Kwang Yul Cha

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.

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.

Does supplementation of in-vitro culture medium with melatonin improve IVF outcome in PCOS?

Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (P<0.05). Pregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer. Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes of IVM IVF-embryo transfer using IVM medium alone or supplemented with melatonin were analysed. In the culture media of GC or COC, the melatonin concentration gradually increased. With human chorionic gonadotrophin priming, the pregnancy and implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control (P<0.05). Our findings suggest that follicular melatonin is released from luteinizing GC during late folliculogenesis and plays a positive role in oocyte maturation. Therefore, addition of melatonin into IVM medium may improve cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes.

Genetic analysis of three polymorphic sites of the luteinizing hormone β-subunit gene in infertile Korean men with nonobstructive azoospermia

OBJECTIVE To investigate the genetic background of nonobstructive male factor infertility. DESIGN Clinical and controlled study. PATIENT(S) Ninety-five nonobstructive male infertile patients (75 with azoospermia, 18 with oligoasthenoteratozoospermia, and two with oligozoospermia) and 200 healthy fertile control men. INTERVENTION(S) Patients were investigated for genetic background including karyotype, Yq chromosome deletion, and three polymorphisms of the LH beta-subunit gene (Trp8Arg, Ile15Thr, and Gly102Ser). MAIN OUTCOME MEASURE(S) To determine three polymorphisms of the LH beta-subunit gene. RESULT(S) An abnormal karyotype was found in 11 of the 75 patients with azoospermia and one of the 18 patients with oligoasthenoteratozoospermia. Eleven (12%) had one or more deleted sites at 13 loci on Yq. The Gly102Ser variant of the LH beta-subunit gene was not detected at all. The frequency of double Trp8Arg and Ile15Thr heterozygotes was similar between the fertile (14.5%, n = 200) and infertile (12.6%, n = 95) groups, with the exception of one homozygous mutation (Arg8 and Thr15) from patient with azoospermia. CONCLUSION(S) Three variants of the LH beta-subunit gene (Trp8Arg, Ile15Thr, and Gly102Ser) may not be associated with male factor infertility. We found one homozygous Arg8 and Thr15 mutation in a patient with azoospermia with normal hormone levels (FSH, LH, PRL, T), a normal karyotype, and no Yq microdeletions.

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.

Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo

We have developed a good manufacturing practice for long‐term cultivation of fetal human midbrain‐derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region‐specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum‐free conditions and standardized operating protocols under clean‐room conditions. Long‐term‐cultivated midbrain‐derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9‐specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain‐derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain‐derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long‐term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high‐content or high‐throughput screening. Stem Cells Translational Medicine 2017;6:576–588

The 2nd International Collaborative Symposium on Stem Cell Research

The Second Biennial International Collaborative Symposium on Stem Cell Research, held in Seoul, Korea, on 18-19 September 2008, showcased talks by a roster of established and emerging leaders in stem cell biology, and demonstrated how far and fast the field has moved in the last 2 years.