The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

study questionAre sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy? SUMMARY ANSWER: Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy. WHAT IS KNOWN ALREADY: While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment. STUDY DESIGN, SIZE, DURATION: A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ. PARTICIPANTS/MATERIALS, SETTING,

methodsWe evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: A significant (P ≤ 0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r = 0.26 and r = 0.43, respectively) and the third cell division (S2) (r = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r = 0.27 and r = -0.27, respectively; P ≤ 0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8-12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P ≤ 0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al., 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones. LIMITATIONS, REASONS FOR CAUTION: The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development. WIDER IMPLICATIONS OF THE

findingsWe found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K., alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST) for J.K. and A.M.A. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.