Epigenomic analyses in sub-populations of spermatozoa from infertile men with short anogenital distance

Abstract

The key developmental steps in the male germ cell lineage include the epigenetic reprogramming of the primordial germ cells, the large-scale replacement of histone by protamine in spermatids and some degree of apoptosis. In past experiments, we prenatally exposed mice to di(2-ethylhexyl)phthalate (DEHP, CAS number 117-81-7). At adulthood, the exposed mice were characterized by reduced anogenital distance (AGD), poor semen quality, and epigenomic abnormalities in their gametes’ DNA. However, the phenotype heavily depended on the genetic background of the mouse strain. We identified SNP-dependent binding motifs for sex hormones signaling pathways varying between the susceptible and the resistant mouse strains. These SNP-dependent motifs were located in the genes that were the most affected one by DEHP, at both DNA methylation and RNA transcription levels. We then tried to replicate these observations into human. We selected 6 infertile patients with short AGD and 5 fertile sperm donors. Testicular volume was significantly reduced in infertile men with shorter AGD. Sub-populations of spermatozoa were FACS-sorted using the chromomycin A3 dye (protamine vs. histone) and with the YO-PRO-1 dye (apoptotic vs. non-apoptotic). In the four sorted sperm populations, we analyzed the epigenomes through reduced-representation bisulfite sequencing (RRBS). The results demonstrated both lower CpG methylation and higher DNA fragmentation in infertile patients. Interestingly, numerous de-methylated regions localized within families of Alu repeats containing estrogen response elements (ERE). We are now in the process of replicating the entire study with a new cohort of five infertile patients and five fertile controls. The aim is to understand if there is a relationship between these epigenomic alterations within Alu repeats containing ERE and fertility, and whether exposures to anti-androgenic endocrine disruptors may alter methylation marks within these Alu-ERE repeats.