From bench to in silico and backwards: What have we done on genetics of recurrent pregnancy loss and implantation failure and where should we go next?

Human reproduction goes through many challenges to its success and in many cases it fails. Cases of pregnancy loss are common outcomes for pregnancies, and implantation failures (IF) are common in assisted reproduction attempts. Although several risk factors have already been linked to adverse outcomes in reproduction, many cases remain without a definitive cause. Genetics of female reproduction is a field that may bring some pieces of this puzzle; however, there are no well-defined genes that might be related to the risk for recurrent pregnancy loss (RPL) and IF. Here, we present a literature review of the studies of genetic association in RPL and IF carried out in the Brazilian population and complemented with a database search to explore genes previously related to RPL and IF, where a search for genes previously involved in these conditions was performed in OMIM, HuGE, and CTD databases. Finally, we present the next steps for reproductive genetics investigation, through genomic sequencing analyses and discuss future plans in the study of RPL genetics. The combined strategy of looking for literature and databases is useful to raise hypotheses and to identify underexplored genes related to RPL and IF.

Genetic and in silico analysis show a role of SMAD3 on recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is one of the most common reproductive failures affecting 1-5% of couples. Smad3 is an effector of signalling of the Transforming Growth Factors-ß superfamily (TGF-ß), regulating the transcription of several target genes of these cytokines. The objective of this study was to evaluate the influence of a variant on SMAD3 (rs17293443) in RPL. A case-control study was carried out with 149 women who experienced RPL and 159 controls, as well as bioinformatics tools to determine the role of this variant in this condition. Our study showed an allelic (p = 0.023) and genotypic (p < 0.01) association of this variant with the RPL. Our functional in silico predictions suggest that this variant causes a change in SMAD3 expression levels. Alterations in the expression of this gene can directly compromise the Smad3-dependent signalling pathway that is fundamental for key processes for gestation such as steroid hormone regulation and implantation, as demonstrated by ontologies analyses performed and the literature. Our findings regarding the involvement of Smad3 on RPL are a novelty in this field, and they seem to be promising to the clinical management of this condition.

Investigating the role of EGF-CFC gene family in recurrent pregnancy loss through bioinformatics and molecular approaches.

Recurrent pregnancy loss (RPL) is the most common reproductive failure, reaching 1-5% of women throughout their lives, and having unknown etiology in 50% of the cases. In humans, EGF-CFC1 (Epidermal Growth Factors & Cripto/FRL-1/Cryptic) gene family is composed by TDGF1 and CFC1, two developmental genes. The aim of this study was to investigate the role of EGF-CFC on RPL. To this, multiple approaches were performed; we conducted an expression analysis of TDGF1 and CFC1 using publicly available data from Gene Omnibus Expression (GEO), systems biology analyses and functional prediction; and a molecular analysis carried out in a case-control study. Our GEO analysis showed a decrease in TDGF1 expression in the endometrium (p=0.049) and CFC1 expression in placenta (p=0.015) of women with RPL. Network analysis, gene ontology and literature pointed to a strong connection between EGF-CFC1 gene family to pathways that play key roles during pregnancy, including TGF-ß, c-Src/MAPK/AKT, Notch, TNFα, IFNγ and IL-6. A pathogenicity score developed for this gene family showed that the c.-14+1429T>C (rs3806702) variant in the TDGF1 and the p.Arg47Gln (rs201431919) variant in CFC1 gene would be the ones with the highest deleterious effect for RPL. In the case-control study, which involved 149 women with RPL and 159 controls, no statistical difference was observed in the allele and genotype distributions of the variants studied in the two groups. In this study, we performed extensive bioinformatics analysis for biomarker prioritization followed by experimental validation of proposed selected markers. Although there is no statistical difference in the frequencies of these variants between RPL and controls, the expression analysis results suggest that TDGF1 and CFC1 genes might play a role in RPL. In addition, systems biology analyzes raise the hypothesis that genes in other signaling pathways that may be related to RPL as good candidates for future studies.Abbreviations RPL: recurrent pregnancy loss; EGF-CFC1: Epidermal Growth Factors - Cripto/FRL-1; GEO: Gene Omnibus Expression; KEGG: Kyoto Encyclopedia of Genes and Genomes.