目的:精子磷脂酶Cζ(PLCζ)谱与胚胎发生和妊娠的质量有关吗?
结论:小鼠和人类中的精子PLCζ水平与理想胚胎发生的指标相关,因此似乎需要最低水平才能成功怀孕。
背景:虽然男性不育的致病因素是多变量的,病例越来越多地与卵母细胞激活的功效相关,在哺乳动物中,这种现象是响应于由精子特异性PLCζ驱动的钙(Ca2)振荡的特定曲线而发生的。尽管精子PLCζ废除与人类男性不育密切相关,但卵母细胞活化不足,尚不清楚精子PLCζ水平或定位是否成为胚胎发育缺陷和生育治疗后妊娠失败的基础。
方法:在费萨尔国王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。利雅得,沙特阿拉伯王国。男性的招募标准是最低精子浓度为5×106精子/ml,而所有女性患者必须至少有五个卵母细胞。精子PLCζ分析在研究实验室进行,在进行精液评估的同时,并获得了延时形态动力学数据,在生育诊所作为常规治疗的一部分。同时使用CRISPR/Cas9系统在Plcζ基因内诱导indel和单核苷酸突变以产生Plcζ突变小鼠的品系。精子PLCζ使用免疫荧光和免疫印迹法用证实对PLCζ具有一致特异性的抗体进行评估。
方法:我们在对结果胚胎进行延时形态动力学分析的背景下,评估了54对接受生育治疗的人类夫妇的精子样本中的PLCζ谱。将这些配置文件与怀孕状态相关联。同时,我们使用CRISPR/Cas9产生了两株突变Plcζ小鼠,并使用野生型(WT)卵母细胞进行IVF,并使用WT或突变Plcζ精子产生胚胎.我们还在延时形态动力学分析和育种结果的背景下评估了WT和突变小鼠精子中的PLCζ状态。
结果:观察到PLCζ相对荧光和相对密度与第二次细胞分裂(CC2)(r=0.26和r=0.43)和第三次细胞分裂(S2)(r=0.26)的时间之间存在显着(P≤0.05)正相关。定位模式的检查还表明精子PLCζ和CC2的存在或不存在之间存在显着相关性(分别为r=0.27和r=-0.27;P≤0.025)。人类精子PLCζ水平在CC2的理想时间(8-12小时)中处于最高水平,而在理想时间范围(<8和>12小时)之外的时间范围内,人类精子PLCζ水平较低。在分配PLCζ水平阈值之后,定量显示两个相对荧光的成功妊娠率显著高于(P≤0.05)指定的临界值(19%vs40%,分别)和相对密度(8%对54%,分别)。免疫印迹显示WT小鼠精子中74kDa的PLCζ单条带,虽然在来自Plcζ突变小鼠精子的杂合精子中也观察到一条条带,但是强度降低了。免疫荧光分析表明先前报道的(Kashir等人。,2021)WT精子的荧光模式,而Plcζ突变小鼠的精子在精子头部的顶体区域表现出明显减少和分散的模式。育种实验表明,与WT小鼠相比,突变Plcζ雄性小鼠的产仔数显着减少,虽然使用来自突变Plcζ小鼠的精子的IVF产生的胚胎表现出很高的多精子率,并导致这些胚胎达到发育里程碑的数量显着减少。
结论:所检查的人群相对较少,并应扩大以检查更大的多中心队列。不孕症通常是多变量的,并且不可能在人类患者中评估所有这些。然而,我们的突变体Plcζ小鼠实验确实表明PLCζ在早期胚胎发育中起着重要作用。
结论:我们发现,最佳早期胚胎发生需要在特定范围内的最小水平的PLCζ,与怀孕增加有关。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有关。尽管在小鼠和人类中都成功受精。据我们所知,这是首次将精子中的PLCζ水平与人类胚胎发生功效和妊娠率的预后指标相关联.我们的数据首次表明,PLCζ的临床利用可能不仅有益于特定的男性不育人群,在这些人群中,卵母细胞激活完全缺乏(其中PLCζ完全有缺陷/被废除),但也许还有更多的夫妇寻求生育治疗。
背景:J.K.由哈利法大学(FSU-2023-015)授予的教师启动补助金支持。这项研究还得到了健康与护理研究威尔士(HCRW)授予J.K.的医疗保健研究奖学金(HF-14-16)的支持。与国家科学一起,技术,和创新计划(NSTIP)项目赠款(15-MED4186-20)由阿卜杜勒阿齐兹国王科技城(KACST)授予J.K.和A.M.A.作者声明没有利益冲突。
背景:不适用。
OBJECTIVE: Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy?
CONCLUSIONS: 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.
BACKGROUND: 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.
METHODS: 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.
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.
RESULTS: 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.
CONCLUSIONS: 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.
CONCLUSIONS: 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.
BACKGROUND: 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.
BACKGROUND: N/A.