光谱核型分析检测人未受精卵细胞非整倍体

目的 建立光谱核型分析检测人未受精卵细胞非整倍体的方法。方法 取试管婴儿术后成熟未受精卵细胞，固定后行光谱核型分析，检测卵细胞各染色体的核型情况。结果 64％的卵细胞核型正常，36％的卵细胞为非整倍体，其中22％为同源染色体不分离，14％为姐妹染色单体非平衡性过早分离。结论 光谱核型分析可用于检测卵细胞各条染色体的异常。同源染色体不分离和姐妹染色单体非平衡性过早分离均参与了卵细胞非整倍体的产生。     

人类减数分裂过程中1号染色体的非同位素检测及二体精子核的显示

本研究运用原位杂交技术和染色体特异探针与精子核的杂交技术,即 pUCl.77特异探针对人类减数分裂、有丝分裂细胞及成熟精子核进行原位杂交,从而确定了1号染色体的非整倍体率。利用 pUCl.77探针进行原位杂交,证实了对1号染色体的着丝粒异染色质之间存在异态性。在间期的外周血淋巴细胞核中,这种异态性表现为两个大小不同的致密杂交信     

人未受精卵胞浆形态与染色体非整倍体关系的实验研究

目的探讨人未受精卵母细胞胞浆形态与其染色体非整倍体间的相关关系。方法采用多色荧光原位杂交法分析未受精卵母细胞中13,16,18,21和22号染色体的核型,并将其与卵母细胞胞浆形态进行相关分析。结果在形态正常的70个卵母细胞中,非整倍体15个(21.4%);在出现胞浆颗粒化的40个卵母细胞中,非整倍体16个(40%),明显高于形态正常卵母细胞中的非整倍体比例(P<0.05)。结论卵母细胞胞浆颗粒化与其染色体非整倍体异常间存在一定的相关性。     

北京地区染色体非整倍体相关因素分析

目的以金标准细胞遗传学染色体核型分析方法为对照,研究应用21、13、18号,X及Y染色体探针检测染色体非整倍体异常;进一步探讨分子细胞遗传学方法产前诊断染色体非整倍体的应用价值。方法应用细胞遗传学方法,常规羊水细胞培养,制备G染带染色体核型,计数分析20个细胞,分析3个分裂期染色体核型,必要时计数100个细胞核型。应用13,18,21,X,Y探针,进行未培养羊水的FISH检测。结果通过Logistic回归分析学历、民族、自然流产次数、产次、孕周以及不同产前诊断指征对染色体结果的影响,以确定造成染色体非整倍体的高危因素。结果表明自然流产次数和超声异常与染色体非整倍体有关(P_10.0001,P_2=0.054)。自然流产次数的优势比为1.423;超声异常的优势比为4.728。通过最佳操作点判定,表明年龄≥33.5岁为染色体非整倍体高发年龄。     

8629例羊水染色体非整倍体异常核型和产前诊断指征分析

目的了解柳州地区行产前诊断胎儿羊水染色体非整倍体异常核型检出情况,分析不同产前诊断指征与染色体非整倍体异常核型的关系。方法选择2017年1月至2018年11月到我院就诊符合产前诊断指征的孕妇8629名,行羊膜腔穿刺检查胎儿染色体核型。结果染色体非整倍体异常核型检出率为3.03%(262/8629),包括203例常染色体非整倍体,59例性染色体非整倍体。其中三体综合征例数最多,占异常核型的77.48%(203/262),以21-三体最常见,为152例,占异常核型比例为58.01%。非整倍体异常核型检出率由高到低的产前诊断指征分别为:高龄妊娠、NIPT高风险、唐氏筛查临界/高风险、B超异常等。结论联合不同产前筛查手段、严格掌握产前诊断指征对提高染色体非整倍体的检出,减少出生缺陷具有重要意义。染色体核型分析目前仍是诊断胎儿染色体非整倍体异常的"金标准"。     

未培养外周血间期分子细胞遗传学研究

近年来，随着分子生物学技术的发展及制备探针技术的提高，荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎ－ｃｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ，ＦＩＳＨ）技术已被广泛应用于遗传学及其它相应学科中。本文选用Ｄ２１Ｚ１／Ｄ１３Ｚ１ＤｘＺ１探针与未培养的外周血间期白细胞进行荧光原位杂交来检测染色体非整倍体。结果表明：通过统计间期核中的杂交信号，能准确检出２１三体及Ｔｕｒｎｅｒ＇ｓ综合征，与常规细胞遗传学结果相符，该法省去了复杂的细胞培养过程和周期，不仅使常规的细胞遗传学得以简化，而且为产前诊断染色体非整倍体及肿瘤病因学的研究提供了捷径     

5959例不同年龄段孕妇孕中期羊水染色体非整倍体结果分析

目的探讨不同年龄段孕妇羊水胎儿染色体非整倍体的发生率。方法回顾分析2012年8月~2014年12月在行羊膜腔穿刺术及羊水细胞染色体核型分析的5959例不同年龄孕妇的病历资料。计算染色体非整倍体发生的概率。将不同年龄孕妇按年龄分成35岁组,35~37岁组,38~40岁组和40岁组,比较4组胎儿染色体21、18以及性染色体非整倍体的异常率。结果共检出胎儿染色体非整倍体87例(含嵌合体),占样本总数的14.6‰(87/5959),其中47,XN,+21最为常见,所占比例为51.7%(45/87),在35岁组,35~37岁组,38~40岁组和40岁组发生率分别为7.5‰,2.6‰,16.1‰和18.1‰,35~37岁组和35岁组比较无显著差异(P0.05),而与38~40岁组比较有显著差异(P0.05)。胎儿47,XN,+18在4组孕妇中无显著差异。性染色体非整倍体在这四组中的发生率分别为3.6‰,1.7‰,10.1‰和18.1‰,35~37岁组和35岁组比较无显著差异(P0.05),而与38~40岁组比较有显著差异(P0.05)。结论不同年龄段孕妇胎儿染色体非整倍体发生率不同,临床可根据孕妇不同年龄运用不同的产前筛查与诊断方法,提高了染色体异常的检出率。     

多色荧光原位杂交检测人卵细胞染色体非整倍体方法的建立

目的 建立多色荧光原位杂交技术检测人卵细胞染色体非整倍体的方法。方法 取试管婴儿助孕技术后未能受精成功的卵细胞，于取卵后13d固定，采用多色荧光原位杂交方法检测卵细胞13，16，18。21和22号染色体的情况。结果 正常未受精卵细胞中期染色体显示一个成对的杂交信号，每条染色单体显示一个单个信号；分裂相中多出或缺少一个成对杂交信号表明多余或缺少一条染色体；分裂相中多出或缺少一个单个信号表明多余或缺少一条染色单体；两个单个信号分离表明两条姐妹染色单体分离。结论 采用多色荧光原位杂交方法可以有效检测人卵细胞染色体非整倍体异常。     

不同产前诊断指征对高龄孕妇的临床意义

目的探讨不同产前诊断指征对高龄孕妇(Advanced maternal age,AMA)胎儿染色体异常核型的预测价值。方法收集2006年1月至2015年12月在我院进行羊膜腔穿刺术的高龄孕妇的病史资料,按不同产前诊断指征分为5组,统计分析各组胎儿染色体异常核型的发生风险。结果 1172例高龄孕妇中,共检出胎儿染色体异常核型42例,异常发生率为3.58%,其中非整倍体31例,占染色体异常总数的73.81%。不同指征组胎儿非整倍体发生率,高龄为唯一指征组最低(0.90%),高龄伴无创DNA检测(NIPT)异常组最高(84.62%)。结论高龄孕妇积极参加血清筛查、超声检查、NIPT等产前筛查,可以有效提高非整倍体胎儿的检出率。     

应用引物原位标记技术快速检测X和18号染色体

目的为发展快速检测人类染色体的技术。方法应用原位引物标记（ＰＲＩＮＳ）在８例女性外周血淋巴细胞培养标本中检测了Ｘ、１８号染色体。结果在间期核及分裂相中均能特异地检测Ｘ、１８号染色体，Ｘ染色体的标记效率为８４％～９２％（平均８９％），１８号染色体为７３％～８７％（平均８４％）。标本经适当浓度的蛋白酶Ｋ处理后可以明显提高标记效率，增强标记信号。标记反应可利用原位ＰＣＲ仪进行，不到一小时即可完成。结论原位引物标记是一种快速、特异的染色体检测方法，利用这一技术将有可能在产前快速诊断染色体非整倍体。     

Chromosome analysis in human oocytes remaining unfertilized after in- vitro insemination: effect of maternal age and fertilization rate

The incidence of chromosomal abnormalities was studied in 719 unfertilized human oocytes obtained from our in-vitro fertilization (IVF) programme. To make chromosome preparations, a gradual fixation/air-drying method was utilized. Of 388 oocytes successfully karyotyped, 70 (18.0%) were abnormal. The abnormalities included 33 aneuploidies (8.5%) (14 hyperhaploidies and 19 hypohaploidies), 25 diploidies (6.4%) and 15 structural abnormalities (3.9%), three of them being accompanied by aneuploidy. Of the 33 aneuploidies, 16 (48.5%) showed the loss or gain of dyads (so-called non-disjunction), while 17 (51.5%) showed the loss or gain of monads (so-called predivision). There was no maternal age-dependent increase in the incidence of aneuploidy. Unfertilized oocytes from patients with a high fertilization rate (>25%) had a significantly higher (11.4%, P < 0.05) incidence of diploidy compared with the oocytes from the remaining patients (4.3 and 4.0%), suggesting that diploid oocytes might have a lower fertilizing ability.      

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.     

Relativity of the concept 'high responder to gonadotrophins'.

Regression analysis of the proportion of unfertilized oocytes on the number of oocytes retrieved per patient was applied to three different ovarian stimulation protocols in order to establish the relativity of the concept 'high responder to gonadotrophins' for in-vitro fertilization (IVF) patients. After fitting the data to the model: probit(proportion of unfertilized oocytes) + 5 = intercept + B Log10(oocytes retrieved per patient), women with a number of oocytes retrieved greater than or equal to the value necessary to obtain 50% fertilization were defined as high responders. Exogenous gonadotrophin stimulation which was commenced after complete suppression of ovarian activity by a gonadotrophin-releasing hormone analogue (GnRHa) (long protocol) resulted in a significantly higher number of oocytes retrieved (10.15) to obtain 50% fertilization compared to a short protocol (6.84) (exogenous stimulation began 2 days after the GnRHa administration). Women stimulated without use of a GnRHa showed an intermediate response. Implantation, pregnancy and miscarriage rates showed no difference between low-moderate and high responders. These results demonstrate the relativity of the concept 'high responder to gonadotrophins' and indicate that the drawback of low fertilization in high responders could be balanced by the high number of oocytes retrieved per patient (and available embryos for transfer) and the selection of the best embryos for transfer.     

The incidence of chromosomal aneuploidy in stimulated and unstimulated (natural) uninseminated human oocytes.

The incidence of chromosomal aneuploidy in human oocytes is higher than for various animal species. Since this estimate for aneuploidies is based on data obtained from in-vitro fertilization (IVF) patients, it is possible that superovulation could be contributing to this phenomenon. In this study we determine the incidence of chromosomal aneuploidy in nonstimulated uninseminated human oocytes donated by IVF patients. Furthermore, we compare this incidence of aneuploidy to that obtained after superovulation using two different protocols for induction of multiple follicular growth. The rate of aneuploidy in non-stimulated oocytes was 20% (4/20). This is not significantly different from the rate of aneuploidy in oocytes obtained after superovulation with clomiphene/human menopausal gonadotrophin (HMG)/(HCG) (15/43 = 35%, chi 2 = 1.11; P > 0.20), buserelin-flare (8/25 = 32%; chi 2 = 0.32; P > 0.05), and the rate of aneuploidy in the total number of superovulated oocytes (23/68 = 34%; chi 2 = 82; P < 0.30). Furthermore, the incidence of chromosome aneuploidy in non-stimulated uninseminated oocytes (20%) was well within the range and not significantly different from that reported in the literature for both superovulated uninseminated oocytes (range, 21-57%; total aneuploidy rate, 67/216 = 31%; P < 0.30) and superovulated inseminated oocytes (range, 3-56%; total aneuploidy, 339/1480 = 23%; P < 0.95). Consequently, the data provide evidence that superovulation protocols used in IVF may not be responsible for the higher rate of aneuploidy in human oocytes. These results are discussed in relation to hypotheses on the occurrence of meiotic non-disjunction.     

女性年龄对卵母细胞纺锤体和染色体构型的影响

目的探讨女性年龄对卵母细胞纺锤体和染色体构型的影响。方法取试管婴儿术后卵龄1天的未受精卵母细胞,采用免疫细胞化学和激光共聚焦术检测卵母细胞纺锤体和染色体构型。结果25~29岁女性卵母细胞Ⅰ级纺锤体和染色体比率分别为33%和31%,显著高于30~34岁(P<0.05)和35~40岁女性(P<0.01);而25~29岁女性卵母细胞的Ⅲ级纺锤体和染色体率均为54%,明显低于30~34岁(P<0.01)和35~40岁女性(P<0.05)。结论卵母细胞纺锤体和染色体构型异常率与女性年龄存在明显的相关性。     

Chromosomal FISH analysis of unfertilized human oocytes and polar bodies

 The incidence of chromosomal aneuploidy was studied in 208 unfertilized metaphase II human oocytes from an in vitro fertilization program by fluorescence in situ hybridization using probes for chromosomes 18, 21, and X. Chromosome spreads were prepared by a gradual fixation–air-drying method. We obtained analyzable results from 183 oocytes and 93 polar bodies; 167 oocytes (91%) were normal, 11 (6%) were diploid, and 5 (3%) were aneuploid. Of the five aneuploid oocytes, four involved chromosome 21, and one involved the X chromosome. In this study, oocyte aneuploidy caused by both nondisjunction of bivalent chromosomes and predivision of univalent chromosomes was observed. The aneuploidy rate (9.8%) in the oocytes from women aged ≧35 years was significantly higher than that (0.7%) in those aged 23 to 34 years (P= 0.0017). Received: February 14, 2002 / Accepted: June 10, 2002     

Karyotyping of human oocytes by cenM-FISH, a new 24-colour centromere-specific technique

BACKGROUND: Metaphase II (MII) chromosome complements are difficult to karyotype. The objective of this study was to investigate the efficiency and limitations of centromere-specific multiplex fluorescence in situ hybridization (cenM-FISH), a new 24 colour FISH technique using centromere-specific probes, to analyse the whole chromosome complement within human oocytes. METHODS: Oocytes were donated by 34 patients undergoing ovarian stimulation and IVF. The MII oocytes were analysed by means of cenM-FISH, while the confirmation of results was performed by FISH and/or by analysing the corresponding first polar bodies using comparative genomic hybridization (CGH). RESULTS: A total of 30 cells, corresponding to 16 oocytes and 14 first polar bodies, were successfully karyotyped by either cenM-FISH or CGH. The incidence of aneuploidy was 25%, and eight out of nine aneuploidy events were confirmed by CGH and FISH. CONCLUSIONS: We demonstrate here for the first time that the identification of any numerical abnormality in oocytes is feasible using cenM-FISH. Despite the fact that the fixation efficiency remains low, the present results confirm the advantage of analysing the whole set of chromosomes to make an accurate estimation of the aneuploidy rate in human oocytes.     

中山地区应用体外受精技术治疗不育症的初步报告

目的：初步报告中山市应用体外受精技术（IVF）治疗不育症的效果。方法：28对不育夫妇的男方在精液常规检查的基础上,进行精子形态学和顶体状态分析。28例受术妇女采用Buserelin/FSH/HCG黄体期长方案控制超排卵,并根据不孕病因、患者年龄及卵巢基础FSH／LH比值指导控制性超排卵用药,比较周期取卵率、受精率、卵裂率及临床妊娠率。结果：基础FSH／LH比值正常反应组问收卵率高于低反应组（P＜0．05）。取卵291个,平均每周期取卵9．6个;受精率81．8％;卵裂率79．8％。9例临床妊娠,5例生化妊娠,妊娠率32％。结论：IVF技术是治疗不育症的有效手段,本文只做丈夫精子IVF,其正常精子形态率和顶体完整率对预测受精有指导意义,根据基础FSH／LH比值判断卵巢储备功能及指导超排卵用药,可避免过大、过少剂量对患者的不利影响。     

Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial

BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and < 0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology.     

The clinical significance of the retrieval of a low number of oocytes following mild ovarian stimulation for IVF: a meta-analysis

BACKGROUND: Milder ovarian stimulation protocols for in vitro fertilization(IVF) are being developed to minimize adverse effects. Mildstimulation regimens result in a decreased number of oocytesat retrieval. After conventional ovarian stimulation for IVF,a low number of oocytes are believed to represent poor ovarianreserve resulting in reduced success rates. Recent studies suggestthat a similar response following mild stimulation is associatedwith better outcomes. METHODS: This review investigates whether the retrieval of a low numberof oocytes following mild ovarian stimulation is associatedwith impaired implantation rates. Three randomized controlledtrials comparing the efficacy of the mild ovarian stimulationregimen (involving midfollicular phase initiation of FSH andGnRH co-treatment) for IVF with a conventional long GnRH agonistco-treatment stimulation protocol could be identified by meansof a systematic literature search. RESULTS: These studies comprised a total of 592 first treatment cycles.Individual patient data analysis showed that the mild stimulationprotocol results in a significant reduction of retrieved oocytescompared with conventional ovarian stimulation (median 6 versus9, respectively, P < 0.001). Optimal embryo implantationrates were observed with 5 oocytes retrieved following mildstimulation (31%) versus 10 oocytes following conventional stimulation(29%) (P = 0.045). CONCLUSIONS: The optimal number of retrieved oocytes depends on the ovarianstimulation regimen. After mild ovarian stimulation, a modestnumber of oocytes is associated with optimal implantation ratesand does not reflect a poor ovarian response. Therefore, thefear of reducing the number of oocytes retrieved following mildovarian stimulation appears to be unjustified.