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。     

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。     

人工皱缩囊胚腔对囊胚玻璃化冷冻后妊娠结局的影响

目的 探讨人工皱缩囊胚腔对囊胚玻璃化冷冻的效果、妊娠结局及新生儿的影响.方法 2006年1月至2009年12月,选择在广西壮族自治区妇幼保健院生殖医学中心接受体外受精-胚胎移植(IVF-ET)或卵母细胞胞质内单精子注射(ICSI)治疗的患者,新鲜胚胎移植妊娠失败后要求行冻融囊胚移植的342个周期,其中,314个周期的囊胚在玻璃化冷冻前,用显微注射针刺入囊胚腔内抽出囊胚液使之皱缩,然后以玻璃微细管为载体行玻璃化冷冻(皱缩组),28个周期未行囊胚腔人工皱缩(未皱缩组).比较两组经冷冻、复苏及移植后的胚胎存活率、种植率、临床妊娠率和取消移植率等.在已妊娠者中,比较两组的流产率、活胎分娩率、分娩孕周、新生儿出生缺陷发生率、新生儿平均出生体质量等,并与同期新鲜移植周期(新鲜周期组,520个周期)进行比较.结果 皱缩组胚胎存活率、种植率及临床妊娠率分别为95.3%(403/423)、38.0%(153/403)、44.6%(140/314),未皱缩组分别为64.3%(27/42)、7.4%(2/27)、7.1%(2/28),分别比较,差异均有统计学意义(P＜0.05);皱缩组取消移植率为0,未皱缩组为25.0%(7/28),两组比较,差异也有统计学意义(P＜0.05).皱缩组的流产率[18.2%(10/55)]、活胎分娩率[80.0%(44/55)]、分娩孕周[(38.2±1.3)周]、新生儿出生缺陷发生率[2.1%(1/47)]、新生儿平均出生体质量[(2989±640)g]与新鲜周期组[17.5%(91/520)、74.0%(385/520)、(37.9±2.3)周、1.7%(8/479)、(2856±640)g]比较,差异均无统计学意义(P＞0.05).结论 人工皱缩囊胚腔能明显提高囊胚玻璃化冷冻效果,并且新生儿先天异常发生率无明显升高.     

玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察

目的 比较玻璃化法冻融小鼠桑葚胚期、囊胚早期和囊胚期胚胎后,胚胎存活及继续发育的能力。方法应用6 mol/L乙二醇和1 mol/L蔗糖的玻璃化冷冻液,冻融小鼠142个桑葚胚期、135个囊胚早期和148个囊胚期胚胎,观察冻融后胚胎存活及继续发育的情况。结果 小鼠桑葚胚期、囊胚早期和囊胚期胚胎的存活率分别为88.0％、73.3％和60.1％,囊胚孵出率分别为73.9%、61.5％和49.3％。桑葚胚期的胚胎存活率及囊胚孵出率均高于囊胚早期,囊胚早期高于囊胚期,各期胚胎存活率、囊胚孵出率比较,差异均有显著性(P<0.05)。结论 玻璃化法冻融桑葚胚期的效果好于囊胚早期及囊胚期,桑葚胚期是卵裂期后胚胎玻璃化冻融的最佳阶段。     

卵子玻璃化冷冻在辅助生殖技术治疗中的临床应用

目的探讨卵子冷冻以及冻卵冻胚(卵裂胚及囊胚)双次冷冻在辅助生殖技术(ART)中临床应用的安全性和可行性。方法回顾性分析2014年1月—2017年12月期间行卵子玻璃化冷冻,随后解冻并后续培养移植共164个周期,其中新鲜胚胎移植组解冻卵88个周期,冻融胚胎移植卵裂胚组冻卵冻胚40个周期,冻融胚胎移植囊胚组冻卵冻囊胚36个周期,并分别以同期未行卵子冷冻的新鲜胚胎(n=1 480)、解冻胚胎(n=246)、解冻囊胚(n=304)移植作为对照,比较卵子受精率、卵裂率及可利用胚胎率,以及各组的复苏率、后续的着床率、临床妊娠率、早期流产率、活产率等指标。结果解冻卵复苏存活率为[94.00%(999/1 063)]。解冻卵受精率[84.00%(838/999)]优于新鲜卵子受精率[72.67%(10 703/14 729)],组间差异有统计学意义(P=0.00),第3日可利用胚胎率、冻融后存活率、着床率、临床妊娠率、早期流产率、活产率差异均无统计学意义(P0.05)。结论卵子冷冻不会降低卵子的发育潜能,冷冻后可获得比较满意的复苏效果,行卵胞质内单精子显微注射(ICSI)仍可获得较好的妊娠和活产结局,对于需要保存生育力的女性来说卵子冷冻是一种较为安全有效的方法。     

不同发育天数冻融单囊胚移植的临床结局比较

目的:研究D5和D6天冷冻胚胎冻融单囊胚移植的临床结局,探讨不同发育天数囊胚的发育潜能,为进一步改进囊胚冷冻方案提供依据。方法:回顾分析922例复苏周期单囊胚移植患者的资料,根据冷冻时间不同,分为D5冷冻组(n=563)和D6冷冻组(n=359),待患者子宫内膜达到8~12mm时,复苏5h后移植。结果:D5冻冻组的生化妊娠率(63.23%)和临床妊娠率(56.48%)均显著高于D6冷冻组(50.97%和44.85%)(P≤0.01)。结论:D5冻融单囊胚移植相较于D6冻融单囊胚移植更有利于胚胎着床,获得更高的临床妊娠率。     

Freezing-thawing of the Blastocysts after Preimplantation Genetic Diagnosis

目的:探讨着床前遗传学诊断(PGD)后囊胚冻融的特点及临床应用.方法:①实验研究:对41枚PGD诊断为遗传学异常的废弃囊胚进行冻融研究,其中27枚囊胚进行程序化冻融,14枚囊胚进行玻璃化冻融.分别观察囊胚冷冻前及解冻后的形态学特点,比较2种冻融方法的囊胚解冻复苏率.②临床应用:对14对夫妇在PGD新鲜周期移植后仍有剩余的遗传诊断正常的40枚囊胚给予冷冻保存.其中,6对夫妇的12枚胚胎进行了解冻移植.结果:①实验研究:27枚进行程序化冻融的囊胚解冻后复苏22枚,复苏率为81.48％(22/27); 14枚进行玻璃化冻融的囊胚解冻后复苏11枚,复苏率为78.57％(11/14),2种冻融方法的囊胚解冻复苏率无统计学差异(P＞0.05).无论何种冻融方法,解冻后复苏存活的囊胚在体外培养30 min均可以观察到复苏现象,体外培养4h囊腔均扩张.②临床应用:14对有剩余囊胚冻存的夫妇中,6对夫妇新鲜周期即获得临床持续妊娠,另外还有1对夫妇早期自然流产,1对夫妇宫外孕.其余新鲜周期未孕的6对夫妇进行了囊胚解冻移植,其中3对夫妇解冻周期获得了临床妊娠.结论:经PGD诊断后的囊胚,无论是程序化冻融还是玻璃化冻融,均可以获得满意的解冻复苏率.PGD诊断后囊胚冻融技术的成熟与发展可以提高PGD夫妇的累积妊娠率.     

三种冻贮细管对人三原核卵裂期胚胎玻璃化冷冻的影响

目的:观察冻贮细管、开放型拉细冻贮细管和闭合型拉细冻贮细管三种冻贮细管对玻璃化冻融胚胎形态和功能的影响。方法:将人三原核受精卵发育形成的200个6-10-细胞胚胎随机分为四组:对照组20个、冻贮细管组(straw,S)、开放型拉细冻贮细管组(openpulledstraw,OPS)和闭合型拉细冻贮细管组(closepulledstraw,CPS)各60个。比较超速玻璃化冷冻胚胎复苏后存活率;并测定复苏后部分存活胚胎线粒体跨膜电位(△Ψm)。结果:CPS超速玻璃化冷冻胚胎存活率最高,与OPS和S组相比,差异具有显著性(P<0.05);后两者间无差异。CPS与OPS组△Ψm均高于S组(P<0.05);前两者间无明显差异(P>0.05);但实验组△Ψm均低于对照组,差异有统计学意义(P<0.05)。结论:CPS对玻璃化冻融胚胎的形态和功能影响最小;OPS组虽未获得比S组更高的形态存活率,但减轻了对胚胎的功能损伤。     

囊胚玻璃化冷冻复融移植的临床效果分析

目的:探讨全囊胚玻璃化冷冻的临床价值。方法:回顾分析2010年1月至2012年12月在河南省人民医院生殖医学研究所行体外受精-胚胎移植(IVF-ET),且因OHSS高危倾向行全胚玻璃化冷冻复融移植周期的不孕患者的临床资料。根据冷冻和培养方式分为A组(复融胚胎移植,784例)、B组(复融囊胚移植,199例)和C组(复融胚胎行囊胚培养后移植,124例)。比较3组患者的基本资料、IVF治疗的妊娠结局。结果:C组患者中3例因未形成囊胚而取消周期,其余121例患者移植后有33枚囊胚需再次冷冻。3组患者的平均移植胚胎数、种植率、临床妊娠率、多胎率、早期流产率比较,差异显著(P0.05)。B、C组患者的种植率、妊娠率、多胎率均显著高于A组(P0.05),平均移植胚胎数、早期流产率均显著少于A组(P0.05)。结论:对可能发生OHSS的全胚冷冻患者,胚胎个数较多时,行囊胚培养后玻璃化冷冻,择期复融移植能提高患者的种植率和妊娠率,同时也减少了患者的费用,是目前最佳的冷冻策略和最有益的冷冻方案。     

三种不同玻璃化冷冻方法对小鼠2-细胞期胚胎冷冻效果的比较

目的:比较三种玻璃化方法冷冻小鼠胚胎的存活率及继续发育能力的差异。方法:将573个小鼠2-细胞期胚胎随机分为四组:Straw冷冻组144个,OPS冷冻组142个,CPS冷冻组149个,比较胚胎复苏后存活率、囊胚形成率及孵化率,并以未冷冻为对照组(138个)。结果:Straw组及CPS组存活率均显著性高于OPS 组(P <0.05);囊胚形成率及孵化率OPS组和CPS组均显著性高于Straw组(P均<0.05),OPS和CPS两组之间无显著性差异(P均>0.05)。结论:OPS与CPS方法能够更好地保持胚胎复苏后的继续发育能力。且CPS方法存活率高于OPS方法,并降低了交叉感染的危险性,更具临床应用潜力。     

Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique

OBJECTIVE: Clinical application of vitrification for the cryopreservation of human blastocysts. DESIGN: Clinical trial of vitrification of human blastocysts. SETTING: Private assisted reproductive technology clinic. PATIENT(S): Supernumerary blastocysts after fresh blastocyst transfer were vitrified for subsequent transfer. INTERVENTION(S): Culture of pronuclear embryos to the blastocyst stage in sequential media and subsequent vitrification of supernumerary blastocysts using a cryoloop technique. MAIN OUTCOME MEASURE(S): Clinical outcome after transfer of vitrified blastocysts. RESULT(S): A total of 60 vitrified blastocysts from 21 patients were warmed, and the survival rate at 2 hours after warming was 63%. Six clinical pregnancies were achieved after 19 transfers. One healthy baby was born, four pregnancies are ongoing, and one ended in miscarriage. CONCLUSION(S): Human blastocysts can be successfully vitrified by suspension on a small nylon loop and a direct plunge into liquid nitrogen. A delivery and ongoing pregnancies prove the safety of this method. This report documents the first successful pregnancy and delivery achieved by blastocyst vitrification using the cryoloop containerless technique.     

Vitrification of human early cavitating and deflated expanded blastocysts: clinical outcome of 474 cycles

Purpose

The present study was undertaken to evaluate and compare the post thaw survival, implantation and pregnancy rates of vitrified human early cavitating blastocysts with deflated expanded blastocysts.

Material and methods

Supernumerary blastocysts were vitrified in 30% ethylene glycol-dimethyl sulphoxide based solution using cryoloop. Fully expanded blastocysts were deflated by gentle aspiration of the blastocoelic fluid using a micromanipulator until the cavity collapses prior to vitrification.

Results

Of the 576 vitrified blastocysts, 545 (94.61%) survived thawing in the early cavitating blastocyst group which was significantly higher than deflated expanded blastocyst group, in which only 370 survived thawing out of 459 (80.62%). However, no significant difference was observed in implantation and pregnancy rates between early cavitating and deflated expanded blastocyst groups.

Conclusions

Early cavitating blastocyst would be the ideal stage for cryopreservation of human blastocysts as it has higher survival rate and avoids additional invasive procedures like deflation of the blastocoele.     

Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination

Survival and development of human embryos was compared following slow cooling versus vitrification involving more than 13,000 vitrified embryos. In addition, the efficacy of an open system, the Cryotop, and a closed vitrification system, the CryoTip(trade mark), were compared using human blastocysts. One hundred percent of vitrified human pronuclear stage embryos survived and 52% developed to blastocysts as compared with 89% survival and 41% blastocyst development after slow cooling. Similar survival rates were seen with vitrification of 4-cell embryos (98%) as compared with slow cooling (91%). Furthermore, 90% of vitrified blastocysts survived and resulted in a 53% pregnancy rate following transfer, as compared with 84% survival and 51% pregnancy rates following slow cooling. All corresponding values were significantly different. When the closed and open vitrification systems were compared, no difference was found with regard to supporting blastocyst survival (93 and 97% for CryoTip and Cryotop respectively), pregnancies (51 versus 59% respectively) and deliveries (48 versus 51% respectively). Vitrification is a simple, efficient and cost-effective way to improve cumulative pregnancy rates per cycle. The use of the closed CryoTip system eliminates the potential for embryo contamination during cryopreservation and storage without compromising survival and developmental rates in vitro and in vivo.     

Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique

Human embryos have been cryopreserved mainly by slow freezing, but vitrification has also proven effective for embryos at early cleavage stages. However, clinical results on blastocyst cryopreservation have not been consistent. A feasible option appears to be ultrarapid vitrification, in which embryos are vitrified with a reduced amount of solution to achieve extremely high rates of cooling and warming. The cryoloop is a tiny nylon loop connected to the lid by a small metal tube; a metal insert on the lid enables the use of a stainless steel handling rod with a small magnet, and the loop can be stored in the cryovial. In the HART Clinic group, of 444 supernumerary human blastocysts that were vitrified by cryoloops 79% survived after warming, and of 126 recipients 36% became pregnant. The outline of ultrarapid vitrification using cryoloops is described.     

Artificial shrinkage of blastocysts prior to vitrification improves pregnancy outcome: analysis of 1028 consecutive warming cycles

Purpose

This study aims to compare implantation, pregnancy, and delivery rates in frozen transfer cycles with blastocysts that were vitrified either with artificial shrinking (AS group) or without (NAS group).

Methods

Retrospective comparative study of artificial shrinking of blastocysts prior to vitrification and frozen embryo transfer cycles in infertile patients undergoing frozen embryo transfer (FET) was done at the Humanitas Fertility Center between October 2009 and December 2013. Main outcome measure(s) were implantation (IR), pregnancy (PR), and delivery rates (DR) between the two groups.

Results

A total of 1028 consecutive warming blastocyst transfer cycles were considered. In 580 cycles (total of 822 blastocysts), artificial shrinking was performed prior to vitrification (AS group), while in the remaining 448 cycles (total of 625 blastocysts), the artificial shrinking was not performed (NAS group). There were no differences in patient age (36.4?±?3.7 vs. 36.3?±?3.9) and number of embryos transferred (1.41?±?0.49 vs. 1.38?±?0.50) between groups. The IR, PR, and DR in the AS group were significantly higher (p?<?0.05) than in the NAS group (29.9 vs. 23.0 %, 36.3 vs. 27.9 %, and 26.5 vs. 18.1 %, respectively).

Conclusions

Performing AS of blastocysts prior to vitrification appears to improve implantation, pregnancy, and delivery rates probably related to a decreased risk of ultrastructural cryodamages, plausible when cryopreserving expanded blastocysts.

     

Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.     

Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome

This report describes a live birth produced from repeat vitrification and thawing of blastocysts derived from in-vitro matured (IVM) oocytes in a woman with polycystic ovarian syndrome. Immature oocyte retrieval was performed on day 12 of her induced menstrual cycle. The patient was administered 10,000 IU of human chorionic gonadotrophin s. c. 36 h before immature oocyte retrieval. A total of 47 immature oocytes were collected. Following IVM of these immature oocytes, 76.6% (36/47) become mature (at metaphase II stage). Thirty oocytes (30/36, 86.1%) were normally fertilized following insemination by intracytoplasmic sperm injection. The fertilized zygotes (two-pronuclear stage) were co-cultured with cumulus cells in YS medium supplemented with 10% human follicular fluid. On day 5 after insemination, three blastocysts were transferred. Unfortunately, fresh embryo transfer did not result in pregnancy. The remaining 10 embryos developed to the expanded blastocyst stage. These remaining blastocysts were vitrified with electron microscope grids following artificial shrinkage. Three months later, three blastocysts were thawed due to a clinical error. Consequently, the embryos were revitrified. After a week, the three blastocysts were warmed again. Two of them developed to hatched blastocysts. Following transfer, a full-term pregnancy resulted in the delivery of healthy twins.     

Slow freezing and vitrification of mouse morula and early blastocysts

Purpose

To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing.

Methods

Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined.

Results

Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively).

Conclusions

Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.