| BackgroundFertilization is the beginning and the most important part of a new life.The general process of fertilization includes:The sperm pass through the cumulus cell layer,recognize and fuse with the sperm,activate the oocyte,and the oocyte processes the sperm;Then,the nuclear membrane reforms and wraps the chromatin to form a male and female pronucleus as a sign of the end of fertilization.In vitro fertilization-embryo transfer(IVF-ET)helps patients who cannot conceive naturally to complete this process outside the body.A high fertilization rate has always been our goal.To detect complete fertilization failure and low fertilization in time,short-term in vitro fertilization is gradually used in clinical practice.Short-term fertilization is performed 4-6 hours after fertilization with premature granular cells to observe the discharge of the second polar body to determine fertilization,which helps to detect complete fertilization failure and low fertilization in time.However,the premature exfoliation of granular cells is in the process of fertilization when the pronucleus has not yet formed.We question our experiments affect the formation of the pronucleus.In addition,the statistical analysis of clinical data from many reproductive centers found that compared with conventional fertilization,the multi-pronucleus rate of short-term fertilization has increased significantly,but there is no basic research on its formation process.PurposeTo observe the occurrence of multiple pronuclei in Short Insemination and early removing the cumulus cell,and the effect on the accumulation of lamin in nuclei formation.And find the influence of early removing the cumulus cell on the formation of pronuclei.It will be as the basis for the wide application of short-term fertilization and early degranulation in clinical practice.MethodsWe Use ICR mice as experimental animal models to establish a short-term in vitro fertilization system.Two time points of 1h and 1.5h were selected for the operation of granular cell stripping.We observe and record the multi-pronuclear zygote 6h after fertilization,and use laminB1 as the labeled protein to observe the pronucleus by immunofluorescence.Use ESCO incubator to observe embryo kinetics in each experimental group.In the study,an experimental group and a control group were set up.The experimental group consisted of zygotes that had early degranulation at 1h(n=24)and 1.5h(n=8)after fertilization,and the control group(n=39)was the same time Proceed to early degranulation cell zygote.Results1.Observing multiple pronuclei of embryos at 6h after fertilization,the rates of multiple pronucleus in the control group?1h and 1.5h were 9.5%?18.4%and 12.8%respectively.Compared with the control group,the 1h group had significant differences(P=0.005).2.Comparing the amount of laminB1 aggregation between each group on each zygote,it was found that there was no statistical difference between 2PN zygote and 3PN zygote(p>0.05).Comparing the accumulation of laminB1 between each group on each pronucleus,it was found that the accumulation of laminB1 on each pronucleus of 3PN zygote(p<0.0001)in the 1h group and each pronucleus of 3PN zygote in the control group(p=0.019)were lower than that of the 2PN zygote in the control group.3.Comparing the total area of laminB1 between each group,it was found that the total area of 2PN zygote laminB1 in the 1h group(p<0.0001)and 1.5h group(p=0.001)was larger than that of the control group,and the total area of 2PN zygote laminB1 in the 1.5h group(p=0.022)was larger than that of the lh group;It was also found that the total area of the 3PN zygote laminBl in the control group(p<0.0001)and the 1h group(p=0.019)was larger than the total area of the 2PN zygote in the control group.Comparing the area of laminB1 on each pronucleus,the laminB1 area of the 2PN zygotic paternal pronucleus and maternal pronucleus in the 1h group(p<0.0001,p<0.0001)and the 1.5h group(p<0.0001,p=0.005)was larger than that of the control group.There was no statistically significant in the increase of the area of laminB1 between the two groups(p>0.05).The laminBl area of 1h group 3PN zygotic male pronucleus(p=0.039)was smaller than that of the 2PN zygotic male pronucleus;The laminBl area of the control group 3PN zygotic male pronucleus was smaller than that of the 2PN zygotic male pronucleus,but there was no significant difference.4.Comparing the differences in the number of NLBs between the groups,it was found that there was no statistical difference in the number of NLBs in the paternal pronucleus and maternal pronucleus of the 1h group and the 1.5h group compared to the control group.5.Comparing the embryo kinetics between the groups,it was found that the pronucleus appearance(p<0.0001)?fading time(p<0.0001)and the two-cell cleavage time(p=0.010)in the 1h group were earlier than those in the control group,and the four-cell cleavage time(p=0.008)and the eight-cell cleavage time(p=0.003)were later than those of the control group,and there were statistically significant.The pronucleus appearance(p<0.0001)and fading time(p=0.003)in the 1.5h group were earlier than those in the control group,and there were statistically significant,and there was no statistical difference in the cleavage time.Conclusion1.Early removing the cumulus cell in short insemination will lead to an increase in the multi-pronuclear rate of the zygotes;2.In the multi-pronuclear zygote,the total accumulation of laminB1 is remains unchanged,but the accumulation on each pronucleus is reduced;3.Early removing the cumulus cell in short insemination makes the area of laminB1 of 2PN zygotes larger,and 1.5h group has a greater impact on it;the total area of laminB1 in multi-pronuclear zygote increases,the area of MAT remains unchanged,and the area of PAT less than 2PN zygotic PAT laminB1 area;4.Early removing the cumulus cell in short insemination has no significant effect on the number of NLBs on the pronucleus;5.Early removing the cumulus cell in short insemination may have an impact on embryo kinetics. |
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