Jung Jae Ko

Live births after vitrification of oocytes in a stimulated in vitro fertilization–embryo transfer program

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.

Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.

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.

Viability of Human Follicular Oocytes Collected from Unstimulated Ovaries and Matured and Fertilized in vitro

Immature human follicular oocytes were collected from unstimulated ovaries, matured and fertilized in vitro and then transferred to patients with no ovarian dysfunction such as premature ovarian failure. From 11 1 consenting donors, 422 immature oocytes were collected from 97 ovaries between January 1990 and October 1991. The number of oocytes collected from ovaries and their development were recorded so that comparisons could be made among donors of different ages and ovarian condition, such as menstrual cycle, cyclic and non-cyclic ovaries. The rate of fertilization in vitro showed a peak in the 31-40-year age group; however, there was no statistical difference in the rate of oocyte maturation and cleavage among the donors in the different age groups. Immature oocytes of the luted phase had a significantly higher maturation rate than those of the follicular phase. There was no significant difference in the number of recovered oocytes, or in the development of immature follicular oocytes, between cyclic and non-cyclic ovaries. Mature follicular fluid and peritoneal fluid had a significant effect on the development of immature follicular oocytes. Also, it was found that fertilized eggs cleaved more frequently in the medium containing hypoxanthine compared with the medium without hypoxanthine. Finally, from 21 transfer cycles, viable embryos were derived from immature follicular oocytes, resulting in two pregnancies, both leading to the birth of normal babies. These findings suggest that culture in vitro of immature follicular oocytes, from unstimulated ovaries, to a suitable condition, could be used optimally for clinical applications such as human ovum donation programmes.

Melatonin prevents cisplatin-induced primordial follicle loss via suppression of PTEN/AKT/FOXO3a pathway activation in the mouse ovary

Premature ovarian failure (POF) is a major side effect of chemotherapy in young cancer patients. To develop pharmaceutical agents for preserving fertility, it is necessary to understand the mechanisms responsible for chemotherapy‐induced follicle loss. Here, we show that treatment with cisplatin, a widely used anticancer drug, depleted the dormant follicle pool in mouse ovaries by excessive activation of the primordial follicles, without inducing follicular apoptosis. Moreover, we show that co‐treatment with the antioxidant melatonin prevented cisplatin‐induced disruption of the follicle reserve. We quantified the various stages of growing follicles, including primordial, primary, secondary, and antral, to demonstrate that cisplatin treatment alone significantly decreased, whereas melatonin co‐treatment preserved, the number of primordial follicles in the ovary. Importantly, analysis of the PTEN/AKT/FOXO3a pathway demonstrated that melatonin significantly decreased the cisplatin‐mediated inhibitory phosphorylation of PTEN, a key negative regulator of dormant follicle activation. Moreover, melatonin prevented the cisplatin‐induced activating phosphorylation of AKT, GSK3β, and FOXO3a, all of which trigger follicle activation. Additionally, we show that melatonin inhibited the cisplatin‐induced inhibitory phosphorylation and nuclear export of FOXO3a, which is required in the nucleus to maintain dormancy of the primordial follicles. These findings demonstrate that melatonin attenuates cisplatin‐induced follicle loss by preventing the phosphorylation of PTEN/AKT/FOXO3a pathway members; thus, melatonin is a potential therapeutic agent for ovarian protection and fertility preservation during chemotherapy in female cancer patients.

Comparison of gene expression at the fetomaternal interface between normal and recurrent pregnancy loss patients

Normal pregnancy requires a series of immunological, metabolic, vascular and endocrine regulating processes. However, the specific genes and proteins involved in these processes are not well defined. Aberration of these processes may lead to problems in pregnancy. One of these problems may be recurrent pregnancy loss (RPL). Little information is available on the level of expression of genes that may play a role in normal pregnancy. Therefore, this study determined whether different levels of gene expression at the feto-maternal interface could be associated with factors for RPL. The expression patterns of genes isolated from subtractive hybridization analysis performed with chorionic villi from normal and abnormal pregnancies were investigated. Eight genes classified into groups, including immunosuppression-related, embryo attachment-related and angiogenesis-related, were isolated.

Increased Expression and Localization of a Serine Protease Inhibitor, Protease Nexin-1 (PN-1), in the Ovary and Uterus During Implantation in Rat

Protease nexin-1 (PN-1) is a serine protease inhibitor (serpin) that inactivates several proteases, including thrombin, urokinase, plasminogen activators (PA), and plasmin. It also plays a role in regulating proteolytic activity generated by PA system. PN-1 is known to be involved in tissue remodeling, cellular invasiveness, matrix degradation, and tumor growth. However, the role of PN-1 in female reproductive tracts, such as the uterus, ovary, and oviduct, during pregnancy is not known. The present study was designed to investigate the changes of PN-1 mRNA level and localization in the tracts during implantation and early pregnancy by using reverse transcription (RT)-polymerase chain reaction (PCR) and in situ hybridization. We found that PN-1 mRNA levels were coordinately regulated during early pregnancy in a stage- and tissue-specific manner, such that an increased expression of PN-1 gene appeared at the time of the implantation period in the uterus and ovary. Both the uterus and ovary synthesized PN-1 mRNA and their maximal PN-1 expression occurred on Day 6.5 postcoitum (p.c.). On 13.5 days of pregnancy, PN-1 level was low in the uterus and ovary. On the other hand, PN-1 mRNA in the oviduct did not show after 6.5 days of pregnancy. It appears that PN-1 mRNA in the uterus and ovary was highly regulated during early pregnancy, which might have an important role in implantation of rat blastocysts. PN-1 was localized in endometrial stromal cells of the uterus and in granulosa cells of the unstimulated primary follicles in the ovary during periimplantation period. Also, PN-1 mRNA expression was higher at implantation period than that at nonimplantation period of pregnancy. In conclusion, PN-1 is expressed in female reproductive tracts and highly regulated during implantation and early pregnancy.

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

3’-UTR Polymorphisms in the MiRNA Machinery Genes DROSHA, DICER1, RAN, and XPO5 Are Associated with Colorectal Cancer Risk in a Korean Population

MicroRNAs play an important role in cancer initiation and development. The aim of this study was to investigate whether polymorphisms in miRNA machinery genes are associated with the development of colorectal cancer (CRC). RAN rs14035 CT heterozygotes and T allele carriers (CT + TT) genotypes had lower risk of CRC, while the DICER1 rs3742330, DROSHA rs10719, and XPO5 rs11077 polymorphisms were not associated with CRC in the full study sample. Specifically, male RAN rs14035 CT heterozygotes and XPO5 rs11077 AA genotype (CT/AA) carriers experienced reduced CRC susceptibility (both colon and rectal). Subgroup analysis demonstrated that the combined RAN rs14035 CT + TT genotype was associated with rectal cancer, but not colon cancer. In addition, the DICER1 rs3742330 AG genotype was associated with a significantly increased risk of colon cancer. Stratified analysis revealed the RAN rs14035 combined CT+TT genotype was associated with decreased CRC risk in male patients without diabetes mellitus (DM) and in patients with rectal cancer. In addition, we found the RAN rs14035 CC genotype was related to a decreased risk of CRC with respect to tumor size and metabolism of homocysteine and folate. Furthermore, patients diagnosed with hypertension or DM who carried the DROSHA rs10719 CC genotype showed increased CRC risk, while the XPO5 rs11077 AC+CC genotype led to increased CRC risk in patients with hypertension only. Our results indicate variations in RAN rs14035, DICER1 rs3742330, XPO5 rs11077, and DROSHA rs10719 of Korean patients are significantly associated with their risk of CRC.

Solute Carrier Family 19, Member 1 (SLC19A1) Polymorphisms (−43T>C, 80G>A, and 696C>T), and Haplotypes in Idiopathic Recurrent Spontaneous Abortion in a Korean Population

The objective was to investigate the association between idiopathic recurrent spontaneous abortion (RSA) and 3 SLC19A1 polymorphisms (−43T>C, 80G>A, and 696C>T). DNA from 269 patients with RSA and 125 controls were genotyped for the 3 SLC19A1 single nucleotide polymorphisms (SNPs) by polymerase chain reaction–restriction fragment length polymorphism. Homocysteine and folate levels of 100 patients with RSA were available for analysis. The combination genotypes of SLC19A1 −43TC/80GG, −43TC/80AA, and −43CC/80GA; 80GA/696TT, 80AA/696CC; and −43TC/696CC were less frequent in patients with RSA compared to controls (P < .05 for each). The −43C/80A/696 T and −43T/80G/696C haplotypes were more frequent in patients than controls, whereas −43T/80A/696C, −43C/80A/696C, −43C/80G/696C, −43C/80G/696T, and −43T/80G/696T haplotypes were less frequent in patients (P < .05 for each). The −43T/80G and 80A/696T haplotypes were more frequent in patients, while −43T/80A, −43C/80G, 80A/696C, 80G/696T, and −43C/696C haplotypes occurred less frequently in patients (P < .05 for each). The associations between idiopathic RSA occurrence and SLC19A1 −43T>C/80G>A/696C>T polymorphisms were identified and can be developed as biomarkers for RSA risk.

Microinjection free delivery of miRNA inhibitor into zygotes

The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.