Effects of Natural Progesterone and Synthetic Progestin on Germ Layer Gene Expression in a Human Embryoid Body Model.

Natural progesterone and synthetic progestin are widely used for the treatment of threatened abortion or in in vitro fertilization (IVF) cycles. This in vitro study aimed to assess whether the treatment with natural progesterone or synthetic progestin influences the germ layer gene expression on the early human embryonic development using human embryonic stem cells (hESCs)-derived embryoid bodies (hEBs) as a surrogate of early stage human embryonic development. Human EBs derived from hESCs were cultured for nine days, and were treated with natural progesterone (P4) or synthetic progestin, medroxyprogesterone acetate (MPA) at 10-7 M for five days. To reverse the effects of treatment, mifepristone (RU486) as progesterone antagonist was added to the hEBs for four days starting one day after the initiation of treatment. Mouse blastocysts (mBLs) were cultured in vitro for 24 h, and P4 or MPA at 10-7 M was treated for an additional 24 h. The treated embryos were further transferred onto in vitro cultured endometrial cells to evaluate chorionic gonadotropin (CG) expression. To analyze the effects of P4 or MPA, the expression of differentiation genes representing the three germ layers was investigated, GATA-binding factor 4 (GATA4), α-fetoprotein (AFP), hepatocyte nuclear factor (HNF)-3ß, hepatocyte nuclear factor (HNF)-4α (endoderm), Brachyury, cardiac actin (cACT) (mesoderm), and Nestin (ectoderm), using quantitative reverse transcription PCR (qRT-PCR) and immunostaining. Significantly lower expressions of HNF-3ß, HNF-4α, Brachyury, and Nestin were observed in MPA-treated hEBs (all p < 0.05), which was negated by RU486 treatment. This inhibitory effect of MPA was also observed in mouse embryos. Conclusively, the effects of natural progesterone and synthetic progestin may differ in the germ layer gene expression in the hEB model, which suggests that caution is necessary in the use of progestogen.

Effects of paclitaxel and cisplatin on in vitro ovarian follicle development.

INTRODUCTION: Despite its importance in pre-chemotherapy counselling, specific reproductive toxicological information about cisplatin and paclitaxel is very rare. This study aimed to investigate the concentrations at which cisplatin and paclitaxel, alone or combined, affect the in vitro development of ovarian follicles. Their differential effects on the oocytes and surrounding granulosa cells was also evaluated. MATERIAL AND METHODS: Ovarian follicles were cultured in vitro using gonadotropins and treated with 10-8-10-10 M of cisplatin, paclitaxel, or both. At day 13, granulosa cells and oocytes were retrieved and used for imaging and functional analyses. RESULTS: Follicular survival and growth was significantly suppressed in all treatment groups at 10-9 M or higher concentrations, and additive effects were observed in the combination group (p < 0.01). Oocyte-specific genes such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) were more suppressed in the paclitaxel group than in the cisplatin group. Granulosa cell-specific gene suppression and its electron microscopic alteration were more prominent in the cisplatin group than in the paclitaxel group. X-linked inhibitor of apoptosis protein (XIAP) expression of granulosa cells was also further down-regulated in the cisplatin group. CONCLUSIONS: These data provide an insight into the critical concentrations regarding in vitro follicular development and the differential effects of chemotherapeutic effects on oocytes and granulosa cells. Further studies are necessary to develop more efficient pre-chemotherapeutic fertility-sparing medical treatment that can evade oocyte-specific damage.

Synergistic regenerative effects of functionalized endometrial stromal cells with hyaluronic acid hydrogel in a murine model of uterine damage.

Clinically intractable infertility and recurrent miscarriage due to irreversible endometrial damage need to be treated with biomaterial- and cell-based therapies. Some previous studies have reported on the efficacy of a collagen scaffold and/or bone marrow-derived mesenchymal stem cells. However, the functional differentiation of grafted cells was uncertain, and the time required for regeneration was long in these studies. Here, we show the synergistic regenerative effects of hyaluronic acid (HA) hydrogel with in vitro decidualized endometrial stromal cells (EMSCs) in a murine uterine infertility (synechiae) model. Decidualized EMSCs (dEMSCs) were encapsulated with HA hydrogel, combined with three different doses of fibrinogen/thrombin (5, 50, and 500 mIU/mL). The HA/fibrin gel showed biocompatibility when mixed with dEMSCs. The addition of thrombin enhanced gel formation (5 and 50 mIU/mL) and engraftment and enabled the effective release of adhesion molecules. Within two weeks, which is a short duration, treatment with hydrogel decreased the fibrous tissue and increased the thickness of the endometrium. The regenerated endometrium demonstrated functional recovery, as evidenced by the expression and secretion of molecules essential for embryonic implantation, such as Desmin, CD44, PECAM, and IGF-1. Transferred embryos successfully implanted and the normal development of implanted embryos (n = 37) were evaluated by co-localization of distinct markers of the three germ layers (Sox2, Nestin, Brachyury, AFP, and HNF4α). Live birth of offspring was achieved in the regenerated endometrium by HA hydrogel. Therefore, HA hydrogel-mixed dEMSCs can be an innovative treatment strategy with rapid recovery of endometrial damage and may also have therapeutic potential in intractable infertility or recurrent miscarriage. STATEMENT OF SIGNIFICANCE: Decidualized EMSCs (dEMSCs) encapsulated with HA hydrogel combined with fibrinogen/thrombin (50 mIU/mL) showed injectability and biocompatibility when mixed with dEMSCs. Hydrogel-encapsulated dEMSCs can be a useful treatment for damaged endometrium in short duration, with successful implantation and normal development in a murine model.

Differential MicroRNA Expression Profile of Human Embryonic Stem Cell-Derived Cardiac Lineage Cells.

MicroRNAs (miRNAs) are small non-coding RNA molecules that participate in transcriptional and post-transcriptional regulation of gene expression. miRNAs have numerous roles in cellular function including embryonic development. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into most of cell types including cardiomyocytes (CMs). These characteristics of hESCs make them considered as an important model for studying human embryonic development and tissue specific differentiation. In this study, we tried to demonstrate the profile of miRNA expression in cardiac differentiation from hESCs. To induce differentiation, we differentiated hESCs into CMs by direct differentiation method and characterized differentiated cells. To analyze the expression of miRNAs, we distinguished (days 4, 8, 12, 16, 20, 24, 28) and isolated RNAs from each differentiation stage. miRNA specific RT-qPCR was performed and the expression profile of miR-1, -30d, -133a, -143, -145, -378a, -499a was evaluated. The expression of all miRs was up-regulated at day 8. miR-143 and -145 expression was also up-regulated at the later stage of differentiation. Only miR-378a expression returned to undifferentiated hESC levels at the other stages of differentiation. In conclusion, we elucidated the expression profile of miRNAs during differentiation into cardiomyocytes from hESCs. Our findings demonstrate the expression of miRNAs was stage-dependent during differentiation and suggest that the differentiation into CMs can be regulated by miRNAs through direct or indirect pathway.

Induction of multiple ovulation via modulation of angiotensin II receptors in in vitro ovarian follicle culture models.

In vitro culture of ovarian follicles is a promising bioengineering technique for retrieving fertilizable oocytes from preserved ovarian tissues of cancer survivors. However, current in vitro follicle culture techniques are labour-intensive and of low efficiency, as only single follicle culture (SFC) has been possible to date. The present study investigated the feasibility of multifollicular cluster culture (MFCC) system using angiotensin II receptor (ATII-Rc) analogues. Ovarian pre-antral follicles isolated from 2-week-old C57BL6 mice were cultured with ATII-Rc agonist or antagonist and their maturation outcomes were compared with control group. When single follicles were cultured, the ovulation and maturation rates were similar in all three groups. When three-follicle clusters were cultured, up to three follicles were ovulated in the ATII-Rc agonist group while none or one follicle ovulated in control or antagonist groups (p < 0.0001). Significantly higher numbers of mature oocytes were obtained in the agonist group (three-follicle 28.2 ± 4.9 vs. SFC 11.0 ± 1.3, per 25 cultured droplets) (p < 0.0001), and the development of each fertilized oocytes was comparable to those from SFC. It is therefore concluded that this novel MFCC system can significantly improve the efficiency of in vitro mature oocyte retrieval via ATII-Rc modulation. Copyright © 2016 John Wiley & Sons, Ltd.

Gonadotropin ratio affects the in vitro growth of rhesus ovarian preantral follicles.

In vitro follicle growth (IVFG) strategy is critical in the fertility preservation of cancer survivors; however, its optimal protocol needs to be developed using primate models since the availability of human samples is limited. Only a few previous studies have reported the successful IVFG of rhesus monkey ovaries using low-dose follicle-stimulating hormone (FSH) (0.3 or 3 ng/mL) and long-term culture (up to 5 weeks) and it is still uncertain in regard to the optimal culture duration and effective dose of treated gonadotropins applicable to the IVFG of rhesus preantral follicles. Recently, we have reported that the FSH to luteinizing hormone (LH) ratio affects the in vitro growth of murine ovarian follicles. We aimed to investigate whether gonadotropin ratios affect the efficiency of rhesus follicular growth in vitro Ovaries were collected from six necropsied rhesus macaques (4-9 years) and preantral follicles were retrieved and cultured for 14 days using 200 mIU/mL FSH. The characteristics of follicular growth were compared between the FSH:LH=1:1 (n=24) and FSH:LH=2:1 (n=24) groups. High concentration gonadotropin treatment shortened the duration required for in vitro maturation of rhesus preantral follicles. The FSH:LH=2:1 group showed a faster follicular growth and enabled the acquisition of mature oocytes, although the expression of growth differentiation factor (GDF)-9 and anti-Müllerian hormone (AMH) did not differ significantly between the two groups. Taken together, high dose gonadotropin treatment can shorten the duration of IVFG and the gonadotropin ratio is important in the IVFG of rhesus monkey ovaries.

Ginsenosides may enhance the functionality of human embryonic stem cell-derived cardiomyocytes in vitro.

Various chemicals have been reported to induce the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs), however, their contributions to the functionality of hESC-derived CMs are still limited. In this study, we evaluated the effects of red ginseng extract (RGE), ginsenoside-Rb1 (gRb1, panaxadiol), and ginsenoside-Re (gRe, panaxatriol) on the differentiation of hESCs and the functionality of derived CMs. Undifferentiated hESCs were treated with 0.25 mg/mL RGE, 10 µmol/L gRb1, or 10 µmol/L gRe for 48 hours at the differentiation induction (early stage) or maturation (late stage) period. The expression of mesodermal and cardiac transcription factor genes was upregulated in the ginsenoside-treated groups from early stage. The expression of cardiac sarcomeric genes was significantly upregulated at the late stage. The gRb1- and gRe-treated groups upregulated the expression of potassium voltage-gated channel subfamily E member 1 (KCNE1) and the gRe-treated group showed a longer beating duration compared to the control. Taken together, ginsenosides may enhance the functionality of hESC-derived CMs in vitro.

MicroRNAs transfected into granulosa cells may regulate oocyte meiotic competence during in vitro maturation of mouse follicles.

STUDY QUESTION: Do microRNAs (miRNAs) in granulosa cells (GCs) affect oocyte maturation during ovarian follicle development? SUMMARY ANSWER: Sophisticated regulation by miRNAs in ovarian GCs may improve oocyte maturation efficiency during ovarian follicle development. WHAT IS KNOWN ALREADY: The meiotic competence of oocytes depends on the follicle's potential to undergo appropriate maturation and is an important factor in infertility therapies such as IVF. The exact function of the GCs during follicular development remains unknown. STUDY DESIGN, SIZE, DURATION: After in vitro maturation (IVM) and ovulation induction of isolated ovarian pre-antral follicles from 12-day-old female C57BL6 mice (n = 40), miRNA expression in the GCs was compared according to the maturity of the oocyte (metaphase I (MI) versus metaphase II (MII)). The miRNAs, which showed notable different expression, were modulated by transfection during IVM of follicles. MATERIALS, SETTING, METHODS: miRNA expression and candidate target gene expression in GCs of isolated murine ovarian pre-antral follicles were evaluated by real-time PCR after IVM. miR mimics and -inhibitors for selected miRNAs were transfected into the in vitro-maturated follicles, and ovulation, oocyte maturation and fertilization rates were compared. Candidate target gene expressions in GC were evaluated by quantitative PCR and immunohistochemistry using confocal microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: The relative expression of mmu-let-7b (0.78 ± 0.10, P = 0.016), mmu-let-7c (0.78 ± 0.12, P = 0.029), mmu-miR-27a (0.57 ± 0.18, P = 0.016) and mmu-miR-322 (0.59 ± 0.14, P = 0.008) was significantly lower in the GCs of follicles containing MII oocytes compared with those of MI oocytes. Transfection with a mmu-miR-27a-mimic sequence decreased the oocyte maturation rate compared with that for the control (9.4 versus 18.9%, P = 0.042), and transfection with mmu-let-7c-, mmu-miR-27a- and mmu-miR-322-inhibitor sequences increased the oocyte maturation rate by 1.5- to 2.0-folds compared with that for the control (40.6, 31.6, and 30.5%versus 18.9%, P < 0.001, P = 0.013, P = 0.021, respectively). The expression of IGFBP-2 was higher in GCs of MII than in the GCs of MI, and higher in miR-inhibitor transfection groups than in miR-mimic transfection groups and controls. LIMITATIONS, REASONS FOR CAUTION: An in vitro model was used in lieu of an in vivo model because of the ease of performing miRNA transfection in cell culture. However, studies have shown similarities and differences in in vivo versus in vitro cultured follicles. The findings of the present study need to be confirmed using in vivo maturation models and extended to evaluate developmental competence. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that sophisticated miRNA regulation in GCs may improve oocyte maturation efficiency during ovarian follicle development. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111539). None of the authors has any conflicts of interest to declare.

Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 µM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

Effects of urinary and recombinant gonadotropins on in vitro maturation outcomes of mouse preantral follicles.

Gonadotropins including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play a crucial role in human-assisted reproduction techniques. Despite wide use of recombinant gonadotropins in clinical practice, the efficacy of urinary gonadotropins and the dosage of LH component have not yet been elucidated. This study was designed to investigate the difference of follicle culture outcomes according to various compositions of gonadotropins during in vitro culture of mouse preantral follicles. Ovaries were obtained from the 14-day-old C57BL/6 mice, and preantral follicles were isolated and cultured in culture media supplemented with human menopausal gonadotropin (hMG) 200 mIU/mL (group 1), recombinant FSH and LH (rFSH + rLH) 200 mIU/mL each (group 2), rFSH 200 mIU/mL + rLH 100 mIU/mL (group 3), or rFSH 200 mIU/mL + rLH 20 mIU/mL (group 4). Follicle survival rate was significantly lower in group 4. Antral follicles in lower doses of LH (groups 3, 4) showed a statistically significant larger diameter and tended to have a higher antral formation rate. However, follicles in group 1 tended to have a higher oocyte maturation rate. Estradiol concentration from conditioned media from 2:1 FSH/LH (group 3) was significantly higher than those from 1:1 FSH/LH (group 2) or 10:1 FSH/LH (group 4). Half dose of rLH to rFSH facilitated upregulation of growth differentiation factor 9 (Gdf9) expression in granulosa cells when compared to 1:1 FSH/LH or 10:1 FSH/LH. Conclusively, recombinant gonadotropins provided a comparable condition to hMG, and half dose of rLH to rFSH seems to be more suitable for follicular development during in vitro culture.

Cryopreservation of human embryonic stem cells derived-cardiomyocytes induced by BMP2 in serum-free condition.

Although previous studies showed that cardiomyocytes (CMs) can be generated from human embryonic stem cells (hESCs), the protocols for cryopreservation of hESC-derived CMs is not available to date. Here, we report on the efficient generation of hESC-derived CMs by direct differentiation using BMP2 in a serum-free condition, along with successful cryopreservation of derived CMs using Rho-associated kinase (ROCK) inhibitor. To induce differentiation, hESCs were treated with activin A and BMP2 for 5 days. A mesodermal gene, Brachyury, was expressed from day 3, and cardiac-specific markers such as Nkx2.5 and cTnI were detected at day 14. Furthermore, these cardiac progenitors expressed ion channel-related transcripts such as HCN1 and HCN2 from day 10. Beating clusters were observed from 14 days of differentiation for up to 35 days. Using mass cryopreservation, we froze hESC-derived CMs at 2 stages, at day 12 and 16 (prebeating and postbeating), after treating with ROCK inhibitor, Y-27632. Postthaw survival of CMs was higher in day 12 group compared to day 16, and some cell clusters from day 12 group recovered their contraction. From transmission electron microscope (TEM) analysis, less ultrastructural alterations were observed in day 12 group. Our results provide an insight into the use of BMP2 for cardiac lineage differentiation in a serum-free condition and a possibility of long-term storage of hESC-derived CMs.

Red ginseng extract facilitates the early differentiation of human embryonic stem cells into mesendoderm lineage.

Human embryonic stem cells (hESCs) have capacities to self-renew and differentiate into all cell types in vitro. Red ginseng (RG) is known to have a wide range of pharmacological effects in vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs), and hESC-derived cardiac progenitors (CPs) were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3ß on EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.

Dicer is a key player in oocyte maturation.

OBJECTIVE: Apply Dicer siRNA to study functions of Dicer and miRNA during oogenesis. MATERIALS AND METHODS: Mouse oocytes were injected with Dicer siRNA and negative control siRNA and then matured in vitro. After IVM, oocytes were examined for maturation rates, spindle and chromosomal organization, and various gene expressions. RESULTS: Dicer siRNA significantly reduced maturation rates, increased abnormal spindle and chromosomal organization, and reduced the transcripts of Dicer miRNAs, spindle formation proteins (plk1 and AURKA) and spindle check points (Bub1, Bublb). Depletion of bulb16 markedly prohibited the first polar body extrusion and increased the incidence of misaligned chromosomes and abnormal meiotic spindle assembly. CONCLUSION: Dicer siRNA triggered a cascade reduction for gene expressions starting from Dicer to miRNAs than to spindle assembly proteins and checkpoints which led to abnormal spindle and chromosomal organization. Thus, Dicer and miRNA appeared to play an important role during oogenesis and were essential for meiotic completion.

MicroRNA expression profiles are altered by gonadotropins and vitamin C status during in vitro follicular growth.

MicroRNAs (miRs) are known to repress target genes at posttranscriptional level and play important roles in the maturation of cells. However, the expression profiles of miRs during follicular maturation have not been fully elucidated. This study was designed to investigate the expression profiles of miRs in murine follicles according to human chorionic gonadotropin (hCG) treatment and vitamin C status during in vitro culture. Ovaries were removed from the 12-day-old wild-type and vitamin C-deficient (L-gulonogammalactone oxidase knockout, Gulo-/-) C57BL6 mice. Preantral follicles were isolated and cultured in 20 µL droplets of culture media supplemented with follicle-stimulating hormone and luteinizing hormone (FSH + LH). After their full maturation, follicles were divided into 2 groups: with and without hCG treatment. Real-time polymerase chain reaction (PCR) was performed using oocytes and granulosa cells (G-cells) to evaluate the miRs known to be expressed mainly in the mouse ovary. After the addition of hCG, miR profiles showed divergent changes between oocytes and G-cells. These profiles significantly differed from those of hCG(-) group. Compared to wild type, Gulo-/- mice showed altered miR profiles in matured oocytes and G-cells. Conclusively, hCG supplementation and vitamin C status alter the miR expression profiles in oocytes and G-cells during in vitro growth of murine follicles.

Expression of the Lycat gene in the mouse cardiovascular and female reproductive systems.

The Lycat homologue in zebrafish maps to the deletion interval of the cloche mutant in which hematopoietic and endothelial cell lineages are affected. However, its definitive relationship to cloche is inconclusive, partly due to inadequate expression data of Lycat from any organisms. We precisely examined the temporal and spatial expression patterns of Lycat in mouse using RNA in situ hybridization, immunostaining, and BAC transgenesis. Lycat is initially expressed in developing heart, lung, and somites, and later becomes progressively restricted to all vascular smooth muscle cells. In adult ovaries, Lycat turns on in oocytes during the transition from primary to secondary follicles. Expression of the Lycat/reporter transgene in the extraembryonic mesoderm, cardiogenic mesoderm, and primitive streak, but not extraembryonic endoderm at E7.5, suggests its potential roles in regulating cardiac, smooth muscle, hematopoietic and endothelial lineages. Promoter mapping assay by transient transgenesis identifies a novel cardiac-specific regulatory region in the Lycat locus.

Serum insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) in IVF patients with polycystic ovary syndrome: correlations with outcome.

OBJECTIVE: To evaluate serum insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) levels during stimulation in polycystic ovary syndrome (PCOS) and control populations as factors predictive of IVF outcome. DESIGN: Observational study. SETTING: Academic medical center-based IVF practice. PATIENT(S): Forty-three PCOS and 33 male-factor control patients undergoing IVF from 2002 to 2004. INTERVENTION(S): Treatment with a dual suppression protocol incorporating oral contraceptive pills (OCPs) and GnRH agonist suppression followed by low-dose gonadotropin therapy. MAIN OUTCOME MEASURE(S): The PCOS and control patients' serum IGF-I and IGFBP-3 levels were compared and correlated with IVF outcome. RESULT(S): PCOS and control patients were comparable in terms of demographics and IVF outcome. In both, mean serum IGF-I levels increased during stimulation. PCOS patients whose IGF-I levels decreased from day 3 to day of hCG had a significantly higher mean number of immature oocytes retrieved (4.8 +/- 1.1 vs. 2.4 +/- 0.4; P=.02). IGFBP-3 levels increased during stimulation in PCOS patients but tended to decrease in control patients. In PCOS patients, an increase in IGFBP-3 levels during stimulation was associated with a greater likelihood of becoming pregnant (P=.03) and of ongoing pregnancy (P=.02). CONCLUSION(S): The bioavailability of IGF-I appears to play a key role in oocyte maturation in PCOS patients. Alterations in IGFBP-3 concentration during stimulation may be a critical mechanism in modulating IGF-I activity.

Correlation of somatic cell steroid secretion and quality of generated oocytes after in-vitro stimulation of mouse follicles.

PURPOSE: To test the possibility of follicular somatic cell steroidogenesis as a marker for quality of their embraced oocytes. METHODS: Mechanically isolated mouse preantral follicles were cultured and matured in-vitro (IVC/IVM) for study. RESULTS: During IVC/IVM, oogenesis occurred concomitantly with folliculogenesis in a coordinated manner and simultaneously with progressive increments of somatic cell steroidogenesis. Follicular E(2) production of matured oocytes were significantly higher than that of immature ones. The majority of MII oocytes (32/36) and all developed blastocysts(12/12) were associated with active E(2) production prior to ovulation. In this study, 18 MII oocytes met both requirements for active and optimal E(2) production. 13 of them were fertilized and 10 developed into blastocysts. CONCLUSION: Active somatic cell steroidogenesis prior to ovulation and an optimal steroid milieu at ovulation are prerequisites for generation of competent oocytes after follicular maturation in-vitro.

Maternal serum vascular endothelial growth factor levels in early ectopic and intrauterine pregnancies after in vitro fertilization treatment.

OBJECTIVE: To determine whether serum levels of vascular endothelial growth factor (VEGF) 11 days after a day-3 embryo transfer were predictive of outcome, in women with normal intrauterine pregnancy (IUP), first-trimester miscarriage (SAB), biochemical (BC), and ectopic pregnancy (EP) after IVF therapy. DESIGN: Retrospective analysis. SETTING: University hospital IVF unit. PATIENT(S): One hundred eight women who underwent IVF therapy and who were subsequently diagnosed with EP, BC, SAB, or a normal IUP (27 in each category). INTERVENTION(S): Serum samples were obtained at 11 days after a day-3 embryo transfer. MAIN OUTCOME MEASURE(S): Serum concentrations of VEGF, P, and beta-hCG. RESULT(S): Serum concentrations of VEGF were similar in women with BC and EP and higher than in women with normal IUP and SAB (571.8 +/- 61.8, 604.4 +/- 73.4 vs. 448.9 +/- 39.9, 461.8 +/- 39.2 pg/mL, respectively). Also, serum beta-hCG and P levels were significantly higher in women with a normal IUP and SAB. Using a cutoff concentration of >700 pg/mL for VEGF, an EP could be distinguished from an IUP (normal and SAB), with a positive predictive value of 64% and a negative predictive value of 71%. CONCLUSION(S): Elevated maternal serum levels of VEGF, as early as 11 days after embryo transfer, are associated with ectopic pregnancies after IVF.

Mouse ovarian tissue cryopreservation has only a minor effect on in vitro follicular maturation and gene expression.

PURPOSE: To establish a protocol for ovarian tissue cryopreservation which can retain fertility potential after thawing and to evaluate the impact of cryopreservation on development and gene expression during folliculogenesis. METHODS: A controlled randomized study in a clinical and academic research setting in a university medical center was conducted to study cryopreservation and in vitro maturation (IVM) of mouse ovarian follicles. Preantral follicles isolated from either fresh (Group A) or cryopreserved (Group B) murine ovarian tissues were used to test their fertility potential by in vitro culture-in vitro maturation (IVC-IVM). Expression of Graafian follicles derived from both groups were detected by DNA microarray techniques for comparison. RESULTS: Although there were no significant differences in IVM outcomes and follicular gene expression between the two experimental groups, cryopreservation appears to induce the expression of heat shock proteins, DNA-damage-inducible protein 45 and death-related apoptosis genes (i.e., Fas and Fas-ligand). CONCLUSION: Cryopreservation may trigger biological events not amenable to normal cell function and follicular development. However, neither follicular development nor gene expression was dramatically changed after cryopreservation. These data suggest that although our current cryopreservation techniques yield competent follicles and mature oocytes, subtle changes observed in gene expression imply that the present cryopreservation techniques need to be further refined.

The role of mitogen-activated protein kinase in insulin and insulin-like growth factor I (IGF-I) signaling cascades for progesterone and IGF-binding protein-1 production in human granulosa cells.

Insulin and IGF-I participate in the regulation of ovulation, steroidogenesis, and IGF-binding protein (IGFBP) production in the ovary. Insulin and IGF-I actions in the ovary are closely related. For example, insulin may amplify IGF-I action in the ovary by up-regulating type I IGF receptors and inhibiting IGFBP-1 production, thus increasing the bioavailability of IGF-I. It is hypothesized that ovarian effects of insulin in insulin-resistant states are mediated via an insulin action pathway(s) distinct from those involved in glucose transport. We previously reported that insulin-induced stimulation of progesterone and inhibition of IGFBP-1 production in the human ovary are mediated by signaling pathways that are independent of phosphatidylinositol 3-kinase, the enzyme whose activation is crucial for glucose transport. We now examined whether activation of MAPK is necessary to mediate insulin-induced or IGF-I-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells. Human granulosa cells were obtained during in vitro fertilization. Cells (0.5-1 x 10(5)) were incubated for 24 h in the presence of 0, 10, 10(2), or 10(3) ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 micro M PD98059, a specific inhibitor of ERK1/2 MAPK. The progesterone concentration in the tissue culture medium was measured by RIA (Pantex, Santa Monica, CA), and the IGFBP-1 concentration was measured by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX). MAPK activity was assessed using the MAPK IP-Kinase assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). ANOVA was used to compare mean values of progesterone or IGFBP-1 concentrations. MAPK was stimulated by insulin up to 350% of the baseline value. Progesterone production in human granulosa cells was stimulated by insulin in a dose-related manner to 123% of the control value (P < 0.001), and IGFBP-1 production was inhibited to 25% of the baseline value (P < 0.001). Despite inhibiting MAPK activity by 99%, PD98059 (1 micro M) did not interfere with insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production. MAPK was stimulated by IGF-I to 730% of the baseline value, with maximal stimulation achieved at 0.5 ng/ml IGF-I. Progesterone production in granulosa cells was stimulated by IGF-I to 130% of the control value (P < 0.001), whereas IGFBP-1 production was inhibited to 44% of the control value (P < 0.001). PD98059 (1 micro M) inhibited IGF-I-induced MAPK activity by 94%. In the presence of 1 micro M PD98059, IGF-I-induced stimulation of progesterone production was inhibited by 96% (P < 0.001). The inhibitory effect of IGF-I on IGFBP-1 production was reduced in the presence of 1 micro M PD98059 by 45% at 5 ng/ml IGF-I and was completely abolished in the presence of 1 micro M PD98059 at concentrations of IGF-I ranging from 0.5-2.5 ng/ml (P < 0.001). We conclude that, under conditions of our experiments, insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells does not require MAPK activation, whereas similar effects of IGF-I are largely MAPK dependent.