| Part I The relationship between CNP levels in human follicular fluid and IVF pregnancy outcomeObjectives: To identify the relationship between CNP levels in follicular fluid and clinical pregnancy outcome in vitro fertilization(IVF),and to explore the possible influencing factors of CNP in follicular fluid,such as IVF type,insemination method and infertility diagnosis,and the correlation between cyclic guanosine monophosphate(c GMP)concentration and CNP concentration in follicular fluid.Methods: We collected mature follicular fluid and clinical data of 158 patients,who who underwent IVF infertility in the reproductive medicine center of Tongji Medical College of Huazhong University of Science and Technology during the period of March 1,2014 to March 1,2015.The concentration of CNP in follicular fluid was measured by ELISA and its relationship with pregnancy outcome,IVF type,insemination method was analyzed in the retrospective study.Futhermore,we also detected the levels of c GMP and CNP in follicular fluid of 30 patients by ELISA and analyzed the correlation between them.Results:Firstly,the patients were arranged into pregnancy group and non-pregnancy group according to pregnancy outcomes,and the pregnancy group included continuing pregnancy group,ectopic pregnancy group and embryo abortion group.Follicular fluid from non-pregnancy patients(17.41±13.15 ng/L,n = 85)contained significant higher CNP levels than follicular fluid obtained from non-pregnancy patients clinical pregnancy patients(13.61±5.31 ng/L,n=73)(p=0.02).Likewise,CNP levels in follicular fluid were significantly higher for non-pregnancy patients compared to continuing pregnancy patients(13.31±5.25 ng/L,n=60)(p=0.02).However,no significant difference was found in the embryo abortion patients,ectopic pregnancy patients(14.33±4.66ng/L,n=5;15.42±6.32ng/L,n=8,respectively)and non-pregnancy patients(p=0.60;p=0.65,respectively)about CNP levels in follicular fluid.When considering IVF type,CNP concentration was significantly higher in follicular fluid samples from husband semen patients(n = 130)than those donor semen patients(n = 28)(16.67 ± 17.19ng/l versus 10.78±1.68 ng/l,p = 0.008,respectively).According to insemination methods,patients were divided into IVF group(n = 106)and ICSI group(n = 52).The concentration of CNP in the follicular fluid was 14.98 ± 10.52 ng / L and 17.25 ± 10.08 ng / L respectively.The CNP concentration in the IVF group was little lower than that in the ICSI group,but the difference was not statistically significant.Additionally,there is a strong positive correlation between CNP and c GMP concentrations in human follicular fluid(R2=0.935,?=2.83).Conclusion : The levels of CNP in follicular fluid of pregnancy patients and continuing pregnancy patients were significantly lower than that of non-pregnancy patients,suggesting that CNP might affect the pregnancy outcome.The CNP concentration in follicular fluid of donor semen patients was significantly lower than that of husband semen patients,which means CNP levels in follicular fluid maybe affected by semen resource.However,we found that insemination methods had no significant effect on CNP levels in follicular fluid because of insufficient patients.The concentration of CNP in follicular fluid was positively correlated with the concentration of c GMP in follicular fluid.Part? The expression of CNP and NPR-B on mouse oocytes and embryosObjectives: To investigate the expression of CNP and NPR-B on mouse oocytes and embryos and their relationship with oocyte function and early embryonic development.Methods: Oocytes of different stages were collected: germinal vesicle(GV;meiotic arrest),germinal vesicle breakdown(GVBD;meiotic resumption),MII(mature oocyte),2-cell embryos,4-cell embryos,8-cell embryos,and blastocysts.CNP and its NPR-B were labeled with immunofluorescence,and their expression was observed under confocal laser scanning microscope.Results: CNP and NPR-B maintained expression during meiotic cell cycle progression and embryo development through GV to blastocyst stageConclusion: CNP and NPR-B expression was consistent during oocyte maturation and embryo development from GV to the blastocyst stage,suggesting that CNP may play an important role in oocyte maturation and early embryo development.Part? Effects of CNP on in vitro fertilization process and early embryonic development of miceObjectives:To observe effects of CNP on in vitro fertilization process and early embryonic development by influence of CNP at sperm capacitation,fertilization and early embryonic development.Methods: This part is performed by three steps.First,based on previous results,we supplement capacitation fluid with CNP(10-6mol / L),incubate mice sperm for 1to1.5hours,thus evaluate fertilization and blastocys rates differences between two groups after IVF.Second,the process of IVF is exposed to different CNP concentrations(0,10-6mol / L,10-7mol / L),which aimed to choose optimized CNP concentration of acquiring highest fertilization rate and blastocyst formation rate.Last,different concentrations of CNP(0,10-6mol / L,10-7mol / L)were added to the embryonic culture medium in vitro,and to identify whether it is beneficial to improve fertilization rate and blastocyst formation rate,if so,the optimal CNP concentration of for early embryonic development is selected.Results: In comparison to the control condition,the rate of 2-cell embryos and blastocysts/2-cell tended to be higher for sperm incubated in medium containing CNP(10-6mol/L)(67.08% vs 79.84% and 50.93% vs 62.14%,respectively).The 2-cell rate of embryos cultured in CNP(10-7mol/L)was superior to control,but not as good as CNP(10-6mol/L)(76.92%,67.95%and 79.24% respectively).Moreover,embryos cultured in CNP showed a higher rate of blastocyst/2-cell embryos than control: the rate was66.32%,65.71% and 54.09% for CNP(10-7mol/L),CNP(10-6mol/L)and control,respectively.Two-cell(fertilization)rate and blastocyst/2-cell embryos rate was not different between the control and the embryo culture medium treated with CNP(10-6mol/L)(68.70%,54.43% for control,69.37%,60.48% for culture medium+CNP(10-6mol/L),respectively).Nevertheless,in comparison to the control condition,the rate of 2-cell embryos and blastocyst/2-cell embryos rate tended to be higher for embryos cultured in medium containing CNP(10-7mol/L)(79.52%,68.86%,respectively)than control.Conclusion: CNP can improve the outcome of mouse in vitro fertilization by promoting the sperm motility and sperm oocyte binding ability,and the optimal concentration is 10-6mol/L CNP.In addition,CNP plays an important role in the early development of embryos,and its effective concentration is 10-7mol/L. |
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