体外受精周期中不同原核数发育囊胚使用价值的探讨

目的:比较3种受精卵来源胚胎的囊胚形成率和冻胚移植周期的临床妊娠率,初步探讨异常受精来源囊胚的使用价值。方法:体外受精(IVF)和卵胞浆内单精子注射(ICSI)周期中,比较2PN、1PN和0PN来源胚胎的囊胚形成率、优质囊胚形成率和在冻融胚胎移植(CET)周期中的临床妊娠率。结果:IVF周期中,2PN来源胚胎的囊胚形成率与0PN比较,无显著差异(P0.05),1PN来源胚胎的囊胚形成率显著低于0PN、2PN来源(P0.01)。ICSI周期中,1PN、0PN来源胚胎的囊胚形成率均显著低于2PN来源(P0.05);3种受精卵来源的优质囊胚在CET周期的临床妊娠率无显著差异(P0.05)。结论:1PN、0PN来源的囊胚可视为与2PN来源的囊胚同等价值,可在患者接受产前检查的基础上用于移植。     

异常受精胚胎临床利用价值探讨

目的:探讨常规体外受精(IVF)和卵胞浆内单精子注射(ICSI)周期中异常受精胚胎的临床利用价值。方法:回顾性分析2013年4月至2015年6月在白求恩国际和平医院生殖中心接受体外受精-胚胎移植(IVF-ET)治疗(包括常规IVF和ICSI)的955个周期的实验室和临床数据。结果:ICSI周期双原核(2 PN)受精率显著高于IVF周期2 PN受精率(88.20%vs 72.04%,P0.01),而ICSI周期的未见原核(0 PN)、单原核(1 PN)及多原核受精率均低于IVF周期(P0.01),差异有统计学意义。第5天(D5)0 PN的囊胚形成率和可利用囊胚率显著高于2 PN和1 PN组(P0.01)。新鲜移植周期和冻融卵裂胚移植周期,0 PN组种植率和临床妊娠率显著低于2 PN组(P0.01),但是冻融囊胚移植周期,0 PN组的种植率和临床妊娠率与2 PN组相比,差异均无统计学意义(P0.05)。结论:IVF/ICSI周期中没有2 PN胚胎可供选择移植时,可选择0 PN胚胎,囊胚培养是合理利用异常受精胚胎的有效手段。     

体外受精中3原核胚胎的发育及可利用价值的探讨

目的:探讨体外受精(IVF)中异常的3原核(PN)胚胎的发育及可利用价值。方法:收集IVF治疗周期中废弃的3PN受精卵204个进行体外培养,观察其发育能力,并与同周期的1 138个2PN受精卵进行比较;采用胚胎植入前遗传学筛查(PGS)技术对由3PN发育成的19枚囊胚进行非整倍体分析。结果:3PN组和2PN组的卵裂率无统计学差异(P0.05);但3PN组囊胚形成率显著低于2PN组[9.6%(19/97)vs 37.9%(204/342),P0.01]。整倍体分析显示,10.5%(2/19)的3PN来源的囊胚为正常二倍体核型。结论:3PN受精卵有继续发育能力;囊胚培养和高通量测序可作为有效筛选异常PN受精卵中正常核型胚胎的一种方法。     

低质量人类胚胎体外发育至囊胚能力的研究

目的:探讨临床上D3丢弃的低质量胚胎培养至囊胚的发育潜能,为从低质量胚胎获得胚胎干细胞提供依据。方法:收集IVF/ICSI35例,184枚低质量胚胎(包括异常受精胚胎),用序贯法微滴培养,D3～D9连续观察囊胚形成情况。结果:(1)184枚低质量胚胎于D4～D6形成23枚囊胚,囊胚形成率12.5%;(2)胚胎卵裂球数越多,囊胚形成率越高,胚胎级别越低,囊胚形成率越低;(3)3PN胚胎囊胚形成率低于2PN囊胚形成率(14.8%vs15.9%),3PN来源的胚胎具有一定的发育潜能。结论:低质量胚胎具有发育至囊胚的潜能,有可能成为分离胚胎干细胞的材料来源。     

体外受精后多原核受精卵的移植价值

目的:对体外受精周期中产生多原核受精卵运用荧光原位杂交技术(FISH)进行非整倍体率检测,分析其移植价值。方法:应用FITC、Texas red标记的X/Y双色染色体着丝粒部位探针对体外受精(IVF)和单精子显微注射(ICSI)周期中的三原核受精卵(3PN)进行荧光原位杂交和分析。结果:分析有杂交信号的3PN卵子68个,IVF后3PN的三倍体率为92.31%,二倍体率为3.85%,单倍体率为3.85%;共存在3种三倍体核型:XXX、XXY、XYY。ICSI后3PN 的三倍体率为70.83%,二倍体率为25%,单倍体率为4.17%;共存在2种三倍体核型:XXX、XXY。结论:IVF后3PN卵子中多余的原核多为精子来源,没有移植价值;ICSI后3PN中多余的原核非精子来源,ICSI后3PN中二倍体率与IVF相比显著增高,在患者ICSI后无2PN卵子时可考虑行植入前遗传学诊断。     

不同精子来源及授精方式对剩余胚胎继续囊胚培养结局的影响

目的:探讨不同精子来源及不同授精方式对胚胎继续发育能力的影响。方法:分析499例患者499个取卵周期剩余胚胎继续培养形成囊胚的情况,按精子来源不同分为供精IVF(D-IVF)组和夫精IVF(H-IVF)组,按授精方式不同分为IVF组和ICSI组,ICSI组按精子来源分为新鲜精液组、附睾精子和睾丸精子组,比较不同精子来源及授精方式获得剩余胚胎的囊胚形成率、胚胎利用率和无囊胚移植率。结果:① D-IVF组和H-IVF组受精率、卵裂率、优质胚胎率、第3日和第5日胚胎种植率、临床妊娠率和流产率均无统计学差异(P0.05),组间剩余胚胎囊胚形成率、胚胎利用率和无囊胚移植率亦无统计学差异(P0.05);②ICSI组与IVF组比较,其受精率较高(P0.05),但优质胚胎率显著下降,有统计学差异(55.11%vs 61.30%,P0.05),组间第3日卵裂期胚胎和剩余胚胎囊胚种植率、临床妊娠率无统计学差异(P0.05),但ICSI组与IVF组比较,其剩余胚胎囊胚形成率、胚胎利用率稍低,无囊胚移植率较IVF组稍高,差异有统计学意义(56.13%vs 65.32%,48.18%vs 55.39%,21.68%vs 13.20%,P0.05)。③新鲜精液组的优质胚胎率、胚胎利用率显著低于附睾精子和睾丸精子组(P0.05),各组囊胚移植的种植率和临床妊娠率无统计学差异(P0.05)。结论:D-IVF可获得H-IVF相似的结局,其剩余胚胎都有较高的发育潜能,ICSI获得的剩余胚胎发育潜能低于IVF组。附睾精子和睾丸精子ICSI后获得的胚胎比新鲜精液精子ICSI后胚胎发育潜能高。针对不同的授精方式可能需要制定相应的剩余胚胎囊胚培养标准。     

人类常规体外受精中3原核(3PN)受精卵及胚胎的染色体组成

目的:分析人类常规体外受精(in vitro fertilization,IVF)中3原核(tripronuclear,3PN)受精卵及胚胎的染色体组成。方法:收集IVF来源的3PN受精卵及胚胎,与秋水仙素共培养14～20 h后制备染色体。结果:共收集到46枚3PN受精卵,277枚3PN来源的胚胎,核型分析显示:受精卵多为三倍体,胚胎可为近单倍体、近二倍体、近三倍体、多倍体等。结论:人类IVF来源3PN受精卵多为三倍体,3PN胚胎染色体紊乱。     

影响冻融胚胎发育潜能及妊娠结局因素的探讨

目的:探讨胚胎冷冻复苏后,卵裂球存活状态对胚胎发育的影响以及体外授精(IVF)和卵胞质内单精子注射(ICSI)2种授精方式对冷冻胚胎复苏后体外发育能力和妊娠结局的影响。方法:回顾性分析142例患者,150个复苏周期,共769个冷冻胚胎复苏后培养至囊胚期进行移植的结果。结果:共复苏胚胎769个,存活胚胎702个,复苏率91.3%。220个胚胎(31%)到达囊胚阶段。在卵裂球全部存活胚胎中,囊胚形成率35%,部分存活胚胎中囊胚形成率24%,两者有统计学差异(P<0.01)。在卵裂球全部存活胚胎中,来源于IVF的胚胎囊胚形成率为40%,来源于ICSI的为26%,两者有统计学差异(P<0.01);部分卵裂球存活胚胎中来源于IVF的胚胎囊胚形成率为26%,来源于ICSI的为19%,差异无统计学意义(P>0.05)。在全部220个囊胚中,IVF组的优质囊胚率为38.6%,ICSI组的优质囊胚率为21.0%,差异有统计学意义(P<0.05)。临床妊娠率IVF组和ICSI组分别为61.05%与61.11%;胚胎种植率分别为37.50%与36.67%,活产率分别为81.03%与78.79%,差异均无统计学意义(P>0.05)。结论:胚胎冷冻复苏后卵裂球的损伤削弱了囊胚形成能力,影响卵裂期胚胎进一步发育,这与ICSI和冷冻胚胎复苏后发育潜能的降低有关,但其对临床结果和妊娠结局的影响不大。     

氧浓度对体外培养胚胎发育的影响

目的:探讨体外受精/卵胞质内单精子注射(IVF/ICSI)后移植前胚胎在不同氧浓度培养条件下的发育情况。方法:将接受IVF或ICSI的患者随机分组成常氧组(体积分数20%的氧浓度,包括IVF卵裂期胚胎患者150例,IVF囊胚期胚胎患者51例,ICSI卵裂期胚胎患者81例,ICSI囊胚期胚胎患者39例)和低氧组(体积分数5%的氧浓度,包括IVF卵裂期胚胎患者108例,IVF囊胚期胚胎患者56例,ICSI卵裂期胚胎患者79例,ICSI囊胚期胚胎患者45例),观察各组胚胎在受精后第3日的卵裂胚和第5日的囊胚发育情况以及临床妊娠结局。结果:在患者年龄、不孕年限、基础FSH值、Gn用量、获卵数和MⅡ卵率组间均无统计学差异的基础上,1与常氧组相比,低氧组的胚胎IVF或ICSI后的受精率、卵裂率以及第3日形成的优质胚胎率无统计学差异(P0.05),临床妊娠率和着床率组间也无统计学差异(P0.05);2低氧组胚胎IVF或ICSI后形成的Ⅴ级囊胚多于常氧组(P0.05),总囊胚形成率组间有显著的统计学差异(P0.01),临床妊娠率和着床率组间无统计学差异(P0.05)。结论:与常氧培养条件相比,低氧培养条件并不能显著促进IVF/ICSI后卵裂期胚胎的发育,但能够促进囊胚的形成,特别是囊胚的孵出,因此低氧培养可以作为囊胚培养的首选条件,但不是卵裂期胚胎培养的必要条件。     

废弃胚胎继续囊胚培养研究

目的:探讨体外受精治疗周期中废弃胚胎的体外发育潜能。方法:通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期。比较不同来源胚胎囊胚形成情况和d3胚胎的卵裂球数、质量分级等;并利用废弃胚胎囊胚形成情况对体外受精妊娠结局进行预测。结果:共收集801个废弃胚胎,经序贯培养,形成209个囊胚(26.09%),其中58个为优质囊胚(27.75%)。1PN胚胎、0PN胚胎、d3卵裂球数为7-9-细胞胚胎、d3评分为Ⅰ-Ⅱ级胚胎以及卵裂球数为偶数的胚胎囊胚形成率较高(P均<0.05)。废弃胚胎中有囊胚形成者的临床妊娠率明显高于无囊胚形成者(P<0.05)。结论:废弃胚胎有不同程度的发育潜能,部分可发育为囊胚;特别是:①0PN和1PN胚胎;②d3的4-9-细胞胚胎,偶数卵裂球者更佳;③I级和Ⅱ级胚胎。     

Factors associated with the formation of triploid zygotes after intracytoplasmic sperm injection

Objective: To determine whether clinical or laboratory factors influence development of triploid (3PN) zygotes after ICSI.

Design: Retrospective review.

Setting: The assisted reproductive technology program of Brigham and Women’s Hospital.

Patient(s): Patients undergoing ICSI.

Intervention(s): None.

Main Outcome Measure(s): Cycles were divided into two groups: group A, cycles with one or more 3PN zygotes after ICSI, and group B, cycles with no 3PN zygotes. Age, amount of gonadotropin administered, peak estradiol levels, number of follicles, number of oocytes retrieved and injected, time between retrieval and injection, oocyte abnormalities, sperm type and motile count, percentage of diploid zygotes, and ongoing pregnancy rates were compared between groups.

Result(s): Compared with patients in group B, those in group A received fewer ampoules of gonadotropins, had higher estradiol levels, and had more follicles on the day of hCG administration, oocytes, immature oocytes and oocytes injected and lower percentages of diploid zygotes. However, ongoing pregnancy rates did not differ between groups.

Conclusion(s): Patients who produce 3PN zygotes after ICSI are high responders to ovarian stimulation. The appearance of such embryos is not associated with lower ongoing pregnancy rates and should not necessarily dictate alterations in ovarian stimulation protocols.     

卵胞浆内单精子显微注射后三原核合子发生相关因素及妊娠结局分析

目的探讨卵胞浆内单精子显微注射(ICSI)后的三原核(3PN)合子发生的相关因素及对妊娠结局的影响。方法对2007年1月至2008年12月在河北医科大学第二医院生殖医学中心行ICSI的287个周期进行回顾性分析。将周期分为2组,A组:至少有1个以上的3PN发生。B组:无3PN的发生。比较两组的年龄、促性腺激素(Gn)用量、雌二醇峰值、获卵数、单精子注射卵数、取卵至ICSI时间、不正常卵子率、精子质量、双原核合子率、种植率、妊娠率、流产率等。结果 A组和B组Gn用量、雌二醇峰值、获卵数、单精子注射卵子数和双原核合子率分别为[(28.45±8.68)支对(33.66±14.02)支,75U/支]、[(10203.04±3402.97)pmol/L]对[(8649.27±3870.19)pmol/L]、[(17.09±8.58)枚对(11.34±6.52)枚]、[(13.61±7.49)枚对(9.26±5.41)枚]、[(66.85±18.69)%对(77.39±19.31)%],差异有统计学意义(P<0.05)。两组种植率、妊娠率、流产率差异无统计学意义。结论卵巢对Gn刺激的高反应所造成的部分卵子质量或内部结构的...     

Correlation between the pronucleus size and the potential for human single pronucleus zygotes to develop into blastocysts

Purpose

In this study, we examined the correlation between pronucleus size and the potential for human single pronucleus (1PN) zygotes to develop into blastocysts after IVF and ICSI.

Methods

This study included 112 patients who underwent a total of 112 cycles of IVF/ICSI. To evaluate embryo development, 1PN zygotes were compared with 2PN zygotes in the same IVF/ICSI cycle (control cycles) using time-lapse live embryo imaging. To assess the potential for blastocyst formation, cutoff values for pronuclear area and diameter were established through receiver operating characteristic curve analysis, after which 1PN zygotes were classified based on those cutoff values.

Results

Among 1PN zygotes cultured to day 5/6, the rate of embryo development was significantly lower than from 2PN zygotes. However, the rates of blastocyst formation and good quality blastocysts from 1PN zygotes with large pronuclear areas (≥?710 μm²) or diameters (≥?31 μm) were significantly higher than from 1PN zygotes with smaller pronuclear areas (≤?509, 510–609, and 610–709 μm²) or diameters (≤?24, 25–27,and 28–30 μm) (P?<?0.01). Moreover, the results for 1PN zygotes with large pronuclei were similar to those for 2PN zygotes.

Conclusions

The developmental potential of 1PN zygotes with large pronuclear areas (≥?710 μm²) or diameters (31 μm) appears to be similar to that of 2PN zygotes, and measurement of pronuclear area or diameter in 1PN zygotes is a simple, potentially useful, clinical method.

     

Rate of blastocyst development from excess embryos remaining in culture after day 3 embryo transfer

OBJECTIVE: To assess the rate of blastocyst development (BDR) from embryos remaining in culture after day-3 embryo transfer (ET), and its relationship to cause of infertility. STUDY DESIGN: Retrospective cohort study in tertiary-care IVF Center. Blastocyst development rate (BDR) after day-3 ET was assessed in 126 women who underwent either conventional IVF or ICSI. RESULTS: Mean age, early follicular FSH levels, number of 2PN zygotes, number of excess embryos, and number of ET were similar between patients that underwent IVF and ICSI. Overall, 20% of extra embryos from conventional IVF patients developed into blastocysts compared to 14% of embryos obtained from ICSI. Cause of infertility did not affect BDR, even in patients who required ICSI due to male factors. CONCLUSIONS: Low rates of blastocyst development from excess embryos are similar between IVF and ICSI patients. Only 15 to 25% of excess embryos left in culture develop into blastocysts, regardless of cause of infertility. Physicians and patients can use this information to adjust both AR treatment protocols and patient expectations.     

Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells

Purpose

This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes.

Methods

After sequential culture for 5–6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis.

Results

The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P?<?0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P?<?0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P?<?0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes.

Conclusions

Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.

     

Triploidy after in vitro fertilization: Cytogenetic analysis of human zygotes and embryos

Tripronuclear zygotes obtained from a clinical IVF program were studied cytogenetically. Successful analysis was possible of 42 specimens at the zygote stage and 21 embryos after the first or second cleavage division. In the majority of zygotes (88%) the expected triploidy was confirmed, whereas only 14% of embryos had solely triploid cells. Therefore it is concluded that after tripolar cleavage division, many different types of mosaicism may originate from irregular chromosome distributions. Since the findings in individual blastomeres in embryos resulting from multipronuclear zygotes do not reflect the genetic content of the whole embryo, these embryos are less suitable in a model system for preimplantion diagnosis. The distribution of the sex chromosomal types (XXX, XXY, and XYY) confirmed theoretical expectations. Since in abortion material or in liveborn triploidy cases, the XYY karyotype is hardly ever observed, this indicates that most likely the 69,XYY karyotype has a very high embryonic mortality.     

Could monopronucleated ICSI zygotes be considered for transfer? Analysis through time-lapse monitoring and PGS

Purpose

The purpose of this study was to investigate the chromosomal constitution and the developmental potential of intracytoplasmic sperm injection (ICSI) deriving embryos displaying a single pronucleus at the zygote stage.

Methods

Eighty-eight embryos from single pronucleus (1PN) two polar bodies (2PB) ICSI zygotes from 64 preimplantational genetic screening (PGS) cycles (October 2012–December 2014), were retrospectively analyzed. Zygotes were cultured in a time-lapse incubator. Embryo biopsy was performed on day 3 and genetic analysis approached by array comparative genomic hybridization.

Results

Chromosomal analysis revealed that 17% (15/88) of embryos derived from 1PN 2PB zygotes were diagnosed as euploid. After blastomere biopsy at day 3, the blastocyst rate at day 5 was 3.4% (3/88). Only 2.3% (2/88) euploid blastocysts were obtained. In two couples and after counseling and patient agreement, the transfer of a euploid blastocyst from a 1PN 2PB ICSI zygote was performed resulting in the birth of a healthy child.

Conclusions

These results open the possibility to consider embryos coming from 1PN 2PB ICSI zygotes for transfer when no other embryos from 2PN 2PB ICSI zygotes are available and if a PGS diagnosis of euploidy is obtained. Confirmation of biparental inheritance is strongly recommended.