Department of Biological Sciences, College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, United Arab Emirates.
Center for Biotechnology, Khalifa University, Abu Dhabi, United Arab Emirates.
Hum Reprod. 2024 Jun 3;39(6):1256-1274. doi: 10.1093/humrep/deae078.
Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy?
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.
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, METHODS: We 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.
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.
We 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.
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精子磷酯酶 C ζ（PLCζ）谱是否与胚胎发生和妊娠质量有关？
无论是在小鼠还是人类中，精子 PLCζ 水平都与理想胚胎发生的衡量标准相关，即最低水平似乎是成功妊娠所必需的。
虽然男性不育的病因是多变量的，但越来越多的病例与卵母细胞激活的功效有关，而在哺乳动物中，卵母细胞激活是通过精子特异性 PLCζ 驱动的特定钙（Ca2+）振荡模式发生的。尽管精子 PLCζ 缺失与卵母细胞激活不足的人类男性不育症广泛相关，但对于 PLCζ 水平或定位是否导致生育治疗后胚胎发生缺陷和妊娠失败的情况，了解较少。
研究设计、规模、持续时间：在沙特阿拉伯利雅得法伊萨尔医院和研究中心的辅助生殖技术实验室招募了 54 对接受生育治疗的夫妇。男性的招募标准是精子浓度至少为 5×106 个/ml，而所有女性患者至少要有 5 个卵母细胞。在研究实验室中进行精子 PLCζ 分析，同时在生育诊所中进行精液评估，并获得延时形态动力学数据，作为常规治疗的一部分。CRISPR/Cas9 系统被同时用于诱导 Plcζ 基因中的插入缺失和单核苷酸突变，以产生 Plcζ 突变小鼠的品系。使用免疫荧光和免疫印迹技术，用经过证实对 PLCζ 具有一致特异性的抗体评估精子 PLCζ。
参与者/材料、地点、方法：我们评估了 54 对接受生育治疗的夫妇的精子 PLCζ 谱，同时对胚胎的延时形态动力学分析进行了评估，将这些谱与妊娠状况相关联。同时，我们使用 CRISPR/Cas9 生成了两种突变 Plcζ 小鼠品系，并使用 WT 卵母细胞和 WT 或突变 Plcζ 精子进行 IVF，以生成胚胎。我们还评估了 WT 和突变小鼠精子中 PLCζ 的状态，同时也对延时形态动力学分析和繁殖结果进行了评估。
观察到第二次细胞分裂（CC2）（r=0.26 和 r=0.43）和第三次细胞分裂（S2）（r=0.26）所需的时间与 PLCζ 相对荧光和相对密度之间存在显著的正相关（P≤0.05）。定位模式的检查也表明，精子 PLCζ 的存在或不存在与 CC2 之间存在显著的相关性（r=0.27 和 r=-0.27，分别；P≤0.025）。与理想时间范围（<8 和>12 小时）相比，人类精子 PLCζ 水平在 CC2 的理想时间（8-12 小时）最高，而在理想时间范围之外的水平较低。在分配 PLCζ 水平阈值后，定量分析显示，相对荧光（19%对 40%，分别）和相对密度（8%对 54%，分别）的较大值的成功妊娠率显著较高（P≤0.05）。免疫印迹分析表明，WT 小鼠精子中 PLCζ 的条带在 74kDa 处为单条带，而杂合子 Plcζ 突变鼠精子中的条带也为单条带，但强度降低。免疫荧光分析表明，WT 精子中存在先前报道的（Kashir 等人，2021）荧光模式，而 Plcζ 突变鼠精子的荧光模式则明显减少且分散在精子头部的顶体区域。繁殖实验表明，突变 Plcζ 雄性小鼠的幼鼠数量明显减少，而使用突变 Plcζ 精子生成的 IVF 胚胎的多精入卵率很高，导致这些胚胎达到发育里程碑的数量明显减少。
局限性、谨慎的原因：检查的人类人群相对较小，应该扩大到检查更大的多中心队列。不孕不育的情况往往是多变量的，无法评估人类患者的所有情况。然而，我们的突变 Plcζ 小鼠实验确实表明 PLCζ 在早期胚胎发育中起着重要作用。
我们发现，在最佳早期胚胎发生中需要特定范围内的最低 PLCζ 水平，这与增加妊娠率相关。低于特定阈值的精子 PLCζ 水平与无效的胚胎发生和较低的妊娠率相关，尽管在小鼠和人类中都成功地引发了受精。据我们所知，这是首次将精子 PLCζ 水平与人类中预测胚胎发生疗效和妊娠率的预后措施相关联。我们的数据首次表明，PLCζ 的临床应用不仅可能对卵母细胞激活完全缺失的特定男性不育症人群（其中 PLCζ 完全缺失/失活）有益，而且可能对寻求生育治疗的更大人群有益。
研究资金/利益冲突：J.K. 得到了 Khalifa 大学（FSU-2023-015）教员启动赠款的支持。本研究还得到了威尔士健康、护理和研究部（HF-14-16）医疗保健研究奖学金的支持（J.K.和 A.M.A.），以及由 King Abdulaziz 城市为科学和技术（KACST）授予的 15-MED4186-20 国家科学、技术和创新计划项目赠款（J.K.和 A.M.A.）。作者没有利益冲突。
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