| 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 developmentObjectives: 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 ofnuclear 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 derivedfrom 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 rateof 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 oforiginally-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 completelymatched 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 vitrofertilization 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, nomatter 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 causefor 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.
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