Monogenic and digenic variants in male PLCZ1, ACTL7A and ACTL9 genes cause fertilization failure after ICSI

Abstract Study question What is the frequency of PLCZ1, ACTL7A and ACTL9 variants in male patients suffering from fertilization failure (FF) after ICSI? Summary answer Male patients with FF after ICSI exhibit variants in PLCZ1 (27,3%), ACTL7A (13,6%) and ACTL9 (4,5%). Some patients present PLCZ1 variants combined with ACTL7A/ACTL9 variants. What is known already FF after ICSI has been often attributed to phospholipase C zeta (PLCζ) deficiency, and consequently to insufficient calcium release, necessary for fertilization. PLCZ1 genetic variants have been detected in one-third of males with FF. Recently, actin-like 7A (ACTL7A) and 9 (ACTL9) variants were also identified, but larger cohort studies are lacking. ACTL7A/ACTL9 variants alter the acrosome structure, resulting in reduced and abnormally localized PLCζ. Assisted oocyte activation (AOA), by calcium ionophore, restores fertilization in patients with PLCZ1 variants, and could also benefit patients with ACTL7A/ACTL9 defects. This study aimed to expand the spectrum of known PLCZ1, ACTL7A and ACTL9 variants. Study design, size, duration A prospective study was conducted from June 2020 to December 2022 including 22 male patients (P1-P22) with FF after ICSI. Patients were only recruited when they had a mean fertilization rate ≤33,33% in at least 1 ICSI cycle. All patients donated a saliva sample for genetic screening of PLCZ1, ACTL7A and ACTL9 genes and a sperm sample for the mouse oocyte activating test (MOAT). All couples with FF after ICSI were offered an AOA treatment. Participants/materials, setting, methods Genetic screening was performed via next-generation sequencing. Identified variants were classified employing Varsome and confirmed by bidirectional Sanger sequencing. Patients in which uncertain significance (VUS), likely pathogenic or pathogenic variants were found underwent additional diagnostic tests, such as the study of the sperm calcium releasing pattern in mouse (MOCA) and in in vitro matured (IVM) human (HOCA) oocytes, immunostaining of PLCζ and ACTL7A, and the analysis of the sperm morphology by transmission electron microscopy (TEM). Main results and the role of chance Genetic screening revealed heterozygous PLCZ1 variants, a novel variant p.Ile379Thr in P10 and the previously published variant p.His233Leu in three patients (P9, P19 and P20). In addition, two novel homozygous ACTL7A variants were detected, p.Ser364GlnfsTer9 in P1 and p.Gly214Ser in P22. Interestingly, digenic variants were observed in P6 (PLCZ1 p.Ile74Thr and ACTL7A p.Tyr183His) and in P8 (PLCZ1 p.His233Leu and ACTL9 p.Arg271Pro). All patients with identified variants showed a high oocyte activation rate (>70%) after MOAT, except P1 and P22 which activation rate was <25%, indicating a more detrimental sperm-related deficiency. While MOCA only detected deficient calcium release in P1 (and not for P6, P8, P9 and P10), HOCA revealed absence of calcium oscillations in all patients analyzed (P1, P8, P9 and P10), except for P6. Immunostaining experiments in P1 revealed no ACTL7A signal and decreased PLCζ signal in patient sperm. Moreover, TEM analysis in P1 confirmed acrosome detachment from nuclear membrane. Overall, AOA increased fertilization rate (from 7,23% to 53,04%) and pregnancy rate (from 7,14% to 46,15%) in patients with identified variants. Specifically, patients with monogenic PLCZ1 (P9 and P19) and ACTL7A (P1) variants, as well as digenic variants (P6 and P8) achieved a live birth after AOA. Limitations, reasons for caution Some patients have not undergone all diagnostic tests yet, thus functional data on all the found variants identified is still ongoing. In addition, patient recruitment for this study is continuing. Genetic screening was carried on exonic and flanking intronic regions, which might have missed additional variants in other intronic regions. Wider implications of the findings The MOAT/MOCA tests cannot detect some subtle sperm-related activation deficiencies. HOCA represents a more sensitive analysis but requires human oocytes. Therefore, genetic screening of PLCZ1, ACTL7A and ACLTL9 could be a faster and more cost-efficient diagnostic test for FF after ICSI. Furthermore, AOA treatment is very efficient for these patients. Trial registration number NA