Estudio de la expresión génica y polimorfismos relacionados con la espermatogénesis y la infertilidad

Résumé

According to the World Health Organisation (WHO), infertility is a global health issue affecting millions worldwide. Infertility is defined as the inability to conceive after 12 months of regular unprotected intercourse. The prevalence of infertility may be as high as 10.5% of the Spanish population, affecting approximately 900,000 couples in this country. Objectives The present thesis is divided into two different chapters, both of which have, as a common aim, the increase of knowledge about the molecular mechanisms involved in reproduction and hence, the improvement of infertility diagnosis and the assisted reproduction rates. The first chapter analyses the gene expression of the testis to understand the molecular processes involved in the development of a cell as specialised as the spermatozoon. For this purpose, we used a non-human primate model, the baboon (Papio anubis). The second chapter intends to improve the methods of selection of the best embryo to transfer, based on the polymorphisms present in genes potentially involved in early human embryonic development. This way we try to increase the chances of success after embryo transfers. Methodology To tackle the first chapter, 8 testicular samples, 4 from immature baboons and 4 from mature baboons, were used. The maturity state of each sample was checked using anatomical and histological characteristics. The RNA of each sample was analysed using RNA-seq technology. The results obtained were then analysed by bioinformatic procedures, where the sequences were pseudoaligned with the reference transcriptomes (Panubis1.0 and hg38) and compared between groups (mature and immature). The differentially expressed genes (logFC≥|2| and FDR<0.01) were used to evaluate their possible role in spermatogenesis. In the second chapter, DNA samples from 131 embryos, that had undergone biopsy, were used. On one hand, we design an Ampliseq panel, choosing 33 genes related to implantation and early gestational development. All the embryos were analysed using this panel, and the polymorphisms detected were evaluated in function of the mutation type, genotype, as well as the location in the gene, the possible pathogenic phenotype or its presence in the population. On the other hand, the KASP technology was used to evaluate the polymorphisms c.677C>T and c.1298A>C of the MTHFR gene in all the embryos transferred. The results from all these genetic analyses were compared with the clinical results after the embryo transfer. Results and conclusions The RNA-seq transcriptomic analysis of baboon testes has annotated a total of 15,297 distinct genes in the Papio anubis genome. The differential expression analysis between mature and immature testis detected 2029 genes overexpressed and 555 underexpressed in mature testis. Some of the novel genes that appeared in this study were SPATA18, SPATC1, TMEM262, ARRDC5, SPATA31D1, TMEM239, LEMD1, FAM187B, C3orf22, C11orf42, TMEM31, TSPAN16, CT83, C7orf61 and SLC9C2. A total of 1897 non-coding genes were detected, where SPACA6, LINC00303 and LINC01121 stand out. After the evaluation of genetic polymorphisms of human embryos (Ampliseq panel), ARHGAP21, ENG, STAT3, PMM2 and GATA4 were highlight as possible genes involved in implantation and early embryonic development that can be used as human embryo selection markers. It has been detected a possible deleterious effect of homozygotic polymorphic variants chr17:40481601 C>T (STAT3) and chr16:8904956 G>A (PMM2). Moreover, a possible deleterious effect in implantation has been detected in other mutations not detected so far in the population even in heterozygosis. They are chr10:24909241 C>T, chr10:24909164 G>A and chr10:24909090 TC>T of ARHGAP21 gene, or chr9:130578295 CAG>C of ENG gene. The relationship between MTHFR gene polymorphisms and the clinical outcome after embryo transfer could not be confirmed. However, an embryo carrying the polymorphic combination c.677TT and c.1298AC of MTHFR gene, which did not implant, has been described for the first time.