Complete phenotypic and metabolic profiles of a large consecutive cohort of untreated Korean women with polycystic ovary syndrome.

OBJECTIVE: To investigate the complete metabolic and phenotypic profiles of a large cohort of untreated, consecutively recruited Korean women with polycystic ovary syndrome (PCOS), for whom a registry for Korean women with PCOS was constructed. DESIGN: Observational study. SETTING: Three infertility clinics and 10 university hospitals. PATIENT(S): Eight hundred sixty-five women with PCOS were recruited using the Rotterdam criteria. INTERVENTION(S): Standardized evaluation protocol and web-based case report form. MAIN OUTCOME MEASURE(S): Metabolic and phenotypic profiles. RESULT(S): The subjects with PCOS mainly consisted of young and nonobese women. The most problematic subjective symptom was menstrual disturbance or infertility, and, on average, the patients seemed to menstruate every 2 months. PCO morphology was observed in 96.5% of the patients. Although few women visited hospitals owing to HA symptoms alone, hirsutism was observed in one-third of the patients (33.9%) and half (47.4%) of the patients had biochemical HA. About one-fifth (20.1%) of the patients had generalized obesity, and one-third (33.2%) had central obesity. Prevalence of dyslipidemia, diabetes, hypertension, and metabolic syndrome were 35.7%, 3.5%, 4.0%, and 13.7%, respectively. Prevalence of prediabetes was 20.8%, and a substantial proportion of additional subjects with normal fasting plasma glucose or oral glucose tolerance tests were identified as having prediabetes by hemoglobin A1C testing. CONCLUSION(S): Our well-defined cohort provided comprehensive estimates of the features of metabolic and phenotypic profiles related to PCOS in Korean women. Further longitudinal follow-up studies are needed to investigate the changes in phenotypic and metabolic markers in this PCOS cohort.

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.

The villous stromal constituents of complete hydatidiform mole differ histologically in very early pregnancy from the normally developing placenta.

Histologic distinction between complete hydatidiform moles and nonmolar abortuses in early pregnancy is often extremely difficult. This study details the chronologic changes that occur in normal placenta, especially in the villous stroma during gestational weeks 4 to 12 and compares the findings in 63 normal placentas and 73 early complete moles. Over time, the chorionic villi in normal placenta showed gradual but recognizable alterations, from basophilic/hypocellular and nonvascular stroma to basophilic/cellular stroma containing angiogenic cell cords (immature blood vessels), and then to loose, edematous/reticular stroma with mature blood vessels containing vascular lumina and hematopoietic components. A basophilic stroma, which was frequently seen in early complete moles, was a constant feature of chorionic villi younger than week 7 in normal placenta, but had disappeared after week 8, except in the newly branched sprouts. Trophoblastic proliferation was virtually unrecognizable in 59% of early complete moles, whereas circumferential trophoblastic sprouts during weeks 4 to 6 of normal placenta can be mistaken as that of complete moles. Few or no mature blood vessels and excessive stromal karyorrhexis are characteristic features of early complete moles, even in the absence of trophoblastic proliferation, but they are occasionally found in normal placenta younger than week 7. Although many features of normal and molar placenta were similar during the earliest weeks (5 to 6) including basophilic stroma, angiogenic cell cords (immature blood vessels), and often circumferential trophoblastic sprouts and proliferation, already by that time early moles showed significantly greater percentages of stromal cells undergoing karyorrhexis and apoptosis compared with normal placenta (37.6% vs. 5.4%). These results indicate the histologic features do exist early on in pregnancy to differentiate normal from molar pregnancy. Further, it points out that complete hydatidiform mole in addition to being disease of trophoblastic proliferation is also associated with defects in vasculogenesis and maturation of villous stromal constituents.

Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation.

Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development. ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures, slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10-15 min equilibration in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose. Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These results suggest that ganglioside GT1b may play an important role in embryo survival or development.

Ex vivo characteristics of human amniotic membrane-derived stem cells.

Cells were isolated from four human amniotic membranes, and their biological characteristics analyzed during ex vivo expansion. Morphologically homogenous populations of fibroblast-like cells were obtained from the second or third passage. Under the appropriate culture conditions, these human amniotic membrane-derived mesenchymal cells (HAM) were shown to differentiate into adipocytes, osteocytes, chondrocytes and neuronal cells, as visualized by Oil Red O, von Kossa, alcian blue, anti-Neu N, and anti-Gal C antibody staining, respectively. Immunophenotype analysis of HAM cells revealed the presence of antigens for SSEA-3, SSEA-4, collagen type-I, -II, -III, -IV, -XII, fibronectin, alpha-SMA, vimentin, desmin, cytokeratin18 (CK18), HCAM-1, fibroblast surface protein, and human leukocyte antigen (HLA) ABC. ICAM-1 protein was weakly detectable, and proteins of TRA-1-60, VCAM-1, von Willebrand factor, PECAM-1, and HLA DR were not detected. HAM cells reached senescence after 14.5+/-0.9 passages, over a period of 146.8+/-8.9 days, and underwent an average of 36.9 4.7 population doublings. RT-PCR analysis showed that all four HAM cell lines consistently expressed genes of Oct-4, Rex-1, SCF, NCAM, nestin, BMP-4, GATA-4, HNF-4alpha, vimentin, and CK18, regardless of the passage number. The genes of Brachyury, FGF-5, Pax-6, and BMP2 were never expressed. Strikingly, alpha-fetoprotein (alphaFP), HLA ABC, and HLA DR genes were expressed in an earlier passage but not expressed in later passages. Telomerase activity of two HAM lines was discernable upon the third passage. These observations strongly suggest that HAM might be immune-privileged and, thus, advantageous as therapeutic cells.

Consideration of aggressive therapeutic strategies for primary testicular lymphoma.

To evaluate the clinical features and treatment of primary testicular lymphoma, 45 cases were retrospectively evaluated. The median age of the patients was 59 years (range, 40-81) and most patients (76%) presented with Stages I-II. All patients underwent an orchiectomy, after which various treatments were given, chemotherapy alone in 37 patients (60%) and chemotherapy with involved field radiotherapy (IFRT) in 15 patients (33%). Prophylactic intrathecal chemotherapy was given to six patients; cranial irradiation was given in two patients. Eleven patients (24%) received prophylactic irradiation or surgery on the contralateral testis. In 40 patients able to be evaluated, complete response (CR) rate was 78%; 11 of 31 CR patients (36%) had relapsed. Relapse or disease progression was observed in 21 patients. The most frequent site (44%) was in the CNS. The median progression free survival and overall survival were 16 and 34 months, respectively. Ten patients who received prophylactic radiation to the contralateral testis had no relapse in this site. In six patients who received prophylactic intrathecal chemotherapy, there was no leptomeningeal progression, but brain parenchymal relapse occurred in two patients. In multivariate analysis, Stage I (P = 0.02) and additional IFRT after orchiectomy (P = 0.01) were found to be good prognostic factors. In conclusion, orchiectomy followed by intensive chemotherapy and IFRT including prophylaxis to the CNS and contralateral testis, should be considered as initial treatment in primary testicular lymphoma.

Selective processing of a follicular matrix metalloproteinase-2 isoform by human oviducal fluid.

The present study demonstrates that a unique isoform of matrix metalloproteinase (MMP)-2 present in human follicular fluid (FF) can be processed selectively by human oviducal fluid (OF). A gelatin zymogram of untreated FF showed distinct 88-, 84- and 62-kDa gelatinases. Treatment of FF with EDTA resulted in the appearance of 110-kDa gelatinase (GA110). Most gelatinases, except for the 88- and 84-kDa gelatinases, were abolished by pretreatment with EDTA or phenanthroline, but not by pretreatment with a serine/threonine protease inhibitor. When EDTA-pretreated FF was mixed with OF, the GA110 of the FF was specifically reduced. The reduction in GA110 was dependent upon the amount of OF protein and the incubation period after mixing. Treatment of FF with aminophenylmercuric acetate reduced GA110 activity, but this reduction was accompanied by a concomitant increase of 62-kDa gelatinase activity. Anti-human MMP-2 antibody strongly reacted with both GA110 and 62-kDa gelatinases of FF, but only GA110 immunoreactivity was abolished when FF was mixed with OF. The results suggest that the GA110 of FF is an MMP-2 isoform that can be processed selectively by OF.

A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.

A three-dimensional (3-D) endometrium culture was established, in which human endometrial stromal cells embedded in a mixture of collagen I, a major component of extracellular matrix, and matrigel, a basement membrane material, supports the epithelial cells seeded on top of the collagen/matrigel matrix. The biological growth and differentiation of the epithelial cells were studied microscopically and immunohistochemically. Transmission electron microscopy showed a polarized columnar epithelium in monolayer with basally positioned nuclei. Scanning electron microscopy revealed a confluent epithelium with an abundance of microvilli and cilia as well as pinopodes on the apical surface. An immunohistochemical staining showed that integrin alpha1, alpha4, and beta3 were co-localized with cytokeratin, confirming the epithelial origin of the cells. In contrast, immunoreactivity against cyclooxygenase-1 or -2 was positive in both epithelial and stromal cells. When epithelial cells were replaced by KLE cells, an endometrial cancer cell of epithelial origin, invasion of KLE cells into the stromal fraction was observed. The invasion was closely correlated to expression of matrix metalloproteinases and their tissue inhibitors of metalloproteinases in a manner consistent with paracrine fashion. The present 3-D culture imitates the normal endometrium physiologically as well as morphologically, thus provides an excellent in vitro tissue suitable for reproducing in vivo physiological processes, including endometrial cancer invasion.

Amelioration of mitochondrial dysfunction and apoptosis of two-cell mouse embryos after freezing and thawing by the high frequency liquid nitrogen infusion.

Liquid nitrogen (LN2) infusions are currently used in a slow controlled-rate freezing during cryopreservation. The effects of two different LN2 infusion frequencies (conventional, slow 50 infusions/min and high 120 infusions/min) were studied with frozen-thawed two-cell mouse embryos and their subsequent development to blastocysts. The embryos that were subjected to the high frequency LN2 infusion (HFLI) showed a significantly higher survival rate over the low frequency LN2 infusion (LFLI) (50.7 vs. 34.6%, P < 0.05). The blastocyst formation was also higher in HFLI (76.7%) than LFLI (44.0%, P < 0.05) with respective to the number of cells in a blastocyst of 71.6 8.0 (n = 20) and 62.5 +/- 4.7 (n = 20) (P < 0.05). The relative amount of H2O2 in an embryo that was assessed by a fluorescence intensity of 2',7'-dichlorofluorecein (DCF) showed a difference between the procedures with 16.6 +/- 1.6 (n = 21) and 23.4 +/- 1.8 (n = 24) for HFLI and LFLI, respectively (P < 0.05). Mitochondrial staining by Rhodamine 123 showed that the number and distribution of viable mitochondria were similar in both procedures, but fewer mitochondria were observed with a marked aggregation in the arrested embryos, indicating a mitochondrial disintegration. The mitochondrial membrane potential was visualized by a membrane potential-sensitive fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). There was a decrease in the number of mitochondria that had a high membrane potential, and they showed a peripheral redistribution along the cell membrane in LFLI. A fluorescent staining of the actin filaments revealed a discontinuity that was noticeably at the peripheral "actin band" in LFLI. The DNA fragmentation was assessed by the dUTP nick end-labeling (TUNEL). The results showed a higher DNA fragmentation of blastocyst nuclei in LFLI compared to HFLI (65.6 vs. 36.0%, P < 0.05). Based on these observations, it was concluded that HFLI was better than LFLI in the case of freezing the mouse 2-cell embryos for preserving cytoskeletons and mitochondrial integrities. This could subsequently lead to a higher survival and developmental rate of the cryopreserved mouse embryos.

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos.

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.