Sook-Hwan Lee

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage

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.

MTHFR C677T polymorphism associates with unexplained infertile male factors

Purpose: To determine whether 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) genotype is associated with male infertility.Methods: Analysis of cytogenetic, Y chromosomal microdeletion assay (Yq), and the C677T and A1298C polymorphisms of the MTHFR gene by pyrosequencing and PCR-Restriction Fragment Length Polymorphism (RFLP) method. SAS 8.1 assessed the statistical risk of MTHFR genotype.Results: The homozygous (T/T) C677T polymorphism of the MTHFR gene was present at a statistically high significance in unexplained infertile men with normal karyotype, instead at no significance in explained infertile men with chromosomal abnormality or Y chromosome deletion. There was no statistically significance of A1298C variation in infertile males.Conclusions: The MTHFR 677TT genotype may be a genetic risk factor for male infertility, especially with severe OAT and non-obstructive azoospermia in unexplained infertile males.

Gene Expression Analysis of Cultured Amniotic Fluid Cell with Down Syndrome by DNA Microarray

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.

Molecular and cytogenetic characterization of two azoospermic patients with X-autosome translocation

AbstractPurpose: To report two azoospermic patients with reciprocal X–autosome translocations. Methods: Cytogenetic analysis utilizing GTG-banding and Yq microdeletions shown by polymerase chain reaction (PCR) with 12 sequence-tagged site (STS) markers for Y chromosome microdeletions. Results: Cytogenetic analysis showed one man with 46,Y,t(X;19)(q22;q13.3) and the other with 46,Y,t(X;8)(p22;q11). Neither had any Yq microdeletions shown. The patient with 46,Y, t(X;8)(p22;q11) showed a slightly lower than normal testosterone level. By NCBI-Blast search, we found four testis-specific genes, t-complex-associated-testis-expressed 1-like (TCTE1L), Ferritin, heavy polypeptide-like 17 (FTHL17), Testis expressed sequence 13A (TEX13A), and Testis expressed sequence 13B (TEX13B) located near breakpoints on X chromosome. FTHL17, TEX13A, and TEX13B are spermatogonially-expressed, germ-cell-specific genes. Conclusion: This is the first clinical report of azoospermia with reciprocal X–autosome translocations on Xp22 and q22. These translocations on Xp22 and q22 may be direct genetic risk factors for azoospermia.

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.

Mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos

OBJECTIVE To compare the level of mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos. DESIGN Reverse transcription polymerase chain reaction was performed in unfertilized oocytes and cleavage-stage embryos derived from tripronucleate embryos to determine ATPase 6 gene expression. SETTING Department of Obstetrics and Gynecology, Human Genetics Laboratory, Infertility Medical Center of CHA General Hospital, College of Medicine, Pochon CHA University, Seoul, Korea. PATIENT(S) Oocytes were obtained from infertile couples undergoing in vitro fertilization. INTERVENTION(S) Unfertilized oocytes collected at 48 hours after retrieval and cleavage-stage embryos derived from tripronucleate embryos were prepared for evaluation of mitochondrial gene expression. MAIN OUTCOME MEASURE(S) Comparison of ATPase 6 gene expression by using single-cell reverse transcription polymerase chain reaction. RESULT(S) Expression of unfertilized oocytes decreased compared with early cleavage-stage embryos. CONCLUSION(S) Our findings of decreased ATPase 6 expression in unfertilized oocytes suggest that there may be a decrease in the mitochondrial functional capacity of oxidative phosphorylation.

Acyl-CoA synthetase long-chain family member 6 is associated with premature ovarian failure

To identify genes that are associated with premature ovarian failure, a linkage disequilibrium-based genome-wide association study with dense single nucleotide polymorphisms as genetic markers was performed. The acyl-coenzyme A synthetase long-chain family member 6 (ACSL6) gene on chromosome 5q31 was associated with premature ovarian failure and identified disease-susceptibility haplotypes.

Expression of Wee1 and Its Related Cell Cycle Components in Mouse Early Stage Follicles

Wee1 is a kinase regulator of the M-phase promoting factor (a complex of cdc2 and cyclin B1). The present study was performed to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. Expression of wee1 and the correlated cell cycle components, namely cdc2, cyclin B1, and cdc25C, was evaluated by immunohistochemistry. In addition, expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2. Each component except cdc25C was found in the oocyte cytoplasm at all follicular stages, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian interstitial cells and to a small extent in granulosa cells of the developing secondary follicles. To further confirm the expression of cell cycle components in the primordial follicular oocytes, day 1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic cells including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. The results of the present study led to the conclusion that wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by inhibitory phosphorylation of cdc2. Expression of all these proteins in the granulosa cells of growing follicles may regulate granulosa cell mitosis concurrently with the growth of oocytes and follicles.

Successful Spouse Pregnancy of Male Patients with Severe Apalstic Anemia and Chronic Myelogenous Leukemia Using Spermatozoa Banked Prior to Bone Marrow Transplantation and Using the ICSI Procedure: Case Reports

AbstractPurpose: To report two cases of successful spouse pregnancies which were conceived with long-term cryopreserved spermatozoa that had been collected prior to the male patients receiving a bone marrow transplant. Methods: The first case is the pregnant wife of a 25-year-old man with chronic myelogenous leukemia, whose semen was collected before bone marrow transplant and then cryopreserved, thawed, and then injected into the wifes eggs via ICSI. The second case is a 28-year-old man with severe aplastic anemia who became a father after his wifes eggs were fertilized via ICSI with thawed spermatozoa. Results: These two cases were achieved pregnancies. Conclusions: These cases support research that men with malignancy have the chance of fathering their own genetic children. Therefore, it is important to increase the awareness of clinicians, oncologists, and patients to the new developments in preserving fertility for cancer patients.