Effect Of Premature Luteinization On IVF-ET Clinical Outcome In Long GnRH Agonist Cycles And Preliminary Study On Its Molecular Mechanism

Objective: To evaluate the effect of premature luteinization (PL)on the clinical outcomes and analyse the cause of PL in long GnRH-a cycles. Methods: 2278 patients with GnRH-a down regulation long protocol in our institution from May 2003 to May 2006 were assessed retrospectively. The patients were grouped (PL group and non-PL group) according to serum P cut-off threshold value on the day of hCG administration (P>1.1 ng/mL or P<1.1 ng/mL) which was determined by receiver operating characteristic (ROC) curve. Of them, 316 patients were sampling randomly from both groups respectively to compare clinical variable. Ovarian down-regulation was achieved using GnRH agonist downregulation long protocol with injection of GnRH-a in the midluteal phase of the previous cycle. Ovarian stimulation was started with daily injections of r-FSH (Gonal F, 75 IU, Serono Switzerland) or human menopausal gonadotrophin (hMG) until the day of hCG administration. The cut-off progesterone threshold was determined by ROC curve. The main outcome measures included fertilization rates and clinical pregnancy rates. Groups were compared by using the unpaired Student t, chi-squared test. The relationships between P level on hCG day and other variables were analyzed by multiple linear regression analysis. P<0.05 was considered statistically significant.Results: The ROC curve showed a progesterone value (P>1.1 ng/mL ) as the most efficient cut-off threshold to discriminated between the pregnant and no pregnant cycles with a 94.6% sensitivity,45.6% specificity. Four hundred forty six (19.6%) of the 2278 patients enrolling in this study demonstrated premature luteinization. The total FSH dose(1949.0±595.8 vs. 1742.9±616.9), E₂(3065.95±1319.8 vs. 2435.3±1063.5), LH (3.13±2.8 vs. 2.36±2.99) level on hCG day, number of mature oocytes retrieved (13.3±5.7 vs. 11.3±5.6)were significantly higher in PL group as compared to the non-PL group. However, the fertilization rate (58.3±0.36% vs69.2±0.24%)and the number of good quality embryo(2.3±3.8vs3.5±2.6)were significantly lower in the PL group. The multiple linear regression analysis did not show a positive and significant association between the total FSH dose, LH, PRL and the serum P levels, but show a positive and significant association between E₂ (r=0.135, P=0.016), number of oocytes retrieved (r=0.239, P<0.01) and the serum P level. The fertilization rate and number of high quality embryos were similar between the two groups. The clinical pregnancy rate (38.6% vs. 48.1%) was significantly lower in the PL group.Conclusions: The best P cut-off value to define premature luteinization may be P>1.1 ng/ml. Premature luteinization could adversely affect the pregnancy rate of IVF-ET in long GnRH-a cycles and it seems to have negative impact on oocytes quality. Premature luteinization could not be related to low ovarian reserve. Premature progesterone elevation of PL is related to E₂ on hCG day and number of mature oocytes retrieved. Objective: A premature serum progesterone (P) elevation untimely before HCG administration during controlled ovarian hyperstimulation (COH) and the concomitant process of granulosa cells differentiate into luteal cells is usually called premature luteinization (PL), which could adversely affect clinical outcomes in IVF-ET. However, the molecular mechanisms of PL are not completely understood. The aim of this stuy is to explore the molecular mechanisms of premature luteinization.Methods: Suppression subtractive cDNA hybridization was performed to identify genes differentially expressed in granulosa cell of premature luteinization. Differentially expressed genes were confirmed by macroarray dot blot analysis and real- time PCR. The differential cDNA fragments was sequenced and further analyzed by bioinformatics.Results: 179 putative positive clones were randomly picked,and 86 clones were sequenced and searched in the GenBank. Among them, 84 clones represented 47 identified genes. These genes were involved in steroidogenesis, translation,cell adhesion,cellular metabolism, lipid metablism and apoptosis.Conclusions: The differentially cDNA subtractive library of premature luteinization was first successfully constructed. The differentially expressed genes may have an important role in process of PL. Further studies of these genes should shed new light on understanding the molecular mechanism of premature luteinization. Objective: To explore the relationship between the differentially expressed genes and the process of premature luteinization.Methods: In the previous chapter we have constructed the differentially cDNA subtractive library of premature luteinization successfully and suggested these differentially expressed genes may have an important role in process of premature luteinization. According to their potential roles in the premature luteinization ,several genes and their encoding proteins were selected from the SSH cDNA library for further studies at mRNA or protein level by real-time PCR and Western blot. And we also examined the effect of growth differentiation facor-9 treatment on the expression of steroidogenic acute regulatory protein (StAR) by Western blot.Results: As the results,we observed a remarkable mRNA up-regulation of steroidogenic acute regulatory protein (StAR), Acetyl-CoA carboxylase (ACC), P450 side chain cleavage enzyme (P450scc), pleckstrin homology- like domain, family A, member l(PHLDAl) and ubiquitin protein ligase E3 in PL granulosa cell and concurrent mRNA down-regulation of growth differentiation factor-9 (GDF9) and bcl-2. Significant up-regulations were also observed in the StAR (30kD), PHLDA1(40kD) protein levels. However, the GDF9(51kD) and bcl-2 (26kDa) protein level significantly reduced in the PL granulosa cells. GDF-9 treatment inhibited premature luteinization granulosa StAR expression but did not affect basal StAR expression.Conclusions: So we concluded the increased expression of StAR, attenuated GDF9 protein and apoptosis induced by increased PHLDA1 and decreased bcl-2 protein may be responsible for the premature luteinization. Objective: To investigate the relationship between premature luteinizaton and granulosa cell apoptosis in controlled ovarian hyperstimulation and to investigate the anti-apoptotic effect of growth differentiation factor-9 (GDF9) in the premature luteinizaton granulosa cells.Methods: Granulosa cell of premature luteinization and non-premature luteinizaton patients were isolated and determined by Annexin-V/ propidium iodide (PI) Double- Staining by flow cytometry or were exposed to 200ng/ml GDF9 and then determined by the same method.Results: The percentage of morphological abnormal and apoptotic granulosa cell was higher in the premature luteinization group(P>1.1ng/ ml) than that of non premature luteinization group(P<1.1 ng/ml).The apoptosis percentage was significantly decreased after treatment by 200ng/ml GDF9.Conclusions: The increased apoptosis activity of premature luteinization granulosa cell may be related to decreased GDF9 level.