Strategy Study On Screening Normal Good-quality Embryos From Human Zygotes With Different Pronuclear Numbers

Part one:Objective By pronuclear grading for zygotes with normal pronuclear numbers, it is aimed to select normal good-quality embryos for transfer in clinical IVF (in-vitro fertilization)/ICSI (intra-cytoplasmic sperm injection). Methods In this retrospective study, a total of 2714 normal zygotes (2 pronuclei) from 434 treatment cycles with IVF/ICSI were analyzed between May 2003 and December 2003. These zygotes were divided into two groups (synchronous group and non-synchronous group) according to the developmental synchrony of pronuclei. The developmental potential of zygotes from these two groups was observed. Embryos for transfer were selected initially by embryo growth rate and morphology, secondarily by zygote grade. The clinical pregnancy rates and implantation rates were compared according to whether the embryos from synchronous group were transferred. Results There were 2714 normal zygotes, the good-quality embryos (≥6 cells, gradeⅠ-Ⅱ) rate of the synchronous group (41.9%,743/1774) was significantly higher (P< 0.001) than the non-synchronous group (34.0%,319/940). Totally 395 cycles transferred at least one embryo from synchronous group, the clinical pregnancy rate was 47.9%(189/395) and implantation rate was 27.5%(273/993). There was no significant difference from 39 cycles which did not transfer embryos from non-synchronous group. The clinical pregnancy rate was 43.6%(17/39) and implantation rate was 25.0% (21/84). Conclusions The data indicate that pronuclear grading can effectively predict the developmental potential and quality of embryos, but there are no significant differences in pregnancy rates and implantation rates between different zygote grades when selecting embryos for transfer by combined grading.Part two:Objective By comparing the chromosomal constitution among the arrested cleavage-stage embryos, blastocysts and human embryonic stem cells (hESCs) which are all derived from monopronuclear (1PN) zygotes, it is aimed to determine whether chromosomally normal embryos can be reliably selected by blastocyst culture. Methods After 1PN zygotes being sequentially cultured for 5 days, the blastocysts and arrested cleavage-stage embryos were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosomes 18, X and Y; G-banding analysis was adopted to analyze the karyotype of 1PN hESCs. Results The diploid rate of blastocysts was 74.6%, which was significantly (P< 0.001) higher than that of arrested cleavage-stage embryos (31.6%), and the diploid rate of hESCs was 97.0%, which was significantly (P< 0.01) higher than that of blastocysts; the haploid embryos were excluded by blastocyst culture; nevertheless, there still existed such chromosomal abnormalities as mosaic and monosomic in blastocysts and trisomic in hESCs. Conclusions Blastocyst culture is an effective method to select against chromosomal abnormalities, especially the haploid in 1PN embryos; however, developing to the blastocyst stage is not a reliable marker against the mosaic or aneuploid.Part three:Objective It is aimed to determine whether chromosomally normal embryos can be reliably selected by the combination of pronuclear removal and blastocyst culture by comparing the chromosomal constitution between the embryos from the microsurgical corrected tripronuclear (3PN) zygotes and intact 3PN zygotes, and make an attempt on deriving human embryonic stem cells (hESCs) from the microsurgical corrected blastocysts. Methods After being sequentially cultured for 5-6 days, the trophoblast of blastocysts and the arrested cleavage-stage embryos developing from microsurgical corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosome 17, X and Y. Intact 3PN from clinical IVF cycles were blastocyst cultured as the control group. The inner cell mass (ICM) of blastocysts developing from the microsurgical corrected 3PN zygotes were used to derive hESC lines in which the characteristics of stem cells were tested. G-banding analysis was adopted to identify the karyotype of hESC lines, and the heteroparental inheritance of hESC line was analyzed by DNA fingerprinting analysis. Results The blastocyst formation rate (13.5%) of the microsurgical corrected 3PN zygotes was significantly higher (P< 0.05) than that of intact 3PN zygotes (8.7%). The triploid rate of the microsurgical corrected 3PN embryos (5.7%) were significantly lower (P <0.01) than that of intact 3PN embryos (19.4%). The diploid rate of blastocysts (55.0%) was significantly higher (P< 0.05) than that of arrested cleavage-stage embryos (18.4%) from the microsurgical corrected 3PN zygotes. Furthermore, we established the first one heteroparental normal karyotypic (46, XX) hESC line from the microsurgical corrected 3PN zygotes. Conclusions pronuclear removal can effectively remove the surplus chromosome set and improve the developmental potential of 3PN zygotes. Combination of pronuclear removal and blastocyst culture can availably pick out the diploidized blastocysts which can derive heteroparental normal karyotypic hESC lines.