Celecoxib attenuates interleukin 33-induced expression of vascular cell adhesion molecule-1 in human ovarian endometriotic stromal cells.

OBJECTIVE: Endometriosis is an estrogen-dependent chronic inflammatory disease in women of reproductive age. A review of the literature revealed that cytokines and inflammatory factors are associated with endometriosis-associated infertility. Interleukin 33 (IL-33) is a strong inducer of other pro-inflammatory cytokines. Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in recruiting inflammatory cells, whose expression facilitates leukocyte adhesion and is rapidly induced by pro-inflammatory cytokines. Many studies have indicated that VCAM-1 expression is high in endometriosis; however, whether the expression of VCAM-1 is related to IL-33 is unclear. MATERIALS AND METHODS: Human ovarian endometriotic stromal cells (hOVEN-SCs) were treated with IL-33 to enable investigation of cell characterization, gene and protein expression, and signal pathways. Proliferation potential was measured using an MTT assay. Gene expression was analyzed using reverse transcription-polymerase chain reaction. Protein expression assay was performed using western blot analysis. RESULTS: This study investigated the effects of IL-33 on VCAM-1 and COX-2 expression in hOVEN-SCs. First, the results revealed that the IL-33/ST2/mitogen-activated protein kinase (MAPK) signaling pathway could increase the expression of VCAM-1 and COX-2 in hOVEN-SCs. Second, we discovered that COX-2 expression was essential for IL-33-induced VCAM-1 expression because the effects could be negated through NS398, a selective COX-2 inhibitor. Finally, treatment of IL-33-treated hOVEN-SCs with celecoxib significantly and dose-responsively decreased VCAM-1 expression. CONCLUSION: Taken together, these results indicate that IL-33 can upregulate VCAM-1 expression in hOVEN-SCs through the IL-33/ST2/MAPK/COX-2 signaling pathway and thereby contribute to endometriosis.

Oct-4 induces cisplatin resistance and tumor stem cell-like properties in endometrial carcinoma cells.

OBJECTIVE: Research has suggested that tumor-initiating tumor stem cells are derived from normal stem cells and that tumor cells undergo progressive de-differentiation to achieve a stem cell-like state. Tumor stem cells are characterized by high proliferation ability, high plasticity, expression of multi-drug resistance proteins, and the ability to seed new tumors. Octamer-binding transcription factor 4 (Oct-4) and its activation targets are overexpressed in the tumor stem cells of various types of tumors, and this expression is associated with the pathogenesis, development, and poor prognosis of tumors. The primary objective of this study was to test if a stably transfected with Oct-4 gene cell line, RL95-2/Oct-4, has the characteristics of tumor stem cells. MATERIALS AND METHODS: Human endometrial carcinoma cells (RL95-2) were transfected with a plasmid carrying genes for Oct-4 and green fluorescent protein (GFP). The stably transfected cells, RL95-2/Oct-4, were selected using G418 and observed to express the GFP reporter gene under the control of the Oct-4 promoter. GFP expression levels of RL95-2/Oct-4 cells were measured using flow cytometry. The proliferation potential of cells was determined according to cumulative population doubling and colony-formation efficiency. Gene expression was analyzed using reverse transcription-polymerase chain reaction. RESULTS: RL95-2/Oct-4 cells not only exhibited increased expression of the three most important stem cell genes, Oct-4, Nanog, and Sox2, but also had increased expression of the endometrial tumor stem cell genes CD133 and ALDH1. Furthermore, enhanced expression of these genes in the RL95-2/Oct-4 cells was associated with higher colony-forming ability and growth rate than in parental RL95-2 cells. We also observed that cisplatin induced less cell death in RL95-2/Oct-4 cells than in RL95-2 cells, indicating that RL95-2/Oct-4 cells were more resistant to chemotherapeutic agents. CONCLUSION: The study findings contribute to investigate the effects of Oct-4 on tumor stem cell origins.

Juniperus communis extract ameliorates lipopolysaccharide-induced acute kidney injury through the adenosine monophosphate-activated protein kinase pathway.

Septic shock can aggravate organ dysfunction and even lead to death. Juniperus communis (JCo) extract has been experimentally demonstrated to have anti-inflammatory and antioxidant effects. We investigated the anti-inflammatory and antioxidant mechanism of JCo extract in vivo and in vitro. In a lipopolysaccharide (LPS)-induced acute kidney injury rat model, JCo extract improved animal survival, reduced kidney injury scores, suppressed kidney injury molecule-1, and preserved E-cadherin expression from LPS damage, as demonstrated by the immunohistochemistry examinations of the rat kidneys. In LPS-stimulated NRK-52E cells, JCo extract inhibited nuclear factor-κB (NF-κB) and increased adenosine monophosphate-activated protein kinase (AMPK) expression, prompting the activation of the antioxidant nuclear factor erythroid 2-related factor-2/heme oxygenase-1 pathway against oxidative stress. JCo extract ameliorated LPS-induced acute kidney injury by suppressing NF-κB signaling and stimulating the release of tumor necrosis factor-α and interleukin-1ß through the AMPK pathway.

Interleukin-33 promotes invasiveness of human ovarian endometriotic stromal cells through the ST2/MAPK/MMP-9 pathway activated by 17ß-estradiol.

OBJECTIVE: Endometriosis is an estrogen-dependent, benign, and chronic gynecological disorder occurring in women of reproductive age. Although the pathogenesis of endometriosis is poorly understood, implantation theory indicates that viable endometrial cells shed from the endometrium into the pelvic peritoneum or ovaries, possibly through retrograde menstruation, and then reattach, invade, and damage other tissues. Interleukin (IL)-33, a new member of the IL-1 superfamily, is mainly upregulated by stromal cells following proinflammatory stimulation. Matrix metalloproteinases (MMPs) are involved in the degradation and reconstruction of the extracellular matrix. MMP-9 participates in the pathogenesis of endometriosis by promoting the invasion of endometriotic cells. This study investigated the effect of IL-33 on the cell invasion ability of and MMP-9 expression in human stromal cells derived from ovarian endometrioma (hOVEN-SCs). MATERIALS AND METHODS: We isolated hOVEN-SCs from human ovarian endometrioma. Gene expression was analyzed using the Illumina Human WG-6 v2 Expression BeadChips microarray platform and through reverse transcription-polymerase chain reaction. Cell migration and invasion were examined by performing the transwell chamber assay. RESULTS: We found that 17ß-estradiol could increase the expression of IL-33 and ST2 through the estrogen receptor pathway in hOVEN-SCs. Moreover, IL-33 upregulated MMP-9 expression in and enhanced the invasion ability of hOVEN-SCs through the ST2/MAPK signaling pathway. Our results showed that MMP-9 expression was essential for IL-33-induced cell invasion. CONCLUSION: Our main finding is that 17ß-estradiol could increase IL-33 expression through the estrogen receptor pathway and activate MMP-9 expression in and invasion ability of hOVEN-SCs through the IL-33/ST2/MAPK signaling pathway. The results of this study and further related studies may provide new strategies for the prevention and treatment of endometriosis.

Downregulation of gap junctional intercellular communication and connexin 43 expression by bisphenol A in human granulosa cells.

Gap junctional intercellular communication (GJIC) is the transfer of ions, metabolites, and second messengers between neighboring cells through intercellular junctions. Connexin 43 (Cx43) was found to be the type of gap junction protein responsible for human granulosa cells (GCs) and oocyte communication, which is required for folliculogenesis and oocyte maturation. Bisphenol A (BPA), an estrogenic-like endocrine-disrupting chemical, is one of the most widely produced chemicals around the world. There are reports that the chemical might cause endometrial tumorigenesis and several female reproductive disorders. This study demonstrated that cell culture medium, containing antioxidants (N-acetyl-l-cysteine and l-ascorbic acid-2-phosphate), was able to enhance the survival and self-renewal of GCs. In addition, we found that BPA at environmentally relevant concentration (10-7  M) reduced Cx43 expression and GJIC in GCs through estrogen receptor and mitogen-activated protein kinase pathways. The results of this study not only reveal the reproductive toxicity of BPA but also provide possible mechanisms by which BPA inhibited GJIC in GCs.

Modulation of tumor stem cell characteristics by 17ß-estradiol in human mesenchymal stem cells derived from ovarian endometrioma.

OBJECTIVE: Ovarian endometrioma is a cyst composed of endometrial tissue and is present in 20%-40% of patients with endometriosis. Endometriosis is an estrogen-dependent benign and chronic gynecological disease that affects women of reproductive age. Studies have reported that tumor stem cells can be isolated from numerous tumor types. Emerging evidence has indicated that tumor stem cells may be responsible for the development of endometriosis and endometrial tumors. The present study investigated the effects of 17ß-estradiol on levels of expression of stem cell markers and cell growth of human mesenchymal stem cells derived from ovarian endometrioma (hOVEN-MSCs). MATERIALS AND METHODS: hOVEN-MSCs were isolated from human ovarian endometrioma. The proliferation potential of hOVEN-MSCs was measured by the cumulative population doubling and colony-formation efficiency. The gene expression of the hOVEN-MSCs was examined by the reverse transcription-polymerase chain reaction analysis. Protein expression assays were performed using flow cytometry and western blot analysis. RESULTS: This study demonstrated that hOVEN-MSCs can be isolated from ovarian endometrioma and that 17ß-estradiol was capable of increasing colony-forming efficiency and cell proliferation of these cells. In addition, we found that 17ß-estradiol not only increased the expression of the stem cell marker OCT-4, but also increased the expression of endometrial tumor stem cell markers CD133 and ALDH1 in hOVEN-MSCs. CONCLUSION: The above results indicate an important role of 17ß-estradiol in cell growth of hOVEN-MSCs concomitant with enhanced expression of stem cell markers. This effect of 17ß-estradiol related to stem cell marker expression, if confirmed by further in vitro, in vivo studies, may be useful for developing new strategies for prevention and treatment of endometriosis and endometrioma.

Pueraria mirifica inhibits 17ß-estradiol-induced cell proliferation of human endometrial mesenchymal stem cells.

OBJECTIVE: The notion that the human endometrium may contain a population of stem cells has recently been proposed. The mesenchymal stem cells (MSCs) in the endometrium are believed to be responsible for the remarkable regenerative ability of endometrial cells. Estrogens influence the physiological and pathological processes of several hormone-dependent tissues, such as the endometrium. Pueraria mirifica (PM) is a herbal plant that contains several phytoestrogens, including isoflavones, lignans, and coumestans, and is known to exert an estrogenic effect on animal models. The present study investigated the effects of PM on the proliferation of human endometrial MSCs (hEN-MSCs). MATERIALS AND METHODS: The hEN-MSCs were isolated from human endometrial tissue. The surface markers of these hEN-MSCs were identified through reverse transcription-polymerase chain reaction analysis. The proliferation potential of hEN-MSCs was measured through a cell proliferation assay. Multilineage differentiation ability was confirmed through Oil red O and von Kossa staining. RESULTS: This study demonstrated that 17ß-estradiol-responsive MSCs with Oct-4, CD90, and CD105 gene expression can be derived from the human endometrium and that PM exerts biological effects on hEN-MSCs, specifically, enhanced cell growth rate, through the estrogen receptor. Furthermore, PM at 1500 and 2000 µg/mL significantly increased cell proliferation compared with the vehicle control, and PM concentration at 1000 µg/mL significantly inhibited the enhanced cell growth rate induced by 17ß-estradiol in hEN-MSCs. CONCLUSION: This study provides new insights into the possible biological effects of PM on the proliferation of hEN-MSCs.

Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants.

We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC) from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate) is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40) of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

Bisphenol A-induced epithelial to mesenchymal transition is mediated by cyclooxygenase-2 up-regulation in human endometrial carcinoma cells.

Many studies have highlighted the correlation between the increase of bisphenol A (BPA) level in the environment and the incidence of tumor in humans. In human carcinogenesis, the overexpression of cyclooxygenase-2 (COX-2) and epithelial-mesenchymal transition (EMT) are closely related with tumor development. In this study, human endometrial carcinoma cells line (RL95-2) was used to investigate whether BPA can induce EMT and COX-2 expression. The results show that BPA increased growth rate and colony-forming efficiency in a dose-dependent manner, induced EMT and COX-2 gene expression and promoted the migration and invasion ability of RL95-2 cells. Furthermore, our study showed that the expression of COX-2 was essential for BPA-induced cell migration and invasion. The results of this study provide new insights into the mechanism of endometrial cancer cell growth and invasion and potential therapeutic strategy.

Upregulation of Nanog and Sox-2 genes following ectopic expression of Oct-4 in amniotic fluid mesenchymal stem cells.

Octamer-binding transcription factor 4 (Oct-4), an important gene regulating stem cell pluripotency, is well-known for its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. The aim of this study was to assess the effect of ectopic expression of Oct human amniotic fluid stem cells. We developed a novel method for isolation of putative human amniotic fluid-derived multipotent stem cells. These cells showing mesenchymal stem cell phenotypes (human amniotic fluid-derived mesenchymal stem cells, hAFMSCs) were transfected with a plasmid carrying genes for Oct-4 and the green fluorescent protein (GFP). The stably transfected cells, hAFMSCs-Oct4/GFP, were selected by using G418 and found to express the GFP reporter gene under the control of Oct-4 promoter. We found that hAFMSCs developed by our method possess very high self-renewal ability (about 78 cumulative population doublings) and multilineage differentiation potency. Significantly, the hAFMSCs-Oct4/GFP cells showed enhanced expression of the three major pluripotency genes Oct-4, Nanog, and Sox-2, and increased colony-forming ability and growth rate compared with the parental hAFMSCs. We demonstrated that the ectopic expression of Oct-4 gene in hAFMSCs with high self-renewal ability could upregulate Nanog and Sox-2 gene expression and enhance cell growth rate and colony-forming efficiency. Therefore, the ectopic expression of Oct-4 could be a strategy to develop pluripotency in hAFMSCs for clinical applications.

Bisphenol A at environmentally relevant doses induces cyclooxygenase-2 expression and promotes invasion of human mesenchymal stem cells derived from uterine myoma tissue.

OBJECTIVE: Uterine myoma is the most common benign reproductive tract tumor in women. Despite its high prevalence, the exact pathogenesis of these benign tumors remains unknown. Toward understanding the pathogenic mechanism of these tumors, we attempted to isolate human uterine myoma mesenchymal stem cells (hUM-MSCs), which may be the target cells for tumorigenesis. Furthermore, we tested the response of these hUM-MSCs to the environmental endocrine disruptor, bisphenol A (BPA), which may mimic the action of estrogen in hormone-sensitive organs such as the uterus. MATERIALS AND METHODS: The hUM-MSC lines were clonally derived from uterine myoma tissue using the MSU-1 medium supplemented with N-acetyl-l-cysteine and l-ascorbic acid-2-phosphate. These hUM-MSCs were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis for the expression of mesenchymal stem cell (MSC) surface markers (e.g., CD90 and CD105) and the transcription factor Oct-4. The proliferation potential was measured by the cumulative population doubling level and the colony-forming efficiency. RESULTS: Putative hUM-MSC lines expressed CD90, CD105, and the stem cell marker gene, Oct-4. The cells were capable of differentiating into adipocytes, osteoblasts, and chondrocytes. Bisphenol A treatment of these hUM-MSCs enhanced cell proliferation and colony-forming efficiency in a dose-responsive manner. At an environmentally relevant concentration (10(-8) M), BPA moreover induced cyclooxygenase-2 (COX-2) gene expression and promoted cell migration and invasiveness. CONCLUSION: The hUM-MSC cell lines can be isolated from uterine myoma tissues. Bisphenol A could enhance cell proliferation and colony-forming efficiency, induce COX-2 gene expression, and promote migration and invasion of hUM-MSCs. The results imply that BPA has a detrimental effect on female health by promoting uterine tumorigenesis.

Promotion of epithelial-mesenchymal transition and tumor growth by 17ß-estradiol in an ER(+)/HER2(+) cell line derived from human breast epithelial stem cells.

A tumorigenic cell line with estrogen receptor and HER2 expression (ER/HER2⁺), R2N1d, was developed from a human breast epithelial cell type with stem cell characteristics in a growth factor/hormone-deprived cell culture condition. This study was undertaken to test whether tumor growth and other biological effects could be induced by estrogen in this cell line. The results clearly show that estrogen treatment greatly promoted the tumor growth of R2N1d cells in immune-deficient mice. Estrogen treatment of R2N1d cells in vitro was also found to induce other phenotypic changes related to breast carcinogenesis, that is, 1) the induction of epithelial-mesenchymal transition (EMT) shown by molecular and functional marker changes; 2) a significant increase of the CD44(high)/CD24(-/low) stem cell population; 3) the enhancement of cell growth rate and colony-forming ability; and 4) the acquisition of metastatic ability, that is, increased cell migration and invasiveness. From these results, we conclude that 1) estrogen could induce EMT and cancer stem cells and promote tumor growth in ER⁺/HER2⁺ cells known to be derived from human breast epithelial stem cells, and 2) normal stem cells could give rise to cancer stem cells.

Simultaneous use of mifepristone and misoprostol for early pregnancy termination.

OBJECTIVE: Simultaneous mifepristone 200mg and vaginal misoprostol 800µg produces a complete abortion rate of approximately 90% at up to 63 days of gestation. The aim of this study was to determine the effectiveness of concurrent administration of mifepristone 200mg and vaginal misoprostol 600µg with respect to early medical abortion. MATERIALS AND METHODS: A total of 254 women with undesired pregnancies of less than 49 days of gestation were enrolled. All women received oral mifepristone 200mg and vaginal misoprostol 600µg concurrently. Follow-up assessment by transvaginal ultrasonography was performed 3 days and 2 weeks after treatment. RESULTS: Efficacy outcome was analyzed for 242 women (95.3%) after excluding 12 individuals lost to follow-up. The complete abortion rate was 92.6%. The mean induction to abortion interval was about 5.8hours. The mean bleeding duration was about 12.6 days. The women indicated that the side effects were tolerable and 90% of them said that their experience was satisfactory. CONCLUSION: Concurrent administration of oral mifepristone 200mg and vaginal misoprostol 600µg is an efficacious regimen for medical abortion of pregnancies up to 49 days of gestation.

A left circum-aortic renal vein aneurysm.

A well-defined, slow-flowing vascular lesion was found incidentally by Doppler abdominal sonography in the left renal hilar region of a 36-year-old Taiwanese woman. Clinically, the physical examination and laboratory screening were unremarkable. A magnetic resonance angiography of the area near the renal hilum showed a saccular mass (3.5 x 3.1 x 2.5 cm) embracing the aorta by the anterior and posterior branch of the aneurysm originating from the left renal vein to the inferior vena cava. However, the patient refused further invasive intervention and has since been examined periodically by ultrasonography for 18 months without increasing size or symptoms.

Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method.

BACKGROUND: Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution. METHODS: Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20-30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180 degrees C in the volume of 2 microl and 6 microl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-microl droplets of alpha-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored. RESULTS: After the vitrification of 14-day-old preantral follicles using 2 microl or 6 microl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 microl and 6 microl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 microl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group. CONCLUSION: Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 microl and 6 microl can be an option.

High-dose misoprostol as an alternative therapy after failed medical abortion.

OBJECTIVE: The aim of this study was to determine the complete abortion rate for the vaginal administration of high-dose misoprostol after a failed medical abortion. MATERIALS AND METHODS: When their medical abortions failed after the conventional oral administration of mifepristone and misoprostol, participants then received 1,000 microg of misoprostol vaginally. The efficacy and side effects of this treatment were evaluated. RESULTS: Twenty-seven women who failed to abort after the conventional administration of mifepristone and misoprostol were enrolled in this trial. Fourteen days after the vaginal administration of 1,000 microg misoprostol, the overall complete expulsion rate had reached 88.8% (24/27). Most adverse effects were mild to moderate and did not require treatment. CONCLUSION: The vaginal administration of 1,000 microg misoprostol as a salvage therapy after a failed medical abortion appears to be a safe and highly effective alternative to surgical intervention.

Abnormal glucose tolerance and insulin resistance in polycystic ovary syndrome amongst the Taiwanese population- not correlated with insulin receptor substrate-1 Gly972Arg/Ala513Pro polymorphism.

BACKGROUND: Insulin resistance and glucose dysmetabolism in polycystic ovary syndrome (PCOS) are related with the polymorphisms in the genes encoding the insulin receptor substrate (IRS) proteins, especially Gly972Arg/Ala513Pro polymorphism being reported to be associated with type-2 diabetes and PCOS. We intended to assess the prevalence of abnormal glucose tolerance (AGT) and insulin resistance in Taiwanese PCOS women. We also tried to assess whether the particular identity of Gly972Arg/Ala513Pro polymorphic alleles of the IRS-1 gene mutation can be used as an appropriate diagnostic indicator for PCOS. METHODS: We designed a prospective clinical study. Forty-seven Taiwanese Hoklo and Hakka women, diagnosed with PCOS were enrolled in this study as were forty-five healthy Hoklo and Hakka women as the control group. Insulin resistance was evaluated with fasting insulin, fasting glucose/insulin ratio, and homeostasis model assessment index for insulin resistance (HOMAIR). The genomic DNA of the subjects was amplified by PCR and digested by restriction fragmented length polymorphism (RFLP) with Bst N1 used for codon 972 and Dra III for codon 513. RESULTS: AGT was found in 46.8% of these PCOS patients and was significantly related to high insulin resistance rather than the low insulin resistance. Those patients with either insulin resistance or AGT comprised the majority of PCOS affected patients (AGT + fasting insulin > or =17: 83%, AGT + glucose/insulin ratio > or =6.5: 85.1%, AGT + HOMAIR > or = 2: 87.2%, and AGT + HOMAIR > or = 3.8: 72.3%). None of the tested samples revealed any polymorphism due to the absence of any Dra III recognition site or any Bst N1 recognition site in the amplified PCR fragment digested by restriction fragmented length polymorphism. CONCLUSION: There is significantly high prevalence of AGT and insulin resistance in PCOS women, but Gly972Arg and Ala513Pro polymorphic alleles of IRS-1 are rare and are not associated with the elevated risk of PCOS amongst Taiwanese subjects. This is quite different from the similar study in phylogenetically diverged Caucasian subjects.

IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line.

BACKGROUND: Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line. RESULTS: In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively). CONCLUSION: IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line.