Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window

Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Michele Gomes Da Broi Jessica Rodrigues Plaça Wilson Araújo da Silva Jr Rui Alberto Ferriani Paula Andrea Navarro About the authors

Abstract

Objective

Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window.

Methods

Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease’s context.

Results

None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment.

Conclusion

Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Keywords:
endometriosis; infertility; eutopic endometrium; RNA-sequencing; mutation

Resumo

Objetivo

Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação.

Métodos

Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença.

Resultados

Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez.

Conclusão

Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

Palavras-chave:
endometriose; infertilidade; endométrio eutópico; sequenciamento de RNA; mutação

Introduction

Endometriosis, a disease characterized by implantation and growth of endometrial tissue outside the uterine cavity,11 Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 2012;98(03):511-519. Doi: 10.1016/j.fertnstert.2012.06.029
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has a high prevalence, affecting between 6 and 10% of women in reproductive age.11 Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 2012;98(03):511-519. Doi: 10.1016/j.fertnstert.2012.06.029
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It is also frequently associated with infertility, being present in between 25 and 50% of infertile women,33 Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility: a committee opinion. Fertil Steril. 2012;98(03):591-598. Doi: 10.1016/j.fertnstert.2012.05.031
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with 30 to 50% of endometriosis patients being infertile.33 Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility: a committee opinion. Fertil Steril. 2012;98(03):591-598. Doi: 10.1016/j.fertnstert.2012.05.031
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However, the mechanisms underlying disease-related infertility are still poorly understood.

Evidence 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.55 Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364(9447):1789- -1799. Doi: 10.1016/S0140-6736(04)17403-5
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The 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.1010 Giudice LC, Telles TL, Lobo S, Kao L. The molecular basis for implantation failure in endometriosis: on the road to discovery. Ann N Y Acad Sci. 2002;955:252-264, discussion 293-295, 396-406. Doi: 10.1111/j.1749-6632.2002.tb02786.x
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It is known that the human endometrium becomes receptive only during the implantation window,1010 Giudice LC, Telles TL, Lobo S, Kao L. The molecular basis for implantation failure in endometriosis: on the road to discovery. Ann N Y Acad Sci. 2002;955:252-264, discussion 293-295, 396-406. Doi: 10.1111/j.1749-6632.2002.tb02786.x
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1313 Kresowik JD, Devor EJ, Van Voorhis BJ, Leslie KK. MicroRNA-31 is significantly elevated in both human endometrium and serum during the window of implantation: a potential biomarker for optimum receptivity. Biol Reprod. 2014;91(01):17. Doi: 10.1095/biolreprod.113.116590
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a 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.1616 Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med. 1999;340(23): 1796-1799. Doi: 10.1056/NEJM199906103402304
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1717 Altmäe S, Esteban FJ, Stavreus-Evers A, Simón CS, Giudice L, Lessey BA, et al. Guidelines for the design, analysis and interpretation of 'omics' data: focus on human endometrium. HumReprod Update. 2014;20(01):12-28. Doi: 10.1093/humupd/dmt048
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Thus, molecular changes in the eutopic endometrium of these patients could impair their endometrial receptivity, contributing to the infertility observed in women with the disease.

However, 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.2323 Da BroiMG, Meola J, Plaça JR, Peronni KC, Rocha CV, SilvaWA, et al. Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window? Hum Reprod. 2019;34(12):2381-2390. Doi: 10.1093/humrep/dez225
https://doi.org/10.1093/humrep/dez225...
Likewise, the miRNA sequencing in the eutopic endometrium of the same patients did not find changes in those post-transcriptional regulatory molecules.2323 Da BroiMG, Meola J, Plaça JR, Peronni KC, Rocha CV, SilvaWA, et al. Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window? Hum Reprod. 2019;34(12):2381-2390. Doi: 10.1093/humrep/dez225
https://doi.org/10.1093/humrep/dez225...
Together, 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.2424 Katsonis P, Koire A, Wilson SJ, Hsu T-K, Lua RC, Wilkins AD, et al. Single nucleotide variations: biological impact and theoretical interpretation. Protein Sci. 2014;23(12):1650-1666. Doi: 10.1002/pro.2552
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2525 Mueller SC, Backes C, Kalinina OV, Meder B, Stöckel D, Lenhof H-S, et al. BALL-SNP: combining genetic and structural information to identify candidate non-synonymous single nucleotide polymorphisms. Genome Med. 2015;7(01):65. Doi: 10.1186/s13073-015-0190-y
https://doi.org/10.1186/s13073-015-0190-...
Since 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.

Total 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.2626 Cirulli ET, Singh A, Shianna KV, Ge D, Smith JP, Maia JM, et al; Center for HIV/AIDS Vaccine Immunology (CHAVI) Screening the human exome: a comparison of whole genome and whole transcriptome sequencing. Genome Biol. 2010;11(05):R57. Doi: 10.1186/gb-2010-11-5-r57
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RNA 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.2727 Han L, Vickers KC, Samuels DC, Guo Y. Alternative applications for distinct RNA sequencing strategies. Brief Bioinform. 2015;16(04): 629-639. Doi: 10.1093/bib/bbu032
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In this sense, the use of data generated by RNA-Seq has been proposed by the literature for the indirect analysis of SNVs and mutations.2828 Sheng Q, Zhao S, Li CI, Shyr Y, Guo Y. Practicability of detecting somatic point mutation from RNA high throughput sequencing data. Genomics. 2016;107(05):163-169. Doi: 10.1016/j.ygeno.2016.03.006
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2929 Quinn EM, Cormican P, Kenny EM, Hill M, Anney R, Gill M, et al. Development of strategies for SNP detection in RNA-seq data: application to lymphoblastoid cell lines and evaluation using 1000 Genomes data. PLoS One. 2013;8(03):e58815. Doi:10.1371/journal.pone.0058815
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3030 Chepelev I, Wei G, Tang Q, Zhao K. Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq. Nucleic Acids Res. 2009;37(16):e106. Doi: 10.1093/nar/gkp507
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3131 Cánovas A, Rincon G, Islas-Trejo A,Wickramasinghe S, Medrano JF. SNP discovery in the bovine milk transcriptome using RNA-Seq technology. Mamm Genome. 2010;21(11-12):592-598. Doi: 10.1007/s00335-010-9297-z
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3232 Peng Z, Cheng Y, Tan BC, Tian Z, Zhu Y, Zhang W, et al. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome. Nat Biotechnol. 2012;30(03):253-260. Doi: 10.1038/nbt.2122
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Thus, 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.

Methods

Study Design

A 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.

From 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.

Patients – Eligibility Criteria

We 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)3333 Fraser IS, Critchley HO, Munro MG, Broder MWriting Group for this Menstrual Agreement Process. A process designed to lead to international agreement on terminologies and definitions used to describe abnormalities of menstrual bleeding. Fertil Steril. 2007; 87(03):466-476. Doi: 10.1016/j.fertnstert.2007.01.023
https://doi.org/10.1016/j.fertnstert.200...
for at least 3 months prior to the study, aged between 18 and 45 years old, body mass index (BMI) ≤ 30 kg/m2, 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.

In 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 Medicine3434 Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril. 1997;67(05):817-821. Doi: 10.1016/s0015-0282(97)81391-x
https://doi.org/10.1016/s0015-0282(97)81...
were 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.

In 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.

Sample Collection and RNA-sequencing

The patients had endometrial samples collected during the implantation window3535 Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122(02):262-263. Doi: 10.1016/s0002- 9378(16)33500-1
https://doi.org/10.1016/s0002-...
(between the 20th and 24th days of the cycle). For data standardization, the ovulation day was considered as the 14th day of a 28-day menstrual cycle.

Eutopic 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).

Total 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.2323 Da BroiMG, Meola J, Plaça JR, Peronni KC, Rocha CV, SilvaWA, et al. Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window? Hum Reprod. 2019;34(12):2381-2390. Doi: 10.1093/humrep/dez225
https://doi.org/10.1093/humrep/dez225...

Mutation Screening and Annotation

Mutation screening was performed on RNA-Seq data generated previously.2323 Da BroiMG, Meola J, Plaça JR, Peronni KC, Rocha CV, SilvaWA, et al. Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window? Hum Reprod. 2019;34(12):2381-2390. Doi: 10.1093/humrep/dez225
https://doi.org/10.1093/humrep/dez225...
The mapping of the generated fragments (reads) was performed with STAR (Spliced Transcripts Alignment to a Reference),3636 Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(01):15-21. Doi: 10.1093/bioinformatics/bts635
https://doi.org/10.1093/bioinformatics/b...
and variant calling was performed using the Genome Analysis Toolkit (GATK; https://gatk.broadinstitute.org/hc/en-us/articles/360035531192?id=3891), following the best practices for variant discovery in RNA-Seq data,3737 Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics. 2013;43 (1110):1-33 filtered 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 (https://varaft.eu/).

In Silico Analysis to Identify Functional Mutations

Functional 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 in silico prediction 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.

With 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 (https://www.ncbi.nlm.nih.gov/snp/), which brings described polymorphisms, and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), which brings disease-associated mutations.

Specifically, 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 endometriosis and/or endometrium.

Statistical Analysis

An 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.

Results

Clinical Characteristics of the Patients

The patients from the endometriosis, infertile control and fertile control groups were similar in relation to age, weight, height and BMI (Supplemental Table S1 (online only).

Table 1
Number and type of variants identified in the transcripts of eutopic endometrium of infertile women with endometriosis, women with tubal and/or male infertility factor (infertile control) and fertile women (fertile control) during the implantation window, from RNA-Seq data before and after application of filters

RNA sequencing

All 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.

Variant Discovery

The 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 Table 1.

After 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 (Fig. 1). 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. Table 2 shows 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.

Table 2
Variants identified after filtering and predicting data obtained from eutopic endometrium RNA-Seq of infertile women with endometriosis, women with tubal and/or male infertility factor (infertile control), and fertile women (fertile control) during the implantation window

Fig. 1
Venn 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.

Targeted Study of Variants Found

The search of functional mutations was, then, performed in the dbSNP and ClinVar databases. The general data for each variant are presented in Table 3. All the mutations found were classified as missense.

Table 3
Data from the dbSNP and ClinVar databases for the predicted pathogenic variants identified in eutopic endometrial RNA-Seq data from fertile women (fertile control; FC), women with tubal and/or male infertility factor (infertile control; IC), and infertile women with endometriosis (END) during the implantation window

According to the findings (Table 3), 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.

Specifically, regarding the endometriosis group, when we performed a search in the PubMed database, by crossing the mutated genes identified with the descriptors endometriosis and/or endometrium, only the CMKLR1 gene was associated with those descriptors. Accordingly, the protein encoded by CMKLR1 is 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.

Discussion

Endometriosis is a disease related to infertility whose underlying mechanisms that impair the fertility of women are still under investigation.11 Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 2012;98(03):511-519. Doi: 10.1016/j.fertnstert.2012.06.029
https://doi.org/10.1016/j.fertnstert.201...
An endometrial factor has been considered, since molecular and functional alterations of the eutopic endometrium could affect embryo implantation.33 Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility: a committee opinion. Fertil Steril. 2012;98(03):591-598. Doi: 10.1016/j.fertnstert.2012.05.031
https://doi.org/10.1016/j.fertnstert.201...
55 Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364(9447):1789- -1799. Doi: 10.1016/S0140-6736(04)17403-5
https://doi.org/10.1016/S0140-6736(04)17...
77 Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility. Fertil Steril. 2006;86(05, Suppl 1):S156-S160. Doi: 10.1016/j.fertnstert.2006.08.014
https://doi.org/10.1016/j.fertnstert.200...
88 Wei Q, St Clair JB, Fu T, Stratton P, Nieman LK. Reduced expression of biomarkers associatedwith the implantationwindow in women with endometriosis. Fertil Steril. 2009;91(05):1686-1691. Doi: 10.1016/j.fertnstert.2008.02.121
https://doi.org/10.1016/j.fertnstert.200...
99 Bulletti C, Coccia ME, Battistoni S, Borini A. Endometriosis and infertility. J Assist Reprod Genet. 2010;27(08):441-447. Doi: 10.1007/s10815-010-9436-1
https://doi.org/10.1007/s10815-010-9436-...
Despite a recent study that evidenced no differential expression in the mRNA and miRNA profile in the endometrium of those patients,2323 Da BroiMG, Meola J, Plaça JR, Peronni KC, Rocha CV, SilvaWA, et al. Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window? Hum Reprod. 2019;34(12):2381-2390. Doi: 10.1093/humrep/dez225
https://doi.org/10.1093/humrep/dez225...
other 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,2626 Cirulli ET, Singh A, Shianna KV, Ge D, Smith JP, Maia JM, et al; Center for HIV/AIDS Vaccine Immunology (CHAVI) Screening the human exome: a comparison of whole genome and whole transcriptome sequencing. Genome Biol. 2010;11(05):R57. Doi: 10.1186/gb-2010-11-5-r57
https://doi.org/10.1186/gb-2010-11-5-r57...
2727 Han L, Vickers KC, Samuels DC, Guo Y. Alternative applications for distinct RNA sequencing strategies. Brief Bioinform. 2015;16(04): 629-639. Doi: 10.1093/bib/bbu032
https://doi.org/10.1093/bib/bbu032...
2828 Sheng Q, Zhao S, Li CI, Shyr Y, Guo Y. Practicability of detecting somatic point mutation from RNA high throughput sequencing data. Genomics. 2016;107(05):163-169. Doi: 10.1016/j.ygeno.2016.03.006
https://doi.org/10.1016/j.ygeno.2016.03....
2929 Quinn EM, Cormican P, Kenny EM, Hill M, Anney R, Gill M, et al. Development of strategies for SNP detection in RNA-seq data: application to lymphoblastoid cell lines and evaluation using 1000 Genomes data. PLoS One. 2013;8(03):e58815. Doi:10.1371/journal.pone.0058815
https://doi.org/10.1371/journal.pone.005...
3030 Chepelev I, Wei G, Tang Q, Zhao K. Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq. Nucleic Acids Res. 2009;37(16):e106. Doi: 10.1093/nar/gkp507
https://doi.org/10.1093/nar/gkp507...
3131 Cánovas A, Rincon G, Islas-Trejo A,Wickramasinghe S, Medrano JF. SNP discovery in the bovine milk transcriptome using RNA-Seq technology. Mamm Genome. 2010;21(11-12):592-598. Doi: 10.1007/s00335-010-9297-z
https://doi.org/10.1007/s00335-010-9297-...
3232 Peng Z, Cheng Y, Tan BC, Tian Z, Zhu Y, Zhang W, et al. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome. Nat Biotechnol. 2012;30(03):253-260. Doi: 10.1038/nbt.2122
https://doi.org/10.1038/nbt.2122...
the 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.3838 Jin CH, Yi KW, Ha YR, Shin J-H, Park HT, Kim T, et al. Chemerin expression in the peritoneal fluid, serum, and ovarian endometrioma of women with endometriosis. Am J Reprod Immunol. 2015;74(04):379-386. Doi: 10.1111/aji.12405
https://doi.org/10.1111/aji.12405...

According 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.

All 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.3939 Stefl S, Nishi H, Petukh M, Panchenko AR, Alexov E. Molecular mechanisms of disease-causing missense mutations. J Mol Biol. 2013;425(21):3919-3936. Doi: 10.1016/j.jmb.2013.07.014
https://doi.org/10.1016/j.jmb.2013.07.01...
It 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.3939 Stefl S, Nishi H, Petukh M, Panchenko AR, Alexov E. Molecular mechanisms of disease-causing missense mutations. J Mol Biol. 2013;425(21):3919-3936. Doi: 10.1016/j.jmb.2013.07.014
https://doi.org/10.1016/j.jmb.2013.07.01...
Nevertheless, 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.4040 Nakayama A, Matsuo H, Ohtahara A, Ogino K, Hakoda M, Hamada T, et al. Clinical practice guideline for renal hypouricemia (1st edition). Hum Cell. 2019;32(02):83-87 Patients are usually asymptomatic, but, in some cases, they may present exercise-induced acute renal failure and nephrolithiasis.4141 Dinour D, Gray NK, Campbell S, Shu X, Sawyer L, Richardson W, et al. Homozygous SLC2A9 mutations cause severe renal hypouricemia. J Am Soc Nephrol. 2010;21(01):64-72. Doi: 10.1681/ASN.2009040406
https://doi.org/10.1681/ASN.2009040406...
4242 WindpesslM, Ritelli M,WallnerM, Colombi M. A novel homozygous SLC2A9 mutation associatedwith renal-induced hypouricemia. AmJ Nephrol. 2016;43(04):245-250. Doi: 10.1159/000445845
https://doi.org/10.1159/000445845...
However, the disease has no relation with the endometrium or with infertility.

Regarding the endometriosis group, there are evidence relating one of the mutated genes (CMKLR1) with endometriosis and/or the endometrium. The CMKLR1 gene encodes a protein called chemerin, which is an adipokine expressed in several human organs.4343 Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, et al. Chemerin is a novel adipokine associated with obesity and metabolic syndrome. Endocrinology. 2007;148(10):4687-4694. Doi: 10.1210/en.2007-0175
https://doi.org/10.1210/en.2007-0175...
4444 Bozaoglu K, Segal D, Shields KA, Cummings N, Curran JE, Comuzzie AG, et al. Chemerin is associated with metabolic syndrome phenotypes in a Mexican-American population. J Clin Endocrinol Metab. 2009;94(08):3085-3088. Doi: 10.1210/jc.2008-1833
https://doi.org/10.1210/jc.2008-1833...
4545 Bozaoglu K, Curran JE, Stocker CJ, Zaibi MS, Segal D, Konstantopoulos N, et al. Chemerin, a novel adipokine in the regulation of angiogenesis. J Clin Endocrinol Metab. 2010;95(05):2476-2485. Doi: 10.1210/jc.2010-0042
https://doi.org/10.1210/jc.2010-0042...
This protein has been associated with several systemic and focal inflammatory processes.4343 Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, et al. Chemerin is a novel adipokine associated with obesity and metabolic syndrome. Endocrinology. 2007;148(10):4687-4694. Doi: 10.1210/en.2007-0175
https://doi.org/10.1210/en.2007-0175...
4444 Bozaoglu K, Segal D, Shields KA, Cummings N, Curran JE, Comuzzie AG, et al. Chemerin is associated with metabolic syndrome phenotypes in a Mexican-American population. J Clin Endocrinol Metab. 2009;94(08):3085-3088. Doi: 10.1210/jc.2008-1833
https://doi.org/10.1210/jc.2008-1833...
4545 Bozaoglu K, Curran JE, Stocker CJ, Zaibi MS, Segal D, Konstantopoulos N, et al. Chemerin, a novel adipokine in the regulation of angiogenesis. J Clin Endocrinol Metab. 2010;95(05):2476-2485. Doi: 10.1210/jc.2010-0042
https://doi.org/10.1210/jc.2010-0042...
4646 Bondue B,Wittamer V, Parmentier M. Chemerin and its receptors in leukocyte trafficking, inflammation and metabolism. Cytokine Growth Factor Rev. 2011;22(5-6):331-338. Doi: 10.1016/j.cytogfr.2011.11.004
https://doi.org/10.1016/j.cytogfr.2011.1...
4747 Roh SG, Song SH, Choi KC, Katoh K, Wittamer V, Parmentier M, et al. Chemerin-a new adipokine thatmodulates adipogenesis via its own receptor. Biochem Biophys Res Commun. 2007;362(04): 1013-1018. Doi: 10.1016/j.bbrc.2007.08.104
https://doi.org/10.1016/j.bbrc.2007.08.1...
It modulates chemotaxis and activates inflammatory macrophages and cytokines.4848 Weigert J, Neumeier M,Wanninger J, Filarski M, Bauer S,Wiest R, et al. Systemic chemerin is related to inflammation rather than obesity in type 2 diabetes. Clin Endocrinol (Oxf). 2010;72(03): 342-348. Doi: 10.1111/j.1365-2265.2009.03664.x
https://doi.org/10.1111/j.1365-2265.2009...
The CMKLR1 gene 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.4949 Carlino C, Trotta E, Stabile H, Morrone S, Bulla R, Soriani A, et al. Chemerin regulates NK cell accumulation and endothelial cell morphogenesis in the decidua during early pregnancy. J Clin Endocrinol Metab. 2012;97(10):3603-3612. Doi: 10.1210/jc.2012-1102
https://doi.org/10.1210/jc.2012-1102...

Interestingly, 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.3838 Jin CH, Yi KW, Ha YR, Shin J-H, Park HT, Kim T, et al. Chemerin expression in the peritoneal fluid, serum, and ovarian endometrioma of women with endometriosis. Am J Reprod Immunol. 2015;74(04):379-386. Doi: 10.1111/aji.12405
https://doi.org/10.1111/aji.12405...
However, there is no data about the expression of CMKLR1 in 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 CMKLR1 gene 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 implantation5050 Maybin JA, Critchley HO, Jabbour HN. Inflammatory pathways in endometrial disorders. Mol Cell Endocrinol. 2011;335(01):42-51. Doi: 10.1016/j.mce.2010.08.006
https://doi.org/10.1016/j.mce.2010.08.00...
5151 King AE, Critchley HO. Oestrogen and progesterone regulation of inflammatory processes in the human endometrium. J Steroid Biochem Mol Biol. 2010;120(2-3):116-126. Doi: 10.1016/j.jsbmb.2010.01.003
https://doi.org/10.1016/j.jsbmb.2010.01....
5252 Roberts M, Luo X, Chegini N. Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-alpha and TGF-beta in human endometrial epithelial and stromal cells. Mol Hum Reprod. 2005;11(10):751-760. Doi: 10.1093/molehr/gah233
https://doi.org/10.1093/molehr/gah233...
and that chemerin plays a direct role in the establishment of pregnancy,4949 Carlino C, Trotta E, Stabile H, Morrone S, Bulla R, Soriani A, et al. Chemerin regulates NK cell accumulation and endothelial cell morphogenesis in the decidua during early pregnancy. J Clin Endocrinol Metab. 2012;97(10):3603-3612. Doi: 10.1210/jc.2012-1102
https://doi.org/10.1210/jc.2012-1102...
it is questioned whether the mutation of the CMKLR1 gene 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.

The 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.

In 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 (CMKLR1) already associated with endometriosis, endometrial function, and initial gestational development.

Conclusion

Considering the aim of the present study of screening analysis and the importance of the CMKLR1 gene in endometrial modulation, CMKLR1 could 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 CMKLR1 mutation 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.

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  • Funding

    The present study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Publication Dates

  • Publication in this collection
    06 Sept 2021
  • Date of issue
    June 2021

History

  • Received
    14 Aug 2020
  • Accepted
    12 Feb 2021
  • Published
    27 July 2021
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