Comparison of the efficacy of three once-weekly bisphosphonates on bone mineral density gains in Korean women

OBJECTIVE: To assess the efficacies of once-weekly bisphosphonates on bone mineral density (BMD) gains in Korean women aged 50 years or more. METHODS: We selected 166 patients who received: alendronate 70 mg (n=48), alendronate 70 mg + cholecalciferol 2,800 IU (n=31) or risedronate 35 mg (n=87) for one year. The baseline BMD and the % changes of BMD at one-year were compared among the three medication groups. RESULTS: The menopausal status and number of women with osteoporosis was not different among the three groups, but mean age of women was significantly lower in alendronate group. Baseline BMD at L1-4 and femur neck (FN) was similar, but baseline BMD at femur total (FT) was significantly lower in alendronate group. After one-year use, the median % changes of BMD at three sites were similar among the three groups; however, the median values were highest in alendronate + cholecalciferol group (L1-4: 4.48%, 6.74%, and 4.50%; FT: 2.09%, 3.70%, and 2.31%; FN: 3.05%, 3.79%, and 2.03%). CONCLUSION: Among three once-weekly bisphosphonates, BMD gains were highest after one-year use of alendronate+cholecalciferol, although statistically not significant.

Effects of insulin-sensitizing agents and insulin resistance in women with polycystic ovary syndrome / 대한생식의학회지

OBJECTIVE: The aim of this study was to investigate the effect of insulin sensitizing agents on hormonal and metabolic parameters as well as menstrual patterns in women with polycystic ovary syndrome (PCOS). METHODS: One hundred and twenty-three patients with PCOS were included. Metformin was administered to patients at 1,500 mg or 1,700 mg daily for 3 months. If the patients had no improvement of the menstrual cycle or metformin-related adverse effects developed, the patients changed medication to a daily dose of either 15 mg pioglitazone or up to 45 mg. Then resumption of a regular menstrual cycle or recovery of ovulation was evaluated. Hormonal and metabolic profiles were compared between the response and non-response group to insulin sensitizing agents. RESULTS: One hundred and five patients with PCOS were treated with metformin for 3 months. Forty-eight patients (45.7%) showed improvement of menstrual cycle regularity after 3 months of metformin use, whereas 57 patients (54.3%) had no change. The mean free testosterone measured after 3 months of treatment was significantly lower in metformin responders than in non-responders. The other parameters did not differ between the groups. Of the 23 patients who used pioglitazone for 3 to 6 months, 19 patients (82.6%) showed improvement in their menstrual cycles. CONCLUSION: Metformin treatment seems to be effective for the improvement of menstrual cyclicity irrespective of insulin resistance in women with PCOS. When metformin related adverse effect occurred, pioglitazone would be effective for aiding the resumption of the menstrual cycle.

Current status of assisted reproductive technology in Korea, 2009

Great advances have been made in the field of assisted reproductive technology (ART) since the first in vitro fertilization (IVF) baby was born in Korea in the year of 1985. However, it deserve to say that the invaluable data from fertility centers may serve as a useful source to find out which factors affect successful IVF outcome and to offer applicable information to infertile patients and fertility clinics. This article intended to report the status of ART in 2009 Korean Society of Obstetrics and Gynecology surveyed. The current survey was performed to assess the status and success rate of ART performed in Korea, between January 1 and December 31, 2009. Reporting forms had been sent out to IVF centers via e-mail, and collected by e-mail as well in 2012. With International Committee Monitoring Assisted Reproductive Technologies recommendation, intracytoplasmic sperm injection (ICSI) and non-ICSI cases have been categorized and also IVF-ET cases involving frozen embryo replacement have been surveyed separately. Seventy-four centers have reported the treatment cycles initiated in the year of 2009, and had performed a total of 27,947 cycles of ART treatments. Among a total of 27,947 treatment cycles, IVF and ICSI cases added up to 22,049 (78.9%), with 45.3% IVF without ICSI and 54.7% IVF with ICSI, respectively. Among the IVF and ICSI patients, patients confirmed to have achieved clinical pregnancy was 28.8% per cycle with oocyte retrieval, and 30.9% per cycle with embryo transfer. The most common number of embryos transferred in 2009 is three embryos (40.4%), followed by 2 embryos (28.4%) and a single embryo transferred (13.6%). Among IVF and ICSI cycles that resulted in multiple live births, twin pregnancy rate was 45.3% and triple pregnancy rate was 1.1%. A total of 191 cases of oocyte donation had been performed to result in 25.0% of live birth rate. Meanwhile, a total of 5,619 cases of frozen embryo replacement had been performed with 33.7% of clinical pregnancy rate per cycle with embryo transfer. When comparing with international registry data, clinical pregnancy rate per transfer from fresh IVF cycles including ICSI (34.1%,) was comparable to clinical pregnancy rate per transfer in European Society for Human Reproduction and Embryology report was 32.5% though lower than 45.0% for USA data. There was no remarkable difference in status of assisted reproductive technology in Korea between the current report and the data reported in 2008. The age of women trying to get pregnant was reconfirmed to be the most important factor that may have impact on success of ART treatment.

Carotid intima-media thickness in mainly non-obese women with polycystic ovary syndrome and age-matched controls

OBJECTIVE: Metabolic disturbances are well-recognized clinical features of polycystic ovary syndrome (PCOS). Carotid intima-media thickness (CIMT) has been widely used as a surrogate marker of atherosclerosis and cardiovascular disease (CVD). CIMT in women with PCOS has been investigated in many studies, but there has been only one report in the Korean population. The aim of the present study was to compare the presence of subclinical atherosclerosis in young untreated Korean women with PCOS and age-matched controls, specifically by measuring their CIMT. METHODS: CIMT was measured by one radiologist in 56 PCOS patients and 56 controls. To compare the CIMT according to PCOS phenotypes, women with PCOS were divided into two subgroups according to the presence of hyperandrogenism. RESULTS: Although PCOS patients were more obese and had higher blood pressure and insulin resistance index than the age-matched controls, the CIMT was not different between the two groups (0.49 +/- 0.09 mm in PCOS patients vs. 0.50 +/- 0.11 mm in controls, respectively, p = 0.562). When the CIMT in the control group was compared with hyperandrogenic and non-hyperandrogenic PCOS groups, also no significant differences were found. CONCLUSION: Despite the significant differences in some vascular risk factors between women with PCOS and controls, PCOS patients did not have a significantly higher CIMT (even in the hyperandrogenic subgroups). Although our study did not show the increased risk of subclinical atherosclerosis in PCOS patients, the role of CIMT continues to be investigated considering the importance of screening and monitoring CVD risk factors in women with PCOS.

The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis / 대한생식의학회지

OBJECTIVE: The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. METHODS: Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. RESULTS: Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). CONCLUSION: The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.

Natural conception rate following laparoscopic surgery in infertile women with endometriosis / 대한생식의학회지

OBJECTIVE: To investigate the influence of laparoscopic surgery on the natural conception rate in infertile women with endometriosis during the first year after the operation. METHODS: We retrospectively studied 43 infertile women with surgically proven endometriosis. The natural conception rate was investigated for the 12 months after the laparoscopy. RESULTS: The overall pregnancy rate was 41.9% (18/43). 66.7% (12/18) and 94.4% (17/18) of the patients conceived within postoperative 3 months and 6 months, respectively. The spontaneous pregnancy rate was not associated with the severity of endometriosis or laparoscopic findings or the type of surgery. The pregnancy rate for stage IV was relatively low (20.0%) compared to stage I, II, and III (35.7%, 44.4%, and 53.3%, respectively), although it did not reach statistical significance. CONCLUSION: Conservative surgical treatment with laparoscopy and a prompt attempt at natural conception may be effective for infertile patients with endometriosis.

Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K) / 대한생식의학회지

Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.

Oocyte Maturity in Relation to Woman's Age in In Vitro Fertilization Cycles Stimulated by Single Regimen

PURPOSE: During stimulated in vitro fertilization (IVF) cycle, up to 30% of the recovered oocytes are immature ones which have poor fertilization capacity; however, the precise influencing factors are largely unknown. Here, we analyzed the association of oocyte immaturity with woman's age in IVF cycles stimulated by single regimen. MATERIALS AND METHODS: A total of one-hundred ninety five IVF cycles stimulated by recombinant FSH and GnRH antagonist protocol between 2003 and 2009 were analyzed retrospectively. The mean age of women was 34.2+/-4.0 (26-45 years). After triggering by exogenous hCG, an ultrasound-guided retrieval of oocytes was performed 35-36 hours later. All clinical data were stratified by woman's age; group I: or =41 (n=19). RESULTS: The total retrieved oocytes, as well as immature oocytes, were significantly lower in group IV, however, the mean % of immature oocytes was significantly higher in group IV than other age groups. Oocyte immaturity tended to decrease as increasing age in women aged 40 years or less. CONCLUSION: In stimulated IVF cycle, much higher oocyte immaturity was noted in women aged 41 years or more.

Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis

PURPOSE: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. MATERIALS AND METHODS: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. RESULTS: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. CONCLUSION: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

The Effect of Tibolone and Estradiol-based Hormone Therapy on Bone Mineral Density and Serum Lipid Profiles / 대한골다공증학회지

OBJECTIVES: To investigate the differences between the effect of tibolone and estradiol (E2)-based hormone therapy (HT) on bone mineral density (BMD) and serum lipid profiles in postmenopausal Korean women. MATERIALS AND METHODS: A retrospective study was conducted with 65 postmenopausal women receiving tibolone or E2-based hormone therapy in university hospital. BMD at lumbar spine (LS) and proximal femur was measured before and after 1 year of therapy and serum total cholesterol, triglyceride (TG) and high-density lipoprotein (HDL) was determined by enzymatic methods. RESULTS: BMD at LS increased after 1 year of tibolone (mean change: 3.0%) or E2-based HT (mean change: 1.6%), and the changes were significant (P=0.002 and 0.04, respectively). In E2 group, serum total cholesterol has decreased significantly after 1 year of therapy (P=0.02). Moreover, the change of HDL level was statistically significant in tibolone group compared to E2 group (P=0.01). The changes of levels of total cholesterol, TG and HDL has demonstrated negative relationship of BMD changes at femur neck and trochanter in tibolone group, whereas only the HDL changes were significantly related to the change of trochanter BMD in E2 group. CONCLUSIONS: Both tibolone and E2-based hormone therapy increased BMD at lumbar spine. The changes of serum lipid levels may be associated with the BMD changes in both groups although the relationships were different according to the regimen.

Soluble Human Leukocyte Antigen G Level in Fluid from Single Dominant Follicle and the Association with Oocyte Competence

PURPOSE: To investigate the direct relationship between the follicular fluid (FF) level of soluble human leukocyte antigen G (HLA-G) and fertilizability of the corresponding oocyte as well as the morphological quality of the corresponding embryo. MATERIALS AND METHODS: Sixty-three patients were stimulated with recombinant FSH combined with gonadotropin-releasing hormone (GnRH) agonist long (n=5) or antagonist protocol (n=58) for standard in vitro fertilization (IVF). At the time oocyte retrieval, follicular fluid was obtained from single dominant follicle in 63 patients, and the level of soluble HLA-G was measured by sandwich enzyme-liked immunosorbent assay (ELISA). Normal fertilization and individual embryo quality were evaluated, and were graded to four categories by morphological criteria (the embryo with symmetrical blastomeres and no fragmentation were assigned as grade A). Good-quality embryo was defined as those with grade A or B. RESULTS: Soluble HLA-G was not detected in 15 FF samples. In the group with positive FF soluble HLA-G (sHLA-G) (n=48), high levels of sHLA-G (>117.758 U/mL) could predict the failure of fertilization with statistical significance {area under the curve (AUC) 0.676, 95% confidence interval (CI) 0.525-0.804}. However, the FF sHLA-G level was not related with the formation of good-quality embryo. CONCLUSION: High level of FF sHLA-G could predict the fertilization failure of the corresponding oocyte, but was not related with the formation of good-quality embryo.

Subretinal transplantation of putative retinal pigment epithelial cells derived from human embryonic stem cells in rat retinal degeneration model / 대한생식의학회지

OBJECTIVE: To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. METHODS: The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells (10(5) cells/5 microL) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. RESULTS: The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. CONCLUSION: Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease.

The Use of Tenaculum During Intrauterine Insemination May Not Affect the Pregnancy Outcome

PURPOSE: The association between tenaculum application to the cervix just before embryo transfer and lower pregnancy rate has been reported. However, studies on the use of tenaculum in intrauterine insemination (IUI) cycles are not available. This study attempted to investigate whether the use of tenaculum affects the clinical outcomes of IUI. MATERIALS AND METHODS: Two hundred and thirty three CC/hMG/IUI cycles of one hundred and forty three couples were recruited at Seoul National University Hospital from October 2006 to December 2008. Mock insemination and IUI with or without tenaculum application to the cervix were also performed, and clinical pregnancy rate was compared. RESULTS: The incidence of difficult mock insemination at the beginning of cycle was higher in the tenaculum use group (p < 0.001). No significant statistical difference in the clinical pregnancy rate was observed between the groups with or without tenaculum application (12.1% vs. 18.5%; p = 0.175), which was not influenced by the difficulty of mock insemination. CONCLUSION: The use of tenaculum during IUI may not affect the pregnancy outcome. Our results need to be confirmed by a prospective study in a larger population.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.

Discrepancy in cytogenetic results between amniotic fluid cells and other fetal tissues: A case report / 대한산부인과학회지

We present two fetuses who were prenatally diagnosed by amniocentesis as having chromosomal mosaicism but who had a normal karyotype in the fetal blood by cordocentesis. One of the both fetuses had Turner and the other had trisomy 20 mosaicism. The prognosis for Turner mosaicism and trisomy 20 mosaicism diagnosed prenatally has yet to be established. The pregnancy with 45,X/46,XX mosaicism was terminated at 23+3 weeks' gestation. Autopsy findings showed no features of Turner's syndrome. Postnatal cytogenetic analysis revealed 45,X[4]/46,XX[52] mosaicism in skin and 46,XX in the lung tissue. The other fetus had amniocytes with trisomy 20 mosaicism and fetal cord blood cells with a normal karyotype. The baby was delivered at 38+2 weeks' gestation. At birth and 3 months after birth, no apparent abnormal findings were found. These cases with chromosomal discrepancy among various fetal tissues are rare. Two cases were discussed with the review of literature.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Non-association Between Polymorphisms of the Frizzled Receptor Genes and Bone Mineral Density in Postmenopausal Korean Women

We investigated the association between single nucleotide polymorphisms (SNPs) in the frizzled (FZD) genes in the Wnt signal pathway and circulating osteoprotegerin (OPG), soluble receptor activator of NF-kappaB ligand (sRANKL) levels, bone turnover markers, and bone mineral density (BMD) in postmenopausal women. The SNPs in the FZD1, FZD5, FZD6, FZD7, and FZD9 genes were analyzed by direct sequencing in 371 postmenopausal Korean women. Levels of serum OPG, sRANKL, osteocalcin, C-telopeptide of type I collagen, calcium, parathyroid hormone and calcitonin, and BMD at the lumbar spine and femoral neck were measured. The SNPs in the FZD1, FZD5, FZD7, and FZD9 genes, and in exon 2 of the FZD6 gene were not observed. No significant differences in the adjusted BMD of lumbar spine and femoral neck and serum levels of OPG, sRANKL, and bone markers were noted among the single or haplotype genotypes of the L345M and E664A SNPs in the FZD6 gene and the distributions of these single or haplotype genotypes were not different according to the bone mass status. In conclusion, the polymorphisms of the FZD genes are not associated with BMD of the lumbar spine and femoral neck, bone turnover markers, or circulating OPG-sRANKL in Korean women.

Non-association Between Polymorphisms of the Frizzled Receptor Genes and Bone Mineral Density in Postmenopausal Korean Women

We investigated the association between single nucleotide polymorphisms (SNPs) in the frizzled (FZD) genes in the Wnt signal pathway and circulating osteoprotegerin (OPG), soluble receptor activator of NF-kappaB ligand (sRANKL) levels, bone turnover markers, and bone mineral density (BMD) in postmenopausal women. The SNPs in the FZD1, FZD5, FZD6, FZD7, and FZD9 genes were analyzed by direct sequencing in 371 postmenopausal Korean women. Levels of serum OPG, sRANKL, osteocalcin, C-telopeptide of type I collagen, calcium, parathyroid hormone and calcitonin, and BMD at the lumbar spine and femoral neck were measured. The SNPs in the FZD1, FZD5, FZD7, and FZD9 genes, and in exon 2 of the FZD6 gene were not observed. No significant differences in the adjusted BMD of lumbar spine and femoral neck and serum levels of OPG, sRANKL, and bone markers were noted among the single or haplotype genotypes of the L345M and E664A SNPs in the FZD6 gene and the distributions of these single or haplotype genotypes were not different according to the bone mass status. In conclusion, the polymorphisms of the FZD genes are not associated with BMD of the lumbar spine and femoral neck, bone turnover markers, or circulating OPG-sRANKL in Korean women.

Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2

PURPOSE: Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs. MATERIALS AND METHODS: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed. RESULTS: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (alphaMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS. CONCLUSION: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.