人类未成熟卵体外成熟培养、雌/雄原核形态学判别和原核期胚胎原核移植及其重构胚后续发育潜能的研究
摘要
研究背景:自体移植是当今最为看好的移植治疗模式。自体胚胎干细胞(ESCs)的自我增殖和多向分化潜能使其成为最有潜力的自体移植供体源。但是,自体ESCs的创建目前还存在明显的“低效率”问题,原因主要是供体细胞核重新编序不完全、重构卵细胞组分的种系特异性丢失、或缺少父源性或母源性基因印记的影响。如此,我们提出了《亲缘性基因印记校正的雌核发育/雄核发育技术创建人自体ESCs》的全面解决方案。该方案选用自体原核作为供体细胞核源,试图解决成体细胞核基因重新编程问题;该方案通过构建雌核发育/雄核发育自体重构胚,试图解决重构胚细胞组分缺失问题;该方案还通过对雌核发育/雄核发育自体重构胚进行基因印记方面的校正处理,试图解决基因印记缺失的问题。本研究就该方案的实施能否使用辅助生殖临床废弃的未成熟卵作为(ESCs研究用)卵源、如何活体判别人类原核的亲缘性(构建雌核发育/雄核发育方式原核期胚胎)以及相关的原核移植技术进行了系列的前期基础研究和探索。
一、卵丘冠颗粒细胞剥除的人未成熟卵体外成熟培养和发育潜能的研究
目的:1、了解卵丘冠颗粒细胞剥除的人未成熟卵(ccdlMOs)体外成熟培养(IVM)和相应受精卵植入前发育潜能。2、评估同步和个体化AutoMFF-IVM(自体成熟卵泡液辅助的IVM)培养系统和AutoMFF&CCs-coIVM(自体成熟卵泡液和自体卵丘冠颗粒细胞共同辅助的IVM)培养系统分别用于M-1期(第一次减数分裂中期)和GV期(第一次减数分裂前期)ccdIMOs的IVM,相应的IVM增进效果。
方法:1、2004年11月-2005年7月间从151对[156个卵巢刺激周期实施卵浆内单精子注射(ICSI)治疗的]不孕夫妇获赠388个ccdIMOs。其中GV期卵196个,M-Ⅰ期卵192个。2、IVM标准方案使用MediCult IVM系统(Cat#:82214010,MediCult公司,丹麦),微滴培养12-48小时(37C,5%CO2,饱和湿度)并作适当改良,作为IVM
Background: In order to establish autologous embryonic stem cells (ESCs) efficiently and promote autologous transplantation, a research project on the establishment of human autologous ESCs in genomic-imprints-rectified gynogenesis or androgenesis was hypothesized. The key step of the hypothesis was the reconstruction of an autologous pronuclear embryo, characteristic of autologous genetic background, diploidization and the matched combination of genomic imprints from both parents, gynogenetically or androgeneticall with the individual pronucleus derivated from her/his own gamete as the donor nucleus. In the present series studies, three aspects on feasibility of the research project were investigated.Part I: Study on in-vitro maturation of human corona-cumulus-denuded immature oocytes and the subsequent preimplantation embryo development
Objectives: 1. To investigate the general pattern of in-vitro maturation (IVM) of corona-cumulus-denuded immature oocyte (ccdIMOs) and the subsequent preimplantation embryonic development. 2. To evaluate the effects of IVM culture system assisted by autologous preovulatory/mature follicular fluid (AutoMFF) on IVM of ccdIMOs in metaphase of meiosis 1 (M1-stage) and the effects of IVM coculture system assisted by both AutoMFF and autologous-corona-cumulus (AutoCCs) on IVM of ccdIMOs in prophase of meiosis 1 (GV-stage).Methods: 1. A total of 196 GV-stage and 192 M1-stage morphologically normal ccdIMOs were recruited from 156 consecutive ovarian stimulation cycles for routine intracytoplasmic sperm injection (ICSI). 2. Two synchronic and individual colVM systems with either 10 (v/v) % AutoMFF alone (AutoMFF-IVM) or both 10 (v/v) % AutoMFF and 1-5 X 10 (5) /ml autoCCs in IVM medium (AutoMFF&CC-colVM) were developed to mature in vitro M1- and GV-stage ccdIMOs respectively. Modified MediCult IVM system (Cat #:82214010, MediCult company, Jyllinge, Denmark) was used as a standard and control IVM system. 3. Stratified and randomized block design was used to evaluate the corresponding outcomes of IVM in different durations (IVM, fertilization, preimplantation development) and different levels (both nuclear and cytoplasmic maturation). 4. The maturity of ccdIMOs during IVM was assessed with photographed evidence every 6-12 hours. The extrusion of the first polar body (PB1) was the marker of
nuclear maturation of ccdIMOs. 5. During embryo culture, observations of embryonic developmental stage and quality were conducted every 24 hours. The corresponding fertilization, cleavage and preimplantation embryonic development were used as the evaluation indices of cytoplasmic maturation of ccdIMOs. 6. Descriptive statistics in proportions were used to describe the general characteristics of M1- and GV-stage ccdIMOs' IVM. Chi-square tests were used to compare IVM outcome and subsequent preimplantation embryonic development in proportions among different IVM protocols. Similar methods were used to show the corresponding indices of autologous in-vivo matured oocytes (vivoMOs) and differences between vivoMOs and in-vitro matured oocytes (vitroMOs).Results: 1. All indices for nuclear maturation time course of M1-stage ccdIMOs (12h-IVM: 63.5%;24h-IVM: 90.1%;36h-IVM: 91.1%;48h-IVM: 92.7%) were significantly higher than those of GV-stage ones (12h-IVM: 0.0%;24h-IVM: 25.5%: 36h-IVM: 61.7%: 48h-IVM: 74.5%: P < 0.01) . 2. All four indices for cytoplasmic maturation of vivoMOs (the rate of ICSI fertilization, the cleavage rate, the rate of 8-cell embryonic development at day 3 and the rate of good quality embryo development at day 3) were 65.6%, 96.9%, 45.7% and 30.9% respectively, significantly higher than those of the vitroMOs derived from M1 -stage ccdIMOs (50.3%, 79.0%, 31.3% and 18.8% respectively;P < 0.01) and those of the vitroMOs derived
from GV-stage ccdIMOs (43.0%, 56.4%, 16.1% and 0.0% respectively;P < 0.01). Among those four indices for cytoplasmic maturation, two indices for the vitroMOs derived from M1-stage ccdIMOs (the cleavage rate and the rate of good quality embryo developmental at day 3) were also significantly higher than those of the vitroMOs derived from GV-stage ccdIMOs (P<0.05). No blastocyst development at day 5 was found from the developed GV-stage ccdIMOs, while 5.8% from the developed M1-stage ccdIMOs (P<0.20). 3. Compared to concurrent autologous autoCCs' growth without AutoMFF in the culture medium, the growth of autoCCs with 10 (v/v) % AutoMFF clearly exhibited bigger cell volume, more dendritic—like cell processes and the extending of cell processes, larger cell confluence and delayed atrophy or degeneration. 4. Among the two IVM protocols used in IVM of M1-stage ccdIMOs, the cumulative maturation rates with AutoMFF-IVM at both 24hr and 36hr IVM durations (96.9%, 96.9%, respectively) were all significantly higher than those with standard IVM (86.7%, 88.3%, respectively, P<0.05). 5. Among the two IVM protocols used in IVM of GV-stage ccdIMOs, the cumulative maturation rates with AutoMFF&CC-colVM at both 36hr and 48hr IVM durations (68.6%, 85.7%, respectively) were all significantly higher than those with standard IVM (53.8%, 61.5%, respectively, P<0.05) . 6. No statistical difference was found in all five indices for cytoplasmic maturation (the fertilization rate, the cleavage rate, the rate of 8-cell embryonic development at day 3, the rate
of good quality embryonic development at day 3, and the rate of blastocyst development at day 5) between the vitroMOs from M1 -stage ccdIMOs with standard IVM and the vitroMOs with AutoMFF-IVM (P>0.05) . Similar situation in this aspect existed between the vitroMOs from GV-stage ccdIMOs with standard IVM and the vitroMOs with AutoMFF&CC-colVM.Conclusions: 1. Most of ccdIMOs acquired from ovarian stimulation cycle could complete their nuclear maturation in IVM. Majority of M1-stage ccdIMOs complete their nuclear maturation over 24hr IVM;while most of GV-stage ccdIMOs complete their nuclear maturation over 36hr IVM. 2. Compared with GV-stage ccdIMOs, M1-stage ccdIMOs are characterized in IVM with greater speed, higher IVM rate and better potential for preimplantation embryonic development. They should be priority to be recommended as the oocyte candidate amongst ccdIMOs in oocyte donation or ESCs research. 3. AutoMFF significantly stimulates the autoCCs' feeder-layer-cell-like growth. 4. The two synchronic and individual IVM culture systems, AutoMFF IVM and AutoMFF&CC colVM, developed here for the first time, are safe from the risk of pathogen transmission, superior for rescue IVM of human ccdIMOs and suitable for IVM of M1- and GV-stage ccdIMOs, respectively, in nuclear maturation, but not cytoplasmic maturation. Further studies are needed to optimize each of the two IVM culture systems.
Part II: Study on kinship significances of morphological characteristics of human pronuclei and their verificationObjectives: 1. To recommend a morphological criteria of human pronuclei (PN) in kinship. 2. To develop a reliable and valid model in this aspect for verification. 3. To verify the effectiveness of recommended morphological criteria of human PN in kinship.METHODS: 1. It was conducted to recommend a morphological criteria of human PN in kinship based on the relative rationale of human PN development in biology and the sporadic description in this aspect in nonprimate. 2. Human 3PN-zygote model constructed by intracytoplasmic 2-sperm injection (2sperm-ICSI) was developed to verify the recommend morphological criteria of human PN in kinship. The correlated rationale of this model in the verification mentioned above was based on facts that the PN kinship in a standard model of this kind was relatively valid, i.e. two male PNs and one female PN with two polar bodies (PB1 and PB2). 3. Eighty-nine mature oocytes (from the in vitro maturation of
originally-donated 108 morphologically normal corona-cumulus-denuded immature oocytes of 68 consecutive ovarian stimulation cycles for routine intracytoplasmic sperm injection) and 68 normal sperm suspension samples (donated by infertile couples for conventional in vitro fertilization) were used to construct human 3PN-zygote model mentioned above. 4. Verification of the effectiveness of recommended morphological criteria of human PN in kinship was conducted on the basis of firstly-validated PN kinship in human 3PN-zygote model. The firstly-validated morphological kinship criteria for male PN of human 3PN-zygote model were that the size and morphology of two male PNs should be mutually matched and uniformed and that the distance to PB2 should be relatively remote. The corresponding criteria for female PN were that the size and morphology of single female PN should be unmatched and ununiformed to the other two PNs and that the distance to PB2 should be relatively near.RESULTS: 1. The recommended morphological criteria of human PN in kinship were that: as to male PN, the morphological characteristics were relative bigger in size and remote to PB2;and as to female PN, the situation was vice versa, i.e. relative smaller in size and near to PB2. 2. The success rate for the construction of a standard 2sperm-ICSI 3PN-zygote model in human was 43.82% (39/89) ? 3. Among 117 PNs of 39 standard models of human 2sperm-ICSI 3PN-zygote, the kinships of 105 PNs (89.7%, including 70 male PNs and 35 female PN) of 35 models were completely
matched with those based on the recommended morphological criteria of human PN in kinship.CONCLUSIONS: 1. Human 2sperm-ICSI 3PN-zygote model developed for the first time in the present study, characteristic of simplicity, economy, feasibility, effectiveness and novelty, is a worthily-recommended model in the verification research on morphological criteria of human PN in kinship. 2. The morphological criteria of human PN in kinship recommended in the present study, provides, for the first time in live and natural state, simple, economic, practical and relatively reliable criteria of the corresponding aspect and makes possible the corresponding applications in research and the clinic.Part III: Study on pronucleus transfer in human zygotes and the subsequent preimplantation development of reconstructed embryosObjectives: 1. To develop a pronucleus transfer (PNT) mode, characteristic of simplicity, naturality, feasibility and effectiveness. 2. To evaluate the subsequent preimplantation development potential of corresponding reconstructed embryos.Methods: 1. Fifty-four human zygotes were donated from 34 consecutive infertile women (age: 29.7+4.2 yrs old) undergoing conventional in vitro
fertilization and embryo transfer (IVF-ET) during from Nov. 2004 to Jul. 2005. 2. Of the donated zygotes, 20 multiple pronucleus (PN) zygotes were conducted to receive a selective PN reduction (SPnR), a kind of micromanipulation to aspirate supernumerary PN or PNs from a multiple PN zygote and to make the zygote into its normal 2PN composition and sexual reproduction mode. The morphological kinship criteria for male PN of human zygotes used in SPnR were that the size was relative bigger and that the distance to PB2 was relatively remote. The corresponding criteria for female PN were that the size was relative smaller and that the distance to PB2 was relatively near. 3. Another 23 multiple PN zygotes of the same origin were used as blank control of SPnR (Control group). 4. The remaining 11 zygotes were divided into 6 groups (including a one-zygote one). Two zygotes in each group were used as a receiver or donor zygote respectively and PNT was conducted between them. As to the one-zygote group, autologous PNT was conducted. The real PNT included three major steps:firstly the SPnR on receiver zygote to make it into the one PN state;secondly the SPnR on donor zygote to get the needed donor PN according to the morphological criteria of human PN in kinship;and lastly the intracytoplasmic PN injection (ICPI) on the one PN receiver zygote prepared before to make it restore its originally normal 2PN state. 5. Routine embryonic culture to day 7 post fertilization on all zygotes, no
matter reconstructed or not. During embryo culture, embryonic developmental stage and quality observations were conducted every 24 hours. 6. Descriptive statistics in proportions were used to describe the duration and success rate for SPnR/ICPI, the embryo potential for preimplantation development. Chi-square tests were used to compare the difference of preimplantation embryonic development between the SPnR-reconstructed zygotes and the control ones.Results: 1. SPnR duration was 5—40 minutes (14.8+8.0 minutes), most(12/20, 60.0%) <10 minutes, majority (18/20, 90.0%) <20 minutes. SPnR success rate was 95% (19/20). 2. All four indices for the preimplantation development of SPnR-reconstructed zygotes (the cleavage rate, the rate of 8—cell embryonic development at day 3, the rate of good quality embryonic development at day 3 and the rate of blastocyst development at day 5) were 85.0%, 25.0%, 10.0% and 5.0% respectively, lower than those of the same origin of control group (91.3%, 34.8%, 13.0% and 17.4% respectively);but there was no significance in statistics (P>0.05). 3. Duration of PNT in SPnR/ICPI was 15-40 minutes (30.8+9.5 minutes), most (4/6, 66.7%) <30 minutes. 4. Among 6 times of ICPI, 3 succeeded and 3 failed. 5. Among 3 zygotes successfully reconstructed in SPnR/ICPI, two were not only alive, but also naturally cleaved and developed to morula on day 5 post fertilization, but not to blastocyst in the end. 6. Incomplete ICPI or real extracytoplasmic PN injection (ECPI) was the collective cause
for PNT failure of all other three zygotes in SPnR/ICPI.Conclusions: 1. The micromanipulation of moving PN out of human zygote, like SPnR on multiple PN zygotes, is feasible, practical and effective. There is no significant impact of SPnR on the preimplantation development of reconstructed embryos. It is worthy of studying further to develop SPnR in the clinic. 2. PNT of human zygotes in SPnR/ICPI is simple, feasible and effective. Most of the successfully-PNT zygotes are alive, cleave naturally and possess considerable potential for preimplantation development. 3. Incomplete ICPI or real ECPI is the main cause for PNT failure in SPnR/ICPI. 4. Further studies are needed for optimization.
一、卵丘冠颗粒细胞剥除的人未成熟卵体外成熟培养和发育潜能的研究
目的:1、了解卵丘冠颗粒细胞剥除的人未成熟卵(ccdlMOs)体外成熟培养(IVM)和相应受精卵植入前发育潜能。2、评估同步和个体化AutoMFF-IVM(自体成熟卵泡液辅助的IVM)培养系统和AutoMFF&CCs-coIVM(自体成熟卵泡液和自体卵丘冠颗粒细胞共同辅助的IVM)培养系统分别用于M-1期(第一次减数分裂中期)和GV期(第一次减数分裂前期)ccdIMOs的IVM,相应的IVM增进效果。
方法:1、2004年11月-2005年7月间从151对[156个卵巢刺激周期实施卵浆内单精子注射(ICSI)治疗的]不孕夫妇获赠388个ccdIMOs。其中GV期卵196个,M-Ⅰ期卵192个。2、IVM标准方案使用MediCult IVM系统(Cat#:82214010,MediCult公司,丹麦),微滴培养12-48小时(37C,5%CO2,饱和湿度)并作适当改良,作为IVM
Background: In order to establish autologous embryonic stem cells (ESCs) efficiently and promote autologous transplantation, a research project on the establishment of human autologous ESCs in genomic-imprints-rectified gynogenesis or androgenesis was hypothesized. The key step of the hypothesis was the reconstruction of an autologous pronuclear embryo, characteristic of autologous genetic background, diploidization and the matched combination of genomic imprints from both parents, gynogenetically or androgeneticall with the individual pronucleus derivated from her/his own gamete as the donor nucleus. In the present series studies, three aspects on feasibility of the research project were investigated.Part I: Study on in-vitro maturation of human corona-cumulus-denuded immature oocytes and the subsequent preimplantation embryo development
Objectives: 1. To investigate the general pattern of in-vitro maturation (IVM) of corona-cumulus-denuded immature oocyte (ccdIMOs) and the subsequent preimplantation embryonic development. 2. To evaluate the effects of IVM culture system assisted by autologous preovulatory/mature follicular fluid (AutoMFF) on IVM of ccdIMOs in metaphase of meiosis 1 (M1-stage) and the effects of IVM coculture system assisted by both AutoMFF and autologous-corona-cumulus (AutoCCs) on IVM of ccdIMOs in prophase of meiosis 1 (GV-stage).Methods: 1. A total of 196 GV-stage and 192 M1-stage morphologically normal ccdIMOs were recruited from 156 consecutive ovarian stimulation cycles for routine intracytoplasmic sperm injection (ICSI). 2. Two synchronic and individual colVM systems with either 10 (v/v) % AutoMFF alone (AutoMFF-IVM) or both 10 (v/v) % AutoMFF and 1-5 X 10 (5) /ml autoCCs in IVM medium (AutoMFF&CC-colVM) were developed to mature in vitro M1- and GV-stage ccdIMOs respectively. Modified MediCult IVM system (Cat #:82214010, MediCult company, Jyllinge, Denmark) was used as a standard and control IVM system. 3. Stratified and randomized block design was used to evaluate the corresponding outcomes of IVM in different durations (IVM, fertilization, preimplantation development) and different levels (both nuclear and cytoplasmic maturation). 4. The maturity of ccdIMOs during IVM was assessed with photographed evidence every 6-12 hours. The extrusion of the first polar body (PB1) was the marker of
nuclear maturation of ccdIMOs. 5. During embryo culture, observations of embryonic developmental stage and quality were conducted every 24 hours. The corresponding fertilization, cleavage and preimplantation embryonic development were used as the evaluation indices of cytoplasmic maturation of ccdIMOs. 6. Descriptive statistics in proportions were used to describe the general characteristics of M1- and GV-stage ccdIMOs' IVM. Chi-square tests were used to compare IVM outcome and subsequent preimplantation embryonic development in proportions among different IVM protocols. Similar methods were used to show the corresponding indices of autologous in-vivo matured oocytes (vivoMOs) and differences between vivoMOs and in-vitro matured oocytes (vitroMOs).Results: 1. All indices for nuclear maturation time course of M1-stage ccdIMOs (12h-IVM: 63.5%;24h-IVM: 90.1%;36h-IVM: 91.1%;48h-IVM: 92.7%) were significantly higher than those of GV-stage ones (12h-IVM: 0.0%;24h-IVM: 25.5%: 36h-IVM: 61.7%: 48h-IVM: 74.5%: P < 0.01) . 2. All four indices for cytoplasmic maturation of vivoMOs (the rate of ICSI fertilization, the cleavage rate, the rate of 8-cell embryonic development at day 3 and the rate of good quality embryo development at day 3) were 65.6%, 96.9%, 45.7% and 30.9% respectively, significantly higher than those of the vitroMOs derived from M1 -stage ccdIMOs (50.3%, 79.0%, 31.3% and 18.8% respectively;P < 0.01) and those of the vitroMOs derived
from GV-stage ccdIMOs (43.0%, 56.4%, 16.1% and 0.0% respectively;P < 0.01). Among those four indices for cytoplasmic maturation, two indices for the vitroMOs derived from M1-stage ccdIMOs (the cleavage rate and the rate of good quality embryo developmental at day 3) were also significantly higher than those of the vitroMOs derived from GV-stage ccdIMOs (P<0.05). No blastocyst development at day 5 was found from the developed GV-stage ccdIMOs, while 5.8% from the developed M1-stage ccdIMOs (P<0.20). 3. Compared to concurrent autologous autoCCs' growth without AutoMFF in the culture medium, the growth of autoCCs with 10 (v/v) % AutoMFF clearly exhibited bigger cell volume, more dendritic—like cell processes and the extending of cell processes, larger cell confluence and delayed atrophy or degeneration. 4. Among the two IVM protocols used in IVM of M1-stage ccdIMOs, the cumulative maturation rates with AutoMFF-IVM at both 24hr and 36hr IVM durations (96.9%, 96.9%, respectively) were all significantly higher than those with standard IVM (86.7%, 88.3%, respectively, P<0.05). 5. Among the two IVM protocols used in IVM of GV-stage ccdIMOs, the cumulative maturation rates with AutoMFF&CC-colVM at both 36hr and 48hr IVM durations (68.6%, 85.7%, respectively) were all significantly higher than those with standard IVM (53.8%, 61.5%, respectively, P<0.05) . 6. No statistical difference was found in all five indices for cytoplasmic maturation (the fertilization rate, the cleavage rate, the rate of 8-cell embryonic development at day 3, the rate
of good quality embryonic development at day 3, and the rate of blastocyst development at day 5) between the vitroMOs from M1 -stage ccdIMOs with standard IVM and the vitroMOs with AutoMFF-IVM (P>0.05) . Similar situation in this aspect existed between the vitroMOs from GV-stage ccdIMOs with standard IVM and the vitroMOs with AutoMFF&CC-colVM.Conclusions: 1. Most of ccdIMOs acquired from ovarian stimulation cycle could complete their nuclear maturation in IVM. Majority of M1-stage ccdIMOs complete their nuclear maturation over 24hr IVM;while most of GV-stage ccdIMOs complete their nuclear maturation over 36hr IVM. 2. Compared with GV-stage ccdIMOs, M1-stage ccdIMOs are characterized in IVM with greater speed, higher IVM rate and better potential for preimplantation embryonic development. They should be priority to be recommended as the oocyte candidate amongst ccdIMOs in oocyte donation or ESCs research. 3. AutoMFF significantly stimulates the autoCCs' feeder-layer-cell-like growth. 4. The two synchronic and individual IVM culture systems, AutoMFF IVM and AutoMFF&CC colVM, developed here for the first time, are safe from the risk of pathogen transmission, superior for rescue IVM of human ccdIMOs and suitable for IVM of M1- and GV-stage ccdIMOs, respectively, in nuclear maturation, but not cytoplasmic maturation. Further studies are needed to optimize each of the two IVM culture systems.
Part II: Study on kinship significances of morphological characteristics of human pronuclei and their verificationObjectives: 1. To recommend a morphological criteria of human pronuclei (PN) in kinship. 2. To develop a reliable and valid model in this aspect for verification. 3. To verify the effectiveness of recommended morphological criteria of human PN in kinship.METHODS: 1. It was conducted to recommend a morphological criteria of human PN in kinship based on the relative rationale of human PN development in biology and the sporadic description in this aspect in nonprimate. 2. Human 3PN-zygote model constructed by intracytoplasmic 2-sperm injection (2sperm-ICSI) was developed to verify the recommend morphological criteria of human PN in kinship. The correlated rationale of this model in the verification mentioned above was based on facts that the PN kinship in a standard model of this kind was relatively valid, i.e. two male PNs and one female PN with two polar bodies (PB1 and PB2). 3. Eighty-nine mature oocytes (from the in vitro maturation of
originally-donated 108 morphologically normal corona-cumulus-denuded immature oocytes of 68 consecutive ovarian stimulation cycles for routine intracytoplasmic sperm injection) and 68 normal sperm suspension samples (donated by infertile couples for conventional in vitro fertilization) were used to construct human 3PN-zygote model mentioned above. 4. Verification of the effectiveness of recommended morphological criteria of human PN in kinship was conducted on the basis of firstly-validated PN kinship in human 3PN-zygote model. The firstly-validated morphological kinship criteria for male PN of human 3PN-zygote model were that the size and morphology of two male PNs should be mutually matched and uniformed and that the distance to PB2 should be relatively remote. The corresponding criteria for female PN were that the size and morphology of single female PN should be unmatched and ununiformed to the other two PNs and that the distance to PB2 should be relatively near.RESULTS: 1. The recommended morphological criteria of human PN in kinship were that: as to male PN, the morphological characteristics were relative bigger in size and remote to PB2;and as to female PN, the situation was vice versa, i.e. relative smaller in size and near to PB2. 2. The success rate for the construction of a standard 2sperm-ICSI 3PN-zygote model in human was 43.82% (39/89) ? 3. Among 117 PNs of 39 standard models of human 2sperm-ICSI 3PN-zygote, the kinships of 105 PNs (89.7%, including 70 male PNs and 35 female PN) of 35 models were completely
matched with those based on the recommended morphological criteria of human PN in kinship.CONCLUSIONS: 1. Human 2sperm-ICSI 3PN-zygote model developed for the first time in the present study, characteristic of simplicity, economy, feasibility, effectiveness and novelty, is a worthily-recommended model in the verification research on morphological criteria of human PN in kinship. 2. The morphological criteria of human PN in kinship recommended in the present study, provides, for the first time in live and natural state, simple, economic, practical and relatively reliable criteria of the corresponding aspect and makes possible the corresponding applications in research and the clinic.Part III: Study on pronucleus transfer in human zygotes and the subsequent preimplantation development of reconstructed embryosObjectives: 1. To develop a pronucleus transfer (PNT) mode, characteristic of simplicity, naturality, feasibility and effectiveness. 2. To evaluate the subsequent preimplantation development potential of corresponding reconstructed embryos.Methods: 1. Fifty-four human zygotes were donated from 34 consecutive infertile women (age: 29.7+4.2 yrs old) undergoing conventional in vitro
fertilization and embryo transfer (IVF-ET) during from Nov. 2004 to Jul. 2005. 2. Of the donated zygotes, 20 multiple pronucleus (PN) zygotes were conducted to receive a selective PN reduction (SPnR), a kind of micromanipulation to aspirate supernumerary PN or PNs from a multiple PN zygote and to make the zygote into its normal 2PN composition and sexual reproduction mode. The morphological kinship criteria for male PN of human zygotes used in SPnR were that the size was relative bigger and that the distance to PB2 was relatively remote. The corresponding criteria for female PN were that the size was relative smaller and that the distance to PB2 was relatively near. 3. Another 23 multiple PN zygotes of the same origin were used as blank control of SPnR (Control group). 4. The remaining 11 zygotes were divided into 6 groups (including a one-zygote one). Two zygotes in each group were used as a receiver or donor zygote respectively and PNT was conducted between them. As to the one-zygote group, autologous PNT was conducted. The real PNT included three major steps:firstly the SPnR on receiver zygote to make it into the one PN state;secondly the SPnR on donor zygote to get the needed donor PN according to the morphological criteria of human PN in kinship;and lastly the intracytoplasmic PN injection (ICPI) on the one PN receiver zygote prepared before to make it restore its originally normal 2PN state. 5. Routine embryonic culture to day 7 post fertilization on all zygotes, no
matter reconstructed or not. During embryo culture, embryonic developmental stage and quality observations were conducted every 24 hours. 6. Descriptive statistics in proportions were used to describe the duration and success rate for SPnR/ICPI, the embryo potential for preimplantation development. Chi-square tests were used to compare the difference of preimplantation embryonic development between the SPnR-reconstructed zygotes and the control ones.Results: 1. SPnR duration was 5—40 minutes (14.8+8.0 minutes), most(12/20, 60.0%) <10 minutes, majority (18/20, 90.0%) <20 minutes. SPnR success rate was 95% (19/20). 2. All four indices for the preimplantation development of SPnR-reconstructed zygotes (the cleavage rate, the rate of 8—cell embryonic development at day 3, the rate of good quality embryonic development at day 3 and the rate of blastocyst development at day 5) were 85.0%, 25.0%, 10.0% and 5.0% respectively, lower than those of the same origin of control group (91.3%, 34.8%, 13.0% and 17.4% respectively);but there was no significance in statistics (P>0.05). 3. Duration of PNT in SPnR/ICPI was 15-40 minutes (30.8+9.5 minutes), most (4/6, 66.7%) <30 minutes. 4. Among 6 times of ICPI, 3 succeeded and 3 failed. 5. Among 3 zygotes successfully reconstructed in SPnR/ICPI, two were not only alive, but also naturally cleaved and developed to morula on day 5 post fertilization, but not to blastocyst in the end. 6. Incomplete ICPI or real extracytoplasmic PN injection (ECPI) was the collective cause
for PNT failure of all other three zygotes in SPnR/ICPI.Conclusions: 1. The micromanipulation of moving PN out of human zygote, like SPnR on multiple PN zygotes, is feasible, practical and effective. There is no significant impact of SPnR on the preimplantation development of reconstructed embryos. It is worthy of studying further to develop SPnR in the clinic. 2. PNT of human zygotes in SPnR/ICPI is simple, feasible and effective. Most of the successfully-PNT zygotes are alive, cleave naturally and possess considerable potential for preimplantation development. 3. Incomplete ICPI or real ECPI is the main cause for PNT failure in SPnR/ICPI. 4. Further studies are needed for optimization.
引文
1. Hochedlinger K, Jaenisch R. Nuclear Transplantation, Embryonic Stem Cells, and the Potential for Cell Therapy [J]. N Engl J Med, 2003, 349(3):275-286.
2. Gurdon J B, Byrne J A, Simonsson S. Nuclear reprogramming and stem cell creation [J]. PNAS, 2003, 100(90001):11819-11822.
3. Mombaerts P. Therapeutic cloning in the mouse [J]. PNAS, 2003,100(90001):11924-11925.
4. Stojkovic M, Lako M, Strachan T, et al. Derivation, growth and applications of human embryonic stem cells [J]. Reproduction, 2004, 128(3):259-267.
5. Chen Y, He Z X, Liu A, et al. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes [J]. Cell Res, 2003, 13(4):251-63.
6. Hwang W S, Ryu Y J, Park J H, et al. Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst [J]. Science, 2004, 303(5664):1669-1674.
7. Hwang W S, Rob S I, Lee B C, et al. Patient-Specific Embryonic Stem Cells Derived From Human SCNT Blastocysts [J]. Obstet Gynecol Surv, 2005, 60(9):587-589.
8. Surani M A, Barton S C, Norris M L. Experimental reconstruction of mouse eggs and embryos: an analysis of mammalian development[J]. Biol Reprod, 1987, 36(1 ): 1-16.
9. Mann J R, Gadi I, Harbison M L, et al. Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting [J]. Cell, 1990, 62(2):251-60.
10. Szabo P, Mann J. Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines [J]. Development, 1994, 120(6):1651-1660.
11. Cibelli J B, Grant K A, Chapman K B, et al. Parthenogenetic stem cells in nonhuman primates [J]. Science, 2002, 295(5556):819.
12. Simerly C, Navara C, Hyun S H, et al. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction [J]. Dev Biol, 2004, 276(2):237-52.
13. Jaenisch R. Human Cloning-The Science and Ethics of Nuclear Transplantation [J]. N Engl J Med, 2004, 351(27):2787-2791.
14. Gurdon J B, Byrne J A. The first half-century of nuclear transplantation [J]. PNAS, 2003,100(14):8048-8052.
15. Mitalipov S M, Yeoman R R, Nusser K D, et al. Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells [J]. Biol Reprod, 2002, 66(5):1367-1373.
16. Simerly C, Dominko T, Navara C, et al. Molecular correlates of primate nuclear transfer failures [J]. Science, 2003,300(5617):297.
17. Takeuchi T, Neri Q V, Palermo G D. Construction and fertilization of reconstituted human oocytes [J]. Reprod Biomed Online, 2005,11(3):309-18.
18. Loebel D A, Tam P P. Genomic imprinting: mice without a father [J]. Nature, 2004, 428(6985):809-11.
19. Reik W, Walter J. Genomic imprinting: parental influence on the genome [J]. Nat Rev Genet, 2001,2(1 ):21-32.
20. Surani M A. Reprogramming of genome function through epigenetic inheritance [J]. Nature, 2001,414(6859):122-8.
21. Morgan H D, Santos F, Green K, et al. Epigenetic reprogramming in mammals [J]. Hum. Mol. Genet., 2005,14(suppl_1):R47-58.
22. Morison I M, Ramsay J P, Spencer H G. A census of mammalian imprinting [J]. Trends in Genetics, 2005,21(8):457-465.
23. Krawetz S A. Paternal contribution: new insights and future challenges [J]. Nat Rev Genet, 2005,6(8):63342.
24. Kono T, Obata Y, Wu Q, et al. Birth of parthenogenetic mice that can develop to adulthood [J]. Nature, 2004,428(6985):860-4.
25. Mikkelsen A L. Strategies in human in-vitro maturation and their clinical outcome [J]. Reprod Biomed Online, 2005,10(5):593-9.
26. Cha K Y, Chian R C. Maturation in vitro of immature human oocytes for clinical use [J]. Hum Reprod Update, 1998,4(2): 103-20.
27. Combelles C M H, Fissore R A, Albertini D F, et al. In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system [J]. Hum. Reprod., 2005,20(5):1349-1358.
28. Chian R C, Tan S L. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles [J]. Reprod Biomed Online, 2002,5(2): 125-32.
29. Goud P T, Goud A P, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium [J]. Hum Reprod, 1998,13(6):1638-44.
30. Hwang J L, Lin Y H, Tsai Y L. In vitro maturation and fertilization of immature oocytes: a comparative study of fertilization techniques [J]. J Assist Reprod Genet, 2000, 17(1):39-43.
31. Janssenswillen C, Nagy Z P, Van Steirteghem A. Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells [J]. Hum Reprod, 1995,10(2):375-8.
32. Cha K Y, Koo J J, Ko J J, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program [J]. Fertil Steril, 1991, 55(1):109-113.
33. Liu J Y, Qian Y, Mao Y D, et al. [In vitro maturation, fertilization and embryo transfer of human immature oocyte] [J]. Zhonghua Fu Chan Ke Za Zhi, 2003, 38(4):230-2.
34. Picton H M, Danfour M A, Harris S E, et al. Growth and maturation of oocytes in vitro [J]. Reprod Suppl, 2003,61:445-62.
35. Moor R M, Dai Y, Lee C, et al. Oocyte maturation and embryonic failure [J]. Hum Reprod Update, 1998,4(3):223-36.
36. Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence [J]. Reproduction, 2001,121(1):51-75.
37. Sutton M L, Gilchrist R B, Thompson J G. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity [J]. Hum Reprod Update, 2003,9(1):35-48.
38. Artini P G, Battaglia C, D'Ambrogio G, et al. Relationship between human oocyte maturity, fertilization and follicular fluid growth factors [J]. Hum Reprod, 1994, 9(5):902-906.
39. Malamitsi-Puchner A, Sarandakou A, Baka S G, et al. Concentrations of angiogenic factors in follicular fluid and oocyte-cumulus complex culture medium from women undergoing in vitro fertilization: association with oocyte maturity and fertilization [J]. Fertil Steril, 2001,76(1 ):98-101.
40. Palan P R, Cohen B L, Barad D H, et al. Effects of smoking on the levels of antioxidant beta carotene, alpha tocopherol and retinol in human ovarian follicular fluid [J]. Gynecol Obstet Invest, 1995, 39(1 ):43-46.
41. Chi H J, Kim D H, Koo J J, et al. The suitability and efficiency of human follicular fluid as a protein supplement in human in vitro fertilization programs [J]. Fertil Steril, 1998, 70(5):871-7.
42. Haberle M, Scheurer P, Lauerer K, et al. Are cumulus cells necessary for the spontaneous maturation of germinal vesicle-stage oocytes to metaphase II [J]. J Assist
Reprod Genet, 1999,16(6):329-31.
43. Fabbri R, Porcu E, Marsella T, et al. Human embryo development and pregnancies in an homologous granulosa cell coculture system [J]. J Assist Reprod Genet, 2000,17(1):1-12
44. Cekleniak N A, Combelles C M H, Ganz D A, et al. A novel system for in vitro maturation of human oocytes [J]. Fertility and Sterility, 2001,75(6):1185-1193.
45. Combelles C M, Cekleniak N A, Racowsky C, et al. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes [J]. Hum Reprod, 2002, 17(4):1006-16.
46. Veeck L. An Atlas of Human Gametes and Conceptuses [M]. New York:Parthenon, 1998:40-45.
47. Huang F-J, Lan K-C, Kung F-T, et al. Human cumulus-free oocyte maturational profile and in vitro developmental potential after stimulation with recombinant versus urinary FSH [J]. Hum Reprod, 2004,19(2):306-315.
48. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte [J]. Lancet, 1992,340(8810): 17-8.
49. WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction [M]. 4th ed:WHO, 1999.
50. Morris R S, Paulson R J, Sauer M V, et al. Predictive value of serum oestradiol concentrations and oocyte number in severe ovarian hyperstimulation syndrome [J]. Hum Reprod, 1995,10(4):811-4.
51. Asch R, Li H, Balmaceda J, et al. Severe ovarian hyperstimulation syndrome in assisted reproductive technology: definition of high risk groups [J]. Hum. Reprod., 1991, 6(10):1395-1399.
52. Orvieto R. Prediction of ovarian hyperstimulation syndrome: Challenging the estradiol mythos [J]. Hum. Reprod., 2003,18(4):665-667.
53. Orvieto R. Can we eliminate severe ovarian hyperstimulation syndrome? [J]. Hum. Reprod., 2005,20(2):320-322.
54. Nikolettos N, Al-Hasani S, Felberbaum R, et al. Gonadotropin-releasing hormone antagonist protocol: a novel method of ovarian stimulation in poor responders [J]. Eur J Obstet Gynecol Reprod Biol, 2001,97(2):202-7.
55. Ubaldi F M, Rienzi L, Ferrero S, et al. Management of poor responders in IVF [J]. Reprod Biomed Online, 2005,10(2):235-46.
56. Tarlatzis B C, Zepiridis L, Grimbizis G, et al. Clinical management of low ovarian response to stimulation for IVF: a systematic review [J]. Hum Reprod Update, 2003, 9(1):61-76.
57. Rowe P J, Comhaire F H, Hargreave T B, et al. WHO manual for the standardized investigation, diagnosis and management of the infertile male [M]. Cambridge:WHO, 2000.
58. De Vos A, Van de Velde H, Joris H, et al. In-vitro matured metaphase-l oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-ll oocytes after intracytoplasmic sperm injection [J]. Hum Reprod, 1999,14(7):1859-63.
59. Strassburger D, Friedler S, Raziel A, et al. The outcome of ICSI of immature Ml oocytes and rescued in vitro matured Mll oocytes [J]. Hum. Reprod., 2004,19(7):1587-1590.
60. Kim B-K, Lee S-C, Kim K-J, et al. In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles [J]. Fertil Steril, 2000, 74(6): 1153-1158.
61. Mrazek M, Fulka Jr J, Jr. Failure of oocyte maturation: possible mechanisms for oocyte maturation arrest [J]. Hum Reprod, 2003,18(11):2249-52.
62. Combelles C M, Albertini D F, Racowsky C. Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation [J]. Hum Reprod, 2003,18(10):2124-30.
63. Gormel V G, Leung P C K. In Vitro Fertilization and Assisted Reproduction [M]. Bologna: Monduzzi, 1997:315-319.
64. Carrell D T, Peterson C M, Jones K P, et al. A simplified coculture system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization [J]. J Assist Reprod Genet, 1999, 16(7):344-9.
65. Joo B S, Kim M K, Na Y J, et al. The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components [J]. Fertil Steril, 2001,75(1): 193-9.
66. Haseltine F P, First N L. Meiotic Inhibition, Molecular Control of Meiosis: Progress in Clinical and Biological Research [M]. New York: Alan, R.Liss, 1988:87-101.
67. Dandekar P V, Martin M C, Glass R H. Maturation of immature oocytes by coculture with granulosa cells [J]. Fertil Steril, 1991,55(1 ):95-9.
68. Schramm R D, Bavister B D. Granulosa cells from follicle stimulating hormone-primed monkeys enhance the development competence of in-vitro-matured oocytes from non-stimulated rhesus monkeys [J]. Hum Reprod, 1996,11(8):1698-702.
69. Egozcue S, Blanco J, Vidal F, et al. Diploid sperm and the origin of triploidy [J]. Hum. Reprod., 2002,17(1):5-7.
70. Zaragoza M V, Surti U, Redline R W, et al. Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole [J]. Am J Hum Genet, 2000,66(6): 1807-20.
71. Rosenbusch B, Schneider M. Hypotriploid tripronuclear oocytes with two polar bodies obtained after ICSI: is irregular chromatid segregation involved? [J]. Hum. Reprod., 2000, 15(8):1876-1877.
72. Rosenbusch B, Schneider M, Kreienberg R, et al. Cytogenetic analysis of human zygotes displaying three pronuclei and one polar body after intracytoplasmic sperm injection [J]. Hum. Reprod., 2001,16(11):2362-2367.
73. Surani M A, Barton S C. Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos [J]. Science, 1983,222(4627): 1034-6.
74. Liu H, Zhang J, Krey L C, et al. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei [J]. Hum Reprod, 2000,15(9):1997-2002.
75. Miller D J, Gong X, Decker G, et al. Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy [J]. J Cell Biol, 1993, 123(6 Pt 1):1431-40.
76. Rosenbusch B, Schneider M, Glaser B, et al. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy [J]. Hum. Reprod., 2002,17(9):2388-2393.
77. Macas E, Imthurn B, Keller P J. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI [J]. Hum. Reprod., 2001,16(1): 115-120.
78. McGrath J, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion [J]. Science, 1983,220(4603): 1300-2.
79. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes [J]. Cell, 1984,37(1): 179-83.
80. Surani M A, Barton S C, Norris M L. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis [J]. Nature, 1984, 308(5959):548-50.
81. Surani M A, Barton S C, Norris M L. Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome [J]. Cell, 1986,45(1):127-36.
82. McGrath J, Solter D. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro [J]. Science, 1984,226(4680): 1317-9.
83. Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals [J]. Reprod Biomed Online, 2005, 10(2):247-88.
84. Lee J W, Wu S C, Tian X C, et al. Production of cloned pigs by whole-cell intracytoplasmic microinjection [J]. Biol Reprod, 2003,69(3):995-1001.
85. Egozcue S, Blanco J, Vidal F, et al. Diploid sperm and the origin of triploidy [J]. Hum Reprod, 2002,17(1):5-7.
86. Macas E, Imthurn B, Rosselli M, et al. The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection [J]. Hum. Reprod., 1996,11(11):2496-2501.
87. Palermo G D, Munne S, Colombero L T, et al. Genetics of abnormal human fertilization [J]. Hum Reprod, 1995,10 Suppl 1:120-7.
88. Kaufman M H. New insights into triploidy and tetraploidy, from an analysis of model systems for these conditions [J]. Hum Reprod, 1991,6(1):8-16.
89. Wakayama T, Tateno H, Mombaerts P, et al. Nuclear transfer into mouse zygotes [J ].Nat Genet, 2000,24(2): 108-9.
2. Gurdon J B, Byrne J A, Simonsson S. Nuclear reprogramming and stem cell creation [J]. PNAS, 2003, 100(90001):11819-11822.
3. Mombaerts P. Therapeutic cloning in the mouse [J]. PNAS, 2003,100(90001):11924-11925.
4. Stojkovic M, Lako M, Strachan T, et al. Derivation, growth and applications of human embryonic stem cells [J]. Reproduction, 2004, 128(3):259-267.
5. Chen Y, He Z X, Liu A, et al. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes [J]. Cell Res, 2003, 13(4):251-63.
6. Hwang W S, Ryu Y J, Park J H, et al. Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst [J]. Science, 2004, 303(5664):1669-1674.
7. Hwang W S, Rob S I, Lee B C, et al. Patient-Specific Embryonic Stem Cells Derived From Human SCNT Blastocysts [J]. Obstet Gynecol Surv, 2005, 60(9):587-589.
8. Surani M A, Barton S C, Norris M L. Experimental reconstruction of mouse eggs and embryos: an analysis of mammalian development[J]. Biol Reprod, 1987, 36(1 ): 1-16.
9. Mann J R, Gadi I, Harbison M L, et al. Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting [J]. Cell, 1990, 62(2):251-60.
10. Szabo P, Mann J. Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines [J]. Development, 1994, 120(6):1651-1660.
11. Cibelli J B, Grant K A, Chapman K B, et al. Parthenogenetic stem cells in nonhuman primates [J]. Science, 2002, 295(5556):819.
12. Simerly C, Navara C, Hyun S H, et al. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction [J]. Dev Biol, 2004, 276(2):237-52.
13. Jaenisch R. Human Cloning-The Science and Ethics of Nuclear Transplantation [J]. N Engl J Med, 2004, 351(27):2787-2791.
14. Gurdon J B, Byrne J A. The first half-century of nuclear transplantation [J]. PNAS, 2003,100(14):8048-8052.
15. Mitalipov S M, Yeoman R R, Nusser K D, et al. Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells [J]. Biol Reprod, 2002, 66(5):1367-1373.
16. Simerly C, Dominko T, Navara C, et al. Molecular correlates of primate nuclear transfer failures [J]. Science, 2003,300(5617):297.
17. Takeuchi T, Neri Q V, Palermo G D. Construction and fertilization of reconstituted human oocytes [J]. Reprod Biomed Online, 2005,11(3):309-18.
18. Loebel D A, Tam P P. Genomic imprinting: mice without a father [J]. Nature, 2004, 428(6985):809-11.
19. Reik W, Walter J. Genomic imprinting: parental influence on the genome [J]. Nat Rev Genet, 2001,2(1 ):21-32.
20. Surani M A. Reprogramming of genome function through epigenetic inheritance [J]. Nature, 2001,414(6859):122-8.
21. Morgan H D, Santos F, Green K, et al. Epigenetic reprogramming in mammals [J]. Hum. Mol. Genet., 2005,14(suppl_1):R47-58.
22. Morison I M, Ramsay J P, Spencer H G. A census of mammalian imprinting [J]. Trends in Genetics, 2005,21(8):457-465.
23. Krawetz S A. Paternal contribution: new insights and future challenges [J]. Nat Rev Genet, 2005,6(8):63342.
24. Kono T, Obata Y, Wu Q, et al. Birth of parthenogenetic mice that can develop to adulthood [J]. Nature, 2004,428(6985):860-4.
25. Mikkelsen A L. Strategies in human in-vitro maturation and their clinical outcome [J]. Reprod Biomed Online, 2005,10(5):593-9.
26. Cha K Y, Chian R C. Maturation in vitro of immature human oocytes for clinical use [J]. Hum Reprod Update, 1998,4(2): 103-20.
27. Combelles C M H, Fissore R A, Albertini D F, et al. In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system [J]. Hum. Reprod., 2005,20(5):1349-1358.
28. Chian R C, Tan S L. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles [J]. Reprod Biomed Online, 2002,5(2): 125-32.
29. Goud P T, Goud A P, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium [J]. Hum Reprod, 1998,13(6):1638-44.
30. Hwang J L, Lin Y H, Tsai Y L. In vitro maturation and fertilization of immature oocytes: a comparative study of fertilization techniques [J]. J Assist Reprod Genet, 2000, 17(1):39-43.
31. Janssenswillen C, Nagy Z P, Van Steirteghem A. Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells [J]. Hum Reprod, 1995,10(2):375-8.
32. Cha K Y, Koo J J, Ko J J, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program [J]. Fertil Steril, 1991, 55(1):109-113.
33. Liu J Y, Qian Y, Mao Y D, et al. [In vitro maturation, fertilization and embryo transfer of human immature oocyte] [J]. Zhonghua Fu Chan Ke Za Zhi, 2003, 38(4):230-2.
34. Picton H M, Danfour M A, Harris S E, et al. Growth and maturation of oocytes in vitro [J]. Reprod Suppl, 2003,61:445-62.
35. Moor R M, Dai Y, Lee C, et al. Oocyte maturation and embryonic failure [J]. Hum Reprod Update, 1998,4(3):223-36.
36. Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence [J]. Reproduction, 2001,121(1):51-75.
37. Sutton M L, Gilchrist R B, Thompson J G. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity [J]. Hum Reprod Update, 2003,9(1):35-48.
38. Artini P G, Battaglia C, D'Ambrogio G, et al. Relationship between human oocyte maturity, fertilization and follicular fluid growth factors [J]. Hum Reprod, 1994, 9(5):902-906.
39. Malamitsi-Puchner A, Sarandakou A, Baka S G, et al. Concentrations of angiogenic factors in follicular fluid and oocyte-cumulus complex culture medium from women undergoing in vitro fertilization: association with oocyte maturity and fertilization [J]. Fertil Steril, 2001,76(1 ):98-101.
40. Palan P R, Cohen B L, Barad D H, et al. Effects of smoking on the levels of antioxidant beta carotene, alpha tocopherol and retinol in human ovarian follicular fluid [J]. Gynecol Obstet Invest, 1995, 39(1 ):43-46.
41. Chi H J, Kim D H, Koo J J, et al. The suitability and efficiency of human follicular fluid as a protein supplement in human in vitro fertilization programs [J]. Fertil Steril, 1998, 70(5):871-7.
42. Haberle M, Scheurer P, Lauerer K, et al. Are cumulus cells necessary for the spontaneous maturation of germinal vesicle-stage oocytes to metaphase II [J]. J Assist
Reprod Genet, 1999,16(6):329-31.
43. Fabbri R, Porcu E, Marsella T, et al. Human embryo development and pregnancies in an homologous granulosa cell coculture system [J]. J Assist Reprod Genet, 2000,17(1):1-12
44. Cekleniak N A, Combelles C M H, Ganz D A, et al. A novel system for in vitro maturation of human oocytes [J]. Fertility and Sterility, 2001,75(6):1185-1193.
45. Combelles C M, Cekleniak N A, Racowsky C, et al. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes [J]. Hum Reprod, 2002, 17(4):1006-16.
46. Veeck L. An Atlas of Human Gametes and Conceptuses [M]. New York:Parthenon, 1998:40-45.
47. Huang F-J, Lan K-C, Kung F-T, et al. Human cumulus-free oocyte maturational profile and in vitro developmental potential after stimulation with recombinant versus urinary FSH [J]. Hum Reprod, 2004,19(2):306-315.
48. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte [J]. Lancet, 1992,340(8810): 17-8.
49. WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction [M]. 4th ed:WHO, 1999.
50. Morris R S, Paulson R J, Sauer M V, et al. Predictive value of serum oestradiol concentrations and oocyte number in severe ovarian hyperstimulation syndrome [J]. Hum Reprod, 1995,10(4):811-4.
51. Asch R, Li H, Balmaceda J, et al. Severe ovarian hyperstimulation syndrome in assisted reproductive technology: definition of high risk groups [J]. Hum. Reprod., 1991, 6(10):1395-1399.
52. Orvieto R. Prediction of ovarian hyperstimulation syndrome: Challenging the estradiol mythos [J]. Hum. Reprod., 2003,18(4):665-667.
53. Orvieto R. Can we eliminate severe ovarian hyperstimulation syndrome? [J]. Hum. Reprod., 2005,20(2):320-322.
54. Nikolettos N, Al-Hasani S, Felberbaum R, et al. Gonadotropin-releasing hormone antagonist protocol: a novel method of ovarian stimulation in poor responders [J]. Eur J Obstet Gynecol Reprod Biol, 2001,97(2):202-7.
55. Ubaldi F M, Rienzi L, Ferrero S, et al. Management of poor responders in IVF [J]. Reprod Biomed Online, 2005,10(2):235-46.
56. Tarlatzis B C, Zepiridis L, Grimbizis G, et al. Clinical management of low ovarian response to stimulation for IVF: a systematic review [J]. Hum Reprod Update, 2003, 9(1):61-76.
57. Rowe P J, Comhaire F H, Hargreave T B, et al. WHO manual for the standardized investigation, diagnosis and management of the infertile male [M]. Cambridge:WHO, 2000.
58. De Vos A, Van de Velde H, Joris H, et al. In-vitro matured metaphase-l oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-ll oocytes after intracytoplasmic sperm injection [J]. Hum Reprod, 1999,14(7):1859-63.
59. Strassburger D, Friedler S, Raziel A, et al. The outcome of ICSI of immature Ml oocytes and rescued in vitro matured Mll oocytes [J]. Hum. Reprod., 2004,19(7):1587-1590.
60. Kim B-K, Lee S-C, Kim K-J, et al. In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles [J]. Fertil Steril, 2000, 74(6): 1153-1158.
61. Mrazek M, Fulka Jr J, Jr. Failure of oocyte maturation: possible mechanisms for oocyte maturation arrest [J]. Hum Reprod, 2003,18(11):2249-52.
62. Combelles C M, Albertini D F, Racowsky C. Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation [J]. Hum Reprod, 2003,18(10):2124-30.
63. Gormel V G, Leung P C K. In Vitro Fertilization and Assisted Reproduction [M]. Bologna: Monduzzi, 1997:315-319.
64. Carrell D T, Peterson C M, Jones K P, et al. A simplified coculture system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization [J]. J Assist Reprod Genet, 1999, 16(7):344-9.
65. Joo B S, Kim M K, Na Y J, et al. The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components [J]. Fertil Steril, 2001,75(1): 193-9.
66. Haseltine F P, First N L. Meiotic Inhibition, Molecular Control of Meiosis: Progress in Clinical and Biological Research [M]. New York: Alan, R.Liss, 1988:87-101.
67. Dandekar P V, Martin M C, Glass R H. Maturation of immature oocytes by coculture with granulosa cells [J]. Fertil Steril, 1991,55(1 ):95-9.
68. Schramm R D, Bavister B D. Granulosa cells from follicle stimulating hormone-primed monkeys enhance the development competence of in-vitro-matured oocytes from non-stimulated rhesus monkeys [J]. Hum Reprod, 1996,11(8):1698-702.
69. Egozcue S, Blanco J, Vidal F, et al. Diploid sperm and the origin of triploidy [J]. Hum. Reprod., 2002,17(1):5-7.
70. Zaragoza M V, Surti U, Redline R W, et al. Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole [J]. Am J Hum Genet, 2000,66(6): 1807-20.
71. Rosenbusch B, Schneider M. Hypotriploid tripronuclear oocytes with two polar bodies obtained after ICSI: is irregular chromatid segregation involved? [J]. Hum. Reprod., 2000, 15(8):1876-1877.
72. Rosenbusch B, Schneider M, Kreienberg R, et al. Cytogenetic analysis of human zygotes displaying three pronuclei and one polar body after intracytoplasmic sperm injection [J]. Hum. Reprod., 2001,16(11):2362-2367.
73. Surani M A, Barton S C. Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos [J]. Science, 1983,222(4627): 1034-6.
74. Liu H, Zhang J, Krey L C, et al. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei [J]. Hum Reprod, 2000,15(9):1997-2002.
75. Miller D J, Gong X, Decker G, et al. Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy [J]. J Cell Biol, 1993, 123(6 Pt 1):1431-40.
76. Rosenbusch B, Schneider M, Glaser B, et al. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy [J]. Hum. Reprod., 2002,17(9):2388-2393.
77. Macas E, Imthurn B, Keller P J. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI [J]. Hum. Reprod., 2001,16(1): 115-120.
78. McGrath J, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion [J]. Science, 1983,220(4603): 1300-2.
79. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes [J]. Cell, 1984,37(1): 179-83.
80. Surani M A, Barton S C, Norris M L. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis [J]. Nature, 1984, 308(5959):548-50.
81. Surani M A, Barton S C, Norris M L. Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome [J]. Cell, 1986,45(1):127-36.
82. McGrath J, Solter D. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro [J]. Science, 1984,226(4680): 1317-9.
83. Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals [J]. Reprod Biomed Online, 2005, 10(2):247-88.
84. Lee J W, Wu S C, Tian X C, et al. Production of cloned pigs by whole-cell intracytoplasmic microinjection [J]. Biol Reprod, 2003,69(3):995-1001.
85. Egozcue S, Blanco J, Vidal F, et al. Diploid sperm and the origin of triploidy [J]. Hum Reprod, 2002,17(1):5-7.
86. Macas E, Imthurn B, Rosselli M, et al. The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection [J]. Hum. Reprod., 1996,11(11):2496-2501.
87. Palermo G D, Munne S, Colombero L T, et al. Genetics of abnormal human fertilization [J]. Hum Reprod, 1995,10 Suppl 1:120-7.
88. Kaufman M H. New insights into triploidy and tetraploidy, from an analysis of model systems for these conditions [J]. Hum Reprod, 1991,6(1):8-16.
89. Wakayama T, Tateno H, Mombaerts P, et al. Nuclear transfer into mouse zygotes [J ].Nat Genet, 2000,24(2): 108-9.