胚胎植入前遗传学诊断10个周期的临床分析

目的:初步探讨使用荧光原位杂交(FISH)方法对染色体异常患者进行胚胎植入前遗传学诊断(PGD)的临床意义。方法:7对不孕夫妇采用长方案控制性超排和卵胞浆内单精子注射,受精后d3胚胎活检、卵裂球固定和FISH,d4或d5择合适胚胎移植。结果:7对夫妇共进行10个PGD周期。获卵251个,可供活检胚胎133个,活检卵裂球207个,胚胎活检成功率为96.2%(128/133)。128个成功活检胚胎的197个卵裂球,其单细胞固定率为93.9%(185/197),FISH信号率为90.8%(168/185)。10个周期共移植22个胚胎,3例获得妊娠,并均足月分娩健康婴儿,其中1例孕妇平衡易位携带者于孕中期时,羊水核型分析为平衡易位携带者。结论:应用FISH方法进行PGD,是遗传病高危夫妇预防流产和染色体异常患儿出生的有效手段。     

培养液中添加不同促性腺激素对卵母细胞体外成熟的影响

目的:比较卵母细胞体外成熟培养液中添加不同促性腺激素对未成熟卵母细胞体外成熟结局的影响。方法:将行卵母细胞体外成熟(IVM)的35例患者共42个新鲜取卵周期,随机分成A组:22个取卵周期将重组人促卵泡激素(果纳芬,rFSH)和重组人绒毛膜促性腺激素(艾泽,hCG)按1∶1的比例混合添加,终浓度为75 mIU/ml;B组:20个取卵周期添加终浓度为75 mIU/ml的尿源性促性腺激素(hMG),进行未成熟卵母细胞体外成熟培养。35例患者中新鲜取卵周期未移植或移植后未孕者行解冻胚胎移植。比较组间患者的卵母细胞成熟率、受精率、卵裂率、优质胚胎率、累计临床妊娠率及胚胎着床率。结果:取卵均于月经周期第12日或最大卵泡发育至10￣12 mm时进行,故所获卵均为未成熟卵。A组获卵181枚,经培养后成熟84枚,行卵胞浆内单精子注射(ICSI)84枚,受精60枚,卵裂55枚,优质胚胎20枚;新鲜胚胎移植9例,获1例临床妊娠,解冻胚胎移植5例,获1例临床妊娠,累计临床妊娠率为14.29%,胚胎着床率为7.14%。B组获卵176枚,经培养后成熟120枚,行ICSI 120枚,受精97枚,卵裂90枚,优质胚胎41枚,新鲜胚胎移植6例,获4例临床妊娠,解冻胚胎移植9例,获3例临床妊娠,累计临床妊娠率为46.67%,胚胎着床率为33.33%。结论:卵母细胞体外成熟培养液中添加尿源性促性腺激素可获得较添加重组人促卵泡激素和重组人绒毛膜促性腺激素高的卵母细胞成熟率、临床妊娠率及胚胎着床率。     

荧光原位杂交技术不同方案用于染色体易位携带者胚胎植入前遗传学诊断的效率比较

目的 探讨荧光原位杂交(FISH)技术不同方案用于染色体易位携带者胚胎植入前遗传学诊断(PGD)的效率.方法 根据FISH检测方案的不同进行分组:采用全染色体涂抹探针对染色体易位携带者8个周期的109个卵母细胞第一极体进行诊断(A组),采用联合端粒和着丝粒探针对染色体易位携带者29个周期的357个卵裂球进行诊断(B组),比较两组的活检成功率、固定时细胞丢失率、无核细胞数等.结果 A组的109个卵母细胞中72个受精,受精率为66.1%(72/109),B组的357个卵裂球中304个受精,受精率为85.2%(304/357),A组的受精率显著低于B组,差异有统计学意义(P<0.05).固定时细胞丢失率A组为9.6%(12/104),B组为1.6%(4/252),两组比较,差异也有统计学意义(P<0.05).杂交后核的无信号率A组为11.2%(10/89),B组为3.0%(7/233),两组比较,差异有统计学意义(P<0.05).A组的诊断率为72.5%(79/109),显著低于B组的89.8%(230/256),差异有统计学意义(P<0.05).A组的临床妊娠率和胚胎植入率分别为3/7和22.2%(4/18),均高于B组的30.4%(7/23)和15.7%(8/51),但差异均无统计学意义(P>0.05).结论 FISH两种方案均可有效地进行染色体平衡易位的PGD,卵裂球PGD的诊断效率更高.     

未成熟卵体外成熟技术在卵巢反应不良和卵泡发育迟缓周期中的应用

目的探讨利用未成熟卵体外成熟技术治疗卵巢反应不良和卵泡发育迟缓周期的可能性。方法2000年10月至2002年7月间,在南京医科大学第一附属医院生殖中心取在常规体外受精(IVF)刺激周期中卵巢反应不良和卵泡发育迟缓患者的生发泡期卵母细胞,体外成熟培养24~48h后,对排出第一极体的卵母细胞采用单精子卵浆内注射法(ICSI)受精,受精3d后移入患者子宫,移植后通过检查血清HCG含量和B超判断妊娠结局。结果8个周期共取生发泡期卵母细胞41枚,成熟培养后有33枚卵母细胞排出第一极体,体外成熟率为805%(33/41)。ICSI后,正常受精率为788%(26/33)。平均每周期移植胚胎25(20/8)枚,获得3例妊娠。结论未成熟卵体外成熟技术可以用来挽救常规IVF治疗中卵巢反应不良和卵泡发育迟缓的周期。     

植入前遗传学诊断中四种卵裂球固定方法的比较

目的:寻求卵裂球固定的最适方法。方法:IVF/ICSI受精治疗周期中不宜移植的24枚胚胎,采用OCTAX-LaserShot胚胎透明带激光打孔,用吸拉法共活检出154个完整卵裂球,分别用4种不同的固定方法(KCl组;Tween-20/HCl组;甲醇/冰醋酸组;Tween-20/HCl+甲醇/冰醋酸组)固定,固定后使用X、Ya-卫星DNA探针进行荧光原位杂交,比较其固定率和出信号率。结果:透明带激光打孔辅助下卵裂球吸拉法活检成功率为96.25%;4种固定方法固定率和出信号率分别为98.4%和95.2%,60.0%和72.2%、63%和73.7%、70%和76.2%。结论:透明带激光打孔辅助卵裂球吸拉法活检有效;第4种卵裂球固定方法能减少卵裂球丢失,简化操作程序,因此两者在植入前遗传学诊断中值得推广。     

冻融胚胎移植周期中胚胎因素的分析研究

目的:分析冻融胚胎移植周期中胚胎卵裂球损伤程度与有丝分裂的恢复对于临床结局的影响。方法:171例患者共进行冻融胚胎移植206个周期,采用慢速冷冻法冻存d3胚胎,快速解冻法复苏后继续培养4h后移植,根据复苏后卵裂球的损伤程度分为完整组、混合组和损伤组,另根据复苏后4h胚胎进行分裂的情况分为分裂组,混合组和未分裂组,比较各组间的临床妊娠率和单胚着床率。结果:206个周期共复苏胚胎632枚,复苏存活率85.44%(540/632),移植胚胎508个,周期妊娠率36.41%(75/206),单胚着床率19.88%(101/508),完整组与损伤组间、分裂组与未分裂组间的临床妊娠率和单胚着床率都有显著性差异(P<0.05)。结论:冻融胚胎移植周期中胚胎卵裂球的损伤程度与有丝分裂的恢复是影响临床结局的重要因素。     

两种指征的卵胞浆内单精子注射结局的比较

目的:探讨常规IVF受精失败患者再次周期行ICSI-ET治疗时,卵子因素对胚胎结局的影响。方法:回顾分析因前次IVF-ET中受精障碍或受精率≤30%而行ICSI治疗的38个周期(A组)和因严重精液异常而行ICSI治疗的181个周期(B组)的ICSI结局。结果:A、B组的受精率(FR)、卵裂率(CR)、胚胎利用率(URE)、胚胎着床率(EIR)、临床妊娠率(CPR)和早期流产率(EAR)分别为82.5%vs78.0%(P<0.05),97.5%vs97.6%(P>0.05),76.4%vs73.1%(P>0.05),10.0%vs19.8%(P<0.05),27.0%vs31.8%(P>0.05),40.0%和16.1%(P>0.05)。结论:常规IVF-ET受精失败的患者,再次周期行ICSI-ET治疗,受精率提高,由于卵子异常对胚胎发育的不利影响,部分胚胎着床和着床后的远期发育潜能降低。     

IVF周期中MⅡ末期卵子形态与胚胎发育潜能的关系

目的:探讨IVF周期中MⅡ末期卵子形态与胚胎发育潜能的关系。方法:收集IVF周期中受精后5～6h脱颗粒细胞MⅡ末期卵子,依据透明带、卵泡浆及两极体形态在光镜下对排出两个极体的卵子进行形态评分分级(Ⅰ～Ⅲ级),统计不同分级组间的高评分原核形成率、卵裂率、优质胚胎率及临床妊娠率和种植率,分析卵子形态与受精及胚胎发育潜能的关系。结果:形态评分较高的卵子与形态评分较低的卵子间受精率和卵裂率均无显著差异(P>0.05),但优质胚胎率显著增高(P<0.05);随着移植胚胎中由高形态评分卵子发育而来的胚胎数目增多,周期临床妊娠率、种植率显著增高(P<0.05)。结论:IVF周期中,早期脱颗粒细胞后MⅡ末期卵子形态对受精率及卵裂率没有影响,但可预测早期胚胎质量及妊娠结局。     

染色体易位的植入前遗传学诊断

目的 观察染色体平衡易位和罗伯逊(罗氏)易位基因携带者夫妇进行植入前遗传学诊断(PGD)后的胚胎染色体遗传特征和胚胎着床、妊娠情况,探讨PGD在染色体易位基因携带者夫妇实现正常生育中的意义.方法 用荧光原位杂交(FISH)技术对36对夫妇的胚胎进行PGD,其中14例为染色体平衡易位(平衡易位组),22例为染色体罗氏易位(罗氏易位组),并对诊断结果和胚胎着床、妊娠情况进行分析.结果 36例患者共活检胚胎253个,成功诊断胚胎225个,成功率为88.9%(225/253),获得可供移植的正常或平衡的胚胎共58个.平衡易位组和罗氏易位组PGD后胚胎着床率分别为36%(5/14)和14%(6/44),临床妊娠率分别为4/9和26%(5/19).结论 PGD可有效诊断胚胎染色体平衡易位和罗氏易位,避免反复流产和不必要的非意愿性终止妊娠,并获得理想的胚胎着床率和临床妊娠率.     

低氧对体外受精-胚胎移植胚胎发育潜能的影响

目的:探讨低氧环境(体积分数5%O2)对体外受精-胚胎移植(in vitro fertilization-em-bryo transfer,IVF-ET)中胚胎发育潜能及临床结局的影响。方法:将接受IVF-ET长方案治疗的265名不孕症患者随机分为研究组(n=156):患者取卵后受精及整个胚胎培养过程全部在三气培养箱(体积分数5%O2)中进行,对照组(n=109):患者取卵后受精及整个胚胎培养过程全部在常规培养箱(体积分数20%O2)内进行,所有患者均移植授精第2日或第3日胚胎。比较组间受精率、正常受精率、卵裂率、正常卵裂率、优质胚胎率、可用胚胎率、生化妊娠率、临床妊娠率和异位妊娠率。结果:组间患者年龄、不孕年限、体质量指数、基础性激素、获卵数、成熟卵数、授精至移植时间和移植胚胎数均无统计学差异(P>0.05)。研究组受精率(84.4%)、正常受精率(72.0%)、卵裂率(97.6%)、优质胚胎率(43.3%)和可用胚胎率(72.5%)均显著高于对照组(分别为80.8%、68.7%、96.1%、35.1%、59.5%)(P<0.01或P<0.05),研究组与对照组正常卵裂率(97.7%vs 98.0%)、生化妊娠率(50.0%vs 39.4%)、临床妊娠率(44.9%vs 35.8%)、异位妊娠率(8.6%vs 12.8%)均无统计学差异(P>0.05)。结论:低氧环境(5%O2)似乎能够提高胚胎的发育潜能,获得更多优质胚胎和可用胚胎。     

应用荧光原位杂交技术进行植入前胚胎染色体诊断的价值

目的 初步探讨应用荧光原位杂交(FISH)技术进行植入前胚胎染色体诊断的价值。方法 对10对不孕夫妇进行植入前遗传学诊断(PGD)周期的超促排卵和卵母细胞浆内单精子注射,于受精后第3天进行胚胎活检及FISH分析,第4天选择染色体组成正常或平衡的胚胎进行移植。结果 10个PGD周期共获卵158个,对其中54个胚胎进行活检,51个胚胎获得明确诊断,诊断率为94％(51／54)。对染色体组成正常或平衡的24个胚胎进行官腔内移植,共4例获得妊娠,其中3例已足月分娩健康婴儿,1例为异位妊娠。结论 应用FISH技术进行植人前胚胎染色体诊断,是预防流产和染色体异常患儿出生的有效手段。     

Modified laser-assisted zonal opening method for human embryo biopsy in preimplantation genetic diagnosis: preliminary experience

OBJECTIVE: To test the safety and efficacy of a modified laser-assisted zonal opening method for human embryo biopsy. STUDY DESIGN: The embryo was treated with a modified method to create an ample perivitelline space between the zona pellucida and underlying blastomeres. This was done to protect the blastomeres from damage by laser treatment of the zona pellucida. Subsequently, the zona pellucida was completely perforated using a diode laser, and the targeted blastomere was aspirated. In vitro embryo development of the 40 biopsied embryos was compared with that of 322 corresponding control embryos without embryo biopsy. RESULTS: Forty targeted blastomeres were successfully extracted from 40 human embryos. The incidence of embryo development to the blastocyst stage was not different between the biopsied and nonbiopsied groups (55% vs. 54.3%). Furthermore, the percentage of top-scoring blastocysts from biopsied embryos was also similar to that of control embryos (77.3% vs. 78.9%). CONCLUSION: These preliminary results demonstrate that the modified technique for human embryo biopsy in preimplantation genetic diagnosis is simple and safe.     

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.     

Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development.     

A simplified technique for embryo biopsy: Use of the same micropipette for zona drilling and blastomere aspiration

Purpose: Using different micropipettes for zona drilling and blastomere aspiration for embryo biopsy is prevalent at centers of preimplantation genetic diagnosis. The purpose of our study was to simplify the technique by using only one micropipette. Methods: In this animal model, ICR mouse embryos at the four-cell stage (n=446) were randomly allocated into two groups: a biopsied group (n=224) for blastomere aspiration and a control group (n=222) without micromanipulation. We used a drilling/biopsy micropipette to drill a hole in the zona by expulsion of acidified Tyrode’s solution and to aspirate the blastomere by gentle suction with the same micropipette and pull it out of the zona. One blastomere was biopsied from each embryo. Results: In all, 222 (99.1%) intact blastomeres were successfully biopsied from 224 embryos. Only two blastomeres were damaged during aspiration. The capacity for blastocyst development (92.4 vs 93.7%) was not different between the two groups, but the percentages of embryos hatching (51.8 vs 18.0%) and hatched (29.9 vs 8.1%) were significantly higher in the biopsied group than in the control group. Conclusions: This simplified technique of embryo biopsy is safe and highly efficient for obtaining blastomeres for preimplantation genetic diagnosis and may also facilitate hatching of the blastocysts.     

Vitrification of biopsied embryos at cleavage,morula and blastocyst stage

This study investigated the effect of vitrification on biopsied embryos at various developmental stages. After biopsy on day 3, embryos were vitrified at cleavage, morula and blastocyst stages using a commercially available kit. Non-biopsied embryos were vitrified as controls. For day-3 cleavage embryo vitrification, embryos from abnormally fertilized oocytes were randomly allocated to the biopsy and control groups. For morula and blastocyst vitrification, the embryos used in the biopsy groups were obtained from aneuploidy or affected embryos diagnosed by preimplantation genetic diagnosis (PGD). After warming, survival, blastulation and development of embryos in different groups were compared. The survival rate after warming in the non-biopsied cleavage control group was significantly higher than in the biopsied cleavage group (92.0% versus 64.0%, P = 0.037). Most of the biopsied embryos were destroyed due to blastomeres escaping. At the morula stage, both biopsied and non-biopsied embryos had similar survival rates. However, a significantly higher survival rate (95.6%) was observed in the biopsied blastocyst group compared with the control group (81.3%, P = 0.035). Biopsied embryos vitrified at an advanced stage had as high survival rates as non-biopsied embryos. Vitrification at the blastocyst stage is a practical and efficient solution for embryo cryopreservation during PGD.     

Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca2+/Mg2+-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos.     

Preimplantation genetic diagnosis for chromosome rearrangements – one blastomere biopsy versus two blastomere biopsy

Purpose

Preimplantation Genetic Diagnosis (PGD) has proven to be a useful reproductive option for carriers of some chromosome rearrangements. The data presented in this study compares the impact of one versus two blastomere biopsy on the likelihood of achieving a PGD result, as well as the effect on subsequent embryo development and clinical outcomes.

Methods

IVF-PGD couples had either one or two blastomeres biopsied from all embryos with ≥7 blastomeres on day 3 post oocyte collection. These blastomeres were assessed for the specific chromosome rearrangement using Fluorescent In-situ Hybridisation (FISH). Further embryo development was monitored on days 4 and 5. Clinical outcomes were assessed retrospectively.

Results

The data shows that statistically more embryos achieved a PGD result following two blastomere biopsy, compared with one blastomere biopsy (92 % versus 88 %, respectively). Furthermore it was found that embryo development and clinical outcomes were similar between the two biopsy groups.

Conclusions

Based on this analysis it appears that the biopsy of two blastomeres from embryos with ≥7 blastomeres on day 3 is a valid and successful approach for couples presenting for IVF-PGD for a chromosome rearrangement.     

Cryopreservation of biopsied cleavage stage human embryos

The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.     

Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.