Impact Of Progesterone Level Of Follicular Phase And The Research On The Application Of Growth Hormone In Controlled Ovarian Hyperstimulation

Controlled ovarian hyperstimulation (COH) is a technique to obtain sufficient number of ovums during a menstrual cycle by using ovulation induction drugs, so that multiple implantable embryos can be ultimately obtained. COH has been widely applied to in vitro fertilization-embryo transfer (IVF-ET) to cure infertility, and has become a key procedure during the infertility cure. With the advance of researches on assisted reproductive technology (ART) in the past two decades, significant progresses have been made in ART drugs and the schemes and monitoring methods for COH. Although the clinical pregnancy rate has been continually increased, over half of the treatment cycles are still unsuccessful. The reasons for the failure of IVF-ET include ovum mature disorder, poor ovarian response, fertilization failure, embryo development disorder and implantation failure etc. This research is separated into two parts. The major research approaches and results are as follows:Part 1 Impact of Progesterone Level of Follicular Phase on Outcome of in Vitro Fertilization in controlled ovarian hyperstimulationThe research in this section is to review and analyze the data for a part of the infertility patients who use IVF to assist pregnancy, and to investigate the impact of early follicular phase progesterone levels on the outcome of IVF. First, the progesterone level of early follicular phase of 538 cycles from May 2003 to January 2005 used down-regulation short protocol were analysised. According to the serum progesterone levels of the patients on the fourth day of gonadotropin stimulation, they were separated into three groups: group A, serum progesterone level≤lng/ml 367 patients; group B, serum progesterone level lng/ml—3 ng/ml 106 patients; group C, serum progesterone level＞3 ng/ml 65 patients, the indication, COH date and the outcome of IVF were analysised retrospectly. The results show that there was no significant difference in theE₂, LH, P concentration on the day of hCG administration, number of oocytes retrieved and the mean number of embryo transfer for each of the group. With an increase in progesterone concentration on D4 of stimulation, the fertilization rate, good-quality embryo rates decreased, but the differences between the patient groups were not significant. We observed on D4 of stimulation the LH, progesterone concentration of group B and group C was significantly higher than that of Group A (P＜0.01), and E₂ concentration of both group B and group C was significantly higher than that of Group A(P＜0.01 and＜0.05 respectively). The pregnancy rate and implantation rate of Group C were significantly lower than that of group A (P＜0.01) as was the miscarriage rate (P＜0.05) and as a result the ongoing pregnancy rate of group C was significantly lower than that of group A (P＜0.01). The implantation rate, for group C was lower than group B (P＜0.05) but the miscarriage rate was not significantly different. Group B miscarriage rate was significantly lower than that of group A (P＜0.05). Conclusion: In patients undergoing gonadotropin-releasing hormone agonist down-regulation short protocols for IVF, if the progesterone concentration＞3ng/ml on D4 of stimulation, the implantation rate and pregnancy rate were lower, if the progesterone concentration＞lng/ml the early miscarriage rate was higher.Next, analysis was conducted on the late follicular phase progesterone levels of 1408 infertility patient from April 2003 to December 2004. 692 cycles were used down-regulation short protocol, patients were divided into three groups according to serum progesterone level on the day of HCG administration,≤lng/ml 462 cycles, lng/ml—2 ng/ml 211 cycles,＞2 ng/ml 19cycles; 716 cycles were used down-regulation long protocol,≤lng/ml 588 cycles, lng/ml—2 ng/ml 108 cycles,＞2ng/ml 20 cycles. The results show that there was no significant difference in the number of oocytes retrieved, fetilization rates, embryos cleavage rates and good-quality embryo rates among three groups in both protocl groups. With the increase of progesterone level, implantation rates and pregnancy rates were decreasing, while the spontaneous abortion rates was increasing, but there were on significant differences. Conclusions: In controlled ovarian hyperstimulation, the increase of progesterone level of late follicular phase have no significant effect on the implantation rate, clinical pregnancy rate, and the spontaneous abortion rate.Part 2 A preliminary research on the application and possible mechanism of growth hormone on poor ovarian response patients in controlled ovarian hyperstimulationIn controlled ovarian hyperstimulation, poor ovarian response is one of the difficulties that hassle the clinicians of assisted reproduction. It is also a common reason for ovulation induction cycle cancellation. People have realized that the gonadotropin is not the only regulative factor of ovarian function, and that the growth hormone(GH) and various polypeptide growth factors also have significant effects on the development of the ovary and ovarian follicle. The research work in this part is to investigate the application value of GH to the patients with poor ovarian response in COH. 38 infertility patients with poor ovarian response were divided into GH group (n=21) and control group(n=17) in random. Each cycle was used down-regulation short protocol, the patients in GH group were administrated GH 4 IU everyday from the day 2-3. the processe of controlled ovarian hyperstimulation and outcome were compared. The results show that there was no significant difference in the the mean age,the length and etiology of infertility,the stimulation date,include the hormone result at any time,the dose od Gn and stimulate time, number of oocytes retrieved fetilization rates,embryos cleavage rates,good-quality embryo rates and the mean number of embryo transfer for every group. Finally, the pregnancy rate was 42.1% in growth hormone group, while it was 13.3% in control group only. A patient in growth hormone group was accepted ectopic pregnancy, the other are in ongoing pregnancy.In the two groups, there was no significant difference in the level of serum GH, inhibinB,IGF-â… , IGF-â…¡and IGFBP3 in D2; there was no significant difference in the level of inhibinB in D6; Compared with control group, the level of serum IGF-â… in the day of hCG was significant higher in GH group (P＜0.000); the level of serum IGFBP3 in the day of hCG was significant lower in GH group (P＜0.000); While there was no significant difference in the level of serum IGF-â…¡on the day of hCG; There was no significant difference in the level of follicular fluid IGF-â… ,IGF-â…¡and IGFBP3 between the two groups.We have made a preliminary research on how the GH could improve the pregnancy rate. By setting up an in vitro culture system of luteinized granulosa cells and adding GH and/or FSH of different concentrations to the culture system, we investigated the in vitro regulation of steroid hormone of grain cell by growth hormone, and provided basis for further examination of its impact on the development of follicle. The results show that, with the concentration of GH increasing, the oestradiol concentration increased also. Compared with control, when the conncentration of GH was 500ng/ml, the culture medium produced a marked seretion of oestradiol (P＜0.05); when the conncentration of GH was 1000ng/ml, the culture medium produced a marked seretion of oestradiol (P＜0.05) also; While ther were no difference of progesteron concentration between every group; When the same FSH concentration, with the concentration of GH increasing, the oestradiol concentration increased; When the same GH concentration, with the concentration of FSH increasing, the oestradiol concentration increased. Compared with FSH control, when the conncentration of FSH was 1000ng/ml, the culture medium produced a marked seretion of oestradiol (P＜0.05) respectively. While ther were no difference of progesteron concentration between every group.We collected the luteinized granulosa cells with and without GH(500ng/ml) treatment. We screened the differentially expressed ESTs by means of suppression subtractive hybridization(SSH) based on SMART (Switching Mechanism At 5' end of the RNA Transcript) technique. The GH treated cells was used as tester, and the control cells was used as driver. We acquired the differentially expressed cDNA products after GH treantment. The amplification of internal contrl of G3PDH gene showed the subtractive efficiency was reached to approximately 10 cycles(about 1000 times). Then the cDNA products were cloned into pUCm-T vector, and blue-white blot screening method and PCR technique were used to selecte out the clones between the luteinized granulosa cells with and without GH treatment. Reverse dot hybridization showed that most clones were expressed differentially in the luteinized granulosa cells with and without GH treatment. 60 clones were selected out for analysis. Among them, 52 clones represented 23 identified genes that share high homology with sequences in GenBank. Four clones represented three hypothetical genes, and four clones were function-unknown EST. Five genes (CSNK2A1, EPS15, SP3, PSME2, RDX) which were represented by five EST were selected out for semi-quantitative RT-PCR. The results showed that the five genes were found expressed up-regulated in the luteinized granulosa cells after GH treatment. Researches on the functions of these genes will provide basis for the explantation of the mechanism of GH to the development of ovarian granulosa cell and follicle.