柴蓓蓓， 于成和，李正偉，許益娟，何培，張若鵬

(1大理大學(xué)臨床醫(yī)學(xué)院，云南大理671000；2大理大學(xué)第一附屬醫(yī)院)

胚胎監(jiān)測(cè)儀延時(shí)攝像間隔時(shí)間對(duì)體外受精胚胎發(fā)育的影響

柴蓓蓓1， 于成和1，李正偉1，許益娟1，何培1，張若鵬2

(1大理大學(xué)臨床醫(yī)學(xué)院，云南大理671000；2大理大學(xué)第一附屬醫(yī)院)

目的 觀察胚胎監(jiān)測(cè)儀延時(shí)攝像間隔時(shí)間對(duì)體外受精胚胎發(fā)育的影響。方法 選取行體外受精治療的受試者24對(duì)，獲得168個(gè)受精胚胎，隨機(jī)分為A、B、C、D組，每組42個(gè)胚胎。四組胚胎均在CO2培養(yǎng)箱中，在37 ℃、6% CO2條件下連續(xù)培養(yǎng)6 d并進(jìn)行觀察。采用Primo vision胚胎監(jiān)測(cè)儀觀察各組胚胎的發(fā)育過(guò)程，A、B、C、D組的拍攝時(shí)間間隔分別為5、10、15、20 min。胚胎監(jiān)測(cè)結(jié)束后，找到每個(gè)胚胎對(duì)應(yīng)的監(jiān)測(cè)照片和模擬視頻。記錄胚胎多細(xì)胞發(fā)育所需時(shí)間,觀察完成受精操作60 h時(shí)胚胎發(fā)育情況，計(jì)算各組形成優(yōu)質(zhì)胚胎和囊胚的比例，記錄各組胚胎初次分裂的卵裂模式。結(jié)果 A組胚胎發(fā)育到5細(xì)胞所需時(shí)間長(zhǎng)于D組，A、B組發(fā)育到6、7、8細(xì)胞所需時(shí)間長(zhǎng)于D組，A組發(fā)育到6、7、8細(xì)胞所需時(shí)間長(zhǎng)于C組(P均<0.05)。完成受精操作60 h時(shí)A組胚胎仍處在6細(xì)胞階段，B組胚胎尚未發(fā)育到7細(xì)胞階段，C組胚胎已分裂至7細(xì)胞階段，D組為8細(xì)胞胚胎。C、D組形成優(yōu)質(zhì)胚胎、囊胚的比例均高于A組，C、D組形成囊胚的比例高于B組(P均<0.05)。C、D組初次正常卵裂胚胎比例高于A組，D組正常卵裂胚胎比例高于B組，C組非軸性卵裂、非二倍性卵裂胚胎比例低于A組，C組不均一性卵裂胚胎比例高于A組，D組非軸性卵裂胚胎比例低于A組和B組(P均<0.05)。結(jié)論 胚胎監(jiān)測(cè)儀延時(shí)攝像間隔時(shí)間對(duì)胚胎發(fā)育存在一定影響，采用15 min和20 min的拍攝間隔較5 min、10 min對(duì)胚胎發(fā)育影響更小。

胚胎監(jiān)測(cè)；延時(shí)攝像技術(shù)；輔助生殖技術(shù)；體外受精；胚胎發(fā)育；多細(xì)胞胚胎

研究[1]表明，通過(guò)體外培養(yǎng)胚胎的形態(tài)和發(fā)育速度可大致判斷胚胎移植后的發(fā)育潛能。高潛能的胚胎相應(yīng)會(huì)有更高的臨床妊娠率和嬰兒出生率，故選擇更加科學(xué)有效的方法評(píng)估和選擇最具發(fā)育潛能的胚胎是臨床研究的熱點(diǎn)[2]。胚胎發(fā)育是一個(gè)連續(xù)的過(guò)程，原核消失的時(shí)間、卵裂時(shí)間、卵裂模式、卵裂同步性對(duì)評(píng)估胚胎發(fā)育潛能有重要指導(dǎo)意義[3，4]。延時(shí)攝像技術(shù)可通過(guò)與培養(yǎng)箱整合在一起的高分辨攝影器材，利用瞬時(shí)曝光拍攝技術(shù)自動(dòng)捕捉胚胎各階段的高清照片并合成動(dòng)態(tài)視頻，用于研究和評(píng)估胚胎的發(fā)育情況。然而延時(shí)攝像技術(shù)在早期胚胎發(fā)育潛能及妊娠率評(píng)估方面的價(jià)值仍不明確[5]。目前各輔助生殖中心主要就延時(shí)攝像技術(shù)對(duì)早期胚胎篩選及其臨床結(jié)局的影響進(jìn)行研究，就延時(shí)攝像技術(shù)對(duì)胚胎發(fā)育的影響關(guān)注較少。所以，本研究觀察了胚胎監(jiān)測(cè)儀延時(shí)攝像間隔時(shí)間對(duì)體外受精胚胎發(fā)育的影響，現(xiàn)報(bào)告如下。

1 資料與方法

1.1 臨床資料 連續(xù)選取2014年6月～2016年5月在大理大學(xué)第一附屬醫(yī)院生殖醫(yī)學(xué)科行體外受精治療的24對(duì)受試者。納入標(biāo)準(zhǔn)：①采用標(biāo)準(zhǔn)長(zhǎng)方案和短方案進(jìn)行促排卵的女受試者年齡25～40歲；②女受試者為輸卵管因素導(dǎo)致的不孕；③男受試者為少弱精或梗阻性無(wú)精癥導(dǎo)致的不育；④所有入選周期取卵數(shù)目≥8個(gè)且受精率≥50%。排除標(biāo)準(zhǔn)：女受試者有子宮肌瘤、子宮腺肌病、子宮內(nèi)膜異位癥等疾?。慌茉囌叽嬖诜磸?fù)胚胎著床失敗、反復(fù)流產(chǎn)等預(yù)后不良狀況。每對(duì)受試者中女方根據(jù)促排卵情況取不定數(shù)量的卵子，男方取適量的精子，最后共取得168個(gè)受精胚胎，隨機(jī)分為A、B、C、D組，每組42個(gè)胚胎。本研究經(jīng)大理大學(xué)附屬醫(yī)院倫理委員會(huì)批準(zhǔn)，所有入組受試者簽署知情同意書。

1.2 胚胎培養(yǎng)、檢測(cè)及延時(shí)攝像方法 四組胚胎均在CO2培養(yǎng)箱中，在37 ℃、6% CO2培養(yǎng)條件下連續(xù)培養(yǎng)6 d并進(jìn)行觀察。采用Primo vision胚胎監(jiān)測(cè)儀對(duì)各組胚胎的發(fā)育過(guò)程進(jìn)行拍攝。Primo vision胚胎監(jiān)測(cè)儀的光源為一個(gè)綠色LED燈，光照波長(zhǎng)550 nm，采集每張照片所需的時(shí)間小于0.5 s。A、B、C、D組的拍攝間隔時(shí)間分別為5、10、15、20 min。

1.3 胚胎發(fā)育觀察及評(píng)價(jià) Primo vision胚胎監(jiān)測(cè)儀監(jiān)測(cè)結(jié)束后，從其配套軟件中找到每個(gè)胚胎對(duì)應(yīng)的監(jiān)測(cè)照片和模擬視頻。記錄胚胎多細(xì)胞發(fā)育所需時(shí)間(即胚胎細(xì)胞發(fā)育到2細(xì)胞、3細(xì)胞、4細(xì)胞、5細(xì)胞、6細(xì)胞、7細(xì)胞、8細(xì)胞所需時(shí)間)；完成受精操作60 h時(shí)胚胎發(fā)育情況；完成受精操作后110～140 h間(由于各胚胎發(fā)育速度不同)多細(xì)胞發(fā)育結(jié)局(參照WHO評(píng)價(jià)標(biāo)準(zhǔn)，計(jì)算各組形成優(yōu)質(zhì)胚胎和囊胚的比例)；胚胎初次分裂的卵裂模式，其中異常卵裂模式包括非軸性卵裂(卵裂球分裂時(shí)分裂溝形態(tài)不規(guī)則，卵膜和卵胞質(zhì)持續(xù)扭轉(zhuǎn))、非二倍性卵裂(一個(gè)卵裂球直接分裂成兩個(gè)以上細(xì)胞)、不均一性卵裂(卵裂球直接分裂成大小不均的兩個(gè)細(xì)胞，且兩細(xì)胞直徑相差五分之一以上)、卵裂球碎(卵裂球完全破裂成碎片)、發(fā)育停滯(卵裂球24 h內(nèi)停止分裂)。根據(jù)Peter卵裂期胚胎評(píng)分系統(tǒng)對(duì)胚胎進(jìn)行分級(jí)，共分為Ⅰ、Ⅱ、Ⅲ、Ⅳ級(jí)，其中Ⅰ、Ⅱ級(jí)胚胎為優(yōu)質(zhì)胚胎。根據(jù)Garnder囊胚分級(jí)法對(duì)形成的囊胚進(jìn)行分級(jí)，先根據(jù)囊胚的擴(kuò)張和孵出程度將囊胚分成1～6級(jí)，3～6級(jí)囊胚需對(duì)內(nèi)細(xì)胞團(tuán)和滋養(yǎng)外胚層細(xì)胞進(jìn)行評(píng)分，囊胚內(nèi)細(xì)胞團(tuán)評(píng)分分級(jí)包括A、B、C級(jí)，將Day 5評(píng)分≥3AA、3AB、3BA、3BB或Day 6～7評(píng)分≥4AA、4AB、4BA、4BB的囊胚判定為優(yōu)質(zhì)囊胚。

2 結(jié)果

2.1 各組胚胎多細(xì)胞發(fā)育時(shí)間比較 A組胚胎發(fā)育到5細(xì)胞所需時(shí)間長(zhǎng)于D組，A、B組發(fā)育到6、7、8細(xì)胞所需時(shí)間長(zhǎng)于D組，A組發(fā)育到6、7、8細(xì)胞所需時(shí)間長(zhǎng)于C組(P均<0.05)。在整個(gè)觀察期內(nèi)，C組、D組胚胎多細(xì)胞發(fā)育時(shí)間無(wú)統(tǒng)計(jì)學(xué)差異。詳見表1。

表1 各組胚胎多細(xì)胞發(fā)育時(shí)間比較±s)

注：與D組相比，*P<0.05；與C組相比，#P<0.05。

2.2 完成受精操作60 h時(shí)各組胚胎卵裂情況比較 完成受精操作60 h時(shí)A組胚胎細(xì)胞大小均勻，仍處在6細(xì)胞階段,并未開始分裂；B組胚胎有兩個(gè)細(xì)胞未完全完成分裂，尚未發(fā)育到7細(xì)胞階段；C組胚胎由明顯的7個(gè)大小較均勻的細(xì)胞組成，已分裂至7細(xì)胞階段；D組胚胎為8細(xì)胞胚胎，但可見部分細(xì)胞碎片。詳見圖1。

注：A為A組6細(xì)胞胚胎；B為B組尚未完全發(fā)育至7細(xì)胞的胚胎；C為C組7細(xì)胞胚胎；D為D組8細(xì)胞胚胎。

圖1 完成受精操作60 h時(shí)各組胚胎發(fā)育情況比較

2.3 各組優(yōu)質(zhì)胚胎、囊胚比例比較 至觀察終點(diǎn)，A組形成優(yōu)質(zhì)胚胎、囊胚的比例分別為59.5%(25個(gè))、47.6%(20個(gè))，B組分別為61.9%(26個(gè))、47.6%(20個(gè))，C組分別為69.0%(29個(gè))、54.8%(23個(gè))，D組分別為64.3%(27個(gè))、52.4%(22個(gè))。C、D組形成優(yōu)質(zhì)胚胎、囊胚的比例均高于A組，C、D組形成囊胚的比例高于B組(P均<0.05)。C、D組優(yōu)質(zhì)胚胎和囊胚的比例差異無(wú)統(tǒng)計(jì)學(xué)意義。

2.4 各組胚胎初次卵裂模式比較 在觀察期內(nèi)，C組有一個(gè)胚胎卵裂球碎，B組有一個(gè)胚胎出現(xiàn)發(fā)育停滯，A組有一個(gè)胚胎未卵裂。C、D組初次正常卵裂胚胎比例高于A組，D組正常卵裂胚胎比例高于B組，C組非軸性卵裂、非二倍性卵裂胚胎比例低于A組，C組不均一性卵裂胚胎比例高于A組，D組非軸性卵裂胚胎比例低于A組和B組(P均<0.05)。詳見表2。

表2 各組初次正常卵裂胚胎比例與異常卵裂胚胎比例比較(例)

注：與A組相比，*P<0.05；與B組相比，#P<0.05。

3 討論

光照條件在體外培養(yǎng)人類胚胎發(fā)育中的影響一直備受爭(zhēng)議。研究[6]表明，僅從波長(zhǎng)角度講，生物細(xì)胞對(duì)于光照波長(zhǎng)敏感度由大到小依次為UV射線(200～400 nm)、藍(lán)光(450～490 nm)、黃光(575～585 nm)、紅光(620～750 nm)、綠光(500～575 nm)。但隨著光通量增加(200、500、900 lux)，胚胎發(fā)育至囊胚期的比例依次減小。雖然延時(shí)攝像技術(shù)相較于傳統(tǒng)的胚胎觀察方式減少了胚胎暴露在光照下的總時(shí)間，但根據(jù)拍攝間隔不同暴露在光下的頻率較普通觀察方法大大提高。本實(shí)驗(yàn)使用Primo vision胚胎監(jiān)測(cè)儀的光源，光照波長(zhǎng)為550 nm，屬低敏感波長(zhǎng)。A、B、C、D組曝光總次數(shù)分別為1 700、860、570、430次，單次曝光時(shí)間平均為0.4 s。有學(xué)者[7]認(rèn)為即便是低敏感波長(zhǎng)光下，極短暫的暴露也足以產(chǎn)生影響胚胎發(fā)育的物質(zhì)。所以拍攝間隔時(shí)間越短，曝光次數(shù)越多，不良物質(zhì)的積累就越多。

受精卵經(jīng)過(guò)卵裂和有絲分裂后形成多個(gè)細(xì)胞，發(fā)育成胚胎，胚胎再經(jīng)過(guò)進(jìn)一步生長(zhǎng)形成囊胚。本研究對(duì)168個(gè)胚胎的發(fā)育過(guò)程進(jìn)行了記錄和分析。目前學(xué)界以卵裂時(shí)間來(lái)判斷胚胎質(zhì)量。研究[8]表明能夠成功達(dá)成妊娠的囊胚有相同的卵裂模式和時(shí)間間隔。Hlinka等[9]發(fā)現(xiàn)第二次卵裂(即形成4細(xì)胞)的時(shí)間間隔為(11±1)h的囊胚有更高的著床率和更好的后期發(fā)展，本研究D組第二次卵裂時(shí)間間隔分別為14、13.3、12.6、12.2 h。Dal Canto等[10]發(fā)現(xiàn)可發(fā)育到囊胚階段的胚胎發(fā)育到7細(xì)胞和8細(xì)胞的時(shí)間較發(fā)育到8細(xì)胞后出現(xiàn)發(fā)育阻滯的胚胎要早。

除正常卵裂外，其他卵裂模式均會(huì)不同程度地對(duì)胚胎發(fā)育潛能產(chǎn)生影響。故卵裂模式亦是選擇優(yōu)質(zhì)胚胎的重要參考因素[11]。非二倍性卵裂和2細(xì)胞、3細(xì)胞、4細(xì)胞出現(xiàn)的時(shí)間也有一定關(guān)系，對(duì)胚胎的發(fā)育速度也存在影響。這種異常的卵裂模式可能與染色體異常有關(guān)[12]。不均一性卵裂是胚胎發(fā)育中最為常見的異常卵裂模式，有研究顯示發(fā)生該種卵裂的胚胎非整倍體率和卵裂球多核率顯著高于正常分裂的胚胎[13，14]。發(fā)育停滯的胚胎也多為非整倍體，其發(fā)育潛能亦較差。相較于其他卵裂模式，非軸性卵裂的胚胎有較好的發(fā)育潛能。

本研究結(jié)果顯示，C組和D組的胚胎發(fā)育速度快于A組和B組，發(fā)育到7細(xì)胞和8細(xì)胞的時(shí)間短于A組，形成囊胚的比例也高于A組。C、D組初次正常卵裂胚胎比例高于A組，其優(yōu)質(zhì)胚胎率和形成囊胚率也較高。C、D組的胚胎較A、B組的胚胎有更好的發(fā)育結(jié)局，說(shuō)明拍攝間隔時(shí)間對(duì)胚胎的發(fā)育存在一定影響。我們推測(cè)，置于光照下對(duì)胚胎發(fā)育產(chǎn)生負(fù)面影響的原因可能是誘發(fā)細(xì)胞膜上的不飽和脂肪酸與類脂產(chǎn)生氧化反應(yīng)，從而導(dǎo)致細(xì)胞內(nèi)活性氧簇(ROS)生成增多，包括過(guò)氧化氫、超氧化物、羥自由基、單線態(tài)氧及其他脂質(zhì)過(guò)氧化反應(yīng)的產(chǎn)物等[15]。大量ROS可導(dǎo)致細(xì)胞內(nèi)重要結(jié)構(gòu)被破壞、DNA損傷，從而對(duì)胚胎發(fā)育產(chǎn)生負(fù)面影響。另外，有研究[16～21]表明，絲氨酸/蘇氨酸蛋白激酶家族中的亞型PLK1生成過(guò)少時(shí)，受精卵無(wú)法及時(shí)從有絲分裂進(jìn)入早期胚胎階段。光照對(duì)胚胎發(fā)育速度、初次卵裂模式的影響可能也與其導(dǎo)致細(xì)胞內(nèi)PLK1生成減少有關(guān)。

綜上所述，胚胎監(jiān)測(cè)延時(shí)攝像的拍攝間隔時(shí)間對(duì)胚胎發(fā)育存在一定影響，選擇15 min和20 min的拍攝間隔可能對(duì)胚胎的發(fā)育影響更小，更有利于提高移植成功率和妊娠率。然而，本研究納入的樣本量較少，關(guān)于最合適的延時(shí)攝影間隔時(shí)間還有待擴(kuò)大樣本量進(jìn)一步證實(shí)。

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Effect of time-lapse interval of embryo monitoring on human embryonic developmentafter in vitro fertilization

CHAIBeibei1,YUChenghe,LIZhengwei,XUYijuan,HEPei,ZHANGRuopeng

(1CollegeofClinicalMedicine,DaliUniversity,Dali671000,China)

Objective To investigate the effect of time-lapse interval of embryo monitoring on human embryonic development after in vitro fertilization.Methods Totally 168 fertilized embryos were obtained from 24 couples who received the treatment of in vitro fertilization (IVF) and then were randomly divided into 4 groups: groups A, B, C and D, 42 embryos in each group. The embryos in the four groups were continuously monitored for 6 days in a CO2incubator at 37℃with 6% CO2. Each group′s embryo development was observed by Primo vision embryo monitor and the time-lapse interval in groups A, B, C and D was 5,10, 15 and 20 min, respectively. After the embryo monitoring, we found the corresponding monitoring photos and analog video for each embryo. We recorded the time of each embryo′s multicellular development and the embryo development at 60 h after in vitro fertilization. The proportion of high quality embryos and blastocysts in each group was calculated, and the first cleavage pattern of embryos in each group was recorded.Results The embryos in the group A needed more time to develop to 5 cells than that of embryos in the group D. The embryos in the groups A and B needed more time to develop to 6 cells, 7 cells and 8 cells than that of the embryos in the group D. The embryos in the group A needed more time to develop to 6 cells, 7 cells, and 8 cells than that of the embryos in the group C (allP<0.05). The embryo in the group A were still in 6-cell stage at 60 h after IVF, the embryo in the group B had not developed to the stage of 7 cells, the embryo in the group C was in the 7-cell stage and the embryo in the group D was 8-cell embryo. The percentages of high quality embryos and blastocysts in the groups C and D were higher than that of group A, the proportion of blastocysts in the group C and group D was significantly higher than that of group B (allP<0.05). The proportion of embryos which had normal cleavage in the group C and group D was higher than that in group A, and the proportion of normal cleavage in the group D was higher than that in the group B. The proportion of non-axis cleavage and non-diploid cleavage in the group C was lower than that in the group A, the proportion of embryos in the group C with an uneven cleavage was higher than that in the group A, and the proportion of non-axis cleavage embryo in the group D was lower than that in the groups A and B (allP<0.05). Conclusion The time-lapse interval of 5 min and 10 min may have some influence on the embryonic development and the effect of using 15 min and 20 min intervals on embryonic development is smaller than that of 5 min and 10 min.

embryo monitoring; time-lapse camera technology; assisted reproductive technology; in vitro fertilization; embryonic development; multicellular embryo

大理大學(xué)博士科研啟動(dòng)費(fèi)項(xiàng)目(KYBS201612);九三學(xué)社大理州委立項(xiàng)調(diào)研課題。

柴蓓蓓(1991-)，女，在讀研究生，主要研究方向?yàn)榕R床檢驗(yàn)診斷學(xué)。E-mail: chaibb1991@sina.com

張若鵬(1974-)，男，博士，副主任醫(yī)師，主要研究方向?yàn)樯翅t(yī)學(xué)與婦科微創(chuàng)。E-mail: zrp263000@163.com

10.3969/j.issn.1002-266X.2017.03.002

R711.6

A

1002-266X(2017)03-0005-04

2016-09-03)