完全不运动精子经冻融后应用激光筛选存活精子行ICSI获临床妊娠*

冯贵雪陈焕华张 波周 红舒金辉甘贤优

1. 广西壮族自治区妇幼保健院（广西南宁 530003）

2. 广西壮族自治区妇产医院

完全不运动精子经冻融后应用激光筛选存活精子行ICSI获临床妊娠*

冯贵雪1△陈焕华1,2△张 波1**周 红1舒金辉1甘贤优1

1. 广西壮族自治区妇幼保健院（广西南宁 530003）

2. 广西壮族自治区妇产医院

目的 报道1例睾丸完全不运动精子经冷冻-解冻后应用激光筛选存活精子行卵胞浆内单精子注射（ICSI）获得临床妊娠。方法 睾丸穿刺获取的完全不运动精子经激光鉴定有存活的精子，立即进行冷冻保存。取卵日，解冻该精子应用激光选择存活的精子进行卵胞浆内单精子注射。结果 睾丸完全不运动精子冷冻前和解冻后经激光鉴定存活率分别为55.75%和39.81%。选择激光鉴定为存活的精子注射到5个成熟卵母细胞中，4个卵子正常受精（受精率80%），培养至第3天均卵裂（卵裂率100%），移植两枚优质胚胎后获得临床妊娠。结论 完全不运动精子是可以冷冻保存的。

激光; 精液保存; 精子注射, 细胞质内

卵胞浆内单精子注射（intracytoplasmic sperm injection, ICIS）技术是目前治疗严重男性不育的理想手段。研究表明，ICSI成功受精的先决条件是需要有存活的精子[1,2]。运动性是精子具有活力的显著标志。已经有研究证实，完全不运动精子ICSI后受精率很低[3]，甚至不受精[4,5]，因此，临床上常选择运动的精子行ICSI。精子冷冻是保存男性生育力最有效的方法，但是冷冻-解冻过程可能对精子造成不可逆的损伤[6,7]。传统的观点认为，精子冷冻保存的前提需要有运动的精子，对于完全不运动的精子是否有冷冻保存价值尚不清楚。我中心对1例冻前和冻后均为完全不运动精子用激光筛选后行ICSI后获得临床妊娠，可能有助于对完全不运动精子的生育力保存价值进行重新认识。

资料与方法

一、临床资料

男方，40岁，女方，38岁，双方原发不孕10年余。男方能顺行射精，无阳痿、早泄。男方查血FSH14.68mIU/ml，Y染色体微缺失检测未见异常，染色体核型分析示46, XY。2次查精均提示“无精子”症。睾丸穿刺结果显示，培养数小时后200倍镜下，每个视野可见3~5条不动精子，激光[8]鉴定的存活率为55.76%。经讨论，可尝试将该患者的完全不运动精子冷冻保存，并用激光选择冻后的精子用于ICSI。

二、睾丸精子抽吸术、精子冷冻和解冻

睾丸精子抽吸术参照文献[9]的报道。获取的睾丸精子悬液与冷冻液按1:1比例稀释后经液氮熏蒸再投入液氮冷冻保存。将冷冻精子从液氮罐中取出，立即投入到37℃的水浴中，轻轻摇动冷冻管使精液快速解冻，然后继续在37℃的水浴中孵育15min。解冻后的精子离心洗涤一次，然后将精子沉淀团加入到Quinn's 1020 操作皿中继续孵育4h。经孵育后，仍未发现有活动精子，经激光鉴定存活率为39.81%，故采用激光法选择存活的精子进行ICSI授精。

三、促排卵方案、取卵

促排卵方案采用本中心常规的长效GnRH-a激动剂长方案[10]，采用达菲林进行垂体降调节后，200IU促卵泡素（Follicle stimulating hormone, FSH）启动，当主导卵泡直径达到18mm时给予10 000IU 人绒毛膜促性腺激素（Human chorionic gonadotropin, hCG）扳机，于hCG 后36h取卵。

四、激光选择完全不动精子方法

参照Aktan等[8]报道的方法，在高倍视野下选择形态较好的精子，在精子尾部靠近尾巴尖端处用激光射击（英国Saturn 5TMLaser Systems），如果激光射击后精子尾部立即出现明显的卷尾现象则判定为存活精子，对激光射击不卷尾的为死精子。选择存活的精子行ICSI受精。

五、胚胎培养和妊娠确定

ICSI后16~18h 观察受精情况，出现两个原核为正常受精，转入含10%血清蛋白替代物（Serum Protein Substitute, SPS）的Quinn's 1026卵裂期培养基中培养至第3天，然后再转入含10% SPS 的Quinn's 1029 囊胚培养液中继续培养至囊胚期。取卵后第1天开始肌内注射黄体酮，60~80 mg/d。移植后14d，查血hCG，若阳性，诊断为生化妊娠； 移植后21~28 d，超声下见妊娠囊诊断为临床。

结 果

患者共获卵8枚。当日将冻存的精子解冻， 200倍镜下8~10个视野见1条完全不运动精子，与新鲜精子评价法一样，用激光打击精子尾部，将发生卷尾的精子立即用于ICSI。8个 卵子中，共有5枚成熟卵子并予ICSI，结果4枚正常受精并正常卵裂。受精后第3天观察形成2枚优质胚胎并移植，剩余两枚非优质胚胎经培养后形成1枚4BB囊胚并冷冻保存。移植后14d，查血hCG 492.6 IU/L，确定生化妊娠，移植后21d超声下检查见宫内1个孕囊，确定临床妊娠。

讨 论

本文首次报道应用激光鉴定冷冻前和冷冻后均为完全不运动精子行ICSI后获临床妊娠。冷冻前，该患者睾丸获取的完全不运动精子经激光鉴定存活率为55.76%；解冻后经孵育仍未找到活动精子，经激光鉴定存活率为39.81%。用激光筛选存活的冷冻-解冻完全不运动精子行ICSI 获得了较好的胚胎发育率和临床妊娠结局。我们的研究首次证明，完全不运动精子经冷冻保存依然能获得正常的体外受精能力，这为完全不动精子症患者的生育力保存提供新视野。

临床上，通常认为只有运动的精子适合冷冻保存，而不运动的精子往往被丢弃[11]，部分患者因此而无法进入辅助生殖助孕治疗。因此，如何有效地保存完全不运动精子症患者的生育力是值得深思的问题。完全不运动精子主要见于Kartagener综合征和睾丸穿刺取精的患者[12-14]，前者是一种原发纤毛运动障碍综合征，是由于精子生成过程中的遗传缺陷而导致精子尾部失去运动能力。完全不运动精子最常见于睾丸穿刺取精的患者[15,16]，而氧化应激、感染等因素是导致睾丸精子不运动的主要原因[17]。按传统的冷冻方法，如果获取的睾丸精子不动则很有可能被废弃，需要重复穿刺，增加了患者远期并发症发生的机会和经济负担。然而，研究已经证实，用激光从新鲜的完全不运动的精子中筛选存活的精子行ICSI后，获得了与活动精子相似的临床结局[11,18]。然而，完全不运动精子是否适合冷冻保存尚不清楚？如何筛选这些经冷冻后的存活但完全不动精子行ICSI是面临的严峻挑战。

目前，鉴别不运动精子存活率的方法有多种。WHO推荐用伊红染色和低渗肿胀试验来鉴别不动精子的存活状态[19]。伊红染色是经典的鉴别不动精子存活率的方法，其原理是基于存活精子对染料的拒染性，而死亡的精子则被染料染色。但是，经伊红染色后的精子无法再用于受精[20]，限制了其在辅助生殖助孕中的应用。有研究发现，低渗肿胀试验（HOS）能有效从新鲜的完全不动精子中鉴别存活的精子，行ICSI后获得良好的受精率及临床结局[21]。但由于精子自发肿胀干扰HOS的准确性，尤其是冷冻-解冻精子自发肿胀率较高，可能导致出现假阳性结果[22]。因此，HOS并不适用于冻融后精子的鉴别。近年来，激光已经被广泛应用于辅助生殖领域中，包括辅助孵化[23]、胚胎活检[24]和精子辅助制动[25]等。ICSI前应用激光辅助制动精子已经诞生了健康的婴儿，并且证实激光制动精子是安全的[26,27]。随后，有研究者证实，激光和低渗肿胀试验在鉴别新鲜完全不运动精子存活率方面具有相似的效能[8]，并已获得了临床妊娠[13,28]。相对于HOS，激光筛选精子的客观性更强，并且简单、快速，且无需借助其他化学制剂。然而，尚无关于激光在筛选冷冻-解冻后完全不运动精子行ICSI获得临床妊娠的报道。本研究证实，经激光鉴定有一定存活率的精子是可以冷冻保存，解冻后用激光筛选存活的精子可获得理想的胚胎发育率和临床结局，证实了激光在完全不运动精子症患者生育力保存中的应用价值。

我们首次报道完全不运动精子经冻融后应用激光筛选存活精子行ICSI后获临床妊娠。这表明，存活但完全不运动精子具有冷冻保存价值，这为完全不运动精子患者的生育力保存提供了新思路。

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（2016-12-15收稿）

Successful pregnancy using the primary completely immotile spermatozoa to cryopreserve and subsequently selected frozenthawed viable spermatozoa for ICSI by laser*

Feng Guixue1△, Chen Huanhua1,2△, Zhang Bo1**, Zhou Hong1, Shu Jinhui1, Gan Xianyou1
1. Reproductive Medicine Center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning,
China; 2. Reproductive Medicine Center, Obstetrics and Gynecology Hospital of Guangxi Zhuang Autonomous Region

Objective TTo report a successful pregnancy using the frozen-thawed completely immotile spermatozoa selected by laser.Methods A single laser shot was used to identify whether viable immotile spermatozoa existed in the fresh and frozen-thawed testicular spermatozoa. The viability rate was 55.76% after identi fi cation by laser, and viable immotile spermatozoa were cryopreserved immediately.Results The thawing test was performed on the day of OPU( oocyte pickup) and none of motile sperm was observed after extending culture for another 4 hours, while 39.81% survival rate was detected by laser. A total of 5 mature oocytes were injected resulting in 4 normal fertilization (80%) on day-1, and two high quality day-3 embryos were transferred, which resulted in a singleton pregnancy.Conclusion Our report demonstrated that completely immotile spermatozoa is worth of cryopreservation for further ICSI, which provide a new insight to male fertility preservation for completely immotile spermatozoa.

lasers; semen preservation; sperm injections, intracytoplasmic

10.3969/j.issn.1008-0848.2017.01.009

R 698.2

资助： 广西自然科学基金（2013GXNSFAA019258)，公益性行业科研专项（项目编号201402004）；广西卫生厅重点科研课题（重2011063，重2011065）；广西卫生厅自筹课题（自2012210）**

，E-mail: cestbon269@sina.com△并列第一作者