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)婦產醫(yī)院
完全不運動精子經(jīng)凍融后應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)婦產醫(yī)院
目的 報道1例睪丸完全不運動精子經(jīng)冷凍-解凍后應用激光篩選存活精子行卵胞漿內單精子注射(ICSI)獲得臨床妊娠。方法 睪丸穿刺獲取的完全不運動精子經(jīng)激光鑒定有存活的精子,立即進行冷凍保存。取卵日,解凍該精子應用激光選擇存活的精子進行卵胞漿內單精子注射。結果 睪丸完全不運動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個成熟卵母細胞中,4個卵子正常受精(受精率80%),培養(yǎng)至第3天均卵裂(卵裂率100%),移植兩枚優(yōu)質胚胎后獲得臨床妊娠。結論 完全不運動精子是可以冷凍保存的。
激光; 精液保存; 精子注射, 細胞質內
卵胞漿內單精子注射(intracytoplasmic sperm injection, ICIS)技術是目前治療嚴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]。運動性是精子具有活力的顯著標志。已經(jīng)有研究證實,完全不運動精子ICSI后受精率很低[3],甚至不受精[4,5],因此,臨床上常選擇運動的精子行ICSI。精子冷凍是保存男性生育力最有效的方法,但是冷凍-解凍過程可能對精子造成不可逆的損傷[6,7]。傳統(tǒng)的觀點認為,精子冷凍保存的前提需要有運動的精子,對于完全不運動的精子是否有冷凍保存價值尚不清楚。我中心對1例凍前和凍后均為完全不運動精子用激光篩選后行ICSI后獲得臨床妊娠,可能有助于對完全不運動精子的生育力保存價值進行重新認識。
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,無陽痿、早泄。男方查血FSH14.68mIU/ml,Y染色體微缺失檢測未見異常,染色體核型分析示46, XY。2次查精均提示“無精子”癥。睪丸穿刺結果顯示,培養(yǎng)數(shù)小時后200倍鏡下,每個視野可見3~5條不動精子,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動精子冷凍保存,并用激光選擇凍后的精子用于ICSI。
二、睪丸精子抽吸術、精子冷凍和解凍
睪丸精子抽吸術參照文獻[9]的報道。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)的長效GnRH-a激動劑長方案[10],采用達菲林進行垂體降調節(jié)后,200IU促卵泡素(Follicle stimulating hormone, FSH)啟動,當主導卵泡直徑達到18mm時給予10 000IU 人絨毛膜促性腺激素(Human chorionic gonadotropin, hCG)扳機,于hCG 后36h取卵。
四、激光選擇完全不動精子方法
參照Aktan等[8]報道的方法,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)兩個原核為正常受精,轉入含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天開始肌內注射黃體酮,60~80 mg/d。移植后14d,查血hCG,若陽性,診斷為生化妊娠; 移植后21~28 d,超聲下見妊娠囊診斷為臨床。
患者共獲卵8枚。當日將凍存的精子解凍, 200倍鏡下8~10個視野見1條完全不運動精子,與新鮮精子評價法一樣,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 卵子中,共有5枚成熟卵子并予ICSI,結果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)質胚胎并移植,剩余兩枚非優(yōu)質胚胎經(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超聲下檢查見宮內1個孕囊,確定臨床妊娠。
本文首次報道應用激光鑒定冷凍前和冷凍后均為完全不運動精子行ICSI后獲臨床妊娠。冷凍前,該患者睪丸獲取的完全不運動精子經(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-解凍完全不運動精子行ICSI 獲得了較好的胚胎發(fā)育率和臨床妊娠結局。我們的研究首次證明,完全不運動精子經(jīng)冷凍保存依然能獲得正常的體外受精能力,這為完全不動精子癥患者的生育力保存提供新視野。
臨床上,通常認為只有運動的精子適合冷凍保存,而不運動的精子往往被丟棄[11],部分患者因此而無法進入輔助生殖助孕治療。因此,如何有效地保存完全不運動精子癥患者的生育力是值得深思的問題。完全不運動精子主要見于Kartagener綜合征和睪丸穿刺取精的患者[12-14],前者是一種原發(fā)纖毛運動障礙綜合征,是由于精子生成過程中的遺傳缺陷而導致精子尾部失去運動能力。完全不運動精子最常見于睪丸穿刺取精的患者[15,16],而氧化應激、感染等因素是導致睪丸精子不運動的主要原因[17]。按傳統(tǒng)的冷凍方法,如果獲取的睪丸精子不動則很有可能被廢棄,需要重復穿刺,增加了患者遠期并發(fā)癥發(fā)生的機會和經(jīng)濟負擔。然而,研究已經(jīng)證實,用激光從新鮮的完全不運動的精子中篩選存活的精子行ICSI后,獲得了與活動精子相似的臨床結局[11,18]。然而,完全不運動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精子行ICSI是面臨的嚴峻挑戰(zhàn)。
目前,鑒別不運動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來鑒別不動精子的存活狀態(tài)[19]。伊紅染色是經(jīng)典的鑒別不動精子存活率的方法,其原理是基于存活精子對染料的拒染性,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法再用于受精[20],限制了其在輔助生殖助孕中的應用。有研究發(fā)現(xiàn),低滲腫脹試驗(HOS)能有效從新鮮的完全不動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局[21]。但由于精子自發(fā)腫脹干擾HOS的準確性,尤其是冷凍-解凍精子自發(fā)腫脹率較高,可能導致出現(xiàn)假陽性結果[22]。因此,HOS并不適用于凍融后精子的鑒別。近年來,激光已經(jīng)被廣泛應用于輔助生殖領域中,包括輔助孵化[23]、胚胎活檢[24]和精子輔助制動[25]等。ICSI前應用激光輔助制動精子已經(jīng)誕生了健康的嬰兒,并且證實激光制動精子是安全的[26,27]。隨后,有研究者證實,激光和低滲腫脹試驗在鑒別新鮮完全不運動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]。相對于HOS,激光篩選精子的客觀性更強,并且簡單、快速,且無需借助其他化學制劑。然而,尚無關于激光在篩選冷凍-解凍后完全不運動精子行ICSI獲得臨床妊娠的報道。本研究證實,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)育率和臨床結局,證實了激光在完全不運動精子癥患者生育力保存中的應用價值。
我們首次報道完全不運動精子經(jīng)凍融后應用激光篩選存活精子行ICSI后獲臨床妊娠。這表明,存活但完全不運動精子具有冷凍保存價值,這為完全不運動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: 廣西自然科學基金(2013GXNSFAA019258),公益性行業(yè)科研專項(項目編號201402004);廣西衛(wèi)生廳重點科研課題(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