玻璃化冷冻法冻融经植入前遗传学诊断后囊胚的应用初探

目的:探讨玻璃化冷冻技术冻融经卵裂球活检后囊胚的可行性。方法:将活检后剩余的可移植囊胚用玻璃化冷冻保存,并在冷冻前人工皱缩囊胚腔,在需要移植时予以解冻囊胚进行移植。结果:24例共进行24个活检周期,活检了159个胚胎,活检后胚胎囊胚形成率60.38%(96/159)。有17个周期共移植26枚新鲜可用囊胚,成功种植13个(50.0%),11例获得临床妊娠(64.71%),7个周期因无可移植胚胎或卵巢过度刺激等因素而取消移植。10例患者(10个周期)有30个可移植囊胚进行了玻璃化冷冻保存,其中6例患者因未成功生育要求解冻其囊胚进行移植。共解冻8枚囊胚,全部存活并移植,5例获单胎妊娠;2例已分娩正常婴儿,3例继续妊娠中。结论:玻璃化冷冻技术结合人工皱缩囊胚腔能冷冻保存经卵裂球活检后的囊胚。     

玻璃化冷冻与程序化冷冻对胚胎发育潜能及临床结局的影响

目的:比较玻璃化冷冻与程序化冷冻对胚胎发育潜能及临床结局的影响。方法:回顾性分析590个复苏周期,比较2种冷冻方法的胚胎复苏率、临床妊娠率、胚胎种植率和流产率等各项指标。结果:玻璃化冷冻组的平均移植胚胎数(2.2±0.5枚)显著少于程序化组(2.5±0.6枚)(P<0.05),复苏率(94.4%)、完整胚胎率(73.7%)、临床妊娠率(50.8%)和种植率(30.2%)显著高于程序化组(77.2%、44.3%、36.2%、21.1%)(P<0.05),而流产率和周期取消率组间均无统计学差异(16.6%vs 27.7%,1.3%vs 2.3%)(P>0.05)。程序化冷冻胚胎的种植率在完整胚胎(13.5%)和非完整胚胎(16.0%)组间无统计学差异(P>0.05);玻璃化冷冻完整胚胎组的种植率(30.4%)显著高于非完整胚胎组(20.1%)(P<0.05);而2种冷冻方法的流产率完整胚胎组(35.7%,15.1%)均显著高于非完整胚胎组(8.7%,2.9%)(P<0.05)。在玻璃化冷冻中,卵裂期胚胎组的各项指标与囊胚期组相比均无统计学差异(P>0.05)。结论:玻璃化冷冻法适用于人类胚胎的保存,对卵裂期和囊胚期胚胎有同样理想的保存效果和临床结局,玻璃化冷冻中,胚胎完整性对胚胎种植率起着重要的作用。     

34个周期玻璃化冷冻卵母细胞临床应用分析

目的:探讨玻璃化冷冻卵母细胞的临床应用可行性。方法:回顾性分析玻璃化冷冻卵母细胞34个周期,对其复苏率、受精率、卵裂率、临床妊娠率等进行统计分析,并筛选出因男方因素不孕的21个周期(研究组)和同期因男方因素进行新鲜卵细胞浆内单精子注射-胚胎移植(ICSI-ET)的78个周期(对照组)进行比较。结果:298枚玻璃化冻存的卵母细胞复苏后存活278枚,对其中成熟的234枚卵母细胞进行ICSI受精后,受精182枚,形成优质胚胎82枚,移植70枚胚胎,11例患者获得成功分娩(10例单胎,1例双胎)。研究组受精率﹑优质胚胎率比对照组低,差异有统计学意义(P0.05),卵裂率、临床妊娠率、种植率、活产率和流产率差异均无统计学意义(P0.05)。结论:玻璃化冻融卵母细胞的技术可以在有适应证的患者中应用,可获得较好的临床结局。     

冷冻前胚胎因素对冻融胚胎移植结局的影响

目的:探讨冻融胚胎移植周期中冷冻前胚胎因素对临床结局的影响。方法:回顾分析本生殖中心2009年1月～9月的589个冻融胚胎移植周期,根据冷冻前受精方式、胚胎培养时间、胚胎卵裂球数目、冷冻前≥6细胞胚胎个数分组。结果:589例冻融移植周期中共解冻胚胎2185枚,复苏率为69.5%,临床妊娠率26.5%。不同受精方式的临床妊娠率分别为23.4%,33.2%,差异有统计学意义;D2胚胎和D3胚胎冷冻后复苏率和临床妊娠率差异有统计学意义(71.4%vs69.1%和20.2%vs30.1%);冷冻前胚胎≥6细胞和6细胞,两组的临床妊娠率(31.8%,22.0%)和卵裂球完全存活复苏率(23.7%,45.4%)比较,差异均有统计学意义;冷冻前3个及以上≥6细胞的胚胎复苏率最高为56.0%、卵裂球完全存活复苏率最低为20.9%,与冷冻前少于3个胚胎组相比差异有统计学意义。若冷冻前仅余1个≥6细胞胚胎,冷冻后复苏率显著高于仅余1个6细胞胚胎,但卵裂球完全存活复苏率显著降低;和仅余2个胚胎相比,组间临床妊娠率无统计学差异。结论:冷冻前≥6细胞胚胎的妊娠结局优于6细胞的胚胎;若冷冻前仅余1个6细胞的胚胎,虽然冻融后复苏率较低,但仍有妊娠的可能,因此仍然建议冻存这部分胚胎,提高患者的累积妊娠率。     

人卵裂期胚胎和囊胚玻璃化冷冻妊娠结局分析

目的探讨人卵裂期胚胎和囊胚玻璃化冷冻解冻后复苏效果及妊娠结局。方法回顾性分析434个卵裂期胚胎（卵裂组）和102个囊胚解冻周期（囊胚组）的临床资料,比较采用玻璃化冷冻解冻后的复苏率及妊娠结局。结果囊胚组患者的临床妊娠率和胚胎种植率分别为63.73%（65/102）和45.79%（87/190）,卵裂组分别为42.82%（185/432）和24.17%（241/997）,两组比较,差异均有统计学意义（P均〈0.05）;囊胚组患者的复苏率、流产率和异位妊娠率分别为98.45%（190/193）、12.31%（8/65）和0（0/65）,卵裂组分别为97.55%（997/1 022）、13.51%（25/185）和2.16%（4/185）,两组比较,差异均无统计学意义（P均〉0.05）。卵裂组中,卵裂球完全存活患者的临床妊娠率和胚胎种植率分别为44.62%（170/381）和25.57%（224/876）,非完全存活者分别为29.41%（15/51）和14.05%（17/121）,两组比较,差异均有统计学意义（P均〈0.05）。结论囊胚解冻移植的妊娠结局优于卵裂期胚胎,卵裂期胚胎解冻后胚胎的完整性影响胚胎种植。     

冷冻膜及冷冻环在人早期胚胎玻璃化冷冻中的应用比较

目的探讨冷冻膜和冷冻环2种载体行玻璃化冷冻对人卵裂期胚胎和囊胚的效果。方法采用回顾性队列研究分析912个解冻复苏周期,比较2种载体行玻璃化冷冻后胚胎复苏结局和临床结局的差异及其相关危险因素。结果 (1) 385枚卵裂期胚胎中,冷冻环组(n=251)和冷冻膜组(n=134)的胚胎复苏率、完整胚胎存活率、临床妊娠率、种植率、流产率、移植周期出生率、双胎出生率、早产率及出生男女比率差异均无统计学意义(P0.05);冷冻环组新鲜周期优质胚胎率(24.78%)、平均移植周期数(1.3±0.6)、复苏后优质胚胎率(40.76%)、复苏后平均移植胚胎数[(2.1±0.4)个]及低体质量儿出生率(19.01%)均明显高于冷冻膜组[18.59%,P=0.002;1.2±0.4,P=0.001;28.13%,P=0.001;(1.9±0.4)个,P=0.000;4.08%,P=0.015]。(2) 527枚囊胚中,冷冻环组(n=287)和冷冻膜组(n=240)的平均移植周期数、胚胎复苏率、完整胚胎存活率、复苏后囊胚扩张率、流产率、双胎出生率、早产率、低体质量儿出生率及出生男女比率差异均无统计学意义(P0.05);冷冻环组的临床妊娠率(67.94%)、种植率(49.72%)、移植周期出生率(52.96%)、新鲜周期优质胚胎率(27.0%)、平均移植胚胎数[(1.9±0.4)个]显著高于冷冻膜组[54.17%, P=0.001;39.58%,P=0.002;41.25%,P=0.047; 23.0%, P=0.002;(1.8±0.5)个,P=0.004]。Logistic回归显示,新鲜周期优质胚胎数、内膜厚度及冷冻胚胎所用载体类型与冷冻囊胚复苏移植后临床妊娠密切相关(P=0.017、P=0.049、P=0.044),而年龄、复苏后平均移植胚胎个数、复苏后囊胚扩张数、平均移植周期数及内膜分型等差异均无统计学意义(P0.05)。结论对于卵裂期胚胎,冷冻膜和冷冻环冷冻胚胎的复苏结局和临床结局均是相当的。对于囊胚,冷冻环组的临床结局优于冷冻膜组。     

开放式和封闭式玻璃化冷冻法对人类胚胎发育潜能的影响

目的:比较开放式与封闭式自制叶片玻璃化冷冻技术对人早期胚胎冷冻复苏的效果。方法:以自制开放式与封闭式的叶片为冷冻载体,用玻璃化冷冻方法冷冻体外受精-胚胎移植第3天移植后剩余的优质胚胎,观察胚胎复苏及临床妊娠情况。结果:开放式冻存组共复苏112个周期、288个胚胎,胚胎复苏率、复苏5h继续卵裂率、空泡出现率、胚胎丢失率、临床妊娠率、植入率及流产率分别为97.2%、30.7%、9.3%、1.7%、50.5%、23.6%、14.5%,相较于封闭式冻存组(共复苏93个周期、222个胚胎)的胚胎复苏率(96.4%)、复苏5h继续卵裂率(34.6%)、空泡出现率(7.9%)、胚胎丢失率(1.4%)、临床妊娠率(59.8%)、植入率(29.0%)、流产率(10.9%)均无统计学差异(P>0.05)。结论:封闭式玻璃化冷冻法能获得与开放式同样的冷冻效率,且能使胚胎与液氮完全隔离,能更好地保护胚胎避免潜在的病原微生物、病毒的污染,是冷冻人早期胚胎理想的方法。     

桑葚期胚胎玻璃化冷冻在体外受精-胚胎移植中的应用

目的:探讨用玻璃化冷冻法冷冻桑葚期胚胎的可行性.方法:选择2010年6月至2012年9月在河北医科大学第二医院生殖医学门诊行体外受精-胚胎移植患者784例(829个周期)为研究对象,对照组(606例)为前期采用玻璃化冷冻法冷冻第3天卵裂期胚胎637个周期;研究组A(145例)采用玻璃化冷冻法冷冻第4天桑葚期胚胎159个周期;研究组B(33例)为前期采用玻璃化冷冻法冷冻胚胎在第3天评分较差,介于冷冻和非冷冻之间的胚胎,通过继续培养至第4天采用玻璃化冷冻法冷冻桑葚期胚胎33个周期.3组均解冻以后行冻融胚胎移植,比较3组复活周期的胚胎完整率、周期临床妊娠率、种植率以及流产率.结果:①研究组A的完整胚胎存活率、周期临床妊娠率和流产率高于对照组,但差异无统计学意义(P＞0.05).而研究组A的胚胎种植率(33.2％)高于对照组(27.0％),差异有统计学意义(P＜0.05);②研究组B和对照组比较,完整胚胎存活率、种植率、周期临床妊娠率和流产率方面,差异均无统计学意义(P＞0.05).结论:桑葚期胚胎较原核期胚胎和早期卵裂期胚胎处于胚胎发育的更晚阶段,是胚胎的进一步选择,其玻璃化冷冻是可行的,能获得较好的妊娠率和种植率,值得临床推广应用.     

透明带薄化法激光辅助孵化对慢速冷冻和玻璃化冷冻卵裂期胚胎临床结局的影响

目的:探讨透明带薄化法激光辅助孵化技术对慢速冷冻和玻璃化冷冻卵裂期胚胎临床结局的影响。方法:回顾性分析复苏后至少有1个胚胎是完整存活的564例卵裂期冻融胚胎移植周期,根据胚胎移植前是否行激光辅助孵化,分为辅助孵化组(研究组)与非辅助孵化组(对照组),分别观察透明带薄化法激光辅助孵化技术对慢速冷冻胚胎及玻璃化冷冻胚胎的临床妊娠率、种植率及流产率的影响。结果:胚胎经慢速冷冻后,研究组胚胎种植率(19.5%)显著高于对照组(13.5%),差异有统计学意义(P<0.05),而临床妊娠率及流产率(37.9%vs 28.5%、15.5%vs 10.8%)无显著差异;胚胎经玻璃化冷冻后,研究组和对照组胚胎的临床妊娠率(38.0%vs 35.6%)、种植率(17.3%vs 15.9%)及流产率(7.6%vs 19.2%)相比较均无统计学差异;研究组中来源于2种冷冻方法的卵裂期胚胎的临床妊娠率(37.9%vs 38.0%)和种植率(19.5%vs 17.3%)无统计学差异(P>0.05)。结论:透明带薄化法激光辅助孵化能够提高慢速冷冻的卵裂期胚胎的种植能力,且不会增加其流产风险,但并不能提高玻璃化冷冻的卵裂期胚胎的种植能力。     

慢速程序化冷冻与玻璃化冷冻对第3天分裂期胚胎发育潜能的影响

目的比较慢速程序化冷冻法（慢速法）和玻璃化冷冻法（玻璃化法）对第3天分裂期胚胎发育潜能的影响。方法选择因输卵管阻塞或男性少弱精因素行体外受精（IVF）或卵母细胞胞质内单精子注射（ICSI）治疗的患者,挑选在行IVF或ICSI后第3天剩余优质胚胎数目超过4个者80例进入本研究。随机将每例患者的2个胚胎用玻璃化法冷冻,另外2个用慢速法冷冻;冷冻复苏后随机抽取40例患者移植慢速法冷冻的胚胎,另40例患者移植玻璃化法冷冻的胚胎,比较2种冷冻方法对胚胎发育潜能的影响。结果冷冻复苏后待移植的胚胎共160个,其中慢速法冷冻80个,复苏后存活73个（91％,73／80）,移植后40例患者中15例（38％,15／40）获得临床妊娠,其中3例（20％,3／15）为双胎妊娠,余为单胎妊娠,胚胎着床率为25％（18／73）;玻璃化法冷冻胚胎80个,复苏后存活71个（89％,71／80）。移植后40例患者中19例（48％,19／40）获得临床妊娠,其中9例（47％,9／19）为双胎妊娠,余为单胎妊娠,胚胎着床率为39％（28／71）,与慢速法相比,玻璃化法的临床妊娠率和双胎妊娠率均有提高,但差异无统计学意义（P〉0．05）,而胚胎着床率则显著提高,差异有统计学意义（P〈0．05）。结论玻璃化法能够更好地保存胚胎复苏后的发育潜能,更适合第3天分裂期胚胎的冷冻保存。     

胚胎冷冻及复苏移植120个周期临床效果分析

目的探讨冷冻胚胎复苏移植后的妊娠结局。方法2003年1月至2007年5月,对2～4细胞期胚胎进行慢速冷冻保存,快速复苏及移植,共有88例患者在哈尔滨医科大学第一临床医学院进行了120个周期的冷冻胚胎复苏移植,观察冷冻复苏移植的效果。结果复苏胚胎370个,存活316个,完整存活胚胎232个,移植316个胚胎,临床妊娠39例,妊娠率为32.5%(39/120)。胚胎种植率16.5%(52/316)。比较妊娠组与非妊娠组在年龄、雌二醇水平、子宫内膜厚度、胚胎复苏存活率及完整存活率上差异均无显著性意义(P>0.05),优质胚胎率差异有极显著性意义(P<0.01)。结论优质胚胎是移植后获得妊娠的关键,采用冻融胚胎移植技术可以提高辅助生殖技术的累计妊娠率。     

Vitrification versus slow freezing gives excellent survival,post warming embryo morphology and pregnancy outcomes for human cleaved embryos

Purpose  The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes. Methods  The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. Result(s)  In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184–10.434) and 8.769 (95% confidence interval; 6.460–11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461–4.033) and 2.726 (95% confidence interval; 1.837–4.046), respectively. Conclusion(s)  Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes. Capsule Cryopreservation of human cleavage stage embryos with vitrification versus slow freezing provides better laboratory and clinical outcomes.     

Human oocyte cryopreservation: comparison between slow and ultrarapid methods

The success of reproductive technologies is facilitated by the cryopreservation of embryos and gametes. In Italy, where legislation prohibits zygote and embryo cryopreservation, clinics have extensively introduced oocyte cryopreservation. Two different strategies of oocyte cryopreservation are available: slow freezing or ultrarapid cooling (vitrification). Although the results are very encouraging with both methods, there is still controversy regarding both the procedure itself and the most suitable method to use. This study reports the routine application of the two different oocyte cryopreservation methods in programmes running in two consecutive periods. The study centre carried out 286 thawing cycles for a total of 1348 thawed oocytes cryopreserved by the slow-freezing method and 59 warming cycles for a total of 285 warmed oocytes cryopreserved by vitrification. Comparison of the outcomes obtained with the slow-freezing method versus vitrification in women who underwent IVF for infertility showed survival, fertilization, pregnancy and implantation rates of 57.9% versus 78.9% (P < 0.0001), 64.6% versus 72.8% (P = 0.027), 7.6% versus 18.2% (P = 0.021) and 4.3% versus 9.3% (P = 0.043) respectively. These results suggest that oocyte vitrification is associated with a better outcome than the slow-freezing method.     

Vitrification demonstrates significant improvement versus slow freezing of human blastocysts

Blastocyst culture has reduced the number of embryos transferred per cycle, whilst simultaneously creating new quandaries regarding supernumerary blastocyst cryopreservation. This retrospective study was undertaken to compare a slow freezing protocol to a vitrification protocol for cryopreservation of day 5 and day 6 human blastocysts. To demonstrate this, the survival, implantation rate and pregnancy rates were compared after thawing, assessment and embryo transfer of 86 consecutive day 5 and day 6 thawed blastocyst transfer cycles from January 1, 2002 to December 31, 2003. Seventy-one day 5 slow-frozen (SF) blastocysts were thawed and 59 embryos survived the thawing (83.1%). An average of 2.5 SF blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 16.7% (4/24). Concurrently, 41 vitrified (VIT) blastocysts were thawed and all 41 survived the thawing process (100%). An average of 2.0 VIT blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 50% (10/20). Survival, pregnancy and implantation rates of day 5 VIT blastocysts have significantly increased (P < 0.01, P < 0.02 and P < 0.01 respectively) over day 5 SF blastocysts. A similar trend was observed with day 6 blastocysts.     

The freezing method of cleavage stage embryos has no impact on the weight of the newborns

Purpose

The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification.

Methods

This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study.

Results

There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4 %). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0 %). The live birth rates were similar (19.5 vs 19.1 %) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9.

Conclusions

The freezing method has no impact on the weight of the newborn.With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

     

激光皱缩法在囊胚玻璃化冷冻中的应用价值

目的：探讨激光打孔使囊胚腔皱缩在体外受精周期囊胚玻璃化冷冻中的应用价值。方法：将常规取卵后第3日移植、冷冻后剩余的形态学评分较差的胚胎发育而来的囊胚,采用或不采用激光打孔使囊胚腔皱缩后冷冻。分析606例解冻囊胚周期,比较采用和未采用激光皱缩2种方法冻存囊胚的效率,分析激光皱缩在囊胚玻璃化冷冻中的价值。结果：激光皱缩组解冻247例,4例（1．62％）取消移植,移植243例;未皱缩组359例,24例（6．69％）取消移植,移植335例。移植患者的年龄、不孕原因、排卵日内膜厚度、平均移植胚胎数组间均无统计学差异（P〉0．05）。激光皱缩组冻融取消率和流产率均显著低于非皱缩组（P〈0．05）,生化妊娠率（45．68％vs35．82％）、种植率（22．54％vs16．56％）和继续妊娠率（87．50％vs75．56％）显著高于非皱缩组（P〈0．05）,临床妊娠率激光皱缩组略高于非皱缩组（32．92％Ⅷ26．87％）,但无统计学差异（P〉0．05）。结论：囊胚冷冻前采用激光打孔皱缩可提高解冻后的胚胎存活率,并能显著降低流产率。     

影响冻融胚胎移植妊娠结局的相关因素分析

目的:探讨影响冻融胚胎移植(FET)妊娠结局的相关因素。方法:回顾性分析324个周期行FET患者的临床资料,分析患者年龄、体质量指数(BMI)、移植日子宫内膜厚度、内膜准备方案、移植胚胎数等相关因素对FET妊娠结局的影响。结果:324个周期共解冻胚胎727个,复苏成活720个(99.0%),临床妊娠144例(44.4%),胚胎植入196例(27.2%)。其中自然内膜准备周期组和激素替代内膜准备周期组患者年龄、不孕年限、基础卵泡刺激素(bFSH)、基础黄体生成素(bLH)、基础雌二醇(bE2)、复苏胚胎数、移植胚胎数、移植日子宫内膜厚度、胚胎种植率及临床妊娠率组间均无统计学差异(P0.05)。年龄≤35岁组的临床妊娠率高于年龄35岁组,差异有统计学意义(P0.05);BMI≥24.0 kg/m2的肥胖组临床妊娠率与正常体质量(BMI=18.5~23.9 kg/m2)组无统计学差异(P0.05);移植日子宫内膜厚度≥7 mm组的临床妊娠率高于移植日子宫内膜厚度7 mm组,但差异无统计学意义(P0.05);各移植胚胎数组间临床妊娠率无统计学差异(P0.05)。结论:年龄是影响FET临床结局的重要因素,内膜准备方案、BMI、移植日内膜厚度、移植胚胎数对冻融胚胎移植临床结局无影响。