Mitochondrial DNA content reduction in the most fertile spermatozoa is accompanied by increased mitochondrial DNA rearrangement

Abstract

Abstract STUDY QUESTION Is there an association between male fertility and spermatozoa mitochondrial DNA (mtDNA) copy number and genome rearrangements? SUMMARY ANSWER Normal spermatozoa not only have a lower mtDNA copy number but also more DNA rearrangements than spermatozoa of men with severe oligoasthenospermia (SOA). WHAT IS KNOWN ALREADY While there is a consensus that mtDNA content is decreased in the most fertile spermatozoa, the role of mtDNA sequence alteration in male infertility is unclear. High-throughput sequencing, which allows an exhaustive analysis of mtDNA rearrangements and mutations, could be helpful in this context, but has yet to be used. STUDY DESIGN, SIZE, DURATION This is an observational study of semen samples obtained from 44 men undergoing ART at an academic infertility centre in France, from October 2018 to November 2020. The men were classified into two groups: 20 men in the SOA group and 24 men with normal semen parameters in the control group. PARTICIPANTS/MATERIALS, SETTING, METHODS For each patient and control, mtDNA was isolated from sperm fractions from the 40% and 90% layers of the density gradient. The average mtDNA content of each sample was assessed using digital PCR. Deep sequencing was performed using next-generation sequencing. Signal processing and base calling were performed via the embedded pre-processing pipeline, the variants were analysed using an in-house workflow and a dedicated tool, based on soft-clipping, was used to study large mtDNA rearrangements. The distribution and the type of rearrangements and variants were compared between patients with SOA and controls on one hand, and between the 40% and 90% gradient layers, on the other hand. MAIN RESULTS AND THE ROLE OF CHANCE The mtDNA content of spermatozoa in the SOA group was significantly higher than in the control group (P < 0.0001). Moreover, mtDNA content was significantly higher in spermatozoa from the 40% layer (the most fertile spermatozoa) compared to the 90% layer, both in the SOA (P = 0.02) and the control group (P < 0.0001). The frequency of large mtDNA deletions and duplications was significantly higher in the control group (P = 0.002). Most of these rearrangements are potentially related to DNA breaks and their number was reduced by the removal of the linear mtDNA from the samples. Heteroplasmic variants were found more frequently in the SOA group (P = 0.05) and in the 40% layer (P = 0.03), but none had any obvious functional consequence. LIMITATIONS, REASONS FOR CAUTION Our findings are novel and significant but should be verified in larger cohorts and other types of male infertility. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that sperm mtDNA rearrangements are not necessarily associated with mitochondrial dysfunction and male infertility. Instead, they seem to be concomitant with the process of mtDNA content reduction in the most potentially fertile spermatozoa. Furthermore, they refute the hypothesis that, in the case of mtDNA alteration, a compensatory mechanism allows an increase in mtDNA copy number to rectify the energy deficit. The increased frequency of mtDNA rearrangements in the most fertile spermatozoa is a novel result that offers new insight into the relation between sperm quality and mtDNA. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Angers University Hospital (grant AOI CHU Angers 2018), Angers University and the French national research centres INSERM and CNRS. There are no competing interests. TRIAL REGISTRATION NUMBER N/A.