馮貴雪陳煥華張 波周 紅舒金輝甘賢優(yōu)

1. 廣西壯族自治區(qū)婦幼保健院（廣西南寧 530003）

2. 廣西壯族自治區(qū)婦產(chǎn)醫(yī)院

完全不運(yùn)動(dòng)精子經(jīng)凍融后應(yīng)用激光篩選存活精子行ICSI獲臨床妊娠*

馮貴雪1△陳煥華1,2△張 波1**周 紅1舒金輝1甘賢優(yōu)1

1. 廣西壯族自治區(qū)婦幼保健院（廣西南寧 530003）

2. 廣西壯族自治區(qū)婦產(chǎn)醫(yī)院

目的 報(bào)道1例睪丸完全不運(yùn)動(dòng)精子經(jīng)冷凍-解凍后應(yīng)用激光篩選存活精子行卵胞漿內(nèi)單精子注射（ICSI）獲得臨床妊娠。方法 睪丸穿刺獲取的完全不運(yùn)動(dòng)精子經(jīng)激光鑒定有存活的精子，立即進(jìn)行冷凍保存。取卵日，解凍該精子應(yīng)用激光選擇存活的精子進(jìn)行卵胞漿內(nèi)單精子注射。結(jié)果 睪丸完全不運(yùn)動(dòng)精子冷凍前和解凍后經(jīng)激光鑒定存活率分別為55.75%和39.81%。選擇激光鑒定為存活的精子注射到5個(gè)成熟卵母細(xì)胞中，4個(gè)卵子正常受精（受精率80%），培養(yǎng)至第3天均卵裂（卵裂率100%），移植兩枚優(yōu)質(zhì)胚胎后獲得臨床妊娠。結(jié)論 完全不運(yùn)動(dòng)精子是可以冷凍保存的。

激光; 精液保存; 精子注射, 細(xì)胞質(zhì)內(nèi)

卵胞漿內(nèi)單精子注射（intracytoplasmic sperm injection, ICIS）技術(shù)是目前治療嚴(yán)重男性不育的理想手段。研究表明，ICSI成功受精的先決條件是需要有存活的精子[1,2]。運(yùn)動(dòng)性是精子具有活力的顯著標(biāo)志。已經(jīng)有研究證實(shí)，完全不運(yùn)動(dòng)精子ICSI后受精率很低[3]，甚至不受精[4,5]，因此，臨床上常選擇運(yùn)動(dòng)的精子行ICSI。精子冷凍是保存男性生育力最有效的方法，但是冷凍-解凍過(guò)程可能對(duì)精子造成不可逆的損傷[6,7]。傳統(tǒng)的觀點(diǎn)認(rèn)為，精子冷凍保存的前提需要有運(yùn)動(dòng)的精子，對(duì)于完全不運(yùn)動(dòng)的精子是否有冷凍保存價(jià)值尚不清楚。我中心對(duì)1例凍前和凍后均為完全不運(yùn)動(dòng)精子用激光篩選后行ICSI后獲得臨床妊娠，可能有助于對(duì)完全不運(yùn)動(dòng)精子的生育力保存價(jià)值進(jìn)行重新認(rèn)識(shí)。

資料與方法

一、臨床資料

男方，40歲，女方，38歲，雙方原發(fā)不孕10年余。男方能順行射精，無(wú)陽(yáng)痿、早泄。男方查血FSH14.68mIU/ml，Y染色體微缺失檢測(cè)未見(jiàn)異常，染色體核型分析示46, XY。2次查精均提示“無(wú)精子”癥。睪丸穿刺結(jié)果顯示，培養(yǎng)數(shù)小時(shí)后200倍鏡下，每個(gè)視野可見(jiàn)3~5條不動(dòng)精子，激光[8]鑒定的存活率為55.76%。經(jīng)討論，可嘗試將該患者的完全不運(yùn)動(dòng)精子冷凍保存，并用激光選擇凍后的精子用于ICSI。

二、睪丸精子抽吸術(shù)、精子冷凍和解凍

睪丸精子抽吸術(shù)參照文獻(xiàn)[9]的報(bào)道。獲取的睪丸精子懸液與冷凍液按1:1比例稀釋后經(jīng)液氮熏蒸再投入液氮冷凍保存。將冷凍精子從液氮罐中取出，立即投入到37℃的水浴中，輕輕搖動(dòng)冷凍管使精液快速解凍，然后繼續(xù)在37℃的水浴中孵育15min。解凍后的精子離心洗滌一次，然后將精子沉淀團(tuán)加入到Quinn's 1020 操作皿中繼續(xù)孵育4h。經(jīng)孵育后，仍未發(fā)現(xiàn)有活動(dòng)精子，經(jīng)激光鑒定存活率為39.81%，故采用激光法選擇存活的精子進(jìn)行ICSI授精。

三、促排卵方案、取卵

促排卵方案采用本中心常規(guī)的長(zhǎng)效GnRH-a激動(dòng)劑長(zhǎng)方案[10]，采用達(dá)菲林進(jìn)行垂體降調(diào)節(jié)后，200IU促卵泡素（Follicle stimulating hormone, FSH）啟動(dòng)，當(dāng)主導(dǎo)卵泡直徑達(dá)到18mm時(shí)給予10 000IU 人絨毛膜促性腺激素（Human chorionic gonadotropin, hCG）扳機(jī)，于hCG 后36h取卵。

四、激光選擇完全不動(dòng)精子方法

參照Aktan等[8]報(bào)道的方法，在高倍視野下選擇形態(tài)較好的精子，在精子尾部靠近尾巴尖端處用激光射擊（英國(guó)Saturn 5TMLaser Systems），如果激光射擊后精子尾部立即出現(xiàn)明顯的卷尾現(xiàn)象則判定為存活精子，對(duì)激光射擊不卷尾的為死精子。選擇存活的精子行ICSI受精。

五、胚胎培養(yǎng)和妊娠確定

ICSI后16~18h 觀察受精情況，出現(xiàn)兩個(gè)原核為正常受精，轉(zhuǎn)入含10%血清蛋白替代物（Serum Protein Substitute, SPS）的Quinn's 1026卵裂期培養(yǎng)基中培養(yǎng)至第3天，然后再轉(zhuǎn)入含10% SPS 的Quinn's 1029 囊胚培養(yǎng)液中繼續(xù)培養(yǎng)至囊胚期。取卵后第1天開始肌內(nèi)注射黃體酮，60~80 mg/d。移植后14d，查血hCG，若陽(yáng)性，診斷為生化妊娠； 移植后21~28 d，超聲下見(jiàn)妊娠囊診斷為臨床。

結(jié) 果

患者共獲卵8枚。當(dāng)日將凍存的精子解凍， 200倍鏡下8~10個(gè)視野見(jiàn)1條完全不運(yùn)動(dòng)精子，與新鮮精子評(píng)價(jià)法一樣，用激光打擊精子尾部，將發(fā)生卷尾的精子立即用于ICSI。8個(gè) 卵子中，共有5枚成熟卵子并予ICSI，結(jié)果4枚正常受精并正常卵裂。受精后第3天觀察形成2枚優(yōu)質(zhì)胚胎并移植，剩余兩枚非優(yōu)質(zhì)胚胎經(jīng)培養(yǎng)后形成1枚4BB囊胚并冷凍保存。移植后14d，查血hCG 492.6 IU/L，確定生化妊娠，移植后21d超聲下檢查見(jiàn)宮內(nèi)1個(gè)孕囊，確定臨床妊娠。

討 論

本文首次報(bào)道應(yīng)用激光鑒定冷凍前和冷凍后均為完全不運(yùn)動(dòng)精子行ICSI后獲臨床妊娠。冷凍前，該患者睪丸獲取的完全不運(yùn)動(dòng)精子經(jīng)激光鑒定存活率為55.76%；解凍后經(jīng)孵育仍未找到活動(dòng)精子，經(jīng)激光鑒定存活率為39.81%。用激光篩選存活的冷凍-解凍完全不運(yùn)動(dòng)精子行ICSI 獲得了較好的胚胎發(fā)育率和臨床妊娠結(jié)局。我們的研究首次證明，完全不運(yùn)動(dòng)精子經(jīng)冷凍保存依然能獲得正常的體外受精能力，這為完全不動(dòng)精子癥患者的生育力保存提供新視野。

臨床上，通常認(rèn)為只有運(yùn)動(dòng)的精子適合冷凍保存，而不運(yùn)動(dòng)的精子往往被丟棄[11]，部分患者因此而無(wú)法進(jìn)入輔助生殖助孕治療。因此，如何有效地保存完全不運(yùn)動(dòng)精子癥患者的生育力是值得深思的問(wèn)題。完全不運(yùn)動(dòng)精子主要見(jiàn)于Kartagener綜合征和睪丸穿刺取精的患者[12-14]，前者是一種原發(fā)纖毛運(yùn)動(dòng)障礙綜合征，是由于精子生成過(guò)程中的遺傳缺陷而導(dǎo)致精子尾部失去運(yùn)動(dòng)能力。完全不運(yùn)動(dòng)精子最常見(jiàn)于睪丸穿刺取精的患者[15,16]，而氧化應(yīng)激、感染等因素是導(dǎo)致睪丸精子不運(yùn)動(dòng)的主要原因[17]。按傳統(tǒng)的冷凍方法，如果獲取的睪丸精子不動(dòng)則很有可能被廢棄，需要重復(fù)穿刺，增加了患者遠(yuǎn)期并發(fā)癥發(fā)生的機(jī)會(huì)和經(jīng)濟(jì)負(fù)擔(dān)。然而，研究已經(jīng)證實(shí)，用激光從新鮮的完全不運(yùn)動(dòng)的精子中篩選存活的精子行ICSI后，獲得了與活動(dòng)精子相似的臨床結(jié)局[11,18]。然而，完全不運(yùn)動(dòng)精子是否適合冷凍保存尚不清楚？如何篩選這些經(jīng)冷凍后的存活但完全不動(dòng)精子行ICSI是面臨的嚴(yán)峻挑戰(zhàn)。

目前，鑒別不運(yùn)動(dòng)精子存活率的方法有多種。WHO推薦用伊紅染色和低滲腫脹試驗(yàn)來(lái)鑒別不動(dòng)精子的存活狀態(tài)[19]。伊紅染色是經(jīng)典的鑒別不動(dòng)精子存活率的方法，其原理是基于存活精子對(duì)染料的拒染性，而死亡的精子則被染料染色。但是，經(jīng)伊紅染色后的精子無(wú)法再用于受精[20]，限制了其在輔助生殖助孕中的應(yīng)用。有研究發(fā)現(xiàn)，低滲腫脹試驗(yàn)（HOS）能有效從新鮮的完全不動(dòng)精子中鑒別存活的精子，行ICSI后獲得良好的受精率及臨床結(jié)局[21]。但由于精子自發(fā)腫脹干擾HOS的準(zhǔn)確性，尤其是冷凍-解凍精子自發(fā)腫脹率較高，可能導(dǎo)致出現(xiàn)假陽(yáng)性結(jié)果[22]。因此，HOS并不適用于凍融后精子的鑒別。近年來(lái)，激光已經(jīng)被廣泛應(yīng)用于輔助生殖領(lǐng)域中，包括輔助孵化[23]、胚胎活檢[24]和精子輔助制動(dòng)[25]等。ICSI前應(yīng)用激光輔助制動(dòng)精子已經(jīng)誕生了健康的嬰兒，并且證實(shí)激光制動(dòng)精子是安全的[26,27]。隨后，有研究者證實(shí)，激光和低滲腫脹試驗(yàn)在鑒別新鮮完全不運(yùn)動(dòng)精子存活率方面具有相似的效能[8]，并已獲得了臨床妊娠[13,28]。相對(duì)于HOS，激光篩選精子的客觀性更強(qiáng)，并且簡(jiǎn)單、快速，且無(wú)需借助其他化學(xué)制劑。然而，尚無(wú)關(guān)于激光在篩選冷凍-解凍后完全不運(yùn)動(dòng)精子行ICSI獲得臨床妊娠的報(bào)道。本研究證實(shí)，經(jīng)激光鑒定有一定存活率的精子是可以冷凍保存，解凍后用激光篩選存活的精子可獲得理想的胚胎發(fā)育率和臨床結(jié)局，證實(shí)了激光在完全不運(yùn)動(dòng)精子癥患者生育力保存中的應(yīng)用價(jià)值。

我們首次報(bào)道完全不運(yùn)動(dòng)精子經(jīng)凍融后應(yīng)用激光篩選存活精子行ICSI后獲臨床妊娠。這表明，存活但完全不運(yùn)動(dòng)精子具有冷凍保存價(jià)值，這為完全不運(yùn)動(dòng)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

資助： 廣西自然科學(xué)基金（2013GXNSFAA019258)，公益性行業(yè)科研專項(xiàng)（項(xiàng)目編號(hào)201402004）；廣西衛(wèi)生廳重點(diǎn)科研課題（重2011063，重2011065）；廣西衛(wèi)生廳自籌課題（自2012210）**

，E-mail: cestbon269@sina.com△并列第一作者