{"paper_id":"19a3e5c3-43a5-4a0c-b795-0240f9af0e3b","body_text":"Endometriosis, a disease characterized by implantation and growth of endometrial tissue outside the uterine cavity,\n 1 \n 2 \nhas a high prevalence, affecting between 6 and 10% of women in reproductive age.\n 1 \nIt is also frequently associated with infertility, being present in between 25 and 50% of infertile women,\n 3 \nwith 30 to 50% of endometriosis patients being infertile.\n 3 \n 4 \n 5 \n 6 \nHowever, the mechanisms underlying disease-related infertility are still poorly understood.\nEvidence have suggested that changes in the endometrial receptivity, due to molecular and functional disorders in the eutopic endometrium, may be related to impaired fertility in women with endometriosis.\n 5 \n 7 \n 8 \n 9 \nThe success of embryonic implantation depends on an adequate embryonic development, on the arrival of a competent embryo to a receptive endometrium, and on an efficient communication between the embryo and the endometrium.\n 10 \n 11 \n 12 \nIt is known that the human endometrium becomes receptive only during the implantation window,\n 10 \n 13 \n 14 \n 15 \n 16 \na certain period that results from the synchronized interaction of a variety of molecules (ovarian hormones, growth factors, transcription factors, cytokines, adhesion molecules), with an important role in establishing uterine receptivity.\n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n 22 \nThus, molecular changes in the eutopic endometrium of these patients could impair their endometrial receptivity, contributing to the infertility observed in women with the disease.\nHowever, a recent comprehensive and integrated evaluation of eutopic endometria of infertile women with endometriosis, infertile and fertile controls during the implantation window through a transcriptome analysis (RNA-Seq), did not identify differentially expressed transcripts among the groups.\n 23 \nLikewise, the miRNA sequencing in the eutopic endometrium of the same patients did not find changes in those post-transcriptional regulatory molecules.\n 23 \nTogether, the findings suggest that the eutopic endometrium of infertile women with the disease is molecularly similar to that of fertile women. However, the absence of alterations in mRNA and miRNA expression does not exclude the possibility of other molecular changes, with consequences for protein synthesis, which could impact the endometrial receptivity of these women. Single nucleotide variants (SNVs) are changes on a DNA sequence basis and comprise both polymorphisms (single-nucleotide polymorfisms [SNPs]) and point mutations, which may result in the wrong translation of transcripts into truncated, inactive and/or altered proteins.\n 24 \n 25 \nSince no study to date has evaluated SNVs in the eutopic endometrium of infertile women with endometriosis, we question whether the occurrence of functional mutations in the eutopic endometrium of those patients could impact the endometrial receptivity and contribute to disease-related infertility.\nTotal genome and/or exome sequencing are methodologies that allow the identification of point mutations in the DNA strands; however, with the disadvantage of having a high cost.\n 26 \nRNA sequencing can be a less costly alternative for the indirect study of mutations in transcripts, with the possibility of analyzing new variations that have occurred as a result of post-transcriptional changes.\n 27 \nIn this sense, the use of data generated by RNA-Seq has been proposed by the literature for the indirect analysis of SNVs and mutations.\n 28 \n 29 \n 30 \n 31 \n 32\nThus, the objectives of the present study were to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis, and of infertile and fertile controls during the implantation window, through the analysis of data previously generated by RNA-Seq, as well as to conduct a targeted study of the changes found in the context of endometriosis.\n\nA prospective case-control study was performed at the Human Reproduction Division of the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (HCFMRP-USP). The study was approved by the Research Ethics Committee of the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (HCFMRP-USP) (grant number 6383/2011). Patients who met the inclusion criteria and expressed their desire to participate in the study signed the informed consent form prior to inclusion.\nFrom November 2011 to November 2014, patients previously submitted to diagnostic videolaparoscopy or tubal ligation procedures in the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (HCFMRP-USP) were evaluated according to the eligibility criteria, and those considered eligible were interviewed. Patients who agreed to participate had an endometrial sample collected during the implantation window.\nWe considered eligible those patients who presented regular cycles (every 24 to 38 days, 4.5 to 8 days of duration and flow up to 80 ml per cycle)\n 33 \nfor at least 3 months prior to the study, aged between 18 and 45 years old, body mass index (BMI) ≤ 30 kg/m\n 2 \n, absence of polycystic ovary syndrome and of other etiologies of chronic anovulation, hydrosalpinx and chronic diseases such as diabetes mellitus or other endocrinopathies, cardiovascular disease, dyslipidemia, systemic lupus erythematosus and other rheumatologic diseases, HIV infection, any active infection, alcohol, drugs or smoking habit, and use of hormonal medication or of anti-inflammatory drugs during the 3 months preceding the beginning of the study were included.\nIn the END group, 6 patients with infertility exclusively associated to pelvic endometriosis diagnosed and classified by videolaparoscopy according to the criteria of the American Society for Reproductive Medicine\n 34 \nwere included. Among them, 2 patients were diagnosed with stage I endometriosis, 1 with stage II endometriosis, 1 with stage III endometriosis and 2 with stage IV endometriosis.\nIn the IC group, 6 patients with infertility attributable to male and/or tubal factors who had ruled out endometriosis and other pelvic diseases by videolaparoscopy were included. The FC group was composed by 5 patients undergoing tubal ligation who were proven fertile (at least one living child) without possible associated endometrial factors.\nThe patients had endometrial samples collected during the implantation window\n 35 \n(between the 20\n th \nand 24\n th \ndays of the cycle). For data standardization, the ovulation day was considered as the 14\n th \nday of a 28-day menstrual cycle.\nEutopic endometrial biopsies were collected during the implantation window from 17 patients (3 infertile women with endometriosis I/II, 3 infertile women with endometriosis III/IV, 6 infertile controls, and 5 fertile controls).\nTotal RNA was extracted with the RiboPure kit (Ambion, Life Technologies, Carlsbad, California, USA), treated with DNase (DNA KIT Free, Ambion - Life Technologies). Total RNA concentration was determined by spectrophotometry (NanoDrop 2000c; Thermo Scientific, Wilmington, DE, USA) at 260 nm, while total RNA integrity was evaluated with Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) according to the instructions of the manufacturer. Samples with RNA Integrity Number (RIN) ≥ 7.0 were considered appropriate. mRNA libraries were prepared using TruSeq RNA Sample Preparation v2 kit (Illumina, San Diego, CA, USA) according to the instructions of the manufacturer. RNA sequencing was performed using the commercial TruSeq SBS kit v5 kit (Illumina Inc.), as instructed by the manufacturer. In total, 17 libraries were distributed in 3 lanes and sequenced paired end (PE 2 × 101pb) in the HISEq. 2500 Illumina Platform, through High Output run. Data regarding the differential expression of transcripts were previously presented.\n 23\nMutation screening was performed on RNA-Seq data generated previously.\n 23 \nThe mapping of the generated fragments (reads) was performed with STAR (Spliced Transcripts Alignment to a Reference),\n 36 \nand variant calling was performed using the Genome Analysis Toolkit (GATK;\n https://gatk.broadinstitute.org/hc/en-us/articles/360035531192?id=3891 \n), following the best practices for variant discovery in RNA-Seq data,\n 37 \nfiltered using the hard filtering method (-window 35 -cluster 3 -FS > 30.0 -QD (Quality By Depth.) < 2.0 -DP (Coverage) > 10.0). The annotation of SNPs and Indels was performed with the VarAFT tool (\n https://varaft.eu/ \n).\nFunctional mutations were selected based on quality and selection criteria (such as: depth > 10, genome region, variant function and register in the NCBI database dbSNP) and on the pathogenicity scores of the following\n in silico \nprediction tools: CADD (Combined Annotation Dependent Depletion); PROVEAN (Protein Variation Effect Analyzer); SIFT (Sort Intolerant From Tolerant) and Polyphen2. Only those classified as damaging, deleterious or possibly damaging in the 4 predictors were considered functional.\nWith the identification of possibly deleterious mutations, in order to interpret the data in the context of the disease, we performed a targeted study of the selected variants in NCBI databases such as Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation (\n https://www.ncbi.nlm.nih.gov/snp/ \n), which brings described polymorphisms, and ClinVar (\n https://www.ncbi.nlm.nih.gov/clinvar/ \n), which brings disease-associated mutations.\nSpecifically, regarding the endometriosis group, in order to target the changes found in the context of the disease, we conducted a search in PubMed crossing the genes related to each mutation with the descriptors\n endometriosis \nand/or\n endometrium \n.\nAn exploratory data analysis was performed by measurements of central position and dispersion and box-plot graphs. The Kruskal-Wallis test was used for the comparison of clinical characteristics (age, height, weight, and BMI) among the groups.\n\nThe patients from the endometriosis, infertile control and fertile control groups were similar in relation to age, weight, height and BMI (\n Supplemental Table S1 \n(online only).\nAbbreviation: SNV, single nucleotide variant.\nAll samples that proceeded to RNA-Seq were evaluated for total RNA integrity in the 2100 BioanalyzerTM (Agilent Technologies) and were considered suitable for the technique (RIN ≥ 7). Paired-end libraries from the 17 RNA samples were sequenced: 6 women with endometriosis (3 with initial endometriosis and 3 with advanced endometriosis), 6 infertile controls and 5 fertile controls, distributed in 3 lanes, yielding ∼ 73 million reads each. Approximately 90% of the reads were mapped, with a phred-score > 30. Of the mapped reads, 1.5% were singleton, and 1% had multiple alignments, which have been removed from the analysis. The uniformity of reads mapped across all samples was considered good.\nThe analyzes performed in the GATK, following the best practices recommended for discovering variants in RNA-Seq data identified 885,515 variants. The detailed data by sample and group are shown in\n Table 1 \n.\nAfter filtering for quality, 793 variants were identified, 225 of which were exclusive to samples from the fertile control group, 261 from the infertile control group, and 170 from the endometriosis group, in addition to the 21 common to the fertile and infertile control groups, 21 to the fertile control and endometriosis groups, 22 common to the infertile control and endometriosis groups, and 3 common to the three groups (\n Fig. 1 \n). According to the predictors of pathogenicity, 42 variants were selected, 14 in the fertile control group, 19 in the infertile control group, and 9 in the endometriosis group.\n Table 2 \nshows the data for the variants in each group after applying the filters. Within the endometriosis group, two samples did not present any mutation predicted as deleterious. In the other groups, all samples showed at least one mutation.\nAbbreviations: Hom, Homozygous; het, heterozygous; 1000 g, frequency described in the 1000 Genomes bank.\nVenn diagram: number of single nucleotide variants (SNV) with depth ≥ 10, located in exonic and splicing regions, not synonymous, found in eutopic endometrial RNA-Seq data from infertile women with endometriosis (END), infertile controls (IC) and fertile controls (FC) during the implantation window.\nThe search of functional mutations was, then, performed in the dbSNP and ClinVar databases. The general data for each variant are presented in\n Table 3 \n. All the mutations found were classified as missense.\nAbbreviations: Chr, chromosome; ID, patient identification; Mut, mutated allele; NR, not reported; Ref, reference allele.\nAccording to the findings (\n Table 3 \n), in the fertile control group, two patients had mutations corresponding to clinical conditions. Among them, patient 1 presented two mutations with associated pathological conditions, being one related to cardiomyopathy and the other to Adams-Oliver syndrome 2, both with benign significance. Patient 2 presented one mutation related to spermatogenic failure and ciliary dyskinesia, also with benign significance. The infertile control group did not have any mutations with an associated clinical condition. In the endometriosis group, only patient 4 presented a mutation associated to a clinical condition (familial renal hypouricemia), with a benign significance.\nSpecifically, regarding the endometriosis group, when we performed a search in the PubMed database, by crossing the mutated genes identified with the descriptors\n endometriosis \nand/or\n endometrium \n, only the\n CMKLR1 \ngene was associated with those descriptors. Accordingly, the protein encoded by\n CMKLR1 \nis increased in the peritoneal fluid of women with endometriosis when compared with controls. In addition, its mRNA protein and receptor appear to be increased in ovarian endometrioma compared with the eutopic endometrium of control women.\n\nEndometriosis is a disease related to infertility whose underlying mechanisms that impair the fertility of women are still under investigation.\n 1 \nAn endometrial factor has been considered, since molecular and functional alterations of the eutopic endometrium could affect embryo implantation.\n 3 \n 5 \n 7 \n 8 \n 9 \nDespite a recent study that evidenced no differential expression in the mRNA and miRNA profile in the endometrium of those patients,\n 23 \nother molecular aberrations could impair protein synthesis and, consequently, endometrial receptivity. However, there is no study to date that evaluated eutopic endometrial mutations in endometriosis patients during the implantation window, which could bring important information regarding functional alterations in their endometrium. Because RNA-Seq data may be useful to identify variants in the transcriptome,\n 26 \n 27 \n 28 \n 29 \n 30 \n 31 \n 32 \nthe aim of the present study was to screen for functional mutations in the transcripts (mRNA) of eutopic endometria of infertile women with endometriosis and of controls during the implantation window, through the analysis of data previously generated by RNA-Seq.\n 38\nAccording to the findings, none of the variants found were common to other samples within the same group, suggesting no pattern of mutations in those patients. Also, no variant was repeated among women with endometriosis, infertile controls, and fertile controls. Interestingly, the endometriosis group had the lower number of variants, followed by the fertile control group, with the infertile control group having the highest number of mutations. However, it is important to highlight the small sample size of the groups, which may represent a bias and precludes groups comparison. Powered studies are necessary to confirm those results.\nAll the filtered mutations were classified as missense, which means that the substitution of a single base pair alters the genetic code and produces an aminoacid which is different from the usual, which is able to affect the protein function.\n 39 \nIt is known that the phenotypic effects of a mutation can be more severe the greater the difference in the chemical nature of the side chains of the aminoacid residues, and that they also depend on the role that this residue plays in the structure and function of the protein.\n 39 \nNevertheless, in the endometriosis group, only one patient presented a mutation associated with a clinical condition (familial renal hypouricemia). Renal hypouricemia is characterized by impaired reabsorption of uric acid in the apical membrane of proximal renal tubule cells caused by dysfunction of renal urate reabsorption transporters.\n 40 \nPatients are usually asymptomatic, but, in some cases, they may present exercise-induced acute renal failure and nephrolithiasis.\n 41 \n 42 \nHowever, the disease has no relation with the endometrium or with infertility.\nRegarding the endometriosis group, there are evidence relating one of the mutated genes (\n CMKLR1 \n) with endometriosis and/or the endometrium. The\n CMKLR1 \ngene encodes a protein called chemerin, which is an adipokine expressed in several human organs.\n 43 \n 44 \n 45 \nThis protein has been associated with several systemic and focal inflammatory processes.\n 43 \n 44 \n 45 \n 46 \n 47 \nIt modulates chemotaxis and activates inflammatory macrophages and cytokines.\n 48 \nThe\n CMKLR1 \ngene is also associated with important endometrial events for pregnancy, such as accumulation of deciduous natural killer (NK) cells and vascular remodeling. In this sense, chemerin levels seems to be higher in stromal endometrial cells of pregnant women compared with nonpregnant or menopausal fertile women, being regulated positively during decidualization.\n 49\nInterestingly, chemerin plays a role in pelvic inflammation related to endometriosis, and its concentration is increased in the peritoneal fluid of women with the disease when compared with controls. In addition, its mRNA, protein and receptor appear to be increased in ovarian endometrioma compared with the eutopic endometrium of control women.\n 38 \nHowever, there is no data about the expression of\n CMKLR1 \nin the eutopic endometrium of women with endometriosis comparing them to fertile controls. In this sense, given its role in the inflammatory process, chemerin could have a role in the impairment of fertility of those patients. The endometrial\n CMKLR1 \ngene mutation could be involved in reduced chemotaxis, less activation of macrophages and decreased release of inflammatory cytokines. Considering that the inflammatory process is important for endometrial receptivity and embryo implantation\n 50 \n 51 \n 52 \nand that chemerin plays a direct role in the establishment of pregnancy,\n 49 \nit is questioned whether the mutation of the\n CMKLR1 \ngene could be related to the impairment of those important events in women with endometriosis, being able to participate in the etiopathogenesis of disease-related infertility. However, this should be clarified in future studies with appropriate methodologies.\nThe present study has limitations, such as the small sample size, which does not allow us to state whether there are differential mutations among women with endometriosis compared with fertile and infertile controls, nor the identification of a pattern of mutations in the endometriosis group. Moreover, the search for variants was performed on RNA-Seq data, which may add bias by evaluating only expressed transcripts. It is unknown whether other mutations, in regulatory regions, for example, may characterize those patients and impact the phenotype.\nIn summary, no pattern of functional mutations was identified in the transcripts of the eutopic endometria from infertile women with endometriosis during the implantation window. However, it is necessary to consider the small sample size and that the analyses were performed on RNA-Seq data. Interestingly, one of the mutations found in one endometriosis patient was related to a gene (\n CMKLR1 \n) already associated with endometriosis, endometrial function, and initial gestational development.\n\nConsidering the aim of the present study of screening analysis and the importance of the\n CMKLR1 \ngene in endometrial modulation,\n CMKLR1 \ncould be suggested as a candidate gene for further studies evaluating mutations in the eutopic endometrium from endometriosis patients. Thus, according to the present findings, future studies with appropriate casuistry, which investigate the\n CMKLR1 \nmutation in DNA samples (and not in transcripts) and evaluate the respective protein (chemerin) in the eutopic endometria of infertile women with endometriosis may clarify this issue and contribute to the understanding of endometriosis-related infertility.","source_license":"CC0","license_restricted":false}