparoscopy and the cases with sono-visible hydrosalpinx, (diameter>10 mm) were included. All patients had normal hormonal [follicular stimulating hormone (FSH), luteinizing hormone (LH), anti-mullerian hormone (AMH), thyroid stimulating hormone (TSH), prolactin (PRL)] profiles, body mass index (BMI: 18-28 kg/m 2 ), and regular menstrual cycles confirmed by mid-luteal progesterone level. None of the women had received any hormonal medication during the last three months. Women with hydrosalpinx underwent laparoscopic salpingectomies to remove the damaged fallopian tube. Mid-luteal-phase endometrial samplings (days 19-21) were performed at the time of surgery and second endometrial biopsies were obtained during the fourth treatment cycle during the midluteal phase of the cycle. The control group was selected from 14 age-matched healthy fertile women with a regular menstrual cycle and a history of successful pregnancy (at least one child) referred to egg donation. The controls were scheduled to undergo endometrial biopsies in the mid-luteal phase. Control and hydrosalpinx endometrial samples were obtained by pipelle. The endometrial samples were immediately transported on ice and samples within the RNA stabilization buffer (AM7020, Ambion, and Austin, TX, US), snap frozen in liquid nitrogen, and stored at -80°Ϲ. A piece of each sample (in the case and control group) was fixed in formalin 10%, embedded in paraffin, sectioned with a microtome to obtain 3 μm-thick paraffin sections, and stained on serial sections with hematoxylin and eosins to perform histological dating according to the criteria of Noyes et al. ( 24 ). Total RNA from endometrial samples was extracted using Trizol reagent according to the manufacturer’s instructions (RiboEX, South Korea, Cat.No: 301-001). Briefly, about 50-100 mg of tissue was thawed and homogenized in 1 ml Trizol. The homogenized sample was mixed well by vortexing and then incubated for 5 minutes at room temperature to permit the complete denature of proteins. Chloroform (0.2 ml per 1 ml of Trizol) was added and shaken vigorously by hand and incubated them at 25°C for 3 minutes. The sample was centrifuged at 12,000 ×g for 15 minutes at 4°C and the colorless upper aqueous phase was transferred to a fresh tube. Then 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent was added and the tube was stored for 1 hour at -20°C. RNA was precipitated by centrifugation at 12 000 X g for 18 minutes at 4°C. The pellet was washed twice with 75% ethanol, centrifuged at7800 X g for 8 minutes at 4°C, briefly dried under air, and dissolved in 20 µl of nuclease-free water. The purity and concentration of RNA were measured using a Nano-drop Microvolume UV–Vis spectrophotometer (Thermo Scientific, USA). To prevent contamination of genomic DNA, extracted RNA was treated with DNase I, as described by the company (Thermo Scientific, USA, Cat.No: EN0521). First-strand cDNA was synthesized using the Reverse transcription kit (SMOBIO, Cat.N0:RP1300), and stored at -20°C. Quantitative real-time polymerase chain reaction (q-PCR) was conducted via specific primers ( Table 1 ). Specific primers of HOXA9, HOXA10, HOXA11, HOXD10, HOTAIR , and GAPDH genes were designed using Perl Primer (version 1.1.21, http://perlp rimer. sourceforge. net/), and checked by Gene Runner software (version 6.0, http://www.generunner.net). To confirm primer sequence specificity, the following public resources were applied: BLAST software (http: //blast.ncbi. nlm.nih.gov/Blast.cgi), ( 2 ) The UCSC genome browser (http://www .genome. ucsc.edu), and ( 3 ) The Ensembl website (http://www. ensembl.org). The qRT-PCR was performed using Step one plus Real-Time PCR system (Applied Biosystems), with the standard fluorescent dye SYBR Green PCR Master) 2xqPCR Master Mix Green-High Rox A32540 ). Real-time PCR was performed under standard conditions in three stages: i. Holding stage: 95°C for 10 minutes (as an initial denaturation phase), ii. Cycling stage: 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute, and iii. Melt curve stage: 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. After each run, a melting curve analysis was carried out to verify the specificity of RT- PCR reaction. The mRNA level of each target gene was normalized to an endogenous reference gene, GAPDH . A healthy fertile control group was considered as the calibrator. Gene expression data analysis was carried out using the 2 -ΔΔCt quantitative method to calculate relative fold change values. The sequences, size of the amplicon, and annealing temperatures for each primer used in this study Data were analyzed using the SPSS software version 24 (IBM, USA). The Kolmogorov-Smirnov test was used to test the normal distribution of data. Independent sample t test was applied for quantitative variables with normal distribution and the Mann-Whitney test was applied for quantitative variables with non-normal distribution. Data were expressed as mean ± standard deviation, and median (interquartile ranges). Non-parametric Wilcoxon’s paired signed ranks test was used to compare endometrial gene expression preand post-salpingectomy. The Non-parametric MannWhitney test was performed to compare the average gene expression between two independent groups. Results of mRNA expression were presented as mean ± SEM. P<0.05 was considered statistically significant.