衰老導(dǎo)致卵巢功能低下研究進(jìn)展

劉傳明，丁利軍,2，李佳音，戴建武，孫海翔

衰老導(dǎo)致卵巢功能低下研究進(jìn)展

劉傳明1，丁利軍1,2，李佳音3，戴建武3，孫海翔1

1. 南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院生殖醫(yī)學(xué)中心，南京 210008 2. 南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院臨床干細(xì)胞研究中心，南京 210008 3. 中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所，分子發(fā)育生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室，北京 100190

由于社會(huì)角色的轉(zhuǎn)變，女性生育延遲現(xiàn)象明顯。女性卵巢功能一般從35歲時(shí)開始下降，主要表現(xiàn)為卵泡數(shù)量減少和卵母細(xì)胞質(zhì)量下降。目前臨床上對于卵巢功能低下的診斷主要依據(jù)血清卵泡刺激素(follicle stimu-lating hormone, FSH)、血清抗苗勒氏管激素(anti-Müllerian hormone, AMH)、竇卵泡計(jì)數(shù)、年齡、月經(jīng)和抑制素B等指標(biāo)。目前研究發(fā)現(xiàn)，伴隨年齡的增加，女性卵巢內(nèi)細(xì)胞會(huì)出現(xiàn)線粒體功能失調(diào)、染色質(zhì)短縮、DNA修復(fù)減少、表觀遺傳學(xué)改變和代謝失序。本文在簡要介紹卵巢功能低下臨床診斷的基礎(chǔ)上，對衰老導(dǎo)致卵巢功能低下的相關(guān)因素進(jìn)行了總結(jié)，并深入探討了其發(fā)生的分子機(jī)制及潛在的干預(yù)靶點(diǎn)，以期為有效改善高齡女性的卵巢功能提供思路。

卵巢；衰老；線粒體；遺傳；表觀遺傳

自20世紀(jì)70年代以來，因經(jīng)濟(jì)、社會(huì)等因素使得女性生育延遲成為一種普遍的社會(huì)現(xiàn)象[1]。根據(jù)中國人口協(xié)會(huì)、國家計(jì)生委聯(lián)名發(fā)布的最新《中國不孕不育現(xiàn)狀調(diào)研報(bào)告》顯示，目前我國的不孕不育率約為12.5%，并有逐年增高的趨勢。美國疾病預(yù)防控制中心2015年的數(shù)據(jù)顯示，美國女性初次生育年齡由21.2歲(1970年)，上升至25.8歲，35歲初產(chǎn)婦超過1/12；2017年韓國女性初次生育年齡平均達(dá)到31歲。研究發(fā)現(xiàn)與小于35歲的女性相比，高齡女性更易出現(xiàn)不孕、流產(chǎn)、死胎和多胎等危險(xiǎn)，而衰老導(dǎo)致的卵巢功能低下可能在其中發(fā)揮關(guān)鍵作用[2~4]。因此，衰老導(dǎo)致卵巢功能低下研究成為生殖醫(yī)學(xué)的熱點(diǎn)之一。本文簡要介紹了卵巢功能低下的診斷標(biāo)準(zhǔn)，并深入探討了衰老影響卵巢功能的可能機(jī)制及已知的治療方法。

1 卵巢功能低下的臨床診斷

卵巢功能低下引起的卵母細(xì)胞數(shù)量和質(zhì)量的下降是影響妊娠的主要因素。卵巢功能低下表現(xiàn)為原始卵泡池的耗竭。卵巢的主要功能在女性約50歲時(shí)基本喪失[5]。女性卵巢功能低下會(huì)增加一系列并發(fā)癥的發(fā)病風(fēng)險(xiǎn)，如骨質(zhì)疏松、心血管疾病、復(fù)發(fā)性抑郁癥和認(rèn)知功能障礙等，從而降低生活質(zhì)量[6,7]。目前卵巢功能低下的主要診斷指標(biāo)包括：年齡、卵泡刺激素(follicle-stimulating hormone, FSH)、抗苗勒氏管激素(anti-Müllerian hormone, AMH)、抑制素B (inhibin B, INHB)、竇卵泡數(shù)(antral follicle count, AFC)、卵巢間質(zhì)血流和基礎(chǔ)卵巢體積等[8]。盡管AMH和AFC被廣泛用于卵巢功能低下的診斷，但是目前尚沒有一個(gè)指標(biāo)被證實(shí)可以獨(dú)立預(yù)測卵巢功能[9]。在既定范圍內(nèi)與年齡相符的功能性卵巢儲(chǔ)備下降稱為生理性卵巢功能衰老(normal ovarian aging, NOA)。另外，研究顯示人群中約10%的女性會(huì)出現(xiàn)與年齡不符的功能性卵巢儲(chǔ)備降低，但沒有表現(xiàn)出顯著的臨床癥狀，被稱為隱匿性卵巢功能低下或隱匿性卵巢功能不全(occult premature ovarian inscuffi-ciency, OPOI)[10,11]。此外，還有約1%女性會(huì)在40歲之前出現(xiàn)卵巢內(nèi)卵泡提前耗竭，完全停經(jīng)等癥狀，被稱為早發(fā)性卵巢功能低下或早發(fā)性卵巢功能不全(premature ovarian inscufficiency, POI)。歐洲人類生殖與胚胎協(xié)會(huì)(ESHRE)規(guī)定了POI的診斷標(biāo)準(zhǔn)：大于4個(gè)月的月經(jīng)稀發(fā)或者停經(jīng)同時(shí)FSH檢測值高于25 U/L。而對于臨床表現(xiàn)更為嚴(yán)重的卵巢早衰(pre-mature ovarian failure, POF)，其診斷標(biāo)準(zhǔn)包括FSH> 40 U/L并伴有超過4個(gè)月的繼發(fā)性閉經(jīng)[12]。

2 衰老導(dǎo)致卵巢功能低下的致病因素

2.1 線粒體功能失調(diào)

線粒體與卵巢功能密切相關(guān)。衰老會(huì)導(dǎo)致線粒體DNA (mitochondrial DNA, mtDNA)不穩(wěn)定性增加，引起卵巢細(xì)胞尤其是卵母細(xì)胞中線粒體DNA突變的積累。線粒體的生物發(fā)生對于卵泡和早期胚胎發(fā)育至關(guān)重要，而卵巢功能下降也會(huì)嚴(yán)重影響卵母細(xì)胞及周圍顆粒細(xì)胞中線粒體的生成及線粒體功能[13]。因此，作為線粒體拷貝數(shù)量最多的細(xì)胞，卵母細(xì)胞中線粒體的功能失調(diào)加速卵巢功能低下，從而導(dǎo)致妊娠失敗。形態(tài)學(xué)和功能學(xué)研究發(fā)現(xiàn)，衰老會(huì)影響細(xì)胞，特別是卵母細(xì)胞線粒體功能，導(dǎo)致線粒體腫脹、空泡化，小線粒體碎片含量增加[14~16]。氧化應(yīng)激(reactive oxidative stress, ROS)被認(rèn)為是衰老相關(guān)的獲得性mtDNA突變的主要來源[17]?！熬€粒體自由基”理論認(rèn)為衰老積聚了高水平的氧自由基和ROS，導(dǎo)致mtDNA突變，進(jìn)而影響功能性電子傳遞鏈(electron transfer chain, ETC)的產(chǎn)生；而mtDNA的突變進(jìn)一步加劇ROS和mtDNA突變的積累，形成惡性循環(huán)，導(dǎo)致ATP產(chǎn)生減少、細(xì)胞周期停滯甚至細(xì)胞凋亡。除ROS外，多種線粒體功能失調(diào)也被證實(shí)與卵巢細(xì)胞的衰老有關(guān)，包括線粒體融合、ETC失活、線粒體代謝改變和鈣穩(wěn)態(tài)失衡等[18]。采用多組學(xué)分析衰老過程中mtDNA變化時(shí)發(fā)現(xiàn)，線粒體單一基因位點(diǎn)的改變即可影響線粒體蛋白穩(wěn)態(tài)、加速活性氧生成、導(dǎo)致端?？s短[19]。作為一種重要的參與線粒體融合的線粒體膜蛋白，線粒體融合蛋白2 (mit-ofusin 2, Mfn2)敲除小鼠出現(xiàn)嚴(yán)重的發(fā)育延遲，并且因胎盤缺陷導(dǎo)致胚胎死亡，卵母細(xì)胞內(nèi)特異性敲除該基因后雌性小鼠不孕[20]。而參與介導(dǎo)線粒體分裂的動(dòng)力相關(guān)蛋白(dynamin-related protein 1, DRP1)也對維持生殖細(xì)胞的正常功能至關(guān)重要，在卵母細(xì)胞內(nèi)特異性敲除DRP1后，小鼠卵泡成熟和排卵均出現(xiàn)障礙[21]。此外，控制線粒體質(zhì)量的相關(guān)蛋白酶在卵巢細(xì)胞中發(fā)揮重要作用，包括CLPP、AFG3L2、PHB、OMA1、LONP1和PARL等在內(nèi)的蛋白酶，其缺陷會(huì)導(dǎo)致相關(guān)線粒體疾病的出現(xiàn)，并加速卵母細(xì)胞的衰老[22~24]。

2.2 遺傳學(xué)改變

卵巢功能下降與卵母細(xì)胞質(zhì)量下降密切相關(guān)。女性卵巢從出生開始在整個(gè)生育周期中不斷受到激素、代謝、免疫等因素的影響，從而導(dǎo)致卵巢內(nèi)卵母細(xì)胞和體細(xì)胞出現(xiàn)DNA損傷。研究表明，隨著年齡增長，卵母細(xì)胞核DNA雙鏈斷裂(double strand break, DSB)明顯增多[25]，并且卵巢中DNA修復(fù)基因的表達(dá)減少，使得DSB不斷累積。而衰老導(dǎo)致的減數(shù)分裂過程中染色體粘結(jié)蛋白的缺失會(huì)影響卵母細(xì)胞染色體分離，導(dǎo)致非整倍體卵母細(xì)胞比例增加，進(jìn)而影響卵母細(xì)胞功能[26]。此外，在姐妹染色單體分離過程中，著絲粒同時(shí)受到來自相反方向的紡錘絲的牽引，引起染色體的錯(cuò)誤分離，這可能是卵母細(xì)胞非整倍體的另一重要原因[27]。紡錘體組裝檢查點(diǎn)(spindle assembly checkpoint, SAC)可以阻止染色體分離，直到姐妹染色單體正確地連接于有絲分裂紡錘體上，其缺失也會(huì)導(dǎo)致非整倍體率顯著提高[28,29]。對CD1小鼠研究表明，12月齡的高齡組小鼠卵母細(xì)胞非整倍體發(fā)生率為31.6%，而年輕組僅為4.9%[30]。老化的卵母細(xì)胞普遍存在端粒的縮短，而縮短的端粒會(huì)引發(fā)DNA損傷反應(yīng)[31]。端粒的縮短主要是由于衰老引起的ROS水平增高所導(dǎo)致的[32]。顆粒細(xì)胞質(zhì)量下降與卵母細(xì)胞質(zhì)量亦密切相關(guān)，隨著年齡增長，顆粒細(xì)胞中同樣存在DNA損傷增多，端?？s短等情況[33,34]。

多組學(xué)研究發(fā)現(xiàn)，衰老可導(dǎo)致卵母細(xì)胞內(nèi)參與細(xì)胞周期信號(hào)轉(zhuǎn)導(dǎo)的基因發(fā)生顯著變化，同時(shí)與SAC、DNA穩(wěn)定性、染色體分離、細(xì)胞分裂、微管和RNA定位等相關(guān)的蛋白質(zhì)表達(dá)也發(fā)生變化[35,36]。通過對高齡和年輕大鼠原始卵泡進(jìn)行微陣列分析，發(fā)現(xiàn)與核苷酸結(jié)合、RNA結(jié)合、核糖體結(jié)構(gòu)成分、轉(zhuǎn)錄因子活性、細(xì)胞周期、同源重組、減數(shù)分裂、DNA復(fù)制和MAPK信號(hào)通路相關(guān)的分子在轉(zhuǎn)錄水平差異顯著[37]。其他物種包括日本黑牛()、C57BL/6小鼠和海門山羊)的研究得到類似的變化趨勢，特別是在高齡母牛卵母細(xì)胞中發(fā)現(xiàn)真核起始因子2 (eukaryotic initiation factor 2, EIf2)信號(hào)通路的相關(guān)分子高表達(dá)[37~39]。已經(jīng)證實(shí)很多基因的改變會(huì)影響卵母細(xì)胞質(zhì)量，加速卵巢衰老。如參與減數(shù)分裂紡錘體組裝的乳腺癌基因1(breast cancer 1,)，其突變會(huì)導(dǎo)致女性卵巢功能加速下降[40,41]。伴隨雌性小鼠年齡的增加，卵母細(xì)胞中染色體結(jié)構(gòu)維持蛋白5/6 (structural maintenance of chromosomes 5/6, SMC5/6)表達(dá)水平下降；其年齡依賴性消耗導(dǎo)致卵母細(xì)胞非整倍體發(fā)生率顯著增加[42]。已知伴隨年齡增加，卵母細(xì)胞內(nèi)發(fā)生顯著性變化的基因如表1所示。

2.3 表觀遺傳學(xué)改變

在卵母細(xì)胞發(fā)生和早期胚胎發(fā)育過程中，建立適當(dāng)?shù)谋碛^遺傳修飾是個(gè)體發(fā)生的一個(gè)重要事件。目前研究已經(jīng)證實(shí)，在衰老的過程中生殖細(xì)胞的DNA會(huì)發(fā)生不正確的表觀遺傳學(xué)修飾，如異常DNA甲基化、組蛋白乙酰化和組蛋白甲基化等。這些異常表觀遺傳修飾可能會(huì)加速衰老[59]。目前認(rèn)為，卵母細(xì)胞中DNA甲基化和組蛋白修飾在生殖發(fā)育過程中發(fā)揮重要作用。Hamatani等[60]比較年輕和高齡C57BL/6雌性小鼠MII期卵母細(xì)胞的mRNA表達(dá)譜時(shí)發(fā)現(xiàn)：5%的轉(zhuǎn)錄本存在明顯差異，其中包括編碼參與表觀遺傳修飾、涉及染色質(zhì)重塑和DNA甲基化的蛋白質(zhì)，如DNA甲基轉(zhuǎn)移酶1、3a、3b、3L和DNMT相關(guān)蛋白-1 (DNA methyltransferase 1-associ-ated protein 1, DAMP1)等。采用其他品系小鼠進(jìn)行微陣列基因表達(dá)分析，獲得類似的轉(zhuǎn)錄本變化[61]。衰老能夠促進(jìn)卵母細(xì)胞DNA甲基轉(zhuǎn)移酶的高表達(dá)，從而催化DNA甲基化。對老齡昆明小鼠的研究發(fā)現(xiàn)，其胚胎致死率和胎兒畸形率均高于年輕組，這與卵母細(xì)胞DNA的甲基化異常密切相關(guān)[62]。而組蛋白乙?；{(diào)控染色體濃縮、DNA斷裂修復(fù)和轉(zhuǎn)錄等細(xì)胞功能[63~65]。在哺乳動(dòng)物卵母細(xì)胞成熟期間，組蛋白H3和H4發(fā)生乙?；揎?。研究發(fā)現(xiàn)，與年輕卵母細(xì)胞相比，高齡動(dòng)物卵母細(xì)胞的基因表達(dá)和組蛋白乙?；揎椌l(fā)生顯著改變[66]。組蛋白3賴氨酸4 (H3K4)的甲基化通常與基因激活和衰老相關(guān)[67,68]。H3K4的二甲基化在年輕動(dòng)物的MII期卵母細(xì)胞中表達(dá)水平更高。而當(dāng)H3K4三甲基化去甲基化酶–視黃醇結(jié)合蛋白2 (retinol binding protein 2, RBP2)缺乏時(shí)，蠕蟲和果蠅的壽命出現(xiàn)縮短[69]。此外，在高齡動(dòng)物的GV期卵母細(xì)胞中，組蛋白甲基化相關(guān)因子(CBX1和SIRT1)的表達(dá)變化趨勢相反，CBX1的表達(dá)顯著升高，而SIRT1的表達(dá)則是降低的[70]。

表1 衰老引起卵母細(xì)胞質(zhì)量降低的基因

2.4 代謝失序

女性隨著年齡的增加會(huì)出現(xiàn)卵巢功能低下，低雌激素血癥，以及一系列的代謝紊亂癥狀。處于絕經(jīng)過渡期的女性會(huì)增加出現(xiàn)代謝綜合征的風(fēng)險(xiǎn)。研究發(fā)現(xiàn)高齡女性的卵泡液內(nèi)脂質(zhì)成分發(fā)生明顯改變，其鞘磷脂、甘油二酯和甘油三酯的豐度更高，而鞘磷脂代謝是凋亡過程中的重要事件，甘油三酯也與卵泡成熟和卵母細(xì)胞質(zhì)量下降相關(guān)[71,72]。同時(shí)，高齡女性的卵泡液中谷胱甘肽過氧化物酶和超氧化物歧化酶含量較低[73,74]。研究表明，晚期糖基化產(chǎn)物增多(advanced glycation end products, AGEs)與卵巢功能下降密切相關(guān)，卵泡內(nèi)AGEs產(chǎn)物積累會(huì)直接損傷蛋白質(zhì)，誘導(dǎo)一系列的氧化應(yīng)激反應(yīng)，并增加炎癥反應(yīng)，引發(fā)早期卵巢功能下降[75,76]。外界環(huán)境可以通過調(diào)節(jié)關(guān)鍵的代謝感知蛋白(如SIRT1和AMPK)來影響衰老，這些蛋白與mTOR和胰島素/胰島素生長因子1相互作用控制能量代謝和細(xì)胞生長。衰老可以通過這種方式降低NAD+/NADH和AMP/ATP比例，損傷線粒體功能，增加氧化應(yīng)激[77~79]。對不同年齡的C57BL/6小鼠的卵母細(xì)胞進(jìn)行單細(xì)胞轉(zhuǎn)錄本測序時(shí)發(fā)現(xiàn)：衰老小鼠卵母細(xì)胞的蛋白質(zhì)代謝發(fā)生變化，與蛋白質(zhì)質(zhì)量控制(蛋白質(zhì)修飾和非折疊蛋白反應(yīng))相關(guān)的基因表達(dá)出現(xiàn)顯著變化，與蛋白質(zhì)代謝相關(guān)的細(xì)胞成分(核仁)出現(xiàn)中斷，同時(shí)代謝相關(guān)蛋白酶的表達(dá)存在差異，而炎癥相關(guān)因子表達(dá)、細(xì)胞質(zhì)中核糖體數(shù)量均顯著增加[80]。近期研究發(fā)現(xiàn)，在衰老小鼠的卵母細(xì)胞內(nèi)核糖體蛋白S2 (ribosomal protein S2, RPS2)表達(dá)增加，進(jìn)一步表明衰老會(huì)導(dǎo)致核糖體數(shù)量的增加[81]。而伴隨著卵巢功能的下降，包括成熟促進(jìn)因子(maturation promoting factor, MPF)、sirtuin家族(SIRT1/2/3)、抗凋亡蛋白B細(xì)胞淋巴瘤-2家族蛋白(B-cell lymphoma-2, BCL-2)和半胱天冬酶(caspase)等蛋白的表達(dá)或修飾發(fā)生顯著變化[82~84]。

3 卵巢功能低下的干預(yù)措施

3.1 藥物干預(yù)

由于線粒體功能障礙與卵巢功能低下有關(guān)，因此線粒體功能的改善可能會(huì)減緩或逆轉(zhuǎn)卵巢功能低下。包括輔酶Q10、白藜蘆醇、雷帕霉素、α硫辛酸和SIRT3等在內(nèi)的線粒體營養(yǎng)藥物被用于改善卵巢功能[85]。此外，有研究表明，ω-3脂肪酸能夠延遲卵巢功能下降，提高卵母細(xì)胞質(zhì)量[86]。而一些具有減少氧化應(yīng)激、抗炎和清除自由基效用的藥物，如C-藻藍(lán)蛋白(C-Phycocyanin, C-PC)、褪黑素等似乎也可以改善卵母細(xì)胞的質(zhì)量，提高女性生育力[86,87]。臨床上對于卵巢功能低下的患者，拮抗劑方案相較于長方案有類似的獲卵數(shù)，而GnRH-a短方案雖然有更多的獲卵數(shù)，但是臨床妊娠結(jié)局與其他方案并無差異[5]。文獻(xiàn)報(bào)道，促排卵周期前8周開始口服脫氫表雄酮(dehydroepiandrosterone, DHEA)可以改善卵巢功能低下患者的臨床結(jié)局[88]。在拮抗劑方案-胞漿內(nèi)單精子注射(intracytoplasmic sperm injection，ICSI)周期中補(bǔ)充生長激素可以顯著增加卵巢反應(yīng)不良患者的獲卵數(shù)、受精卵數(shù)和可移植胚胎數(shù)，但是妊娠率和活產(chǎn)率并沒有改善[89]。衰老導(dǎo)致的卵巢功能低下原因多樣，機(jī)制復(fù)雜，雖然很多藥物被證實(shí)能夠緩解卵巢功能的減退，但到目前為止，尚無有效藥物可以完全延遲卵巢功能的下降。

3.2 線粒體移植

線粒體在卵母細(xì)胞中發(fā)揮著重要作用，并且是植入前胚胎發(fā)育過程中ATP的主要來源。卵母細(xì)胞線粒體功能障礙被認(rèn)為是高齡女性卵母細(xì)胞發(fā)育潛能差的關(guān)鍵因素。包括藥物治療、細(xì)胞質(zhì)移植、細(xì)胞核移植及線粒體移植在內(nèi)的多種方法被用來增強(qiáng)衰老卵母細(xì)胞中線粒體的完整性、活性和數(shù)量[90]。研究表明卵母細(xì)胞胞質(zhì)移植可以明顯改善胚胎發(fā)育情況，促進(jìn)妊娠并獲得健康胎兒[90]。然而該技術(shù)移植成分復(fù)雜，線粒體基因存在異質(zhì)性，會(huì)造成“三個(gè)遺傳親本”的倫理問題，已于2002年被美國食品和藥物管理局(FDA)暫停[91]。而異體線粒體移植，即從攜帶異常線粒體的患者的未受精卵母細(xì)胞中取出核DNA，然后轉(zhuǎn)移到含有健康線粒體的去核供體卵母細(xì)胞內(nèi)，此種方法臨床效果不佳，并且存在兩種mtDNA基因組的倫理爭議[92]。文獻(xiàn)報(bào)道稱自體線粒體移植可以顯著改善卵母細(xì)胞質(zhì)量，提高高齡女性的妊娠成功率[93]，但是亦有研究指出自體線粒體移植并不能改善卵母細(xì)胞質(zhì)量[94]。因此，線粒體移植的安全性和有效性需要進(jìn)一步證實(shí)。

3.3 卵巢內(nèi)干細(xì)胞移植

已經(jīng)證實(shí)，卵巢內(nèi)間充質(zhì)干細(xì)胞(mesenchymal stem cell, MSCs)移植可以恢復(fù)卵巢功能，并提高嚙齒動(dòng)物的生育力[95,96]。而膠原蛋白支架能夠支持細(xì)胞的附著、增殖和分化，研究表明膠原蛋白支架可以將間充質(zhì)干細(xì)胞錨定于支架網(wǎng)絡(luò)中，從而增加MSCs的卵巢滯留時(shí)間[97,98]。我們前期研究表明，脂肪組織來源的MSCs聯(lián)合膠原支架移植可以促進(jìn)顆粒細(xì)胞增殖，改善卵巢早衰大鼠的卵泡發(fā)育和生育力[98]。但是MSCs改善卵巢功能的具體機(jī)制尚不清楚。原始生殖細(xì)胞(primordial germ cell, PGCs)和卵巢干細(xì)胞(ovarian stem cell, OSCs)是重要的卵母細(xì)胞前體細(xì)胞[99]。在小鼠中，移植的OSCs所形成的卵母細(xì)胞可以完全成熟并且能夠形成胚胎和后代[100]。在我們最近的一項(xiàng)研究中發(fā)現(xiàn)，通過采用卵巢、膠原/臍帶間充質(zhì)干細(xì)胞共培養(yǎng)的方法，可以促進(jìn)小鼠卵巢內(nèi)FOXO3a和FOXO1的磷酸化，進(jìn)而促進(jìn)原始卵泡的激活，其機(jī)制如圖1所示[9,101,102]。共培養(yǎng)后的卵巢移植到受體小鼠腎被囊內(nèi)，在給予FSH和HCG刺激后，原始卵泡可發(fā)育到排卵前卵泡階段[101]。在通過國家衛(wèi)計(jì)委干細(xì)胞臨床研究備案和醫(yī)院倫理委員會(huì)批準(zhǔn)后，本課題組進(jìn)行了POF合并不孕癥患者臍帶間充質(zhì)干細(xì)胞移植干預(yù)的臨床研究，移植后3個(gè)月，相較于對照組，實(shí)驗(yàn)組FSH水平明顯降低，雌激素水平、卵巢體積和卵巢血流明顯增加[101]。項(xiàng)目實(shí)施至今，在前期入組的23人中，隨訪發(fā)現(xiàn)9人有優(yōu)勢卵泡活動(dòng)，已有2位患者獲得臨床妊娠，另有2位患者已獲得可移植胚胎。首例健康嬰兒于2018年1月12日在南京鼓樓醫(yī)院順利誕生。這些研究表明，干細(xì)胞可以有效提高POF患者的妊娠成功率，具有良好的臨床應(yīng)用前景，但是仍需進(jìn)一步的大樣本、多中心的干細(xì)胞臨床研究加以驗(yàn)證。

圖1 膠原/臍帶間充質(zhì)干細(xì)胞促進(jìn)顆粒細(xì)胞增殖和卵母細(xì)胞激活的分子機(jī)制

臍帶間充質(zhì)干細(xì)胞在三維支架中分泌多種生長因子，包括IGF/bFGF/PDGF/BDNF等，這些因子可以與顆粒細(xì)胞和卵母細(xì)胞表面受體結(jié)合，一方面促進(jìn)顆粒細(xì)胞增殖；另一方面激活卵母細(xì)胞內(nèi)PI3K/AKT通路，使得PIP2磷酸化成為PIP3聚集在卵母細(xì)胞膜上，進(jìn)而導(dǎo)致AKT的活化，通過mTOR增加FOXO3a出核，從而激活卵母細(xì)胞，使得原始卵泡發(fā)育至生長卵泡階段。

4 結(jié)語與展望

卵巢功能低下是影響妊娠的重要因素。衰老會(huì)影響卵巢內(nèi)多種細(xì)胞的質(zhì)量和功能，導(dǎo)致線粒體功能障礙、非整倍體性、代謝紊亂和表觀遺傳修飾改變等。包括改善線粒體功能、減少氧化應(yīng)激、清除自由基的藥物對于卵巢功能低下的治療作用較小，臨床干預(yù)效果并不顯著。線粒體移植的效果存在爭議，并且倫理上有待商榷。而前期實(shí)驗(yàn)表明間充質(zhì)類干細(xì)胞能夠改善卵巢功能低下患者的卵巢功能，提高妊娠率，具備良好的臨床應(yīng)用前景，但是仍需擴(kuò)大臨床研究，證實(shí)干細(xì)胞對卵巢功能低下患者的有效性和安全性。隨著生育延遲及二胎政策的放開，衰老所導(dǎo)致的卵巢功能低下正在影響越來越多育齡婦女的生育需求。目前仍有兩個(gè)主要問題尚未解決，一是衰老引起卵母細(xì)胞質(zhì)量下降的具體機(jī)制，二是尋求切實(shí)有效的治療方法改善高齡引起的卵巢功能衰退。伴隨高通量技術(shù)(包括表觀遺傳組學(xué)、轉(zhuǎn)錄組學(xué)、蛋白組學(xué)和代謝組學(xué)等)的迅速發(fā)展，尤其是檢測單個(gè)細(xì)胞中各組學(xué)變化相關(guān)技術(shù)的成熟，將加速我們對衰老相關(guān)的卵巢功能低下的認(rèn)識(shí)。卵巢功能低下的機(jī)制復(fù)雜，臨床干預(yù)困難，如何改善生理性和早發(fā)性卵巢功能低下患者的卵巢功能、明確衰老影響卵巢內(nèi)細(xì)胞功能的機(jī)制仍任重道遠(yuǎn)。

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Advances in the study of ovarian dysfunction with aging

Chuanming Liu1, Lijun Ding1,2, Jiayin Li3, Jianwu Dai3, Haixiang Sun1

Societal changes regarding the role of women have significant impacts on women’s willingness and the timing of childbearing. Ovarian reserve in woman typically begins to decline at the age of 35, and it is primarily characterized by a reduction in the number of ovarian follicles and a decline in oocyte quality. The clinical diagnosis of ovarian insufficiency relies on multiple variables including changes of follicle stimulating hormone (FSH), serum anti-Müllerian hormone (AMH), inhibin B, antral follicle count, menstruation and age. It is proven that ovarian cells demonstrate dysfunction associated with aging including mitochondrial dysfunction, telomere shortening, impaired DNA repair, epigenetic changes and metabolic/energetic disorders. In this review, we introduce the clinical diagnosis and management of ovarian insufficiency. We mainly discuss the molecular mechanism and potential interventions.We are optimistic that this information and knowledge will inform the important decisions for women and society regarding childbearing.

ovarian; aging; mitochondrion; heredity; epigenomics

2019-06-28;

2019-07-16

國家重點(diǎn)研發(fā)計(jì)劃“生殖健康及重大出生缺陷防控研究”專項(xiàng)(編號(hào)：2018YFC1004700)和中科院戰(zhàn)略先導(dǎo)科技專項(xiàng)(編號(hào)：XDA01030501)資助[Supported by the National Key Research and Development Program of China (No. 2018YFC1004700) and the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA01030501)]

劉傳明，博士，研究方向：生殖醫(yī)學(xué)。E-mail: 15950562099@163.com

孫海翔，博士，教授，博士生導(dǎo)師，研究方向：生殖醫(yī)學(xué)。E-mail: stevensunz@163.com

戴建武，博士，研究員，博士生導(dǎo)師，研究方向：再生醫(yī)學(xué)。E-mail: jwdai@genetics.ac.cn

10.16288/j.yczz.19-134

2019/8/12 14:49:54

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190812.1449.002.html

(責(zé)任編委: 史慶華)