第一部分 原核期形态特征对胚胎体外发育能力和体内着床潜能的影响 目的： 探讨原核期评分系统与受精后第三天胚胎形态、受精后第六天囊胚形成和着床率之间的关系，以评估其在IVF-ET中的应用价值。 方法： 于常规IVF或ICSI授精后16-20小时观察受精情况，将正常受精卵母细胞分为Z1-Z4四种类型，比较不同原核类型之间的卵裂率，D3优质胚胎率和D6囊胚形成率。并通过分析着床结局明确的移植胚胎，比较各种类型受精卵之间的着床率。 结果： Z4组受精卵的卵裂率(P<0.001)，D3优质胚胎率(P<0.001)，D6囊胚形成率(P<0.01)均明显低于其它各组。各组的着床率之间差异无显著性(P=0.053)，但Z4组的着床率(5.88%)很低。Z1，Z2, Z3三组间的卵裂率，D3优质胚胎率， D6囊胚形成率和着床率之间差异均无显著性。按原核内NPBs的数量进行分组研究Z1+Z2组以及分别分析Z3亚组也得到同样的结果。 结论： 异常的原核形态(Z4型)可预测胚胎的体内外发育潜能，但原核内NPBs的形态特征并不一定能预测胚胎的活力，Z1，Z2和Z3胚胎可能具有相同的发育潜能。 关键词：体外授精-胚胎移植；形态评估；胚胎质量；囊胚形成；着床能力；原核评估 第二部分 D2形态特征对胚胎体外发育能力和体内着床潜能的影响 目的： 研究D2胚胎的各种形态特征对D3胚胎质量，D6囊胚发育和胚胎着床潜能的影响，以评估D2形态评估在体外受精-胚胎移植(IVF-ET)中的临床应用价值。 方法： 于常规IVF或ICSI授精后42-44小时观察D2胚胎形态特征，按D2细胞数，碎片比例，卵裂球大小均匀程度和卵裂球内的核特征进行分组，比较各组间的D3优质胚胎率，D6囊胚形成率和着床率。 按卵裂球内的核特征分组，将胚胎分为A级(胚胎的每个卵裂球内均可见单核)、B级(胚胎至少有1个卵裂球内未见核)，C级(胚胎每个卵裂球均未见核)和D级(胚胎至少有1个卵裂球内见多核(≥2个核))。其中D级胚胎按卵裂球内核的特征再分为D1和D2二个亚组，D1组为双核胚胎，即胚胎卵裂球内见到2个大小较均匀的核；D2组为多核或微核胚胎，即胚胎卵裂球内见到2个以上的核或卵裂球内见多个大小不等的小核。 结果： 1.4细胞胚胎的体外发育能力和体内着床潜能优于另外两组，4细胞以上胚胎的D3优质胚胎率和着床率高于4细胞以下的胚胎。 2.碎片≤10%的D2胚胎的体外发育能力和体内着床潜能优于碎片>10%的胚胎。 3.与其他各组相比，A组具有最高的体外发育能力和着床潜能。在形态优质的D2胚胎中，也得到同样的结果。而D组胚胎中，第三天的优质胚胎率明显低于其它各组，而且在移植的27个D组胚胎中，没有胚胎着床。 4.D1组的D3优质胚胎率和囊胚形成率均高于D2组。单独分析D2形态优质的D级胚胎，仍发现D1组的囊胚形成率高于D2组。 结论： 碎片≤10%的4细胞胚胎具有较高的体外发育能力和体内着床潜能；同时， D2核评估是独立的预测胚胎活力的可靠指标，选择时应优先考虑每个卵裂球上均可见单核的胚胎；胚胎中多核的存在是发育潜能受损的表现，但多核胚胎中的双核类型有着相对较高的发育潜能。 关键词：体外受精-胚胎移植；形态评估；胚胎质量；着床能力；单核卵裂球；多核卵裂球 第三部分 D3低形态学评分胚胎的继续培养及其意义 目的： 研究和评估D3低形态学评分胚胎的囊胚形成率及其价值。 方法： 将IVF/ICSI患者多余的D3低形态学评分胚胎，转移至G2液中培养至D6，观察囊胚形成情况，并分析囊胚形成率与D1，D2和D3形态特征之间的关系。 结果： 840个低形态学胚胎继续培养至D6，共有185个发育成囊胚，囊胚形成率为22%。其囊胚形成率与D1原核形态，D2和D3胚胎形态均明显相关。D1的Z4型受精卵的囊胚形成率明显低于Z1，Z2和Z3组，D2的多核或微核胚胎的囊胚形成率较低。D3的<4细胞胚胎或碎片>20%的胚胎的囊胚形成率均低于其它组。 结论： 低形态学评分的胚胎具有一定的发育潜能和利用价值，其继续发育能力与D1，D2和D3的形态特征密切相关。 关键词：囊胚；继续培养；形态学评分 第四部分 体外授精-胚胎移植中卵裂期胚胎凋亡的研究 目的： 评估体外授精的人卵裂期胚胎中是否存在凋亡。探讨卵裂期胚胎的凋亡与胚胎形态之间的关系。 方法： 将末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)和测定caspase-3的表达这两种方法相结合，检测人卵裂期胚胎中的凋亡。并分析卵裂期胚胎中不同的碎片比例和发育速度与TUNEL阳性率和caspase-3的表达强度之间的关系。 结果： 碎片>20%的胚胎的凋亡率明显高于碎片≤20%的胚胎，Caspase平均荧光强度在碎片>50%组高于另外三组。发育正常、迟缓和停滞胚胎的凋亡率之间差异无显著性。发育停滞组的Caspase平均荧光强度高于发育正常和迟缓组。 结论： 人体外授精的卵裂期胚胎中存在凋亡，凋亡主要与胞质碎片有关，胚胎发育停滞可能是胚胎凋亡的早期表现。 关键词：体外授精 卵裂期胚胎 凋亡 胚胎形态 TUNEL

 Part Ⅰ Effects of Pronuclear Morphological Characteristics on in-vitro Development and Implantation Potential in IVF-ET Objective To assess the relationship between pronuclear scoring and day-3 embryo quality and day-6 blastocyst rate and implantation rate to determine the clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer (IVF-ET) program. Methods A pronuclear scoring system was used to score zygotes 16-20 hours after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). The embryos were classified into group Z1, Z2, Z3 and Z4. Comparisons were made of the rates of cleavaged embryos and excellent embryos on day 3 and blastocyst on day 6. Comparisons of impantation rate were made between the transferred embryos which had the proven implantation behaviour. Results There were less cleavaged embryos and less excellent embryos on day 3 and less blastocysts on day 6 in group Z4 than those in group Z1, Z2 and Z3. There were no statistical differences on implantation rate among the four groups, though the implantation rate was very low in group Z4. There were no difference in excellent embryos rate and blastocyst rate and implantation rate among Z1, Z2 and Z3. The similar results came from the subgroups of Z1+Z2 and Z3. Conclusion These data suggests that abnormal pronuclear morphology (Z4) can predict developmental potential. The morphological characteristics of nucleolar precursor bodies defined by Scott did not predict the embryo viability. Z1, Z2 and Z3 zygotes may have the same development potential. Key words In vitro fertilization-embryo transfer; morphology scoring; embryo quality; blastocyst development; implantation potential; pronuclear scoring Part Ⅱ Effects of Morphological Characteristics of Day 2 Preembryos on in-vitro Development and Implantation Potential in IVF-ET Objective To assess the relationship between morphological characteristics of day 2 preembryos and day-3 embryo quality and day-6 blastocyst rate and implantation rate and to determine the clinical value of day 2 morphological scoring in human in vitro fertilization-embryo transfer (IVF-ET) program. Methods Day 2 morphological scoring, based on blastomere numbers, fragmentation rate, blastomere size and the nuclear characteristics, was made 42 - 44 hours after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). Day 2 preembryos were classified according to the number of nuclei in each blastomere into 4 groups. Grade A embryos having only mononucleated blastomeres; grade B embryos having one or more blastomeres containing no visible nucleus; grade C embryos having no visible nuclei and grade D embryos having one or more multinucleated blastomeres. Grade D embryos were classified into 2 groups: D1 embryos (binucleated, BN embryos) were defined as those with two nuclei of relatively the same size in one or more blastomeres; D2 embryos (multinucleated or micronucleated, MN embryos)were defined as those with more than two nuclei in one or more blastomeres. Comparisons were made of the rates of excellent embryos on day 3 and blastocyst rate on day 6. Comparisons of impantation rate were made between the transferred embryos which had the proven implantation behaviour. Results 1.On day 2 after fertilization, 4 cell embryos were predictive of the highest in-vitro developmental and implantation potential. 2.The in-vitro developmental and implantation potential of embryos with ≤ 10% fragmentation was better than those with ＞ 10% fragmentation. 3.Group A embryos had the highest in-vitro developmental and implantation potential. Exclusive of the effects of the cell number and fragmentation rate, visible mononuclear in all blastomeres remain a strong predictor of high developmental potential. With the lowest day 3 excellent embryos rate, group D had zero implantation of 27 transferred embryos. 4.Day 3 excellent embryos rate and blastocyst rate were significantly higher with subgroup D1 embryos than subgroup D2 embryos. The analysis in day 2 excellent embryos concluded that blastocyst rates of subgroup D1 embryos were higher than subgroup D2 embryos. Conclusion The 4 cell embryos with ≤10% fragmentation predict the high in-vitro developmental and implantation potential. Day 2 nuclear scoring is the independent predictors of embryo viability. Embryos with single nuclear in all blastomeres are preferable for transferring. Multinucleated blastomeres in day 2 embryos imply the impaired developmental potential. But compared with the multinucleated and micronucleated embryos, the binucleated embryos may have the higher developmental potential. Key words In vitro fertilization-embryo transfer; morphology scoring; embryo quality; blastocyst development; implantation potential; mononucleated blastomere; multinucleated blastomere Part Ⅲ Evaluation of the value of poor morphology embryos in extended culture Objective To investigate the blastocyst rate of poor morphology embryos and evaluate the clinical value of poor morphology embryos. Material and methods A total of 840 day 3 poor morphology embryos from the patients undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), were cultured in G2 medium. Blastocyst evaluation was performed on day 6. Additionally, blastocyst rate were analyzed with regard to early embryo development parameters in D1, D2 and D3. Results There were 185 blastocysts developed from 840 day 3 poor morphology embryos on day 6. The blastocyst rate was 22%. The blastocyst rate was significantly related to the pronuclear morphology, D2 and D3 morphological characteristics. There was lower blastocyst rate in group Z4 than that in other groups. The blastocyst rate was relatively low for multinucleated and micronucleated embryos on day 2. The embryos with ＜4 cells or embryos with ＞20% fragmentation developed less blastocysts than the other embryos. Conclusion The poor morphology embryos on day 3 possess certain developmental potential and utilized value. The developmental potential of D3 poor morphology embryos is related to the morphological characteristics of D1, D2 and D3. Key words blastocyst, extended culture, morphology scoring Part Ⅳ The research on apoptosis of cleavage stage embryos in IVF-ET Objective To evaluate whether apoptosis exist in human cleavage stage embryos from in -vitro fertilization and the relationship between apoptosis in cleavage stage embryos and morphological characteristics on D3. Material and methods Detection of apoptosis in human cleavage stage embryos was performed using the terminal transferase-mediated DNA end labelling (TUNEL) assay and the expression of caspase-3. The TUNEL-positive rate and the average fluorescent intensity were determined according to the fragmentation rate and developmental rate. Results Apoptosis rate in embryos with ＞20% fragmentation was significantly higher than that in other groups. The average fluorescent intensity was significantly increased in embryos with ＞50% fragmentation. There was not significant difference in apoptosis rate among the normal developmental embryos, slow developmental embryos and arrested embryos. The average fluorescent intensity in arrested embryos was higher than that in the normal developmental embryos and slow developmental embryos. Conclusion Apoptosis does exist in cleavage stage embryos from in-vitro fertilization. Apoptosis in cleavage stage embryo is strongly correlated with cellular fragments. The early developmental arrest of the human preimplantation embryo may be the early phenomenon of apoptosis. Key words In-vitro fertilization; cleavage stage embryo; apoptosis; embryo morpholonogy; TUNEL