{"paper_id":"d54c90b1-c891-42b4-8ad2-5f6f22305a54","body_text":"Tubal factor is the most common cause of female\ninfertility, responsible for approximately 25 to 30% of all\nfemale infertility problems ( 1 ). The most severe form of\ntubal pathology is hydrosalpix, characterized by obstruction\nand accumulation of watery fluid in the distal part of one or\nboth fallopian tubes. Hydrosalpinx accounts for 10-30% of\ntubal disorders, according to the diagnostic modality used\n( 2 ). The most common cause for hydrosalpinx formation is\npelvic inflammatory disease resulting from prior sexually\ntransmitted diseases. Other causes include endometriosis,\nprevious pelvic or abdominal surgery due to appendectomy,\nmyomectomy, and ectopic tubal pregnancy ( 2 ).\nSeveral studies have demonstrated that the presence of a hydrosalpinx is associated with an\nadverse effect on fertility outcomes in patients undergoing  in vitro \nfertilization (IVF) cycles, leading to a 50% reduction in implantation and pregnancy rates\nand a two-fold increase in spontaneous miscarriage ( 1 ,  3 - 5 ).\nEvidence from prospective trials strongly suggests that\nlaparoscopic salpingectomy before IVF in patients with\nultrasound-visible hydrosalpinges significantly improves\nimplantation and pregnancy rates ( 4 - 8 ).\nVarious theories have been proposed to explain the\nunderlying mechanisms by which hydrosalpinges fluid can\nhave detrimental impacts on implantation and pregnancy,\nhowever, the exact mechanism is not well understood ( 8 - 11 ).\nA combination of mechanical and chemical factors is thought\nto interfere with embryo implantation ( 8 ). Some authors have\nsuggested that fluid may potentially be embryotoxic ( 9 ,  10 ),\ndisturb sperm motility ( 9 ), and early embryo development ( 9 ,\n 10 ). In contrast, others proposed that the reflux of fluid into\nthe uterus could produce a flushing effect that mechanically\ninhibits implantation ( 11 ). The most accepted theory is poor\nendometrial receptivity caused by altered expression levels of\ngrowth factors, inflammatory cytokines, enzymes, peptides,\nsteroid hormones, and significantly lower expression of\nkey receptivity molecules such as HOXA10 ( 12 ,  13 ), αVβ3\nintegrin ( 14 ), and leukemia inhibitory factor (LIF) ( 15 ) in the\nendometrium exposed to hydrosalpinges fluid at the time of\nimplantation.\nHOX genes are a family of transcriptional regulators that play critical roles in embryonic\ndevelopment and adult functional tissue differentiation ( 16 ). The paralogs of Hox genes\ndisplay similar functions and overlapping expression patterns during embryogenesis in mice\nand humans ( 17 ). Specifically,  HOXA10  is a well-known endometrial marker\nfor both the development and receptivity of the endometrium. It is expressed at the highest\nlevel in the midsecretory phase in response toHOXA10, several HOX sex hormones ( 16 ).\nIn addition to  HOXA10 , several HOX family member genes including\n HOXA9, HOXA11 , and  HOXD10  show significant upregulation\nduring the mid-secretory phase, suggesting a similar role in the process of decidualization\nand implantation ( 17 ).\nHOX gene expressions are regulated by long noncoding RNAs ( lncRNA ), a\nclass of genes with longer than 200 nucleotides in length or little and no protein-coding\npotential ( 18 ). In the last twenty years, the role of lncRNA in the epigenetic regulation of\nwide biological processes such as differentiation, development, and multiple human diseases\nhas been confirmed ( 18 ,  19 ).\nHuman HOX transcript antisense intergenic RNA ( HOTAIR ) is the first\nidentified trans-acting repressor lncRNA located between HOXC11 and HOXC12 on chromosome 12\nand acts in trans leading to epigenetic silencing of posterior HOXD genes located in\nchromosome 2 ( 20 ).\nRecent studies have shown that HOTAIR is involved\nin inflammatory cytokines production, and inflammation\n( 20 ,  21 ). Research shows that HOTAIR plays an important\nrole in promoting endometriosis, endometrial fibrosis,\nand intrauterine adhesion ( 22 ,  23 ).\nTo the best of our knowledge, no studies have been performed to evaluate the impact of\nsalpingectomy on the expression level of a panel of homeobox genes and  lnc\nHOTAIR  in the endometrium of infertile patients with hydrosalpinx. Therefore, in\nthis prospective study, we first investigated the alteration of these genes in the\nendometrium of infertile patients exposed to hydrosalpinx fluid before and after\nsalpingectomy. We next predicted the potential functions of HOTAIR by analyzing its cisand\ntrans-regulating protein-coding genes.\n\nIn this prospective study, 14 infertile women aged 18- 40 years- old with unilateral\ncommunicating hydrosalpinx detected by hysterosalpingography or laparoscopy who underwent\nlaparoscopic salpingectomy from May 2022 to September 2023 were evaluated. Royan Institute\nEthics Committee approved the study for Research on Human Subjects\n(IR.ACECR.ROYAN.REC.1401.015) and all participants signed written informed consent before\nthe collection of tissue samples. The etiology for hydrosalpinx was a history of pelvic\ninflammatory disease, appendectomy, previous abdominal surgery, ectopic pregnancy, and\nperitubal adhesions. Exclusion criteria were the presence of endometriosis, intrauterine\npathologies such as a polyp, submucous or intramural myoma, Asherman syndrome, history of\nprevious uterine surgery, Mullerian anomaly, habitual miscarriage, and etiology other than\nhydrosalpinx. Male factors were excluded from the study (normal semen analysis according\nto World Health Organization criteria, 1992).\nDiagnosis of hydrosalpinx was made based on the presence of obstruction, enlargement, and\na fluid-filled ampullary portion of fallopian tubes with no free intraperitoneal spill of\ncontrast on hysterosalpingography or laparoscopy and the cases with sono-visible\nhydrosalpinx, (diameter>10 mm) were included. All patients had normal hormonal [follicular\nstimulating hormone (FSH), luteinizing hormone (LH), anti-mullerian hormone (AMH), thyroid\nstimulating hormone (TSH), prolactin (PRL)] profiles, body mass index (BMI: 18-28\nkg/m 2 ), and regular menstrual cycles confirmed by mid-luteal progesterone\nlevel.\nNone of the women had received any hormonal\nmedication during the last three months. Women with\nhydrosalpinx underwent laparoscopic salpingectomies\nto remove the damaged fallopian tube. Mid-luteal-phase\nendometrial samplings (days 19-21) were performed at\nthe time of surgery and second endometrial biopsies were\nobtained during the fourth treatment cycle during the midluteal phase of the cycle. The control group was selected\nfrom 14 age-matched healthy fertile women with a regular\nmenstrual cycle and a history of successful pregnancy (at\nleast one child) referred to egg donation. The controls\nwere scheduled to undergo endometrial biopsies in the\nmid-luteal phase. Control and hydrosalpinx endometrial\nsamples were obtained by pipelle. The endometrial samples\nwere immediately transported on ice and samples within the\nRNA stabilization buffer (AM7020, Ambion, and Austin,\nTX, US), snap frozen in liquid nitrogen, and stored at -80°Ϲ.\nA piece of each sample (in the case and control group) was fixed in formalin 10%,\nembedded in paraffin, sectioned with a microtome to obtain 3 μm-thick paraffin sections,\nand stained on serial sections with hematoxylin and eosins to perform histological dating\naccording to the criteria of Noyes et al. ( 24 ).\nTotal RNA from endometrial samples was extracted using Trizol reagent according to the\nmanufacturer’s instructions (RiboEX, South Korea, Cat.No: 301-001).\nBriefly, about 50-100 mg of tissue was thawed and\nhomogenized in 1 ml Trizol. The homogenized sample was\nmixed well by vortexing and then incubated for 5 minutes at\nroom temperature to permit the complete denature of proteins.\nChloroform (0.2 ml per 1 ml of Trizol) was added and shaken\nvigorously by hand and incubated them at 25°C for 3 minutes.\nThe sample was centrifuged at 12,000 ×g for 15\nminutes at 4°C and the colorless upper aqueous phase\nwas transferred to a fresh tube. Then 0.5 ml of isopropyl\nalcohol per 1 ml of TRIZOL Reagent was added and the\ntube was stored for 1 hour at -20°C. RNA was precipitated\nby centrifugation at 12 000 X g for 18 minutes at 4°C. The\npellet was washed twice with 75% ethanol, centrifuged\nat7800 X g for 8 minutes at 4°C, briefly dried under air,\nand dissolved in 20 µl of nuclease-free water.\nThe purity and concentration of RNA were\nmeasured using a Nano-drop Microvolume UV–Vis\nspectrophotometer (Thermo Scientific, USA). To prevent\ncontamination of genomic DNA, extracted RNA was\ntreated with DNase I, as described by the company\n(Thermo Scientific, USA, Cat.No: EN0521). First-strand\ncDNA was synthesized using the Reverse transcription kit\n(SMOBIO, Cat.N0:RP1300), and stored at -20°C.\nQuantitative real-time polymerase chain reaction (q-PCR) was conducted via specific\nprimers ( Table 1 ). Specific primers of  HOXA9, HOXA10, HOXA11, HOXD10,\nHOTAIR , and  GAPDH  genes were designed using Perl Primer\n(version 1.1.21, http://perlp rimer. sourceforge. net/), and checked by Gene Runner\nsoftware (version 6.0, http://www.generunner.net). To confirm primer sequence specificity,\nthe following public resources were applied: BLAST software (http: //blast.ncbi.\nnlm.nih.gov/Blast.cgi), ( 2 ) The UCSC genome browser (http://www .genome. ucsc.edu), and\n( 3 ) The Ensembl website (http://www. ensembl.org). The qRT-PCR was performed using Step\none plus Real-Time PCR system (Applied Biosystems), with the standard fluorescent dye SYBR\nGreen PCR Master) 2xqPCR Master Mix Green-High Rox  A32540 ).\nReal-time PCR was performed under standard conditions in three stages: i. Holding stage:\n95°C for 10 minutes (as an initial denaturation phase), ii. Cycling stage: 40 cycles of\n95°C for 15 seconds, and 60°C for 1 minute, and iii. Melt curve stage: 95°C for 15\nseconds, 60°C for 1 minute, and 95°C for 15 seconds. After each run, a melting curve\nanalysis was carried out to verify the specificity of RT- PCR reaction. The mRNA level of\neach target gene was normalized to an endogenous reference gene,  GAPDH . A\nhealthy fertile control group was considered as the calibrator. Gene expression data\nanalysis was carried out using the 2 -ΔΔCt  quantitative method to calculate\nrelative fold change values.\nThe sequences, size of the amplicon, and annealing temperatures for each primer used in this study\nData were analyzed using the SPSS software version 24 (IBM, USA). The Kolmogorov-Smirnov\ntest was used to test the normal distribution of data. Independent sample t test was\napplied for quantitative variables with normal distribution and the Mann-Whitney test was\napplied for quantitative variables with non-normal distribution. Data were expressed as\nmean ± standard deviation, and median (interquartile ranges).\nNon-parametric Wilcoxon’s paired signed ranks test\nwas used to compare endometrial gene expression preand post-salpingectomy. The Non-parametric MannWhitney test was performed to compare the average\ngene expression between two independent groups.\nResults of mRNA expression were presented as mean\n± SEM. P<0.05 was considered statistically significant.\n\nThe flow diagram clearly illustrates the enrollment,\nfollow-up, and analysis. As the flow diagram shows, 17\npatients with hydrosalpinx underwent salpingectomy\nand were allocated for the first endometrial biopsy in the\nmid-luteal phase before salpingectomy. In the control\ngroup, 15 healthy fertile women who were candidates\nfor egg- donation were recruited for the first biopsy in\nthe mid-luteal phase. In the hydrosalpinx group, three\npatients were excluded from the study due to incomplete\ndata and 14 patients completed the study. In the control\ngroup, 1 woman was excluded from the study due to\ninsufficient RNA isolation and 14 women completed the\nstudy ( Fig .1 ).\nStudy flow chart illustrates the participants in hydrosalpinx and nonhydrosalpinx groups.\nTable 2 shows the demographic characteristics and\nthe hormonal profile of hydrosalpinx and control group.\nThere were statistically no significant differences\nbetween the hydrosalpinx and healthy fertile women in\nthe mean of age, BMI, basal hormonal profiles, basal\nFSH, LH, AMH, prolactin (PRL), and mid-secretory\nprogesterone levels (P>0.05).\nClinical and hormonal characteristics of the hydrosalpinx and non-hydrosalpinx groups\nt test was applied for quantitative variables with non-normal distribution. P<0.05 is\naccepted as statistically significant. BMI; Body mass index, FSH; Follicular\nstimulating hormone, LH; Luteinizing hormone, AMH; Anti-mullerian hormone, PRL;\nProlactin,  a  ; Mann-Whitney test was applied for quantitative variables\nwith non-normal distribution, and IQR; Interquartile range.\nFirst, we investigated and compared endogenous  HOXA9, HOXA10, HOXA11 ,\nand  HOXD10  mRNA expression levels in parallel with the expression of lnc\nRNA HOTAIR in the endometrium of infertile women with hydrosalpinx before and four cycles\nafter salpingectomy during the mid-luteal phase ( 13 ). Furthermore, we predicted the\npotential functions of HOTAIR by analyzing its co-expressed protein-coding genes.\nCompared with fertile control subjects, the expression levels of  HOXA9 \n(P<0.001),  HOXA10  (P=0.001),  HOXA11  (P=0.003),\nand  HOXD10  (P=0.004) mRNA were significantly lower during the\nmid-secretory phase in the endometrium of patients with hydrosalpinges.\nTo investigate the effect of surgical removal of\nhydrosalpinx on the expression of endometrial receptivity\nHOX genes in patients with hydrosalpinges pre- and postoperative mRNA expression levels of each gene in each\nsubject were analyzed ( Fig .2A-D ).\nSurgical removal of hydrosalpinx restored impaired endometrial expression of\n HOXA9  and  HOXA10 , in the endometrium of 78.57 % (11 of\n14) and 71.4% (10 of 14) of infertile patients with hydrosalpingies respectively. After\nsalpingectomy, we observed a 6-fold increase in the expression of  HOXA9 \nand a 7.64-fold increase in the expression of  HOXA10  mRNA levels in\ncomparison with before salpingectomy samples (P=0.006 and P=0.023 respectively, Fig.2A,\nB).\nSimilarly,  HOXA11  and  HOXD10  levels increased in 71.4%\n(10/14) of patients after surgery. Data obtained in the present study showed a significant\nraising in endometrial  HOXA11  (6.78-fold increase, P=0.012) and\n HOXD10  mRNA expression levels (5-fold increase, P=0.013) in\npostoperative samples compared to preoperative tissue ( Fig .2C, D ).\nMoreover, there were no significant differences in endometrial expression levels of\n HOXA9, HOXA10, HOXA11 , and  HOXD10  mRNA between\npatients undergoing salpingectomy and control groups (P=0.462, P=0.494, P=0.631 and\nP=0.767, respectively).\nInterestingly, lnc HOTAIR expression level was significantly higher in the endometrium of\nwomen with hydrosalpinges compared to control groups (P=0.020,  Fig .2E ). After\nsalpingectomy, we found a significant decrease in the mean relative expression level of\nHOTAIR (2.89-fold decrease) compared to before salpingectomy samples (P=0.010).\n HOTAIR  expression decreased in the endometrium of 71.4% (10/14) of the\nendometrium exposed to hydrosalpinges fluid. Our findings showed no evidence for an\ninverse correlation between  HOTAIR  and  HOXD10  expression\ndue to limited sample size (r Pearson=-0.067, P=0.821).\nRelative gene expression levels of  HOX-9/HOXA10/HOXA11/ HOX-D10  and \nlncRNA  HOTAIR in infertile women with Hydrosalpinx before and after\nsalpingectomy (n=14) compared to healthy fertile women (n=14).  A-D.  In\nthe hydrosalpinx group, the surgery restored a 6-fold increase in\n HOXA9 , a 7.64-fold increase in  HOXA10 , a 6.78-fold\nincrease in  HOXA11 , and a 5-fold increase in  HOXD10 \nmRNA expression levels.  E.  The expression level of lnc HOTAIR was found\nto be significantly higher in the endometrium-induced hydrosalpinx fluid compared to\ncontrols (P=0.020), which had a 2.89-fold decrease following salpingectomy (P=0.010).\nThe values are expressed as mean ± SEM. The data were analyzed by nonparametric\nWilcoxon’s rank test.  *  ; P<0.05,  ** ; P<0.01,\n *** ; P<0.001, B.S; Before salpingectomy, A.S; After salpingectomy,\nand FC; Fertile control.\n\nThe cross-talk between trophoblast and endometrium is\nhighly mediated by the expression of specific receptivity\ngenes and inflammatory cytokines. A complex network\nof signaling is required for implantation. Alteration of\nthese signaling pathways often results in pathological\nconditions contributing to infertility ( 25 ).\nIn this study, we represent the first report of remarkable disruption in the expression\npattern of the lnc RNA HOTAIR and endometrial receptivity HOX genes mRNA ( HOXA9,\nHOXA11 , and  HOXD10  mRNA, in addition to\n HOXAl0 ), during implantation window in infertile patients with\nhydrosalpinges compared to samples derived from fertile healthy women. Our findings revealed\nthat cyclic upregulation of HOX genes involved in the receptivity of endometrium does not\noccur during the implantation window in infertile women with hydrosalpinges and\nsalpingectomy can restore expression of these genes to physiological levels in two-thirds of\npatients.\nIn line with our findings, several studies have shown a detrimental effect of hydrosalpinx\nfluid on endometrial receptivity markers, for instance,  HOXA10, LIF, Integrin αʋβ3,\n MUC1, and pinopodes expression levels and the benefit of salpingectomy as a\ntherapeutic option for management of hydrosalpinx prior IVF programs ( 13 -  15 ,  26 ,  27 ). In an\n in vitro  study, Daftary and Taylor ( 12 ), reported significantly lower\nexpression levels of  HOXA10  mRNA using hydrosalpinx fluid in a\nconcentrationdependent manner in Ishikawa cells. Subsequently, the authors in a prospective\nstudy examined the expression of endometrial  HOXA10  mRNA levels in women\nwith hydrosalpinges before and after salpingectomy and found that  HOXA10 \nmRNA levels returned to normal physiological levels (15-fold increase) after salpingectomy\n( 13 ), indicating surgical removal of hydrosalpinx restores  HOXA10 \nexpression to normal levels. Increased  HOXA10  mRNA levels were detected in\nboth glandular cells and endometrial stroma. Similarly, the result of two other studies\ndemonstrated the detrimental effect of hydrosalpinx on endometrial expression of integrin\nανβ3 and its restoration in 70% of patients following salpingectomy ( 14 ,  26 ). Likewise, Li\net al. ( 25 ) noted that the remarkable reduction of LIF, integrin b3, and MUC1 expression\nduring the mid-secretory phase might be one of the reasons for the low pregnancy rate in\nwomen with hydrosalpinx.\nIn women with normal cyclicity, expression of HOX genes ( HOXA9, HOXA10,\nHOXA11 , and  HOXD10 ) involved in endometrial development,\nreceptivity, decidualization, and implantation increase under the control of ovarian\nsteroids, especially progesterone and reach to the maximal level in mid-secretory phase. In\nhumans, the above-mentioned genes are important transcriptional regulators, which mediate\n(activate or repress) the multiple downstream target genes; including EMX2, IGFBP1, ITGB3,\nand are important for decidualization, and embryo implantation ( 17 ).\nHOXA10  dramatically regulates the expression of integrin αvβ3 and pinopode\nimplantation efficiency ( 27 ). A defect in the endometrial expression of the  HOXA10/\nHOXA11  has previously been described in several pathological conditions related\nto endometrium such as recurrent implantation failure, endometriosis, submucosal leiomyomas,\nand polycystic ovarian syndrome ( 28 - 30 ).\nThe hydrosalpingeal fluid in the damaged tubes is a\nresult of pelvic inflammatory disease and consists of\ninflammatory cells and pro-inflammatory cytokines,\nwhich prevents the formation of a receptive endometrium\n( 31 ,  32 ). Although acute inflammation of endometrium\nis a part of the embryo-uterine “dialogue” and promotes\nsuccessful trophoblast invasion ( 33 ), chronic inflammation\nis a pathological condition that may adversely affect\nuterine receptivity expression markers involving in\nendometrial apposition and embryo adhesion ( 31 ,  32 ).\nThe nuclear factor -Kappa B (NF-κB) pathway has\nshown that mainly regulates inflammatory processes in\nthe endometrium-induced hydrosalpinx fluid. We did not\nmeasure the expression level of NF-KB in our samples;\nhowever, several studies have strongly suggested that the\npresence of hydrosalpinx or endometrioma is associated\nwith considerably increased endometrial NF-κB ( 31 ,  32 ).\nMoreover, the result of a more recent study demonstrated that overexpression of\n TNF-α, IL-7 , and  NF-κB  genes may induce potentiation of\nthe proinflammatory environment in the endometrium, leading to the following downregulation\nof HOX genes involved in implantation ( 32 ).\nOn the other hand, the research on HOX genes and NF-KB pathways in inflammatory conditions\nindicates a negative relationship between NF-kB and receptivity genes ( 34 - 36 ). Based on the\nliterature, activation of the canonical NF-κB pathway can result in transcriptional\nrepression of HOX genes and the cross-talk between these two pathways plays an important\nrole in pathogenic inflammatory microenvironments such as breast cancer, atherosclerosis,\nand endometrioma ( 34 ,  35 ). Trivedi et al. ( 34 ) reported that HOXA9 expression rapidly\ndownregulates in response to TNF-α and NF-KB expressions in endothelial cells. Their\nfindings indicated the presence of an NF-κB binding site in the  HOXA9 \npromoter proximal to the transcription start site. Similar to these findings, Dokuzeylül\nGüngör et al. ( 36 ) found a significant inverse correlation between endometrial upregulated\nNF-kB and downregulation of  HOXA10, HOXA11 , and LIF expression levels in\nthe presence of type 0 or 1 fibroid that returned to normal values after hysteroscopic\nmyomectomy.\nAberrant DNA methylation is a possible epigenetic\nmechanism that is responsible for the aberrant expression\nof endometrial receptivity genes ( 37 ).\nA growth body of evidence supports NF-kB regulates\nthe epigenetic changes associated with inflammation\nthrough DNA methylation or demethylation in the\npromoter regions of HOXA10/HOXA11 in endometriosis,\nso increased endometrial NF-kB expression may be a\nprobable mechanism, which suppresses the expression of\nHOX genes through hypermethylation ( 37 ,  38 ).\nBesides, the retrograde flow of fluid into the uterine cavity is accompanied by the\naberrant expression of endometrial inflammatory microRNAs for example; miR-135a, miR-135b,\nand miR-145 ( 39 ,  40 ). Specifically, miR-135a and miR-135b suppress  HOXA10 \nexpression leading to impaired endometrial receptivity in women with endometriosis ( 39 ).\nLikewise, overexpression of miR-145 in hydrosalpinx-induce defective endometrium is\nsignificantly associated with down-regulation of  HOXA10  through\nfacilitating the secretion of TGF- β1, TNF-α, IL-6, and IL-8 by endometrial cells ( 40 ).\nAdditionally, we evaluated and compared HOTAIR\nexpression levels before and after surgery. We observed\nthat the endometrial HOTAIR expression level in patients\nwith hydrosalpinx was significantly higher than in controls\nand salpingectomy returned pre-implantation endometrial\nHOTAIR expression to physiological level.\nIn the current study, we selected HOTAIR because of its inflammatory nature and epigenetic\nmodulatory function ( 20 ). The aberrant endometrial HOTAIR expression has been seen in severe\nendometriosis and polycystic ovarian syndrome, which leads to downregulation of\n HOXD10  and alterations of the microenvironment of endometrium and\nreceptivity ( 22 ). However, in our study, the differences failed to show a statistically\ninverse correlation between  HOTAIR  and  HOXD10  expression\nlevels due to the limited sample size.\nBeyond its role in chromatin modification and gene repression, HOTAIR was found to promote\nan inflammatory response in different pathological situations such as diabetes, arthritis,\nacute myocardium infarction, intrauterine adhesion, endometriosis, cancer, and other\ninflammatory diseases ( 20 - 23 ). HOTAIR triggers the NF-KB pathway and immune response through\nthe reduction of its inhibitor, IκBα. Upon activation, NF-κB translocates to the nucleus and\nbinds to the promoters of pro-inflammatory cytokines, resulting in an upregulation of their\nexpression. The overexpression of  HOTAIR  has been reported with an\nincreased expression of  IFN-γ, IL6, IL-17, TNF-α, IL-1β , and  IL-6R\n in monocytes ( 20 ).\nThe aberrant endometrial  HOTAIR  expression has been seen in severe\nendometriosis, which leads to the downregulation of  HOXD10  and alterations\nof the microenvironment of endometrium and receptivity ( 22 ).\nThe possible limitation of our study could be the small number of patients. Although our\nfindings showed a significant increase in  HOTAIR  expression levels in\npreoperative samples compared to postoperative tissue, the results failed to show a\nstatistically inverse correlation between  HOTAIR  and\n HOXD10  expression levels due to the limited sample size.\n\nOur findings suggest that the presence of hydrosalpinx\nis associated with overexpression of the pro-inflammatory\nlncRNA HOTAIR, which may impair the regulation\nof HOX genes involved in endometrial receptivity.\nNevertheless, our current knowledge about the role of\nlncRNAs in endometrium exposed to hydrosalpinx fluid\nis limited. Additional research with more sample sizes\nis required to explore the HOTAIR pro-inflammatory\nsignaling pathway and its inverse co-expression with\nendometrial receptivity HOX genes in these subjects.","source_license":"CC0","license_restricted":false}