The Effect Of Follicular Fluid From The Endometriosis Patients On Growth And Fertilization Of The Oocyte From The Mice And The Level Of ENA-78 Of The Follicular Fluid

BackgroundEndometriosis is a kind of benign disease which behavior as malignant tumor.Its common symptoms includes pelvic pain, dysmenorrhoea and infertil-ity. Studies estimate that endometriosis may affect around 25 percent women of reproductive age, and the incidence of infertility in these women varied from about 30 percent to 50 percent. To date, there are no ideal treatments for endometriosis and endometriosis-associated infertility, which associated with uncertain pathogenesis and mechanism of the endometriosis related to infertility. A number of studies on endometriosis have been done ,but the researchers and the doctors still can not completely illuminate it. The widespread development of the assisted reproductive techniques provides a better way to conceive for the endometriosis-associated infertility patients. At the same time, it is possible to study the mechanism of infertility caused by endometriosis from different angels. Most studies suggested that compared with the tubal factor infertility groups, the success of the endometriosis-associated infertility groups with IVF-ET was lower, which associated with the adverse effects of the endometriosis on the fertilizability of the sperm and oocyte, the quality of the embryo and the implanted environment of the embryo. In order to improve the success of the endometriosis-associated infertility groups with IVF-ET, it is very important to explore the mechanism of infertility caused by endometriosis from different decks.ObjectiveTo observe the effect of the follicular fluid from the endometriosis- associated infertile patients on in vitro fertilization of oocytes from the mice ,and ,on maturation and fertilization in vitro of immature oocytes , explore the mechanism of infertility induced by endometriosis. To test the level of ENA-78 in the follicular fluid of the endometriosis patients ,identify the microenvironment of the follicular fluid.Methods1.The effect of the follicular fluid from the endometriosis-associated infertility on the fertilization in vitro of the oviums from the mice a.Kunming mouse was induced super-ovulate by injected with HMG and HCG after 44～48 hours. We killed the mouse by decapitation after 15～17 hours, took out of the bilateral oviducts, and collected the oocytes under the anatomical microscope. The collected oocytes were arranged randomly in three groups to fertilize with sperm from Kunming mice in vitro: blank control group:DMEM +10%FCS+50IU/ml penicillin G+50μg/ml streptomycin sulfate; study group: DMEM+10%FCS+50IU/ml penicillin G+50μg/ml streptomycin sulfate+40% follicular fluid of the endometriosis-associated infertility;control group: DMEM+10%FCS+50IU/ml penicillin G+50μg/ml streptomycin sulfate+40% follicular fluid of the tubal-associated infertility. b.Statistic the fertility rate of three groups.2.The effect of the follicular fluid from the endometriosis-associated infertility on maturation and fertilization in vitro of the immature oocytes from the micea. Kunming mouse was induced super-ovulate by injected with HMG.We killed the mouse by decapitation after 44～48 hours, took out of the bilateral ovaries, then lacerated and collected the immature oocytes under the anatomical microscope. The collected oocytes were arranged randomly in three groups to cultured in vitro: blank control group: DMEM+10%FCS+1IU/mlFSH+ 0.5IU/mlLH+0.5IU/mlHCG+50IU/ml penicillin G+50μg/ml streptomycin sulfate; studygroup:DMEM+10%FCS+1IU/mlFSH+0.5IU/mlLH+0.5IU/mlHCG+50IU/ml penicil- lin G+50μg/ml streptomycin sulfate+40% follicular fluid of the endometriosis -associated infertility; control group: DMEM+10%FCS+ 1IU/ml FSH+0.5IU/mlLH+ 0.5IU/mlHCG+50IU/ml penicillin G+50μg/ml streptomycin sulfate+40% follicular fluid of the tubal-associated infertility. The stages of the oocytes were observed with invert microscope during maturation at the 0,24,36,48 and 54 hours.The oocytes were classified as MⅡ(nuclear maturation) when the first polar body(PBI)was found in the perivitelline space.b.Fertilized MⅡoocytes in vitro with sperm.The sperm were taken from Kunming mice.The fertilization in vitro fluid: DMEM+10%FCS+50IU/ml penicillin G+50μg/ml streptomycin sulfate.c.Statistic the mature rate and the fertility rate of three groups.3.The level of epithelial neutrophil-activating peptide-78(ENA-78) in follicular fluid from endometriosis-associated infertilitya.The endometriosis patients confirmed by operation or puncturing the ovarian cyst and the salpingitis patients confirmed by operation all were super-ovulated applied with long protocol. The first tube of follicular fluid without blood were collected during retrieving eggs,then clarification by centrifugation at 1000×g for 10 minutes, and the supernatants were stored at -20℃.b.The collected follicular fluid were measured using enzyme-linked immunosorbent assay(ELISA).Results1. The effect of the follicular fluid from the endometriosis-associated infertility on the fertilization in vitro of the oviums from the mice The in-vivo mature oocytes of mice were fertilized in vitro in three kinds environment, we found that the fertility rate of the study group, control group and blank control group were 68.28%,74%,78.5%.There was no difference in fertilization in the three groups(P>0.05). 2. The effect of the follicular fluid from the endometriosis-associated infertility on maturation and fertilization in vitro of the immature oocytes from the micea.The effects of in-vitro maturation of different stages oocytes The in-vitro maturing rate of GV stage oocytes in the study group, control group and blank control group were 25.81%,34.18%,38.38%.The maturing rate of the study group was decreased significantly as compared to that of other groups(P<0.05).There was no difference in the control and blank control groups(P>0.05). The in-vitro maturing rate of MⅠstage oocytes in the study group, control group and blank control group were 46.33%,58.02%,62.83%.The maturing rate of the study group was decreased significantly as compared to that of other groups(P<0.05).There was no difference in the control and blank control groups(P>0.05).At the same time, the maturing rate of MⅠstage oocytes was increased significantly as compared to that of GV stage in each same group(P<0.05).b. The effects of in-vitro fertilization of oocytes in-vitro-matured from different stagesThe fertility rate of oocytes in-vitro-matured from GV stage in the study group, control group and blank control group were 41.67%,58.33%,58.77%,the fertility rate of the study group was decreased significantly as compared to other groups(P<0.05).There was no difference in the control and blank control groups(P>0.05). The fertility rate of oocytes in-vitro-matured from MⅠstage in the study group, control group and blank control group were 57.43%,70.73%,73.33%.The fertility rate of the study group was decreased significantly as compared to other groups(P<0.05).There was no difference in the control and blank control groups(P>0.05).At the same time, the fertility rate of oocytes in-vitro-matured from MⅠstage was increased significantly as compared to that of GV stage in each same group(P<0.05).3.The level of ENA-78 in follicular fluid of the endometriosis-associated infertilityThe average concentration of EAN-78 of the study group and control group were 185.55±71.75 pg/ml and 130.79±55.13pg/ml,the level of the study group was significant higher than that of the control group(P<0.05).Conclusion1. There is no obvious effect of follicular fluid from endometiosis - associated infertility on fertilizability of mature oocytes from mice;2. Follicular fluid from endometiosis-associated infertility not only brings harmful effects to the maturation in vitro of oocytes ,but also decreases the fertilizability of the oocytes matured in vitro.Adverse effect on the development of oocytes may play an important role in the mechanism,which impairs the fertilizability of them;3. The level of ENA-78 in the follicular fluid of the endometiosis-associated infertility patients is significant higher than that of the tubal- associated infertility patients.The high level of ENA-78 is one of the reasons that cause poorer quality and lower fertilizability of oocytes of the endometiosis.