The Effects Of RhAMH On The Function Of Human Granulosa Cells And Its Relationship With The Ovary Reserve And Response In Infertile Women

Anti-müllerian hormone(AMH)belongs to the transforming growth factor-βsuperfamily.The existence of anti-Mulerian hormone,also known as Mulerian inhibiting substance(MIS)was first suggested by the pioneering experiments performed by the French scientist Alfred Jost in the 1940s.At the early stages of development in mammals,foetuses of both sexes have two pairs of ducts:the mesonephric or Wolffian ducts and the paramesonephric or Müllerian ducts.In the male foetus,the development of Wolffian ducts into the epididymes,vasa deferentia and seminal vesicles was already known to occur under the influence of testicular androgens.Jost had noticed that a crystal of testosterone propionate was capable of masculinising Wolffian ducts in the female foetus but it did not affect Müllerian ducts,which formed the Fallopian tubes,the uterus and the upper third of the vagina.Intrigued by his observation,Jost developed refined techniques of foetal surgery allowing him to show that a testicular product different from testosterone,that he named "hormone inhibitrice"or "Müllerian inhibitor",was responsible for the regression of Müllerian ducts in the male foetus.The identification of the "Müllerian inhibitor" did not prove easy.In 1969,one of Jost's pupils,Reine Picon,developed a test for the detection of anti-Müllerian activity.In 1972,another disciple of Jost's,NathalieJosso,showed the macromolecular nature of AMH/MIS,and in 1978,the first evidence was produced indicating its glycoprotein dimeric nature.Picon's test,as well as antibodies developed by a third former member of Jost's laboratory,Bernard Vigier,and by Patricia Donahoe's team in Boston,helped to localise AMH/MIS activity to immature Sertoli cellsand to granulosa cells of the postnatal ovary.We now know that the human AMH/MIS gene maps on chromosome 19 p13.2-13.3.In the female,it is a product of the granulosa cells from pre-antral and small antral follicles.Together with two other key players(growth and differentiation factor-9 and bone morphogenetic protein-15),it inhibits the initiation of premature follicle growth and decreases the sensitivity of follicles for the FSH-dependent selection process.AMH/MIS levels are characterized by a steady decline with age from adulthood toward menopause reflecting the size of the ovarian follicle pool.Therefore,AMH/MIS seems to be a promising parameter for early detection of reduced ovarian reserve as well as ovarian dysfunction.Epidemiological surveys suggested that an increasing number of female patients in their late 30s seek help for infertility problems,meaning that diminished ovarian reserve and age-related ovarian dysfunction have become a major cause of infertility.Furthermore,approximately as many as 10%of women in their early 30s could be approaching their perimenopause transition.Most women with reduced ovarian reserve are still displaying regular menstrual cycle characteristics that will continue normally with deterioration only becoming clinically apparent when ovarian function is already severely impaired.Thus ovarian aging and a reduced ovarian reserve can become a critical factor in infertility,without any obvious clinical symptoms,he chronological age of the ovary does not always reflect its biological and reproductive age.Because traditional assessments of ovarian reserve such as early follicular phase FSH,basic Estradiol,inhibin B,FSH/LH and the clomifen challenge test have low sensitivity in the early stages of reduced ovarian reserve,there is an urgent need for a reliable and early marker for the detection of a declining number of follicles,and prediction of spontaneous pregnancy potential and assisted reproduction technology outcome.From the current literature,AMH/MIS is such a promising and reliable marker,corresponding to the number of small antral follicles with essentially constant levels across the cycle and a superior intercycle reproducibility compared with that of FSH and early antral follicle count,Very recently some studies reported a possible benefit of serum AMH/MIS measurement in assisted reproductive programs.Diminished ovarian reserve has become a major cause of infertility.Anti-Mullerian hormone seems to be a promising candidate to assess ovarian reserve and predict the response to controlled ovarian hyperstimulation (COH).This prospective study was conducted to evaluate the relevance of AMH/MIS in a routine IVF program(2008).In conclusion,the author arrived at the AMH/MIS is a predictor of ovarian response and ovarian reserve. Measurement of AMH/MIS supports clinical decisions,but alone it is not a suitable predictor of IVF success.However,urgently needed cut-off levels of AMH/MIS for supporting clinical decisions are still missing.We therefore carried out a study on the effects of recombinant human anti-müllerian hormone(rhAMH)on the E₂ secretion of human granulosa cells in vitro,and furthere more a larger prospective trial calculating the predictive value of anti-müllerian hormone for ovary reserve and response during the control ovary hyperstimulation in in vitro fertilization and embryo transfer patients and evaluating the relevance of AMH/MIS measurement for treatment strategies and outcome in a routine IVF program.The methods involves in this study including:(1)Human ovary granulosa cells were obtained from infertile patients undergoing IVF-ET cycles and cultured in M199+10%FFCS medium.In the absence or presence of rhFSH,granulosa cells were treated by 10ng/ml rhAMH in different groups,the culture media were collected,and the E₂ and progesterone level were detected by ELISA kit.And also the AMH/MIS secretion by granulosa cells in vitro under different concentration of FSH(0IU/ml,0.02IU/ml,0.2IU/ml,2U/ml,20IU/ml)during diffenrent times(24 hours and 48 hours respectively)were measured by ELISA.(2) The level of AMH/MIS protein secretion in serum was examined by ELISA in 228 cases of infertile woman,and the level of AMH/MIS protein in follicular fluid and the AMH/MIS mRNA relative concentration in granulosa cells were examined by ELISA and RT-real-time-PCR in 71 cases who received IVF-ET treatment in the total 228 cases.The relationship between the expression of AMH/MIS and ovarian reserve were analyzed by Pearson's correlation,Spearman's correlation and T-test.The results showed that:(1)There was no great significance of rhAMH on the E₂ secretion in the absence of rhFSH(P＞0.05),while it obviously decreased the E₂ secretion with the co-effect of rhFSH(P＜0.05).There were no significance of rhAMH on the P secretion in the in vitro culture granulosa cells either in the absence or presence of rhFSH(P＞0.05).The secretion of AMH/MIS protein by the in vitro cultured granulosa cells was not significantly infuluenced by the dose and time dependent FSH stimulation(P＞0.05).(2)A positive correlation was found between serum AMH/MIS and AFC(r=0.907,p＜0.01)in 228 cases of infertile women,and it correlated negatively with Age,Age at Menarche and Years Since Menarche(r=-0.833,p＜0.01;r=-0.580,p＜0.01;r=-0.783,p＜0.01 respectively), serum AMH/MIS level and AFC correlated each other and declined significantly with age.(3)There are no significant difference between the early folliculiar phase (Day 3)serum AMH/MIS protein level with the AMH/MIS protein on day 5 after Gn injection,the day of HCG injection and the oocyte retrieval day(p＞0.05)in 71 cases of IVF-ET treatment patients.(4)A positive correlation was found between serum AMH/MIS with FF AMH/MIS and relative concentration of AMH/MIS mRNA in GC(r=0.779,p＜0.01;r=0.838,p＜0.01 respectively)in 71 cases of IVF-ET treatment patients,there are significant differences comparing serum,FF AMH/MIS level and GC AMH/MIS mRNA relative concentration with the ovarian response(p＜0.05).(5)A positive relationship was found between the early follicular phase AMH/MIS and the number of oocytes retrieved(r=0.527, p＜0.01);and there were positive correlations between the AMH/MIS mRNA level with the number of fertilized oocytes and the number of cleavage(r=0.529, p＜0.01,r=0.630,p＜0.01).(6)The early follicular phase serum AMH/MIS level in 12 OHSS patients was significantly higher than the 28 ovary poor response cases (p＜0.01).(7)A calculated cut-off level＜0.81 ng/ml AMH/MIS alone detected poor ovary response with a sensitivity of 83.6%and a false positve rate of 17.9%,and the AFC＜5 was 70.1%and 29.8%respectively.(8)The early follicular phase serum AMH/MIS protein level was negtively related to the doses of gonadotropins used in COH protocol(r=-0.323,P＜0.0),and positively related to the number of bigger than 11 mm follicles on the day of HCG injection(r=0.229,P＜0.01).In conclusions:(1)The successful completion of the culture and identification of human ovarian luteinized granulosa cells in vitro.(2)AMH/MIS influenced the steroidogenesis of granulosa cells in vitro with or without additional FSH stimulation,and may play an important role on the growth and development of the human ovarian follicles.(3)There were obvious relationships between serum AMH/MIS level with ovarian reserve and response.(4)The early follicular phase serum AMH/MIS protein level was siginificantly up-regulated in the OHSS cases comparing with the ovarian low response cases.(5)A calculated cut-off level＜0.81 ng/ml srum AMH/MIS alone detected poor ovary response with a sensitivity of 83.6%and a false positve rate of 17.9%,which is better than the predit value of AFC.(6)The first time that proof the granulosa cells AMH/MIS protein secretion was independent of the regulation of FSH through in vitro and in vivo study at the same time,it may be utilized to be a non-menstrual cycle ralated serum marker for predicting the ovarian reserve and response.(7)The early follicular phase serum AMH/MIS protein level was negtively related to the doses of gonadotropins used in COH protocol,and may be expect to apply in the clinic to guide the gonadotropin doses during the COH,in order to develop the individualized treatment plan for infertile patients involved in clinical practise.