人成熟卵母细胞玻璃化冷冻技术的临床应用

目的探讨玻璃化冷冻技术应用于人成熟卵母细胞冷冻保存的临床效果。方法使用开放式载体cryotop进行人成熟卵母细胞的玻璃化冻存,对10例患者的冷冻卵子复苏后进行卵母细胞浆内单精子（ICSI）注射后行胚胎移植,观察复苏卵母细胞受精、卵裂、胚胎发育及临床妊娠情况。结果 10例患者共86枚玻璃化冻存的成熟卵母细胞复苏后77枚存活,69枚正常受精,获得65枚胚胎,共移植17枚胚胎,3例患者获临床妊娠,其中2例双胎妊娠。结论玻璃化冷冻技术是有效地保存人成熟卵母细胞的方法,有一定的临床应用价值。     

不同方法冻融山羊卵巢组织的研究

目的探讨山羊卵巢组织不同玻璃化冷冻方法对其卵母细胞体外成熟、体外受精及胚胎发育的影响。方法采用三种不同的冷冻保护液(Ⅰ、Ⅱ、Ⅲ)保存的卵巢组织吸入0.25ml麦管中直接投入液氮中冷冻保存。采用慢速解冻法解冻后对卵巢组织进行形态学观察;收集卵母细胞进行体外培养。结果三种不同冷冻方法冷冻卵巢组织解冻后收集的卵母细胞存活率分别为55.2%、48.9%和46.7%,组间比较无显著性差异(P>0.05);成熟率分别为58.5%(I组)、28.9%(II组)、33.3%(III组),I组显著高于Ⅱ、Ⅲ组(P<0.01);三组的受精率、卵裂率、8细胞胚胎的形成率均无显著性差异(P>0.05);卵巢组织解冻后形态学分析表明,I组效果好于Ⅱ、Ⅲ组。结论采用玻璃化方法冷冻卵巢组织,在冷冻保护液中加入卵黄可提高冷冻保护效果。     

Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation

Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development. ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures, slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10–15 min equilibration in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose. Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These results suggest that ganglioside GT1b may play an important role in embryo survival or development. These authors contributed equally to this work.     

卵裂期胚胎玻璃化冷冻解冻后继续培养至囊胚的妊娠结局分析

目的了解卵裂期胚胎玻璃化冷冻解冻后行囊胚培养的妊娠结果。方法回顾性分析2013年5月-2013年9月在郑州大学第二附属医院生殖中心行玻璃化冷冻解冻后移植的患者共202周期的妊娠情况;其中卵裂期胚胎冷冻解冻后移植者136周期,卵裂期胚胎解冻后行囊胚培养后移植者66周期。结果卵裂期胚胎冻融移植者136周期,均有胚胎可移植,获得妊娠45周期,妊娠率为33．08％（45／136）、着床率为17．35％（55／317）,早期流产率为17。78％（8／45）;卵裂期胚胎解冻后行囊胚培养者66周期212个胚胎,其中43个周期共形成囊胚87个,囊胚形成率为41．03％（87／212）、获得妊娠22周期,妊娠率为51．16％（22／43）、着床率为32．94％（28／85）、早期流产率14．29％（3／22）。囊胚期移植者的着床率及临床妊娠率显著高于卵裂期移植者,差异有统计学意义（P〈0．05）。结论卵裂期胚胎解冻后培养至囊胚能获得较高的着床率和妊娠率。     

Is there an optimal timing interval between hCG trigger and oocyte vitrification?

ObjectiveOocyte vitrification has been developed as a promising alternative to slow freezing; however, the clinical outcome is highly operator dependent. From the past study, we know the timing of cryoprotectant exposure and understand that the intervals between the application of liquid nitrogen and thawing solution are crucial factors in the vitrification process. However, the optimal time intervals between hCG trigger and oocyte vitrification and equilibration remain unknown. This study aimed to evaluate the optimal intervals before and during modified vitrification.Materials and methodsThis retrospective study included 66 patients undergoing vitrified-thawed oocyte cycles from June 2018 to May 2019. Oocyte in vitro maturation (IVM) is defined as the maturation in vitro of an immature oocyte collected using a standard pick up procedure. Oocytes were grouped into the following intervals: (1) human chorionic gonadotropin (hCG) trigger to oocyte vitrification (<38 h; 38–39 h; >39 h; IVM) (2) oocyte equilibration time (<10 min; 10–12 min; 12–15 min). The vitrification and warming procedures were performed following the steps as shown in the Cryotec method.ResultsA total of 390 mature oocytes were vitrified with the Cryotec method. The survival rates were not significantly different among the various intervals after the hCG trigger (97.59%; 95.54%; 100%); however, there was a trend of decreased survival rate in IVM group (66.67%). The oocyte survival rates were not significantly different among the various times of oocyte equilibration (96.77%; 97.33%; 95.42%).ConclusionsThis was the first study to demonstrate no correlation between oocyte survival rate and the time intervals between hCG trigger and oocyte vitrification. Nor did the oocyte survival rate correlate with the various equilibration times during vitrification, as long as the oocyte was mature. In the future, large, prospective, randomized controlled studies will be required to confirm the clinical outcomes.     

玻璃化法保存异体动脉的实验研究

目的　探讨玻璃化法保存动脉的可行性和异体移植的有效性。　方法　家兔6 0只。其中2 4只切取双侧股动脉共计4 8条,平分为2组,分别采用玻璃化法和低温冷冻法保存14 d,作为移植材料。其余36只作为移植对象,平分为3组,每组12只2 4条动脉。A组行新鲜家兔动脉自体移植,B组行玻璃化保存家兔动脉异体移植,C组行低温冷冻保存家兔动脉异体移植。移植前对动脉进行大体和组织学观察。术后行动脉造影了解移植后动脉的通畅率,并分别于术后14、30、6 0和12 0 d取材,对动脉进行大体和组织学观察,测定内膜/中膜比值,并进行组间比较。　结果　移植前B组的动脉完整率为91.6 7% ,优于C组的5 4 .17% ,二者差异有统计学意义(P<0 .0 1)。移植后B组的累计通畅率达到87.5 0 % ,与A组差异无统计学意义,但优于C组的6 6 .6 7% ,二者差异有统计学意义(P<0 .0 5 )。B组的内膜/中膜比值与A、C组比较,差异有统计学意义(P<0 .0 5 )。　结论　玻璃化法保存动脉在降温和复温过程中冰晶较少,对动脉整体结构和活性影响较小,能较好地保存动脉的组织结构;玻璃化法保存的动脉异体移植的效果优于低温冷冻法保存的动脉异体移植。     

In Vivo Evaluation of the Effects of a New Ice-Free Cryopreservation Process on Autologous Vascular Grafts

John B. Murphy (1857–1916), one of the most outstanding and controversial surgeons of his age, was a product of the strong clinical programs that developed in Chicago in the late 19th century. Heavily influenced by Christian Fenger, surgeon and surgical pathologist, he went to Europe for training under Theodore Billroth and others. He applied results of his dog lab experiments to human problems while maintaining a large private practice. He is best known for his teaching clinics, for his groundbreaking work with a button for intestinal anastomosis, and for encouraging early operation in appendicitis. Though often in trouble with his fellow surgeons who accused him of stealing patients, especially in the Haymarket affair and when Theodore Roosevelt was shot, he nevertheless made many contributions to the care of surgical patients and development of academic surgery.     

冷冻方法对人卵裂期冷冻胚胎移植周期妊娠结局的影响

目的探讨慢速冷冻和玻璃化冷冻方法对人卵裂期冷冻胚胎移植周期妊娠结局的影响。方法回顾性分析2011年1月~2015年12月在北京大学第一医院进行卵裂期冷冻胚胎移植的1331个周期,包括慢速冷冻周期431个和玻璃化冷冻周期900个,比较两种不同冷冻方法的胚胎复苏率、完整胚胎率、临床妊娠率、种植率、流产率等各项指标,并分析卵裂球损伤对胚胎发育潜能的影响。结果玻璃化冷冻的胚胎复苏率(92.53%)、完整胚胎率(75.43%)、临床妊娠率(45.72%)、种植率(27.41%)高于慢速冷冻(76.93%、48.46%、39.52%、19.88%,P0.05);而流产率分别为12.20%、12.56%(P0.05);周期取消率分别为3.71%、1.33%(P0.05)。慢速冷冻移植0、1、2、3个无卵裂球损伤胚胎的临床妊娠率分别为:36.2%、37.7%、42.0%、44.3%(P0.05);玻璃化冷冻移植0、1、2、3个无卵裂球损伤胚胎的临床妊娠率分别为:20.5%、42.3%、48.8%、54.1%(P0.05)。结论玻璃化冷冻法更适合于人卵裂期胚胎冷冻保存,其冷冻胚胎移植周期的妊娠结局要优于慢速冷冻法;卵裂球损伤对玻璃化冷冻复苏胚胎的发育潜能影响较大。     

Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: an analysis of 5613 frozen cycles

Purpose

This study aimed to compare slow freezing (SF) and vitrification (VT) techniques for day 3 embryo cryopreservation in infertile couples.

Methods

This retrospective cohort study enrolled 5613 infertile patients, with 7862 frozen-thawed day 3 embryos and 3845 vitrified-warmed day 3 embryos, from 2010 to 2014, at a single center. The rates of embryo survival, pregnancy, implantation, miscarriage, live birth, and live birth weight were compared between the two groups.

Results

A total of 5613 cycles with 5520 transfers were analyzed. Using SF, the rates of overall embryo survival and fully intact blastomeres were lower than those in VT (91.5 vs. 97.4 % and 68.7 vs. 92.3 %, respectively). The rate of good quality embryos after thawing/warming was lower in SF than in VT. In single frozen embryo transfer cycles (FETs), the pregnancy and implantation rates were similar between the two groups (35.0 vs. 40.8 % and 34.6 vs. 35.9 %, respectively). In double FETs, the pregnancy rate per cycle was also similar between the groups (58.8 vs. 58.4 %). The implantation rate per embryo transfer was significantly higher with SF than with VT (38.8 vs. 34.6 %). With adjustment for maternal age and the number of good quality embryos, differences in implantation rate remained significant (adjusted P value, SF vs. VT P < 0.05). No independent effect was found for the method of cryopreservation on the pregnancy rate. No significant differences in the rates of miscarriage, live birth, and live birth weight were observed between the two techniques.

Conclusions

Despite the significantly low embryo survival rate, fully intact blastomere rate, and good quality embryo rate in SF, the pregnancy and implantation rates were not adversely affected in single and double FETs. SF yielded an equivalent miscarriage rate, live birth rate, and live birth weight compared with VT. The SF protocol to cryopreserve day 3 embryos still should be considered.     

不同载体和冷冻保护剂用于人胚胎玻璃化冷冻的初步研究

目的：比较分析冷冻环、自制叶片、冰珠3种载体,以及5．5mol／LEG、15％PROH＋15％EG2种冷冻保护剂用于人胚胎玻璃化冷冻的效果。方法：①选择131个6-10细胞,Ⅰ-Ⅱ级胚胎,按载体不同随机分为3组,将胚胎解冻后用于移植,比较3组的复苏率、植入率、临床妊娠率;②部分患者成功妊娠分娩后,自愿放弃剩余冻存胚胎,选择6-10细胞,Ⅰ-Ⅱ级,以叶片为载体冷冻的胚胎52个,按照冷冻溶液的不同分为2组,解冻后将胚胎继续培养3天,比较2组的复苏率和囊胚形成率。结果：①冰珠组的复苏率显著低于冷冻环和自制叶片组,3个组的植入率、临床妊娠率无明显差异;②15％PROH＋15％EG冻存的胚胎解冻后复苏率和囊胚形成率显著高于5．5mol/L EG。结论：冷冻环和自制叶片是较为理想的冷冻载体,二者用于玻璃化冷冻效果相近,自制叶片具有成本低的优点;联用15％PROH＋15％EG能降低冷冻保护剂对胚胎的毒性,玻璃化冷冻效果好于5．5mol／LEG。