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。     

IVF和ICSI中多原核受精卵的发育及遗传多态性分析

目的:了解IVF和ICSI治疗周期中多原核受精卵的发育情况及其基因座遗传多态性。方法:分别收集IVF和ICSI治疗中的废弃多原核(≥3PN)受精卵315个和67个,进行体外培养,观察比较其发育能力,并复合扩增多原核来源的1株胚胎干细胞和2个囊胚细胞DNA的15个短串联重复序列(STR)基因座,利用3100遗传分析仪对其进行STR基因座多态性检测。结果:IVF组和ICSI组的多原核受精卵卵裂率无显著性差异(P>0.05),但IVF组胚胎停育率和囊胚形成率显著低于ICSI组(P<0.01)。STR基因座多态性检测显示,IVF组3PN受精卵来源的胚胎干细胞为典型的三倍体特征,但ICSI组4PN受精卵来源的2个囊胚未表现多倍体特征。结论:多原核受精卵有继续发育能力,其中IVF组多原核来源胚胎干细胞表现遗传物质倍性改变,而ICSI组多原核来源囊胚无遗传物质倍性变化。     

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。     

体外受精治疗周期中出现过多的多原核合子5例分析

在体外受精中异常受精的现象，已经引起越来越多胚胎学家的关注。在常规体外受精（IVF）中多原核一般认为是由于多精受精引起的，而对于卵胞浆内单精子注射（ICSI），研究者则认为多原核的形成是由于第二极体不能排出，也可能是二倍体的精子或卵母细胞参与了受精。文献报道在体外受精周期中，受精卵中出现多原核的比例约为3％-10％。然而在一些体外受精治疗周期中，受精卵中存在过多多原核形成的现象。本文对体外受精（IVF和ICSI）治疗周期中，部分患者的大部分受精卵中出现多原核的现象进行汇总分析，以更好地了解多原核形成的原因及其机制，为临床上体外受精方法的选择提供依据。     

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。     

体外受精失败后补行卵胞浆内单精子注射的临床价值探讨

目的:探讨卵胞浆内单精子注射(ICSI)在体外受精完全失败或受精率低于25%的常规IVF周期中的临床价值。方法:回顾分析2001.01-2004.12在我院生殖医学中心接受常规IVF治疗的35例非男性因素不育患者,取卵后体外受精培养16-18h,发现卵母细胞完全未受精或受精率低于25%,立即行ICSI再授精。结果:在24个常规IVF低于25%的周期中,共有197个未受精卵,其中159个MⅡ期卵,显微注射159个,受精123个,最终形成胚胎96个,受精率为77.4%,卵裂率为78.1%,在22个新鲜移植周期(每周期的移植胚胎由来源于常规体外受精卵和补救ICSI后受精卵的胚胎组成),共有4例临床妊娠;在8个冷冻移植周期中(每周期的移植胚胎完全来源于补救ICSI后的受精卵),有1例临床妊娠。在11个常规IVF完全失败周期中,共有89个未受精卵,其中78个MⅡ期卵,显微注射78个,受精63个,卵裂51个,受精率为80.7%,卵裂率为80.9%,在10个新鲜移植周期(每周期的移植胚胎完全来源于补救ICSI后的受精卵)中共有2例临床妊娠;在2个冷冻移植周期(每周期的移植胚胎完全来源于补救ICSI后的受精卵)中有1例单胎妊娠,妊娠早期流产。胚胎来源于常规体外受精卵和补救ICSI后受精卵的移植周期临床妊娠率为18%;胚胎完全来源于补行ICSI后受精卵的移植周期临床妊娠率为20%。结论:ICSI可作为常规IVF失败后的有效补救措施。     

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。     

受精方式、女方年龄、生育史和取卵数对体外异常受精的影响

目的:探讨人卵子体外受精后单原核(PN)和多原核胚胎形成的影响因素,为提高正常受精率探寻可行的方法。方法:回顾性分析1 004个IVF周期和250个ICSI周期,共计15 364个卵细胞资料,研究胚胎原核形成与体外受精方式、女方年龄、生育史、获卵数的关系。结果:①ICSI周期的1PN率明显高于IVF周期,而3PN和4PN形成率则明显低于IVF(P<0.05)。②当女方年龄>35岁时,1PN、3PN和4PN形成率均显著高于年龄≤35岁者(P<0.05);当女方年龄在28￣35岁时2PN(正常受精率)生成率最高(P<0.05),此年龄段的患者施行ICSI时3PN生成率也最高(P<0.05)。③女方无生育史的1PN形成率上明显高于有生育史患者(P<0.05)。④获取6￣10枚卵组,2PN形成率明显高于1￣5枚卵组和>20个卵组(P<0.05);而对于1PN、3PN、4PN形成率,获11￣15枚卵组明显高于其他获卵数组(P<0.05);而在获卵数>20枚时ICSI组2PN(正常受精率)生成率显著降低。结论:人体外受精正常与否受到受精方式、女方年龄、生育史和取卵数目等因素的影响,因此,在进行辅助生殖技术(ART)时应综合考虑这些因素,有助于提高正常受精率,降低异常受精率。     

同周期同胞卵研究较早期实施补救ICSI价值的探讨

目的:比较同周期同胞卵不同时间补救卵细胞浆内单精子显微注射(ICSI)的结果。方法:选取常规体外受精(IVF)治疗失败的26个周期312枚卵子(每周期未受精卵≥8枚,受精率<25%),分为两组:观察组受精后8h实施补救ICSI,对照组20h实施补救ICSI,对比两组受精率、优质胚胎形成率、囊胚形成率。结果:观察组和对照组受精率分别为81.4%、74.36%,两者差异无统计学意义(P>0.05),观察组优质胚胎形成率和囊胚形成率分别为48.82%、38.58%,明显高于对照组的33.62%,22.41%(P<0.01)。结论:对常规IVF受精失败及受精率较低的周期受精后8h实施补救ICSI得到的结果优于受精后20h的结果。     

自体颗粒细胞线粒体移植对胚胎发育质量的影响

目的　探讨自体颗粒细胞线粒体移植 (线粒体移植 )对改善胚胎发育质量的影响。方法　对 3次行体外受精 胚胎移植 (IVF ET)治疗未获妊娠或年龄大于 37岁的 18例患者 ,采用长周期超促排卵方案促排卵。当患者取出的成熟卵子多于 4个时 ,采用常规方法 ,进行卵母细胞浆内单精子注射 (ICSI) ;或进行线粒体移植。线粒体移植的方法为收集卵丘复合体上和卵泡液中的颗粒细胞 ,经匀浆离心提取线粒体 ,应用显微操作技术将线粒体和精子一并注入卵母细胞浆内。观察、比较两种方法获得卵子的受精和胚胎发育情况。　结果　 18例行超促排卵方案后 ,获得成熟卵子 16 8个 ,其中行线粒体移植卵子 86个 ,受精 6 4个 ,受精率为 74 4 % ;行ICSI卵子 82个 ,受精 6 3个 ,受精率为 76 8%。两种方法比较 ,差异无显著意义 (P >0 0 5 ) ;行线粒体移植的良好胚胎形成率为 5 9 4% ,行ICSI的良好胚胎形成率为 34 9% ,两种方法比较 ,差异有显著意义 (P <0 0 5 )。 18例中 7例临床妊娠 ,妊娠率为 38 9%。　结论　自体颗粒细胞线粒体移植可明显改善胚胎发育质量 ,提高妊娠率。     

Chromosomal constitution of embryos derived from tripronuclear zygotes studied by fluorescence in situ hybridization using probes for chromosomes 4, 13, 18, 21, X, and Y

This study was designed to assess the chromosomal constitution and segregation patterns of cleaving embryos derived from tripronuclear zygotes. Thirty-two embryos obtained from 19 conventional IVF patients were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 4, 13, 18, 21, X, and Y. Sixteen embryos (50.0%) exhibited uniform, non-mosaic patterns. These embryos showed pure triploid (n = 7), pure diploid (n = 7), or pure haploid (n = 2). The remaining 16 embryos showed mosaic patterns; 1 was triploid-diploid mosaics, 9 were diploid-haploid, and 4 were haploid only. Autosomal aneuploidy occurred in 2 embryos showing a triploid complement. The sex chromosomal constituent XXX:XXY:XYY was 3:4:2 in embryos showing a pure triploid complement (including 2 embryos with aneuploidy). This ratio was not significantly different from the expected 1:2:1 (p = 0.96). Pure triploid was found in 41.7% of 2-cell embryos, but no triploid complement was found in 3-cell embryos. The present study also supports the diandric origin of tripronuclear zygotes in the conventional IVF, and, to our knowledge, is the first study to use simultaneous six-color FISH for chromosomes 4, 13, 18, 21, X, and Y in human embryos. However, no additive information was obtained about chromosome 4.     

Aneuploid analysis of tripronuclear zygotes derived from in vitro fertilization and intracytoplasmic sperm injection in humans

This study demonstrates that the diploid ratio of tripronuclear zygotes after intracytoplasmic sperm injection (ICSI) is significantly higher as compared with that after conventional IVF; the extra pronucleus of tripronuclear zygotes after ICSI are mostly from the second polar body, not from sperm.     

Unequal Pronuclear Size—A Powerful Predictor of Embryonic Chromosome Anomalies

Purpose: Our purpose was to evaluate whether pronuclei of unequal size, observed in 13.7% of zygotes evaluated after in vitro fertilization (IVF), are predictive of chromosome anomalies in the developing embryo. Methods: The ploidy of 38 embryos grown from zygotes with unequal-sized pronuclei was assessed by fluorescent in situ hybridization (FISH). Twenty-six embryos developed after intracytoplasmic injection of sperm (ICSI) and 12 embryos were derived from conventional IVF. Results: Chromosome anomalies were documented in the ICSI and IVF groups in 88.5 and 50% of cases, respectively. Conclusions: We suggest that FISH should be employed to examine the ploidy of zygotes with unequal pronuclei, prior to embryo transfer.     

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.     

Normal birth after single-embryo transfer in a patient with excessive polypronuclear zygote formation following in vitro fertilization and intracytoplasmic sperm injection

OBJECTIVE: To report an unusual case of idiopathic zygotic polypronuclei. DESIGN: Case report. SETTING: Major urban infertility referral center. PATIENT(S): A 34-year-old woman with unexplained infertility. INTERVENTION(S): The patient underwent two cycles of controlled hyperstimulation and oocyte retrieval followed by in vitro insemination and intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Pregnancy and delivery of a normal infant following transfer of a single preembryo from an ICSI cycle in which only one zygote showed a normal pronuclear number (2PN) and 12 zygotes appeared polypronucleated (> or =3PN). RESULT(S): On the first IVF cycle, 23 oocytes were retrieved and inseminated with 240 x 10(3) motile sperm/mL, after which two zygotes showed a normal pronuclear number and 20 zygotes appeared polyploid with three to seven pronuclei. Transfer of two poor-quality day-3 preembryos following assisted hatching did not achieve pregnancy. On the subsequent ICSI cycle, 33 oocytes were retrieved, and 17 mature oocytes were subjected to ICSI, after which only one zygote showed 2PN and 12 zygotes appeared polyploid with three to eight pronuclei. The normally fertilized zygote developed into a poor-quality, day-3 embryo and was subjected to assisted hatching. Transfer of this preembryo resulted in an uneventful pregnancy and birth of a normal infant. CONCLUSION(S): Mechanisms other than polyspermia may result in polypronuclear development in some patients.     

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.     

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.     

Detection of numerical chromosome abnormalities in human spermatozoa by three-color fluorescence in situ hybridization

Three-color fluorescence in situ hybridization (FISH) was used to detect numerical X, Y, and 17 chromosomal aberrations in human sperm nuclei. Digoxigeninlabeled alpha satellite chromosome X-specific probe DXZ1, biotin-labeled classical satellite chromosome Y-specific probe DYZ1, and biotin plus digoxigenin-labeled alpha satellite chromosome 17-specific probe D17Z1 were simultaneously hybridized to sperm preparations from donors with normal semen (group A) and abnormal semen (group B) characteristics. The proportions of haploid X, Y, 17 and disomy, diploidy of them before and after swim-up were determined. At least 3,000 sperm were analyzed for each sample. Overall, up to 98% of sperm were labeled with the probes, and all statistical analyses were performed using chi 2 tests. A significant difference was observed between group A and group B in frequency of sex chromosome disomy (p < 0.05). In group B, there were significant differences in frequencies of sex chromosomes disomy (p < 0.05) and diploidy (p < 0.01) before to after swim-up. There was no significant difference in frequency of disomy 17 between the 2 groups. In group A and B, the ratios of X- to Y-bearing sperm were 1:1 (neat semen), but in both groups there was a significant increase in Y-bearing sperm after swim-up. The results of this study demonstrated that abnormal semen has sex chromosome disomy more frequently than does normal semen and that portion of sex chromosome disomic and diploid sperm is removed by swim-up, especially for abnormal semen. These findings suggest that we should be careful in using abnormal semen for IVF, especially for ICSI, and should perform swim-up if possible.     

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.