| Background and Objective In the procedure of in vitro fertilization and embryo transfer of human,embryologists always check the zygotes at 16-18 h post-insemination. In the normal condition, two pronuclei and two polar body are present at the fertilization check.Bi-pronuclear zygotes with good morphology patterns are selected for further culture because these zygotes have the highest development potential. Meanwhile, a few zygotes present no, one, or more than two pronuclei at the check time. The non-,mono-, tri- or poly-pronuclear zygotes are classified as abnormal zygotes, which are discarded. However, previous reports have shown that non-pronuclear zygotes and mono-pronuclear zygotes can be fertilizated normal just because we missed the appropriate check time. It will be unfortunate if we discard these zygotes or embryos derived from them. A few number of reports have shown that non-pronuclear oocytes and mono-pronuclear oocytes can give rise to healthy infants.In in-vitro fertilization(IVF), to select embryos with the highest implantation potential for transfer is crucial.With the progress of genetic diagnosis methods,preimplantation genetic diagnosis improved a lot in recent years. Nowadays,preimplantation genetic diagnosis(PGD) has became an important aspect of assisted reproduction technology. Continuous appearances and applications of new methods and technologies, such as fluorescence in situ hybridization, array-comparative genome hybridization, single nucleotide polymorphism microarray and next-generation sequencing amplification in PGD. The array-comparative genome hybridization shows its obvious advantage in high sensitivity, accuracy and flexibility,which has become a powerful detection means in PGD.We study the mono-pronuclear zygotes and nonopronuclear zygotes of 299 patients in ICSI cycles and 788 patients in IVF cycles at 16-18 h post-insemination,recording pronuclear, cleavage as well as blastocyst to investigate the developmental potential and the influencing factor of these abnormal zygotes. Meanwhile, the pronuclear zygote morphology and cleavage of 788 IVF cycles were evaluated, which subsequently cultured to day 6. Then the chromosomal constitution of blastocysts derived from non-pronuclear and mono-pronuclear was checked to find out a good method to select the highest development potential embryos exhibiting non-pronuclear and mono-pronuclear at the fertilization check.Methods This prospective study was supported by the Ethics Committee of Zhengzhou University. A total of 788 IVF and 298 ICSI fresh cycles in the Reproductive Medicine Center of People’s Hospital of Zhengzhou University from July 2014 to October 2014 were enrolled in this study. We study the mono-pronuclear zygotes and nonopronuclear zygotes at 16-18 h post-insemination, recording pronuclear, cleavage as well as blastocyst, and cultured into blastocyst by the sequential method to confirm the developmental potential of these abnormal zygotes. Meanwhile, data of the patients were retrospectively analyzed to investigate the factors affecting embryos derived from monopronucleated zygotes or two polar bodies zygotes in in vitrofertilization and intracytoplasmic sperm injection cycles. It provides guidance on how to decrease the incidence of abnormal zygotes and improve the utilization rate of oocytes. The pronuclear zygote morphology and cleavage of 788 IVF cycles were evaluated, which subsequently cultured to day 6. Then the chromosomal constitution of blastocysts derived from non-pronuclear and mono-pronuclear was checked by trophectoderm cell biopsy and array-comparative genome hybridization to find out a good method to select the highest development potential embryos exhibiting non-pronuclear and mono-pronuclear at the fertilization check.Analyses were performed using the Statistical Package for Social Sciences version 17.0. Frequencies and proportions were reported for categorical outcomes,and means and standard deviations or median(min-max) were reported for continuous outcomes. One-way ANOVA, Kruskal-Wallis tests or c2 tests were used to ascertain the significant differences among groups, as appropriate. P <0.05 was considered as statistically significant.Results1. The development potential of embryos derived from non- and mono-pronuclear zygotes compared with the embryos come from bi-pronuclear zygotes Both non- and mono-pronuclear zygotes can cleavage and form blastocysts eventually. However, the cleavage rate(90.16% and 88.11%, respectively) and blastocyst formation rate(17.09% and 30.03%) were significantly lower than bi-pronuclear zygotes(98.40% and 45.71%, respectively)(both P < 0.05).It telled that maybe the embryos derived from non- and mono-pronuclear zygotes have certain development potential, but lower than that of bi-pronuclear embryos.2. The development of embryos derived from non- and mono-pronuclear zygotes of women at different age Compared with woman older than 35 years, the cleavage rate of non- and bi-pronuclear zygotes and the incidence of non-pronuclear zygotes were slightly higher than women who are 30 to 35 years old while the differences of the incidenceof bi- and mono-pronuclear zygotes and the cleavage rate of mono-pronuclear zygotes were not significant. However, for the women who is younger than 30 years old, only the cleavage rate of non-pronuclear zygotes were significantly higher than the advanced(P<0.05)。3. The development of embryos derived from non- and mono-pronuclear zygotes in IVF cycles and ICSI cycles In IVF cycles, the incidence of non- and mono-pronuclear zygotes were slightly higher than those of ICSI cycles(3.65% vs. 3.01%,P<0.05; 4.93% vs. 1.07%,P<0.05), while the cleavage rate(90.16% vs. 92.96%,P>0.05;88.11% vs. 84.0%,P>0.05) and blastocyst formation rate(17.09% vs. 7.58%,P>0.05;30.03%vs.14.29%,P>0.05)of non- and mono-pronuclear zygotes had no differences.4. The development of embryos derived from non- and mono-pronuclear zygotes in different number of retrieved oocytes The demarcation points of retrieved oocytes are 5,10,15,20. The incidence of non- and mono-pronuclear zygotes and cleavage of mono-pronuclear zygotes are similiar. The incidence, cleavage rate, high quality embryo obtained rate of bi-pronuclear zygotes and cleavage rate of mono-pronuclear zygotes were significant difference(P < 0.05) among different groups.5. To analysis the influencing factors on the formation of non- and mono-pronuclear zygotes using Logistic regression analysis Multivariate analysis showed that the insemination method, M Ⅱ oocytes,bi-pronuclear zygotes have related to the formation of non- and mono-pronuclear zygotes(P<0.05), while the maternal age, body mass index, basal hormone level and the number of oocytes obtained has nothing to do with the formation of these abnormal zygotes(P<0.05). In IVF cycles, it is likely to appearance non-pronuclear zygotes than mono-pronuclear zygotes while in ICSI cycles it reverse.6. Comparison of the percentage of blastocysts with normal chromosome constitution among non-/mono-pronuclear zygotes groups and bi-pronuclear blastocysts The comparative genome hybridization array(a CGH) results showed that 64.71%and 50.0% of the blastocysts derived from non- and mono-pronuclear zygotes,respectively, had normal chromosomal compositions. They were lower than that of blastocysts from bi-pronuclear zygotes(69.39%),and the difference was statistically significant(P<0.05). The percentage of non-pronuclear blastocysts with normal chromosomal composition was slightly higher than that of mono-pronuclear blastocysts,but there was no statistical difference.7. The incidence of blastocysts with normal chromosome constitution on D5 and D6We also found that the percentages of D6 blastocysts with a normal chromosome constitution were higher than that of D5 blastocysts for non- and mono-pronuclear zygotes, the differences of mono-pronuclear zygotes were significant(72.22% vs.37.50 %, P<0.05) while the differences of mono-pronuclear zygotes were not significant(71.79% vs. 55.17%, P>0.05). The percentage of normal blastocysts on D5 was slightly higher than on D6(82.14%vs. 52.38%, P<0.05)in blastocysts from bi-pronuclear zygotes.Conclusion1 Embryos derived from non- and mono-pronuclear zygotes have certain development potential, but lower than that of bi-pronuclear embryos. The percentage of blastocysts with normal chromosome constitution among non-/mono-pronuclear zygotes group is slightly lower than bi-pronuclear ones;2 Different insemination methods may lead to different incidence of non- and mono-pronuclear zygotes;3 The percentage of blastocysts with normal chromosome constitution among non-pronuclear zygotes group is higher than mono-pronuclear ones, and the percentage of blastocysts with normal chromosome constitution among non-pronuclear zygotes group on D6 is higher than D5, which need further study. |
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