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通讯作者:

王磊(1982-),男,陕西西安人,教授,博士生导师,主要从事人类生殖疾病的遗传学基础研究。E-mail:wangleiwanglei@fudan.edu.cn

中图分类号:[R715.5]

文献标识码:A

文章编号:2096-8965(2022)04-0045-06

DOI:10.12287/j.issn.2096-8965.20220406

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目录contents

    摘要

    不孕不育已成为全世界人类第三大疾病,随着辅助生殖技术的广泛应用,每年有上百万人借助辅助生殖技术解决不孕不育问题。然而临床中有很多患者辅助生殖反复失败,原因包括配子发育异常、受精障碍及早期胚胎停滞等。随着基因测序技术的发展及生殖遗传学研究的深入,越来越多的遗传致病因素被发现。针对这些疾病表型的基因检测应运而生,通过基因检测可以帮助患者早期诊断及明确病因,避免过度的反复无效尝试。然而如何在基因检测的同时对反复辅助生殖失败患者进行治疗已成为新的关注热点。本文就近年来反复辅助生殖失败的遗传因素、治疗现状及潜在的干预策略进行汇总,为个体化诊疗提供参考。

    Abstract

    Infertility has become the third largest human disease in the world. With the wide application of in vitro fertilization (IVF), millions of people solve the problem of infertility by IVF every year. However, many patients experience recurrent IVF failures, displaying abnormal reproductive phenotypes (such as gamete development abnormalities, fertilization disorders, and early embryonic discontinuation). With the development of gene sequencing technology and the deepening of reproductive genetics research, more and more pathogenic genetic factors have been discovered. Genetic testing for these reproductive phenotypes can help patients make an early diagnosis and avoid repeated ineffective attempts. However, the treatment of the patients with recurrent IVF failures after genetic testing has become a new focus of attention. In this paper, the genetic factors, the treatment and potential intervention strategies of recurrent IVF failures in recent years are summarized to provide a reference for individualized diagnosis and treatment.

  • 不孕不育已成为全世界第三大疾病,是影响人类生殖健康的重大全球卫生问题。世界范围内,受不孕不育困扰的夫妇超过5 000万对[1]。在发达国家不孕不育的发病率为15%,而发展中国家的发病率更达到 9%~30%[2]。自 1978 年第一例试管婴儿诞生以来,每年上百万人借助辅助生殖技术解决不孕不育问题,周期数逐年攀升,大概有600多万试管婴儿出生[13]。发达国家辅助生殖技术的使用率达 40%~50%,我国的总体使用率也在逐渐增加,我国每年约有30万例新生儿通过辅助生殖技术出生。辅助生殖技术主要包括体外受精-胚胎移植 (In Vitro Fertilization,IVF) 又称第一代辅助生殖技术,主要解决因女方因素造成的不孕,男方正常或轻度异常;卵胞浆内单精子注射(Intracytoplasmic Sperm Injection,ICSI) 又称第二代辅助生殖技术,主要解决男性精子质量差的问题;胚胎植入前遗传学诊断 (Preimplantation Genetic Diagnosis,PGD) 又称第三代辅助生殖技术,主要针对胚胎的遗传性疾病进行检测,排除染色体异常或有致病基因的胚胎,实现优生优育[4-6]。然而,有相当部分的患者行辅助生殖技术反复失败,临床表现为空卵泡综合症、卵母细胞成熟障碍、卵子死亡、卵子透明带缺失、受精失败、早期胚胎停滞和植入失败等。

  • 生殖过程包括精子和卵子生成、受精、受精卵发育形成胚胎等环节,其中任何步骤异常都会导致不孕不育。这一过程的分子调控也是生殖医学及发育生物学最重要的科学问题之一。随着生殖遗传学的研究及基因测序技术的发展,越来越多的导致反复辅助生殖失败的遗传因素被报道。通过基因检测,可以帮助患者早期诊断,避免过度的反复无效尝试。本文主要针对临床中反复辅助生殖失败相关的遗传疾病,从卵母细胞发育、受精、合子分裂及早期胚胎发育异常三个方面,在基因检测结果的指导下对治疗方案的调整、新的治疗方法的研究及应用进展进行汇总。

  • 1 卵母细胞发育异常

  • 成熟卵子的获得是正常受精、胚胎发育及后续成功妊娠的关键。人类卵母细胞经过减数分裂过程后由生发泡期 (Germinal Vesicle,GV),减数第一次分裂中期 (Metaphase Ⅰ,MⅠ),发育为减数第二次分裂中期 (Metaphase Ⅱ,MⅡ) 的成熟卵子,而后才能与精子受精。这一过程出现任何异常会导致成熟障碍,从而影响后续的受精和胚胎发育过程。目前,已报道在反复辅助生殖失败的患者中因为卵母细胞发育异常相关的突变基因包括LHCGR[7]TUBB8[8]PATL2[9]TRIP13[10]TBPL2[11]PANX1[12]LHX8[13] 等。对于反复尝试IVF/ICSI无效后,只有少部分有比较明确的治疗方案,大多数只能采用供卵的方式进行妊娠。

  • 临床因 LHCGR 基因突变的患者常表现为月经紊乱、卵巢多发囊肿、IVF无法获得卵子,表现为 “空卵泡综合症”[714]LHCGR在卵泡发育、雌激素合成及排卵过程中起着重要作用,在此之前,具有 LHCGR 突变的不孕症患者没有成功妊娠的报道。直到2019年,Lu等[15] 报道并验证了一种新的治疗方法,通过增加配体人绒毛膜促性腺激素(human Chorionic Gonadotropin,hCG)的剂量到5 000~20 000 IU,结合延长诱发排卵时间至 33~50 h,使患者获得成熟卵子,成功怀孕并诞下健康婴儿。该方法为 LHCGR 突变的患者的治疗提出了一项新的行之有效的 IVF 治疗策略。

  • 2016 年,Feng 等[8] 发现人类卵母细胞 MⅠ阻滞的第一个致病基因TUBB8,该基因突变类型和患者表型具有多样性,可导致卵母细胞不成熟、部分成熟但不受精、受精差或不卵裂、早期胚胎发育阻滞等多种表型[1617]TUBB8 突变遵循显性或隐性遗传模式,新发和遗传的错义杂合突变通过显性负效应引起疾病表型,而纯合缺失和移码突变导致疾病表型通过单倍剂量不足效应,破坏 β8 微管蛋白的组装过程并影响卵子减数分裂纺锤体组装过程及早期胚胎发育停滞从而导致女性不孕[1617]TUBB8 突变可以解释约30%的典型卵母细胞MⅠ期阻滞患者的病因,是导致此表型的主效致病基因[17],但目前有效的治疗方法尚未有报道。2020 年,Jia 等[18] 在小鼠模型中进行了挽救治疗的探索,他们通过将野生型 TUBB8 cRNA 注射到表达突变型 TUBB8 的小鼠卵母细胞中进行了拯救实验,发现补充外源性的野生型 TUBB8 cRNA可以有效改善突变引起的纺锤体组装异常,改善体外和体内的胚胎发育,并成功获得了足月小鼠后代。尽管缺乏在人体的治疗探索以及后续的安全性、有效性的研究,但这一研究为治疗由 TUBB8 突变引起的不孕症患者提供了理论参考。

  • 2020年,Zhang等[10] 发现 TRIP13的双等位基因突变会导致卵母细胞成熟障碍,该基因突变影响TRIP13 蛋白的表达、 ATP 酶活性及下游分子 HORMAD2的表达,研究团队因此探索了患者的治疗方法。在患者不成熟的卵母细胞中注射了 TRIP13 cRNA,能够挽救卵母细胞MⅠ阻滞的表型,所有被注射的卵母细胞均可成熟,部分可以成功受精并发育成囊胚[10],该干预方法对将来治疗携带 TRIP13基因变异的女性不孕患者提供了潜在方法。

  • 2 受精异常

  • 受精过程涉及一系列复杂的生物学事件,主要包括:精卵结合、Ca2+ 振荡、皮质颗粒释放、第二次减数分裂完成、母源 mRNAs 翻译与降解等。以上环节出现异常均可能导致受精障碍并引起不孕。受精失败的原因分为精子因素和卵子因素。

  • 2.1 精子因素

  • 临床中男性不育通过精液检查诊断出无精子症及少、弱、畸形精子症,可以通过ICSI或者捐精来克服。但是,对于精液常规参数正常的反复受精失败患者,往往难以明确原因。PLCZ1是2012年[19] 最早发现的男性因素受精失败的致病基因,是该疾病的主效基因,可解释 30% 左右的病例。磷脂酶 C-zeta (Phospholipase C-zeta,PLCζ) 是精原性卵母细胞激活因子,可通过诱发 Ca2+ 振荡从而启动卵母细胞激活,在人体中的主要编码基因为 PLCZ1。已报道的 PLCZ1 基因突变包括无义突变、错义突变、缺失及重复等多种类型[20-24],对于发现的 PLCZ1 突变家系,研究者们[202224] 大多采用辅助卵母细胞激活 (Assisted Oocyte Activation,AOA) 技术+ICSI的策略,可以获得优质胚胎以及成功妊娠。也有研究者发现,对人卵母细胞注射重组 PLCZ1 RNA 可以延长 Ca2+ 振荡幅度和时间[25],有望以后成为一个治疗方案的选择。除了 PLCZ1,在 2021年, Dai 等[26] 首次报道 ICSI 受精失败男性患者携带 ACTL9基因突变,该基因突变导致精子呈现“褶皱状”顶体,PLCζ 无法正常的定位于核周鞘中,从而无法释放于卵胞浆内激发 Ca2+ 振荡最终引起受精失败。研究人员对患者调整治疗方案,采用钙离子辅助激活手段可以有效干预,患者可获得正常受精和优质胚胎,移植后获得健康活产。因此,对于携带PLCZ1、ACTL9基因突变的男性不育患者,可以采用AOA+ICSI的方式正常生育。

  • 2.2 卵子因素

  • 成熟的卵母细胞被细胞外透明带包围,透明带介导精子结合和穿透,是受精所必需的。人类的透明带是由 ZP1、2、3、4 四种糖蛋白组成的,目前已报道的遗传突变导致不育病例包括透明带家族 ZP1[27]ZP2 [28-30]ZP3[3132],这些基因的突变会导致受精失败及多精受精。2017 年,Liu 等[28] 首次报道了由 ZP2ZP3双杂合突变导致患者卵子透明带极薄或完全缺失,体外受精后发生多精受精而不育的病例,并通过动物及细胞模型阐明了其致病机制,发现双杂合会导致剂量效应而使透明带异常程度加重。患者经过ICSI干预,最终成功受孕并产下健康男婴。因此,对于卵子透明带异常的患者,采用ICSI助孕能够显著改善受精率及临床妊娠率。

  • 另外,有相当部分受精障碍患者是由于 WEE2[33]TLE6[34]CDC20[35] 突变导致。2018 年, Sang 等[33] 报道了第二个引起受精失败的致病基因 WEE2WEE2 突变通过破坏 CDC2WEE2 蛋白自身的磷酸化使得成熟促进因子 (Maturation-Promoting Factor,MPF) 活性升高,进而导致受精后无法形成雌雄原核。研究者还进一步探索了潜在的分子干预方法:给予一名突变患者的卵子体外注射一定剂量的野生型 WEE2 cRNA,可使得卵子成功受精,并产生了基因组水平相对正常的囊胚。 2020 年,Zhao 等[35] 报道了 CDC20 基因突变可导致受精障碍,该基因突变会引起 CDC20 蛋白剂量降低、着丝粒定位丧失以及MⅠ阻滞表型回补能力降低,同时其下游底物CyclinB1的表达量发生异常累积,最终导致卵子及胚胎发育异常。作者还探索了突变携带患者的干预治疗措施,通过在临床中对两名携带有 CDC20 突变患者 (家系 3 中个体Ⅱ-Ⅰ和家系 4 中个体Ⅱ-Ⅰ) 的卵子干预注射了 CDC20 cRNA,成功挽救了其受精失败的表型,并在体外培养过程中获得了发育良好的囊胚。胚胎移植前诊断结果显示,得到的囊胚不存在大片段缺失及拷贝数变异,进一步说明了该干预过程并不影响胚胎的最终质量。该研究为今后的临床干预提供了科学的指导依据,为未来开展患者的临床基因治疗提供了理论参考并奠定了基础。

  • 3 合子分裂失败及早期胚胎停滞

  • 临床有一类辅助生殖反复失败患者,可以获得成熟卵子,卵子也可以受精,但是受精后得到合子无法分裂,称为合子分裂失败,或者即使分裂,胚胎最终停滞在8细胞之前,称为早期胚胎停滞。在2016 年,Xu 等[36] 首次明确人类早期胚胎停滞为新孟德尔隐性遗传病及第一个突变基因 PADI6。 PADI6蛋白为皮质母源复合体 (Subcortical Maternal Complex,SCMC) 的重要组成部分,该基因突变通过破坏胚胎基因组激活过程,导致人类胚胎早期停滞。至此确定了遗传突变基因可能是导致早期胚胎停滞的重要因素,之后陆续研究表明女性或男性均有可能携带可遗传的基因突变引起早期胚胎停滞[36-38]。而合子分裂失败的致病基因到2020年才被首次报道[39]。目前已经明确的导致合子分裂失败和早期胚胎停滞的致病基因包括PADI6[36]KHDC3L[40]NLRP2[41]NLRP5[41]BTG4[39]ACTL7A[37]REC114[42]MEI1[43]FBXO43[44]CHK1[45]MOS[38] 等。研究者对其中的 ACTL7ACHK1 两个突变基因进行了探索干预。

  • 早期胚胎停滞一直是生殖医学领域的一大难题,对于看似“正常”的精子和卵母细胞,反复出现的早期胚胎停滞,临床医生一般归因于女性因素而非男性因素[37],但在 2020 年,Xin 等[37] 发现了男性遗传因素导致早期胚胎停滞的第一个突变基因 ACTL7A,两对患者来自一个具有兄弟两人的近亲家系,精子形态和精液常规都表现正常,但多次 IVF治疗均呈现胚胎发育停滞,最后都只能通过供精IVF的方式获得后代。研究人员发现兄弟两人均携带 ACTL7A 的纯合错义突变,于是构建了与患者一致的点突变小鼠模型进行机制及治疗的研究。他们发现利用氯化锶 (SrCl2) 进行 ICSI-AOA,可成功使得突变小鼠精子与卵子受精并发育成囊胚,经胚胎移植后突变小鼠可以生育健康子代,这一研究为这类患者的临床治疗奠定了基础并提供了方向。

  • 2021 年,Zhang 等[45] 发现 CHK1 显性突变可导致原核融合失败与受精卵发育阻滞,并阐明了突变引起的 CHK1 激酶活性增加阻碍了受精卵的 G2/M 细胞周期转换,使受精卵无法从减数分裂恢复有丝分裂。值得一提的是,研究者使用CHK1抑制剂可分别有效治疗小鼠和人类该异常表型,可成功获得囊胚,小鼠能恢复生育,治疗后的人类囊胚可诱导获得核型正常的胚胎。该研究为此类不孕症患者的精准诊断和未来治疗提供了可能性。

  • 4 总结和展望

  • 随着高通量基因检测技术的迅猛发展,疾病的基因诊断受到了人们的高度重视和广大患者的接受,同时在基因诊断指导下的各种疾病治疗方案及新药开发也成了生物医药领域研究的热点。在辅助生殖领域,对于反复辅助生殖失败患者,早期进行遗传学检测明确病因,同时结合相应的治疗方案如调整刺激配体 hCG 的剂量和时间、采用人工 AOA 激活卵母细胞、ICSI-AOA、ICSI 等可以改善临床治疗结局。基因突变导致的剂量不足而致病的情况可以通过补充野生型 cRNA或者蛋白进行挽救,或者突变导致基因活性增强可用小分子抑制剂进行治疗,目前大多都还处于实验探索阶段。这些方法仍存在一些不足及有待进一步研究的必要,例如,虽然有些已可以获得囊胚,但后期发育结局如何,除了挽救表型是否会有其他的副作用及风险,补充的剂量、干预的窗口期如何确定等一系列问题需要考虑。而且,在对有效性和安全性充分论证后,也需要经过伦理的审核才能在临床中应用,从理论实践到临床应用这中间还需要较长时间的摸索。 CRISPR/Cas9等基因编辑技术的迅猛发展,使得修复遗传缺陷成为可能,这也将成为未来反复辅助生殖失败治疗策略研究的重要方向。

  • 随着广大学者的不断研究和对反复辅助生殖失败中遗传疾病治疗的进一步探索,会有更多的IVF/ ICSI反复失败的治疗方法应用到临床中,基于基因检测指导下的精准治疗也将进一步提高不孕不育的诊治率,为广大患者带来福音。

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