ICSI后复发性胚胎发育问题是否可以通过辅助卵母细胞激活(AOA)来克服?
在ICSI后复发性胚胎发育问题的患者队列中,AOA并未改善胚泡形成。
据报道,使用AOA通过使用Ca2离子载体(主要是钙霉素和离子霉素)人工诱导钙(Ca2)升高在克服ICSI后受精失败方面非常有效,尤其是在受精过程中Ca2动力不足的患者中。然而,关于ICSI后使用AOA克服胚胎发育问题的文献很少且相互矛盾,尚不清楚受精过程中异常的Ca2模式是否会干扰人类植入前胚胎的发育。此外,ICSI后胚胎发育不良也与皮质下母体复合体(SCMC)基因的遗传缺陷有关.
这项前瞻性队列单中心研究比较了在至少两个先前ICSI周期中,受精率正常(≥60%)但胚胎发育受损(胚泡形成≤15%)的夫妇的ICSI-AOA周期和先前的ICSI周期。总的来说,从2018年1月到2021年1月,有42对胚胎发育问题的夫妇被纳入这项研究。
包括42对夫妇,17经历了由CaCl2注射和双离子霉素暴露组成的ICSI-AOA循环。施肥,囊胚发育,怀孕,将ICSI-AOA后的活产率与之前的ICSI周期进行比较.此外,通过小鼠卵母细胞钙分析研究了男性患者精子诱导的钙模式。此外,所有42对夫妇都接受了基因筛查.女性患者筛选SCMC基因(TLE6,PADI6,NLRP2,NLRP5,NLRP7和KHDC3L),男性患者筛选精子-卵母细胞活化因子PLCZ1。
我们比较了来自同一患者队列的17个AOA周期和44个之前的ICSI周期。AOA之后,总受精率为68.95%(131/190),囊胚发育率为13.74%(18/131),妊娠率为29.41%(5/17),活产率为23.53%(4/17),与之前的ICSI周期没有差异(76.25%(321/421,P值=0.06);9.35%(30/321,P值=0.18),25.00%(11/44,P值=0.75),15.91%(7/44,P值=0.48),分别)。钙分析显示,患者精子诱导的钙模式与对照精子样本相似,显示出正常的胚胎发育潜力。遗传筛选揭示了14名雌性中具有不确定意义(VUS)的10个独特杂合变体(在NLRP2,NLRP5,NLRP7,TLE6和PADI6中)。变体NLRP5c.623-12_623-11insTTC(第?)在两个无关的个体和变体NLRP2c.1572T>C(p。Asp524=)在四只雌性中鉴定。有趣的是,我们鉴定了一个先前报道的纯合突变PLCZ1,c.1499C>T(p。Ser500Leu),一名男性患者胚胎发育受损,但没有表现出典型的受精失败。
我们严格的纳入标准,需要至少两个ICSI周期的胚胎发育受损,减少周期到周期的可变性,虽然不受受精不良影响的较低胚泡发育的要求排除了否则将是AOA选择性病例的夫妇;然而,这些标准限制了本研究的样本量.靶向遗传筛选可能太受限制,无法确定所有患者胚胎发育不良表型的遗传原因。此外,应进一步确定已识别的VUS的因果关系。
文献中仍然缺乏AOA克服受损胚胎发育的有力证据。到目前为止,只有一篇文章报道了AOA(使用钙霉素)在该患者人群中与以前的ICSI周期相比的有益效果,而最近的两项同胞卵母细胞对照研究(一项使用钙霉素,另一项使用离子霉素)和我们的研究(使用离子霉素)无法证实这些发现.尽管在AOA后出生的儿童中没有发现重大异常,该技术应保留给具有明显Ca2+释放缺陷的夫妇。最后,通过全外显子组测序进行的基因筛选可能揭示与胚胎发育问题相关的新基因和变异,并允许设计更个性化的治疗方案,例如野生型互补RNA或重组蛋白注射。
这项研究得到了佛兰德科学研究基金(FWO。OPR.2015.0032.01至B.H.并授予编号1298722N至A.B.)。A.C.B.,D.B.,A.B.,V.T.,R.P.,F.M.,I.D.C.,L.L.,D.S.,P.D.S.,P.C.,F.V.M.没有什么可透露的.B.H.报告了佛兰德科学研究基金的研究经费,并报告是比利时生殖医学学会和比利时胚胎研究伦理委员会的董事会成员。
NCT03354013。
Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)?
AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI.
The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes.
This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021.
Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient\'s sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1.
We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient\'s sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure.
Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined.
Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection.
This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research.
NCT03354013.