Timing of hormone therapy and its association with cardiovascular risk and metabolic parameters in 4-vinylcyclohexene diepoxide-induced primary ovarian insufficiency mouse model.

OBJECTIVE: To evaluate the effects of various initiation time points and durations of hormone therapy (HT) on cardiovascular and metabolic parameters of premenarche, primary ovarian insufficiency (POI) mouse model, induced by 4-vinylcyclohexene diepoxide. METHODS: A total of 50 mice at 4 weeks of age were developed into POI mouse model, further randomly categorized into 5 groups: control group without any intervention; no HT group with only high-fat diet (NT); group 1 with delayed estradiol treatment (T1); group 2 with on-time, continuous estradiol treatment (T2); and group 3 with on-time estradiol treatment but early stop (T3). Cardiovascular risk and metabolic parameters were measured. RESULTS: Presenting with similar body weights, blood glucose levels of T1, T2, and T3 were all significantly lower than NT (p < .001). Serum total cholesterol and insulin were also significantly lower in all HT groups than in NT, especially in T2 (p < .001). For serum low-density lipoprotein-cholesterol, only T2 resulted in the statically lower level than those of NT, T1, and T3 (p < .001). Aortic thickness was significantly increased with aggravated fibrotic change of the intima in NT, and such consequence was significantly ameliorated in HT groups, mostly lowered in T2 (p < .05). Last, serum pro-inflammatory cytokines were significantly low in the HT groups than in NT, especially in T2 with the lowest level (p < .05). . CONCLUSIONS: On-time, continuous E2 treatment immediately after a biologic estrogen deprivation event significantly reduced metabolic and cardiovascular risks in young, pre-menarche female mouse models of POI, confirming decreased serum levels of pro-inflammatory cytokines.

Effects of coenzyme Q10 on ovarian surface epithelium-derived ovarian stem cells and ovarian function in a 4-vinylcyclohexene diepoxide-induced murine model of ovarian failure.

BACKGROUND: Several studies have shown that coenzyme Q10 (CoQ10) can rescue ovarian aging and that ovarian surface epithelium (OSE)-derived ovarian stem cells (OSCs) are useful for treating infertility due to ovarian aging. However, few studies have examined the effect of CoQ10 on OSCs. This study was aimed to investigate whether CoQ10 activates OSCs and recovers ovarian function in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse model of ovarian failure. METHODS: Forty female C57BL/6 mice aged 6 weeks were randomly divided into four groups (n = 10/group): a control group administered saline orally, a CoQ10 group administered 150 mg/kg/day of CoQ10 orally in 1 mL of saline daily for 14 days, a VCD group administered 160 mg/kg/day of VCD i.p. in 2.5 mL of saline/kg for 5 days, and a VCD + CoQ10 group administered VCD i.p. for 5 days injection and CoQ10 (150 mg/kg/day) orally for 14 days. After treatment, follicle counts were evaluated by hematoxylin and eosin (H&E) staining, and ovarian mRNA expressions of Bmp-15, Gdf-9, and c-Kit were examined by quantitative real-time PCR. Serum FSH, AMH, and ROS levels were also measured. Oocyte-like structure counts and the expressions of Oct-4 and MVH were also evaluated after culturing OSE for 3 weeks. In a second experiment, 32 female mice were administered CoQ10 as described above, induced to superovulate using PMSG and hCG, and mated. Numbers of zygotes and embryo development rate were examined. RESULTS: Postcultured OSE showed significant increases in the numbers of oocyte-like structure and that the expression of Oct-4 and MVH were higher in the VCD + CoQ10 group than in the VCD group (p < 0.05). Numbers of surviving follicles from primordial to antral follicles, numbers of zygotes retrieved and embryo development rate to blastocyst were significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.01). Serum AMH level and ovarian expressions of Bmp-15, Gdf-9 and c-Kit were also significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.05). In contrast, serum ROS level was significantly lower in the VCD + CoQ10 group than in the VCD group (p < 0.05). CONCLUSION: This study shows that CoQ10 stimulates the differentiation of OSE-derived OSCs and confirms that CoQ10 can reduce ROS levels and improve ovarian function and oocyte quality in mice with VCD-induced ovarian failure.

Differential expression of visfatin, leptin, stromal cell derived factor-1α, endothelial nitric oxide synthase, and vascular endothelial growth factor in human leiomyomas.

AIM: This study was aimed to understand expressions of the visfatin, leptin, stromal cell derived factor (SDF)-1α, endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) in human uterine leiomyomas (UL) and normal myometrium. METHOD: This study investigated expression of visfatin, leptin, SDF-1α, eNOS and VEGF in 23 uterine leiomyoma patients and 10 normal myometrium by RT-PCR and western blot. Messenger RNA transcripts of SDF-1α, eNOS, VEGF and hypoxia inducible factor-1α (HIF-1α) were analyzed according to the size of UL by real-time PCR. RESULTS: There were no significant differences in expressions of visfatin and leptin between UL compared with normal myometrium. However, expressions of eNOS, SDF-1α and VEGF were significantly higher in both intramural and subserosal UL compared with normal myometrium. The expression of SDF1-α was significantly increased in small UL (<5 cm) compared to the large UL (≥5 cm), whereas the expressions of eNOS, VEGF and HIF-1α were higher in large UL than small UL. CONCLUSIONS: This study shows that expression of SDF-1α, eNOS and VEGF were significantly higher in UL than myometrium with a different expression pattern according to the size of UL. However, expressions of visfatin and leptin had no significant differences between the two groups.

Prediction of preterm delivery using levels of vascular endothelial growth factor and leptin in amniotic fluid from the second trimester.

PURPOSE: Impaired angiogenesis of the developing placenta in the early pregnancy is one etiology of preterm delivery. Vascular endothelial growth factor (VEGF) is a key regulator of normal angiogenesis. Leptin stimulates other angiogenic factors, including VEGF. In this study, we aimed to investigate whether levels of VEGF and leptin in amniotic fluid during the second trimester could serve as markers for preterm delivery. METHODS: This study was conducted on second trimester amniotic fluid samples obtained from women undergoing genetic amniocentesis at 16-20 weeks of gestation. VEGF and leptin levels were measured by enzyme-linked immunosorbent assay in every case of delivery at <37 weeks' gestation (n = 36) and in 36 matched controls who delivered at ≥ 37 weeks' gestation. RESULTS: Amniotic fluid VEGF levels in the preterm group (32.24 ± 4.87 pg/ml) were significantly higher than those in the control group (23.49 ± 2.09 pg/ml) (p < 0.05). Leptin levels in the amniotic fluid were higher in the preterm group (6.64 ± 0.68 ng/ml) compared to the control group (5.35 ± 0.59 ng/ml), but this difference was not significant. Amniotic fluid VEGF and leptin levels were highest in women with placenta previa and were lowest in women with intrauterine growth retardation and pregnancy-induced hypertension. CONCLUSIONS: These results show that amniotic fluid VEGF levels in the second trimester are more predictive of preterm delivery than leptin levels. This study also demonstrates that VEGF levels vary depending on the cause of preterm delivery.

Differential expression of pluripotent and germ cell markers in ovarian surface epithelium according to age in female mice.

BACKGROUND: Many studies have proposed that putative ovarian stem cells (OSCs) derived from the ovarian surface epithelium (OSE) layer of adult mammalian ovaries can produce oocytes. Few studies have reported that ovaries of aged mammalian females including mice and women possess rare premeiotic germ cells that can generate oocytes. However, no studies have reported the changes of OSCs according to the age of the female. Therefore, this study evaluated pluripotent and germ cell marker expression in the intact ovary, scraped OSE, and postcultured OSE according to age in female mice. METHODS: C57BL/6 female mice of 2 age groups (6-8 and 28-31 weeks) were superovulated by injection with 5 IU equine chorionic gonadotropin (eCG). Both ovaries were removed after 48 hours and scrapped to obtain OSE. Gene expressions of pluripotent (Oct-4, Sox-2, Nanog) and germ cell markers (c-Kit, GDF-9, and VASA) were evaluated by RT-PCR. VASA and GDF-9 were immune-localized in oocyte-like structures. RESULTS: Expressions of germ cell markers in the intact ovary were significantly decreased in aged females, whereas expressions of pluripotent markers were not detected, regardless of age. Scraped OSE expression of all pluripotent and germ cell markers, except for c-Kit, was similar between both age groups. Three weeks postcultured OSE had significantly decreased expression of GDF-9 and VASA , but not c-Kit, in old mice, as compared to young mice; however there was no difference in the expression of other genes. The number of positively stained Oct-4 by immunohistochemistry in postcultured OSE was 2.5 times higher in young mice than aged mice. Oocyte-like structure was spontaneously produced in postcultured OSE. However, while that of young mice revealed a prominent nucleus, zona pellucida-like structure and cytoplasmic organelles, these features were not observed in old mice. CONCLUSIONS: These results show that aged female mice have putative OSCs in OSE, but their differentiation potential, as well as the number of OSCs differs from those of young mice.

Effect of propofol on prostaglandin E2 production and prostaglandin synthase-2 and cyclooxygenase-2 expressions in amniotic membrane cells.

PURPOSE: Surgery during pregnancy can be a cause of preterm labor or birth, possibly resulting from anesthetic agents or direct effects of surgery. This study was aimed to investigate the effect of propofol on uterine contractility by examining prostaglandin E2 (PGE2) production and the expression of PGE synthase 2 (PGES2) and cyclooxygenase-2 (COX-2) in amniotic membrane cells. METHODS: Amniotic membranes were collected from healthy full-term women who underwent cesarean section at 37-40 weeks of gestation. The amniotic cells were cultured in α-modified-Eagle's medium with 10% fetal bovine serum for 24 h at 5% CO2 in a 37 °C incubator. Then, various doses of propofol (0.01-10 µg/ml) were used for treatment for 3 h. PGE2 concentrations in conditioned media were evaluated using ELISA. PGES2 and COX-2 expression were examined using RT-PCR and Western blot. Cell viability and apoptosis were examined by MTT, ATP assays, and the TUNEL method. RESULTS: PGE2 production significantly decreased at 0.1 and 1.0 µg/ml propofol concentrations compared to controls. COX-2 and PGES2 mRNA expression was decreased in a dose-dependent manner with a significant difference at 0.1 µg/ml propofol compared to controls. The protein expression of COX-2 showed a similar result to mRNA expression, but protein expression of PGES2 was not significantly decreased. No effect of propofol was found in cell viability. CONCLUSIONS: This study showed that propofol reduced the production of PGE2 and the expression of COX-2 and PGES2 without affecting cell viability.

Effect of phorbol 12-myristate 13-acetate on the differentiation of adipose-derived stromal cells from different subcutaneous adipose tissue depots.

Human adipose-tissue-derived stromal cells (hADSCs) are abundant in adipose tissue and can differentiate into multi-lineage cell types, including adipocytes, osteoblasts, and chondrocytes. In order to define the optimal harvest site of adipose tissue harvest site, we solated hADSCs from different subcutaneous sites (upper abdomen, lower abdomen, and thigh) and compared their proliferation and potential to differentiate into adipocytes and osteoblasts. In addition, this study examined the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, on proliferation and differentiation of hADSCs to adipocytes or osteoblasts. hADSCs isolated from different subcutaneous depots have a similar growth rate. Fluorescence-activated cell sorting (FACS) analysis showed that the expression levels of CD73 and CD90 were similar between hADSCs from abdomen and thigh regions. However, the expression of CD105 was lower in hADSCs from the thigh than in those from the abdomen. Although the adipogenic differentiation potential of hADSCs from both tissue regions was similar, the osteogenic differentiation potential of hADSCs from the thigh was greater than that of hADSCs from the abdomen. Phorbol 12-myristate 13-acetate (PMA) treatment increased osteogenic differentiation and suppressed adipogenic differentiation of all hADSCs without affecting their growth rate and the treatment of Go6983, a general inhibitor of protein kinase C (PKC) blocked the PMA effect. These findings indicate that the thigh region might be a suitable source of hADSCs for bone regeneration and that the PKC signaling pathway may be involved in the adipogenic and osteogenic differentiation of hADSCs.

Where Microsurgical Tubal Reanastomosis Stands in the In vitro Fertilization Era.

Among various options of contraception, bilateral tubal ligation (BTL) remains the most frequently used method for women worldwide even at present. However, up to 30% of those who undergo BTL eventually change their minds and wish to conceive again for a variety of reasons, such as a change in marital status or simply wanting more children. In this case, we can either approach it surgically with tubal re-anastomosis (TA) or by in vitro fertilization (IVF)-embryo transfer. Despite the many advantages of TA which lead the American Society of Reproductive Medicine Committee Opinion to recommend it as the primary choice of treatment in posttubal ligation infertility in 2012, IVF is widely being chosen as the first-line treatment nowadays. This study will review the efficacy of TA in various aspects, including pregnancy rate, cost-effectiveness, feasibility, and accessibility, based on review of the literature and our experience. Through this study, we intend to provide a basis for gynecologists to consider TA as the first option in women who wish to conceive again after BTL in this day and age of IVF.

Effects of Perilla frutescens Var. Acuta in Busulfan-Induced Spermatogenesis Dysfunction Mouse Model.

PURPOSE: The leaves of Perilla frutescens var. acuta (PFA) are generally reported to have antioxidant, anti-allergic, anti-inflammatory, and antitumor effects and commonly used as a traditional medicine in East Asia. This study aimed to investigate the protective effect and antioxidant activity of PFA on busulfan-induced testicular dysfunction, histological damage, oxidative stress (OS), sperm quality, and hormone levels using a mouse model. MATERIALS AND METHODS: C57BL/6 male mice were divided into four groups: control, busulfan-only treated, and varying concentrations of PFA (100 and 200 mg/kg) with busulfan. In the busulfan group, 40 mg/kg of busulfan was intraperitoneally injected to induce azoospermia. Mice were orally administered PFA for 35 consecutive days after busulfan administration. Samples were collected and assessed for testis/body weight, testicular histopathology, sperm quality, serum hormone levels, and OS to evaluate the effects of PFA treatment on spermatogenesis dysfunction induced by busulfan. RESULTS: The busulfan-induced testicular dysfunction model showed reduced testis weight, adverse histological changes, significantly decreased sex hormones and sperm quality, and attenuated OS. These results indicate that PFA treatment significantly increased testis weight, testis/body weight, epididymal sperm count, motility, and testosterone level compared with busulfan alone. PFA treatment also attenuated the busulfan-induced histological changes. Furthermore, compared with mice treated with busulfan alone, PFA supplementation upregulated the testicular mRNA expression of the antioxidant enzymes superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), with a decrease in malondialdehyde (MDA) production and an increase in SOD and GPx activities. CONCLUSIONS: This study shows that PFA exerts a protective effect against testicular damage by attenuating OS induced by busulfan. Our results suggest that PFA is a potentially relevant drug used to decrease the side effects induced by busulfan on testicular function and sperm during cancer chemotherapy.

Computer simulation approach to the identification of visfatin-derived angiogenic peptides.

Angiogenesis plays an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and skin regeneration. Visfatin is a 52 kDa adipokine secreted by various tissues including adipocytes. It stimulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis. However, there are several issues in developing full-length visfatin as a therapeutic drug due to its high molecular weight. Therefore, the purpose of this study was to develop peptides, based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques.Initially, the active site domain (residues 181∼390) of visfatin was first truncated into small peptides using the overlapping technique. Subsequently, the 114 truncated small peptides were then subjected to molecular docking analysis using two docking programs (HADDOCK and GalaxyPepDock) to generate small peptides with the highest affinity for visfatin. Furthermore, molecular dynamics simulations (MD) were conducted to investigate the stability of the protein-ligand complexes by computing root mean square deviation (RSMD) and root mean square fluctuation(RMSF) plots for the visfatin-peptide complexes. Finally, peptides with the highest affinity were examined for angiogenic activities, such as cell migration, invasion, and tubule formation in human umbilical vein endothelial cells (HUVECs). Through the docking analysis of the 114 truncated peptides, we screened nine peptides with a high affinity for visfatin. Of these, we discovered two peptides (peptide-1: LEYKLHDFGY and peptide-2: EYKLHDFGYRGV) with the highest affinity for visfatin. In an in vitrostudy, these two peptides showed superior angiogenic activity compared to visfatin itself and stimulated mRNA expressions of visfatin and VEGF-A. These results show that the peptides generated by the protein-peptide docking simulation have a more efficient angiogenic activity than the original visfatin.

High pregnancy rate after microsurgical tubal reanastomosis by temporary loose parallel 4-quadrant sutures technique: a long long-term follow-up report on 961 cases.

BACKGROUND: Only a limited portion of sterilized women undergo tubal reanastomosis due to high costs, limited availability of qualified practitioners willing to perform the procedure and increasing success rates with IVF. However, IVF has complications and an increased risk of ectopic pregnancy and multiple pregnancies. Recently, the importance of specialized training for tubal anastomosis has been re-emphasized. This study aimed to report the procedure of our microsurgical tubal reanastomosis by a temporary loose parallel 4-quadrant suture technique and its high pregnancy outcome over the last 20 years. METHODS: This clinical study retrospectively analyzed data on 961 consecutive patients who underwent tubal reversal between March 1988 and August 2007 in a large urban medical center. All surgical operations were performed by microsurgical tubal reanastomosis using a temporary loose parallel 4-quadrant suture technique by a single surgeon. Subsequent pregnancy outcomes were evaluated. RESULTS: The overall pregnancy rate was 85.1, 82.6 being intrauterine and 2.5% ectopic. The pregnancy rate was significantly reduced in patients over 40 years old (53.9%) compared with patients aged 40 years or less (90.3%) (P < 0.05). Repair done at the interstitial-ampulla site yielded a significantly higher ectopic pregnancy rate (20.0%) compared with other anastomosis sites (0-3.2%) (P < 0.001). CONCLUSIONS: This study shows that our technique resulted in a high pregnancy rate comparable with the level of natural fertility. The study also reveals that ectopic pregnancy frequently occurs in tubal reanastomosis of the interstitial-ampulla site compared with other sites.

Decreased expressions of vascular endothelial growth factor and visfatin in the placental bed of pregnancies complicated by preeclampsia.

AIM: The aim of the present study was to examine the expression of vascular endothelial growth factor (VEGF) and visfatin in the third trimester placental bed of pregnancies with and without preeclampsia (PE). MATERIAL AND METHODS: The study group consisted of placental bed biopsy tissues obtained from pregnancies with (n = 20) and without (n = 20) PE. The normotensive controls without PE were matched for gestational age at delivery with patients with PE. The expression of VEGF and visfatin in the placental bed tissues were evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot. RESULTS: There was no statistical difference between the PE group and the normotensive control group in age and body mass index (BMI). The expression of VEGF and visfatin was significantly decreased in the PE group compared with the normotensive control group (P < 0.05). CONCLUSION: This study showed decreased expressions of VEGF and visfatin in the third trimester placental bed of pregnancies with PE compared with the normotensive controls. This result suggests that decreased expression of these angiogenic factors in placental bed may be associated with the pathogenesis of PE.

Up-regulation of inhibitors of DNA binding/differentiation gene during alendronate-induced osteoblast differentiation.

PURPOSE: To investigate the effect of alendronate on the expression of Id genes in osteoblast differentiation. METHODS: C2C12 cells were treated with alendronate for various concentrations and time periods. For evaluation of alendronate-induced osteoblast differentiation in C2C12 cells, alkaline phosphatase (ALP) activity was measured. The expression of osteoblast differentiation markers such as ALP, type-1 collagen (Col 1), and osteocalcin (OCN), and the expression of Id-1 and Id-2 were measured by RT-PCR. In order to understand the mechanism underlying the regulation of Id genes, the promoter region of the Id-1 gene was identified. Database analysis of the promoter region for Id-1 using known consensus sequences identified several putative response elements, including CCAAT/enhancer-binding protein beta (C/EBPß). RESULTS: Alendronate treatment significantly increased not only ALP activity but also the expression of ALP, Col 1, and OCN, Id-1 and Id-2. C/EBPß and alendronate cooperatively increased the promoter activity and expression of Id-1. CONCLUSIONS: These results suggest that C/EBPß-mediated Id-1 transcriptional activation may regulate alendronate-induced osteoblast differentiation of C2C12 cells.

A Combination of Pharmacophore-Based Virtual Screening, Structure-Based Lead Optimization, and DFT Study for the Identification of S. epidermidis TcaR Inhibitors.

The transcriptional regulator (TcaR) enzyme plays an important role in biofilm formation. Prevention of TcaR-DNA complex formation leads to inhibit the biofilm formation is likely to reveal therapeutic ways for the treatment of bacterial infections. To identify the novel ligands for TcaR and to provide a new idea for drug design, two efficient drug design methods, such as pharmacophore modeling and structure-based drug design, were used for virtual screening of database and lead optimization, respectively. Gemifloxacin (FDA-approved drug) was considered to generate the pharmacophore model for virtual screening of the ZINC database, and five hits, namely ZINC77906236, ZINC09550296, ZINC77906466, ZINC09751390, and ZINC01269201, were identified as novel inhibitors of TcaR with better binding energies. Using structure-based drug design, a set of 7a-7p inhibitors of S. epidermidis were considered, and Mol34 was identified with good binding energy and high fitness score with improved pharmacological properties. The active site residues ARG110, ASN20, HIS42, ASN45, ALA38, VAL63, VAL68, ALA24, VAL43, ILE57, and ARG71 are playing a promising role in inhibition process. In addition, we performed DFT simulations of final hits to understand the electronic properties and their significant role in driving the inhibitor to adopt apposite bioactive conformations in the active site. Conclusively, the newly identified and designed hits from both the methods are promising inhibitors of TcaR, which can hinder biofilm formation.

Efficacy of bleeding control using a large amount of highly diluted vasopressin in laparoscopic treatment for interstitial pregnancy.

OBJECTIVE: We sought to report the safety and effectiveness of bleeding control using a large amount of highly diluted vasopressin in laparoscopic management of interstitial pregnancy. STUDY DESIGN: This was an uncontrolled retrospective review of 20 patients who were laparoscopically treated for interstitial pregnancy using a large amount of highly diluted vasopressin. For hemostasis, 1 ampule of vasopressin was diluted in 1000 mL of normal saline (1000-fold) and 150-250 mL of diluted vasopressin was injected in the uterus below interstitial pregnancy. RESULTS: Mean patient age and gestational age was 33.5 years and 6.7 weeks, respectively. Mean blood loss was 24 mL. The mean serum human chorionic gonadotropin level was 10,950, 4065, and 959 mIU/mL on the day of operation and postoperative days 1 and 4, respectively. CONCLUSION: Laparoscopic management of interstitial pregnancy using a large amount of highly diluted vasopressin is safe and effective in hemostasis with minimal blood loss and no complications.

Estrogen administration during superovulation increases oocyte quality and expressions of vascular endothelial growth factor and nitric oxide synthase in the ovary.

AIMS: This study investigated whether estrogen administration during superovulation enhances oocyte quality using a mice model. We also investigated whether this estrogen treatment regulates the expressions of angiogenic factors, such as vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), in the ovary. METHOD: Female mice were co-injected with various doses of estrogen (1 microM, 10 microM and 100 microM) and pregnant mare serum gonadotrophin during superovulation, followed by human chorionic gonadotrophin injection 48 hours later. Then they were mated with individual males. After 18 hours, zygotes were flushed and cultured to blastocyst. The expression of VEGF and eNOS in the ovary was examined using Western blot and immunohistochemistry. The control group was superovulated without estrogen. RESULTS: Both numbers of ovulated zygotes and the rate of embryo development to blastocyst were significantly increased in the 1-microM estrogen dose compared to the control group. VEGF and eNOS expressions were stimulated by estrogen treatment. In particular, VEGF expression was significantly increased at 1-microM estrogen concentration, whereas, eNOS expression was significantly increased in all estrogen concentrations compared to controls. CONCLUSIONS: The study showed that estrogen co-injection during superovulation increased the ovarian response, embryo developmental competence and expressions of VEGF and eNOS in the ovary.

Effects of Oxysterols on Chondrogenesis of Human Adipose-derived Stem Cells.

Human adipose-derived stem cells (hADSCs) have been implicated as a high potency source of chondrocytes in cell therapy for cartilage defects. However, appropriate stimulators for the chondrogenesis of hADSCs are needed. Oxysterols have the potential to act as a stimulator. This study aims to investigate the effect of oxysterols on the chondrogenesis of hADSCs. hADSCs were collected from the abdominal subcutaneous tissue samples of patients undergoing caesarean section, and were cultured to passage 5. Mesenchymal stem cell markers were examined by flow cytometry. After the cells were subjected to adipogenic, osteogenic, and chondrogenic induction, the differentiation of each cell lineage was evaluated by RT-PCR and specific staining (Oil red O, Alizarin red S, and Alcian blue, respectively). The cell pellets of hADSCs were cultured in chondrogenic induced media containing 2µM 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, or 25-hydroxycholesterol for 4 weeks. At 3 and 4 weeks of culture, the size and wet weight of the pellets were measured. The expressions of chondrogenesis-related genes and glycosaminoglycans production were examined by quantitative real-time PCR and Alcian blue staining. hADSCs were positive for the mesenchymal markers CD75, CD90, and CD105, while being negative for the hematopoietic markers CD31, CD34 and CD45. The multilineage potential of hADSCs was confirmed by the expression of adipogenic-, osteogenic-, and chondrogenic-specific genes, along with specific staining. 22(R)-hydroxycholesterol treatment significantly increased the size and wet weight of the pellet, glycosaminoglycans production, and expression of chondrogenic-related genes compared to the control group and other oxysterols (P<0.05). These results indicate that 22(R)-hydroxycholesterol can be effective as a stimulator for the chondrogenesis of hADSCs.

Combination of Korean Red Ginseng Extract and Hydrogen-Rich Water Improves Spermatogenesis and Sperm Motility in Male Mice.

OBJECTIVE: To investigate the effect of hydrogen-rich Korean Red Ginseng (KRG) water (HRGW) mixture on the spermatogenesis and sperm motility of mice of different ages. METHODS: Eighty young (3 month-old) and aged (12 month-old) male mice were randomly assigned to 4 groups (n =10 per group) including control group, hydrogen-rich water (HRW) group (10 mL/kg daily), KRG group (50 mg/kg daily) and HRGW group (10 mL/kg and 50 mg/kg daily) by an oral zoned needle for 4 weeks. Sperm count and motility were measured using sperm suspension released from cauda epididymis. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and reactive oxygen species (ROS) in serum have also been estimated. Tubular changes were examined through histological hematoxylin and eosin staining. Expression of antioxidation (PPx3, PPx4, GSTm5 and GPx4), spermatogenesis (inhibin-a, neptin-2 and CREM), antiaging (SIRT1 and SIRT2), and angiogenesis [visfatin and vascular endothelial growth factor (VEGF)] related genes were examined through real-time polymerase chain reaction. RESULTS: HRW and KRG treatment stimulated spermatogenesis followed by increasing sperm production and sperm motility (P <0.05). These effects were strengthened synergistically by a HRGW mixture (P <0.05 or P <0.01). HRGW greatly increased the expressions of antioxidation, antiaging, spermatogenesis related genes and VEGF especially in aged mice (P <0.05). Serum testosterone and FSH levels also increased, while serum ROS level decreased (all P <0.05). CONCLUSION: HRGW increases sperm production and motility by enhancing antioxidation and stimulating spermatogenesis and sex hormone production, particularly in aged mice.

Role of Visfatin in Restoration of Ovarian Aging and Fertility in the Mouse Aged 18 Months.

The activation of dormant primordial follicles and ovarian angiogenesis has been attempted as a new treatment strategy for age-related ovarian aging. This study examined whether visfatin rescues age-related fertility decline in female mice aged 18 months, and whether this effect relates to the mTOR/PI3K signaling pathways for activation of primordial follicles and ovarian angiogenesis. Female mice were intraperitoneally injected with 0.1 ml of 500 ng/ml or 1000 ng/ml of visfatin three times at intervals of 2 days, and both ovaries were provided for H&E staining. In another experiment, the mice were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin, and were mated with males. After 18 h, zygotes were collected and cultured for 4 days, and numbers and embryo developmental competency of zygotes retrieved were evaluated. The expression of mTOR/PI3K signaling pathway regulated genes (4EBP1, S6K1, and RPS6) and angiogenic factors (VEGF, visfatin, and SDF-1α) in the ovary were examined. As well, visfatin-treated mice were mated with male mice for 2 weeks, and the pregnancy outcome was monitored up to 3 weeks. Visfatin significantly increased the total numbers of follicles compared with control. Numbers of zygotes retrieved, blastocyst formation rate, and pregnancy rate were significantly increased at 500 ng/ml of visfatin (2.83%, 40.0%, and 80%, respectively) compared with control (0, 0, and no pregnancy). Ovarian expressions of S6K1, RPS6, VEGF, visfatin, and SDF-1α were significantly stimulated at 500 ng/ml of visfatin. These results show that visfatin treatment of an optimal dose rescues age-related decline in fertility, possibly by stimulating mTOR/PI3K signaling.

Prediction of ovarian aging using ovarian expression of BMP15, GDF9, and C-KIT.

IMPACT STATEMENT: Ovarian aging is becoming a more important issue in terms of fertility preservation and infertility treatment. Serum anti-Mullerian hormone (AMH) level and antral follicle count (AFC) are being practically used as markers of ovarian aging as well as ovarian reserve in human. However, these factors have some drawbacks in assessing ovarian aging and reserve. Therefore, the identification of ovarian expressions of BMP15, GDF9, and C-KIT according to female could be applied as a potent predictor of ovarian aging. This work provides new information on the development of diagnosis and treatment strategy of age-related fertility decline and premature ovarian insufficiency.