| PART ONE ANALYSING ZONA PELLUCIDA OF ZYGOTE BY POLSCOPE AFTER FERTILIZATIONObjectiveIn human in-vitro fertilization (IVF), the failure and low fertilization rate is about 10%~30% and the abnormal fertilization rate is about 2%~9%. Oocyte quality plays an important role in fertilization. Zona pellucide (ZP), the transparent extracellular matrix, the way where sperm must penetrate thereafter form a barrier arrested more sperm penetrate. The characteristic of ZP may be related to fertilization outcome. The polarized light microscope (Polscope) was used in this study to investigate the difference of zona pellucida between germinal vesicle present (GV), metaphase I (MI), approaching metaphase II (AMII), metaphase II (MII); two-pronuclear (2PN) from intracytoplasmic sperm injection (ICSI) and 2PN from IVF; 2PN, three- pronuclear (3PN), and zero-pronuclear (0PN) from IVF.MethodsThe zona pellucida of 127 good quality matured oocytes, 80 unmatured oocytes, 69 2PN zygotes, 26 0PN zygotes, 93 3PN zygotes from IVF and 61 2PN zygotes from ICSI were imaged by Polscope.ResultsZPT and ZPD of inner layer (IL) and ZPT of total layer (TL) of oocyte after full grown gradually decreased until MII. The ZPT and ZPD of IL and ZPT of the TL of 2PN derived from IVF was higher than MII (2.49±0.49, 8.79±1.91, 19.33±2.28; 2.36±0.65, 8.80±1.54, 19.37±2.38 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05) ZPT of 2PN derived from IVF was higher than 2PN derived from ICSI too. In IVF the ZPT and ZPD of IL and ZPT of the TL of 0PN zygotes were higher than 2PN (2.49±0.49, 8.79±1.91, 19.33±2.28 vs. 2.14±0.52, 7.98±1.73, 16.54±2.43; p<0.05), whereas the IL ZPT of 3PN zygotes was lower than 2PN (8.19±1.68 vs. 8.79±1.91; p<0.05).ConclusionsAfter oocyte full grown, ZP was gradually thinner and looser until MII. After ZP reaction, the ZP structure changed. There are different between IVF and ICSI. The status of ZP was the cause of abnormal fertilization even when the sperm were considered normal. PART TWO CHANGE OF ZONA PELLUCIDA BETWEEN 6H SHORTEN GAMETS CO-INCUBATION AND 18H GAMETS CO-INCUBATION?ObjectiveThe positive value of the short gametes co-incubations on embryo quality is still controversial. A study showed that short gametes co-incubations reduced zone pellucida thickness (ZPT) which was evidence of better culture condition. To further investigate these finding, sibling oocytes (n=258) from 21 IVF couples were randomized allocated to a 6 h or 18 h co-incubations group to compare zone pellucida (ZP) and embryo quality (EQ).MethodOocytes (n=258) from 21 IVF couples were randomly allocated to 6 h group or 18 h group. Mean ZPT and ZP density (ZPD) of zygote were measured by polarized light microscope (Polscope) 20 h after fertilization. Other targets were normal fertilization (two-pronuclear, 2PN), EQ, clinical pregnancy (CP) and implantation (IR).ResultRate of normal fertilization and 3PN and proportion of high quality embryo were not statistically different between two groups. However, ZPD and ZPT of inner layer and ZPT of full layers for 2PN zygote were significantly thinner in 6 h than 18 h of incubations (2.09±0.50, 8.28±1.28, 18.97±2.64 vs. 2.24±0.67 8.86±1.52 19.57±2.66; P<0.05). ZPD of inner layer for grade 1-3 embryo was also lower from 6 h incubation. In contrast, ZPT of outer layer for grade 4 embryos was thicker at 6 h incubation. Overall, ZPT of inner layer was negatively related to embryo quality.ConclusionsShort gametes co-incubations reduced ZPT and ZPD of zygote which may be benefit to embryo hatching. |
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