| BackgroundHydrosalpinx is abnormal fluid accumulation within the fallopian tube caused by the distal tubal obstruction, which mostly occurs after the acute or chronic pelvic infections, usually no clinical symptoms except vaginal discharge occasionally. Hydrosalpinx is a common cause of infertility and in women of childbearing age the incidence rate showed an increasing trend. In vitro fertilization-embryo transfer(IVF-ET)played a significant role in solution of tubal infertility. But many clinical studies have shown that hydrosalpinx affects IVF outcome and the exact mechanisms remain unclear. Therefore, the study of hydrosalpinx on IVF pregnancy outcome mechanism, for guiding clinical hydrosalpinx patients with appropriate pretreatment is essential to further improve the success rate of IVF.PurposeThrough the effects of different concentrations of hydrosalpinx in embryo growth and development, to understand whether the hydrosalpinx has a toxic effect on embryo. Through the differental staining of expanded blastocyst of different concentrations of hydrosalpinx, to identified the quality of the expansion of blastocysts in each group and the potential impact on embryo. Through the detection of growth factor IGF-Ⅱand EGF of each group at different growth stages of blastocyst culture medium to further discuss the reasons of the embryonic growth or growth restriction in hydrosalpinx.Methods1. Embryo culture and observation in different concentrations of hydrosalpinx groupCollected the fresh discarded embryos of IVF-ET/ICSI-ET conventional treatment in the reproductive center from June 2009 to December 2009. Randomly divided into hydrosalpinx concentrations were 0%, 20%, 40%, 60%, 80%, 100% of the blastocyst culture medium, and cultured to D5~D8, daily observed the development of embryos at the same time and record the situation. Compare the cells number of D5 of early blastocyst formation, D6 of blastocyst expansion, D7 of blastocyst hatching, and the total cells number of blastocyst formation, blastocyst expansion and blastocyst hatching of each different concentrations of hydrosalpinx group to understand the different concentrations of hydrosalpinx on blastocyst growth rate and on the stages of blastocyst development situation.2. Differental staining of expanded blastocyst to identified the blastocyst quality in each groupUsing two fluorescent dyes Bisbenzimide (Hoechst33342) and PI (Propidium iodide, PI) to stained the expanded blastocyst of 0% to 80% hydrosalpinx group and photographed under fluorescent microscope. Determined the number of internal cell mass (ICM) and trophectoderm (TE) cells of blastocyst. Compared the ICM cells and ICM cells/Total cells ratio to identify the quality blastocyst. 3.Enzyme-linked immunosorbent assay for detecting embryo culture medium IGF-Ⅱand EGF levelsDetermined the content of IGF-Ⅱand EGF by enzyme-linked immunosorbent assay with different concentrations of hydrosalpinx in the embryo culture medium, each group of expanded blastocyst and hatching medium. Using microplate Reader(450nm) to determine the value, each of 3 holes. 4. Statistical AnalysisThe data were analyzed with SPSS 17.0(SPSS Inc, Chicago, IL, USA). Measurement data were presented as mean±standard deviation (x±s). Multiple comparisons between groups using analysis of variance. P <0.05 as statistically significant difference.Results1. The groups of concentrations of hydrosalpinx with 0%~80%, the cells number of D5 early blastocyst formation, D6 of blastocyst expansion, D7 of blastocyst hatching were no significant difference (P>0.05); concentration of hydrosalpinx with 100%, the observation of indicators was significantly reduced than 0% to 80% group(P<0.05). The groups of concentrations of hydrosalpinx with 0% ~ 80%, the total cells number of blastocyst formation, blastocyst expansion and blastocyst hatching were no significant difference (P>0.05); concentration of hydrosalpinx with 100%, the observation of indicators was significantly reduced than 0% to 80% group(P<0.05).2. The groups of concentrations of hydrosalpinx with 0% ~ 60%, the total number of blastocyst and ICM/Total cells ratio was no significant difference (P>0.05); concentration of hydrosalpinx with 80%, the total number of blastocyst was reduced, but the result of ICM/Total cells ratio was no significant difference than previous group(P>0.05).3. Blastocyst culture medium contained basic concentrations of IGF-Ⅱand EGF of hydrosalpinx with different concentrations, the reaults among 0%~40% group, 60% to 80% group and 100% group were significant difference(P<0.05); the reaults between 60% to 80% group and 100% group was significant difference(P<0.05). At the stage of blastocyst expansion the results among 0%~60% group, 80% group and 100% group were significant difference(P<0.05); the reaults between 80% group and 100% group was significant difference(P<0.05). At the stage of blastocyst hatching the reaults between 0%~80% group and 100% group was significant difference(P<0.05). Conclusion1. Low concentration (20%~60%) of hydrosalpinx has no embryotoxicity and does not affect embryonic development; high concentration (80%) of hydrosalpinx reduce the total number of blastocyst; high concentration (100%) of hydrosalpinx affect the development of embryos.2. Hydrosalpinx contains a certain concentration of growth factors, such as IGF-Ⅱ, EGF. Its concentration reduced by the concentration of hydrosalpinx increased. Embryos increase the concentration of IGF-Ⅱand EGF through its autocrine effect in 20% to 80% group to to continue development of embryos while the increased secretion of IGF-Ⅱand EGF can not meet the embryo continued growth and development needs in 100% groups.3. Morphology of the embryo may not fully reflect the embryonic situation, not able to accurately predict the developmental potential of embryos. The concentration of growth factors in embryo culture medium were correlated with the blastocyst expansion and hatching. Detect the concentration of growth factors in embryo culture medium can effectively assess the quality and developmental potential of embryos.4. In the research of whether hydrosalpinx affect human embryonic development, they can not replaced by animal experiments. Human embryonic laboratory experiments and animal embryos may be totally different conclusions. |
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