卵母细胞同源染色体分离及其调控

同源染色体分离是卵母细胞减数分裂过程中的重要环节,染色体错误分离会形成非整倍体,可能导致不育不孕、自发流产和先天出生缺陷.因此研究卵母细胞染色体分离的调控机制对防止非整倍体的发生、减少人类生殖相关非整倍体疾病的发生提供理论基础.本文对卵母细胞第一次减数分裂的染色体分离调控机制进行综述。主要针对后期促进复合体或称环体(APC/C)、纺锤体组装检查点(SAC)及其相关因子总结目前研究进展。     

氯化锶不同浓度及作用时间对小鼠孤雌胚类原核数量的影响

目的:探讨不同浓度及作用时间的氯化锶(SrCl2)对小鼠卵母细胞孤雌激活胚胎中出现类原核数量及发育潜能的影响,建立能模拟精子效应的小鼠孤雌的激活体系。方法:分别用5 mmol/L、10 mmol/L、15 mmol/L、20 mmol/L浓度的SrCl2激活小鼠卵母细胞,观察激活胚的类原核数量及发育潜能,然后在最佳刺激浓度的SrCl2的激活液中分别处理4 h、6 h、8 h,观察类原核数目及形态,分析SrCl2浓度对小鼠卵母细胞激活效果的影响。结果:hCG注射后18 h取小鼠卵母细胞,在10 mmol/L SrCl2浓度下激活6 h,出现双原核(2PN)百分比显著高于其它浓度SrCl2处理组(P0.01),且与小鼠体外受精6 h后的2PN率及囊胚形成率无统计学差异(P0.05)。结论:10 mmol/L SrCl2条件下激活小鼠卵子6 h产生的孤雌胚胎中出现2PN率及发育潜能最高,提示10 mmol/L SrCl2激活处理6 h可以模拟精子对卵子的激活条件。     

双胎妊娠胎儿染色体非整倍体产前筛查的研究进展

双胎妊娠胎儿染色体非整倍体疾病的发生率较单胎妊娠高,同时其筛查受到很多因素的影响。双胎妊娠的合子性质非常重要,直接影响风险率的计算模式,临床实践中多以妊娠早期绒毛膜性间接判断。目前双胎妊娠染色体非整倍体疾病筛查方案集中在妊娠早期胎儿颈部半透明层厚度(nuchal translucency,NT)筛查、妊娠早期联合筛查(NT+血清学筛查)、妊娠中期血清学筛查以及利用无创产前检测技术(noninvasive prenatal test,NIPT)进行胎儿染色体非整倍体疾病筛查。这些方案主要涉及染色体疾病中发病率较高的21-三体综合征和18-三体综合征。考虑到检出率、假阳性率以及经济学效应,妊娠早期联合筛查为目前双胎妊娠染色体非整倍体疾病筛查的主要方案,对近年来双胎妊娠染色体非整倍体产前筛查研究进展进行综述。     

新生小鼠原始卵泡成熟的诱导

目的:建立人工诱导新生小鼠原始卵泡成熟的方法。方法:30只新生小鼠卵巢同种异体移植于去势的10只成年雌小鼠肾被膜下,14d后分离窦前卵泡进行体外成熟培养。结果:卵泡成活率为50.6%,窦腔形成率为22.3%,卵母细胞成熟率为10.3%,MⅡ期卵母细胞平均直径为71.8±5.6μm。结论:本方法可以诱导新生小鼠原始卵泡发育,从而得到第二次减数分裂中期(MⅡ期)的卵母细胞。     

细胞松弛素B预处理人卵行玻璃化冻融后纺锤体与染色体变化的研究

目的:探讨细胞松弛素B(cytochalasin B,CCB)预处理对改善人卵玻璃化冷冻的效果。方法:收集常规IVF-ET或ICSI-ET周期中成熟卵母细胞71枚,随机分成4组行玻璃化冷冻。A组(17枚)、B组(20枚)、C组(17枚)分别用CCB预处理20min、30min、40min后行玻璃化冷冻,D组(17枚)不用CCB处理直接玻璃化冷冻。冻存3周后融冻卵母细胞,复苏的卵母细胞行荧光染色固定,在共聚焦显微镜下观察各组卵母细胞纺锤体及染色体形态结构。结果:A、B、C、D各组间的复苏率(70.59%、64.71%、50.00%和64.71%)无统计学差异(P>0.05),4组间的纺锤体及染色体正常形态率(27.27%,30.00%,45.46%及33.33%)亦无统计学差异(P>0.05)。结论:冷冻使成熟卵母细胞纺锤体及染色体的正常形态结构受到一定程度的破坏,CCB并不能保护卵母细胞纺锤体及染色体在冻融过程中的损伤。     

染色体微阵列分析在无创产前筛查非整倍体高风险中的应用价值

目的:探讨染色体微阵列分析(CMA)技术在无创产前筛查(NIPT)提示非整倍体高风险胎儿的产前诊断中的应用价值。方法:241例NIPT提示胎儿染色体非整倍体高风险的孕妇,经羊膜腔或绒毛穿刺获取胎儿细胞,进行CMA分析。对于NIPT提示21-三体和性染色体非整倍体高风险的孕妇根据筛查原因(高龄筛查组、唐筛异常或超声软指标异常组、非高龄筛查组)进行组间NIPT阳性预测值的比较,并对CMA检测提示有染色体拷贝数变异(CNV)的病例进行致病性分析。结果:NIPT对于染色体非整倍体总的阳性预测值为76.3%。NIPT对于21-三体、18-三体、13-三体和性染色体非整倍体的阳性预测值分别为95.0%、88.8%、75.0%和53.3%。经CMA分析确认,在101例NIPT提示21-三体高风险病例中,以高龄筛查组阳性预测值最高(100.0%),3组间比较,差异有统计学意义(P=0.001)。在90例NIPT提示性染色体非整倍体高风险病例中,仍以高龄筛查组阳性预测值最高(61.9%),3组间比较,差异有统计学意义(P=0.002)。12例NIPT提示染色体非整倍体高风险经CMA检测存在CNV。结论:对于21-三体和性染色体非整倍体,不同人群中高龄组的NIPT阳性预测值最为显著,临床上对于此类人群,更要引起足够的重视,做好跟踪随访。CMA较传统的染色体核型分析能额外检出CNV,更能提高染色体结构异常的检出率。     

妇科恶性肿瘤的细胞遗传学研究

本研究采用新的改良的染色体制备法,对20例妇科实体肿瘤,包括12例宫颈癌,4例宫体癌和4例卵巢癌进行了细胞遗传学分析。结果显示:几乎所有肿瘤中均存在染色体数量异常和结构畸变。非整倍体发生于所有病例,变异范围由低二倍体至高四倍体。结构畸变最多见于第1,2,3,4,5,11和17号染色体,第1号染色体长臂及第8和11号染色体短臂的部分缺失见于不同的病例中。发生断裂点的某几个染色体区带中,可能是一些瘤基因的位点所在。     

有无卵丘细胞对玻璃化冷冻后小鼠卵母细胞细胞骨架及体外发育能力的初步研究

目的:探讨卵丘细胞在玻璃化冷冻中对小鼠卵母细胞发育潜能及细胞骨架的影响。方法:采用玻璃化冷冻技术,保存带卵丘细胞或完全剥除卵丘细胞的小鼠MⅡ期和GV期卵丘复合体/裸卵(COC/DO),复苏且GV卵体外培养成熟后分别作体外受精或免疫荧光标记检查纺锤体和染色体的完整性。结果:MⅡ-COC和GV-COC的复苏率均显著高于MⅡ-DO和GV-DO(分别为86.49%vs60.92%和85.94%vs64.93%,P<0.01)。GV-COC组的受精率、囊胚率均高于GV-DO组,且MⅡ-COC组和GV-COC组的纺锤体和染色体均正常率均分别高于MⅡ-DO组和GV-DO组,但无显著性差异(P>0.05)。结论:卵丘细胞在玻璃化冷冻卵母细胞中能有效减少冷冻对细胞骨架的损伤,并改善卵子复苏及胚胎发育潜能。     

胎儿染色体疾病及单基因疾病非侵入性产前诊断

胎儿染色体疾病及单基因疾病是较常见的出生缺陷。染色体病是由于染色体数目或结构异常而发生的疾病,其中染色体数目异常比结构异常更为常见。染色体的数目异常可表现为非整倍体及多倍体。常见的结构异常有缺失、环状染色体、易位、重复、倒位和等臂染色体。细胞遗传学是经典的诊断方法。胎儿单基因病可因一个(对)基因的突变,或由双亲单基因遗传病按孟德尔法则遗传至后代。目前已知的单基因遗传病有3000余种。分子遗传学、免疫生化学等是目前常用的诊断方法。非侵入性产前诊断胎儿染色体疾病及单基因疾病有赖于可靠的实验室方法及获取可用于…     

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。     

Embryonic development after microsurgical repair of polyspermic human zygotes

Correction of polyspermy through pronucleus extraction in the absence of membrane relaxants was applied to 25 polyspermic human zygotes. Nine zygotes survived the procedure, and seven cleaved normally (two of which were fixed for chromosome analysis); two proceeded to compact and one cavitated. Eighteen of the polyspermic zygotes (14 with three pronuclei and 4 with four pronuclei) were obtained from zona pellucida-intact oocytes, and seven (1 five-pronucleate 3 four-pronucleate, and 3 three-pronucleate) from previously zona-drilled oocytes. Survival and cleavage occurred in all groups except in four- and five-pronucleate zona-drilled zygotes. Criteria used to identify male pronuclei were (1) pronucleus-associated sperm tails, (2) increased pronucleus size, and (3) greater distance (relative to female pronuclei) from the second polar body. Sperm tails were never seen and pronucleus size usually was identical. Therefore, the third criterion was used, although its reliability should be further evaluated. Until complete pronucleus removal techniques and reliable pronucleus selection criteria are perfected, embryo replacement after polyspermy correction could result in aneuploidy and molar pregnancy.     

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation.Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19-25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART.     

Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity.

OBJECTIVE: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization and cleavage of human in vitro matured (IVM) oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): The experimental group consisted of 73 spare germinal vesicle-stage oocytes from 25 patients. The control group consisted of sibling in vivo matured oocytes from the same patients that were subjected to ICSI in the clinical program. INTERVENTION(S): Equal numbers of sibling IVM oocytes were subjected to ICSI either soon after maturation (30 hours, group 1) or after a 6-hour delay (36 hours, group 2). In a subsequent set of experiments, spermatozoa were labeled with a fluorescent mitochondria-specific vital dye and injected into 17 IVM oocytes that were intentionally aged by 6-8 hours. The resultant zygotes were fixed. MAIN OUTCOME MEASURE(S): Incidence of fertilization and cleavage, numbers and mean diameters of pronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the male pronucleus by its proximity to the fluorescent sperm midpiece remnant. RESULT(S): Group 2 had significantly lower rates of normal fertilization (60%) than the control group (82.9%) and significantly lower cleavage rates (46.7%) than both group 1 (85%) and the control group (98.1%). The incidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higher in group 2 (32% and 40%, respectively) than in group 1 (4% and 5%, respectively) and in the control group (4.1% and 4.4%, respectively). All the zygotes with significant pronucleus size asynchrony that developed after delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant to the smaller pronucleus. CONCLUSION(S): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possibly related to a smaller male pronucleus), and cleavage failure increase when ICSI is delayed after maturation. Thus, the timing of ICSI is critical for optimum fertilization of IVM oocytes.     

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome-and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. Conclusions: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation

Purpose

We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation.

Methods

Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient.

Results

There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient’s own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts.

Conclusions

Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

     

How Long Do Parthenogenetically Activated Mouse Oocytes Maintain the Ability to Accept Sperm Nuclei as a Genetic Partner?

Purpose: Cytogenetic risk of intracytoplasmic sperm injection (ICSI) after artificial oocyte activation (post-activation ICSI) was evaluated in the mouse.Methods: Mouse zygotes were produced by ICSI into eggs at various intervals after parthenogenetic exposure to strontium (Sr) for 30 min. Male pronucleus formation and the chromosome constitution were studied.Results: Sperm nuclei injected into oocytes within 1 h after Sr exposure (from early through mid-telophase) transformed normally into male pronuclei, and the number of chromosome aberrations did not significantly increase in the resultant zygotes. When sperm nuclei were injected into eggs at intervals beyond 1 h after Sr exposure (from late telophase through the G₁ pronuclear stage), the rate of male pronucleus formation was significantly reduced. The incidence of chromosome aberrations increased with time between oocyte activation and ICSI.Conclusions: ICSI into oocytes within 1 h after parthenogenetic activation produces cytogenetically competent embryos in the mouse.     

Karyotyping of human metaphase II oocytes by multifluor fluorescence in situ hybridization

OBJECTIVE: To quantify aneuploidy in inseminated, injected, and noninjected oocytes from infertility patients using Multifluor fluorescence in situ hybridization (M-FISH). DESIGN: Prospective study. SETTING: Reproductive biology group, academic unit of pediatrics, obstetrics, and gynecology. PATIENT(S): Forty-eight patients undergoing ovarian stimulation and either intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): M-FISH karyotyping of 67 metaphase II oocytes, including noninjected in vitro matured oocytes, and injected inseminated-failed fertilized oocytes. RESULT(S): Thirty-nine percent of oocytes were aneuploid, with nondisjunction of chromosomes in 34% of oocytes and predivision of chromatids in 10%. There was no difference in aneuploidy rates between ICSI noninjected in vitro matured oocytes and injected, failed fertilized oocytes. Chromosomes most frequently involved in aneuploidy were 15, 18, 19, 22, and X. In seven injected ICSI MII oocytes, the prematurely condensed sperm chromatin was karyotyped by M-FISH. CONCLUSION(S): M-FISH was used to diagnose aneuploidy at maternal meiosis I in 39% of oocytes, and M-FISH karyotyping of sperm was demonstrated.     

Analysis of human unfertilized oocytes and pronuclear zygotes--correlation between chromosome/chromatin status and patient-related factors

OBJECTIVE: The objective was to evaluate the relationship between ploidy and chromatin status of human unfertilized oocytes/zygotes and infertility history, female age, and stimulation regimens. STUDY DESIGN: Two hundred and eighty-nine unfertilized oocytes and 63 zygotes were subjected to cytogenetic analysis: karyotyping for oocytes and fluorescent in situ hybridization (FISH) analysis for zygotes. Ploidy and chromosome/chromatin status were analyzed according to stimulation regimen, female age, and infertility history. The correlation coefficient was estimated and data were interpreted using a five-grade scale. RESULTS: Aneuploidy in karyotyped oocytes (19.7% hyperhaploidy, 18.8% hypohaploidy, and 6.3% haploid abnormal) was associated with chromosome fragmentation and lesions due to chromosome aging in culture. Premature chromosome condensation and cytoskeletal defects were significantly higher in unexplained infertility (34.7% and 52.9%, respectively; p<0.05). Chromatin quality was most important for successful ploidy analysis of zygotes. FISH analysis of abnormal zygotes elucidated genetic aspects of pronuclear number aberrations and raised questions about the current selection criteria. Abnormalities were found to correlate moderately with stimulation strategy and female age and significantly with infertility history. CONCLUSION: Genetic analysis of human oocytes and zygotes showed that poor chromatin quality and patient-related factors contribute to aneuploidy and pronuclear number aberrations.     

The chromosome complement of first-cleavage mouse embryos after in vitro fertilization

A comparison of the first cleavage-stage chromosome complements of 1022 in vivo fertilized mouse embryos and 1033 in vitro fertilized mouse embryos is reported. The chromosome analysis of first-cleavage embryos allows us to study directly the chromosome complement of the sperm and oocyte that contribute to the embryo, since both chromosome clusters remain separate when an antimitotic agent is used to prevent syngamy. In this paper we show that the sex ratio and the incidence of aneuploidy are similar, irrespective of the fertilization system used. Male and female gametes have the same levels of aneuploidy. Triploidy is more frequent in the in vitro fertilized embryos and the difference can be ascribed to a higher incidence of polyspermy and diploid spermatozoa.