RBGO

Volume 43 - Junho 2021

DOI: 10.1055/s-0041-1730287

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, Rui Alberto Ferriani, Paula Andrea Navarro

92 Visualizações

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

Introduction

Endometriosis, a disease characterized by implantation and growth of endometrial tissue outside the uterine cavity,1 2 has a high prevalence, affecting between 6 and 10% of women in reproductive age.1 It is also frequently associated with infertility, being present in between 25 and 50% of infertile women,3 with 30 to 50% of endometriosis patients being infertile.3 4 5 6 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.5 7 8 9 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.10 11 12 It is known that the human endometrium becomes receptive only during the implantation window,10 13 14 15 16 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.16 17 18 19 20 21 22 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.23 Likewise, the miRNA sequencing in the eutopic endometrium of the same patients did not find changes in those post-transcriptional regulatory molecules.23 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.24 25 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.26 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.27 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.28 29 30 31 32

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)33 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 Medicine34 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 window35 (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.23

Mutation Screening and Annotation

Mutation screening was performed on RNA-Seq data generated previously.23 The mapping of the generated fragments (reads) was performed with STAR (Spliced Transcripts Alignment to a Reference),36 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,37 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

Group Pacient ID Variants Indel SNV Total after filtering/ prediction
Before filtering After filtering/ prediction Before filtering After filtering/ prediction Before filtering After filtering/ prediction
Endometriosis 1 72239 5 1286 0 70953 5 9
2 16482 0 975 0 15507 0
3 14955 0 210 0 14745 0
4 84156 1 4743 0 79413 1
5 69363 2 1111 0 68252 2
6 146610 1 8595 0 138015 1
Fertile control 1 79967 4 4694 0 75273 4 14
2 66279 5 1505 0 64774 5
3 98901 2 5775 0 93126 2
4 157215 1 9525 0 147690 1
5 84380 2 4940 0 79440 2
Infertile control 1 149952 2 9262 0 140690 2 19
2 118616 4 7285 0 111331 4
3 97232 2 5600 0 91632 2
4 89246 1 5148 0 84098 1
5 88790 7 1906 0 86884 7
6 84869 3 4976 0 79893 3
  • Abbreviation: SNV, single nucleotide variant.

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

Group Patient ID Chromosome Reference allele Mutant allele Genotype Depth SNV score Gene 1000 g dbSNP NCBI CADD
CF 1 2 C T het 10 62.77 TTN 0.076877 rs4894028 24.0
3 A G het 10 52.77 ZNF502 0.10603 rs56084453 17.61
17 G A het 10 109.77 EVPL 0.0081869 rs150149800 33.0
19 G A het 10 106.77 DOCK6 0.519569 rs12978266 22.9
5 1 G A het 10 103.77 ATAD3B 0.00239617 rs141377718 23.5
3 C T het 10 32.77 DNAH1 0.0299521 rs419752 34.0
6 T C het 10 66.77 GSTA3 0.000199681 rs139422505 21.8
8 C A het 10 58.77 MAPK15 0.095647 rs60732298 28.2
12 A C het 10 71.77 CLEC7A 0.00858626 rs16910527 25.2
8 1 C T het 10 124.77 OXCT2 rs150795467 22.6
19 T C het 10 81.77 ZNF836 0.0129792 rs61739527 18.91
9 1 A C het 10 24.78 PLEKHN1 rs181207265 20.5
32 1 G C het 10 224.77 ANKRD45 0.00199681 rs191985325 24.7
10 A G het 10 30.77 PPP1R3C 0.00199681 rs143318107 24.6
CI 2 1 C T het 10 127.77 KMO 0.000798722 rs200044625 28.8
11 A T het 10 166.77 CCDC88B 0.000399361 rs572682028 29.4
6 5 G A het 10 93.77 PCDHB5 0.0297524 rs17844422 18.71
11 G A het 10 54.77 SLC25A45 0.0101837 rs34400381 26.0
16 C A het 10 204.77 MT1A 0.470647 rs11640851 18.37
18 G A het 10 69.77 ALPK2 0.0203674 rs79863383 24.1
7 1 C G het 10 56.77 TRAF3IP3 0.00139776 rs147791408 22.8
10 G A het 10 31.77 CFAP58 rs143080879 29.2
17 1 G A het 10 67.77 C1orf87 rs772501233 26.5
19 3 G A het 10 234.77 CCDC13 0.167732 rs17238798 24.8
C G het 10 59.77 IQCG 0.281749 rs67877771 26.2
5 C T het 10 91.77 C5orf51 0.00159744 rs151191974 33.0
6 T C het 10 190.77 CRYBG1 0.0201677 rs61741114 27.0
G A het 10 113.77 LAMA4 0.0309505 rs11757455 34.0
11 C T het 10 152.77 RIN1 0.0183706 rs140145986 24.7
17 G A het 10 94.77 ITGAE 0.265375 rs1716 25.0
22 8 C T het 10 184.77 MICU3 0.000399361 rs201776772 26.8
9 G A het 10 140.77 FAM166B 0.0333466 rs75679360 33.0
12 G C het 10 49.77 CAPRIN2 0.0111821 rs73079976 28.0
END 3 4 C T het 10 136.77 NSG1 0.00139776 rs142822048 32.0
12 G A het 10 111.77 CMKLR1 0.000199681 rs201809939 29.0
14 G A hom 10 241.41 AHNAK2 0.538538 rs10438247 24.7
17 A T het 10 108.77 EFCAB13 0.0892572 rs72825679 24.7
20 T C het 10 97.77 DHX35 0.014976 rs36053162 23.0
27 4 C T het 10 227.77 SLC2A9 0.294129 rs3733591 22.8
28 17 G A het 10 44.77 ASB16 0.0141773 rs74491716 24.2
19 A T het 10 131.77 IZUMO4 0.0107827 rs45506200 25.6
31 5 C T het 10 224.77 JMY 0.0141773 rs116121324 24.5
  • Abbreviations: Hom, Homozygous; het, heterozygous; 1000 g, frequency described in the 1000 Genomes bank.

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

Group ID Chr Ref Mut NCBI register Gene Symbol Official name Codon impact Molecular consequence (dbSNP) Interpretation(ClinVar) Associated condition (ClinVar)
CF 1 2 C T rs4894028 TTN titin R (Arg) > H (His) Missense variant Benign / Likely benign Dilated Cardiomyopathy, Myopathy, Hypertrophic cardiomyopathy, Limb-Girdle Muscular Dystrophy, Distal myopathy Markesbery-Griggs type
3 A G rs56084453 ZNF502 zinc finger protein 502 Q (Gln) > R (Arg) Missense variant NR
17 G A rs150149800 EVPL envoplakin R (Arg) > C (Cys) Missense variant NR
19 G A rs12978266 DOCK6 dedicator of cytokinesis 6 P (Pro) > L (Leu) Missense variant Benign Adams-Oliver syndrome 2
2 1 G A rs141377718 ATAD3B ATPase family AAA domain containing 3B V (Val) > M (Met) Missense variant NR
3 C T rs419752 DNAH1 dynein axonemal heavy chain 1 R (Arg) > C (Cys) Missense variant Benign • Ciliary dyskinesia, Spermatogenic failure
6 T C rs139422505 GSTA3 glutathione S-transferase α 3 N (Asn) > S (Ser) Missense variant NR
8 C A rs60732298 MAPK15 Mitogen-activated protein kinase 15 T (Thr) > K (Lys) Missense variant NR
12 A C rs16910527 CLEC7A C-type lectin domain containing 7A I (Ile) > S (Ser) Missense variant NR
3 1 C T rs150795467 OXCT2 3-oxoacid CoA-transferase 2 D (Asp) > N (Asn) Missense variant NR
19 T C rs61739527 ZNF836 zinc finger protein 836 E (Glu) > G (Gly) Missense variant NR
4 1 A C rs181207265 PLEKHN1 pleckstrin homology domain containing N1 T (Thr) > P (Pro) Missense variant NR
5 1 G C rs191985325 ANKRD45 ankyrin repeat domain 45 L (Leu) > V (Val) Missense variant NR
10 A G rs143318107 PPP1R3C protein phosphatase 1 regulatory subunit 3C F (Phe) > S (Ser) Missense variant NR
CI 1 1 C T rs200044625 KMO kynurenine 3-monooxygenase T (Thr) > I (Ile) Missense variant NR
11 A T rs572682028 CCDC88B coiled-coil domain containing 88B E (Glu) > V (Val) Missense variant NR
2 5 G A rs17844422 PCDHB5 protocadherin β 5 S (Ser) > N (Asn) Missense variant NR
11 G A rs34400381 SLC25A45 solute carrier family 25 member 45 R (Arg) > C (Cys) Missense variant NR
16 C A rs11640851 MT1A metallothionein 1A T (Thr) > N (Asn) Missense variant NR
18 G A rs79863383 ALPK2 α kinase 2 T (Thr) > I (Ile) Missense variant NR
3 1 C G rs147791408 TRAF3IP3 TRAF3 interacting protein 3 D (Asp) > E (Glu) Missense variant NR
10 G A rs143080879 CFAP58 cilia and flagella associated protein 58 R (Arg) > H (His) Missense variant NR
4 1 G A rs772501233 C1orf87 chromosome 1 open reading frame 87 A (Ala) > V (Val) Missense variant NR
5 3 G A rs17238798 CCDC13 coiled-coil domain containing 13 R (Arg) > W (Trp) Missense variant NR
3 C G rs67877771 IQCG IQ motif containing G D (Asp) > H (His) Missense variant NR
5 C T rs151191974 C5orf51 chromosome 5 open reading frame 51 P (Pro) > L (Leu) Missense variant NR
6 T C rs61741114 CRYBG1 crystallin β-gamma domain containing 1 L (Leu) > P (Pro) Missense variant NR
6 G A rs11757455 LAMA4 laminin subunit α 4 R (Arg) > W (Trp) Missense variant Benign
11 C T rs140145986 RIN1 Ras and Rab interactor 1 A (Ala) > T (Thr) Missense variant NR
17 G A rs1716 ITGAE integrin subunit α E R (Arg) > W (Trp) Missense variant NR
END 1 4 C T rs142822048 NSG1 neuronal vesicle trafficking associated 1 P (Pro) > S (Ser) Missense variant NR
  12 G A rs201809939 CMKLR1 chemerin chemokine-like receptor 1 R (Arg) > C (Cys) Missense variant NR
  14 G A rs10438247 AHNAK2 AHNAK nucleoprotein 2 P (Pro) > L (Leu) Missense variant NR
  17 A T rs72825679 EFCAB13 EF-hand calcium-binding domain-containing protein 13 D (Asp) > V (Val) Missense variant NR
  20 T C rs36053162 DHX35 DEAH-box helicase 35 I (Ile) > T (Thr) Missense variant NR
4 4 C T rs3733591 SLC2A9 solute carrier family 2 member 9 R (Arg) > H (His) Missense variant Benign Familial renal hypouricemia
5 17 G A rs74491716 ASB16 ankyrin repeat and SOCS box containing 16 A (Ala) > T (Thr) Missense variant NR
  19 A T rs45506200 IZUMO4 IZUMO family member 4 Y (Tyr) > F (Phe) Missense variant NR
6 5 C T rs116121324 JMY junction mediating and regulatory protein, p53 cofactor P (Pro) > L (Leu) Missense variant NR
  • Abbreviations: Chr, chromosome; ID, patient identification; Mut, mutated allele; NR, not reported; Ref, reference allele.

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.1 An endometrial factor has been considered, since molecular and functional alterations of the eutopic endometrium could affect embryo implantation.3 5 7 8 9 Despite a recent study that evidenced no differential expression in the mRNA and miRNA profile in the endometrium of those patients,23 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,26 27 28 29 30 31 32 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.38

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.39 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.39 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.40 Patients are usually asymptomatic, but, in some cases, they may present exercise-induced acute renal failure and nephrolithiasis.41 42 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.43 44 45 This protein has been associated with several systemic and focal inflammatory processes.43 44 45 46 47 It modulates chemotaxis and activates inflammatory macrophages and cytokines.48 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.49

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.38 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 implantation50 51 52 and that chemerin plays a direct role in the establishment of pregnancy,49 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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