DataSheet1_Novel mutations in PLCZ1 lead to early embryonic arrest as a male factor.pdf

Early embryonic arrest is one of the causes of assist reproduction technology (ART) failure. We have previously reported that the first sperm-derived genetic factor, ACTL7a mutations, could lead to early embryonic arrest. However, whether there are other male genetic factors associated with early embryonic arrest remains elusive. Here, we reported bi-allelic mutations in PLCZ1, a well-known causal gene of total fertilization failure, in four infertile males. Among these mutations, p.403_404del, p.I489S, and p.W536X were newly reported in this study. Histological and Western blotting analysis of the patients’ sperm indicated these variants as loss-of-function mutations. These patients manifested normal conventional semen parameters and ultra-structures in sperm heads. However, among four in vitro fertilization (IVF) cycles, 81.8% (18/22) of the oocytes were polyspermic fertilized, which was rarely reported in PLCZ1-related male patients. In the following six ICSI cycles, artificial oocyte activation (AOA) was applied and successfully rescued the fertilization failure and polyspermy phenotypes, with 31.3% (15/48) of the MII oocytes normally fertilized. However, 60.0% (9/15) of these normally fertilized zygotes were arrested at 2–5-cell stage, with one failing to cleave, indicating that PLCZ1 was not only necessary for fertilization, but also crucial for early embryonic development. However, these rescued zygotes showed a lower potential in developing into blastocysts when cultured in vitro. Thus, fresh cleavage transfer was tried and two live births were successfully achieved thereafter. In conclusion, this study provided novel mutations in PLCZ1 gene to expand the pathogenic mutational spectrum in male infertility and demonstrated that PLCZ1 was a crucial sperm-related genetic factor for early embryonic arrest. We also proposed that cleavage transfer after ICSI and AOA treatment could be a potential treatment method for male patients carrying bi-allelic mutations in PLCZ1.

早期胚胎停滞是辅助生殖技术（ART）失败的原因之一。我们先前报道，第一精子衍生的遗传因子ACTL7a突变可能导致早期胚胎停滞。然而，是否存在与早期胚胎停滞相关的其他男性遗传因素尚不明确。在本研究中，我们报道了四名不育男性中PLCZ1基因的双等位基因突变，PLCZ1是已知的全 fertilization失败因果关系基因。在这些突变中，p.403_404del、p.I489S和p.W536X是新报道的。通过对患者精子的组织学及蛋白质印迹分析，这些变异被确认为功能丧失突变。这些患者表现出正常的传统精液参数和精子头部的超微结构。然而，在四轮体外受精（IVF）周期中，81.8%（18/22）的卵母细胞发生了多精子受精，这在PLCZ1相关男性患者中罕见报道。在随后的六轮卵胞浆内单精子注射（ICSI）周期中，应用人工卵母细胞激活（AOA）成功挽救了受精失败和多精子受精现象，其中31.3%（15/48）的MII期卵母细胞正常受精。然而，60.0%（9/15）的正常受精原核在2-5细胞期停滞，其中有一个未能分裂，表明PLCZ1不仅对受精至关重要，而且对早期胚胎发育同样关键。然而，这些挽救的受精原核在体外培养时表现出较低的发育成囊胚的潜力。因此，尝试了新鲜分裂转移，并成功实现了后续的两个活产。总之，本研究提供了PLCZ1基因的新突变，以扩展男性不育的致病突变谱，并证明了PLCZ1是早期胚胎停滞的关键精子相关遗传因子。此外，我们还提出，ICSI和AOA治疗后进行分裂转移可能成为携带PLCZ1双等位基因突变的男性患者的潜在治疗方法。