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Objective: To identify whether epistasis between TG and HSD17B4 and whether polymorphisms in HSD17B4 are associated with premature ovarian failure (POF)., Design: Case-control genetic association study., Setting: Research laboratory of a university., Patient(s): Female patients with POF (98) and controls (218) of Korean ethnicity participated in this study., Intervention(s): None., Main Outcome Measure(s): Genotype distribution, haplotype (HT) inference, and gene-gene interaction., Result(s): Distribution of one haplotype (A-G-A-A-G-G) on the HSD17B4 gene was significantly different between the POF group and the control group in a dominant model. In addition, the combined effect of the single nucleotide polymorphisms (SNPs) HSD17B4 rs28943592 and TG rs2076740 was significantly associated with POF (odds ratio = 7.74, 95% confidence interval = 1.67-35.94), although a significant association was not observed in the single SNP model., Conclusion(s): A haplotype in the HSD17B4 gene was identified that was significantly associated with resistance to POF. In addition, epistasis between two missense SNPs (rs28943592, rs2076740) located in HSD17B4 and TG was significantly associated with susceptibility to POF., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Follicle-stimulating hormone secreted from the pituitary gland plays a key role in human reproduction and regulates estrogen production by acting on the regulatory region of CYP19A1. We observed a significant association between premature ovarian failure and the combined genetic effect of single nucleotide polymorphism (SNP) rs4646 (CA+AA) in the 3' untranslated region of CYP19A1 and the missense FSHR SNP rs6166 (AG+GG) genotype (odds ratio 5.42, 95% confidence interval 1.96-14.98), and we identified a significant association between premature ovarian failure and the combined genetic effect of the FSHR missense SNP rs6166 (AA) and the rs4646-rs10046 haplotype (C-T)+(C-C) (odds ratio 5.47, 95% confidence interval 2.03-14.75), suggesting that two biochemical pathways may be involved in the regulation of folliculogenesis., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the association of vascular endothelial growth factor (VEGF) polymorphisms (-2578C>A, -1154G>A, -634G>C, 936C>T) with idiopathic recurrent spontaneous abortion (RSA) in Koreans., Design: Prospective case-control study., Setting: University-based hospital., Patient(s): Two hundred and fifteen patients with a history of two or more unexplained consecutive pregnancy losses and 113 healthy controls with at least one live birth and no history of pregnancy loss., Intervention(s): None., Main Outcome Measure(s): Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses were performed for the -2578C>A and 936C>T genotypes. Real-time PCR was also used to analyze the -1154G>A and -634G>C genotypes., Result(s): The GA (adjusted odds ratio [AOR] 2.774; 95% confidence interval [CI] 1.512-5.092) genotype of the VEGF -1154G>A polymorphism was significantly different between women with idiopathic RSA and controls. The difference in overall (GA + AA) frequency was also marginally significant between the controls and patients with idiopathic RSA (AOR, 2.006; 95% CI, 1.158-3.473). The differences in frequencies of the A-A-G-T and C-A-G-T haplotypes of the VEGF polymorphisms (-2578C>A, -1154G>A, -634G>C, 936C>T) were marginally significant between the patient and control groups., Conclusion(s): This study suggests that VEGF polymorphisms and haplotypes are a genetic determinant for the risk of idiopathic RSA in Korean women., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the usefulness of the vitrification method in clinical practice., Design: Clinical study of vitrification of human oocytes., Setting: A university-affiliated hospital., Patient(s): Thirty-four patients who agreed to undergo additional IVF-ET after the failed fresh cycle using the previously vitrified/thawed oocytes., Intervention(s): Surplus oocytes from the IVF-ET patients were vitrified for the next cycle., Main Outcome Measures: Morphologic normality of post-thawed oocytes, fertilization, cleavage, pregnancy, and implantation rate., Result(s): Overall morphological survival and fertilization rates of vitrified/liquefied oocytes were 68.6% (325/474) and 71.7% (142/198), respectively. Pregnancy rate and implantation rate per embryo transfer were 21.4% (6/28) and 6.4% (8/125), respectively. All pregnancies resulted in the delivery of healthy babies (1 twin and 5 singletons/6 pregnancies)., Conclusion(s): The feasibility of the vitrification method for human oocytes was confirmed by our clinical results. Subsequent studies on vitrification and thawing procedures should be undertaken for further optimizing the vitrification method.

Objective: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development., Design: In vitro model study using mouse embryos., Setting: University affiliated hospital, Pochon CHA University., Animals: Four-week-old block strain ICR mice naturally mated after superovulation., Intervention(s): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 microg/ml Hb and/or 0.1 mM EDTA were added., Main Outcome Measure(s): Preimplantation development and blastomere number., Result(s): More (P<.05) 1-cell embryos developed to the 4-cell (52% vs. 67%-84%), 8-cell (48% vs. 65%-81%), and blastocyst (40% vs. 61%-79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb+EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb+EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb+EDTA than after no addition (0.76 vs. 0.96-0.98). Significant increases in the cell number of blastocysts (46.5-47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7-17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3-0.37 to 0.39) were found after the combined addition of Hb+EDTA, compared with no addition or with the addition of EDTA or Hb alone., Conclusions: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts.

Objective: To improve the efficacy of an IVF-ET program for unstimulated patients with polycystic ovary syndrome (PCOS) with the use of culture for oocyte maturation., Design: Prospective studies with the comparison of different ET procedures from March 1995 through February 1998., Setting: University-affiliated hospital., Patient(s): Ninety-four cycles in 64 consenting patients with PCOS., Intervention(s): Immature oocytes were retrieved from unstimulated patients with PCOS and subsequently cultured and fertilized in vitro. Zygote intrafallopian transfer (ZIFT), uterine ET, or a combined approach of ZIFT + uterine ET was subsequently performed., Main Outcome Measure(s): Laboratory and clinical data., Result(s): Among 1, 280 immature oocytes (13.6 +/- 7.5 oocytes per patient) retrieved, 89% (1,139) were morphologically normal, and 62.2% (708/1,139) of the normal oocytes matured in vitro after culture for 48 hours. When intracytoplasmic sperm injection was performed, 68% (481/708) developed to the normal pronuclear stage, and 88.1% of the embryos cocultured with Vero cells (266/302) cleaved. Eighty-five ET cycles were conducted and pregnancy was established in 23 cycles (27.1%), which consisted of 8 after uterine ET and 15 after a combined approach. Seventeen patients delivered 20 normal infants., Conclusion(s): The IVF-ET method using no ovarian stimulation followed by in vitro maturation culture can be a feasible assisted reproductive technology for treatment of PCOS with various complications.

Objective: To evaluate the developmental competence and chromosomal normality of oocytes vitrified at various times after maturation culture., Design: In vitro model study., Setting: A university-affiliated hospital., Patient(s): Unstimulated women who underwent cesarean section or oophorectomy and infertile women who underwent a long protocol of GnRH stimulation., Intervention(s): Retrieved oocytes were vitrified at 0 or 48 hours after culture in unstimulated cycles and at 0, 8-15, or 24-28 hours after culture in stimulated cycles., Main Outcome Measure(s): Postthaw morphologic normality, maturation, fertilization, cleavage, blastocyst formation, and chromosome number., Result(s): In the 53 oocytes that were obtained from unstimulated cycles, no statistically significant differences were found in rates of morphologic normality (range, 56%-63%) or fertilization (range, 31%-37%) according to the time of vitrification. In the 50 oocytes that were obtained from stimulated cycles, more of those that were vitrified at 24-28 hours were morphologically normal than those that were vitrified at 0 or 8-15 hours. Regardless of these differences, high cleavage rates (83%-100%) were obtained that did not differ significantly among the treatment groups. In both cycles, 20%-43% of cleaved oocytes developed to the blastocyst stage by 6 days after IVF. All the karyotyped blastocysts, three from unstimulated cycles and four from stimulated cycles, had a normal number of chromosomes., Conclusion(s): Vitrified and thawed oocytes from unstimulated or stimulated cycles developed to the blastocyst stage, regardless of when vitrification occurred; the number of chromosomes in the blastocysts was normal.

Objective: To evaluate the developmental competence of vitrified human oocytes thawed using two different methods to establish an effective cryopreservation protocol., Design: In vitro model study., Setting: University-affiliated hospital., Patient(s): Patients who underwent a long protocol of ovarian stimulation with GnRH and gonadotropins., Intervention(s): Vitrified oocytes from the patients were thawed using either a four-step method with 2.5-minute intervals or a four-step method with 5-minute intervals., Main Outcome Measure(s): Morphologic normality, maturation, fertilization, and development of the oocytes to the blastocyst stage., Result(s): The two thawing methods did not significantly affect the morphologic normality (84%-100%), maturation (75%-100%), fertilization (38%-71%), polyspermy (more than three pronuclei; 0%-20%), or parthenogenetic activation (only female pronucleus; 0%-8%) of the vitrified oocytes. However, more of the vitrified oocytes developed to the two-cell (71%-100% versus 50%-67%), four-cell (71%-93% versus 0%-50%), eight-cell (46%-71% versus 0%), and blastocyst (23%-36% versus 0%) stages after thawing using the four-step method with 2.5-minute intervals than using the four-step method with 5-minute intervals., Conclusion(s): Vitrified human oocytes developed to the blastocyst stage with IVF. A four-step thawing method with 2.5-minute intervals was more effective in supporting preimplantation embryo development than a four-step thawing method with 5-minute intervals.

Objective: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes., Design: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours., Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea., Patient(s): Oocytes were obtained from patients undergoing gynecologic surgery., Main Outcome Measure(s): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin., Result(s): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3., Conclusion(s): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.

Objective: To investigate effects of 1,2-propanediol and freezing-thawing treatment on the maturation and developmental capacity of the human immature oocytes obtained from unstimulated ovaries., Design: Intact cumulus-enclosed immature oocytes collected from unstimulated ovaries were divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol-treated (group 2), and cryopreserved group (group 3). Oocytes in group 1, group 2, and survived oocytes from cryopreservation in group 3 were cultured for 48 hours. A random selection of matured oocytes was inseminated with normal donor sperm to evaluate the fertilization and developmental capacity., Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea., Patient(s): Oocytes were obtained from patients undergoing gynecological surgery., Main Outcome Measure(s): Rates of survival, maturation to metaphase II, fertilization, and cleavage., Result(s): Survival rate after freezing-thawing in group 3 was 55.1% (54/98). Oocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL hCG. Maturation rates were 76.8% (63/82), 67.1% (47/70), and 59.3% (32/54) in the groups 1, 2, and 3, respectively. Maturation rate in group 3 was significantly lower than that of group 1. Fertilization rates were 90.5% (19/21), 81.0% (17/21), and 42.9% (6/14), and cleavage rates were 94.7% (18/19), 88.2% (15/17), and 16.7% (1/6) in groups 1, 2, and 3, respectively. Fertilization and cleavage rates of survived oocytes in group 3 also were significantly lower than those of groups 1 and 2., Conclusion(s): Results suggest that the pretreatment with 1.5 M 1,2-propanediol itself before the freezing has no inhibitory effect on the maturation, fertilization, and cleavage of human immature oocytes in vitro. However, the freezing-thawing procedure used had detrimental effects on the maturation and developmental capacity.

This study describes the results with immature human follicular oocytes harvested from unstimulated ovaries, matured in vitro, fertilized, and transferred to an agonadal recipient. Two hundred seventy immature oocytes were aspirated from 23 ovaries removed for various gynecological indications from August 1988 to October 1989. The numbers of follicular oocytes collected from ovaries were compared by patients' ages and the stages of menstrual cycle. Immature oocytes in vitro were incubated with either mature follicular fluid (FF) or fetal cord serum (FCS). The maturation rate in the mature FF group was 55.8%, significantly higher than the 35.9% in the FCS group. In addition, mature FF group was shown to provide a significantly higher fertilization rate than the FCS group (81.0% versus 31.6%). More fertilized eggs developed into normal embryos in the nonstimulated cycle group than in stimulated cycles with routine treatment. Finally, five embryos were transferred to a woman with premature ovarian failure on day 18 of a steroid replacement cycle. She subsequently delivered healthy triplet girls. These results suggest that in vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in a donor oocyte program after in vitro fertilization.