| Endometriosis (EMs) is a common and frequently occurring disease withwomen. It is one of the most common cause of female infertility and it refersto the endometrium (glands and stroma) in any position outside the uterinecavity. In vitro fertilization and embryo transfer (IVF-ET) is an effectivemethod to solve the infertility. But compared with other factors of infertilitypatients undergoing IVF-ET, the poor pregnancy outcome in patients withendometriosis is great mental pressure and economic burden to them. Thecause of EMs leading to the adverse outcome of IVF-ET is complex, includingthe regulation of reproductive endocrine system, egg quality, embryodevelopment, embryo implantation and endometrial receptivity change etc. Atpresent a lot of studies have been reached the relationship betweenendometriosis and pregnancy outcome of IVF-ET through the above reasons,and the decline of the oocyte quality may be one of the key factor that affectthetreatment outcome.Oocyte quality may play an important role in the normal fertilization, theembryo development and implantation, also it may has the profound influenceof the establishment and maintenance of pregnancy and fetal development.Therefore, to improve the quality of oocytes in IVF-ET has a greatsignificance. The development of oocytes has two stages: growth and maturity,the mature concomitant with follicular growth. At the same time, oocytescommunicate with granulosa cell for information exchange through a widegap juction, the autocrine and paracrine function of local growth factors, theyare indivisible and jointly for a integrity. Therefore, study on the evaluation ofthe quality of oocytes from aspects of follicular fluid and granulosa cells caused a widespread concern. The discovery of oocyte secreted factors (OSFs)provides a new way of thinking for study the quality of oocyte, predicting theembryo quality and pregnancy outcome. In the numerous oocyte factors,growth differentiation factor-9(GDF-9) is the first discovered which plays animportant role in follicle development. Many studies have found that, innormal women as control, follicular fluid of severe endometriosis in womenwith laparoscopic puncture of large follicles, the level of GDF-9wasdecreased significantly.To sum up, the first part of this study is to collect the clinical andlaboratory dates of endometriosis patients undergoing IVF-ET in order toanalyze the factors related with the pregnancy outcome of IVF-ET inendometriosis. The second part is to measure the levels of GDF-9in serumand follicular fluid and the expressions of GDF-9protein and mRNA onluteinized granulosa cells with endometriosis patients, to investigate therelationship between the levels of GDF-9and outments in patients ofendometriosis, to explore the possible causes of oocyte quality, and to providenew ideas and theoretical basis for guiding clinical treatment.Part ⅠRelated factors affecting the outcome of patients ofendometriosis in vitro fertilization and embryo transferObjective: Compared with patients of tubal factors, to analysis theclinical and laboratory data of endometriosis underwent in vitro fertilizationand embryo transfer retrospectively, and to explore the relating clinical andlaboratory factors of pregnancy outcome of endometriosis in IVF-ET.Methods: Collecting the clinical and laboratory information ofconventional IVF cycles in the reproductive medicine of the second hospital ofHebei Medical University from January2011to March2013. According to thedifferent etiologys, they were divided into two groups: the group of endometriosis is Group A and the group of fallopian tube reason is Group B.According to the pregnancy outcome, Group A was divided into pregnantgroup (Group A1), nonpregnancy group (Group A2). In Group A, there was129cycles, including60cases of Group A1,54cases in Group A2and1660cycles in Group B. Compare the following contents:①the female age,duration of infertility, body mass index, basal levels of follicle stimulatinghormone (FSH), luteinizing hormone (LH) and estradiol (E2), the proportionwith primary infertility and secondary infertility in group A and group B;②ovarian stimulation protocol, duration of Gn, Gn consumption; the hormonelevels at hCG injection day in group A and group B;③the average numberof retrieved oocytes, number of available embryos, number of transferableembryos, number of good quality embryos, fertilization rate, normalfertilization rate, cleavage rate, the rate of frozen embryos, and the pregnancyrate of total freezing embryo in group A and group B;④the implantationrate, clinical rate, multiple pregnancy rate, abortion rate in group A and groupB;⑤the femal age, duration of infertility, levels of basal FSH, LH and E2, theproportion with primary infertility and secondary infertility, ovarianstimulation protocol, duration of Gn, Gn consumption, the hormone levels athCG injection day in group A1and group A2;⑥the average number ofretrieved oocytes, number of available embryos, number of transferableembryos, embryos of good quality, fertilization rate, normal fertilization rate,cleavage rate, the rate of frozen embryos in group A1and group A2.Results: In1789IVF cycle, there were129cycles with endometriosisgroup (group A),1660cycles with control group (group B).①The mean age of the femal patients in the two group were31.29±4.31years,29.21±4.04years, group A was significantly higher than that of groupB, there was a significant difference between two groups (Z=-5.2750,P<0.05); the duration of infertility of the group A, B were4.57±3.99years,3.80±2.73years, there was a significant difference between two groups (Z=-2.064, P<0.05); the primary infertility ratio of two groups were62.80%,52.53%, there was significant difference between two groups (χ2=5.035, P<0.05);②there was no significant differences between the two groups in bFSH, bLH levels (Z=-1.145, P>0.05; Z=1.176, P>0.05);The E2levels was57.47±53.01pg/ml,44.92±32.32pg/ml, the difference between the two groups were significantly (Z=-3.011, P<0.05)；The rateof ovarian stimulation protocol（long protocol, short protocol, GnRH-antagonist regimen, exceed long protocol) were26.4%,4.7%,10.9%,58.1%and78.4%,5.2%,12.9%,3.4%, there was significant difference in theabove four protocols between the group A and B (χ2=529.442, P<0.05); duration of Gn, Gn consumption of the two group were10.78±2.25days,10.13±2.12days and2896.90±824.71IU,2375.85±764.96IU, difference between the two groups was significantly (Z=-3.610, P<0.05, Z=-7.548, P<0.05); The levels of LH, E2, P on hCG injection day were1.69±1.77mIU/ml,2543.88±1547.71pg/ml,1.04±0.56ng/ml and2.23±2.54mI/mlU,3407.31±1444.48pg/ml,1.17±0.69ng/ml, the difference between the two groups were statistically significant (Z=5.715, P<0.05; Z=6.16, P<0.05; Z=2.430, P<0.05);③the average number of oocytes retrieved of group A and B were9.69±7.17,13.67±7.83, average number of oocytes of two groups had significant difference (Z=6.508, P<0.05); the number ofgood quality embryos of two groups of patients was2.39±3.32,3.86±3.91, there was a significant difference (Z=4.712, P<0.05); the number ofavailable transferable embryos of two groups was4.01±3.82and5.94±4.27, there was statistically significant difference (Z=5.704, P<0.05); thenumber of transferred embryos of the two groups were2.00±0.51and1.70±0.89, there was all similar between the two groups.(Z=0.634, P>0.05); the fertilization rate and the normal fertilization rate were73.60%,84.32%and60.08%,63.54%, the differences were significant (χ2=100.13, P<0.05, χ2=6.130, P<0.05); the ratio of cycles with freezing was44.96%,64.76%respectively, there was significant difference between twogroups (χ2=20.203, P<0.05), but the whole embryo freezing rate of thetwo groups was10.08%and16.20%, no significant difference(χ2=3.216,P>0.05);④the cleavage rates of two groups were98.91%and98.90 %, Late-ICSI ratio was2.29%and2.97%, implantation rate was35.65%and36.69%, multiple pregnancy rate was33.33%and30.11%, pregnancy rate and abortion rate were52.63%,53.89%and11.67%,6.80%. There were not statistically significant all above index of the two groups(χ2=3.617, P>0.05; χ2=0.32, P>0.05; χ2=0.068, P>0.05; χ2=0.280, P>0.05; χ2=0.099, P>0.05; χ2=2.044, P>0.05);⑤In groupA1and A2, the mean age, duration of infertility were30.33±3.72years,3.93±2.74years and32.33±4.72years,5.18±5.26years, there were no significant differencesbetween the two groups (Z=1.766, P>0.05; Z=0.935, P>0.05); the primary infertility rates of two groups of patients was71.67%,33.33%, groupA1was significantly higher than that of group A2, there was significant difference between two groups (χ2=16.788, P<0.05); the levels of bFSH, bE2in two group were8.31±3.30mIU/ml,59.46±62.22pg/ml and8.74±3.27mIU/ml,55.15±51.52pg/ml, differences between the two groups had no statistical significance (Z==1.086, P>0.05; Z=-0.738, P>0.05); the levels of bLH in the two groups were3.55±1.75mIU/ml,4.31±1.91mIU/ml, the difference was significant (t=-2.082, P<0.05); the average Gndays of two groups were10.17±3.84days,10.29±2.54days, there no statistical difference significance (Z=-0.609, P>0.05); but the Gn dosagewere2699±676.91IU,3132.1±923.58IU in group A1and group A2, there was significant difference between two groups (t=2.758, P<0.05); the levels of E2at hCG injection day were2691.90±1415.73pg/ml,2191.22±1513.05pg/ml in two groups, group A1was significantly higher than that in group A2, there was significant difference between the two groups(Z=-2.018, P<0.05);⑥the average number of oocytes, the number of good quality embryos, the number of available embryos were9.50±5.18,2.68±2.90,4.30±3.00and7.72±5.14,1.38±1.83,2.81±2.12in groupA1and group A2, group A1were higher than those of group A2, thedifferences between the two groups were sgnificantly (Z=-2.364, P<0.05; Z=-2.401, P<0.05; Z=-3.023, P<0.05); the fertilization rate, normal fertilization rate of two groups were74.56%,61.75%and74.10%,58.03 %, although group A1were higher than group A2, no statistically significant difference compared with the two groups (χ2=0.027, P>0.05; χ2=1.391, P>0.05); cleavage rate was98.82%and98.06%, no significant differences between group A1and group A2(χ2=0.710, P>0.05); the freezing cycle ratio in group A1and group A2was53.33%,24.07%respectively, there was statistically significant difference(χ2=10.184, P<0.05).Conclusion: Patients of endometriosis may have older age, longerinfertility duration, and higher primary infertility ratio. Poor ovarian responsein COH, abnormal fertilization of oocyte, may eventually lead to the lowerclinical pregnancy rate, implantation rate and the higher rate of abortion. Thepregnancy patients may have the feactures of good response of ovarianstimulation and good embryo quality.Part Ⅱ The expression of GDF-9in the follicular fluid andgranulosa cells in vitro fertilization and embryo transfer inendometriosis patientsObjective: To measure the levels of GDF-9in serum and follicular fluidand the expression of GDF-9protein and mRNA on luteinized granulosa cellswith endometriosis patients, to explore the relationship between GDF-9andthe oocyte quality with endometriosis patients and pregnancy outcome in IVF,in order to find the effect of possible ways of oocyte quality.Methods:30patients with endometriosis (group A) and30patients withobstruction of fallopian tube undergoing IVF cycles (group B) were collected.All these patients were selected from the department of reproductive medicineof the second hospital of Hebei Medical University from August2012toMarch2013. Serum, follicular fluid and luteinized granulosa cells werecollected. Compared with the general index of two groups such as age,duration and dosage of Gn, the number of oocytes, the number and the rate oftransferable embryos, the number of good quality embryos and the fertilizationrate, cleavage rate, implantation rate, clinical pregnancy rate; the levels of linked immunosorbent assay (ELISA); the expression of GDF-9protein inhuman luteinized granulosa cells in two group were measured by Western blot;the GDF-9mRNA expression in granulosa cells were measured by reversetranscriptase polymerase chain reaction (RT-PCR). The levels of GDF-9inserum, follicular fluid, the expression GDF-9protein and mRNA on luteinizedgranulose cells in two groups were compared. The relationship between levelsof GDF-9in serum and follicular fluid, the expression of GDF-9protein andmRNA on luteinized granulose cells with patients’age, Gn duration, dosage ofGn, the number of oocytes, the number and rate of transferable embryos andgood quality embryos, fertilization rate and cleavage rate.Results: Group A and Group B is30cycles respectively.①The averagefemal age of two groups were31.50±4.83years and28.15±4.27years, group Awas significantly higher than that of group B, there was a significantdifference between two groups (Z=-3.080, P<0.05); the levels of bFSH, bLH,bE2were8.31±3.62mIU/ml,4.30±1.78mIU/ml,58.88±49.27pg/ml and7.34±1.69mIU/ml,4.42±1.76mIU/ml,43.72±36.81pg/ml, differences betweenthe two groups had no statistical significance (Z=-0.412, P>0.05; t=-0.249,P>0.05; Z=-1.224, P>0.05);②The days of Gn and Gn dosage of two groupswere11.17±1.42days,3010.83±655.19IU and9.55±1.28days,2184.20±580.00IU, the difference between the two groups were significant(Z=-4.080, P<0.05; Z=-4.699, P<0.05); the levels of LH, E2at hCG injectionday were1.38±1.20mIU/ml,3066.37±1612.51pg/ml and2.02±1.26mIU/ml,3846.06±1216.97pg/ml, group A were lower than those of group B, twogroup differences were significant (Z=3.142, P<0.05; Z=2.016, P<0.05); butthe levels of P at hCG injection day were1.18±0.47ng/ml,1.14±0.69ng/ml, nosignificant differences between the two groups (Z=0.606, P>0.05);③Theaverage number of oocytes, the number of good quality embryos, the numberof available embryos were10.63±8.10,3.27±4.82,4.47±4.90and15.15±8.23,5.28±5.12,7.03±5.46, group A were lower than those of group B, thedifference in the two groups were statistically (Z=2.792, P<0.05; Z=2.295, P<0.05, Z=2.380, P<0.05); the average fertilization rate was81.63%±15.72%,77.84±17.52%in the two groups, there was no significant difference(Z=-0.700, P>0.05); the average normal fertilization rate were55.93%±22.05%,67.34%±15.67%, there was significant difference betweentwo groups (t=-2.331, P<0.05); the average cleavage rate in patients of thetwo groups were97.46%±9.74%,97.67%±5.73%, there was no significantdifferences between the two groups (Z=-1.351, P>0.05); the embryotransferred number of two groups were1.73±0.94,1.76±0.79, no significantdifferences between the two groups (Z=-0.142, P>0.05);④The freezing cyclein two groups were40%and66.66%, group A was lower than that of group B,there was significant difference between two groups (χ2=20.203, P<0.05); theembryo implantation rates were30.77%and34.48%, the clinical pregnancyrate were53.33%and60.60%, multiple pregnancy rate were60%and66.67%,abortion rates were20%and8.33%, there were not statistically significant allabove in the two groups (χ2=0.172, P>0.05; χ2=0.063, P>0.05; χ2=0.105,P>0.05; χ2=0.630, P>0.05);⑤The levels of GDF-9in serum, follicular fluidof two groups were153.47±9.81pg/ml,143±90.28pg/ml and204.40±137.65pg/ml,236.85±163.40pg/ml, group A were significantly lowerthan those in group B, there were significant difference between two groups(Z=2.047, P<0.05; Z=2.216, P<0.05;); the expression of GDF-9protein andmRNA on luteinized granulosa cells of two groups were0.53±0.17,0.57±0.21and0.72±0.19,0.75±0.18, group A was significantly lower than those ingroup B, there were significant difference between two groups (Z=3.615,P<0.05; t=-3.699, P<0.05);⑥In group A, there were positive correlationbetween the GDF-9protein on luteinized granulosa cells and the number ofoocytes, the number of available embryo, the number of good quality embryos(r=0.703, P<0.05; r=0.644, P<0.05; r=0.665, P<0.05); there were positivecorrelation between the expression of GDF-9mRNA on luteinized granulosacells and the number of oocytes, the number of available embryo, thenumber of good quality embryos (r=0515, P<0.05; r=0.569, P<0.05; r=0.526,P<0.05); In group B, there were positive correlation between the GDF-9 protein and mRNA with fertilization rate(r=0.442, P<0.05; r=0.430, P<0.05);⑦In group A1and group A2, the levels of GDF-9in serum and follicularfluid were191.63±102.04pg/ml,196.43±116.83pg/ml and134.35±95.45pg/ml,124.50±99.70pg/ml, group A1were significantly higher than those in groupA2, the difference between the two groups were statistically significant(Z=-2.288, P<0.05; Z=-2.332, P<0.05); the expressions of GDF-9protein andmRNA in group A1and group A2were0.63±0.15,0.70±0.17and0.48±0.16,0.50±0.20, group A1were higher than those in group A2, there weresignificant difference between two groups (t=2.485, P<0.05; Z=-2.772,P<0.05); the levels of GDF-9in serum and follicular fluid in group B1andgroup B2were256.52±157.45pg/ml,279.24±129.78pg/ml and172.89±119.48pg/ml,190.43±169.01pg/ml, group B1was significantly higherthan those in group B2, there were significant difference between two groups.(Z=-2.156, P<0.05; Z=-2.090, P<0.05); the levels of GDF-9protein andmRNA on granulosa cells in group B1and group B2were0.76±0.22,0.68±0.22and0.70±0.17,0.78±0.14, there were no significant differencesbetween the two groups (Z=-1.210, P>0.05; t=-1.534, P>0.05).Conclusion: The levels of GDF-9in serum and follicular fluid of theendometriosis patients were lower than those of the control group. And theexpression of GDF-9protein and mRNA on the granulosa cells of theendometriosis patients were lower than those of control group. The lowerexpressions of GDF-9were positive correlations with the number of oocytes,the number of available embryo, the number of good quality embryos, whichmay affect the follicular development, oocyte and embryo quality, lead to thelow clinical pregnancy rate, implantation rate and high abortion rate inpatients with endometriosis. |
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