Clínica EUGIN, Barcelona, Spain.
Faculty of Medicine, University of Barcelona, Barcelona, Spain.
Hum Reprod. 2019 Aug 1;34(8):1494-1504. doi: 10.1093/humrep/dez094.
Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI?
New mutations in the PLCZ1 sequence are associated with FFs after ICSI.
FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated.
STUDY DESIGN, SIZE, DURATION: We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection.
In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth.
LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis.
PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.
PLCZ1 基因突变是否与 ICSI 后受精失败（FF）相关？
PLCZ1 序列中的新突变与 ICSI 后的 FF 相关。
FF 发生在 1%-3%的 ICSI 周期中，主要是由于卵母细胞激活失败（OAF）。精子 PLCζ/PLCZ1 蛋白在卵母细胞中水解磷脂酰肌醇（4,5）-二磷酸，导致细胞内钙释放和卵母细胞激活。迄今为止，已经发现一些 PLCZ1 点突变导致蛋白水平或活性降低与 FF 相关。然而，尚未研究 PLCζ/PLCZ1 的功能改变对已描述和新突变的反应。
研究设计、规模、持续时间：我们进行了一项研究，纳入了 2014 年至 2018 年间发生的总受精率（FR，≤25%）或部分 FF（FF，≤25%）的 37 名患者。
参与者/材料、设置、方法：根据 ICSI 后 19 小时的卵母细胞评估，将患者分为两组：由于卵母细胞激活缺陷导致的 FF（OAF，n=22）和由于其他原因导致的 FF（“非-OAF”，n=15）。将 13 名具有良好受精率（FR，>50%）的男性的样本用作对照。通过免疫荧光和western blot 分别评估精子中 PLCζ/PLCZ1 蛋白的定位和水平。使用 Sanger 测序对基因组 DNA 进行测序，以鉴定外显子区域的 PLCZ1 突变。使用 MODICT 算法预测突变对蛋白功能的影响。通过将野生型和突变型 PLCZ1 的 cRNA 注射到人体外成熟的中期 II 卵母细胞中，进行功能测定，并在注射后 19 小时评估第二极体排出和原核出现的受精结果。
在 OAF 组中，12 名（54.6%）患者的 PLCZ1 编码序列中至少携带一个突变，15 名患者中的 1 名（6.7%）在非-OAF 组中携带突变（P<0.05），而 13 名对照者中无一例携带突变（P<0.05）。共鉴定出六种不同的突变。其中 5 种是单核苷酸错义突变：位于 EF 手结构域末端的 p.I120M；位于 X 催化结构域的 p.R197H、p.L224P 和 p.H233L；以及位于 C2 结构域的 p.S500L。第六种突变为移码变异（p.V326K fs*25），在 X-Y 连接区产生截短蛋白。MODICT 分析预测除了 p.I120M 以外的所有突变都可能对 PLCζ/PLCZ1 活性具有潜在的有害影响。在注射 PLCZ1 cRNA 后，对于三个突变（p.R197H、p.H233L 和 p.V326K fs*25），观察到激活卵母细胞的百分比显著降低，表明酶活性具有有害影响。各组之间 PLCZ1 蛋白在精子中的定位和表达水平相似。在携带 PLCZ1 突变的患者（n=10）中，辅助卵母细胞激活（AOA）后 FR 恢复（>60%），其中 7 名患者成功妊娠并分娩。
局限性、谨慎的原因：特别是在杂合子携带突变的患者中，在比较卵母细胞注射结果与 ICSI 后的受精结果时应谨慎。
在出现 OAF 的患者中，PLCZ1 突变的频率很高。在人卵母细胞中对三种突变的功能分析证实了 PLCζ/PLCZ1 活性的改变，并可能涉及卵母细胞激活受损。我们的结果表明，PLCZ1 基因突变测序可作为诊断和咨询 ICSI 后 OAF 导致 FF 患者的工具。
研究基金/利益冲突：这项工作得到了 Clínica EUGIN 的内部资金、加泰罗尼亚经济和知识部的大学和研究秘书（GENCAT 2015 DI 049 给 M.T.-M.和 GENCAT 2015 DI 048 给 D.C.-B.）以及西班牙经济和竞争力部的 Torres Quevedo 计划的支持。A.F.-V. 没有竞争利益声明。
