Profiling the male germline genome to unravel its reproductive potential

Over the years, genetic testing has become increasingly relevant in reproductive medicine. Genetic assessments in male infertility include testing for whole chromosomal structural aberrations, partial chromosomal defects, and monogenic diseases. These genetic tests are exclusively performed on peripheral blood samples, and although these tests are focused on identifying the etiology of compromised sperm production and preventing the transmission of inherited defects, they do not provide any information on gamete competence.

More intriguing is the identification of a subtle male factor or the detection of gamete function, particularly in individuals with normal semen parameters. There has recently been a renewed effort to identify superimposed occult factors that may impair a man’s reproductive potential and affect assisted reproductive technology (ART) related clinical outcomes.

Sperm chromatin fragmentation assays have become widely popular and can detect elevated sperm DNA fragmentation, which is correlated with poor embryo development, low implantation, and high miscarriage rates.

Regardless of the available ancillary assessments and genetic tests, the sperm-related reasons for poor ART outcome, occurring despite a normal semen analysis, remain largely unknown. Stephanie Cheung et al hypothesized that detectable genetic differences exist in spermatozoa obtained from men with unexplained infertility, and that these differences can be used to understand the specific aspects of their suboptimal ART outcomes. Therefore, in the study they investigated the relationship between the genetic profile of spermatozoa and the reasons for reproductive failure, after ICSI, in couples with negative infertility workups and normal sperm parameters.

In this retrospective study, couples were divided according to whether they had successful intracytoplasmic sperm injection outcomes (fertile) or not (infertile). Ancillary sperm function tests were performed on ejaculates, and whole exome sequencing was performed on spermatozoal DNA. Sperm aneuploidy and gene mutation profiles were compared between the 2 cohorts as well as according to the specific reasons for reproductive failure.

Thirty-one couples with negative infertility workups and normal semen parameters were included. Couples with mutations on fertilization- or embryo development-related genes were subsequently treated by assisted gamete treatment or microfluidics, respectively. Intracytoplasmic sperm injection cycle outcomes including fertilization, clinical pregnancy, and delivery rates.

Sperm aneuploidy was lower in the fertile group (4.0% vs. 8.4%). Spermatozoa from both cohorts displayed mutations associated with sperm–egg fusion (ADAM3A) and acrosomal development (SPACA1), regardless of reproductive outcome.

The infertile cohort was then categorized according to the reasons for reproductive failure: absent fertilization, poor early embryo development, implantation failure, or pregnancy loss.

Spermatozoa from the fertilization failure subgroup (n = 4) had negligible PLCz presence (10%± 9%) and gene mutations (PLCZ1, PIWIL1, ADAM15) indicating a sperm-related oocyte-activating deficiency. These couples were successfully treated by assisted gamete treatment in their subsequent cycles.

Spermatozoa from the poor early embryo development subgroup (n = 5) had abnormal centrosomes (45.9%± 5%), and displayed mutations impacting centrosome integrity (HAUS1) and spindle/microtubular stabilization (KIF4A, XRN1).

Microfluidic sperm processing subsequently yielded a term pregnancy. Spermatozoa from the implantation failure subgroup (n = 7) also had abnormal centrosomes (53.1%± 13%) and carried mutations affecting embryonic implantation (IL9R) and microtubule and centrosomal integrity (MAP1S, SUPT5H, PLK4), whereas those from the pregnancy loss subgroup (n = 5) displayed mutations on genes involved in trophoblast development (NLRP7), cell cycle regulation (MARK4, TRIP13, DAB2IP, KIF1C), and recurrent miscarriage (TP53).

The lack of assessments on the male gamete is partially attributed to the fact that spermatogenesis is a complex process, controlled by well-coordinated transcriptional and posttranscriptional regulators. Nevertheless, the spermatozoon is not simply a carrier that delivers the male genome to the oocyte and should therefore not be overlooked. Although an array of ancillary tests can be used to evaluate spermatozoa, they can be tedious and inaccurate, and are each capable of assessing only a single facet of sperm reproductive potential.

By sequencing the sperm exome, authors identified candidate genes associated with the different causes of reproductive failure in couples with normal infertility workups and semen parameters. In addition, the novel variants identified in the study population may lay the foundation for future gene therapy research. Furthermore, these findings would allow for the design of a custom gene panel for targeted sperm DNA sequencing. This clinical panel, used prospectively at the time of semen analysis and before the start of the patients’ cycles, would encompass all of the altered genes identified in this study and could discern relevant genetic changes that can streamline male infertility clinical management. Most importantly, screening spermatozoa for these mutations would serve as a useful precision medicine tool to enhance the diagnosis, treatment, and prediction of clinical outcome for couples with unexplained infertility.

Source: Stephanie Cheung, Philip Xie, Zev Rosenwaks; Fertility and Sterility® Vol. 119, No. 2, February 2023 0015-0282 https://doi.org/10.1016/j.fertnstert.2022.11.006