Objective To investigate the treatment options of clomiphene(CC) resistant polycystic ovary syndrome (PCOS) related infertility. Methods Figty nine PCOS patients with CC resistant anovulation were accepted and treated by the following three protcols :(1)follicle stimulating hormone (FSH) group, 49 cycles; (2)FSH +pulsatile gonadotropin releasing hormone(GnRH) group, 13 cycles; (3)conventional in vitro fertilization (IVF)_embryo transfer(ET) group, 19 cycles. Suppressive treatment on serum luteinising hormone (LH)and testosterone (T) levels was given in the first and second groups in advance. Serum estradiol level; pregnancy rate; miscarriage rate, ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy rate were compared among the three groups. Results The pregnancy rate in FSH group, FSH+GnRH group and IVF ET group werte 37%,54% and 22% respectively. The highest rate and multiple pregnancy rate was found in IVF ET group. Conclusion The rationale treatment options for CC resistant PCOS related infertility was the addition pre suppressive treatment, low dose FSH stimulated regimen subsequent pulsatile GnRH infusion. IVF were only accepted after failure with gonadotropin therapy.

Objective To investigate the premature spontaneous ovulation rates in in vitro fertilization-embryo transfer (IVF-ET) cycles using gonadotropin-releasing hormone antagonist (GnRH-ant) and gonadotropin-releasing hormone agonist (GnRH-a), as well as the risk factors for premature spontaneous ovulation. Methods The rates of premature spontaneous ovulation in a total of 10 612 cycles using GnRH-ant or GnRH-a were compared. Matched case-controlled study and binary logistic regression model were conducted to analyze the risk factors for premature spontaneous ovulation. Results The spontaneous ovulation rate in the whole for GnRH-a cycles was 0.15%(13/8 514), compared with a 1.62%(34/2 098) in GnRH-ant cycles (P<0.01). Further matched controlled study and regression analyze found out that higher basal FSH level was a predominant risk and prediction factor for spontaneous ovulation (OR=1.20, P=0.009). Conclusions In GnRH-ant cycles, spontaneous ovulation rate is about 10 times than which in GnRH-a cycles. Diminished ovarian function is a predominate risk factor for premature spontaneous ovulation.

AIM: To explore the activation effect of calcium ionophore A23187 on unfertilized human mature oocytes after conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Thirty-seven unfertilized mature oocytes from IVF and 41 after ICSI were included in our experiment. They were incubated in 5 ?mol/L calcium ionophore A23187 for 5 minutes. Second polar body extrusion and pronuclear formation were recorded 12-16 hours later. The activated oocytes were cultured for another 2 days in vitro. RESULTS: Activation rate of unfertilized oocytes from conventional IVF and ICSI were 64.9%(24/37)and 73.2%(30/41), respectively. Among 41 unfertilized oocytes after ICSI treated with A23187, 30 were activated and 24 had 2 polar body (PB) and 2 pronuclear (PN). But for the unfertilized oocytes from conventional IVF only 20% activated oocytes had 2 PN and 2 PB. The percentage difference of oocytes containing 2 PB and 2 PN between the two groups was significant ( P

AIM: To investigate the optimal materials and culture system of human primordial germ cells (PGCs) in order to establish human embryonic germ (EG) cell lines. METHODS: Human embryos of different gestational age were collected to isolate human PGCs. The isolated human PGCs were cultured in different medium and on different feeder layers, then their growth, proliferation and differentiation in different culture systems were observed. RESTILTS: The formation rate of primary colonies was higher when human PGCs were obtained with enzyme-mechanical method from 8-and 9-weeks gestational age human embryos than that from 7-weeks. Human PGCs grew better and maintained undifferentiating when mouse embryonic fibroblast or STO cells served as feeder layers and in conditional medium with hLIF, hbFGF, hSCF. CONCLUSION: 8-and 9-week gestational age human embryo are optimal material for isolating human PGCs. Enzyme-mechanical method is simple and available to isolate human PGCs. Feeder layer and growth factors are necessary for human PGCs culture in vitro.

[Objective] The aim was to choose the best feeder layer by observing the effects of various human feeders supporting human embryonic stem cells (hESCs), and to probe the correlation between the levels of basic fibroblast growth factor (bFGF) secreted by feeders and the growth of the hESCs. [Methods] The primary cells from various tissues were cultured, including foreskin, stromal endometrium, villus, adult fallopian tubal, fetal skin, fetal muscle and mouse embryonic fibroblasts (MEFs). The hESCs were transferred to various feeders, and then the best condition was probed, which was based on the feeder density and the time of mitomycin-C acting on the feeder. Comparing the characteristics of the hESCs, the best feeder was chosen of all kinds of feeders from various tissues that support the hESCs. The level of bFGF secreted by various feeders was detected using ELISA. [Results] All of tested feeders could support the hESCs growth for over 10 passages in the culture, especially the foreskin and the adult fallopian tubal. The density of feeders was related with the morphology and the differentiation rate of the hESCs. According to the characteristics of feeder, the feeder ranking was as follows: foreskin, stromal endometrium, villus, adult fallopian tubal, fetal skin and fetal muscle. Based on the characteristics of the hESCs, the order of feeders was: foreskin, adult fallopian tubal, stromal endometrium, villus, fetal muscle and fetal skin. The levels of bFGF (pg·10~(-5)·mL~(-1)) secreted by various feeders were as follows: adult fallopian tubal (13.23±3.39), foreskin (1.99±0.17), villus (1.40±0.17), fetal muscle (2.02 ±1.59), stromal endometrium (0.38±0.28), and fetal skin (0.29±0.29). [Conclusion] The foreskin and the adult fallopian tubal could support the hESCs better than others though all of them could;do, especially the, foreskin. The bFGF that secreted by the adult fallopian tubal was the highest of all. The correlation was not obvious .between the levels of bFGF secreted by feeders and the growth of the hESCs.

AIM: To study the effect of double gene transduction mediated by lentiviral vectors on the characteristics of human embryonic stem cells (hESCs). METHODS: Using the backbone from inducibal dual and excisable transgene vector (iDuet101) , lentiviral vectors overexpressing cytotoxic T lymphocyte - associated molecule - 4 immuno-globulin (iDuet101 - CTLA4Ig) , indoleamine 2, 3 dioxygenase (iDuet101 - IDO) , and ubiquitin - C promoter - lueifer-ase - ires - puromycin ( ULIP) were constructed and packaged according to the standard recombinant techniques. Human embryonic stem cells (hESCs) were first transduced with iDuet101, iDuet101 - CTLA4Ig or iDuet101 - IDO, then, after the selection, were transduced again with ULIP. The expression and function of the exogenous genes were detected. Immu-nohistochemistry, RT - PCR and flow cytometry were applied for detection of embryoid bodies ( EB) formation in vitro and in vivo teratoma formation. RESULTS: Double - transduced hESCs showed typical shape of cell clones and positive staining of tumor rejection antigen -1 - 60 ( Tra -1 - 60 ) and octomer transcription factor - 4 ( OCT - 4 ). The formation of EB was observed, in which a - fetoprotein (AFP), paired box gene 6 ( Pax6) and Musashi 1 ( MSI1) were positively expressed. The cells formed teratomas, and the luciferase signals existed until 28 days after xeno - transplantation. CONCLUSION : Double transduction of non - transcriptional factors mediated by lentiviral vectors does not affect the cell growth rate and their differentiation ability.

[Objective] This study compared outcomes of in vitro maturation (IVM) and in vitro fertilization (IVF) intracytoplasmic sperm injection (ICSI) cycles after IVM of immature germinal vesicle (GV) oocytes.[Methods] ICSI was performed on metaphase II (MII) oocytes retrieved in 163 IVF-ICSI cycles (group I;n ＝ 987) or matured from GV stage oocytes in IVF-ICSI ( group II;n ＝ 132) and 37 IVM cycles ( group III;n ＝ 235).Fertilization and cleavage rates and embryo quality were compared among the three groups.[Results] The fertilization rate,cleavage rate and top quality embryos rate were higher in group I than group II and group III (84.9%,98.1%,and 61.6%;72.0%,90.5% and 22.1%l;75.3%,94.4%,and 25.1%,respectively).Blastomere numbers and morphology scores were highest in group I (P ＜ 0.05),but no significant differences existed between group II and group III.[Conclusion] The morphology of embryos developed from in vivo MII oocytes was superior to those from in vitro matured MII oocytes.No significant difference was observed in embryo morphology from immature GV oocytes in IVF and IVM cycles.

Objective Try to determine the relationship between blastocyst quality,the clonal growth of inner cell mass(ICM)and the establishment of human embryonic cell line. Methods Coculture D 3 discarded embryos with mouse embryonic fibroblast cells(MEFs).Then remove trophoectoderm by immunosurgery after getting different quality blastocysts.Culture ICM and passage these cells on MEFs. Results Human embryonic stem cells derived from good quality blastocysts could be passaged further than that from poor quality blastocysts,and ICMs growing fast could be passaged more quickly and efficiently.Conclusion The establishment of human embryonic stem cells is closely related with blastocyst quality and the original growth of ICM.

【Objective】To perform preimplanation genetic diagnosis by using dual color fluorescent in-situ hybridization (FISH).【Methods】The chemical and mechanical division methods were used to perform embryo biopsy in 3 cases of Robertsonian translocation t (13q14q).Vysis LSI 13q14 and Tel Vysion 14q probes were used to detect the blastomeres biopsied from the IVF embryos of the patients.FISH analysis was performed to select normal or balanced karyotype embryos ,which then were transfered into the uterus.【Results】Total of 23 oocytes were retrieved in 3 treatment cycles.Fertilization rate was 79%.14 embryos were available for embryo biopsy.Among them,9 embryos were biopsied by chemical division method,with further cleavage rate of 67%;5 embryos were biopsied by mechanical division method,with further cleavage rate of 40%.Single embryo was diagnosed as normal karyotype or balanced respectively in 2 treatment cycles.Both of them were transfered into the uterus.One clinical normal on-going pregnancy was achieved,the diagnosis was confirmed by amniocyte karyotype analysis.【Conclusion】Preimplanation genetic diagnosis can be used to resolve the problem of fertility for Robertsonian Translocation Carriers.

Objective To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD)techniques through clinical analysis on PGD cycles. Methods Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Results Among 354 embryos biopsied by PGD for translocations, 321 (90. 7% ) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38. 3% (64/167),which was significantly higher than 20. 8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82. 5% ( 443/537 ). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34. 4% (77/224), which was significantly higher than 19. 6% ( 19/97 ) of biopsed embryos with ＜ 7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% ( 57/96 ), which was significantly higher than 34. 2% ( 77/225 ) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43. 1% ( 25/58 ) . Conclusions PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.