Shuichiro Hara

Poor Embryo Development in Mouse Oocytes Aged In Vitro Is Associated with Impaired Calcium Homeostasis

Abstract We examined whether impairment of intracellular Ca2+ homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca2+ oscillations at fertilization: these Ca2+ oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca2+ store was decreased in in vitro-aged oocytes. A decrease in the Ca2+ store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca2+-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca2+ oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca2+ homeostasis (such as a decrease in the ER Ca2+ store) caused an alteration in Ca2+ oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H2O2 or thapsigargin. Acting together, alteration in Ca2+ oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

Molecular mechanism of poor embryo development in postovulatory aged oocytes: Mini review

Oocyte quality is a key factor in determining embryo development; however, we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. Postovulatory aging of oocytes that have not been fertilized for a prolonged time after ovulation is known to significantly impair oocyte quality and subsequent embryo development after fertilization. Embryos derived from postovulatory‐aged oocytes are prone to undergo apoptosis due to the decreased Bcl‐2 expression. Postovulatory aging of oocytes changes the patterns of Ca2+ oscillations at fertilization as a result of impaired Ca2+ regulation in the endoplasmic reticulum. Moreover, postovulatory aging of oocytes impairs mitochondrial adenosine triphosphate production as a result of increasing oxidative stresses. Oxidative stresses also affect intracellular Ca2+ regulation and impair embryo development after fertilization. Collectively, the mechanism of postovulatory oocyte aging might be involved in reactive oxygen species‐induced mitochondrial injury followed by abnormal intracellular Ca2+ regulation in the endoplasmic reticulum.

Molecular Mechanism of the Inhibition of Estradiol-Induced Endometrial Epithelial Cell Proliferation by Clomiphene Citrate

We examined the molecular mechanisms of the antiestrogenic effects of clomiphene citrate (CC) in the endometrium using two types of cell lines, Ishikawa and EM-E6/E7/hTERT cells. CC or ICI182780 inhibited 17beta-estradiol (E2)-induced endometrial cell proliferation and transcriptional activation of the estrogen response element (ERE) gene. We directly visualized the ligand-estrogen receptor (ER)alpha interaction using green fluorescent protein (GFP)-tagged ER alpha in a single living cell. Whereas E2 changed the nuclear localization of GFP-ER alpha to a punctate distribution within 5 min, CC or ICI182780 changed the slower and less mobilization of GFP-ER alpha compared with E2. Pretreatment with CC or ICI182780 partly prevented the E2-induced nuclear redistribution of GFP-ER alpha. Fluorescence recovery after photobleaching revealed that GFP-ER alpha mobility treated with E2 was more rapid than that treated by CC or ICI182780. As coactivator recruitment to the ER is essential for ER-dependent transcription, we examined the interaction between ER alpha and steroid receptor coactivator-1 (SRC-1). The complex formation between ER alpha and SRC-1 was significantly increased by E2 but was prevented in the presence of CC or ICI182780 by coimmunoprecipitation. Moreover, the E2-induced colocalization of GFP-ER alpha and SRC-1 was prevented in the presence of CC or ICI182780 according to an immunofluorescence assay. We also observed that the reduction of SRC-1 using small interfering RNA for SRC-1 resulted in the inhibition of E2-induced cell proliferation and transcriptional activation of the ERE gene. Collectively, these results suggest that CC may inhibit E2-induced endometrial epithelial cell proliferation and ERE transactivation by inhibiting the recruitment of SRC-1 to ER alpha.

Bezafibrate Restores the Inhibition of FSH-Induced Follicular Development and Steroidogenesis by Tumor Necrosis Factor-Alpha Through Peroxisome Proliferator-Activated Receptor-Gamma Pathway in an In Vitro Mouse Preantral Follicle Culture

We recently reported that bezafibrate, a lipid-lowering drug of the fibrate class, administered in addition to clomiphene citrate (CC) successfully induced ovulation in CC-resistant polycystic ovary syndrome (PCOS) patients. We hypothesized that bezafibrate may directly affect ovarian follicle development. Insulin resistance and compensatory hyperinsulinemia are important for the pathogenesis of PCOS. In this study, we first examined the effects of tumor necrosis factor-alpha (TNF), which plays a role in insulin resistance, on follicle development by using the follicle culture system. TNF significantly inhibited follicle-stimulating hormone (FSH)-induced follicle development, 17beta-estradiol (E2) secretion, and ovulation rate in a dose-dependent manner. We then examined whether bezafibrate treatment could rescue the inhibition of FSH-induced follicle development and steroidogenesis by TNF. Bezafibrate treatment rescued inhibition of follicle development, secretion of E2, and ovulation rate by TNF. We examined the expression of peroxisome proliferator-activated receptor (PPAR) subtypes in mouse preantral follicles. As the protein expression of only PPARG was observed in mouse preantral follicles, we examined whether bezafibrate could affect follicle development and steroidogenesis through PPARG pathways. Treatment with GW1929, a selective PPARG agonist, restored inhibition of FSH-induced follicle development and steroidogenesis by TNF, whereas treatment with GW9662, a selective PPARG antagonist, canceled the restorative effects of bezafibrate. Collectively, the results in this study suggest that bezafibrate may directly exhibit a restorative effect on the inhibition of ovarian follicle development and steroidogenesis by TNF through the PPARG pathway.

Usefulness of Bezafibrate for Ovulation Induction in Clomiphene Citrate-Resistant Polycystic Ovary Syndrome Patients with Dyslipidemia: A Prospective Pilot Study of Seven Cases

Background: Dyslipidemia is commonly observed in polycystic ovary syndrome (PCOS) patients. Bezafibrate is a drug for dyslipidemia acting through peroxisome proliferator-activated receptors. We investigated the effects of bezafibrate for ovulation induction in patients with PCOS with dyslipidemia who were resistant to clomiphene citrate (CC). Methods: This was a prospective pilot study. Seven infertile, CC-resistant, PCOS patients with dyslipidemia were enrolled in this study. The participants received bezafibrate at 400 mg/day from day 1 of menses and CC at 100 mg/day from day 5 of menses simultaneously until one follicle measuring at least 18 mm in diameter was found by transvaginal ultrasound. The main outcome was ovulation rate. Results: Five of 7 patients successfully ovulated. The mean number of days of menses until the follicle reached 18 mm in diameter was 16 ± 3 (range 13–20). Monofollicular development was observed in all patients that ovulated. One woman became pregnant and delivered a healthy baby. Conclusion: Bezafibrate may be effective for ovulation induction in CC-resistant PCOS patients with dyslipidemia.

Effects of different trehalose concentrations in a warming medium on embryo survival and clinical outcomes in vitrified human embryos.

Background/Aims: In the present study, we examined the effects of different concentrations of trehalose in a warming medium on both embryo survival and clinical outcomes in vitrified-warmed embryo transfer cycles. Methods: We retrospectively analyzed a total of 209 vitrified-warmed cycles from 177 patients who underwent in vitro fertilization or intracytoplasmic sperm injection and embryo transfer. Embryos were cryopreserved by the vitrification method and warmed in solutions containing either 0.5 or 1.0 <smlcap>M</smlcap> trehalose. We compared the 0.5 and 1.0 <smlcap>M</smlcap> trehalose warming solution groups with respect to the embryo survival rate after warming and clinical outcomes. Results: The embryo survival rate in the 1.0 <smlcap>M</smlcap> trehalose group (96.5%) was significantly higher than that in the 0.5 <smlcap>M</smlcap> trehalose group (57.0%). The percentage of embryo transfers after warming in the 1.0 <smlcap>M</smlcap> trehalose group (94.3%) was significantly higher than that in the 0.5 <smlcap>M</smlcap> trehalose group (83.7%). The clinical pregnancy rate in the 1.0 <smlcap>M</smlcap> trehalose group (25.0%) was significantly higher than that in the 0.5 <smlcap>M</smlcap> trehalose group (11.1%). Conclusion: Embryo survival and clinical pregnancy rates were higher when a 1.0 <smlcap>M</smlcap> trehalose solution was used than when a 0.5 <smlcap>M</smlcap> trehalose solution was used during the embryo warming process.

Dizygotic twin pregnancy after single embryo transfer: a case report and review of the literature

IntroductionElective single embryo transfer (SET) is a reasonable optionfor avoiding multiple pregnancies in patients who undergoassisted reproductive technology (ART) treatment [1, 2]. TheJapan Society of Obstetrics and Gynecology (JSOG) recom-mends SET for patients under 35 years of age and for thosewho have undergone ≤2 treatment cycles since 2008. TheJSOG recommendation increased the use of the SET tech-nique from 49.9 % (52,800/105,812) of all embryo transfercyclesin2007to73%(106,758/146,286)in2010[3,4].Asaresult, the proportion of all embryo transfer cycles that result-ed in multiple pregnancies decreased from 3.0 % in 2007 to1.3 % in 2010 [3, 4].Multiplepregnancies may occur evenifSETisperformed.Todate,17casesinvolvingtwinortripletpregnanciesaftertheSEThavebeenreported[5].Inthe general population,mono-zygotic twins have been calculated to account for 0.4–0.45 %ofalllivebirths[6,7].InpatientsundergoingART,thisrateisapproximately twice that of spontaneous pregnancies [8, 9].Theoretically,twinsresultingfromaSETprocedureshouldbemonozygotic.However,areviewoftheliteraturesuggeststhat4 cases of dizygotic twin pregnancies have been reported inpatients undergoing the SET technique [10–12]. Here, wepresent a fifth case and a review of the literature concerningthis phenomenon.Case reportA 39-year-old, nulligravida woman and her 44-year-old hus-band had an 8-year history of infertility. She had regular men-strual cycles occurring every 28 days and lasting 7 days, andwas negative for Chlamydia trachomatis IgAandIgGantibod-ies. She had bilateral tubal patency by hysterosalpingography.Her husband’s semen analysis showed normozoospermia,based on the 2010 World Health Organization criteria.The woman underwent in vitro fertilization (IVF) and em-bryo transfer with a long-protocol, gonadotropin-releasing hor-mone agonist because of unexplained infertility. Nine cumulusoocytecomplexes(COCs)wereaspiratedandtheseCOCsweresubjected to IVF. Six fertilized oocytes with two pronuclei wereobserved 20 h after insemination. Although one embryo at themorulastagewastransferredtotheuterusondayfourafterIVF,she did not become pregnant. The remaining four morula-stageembryos with good morphology were subjected to vitrificationin a solution containing 40 % (v ol/vol) ethylene glycol, 18 %(wt/vol) Ficoll 70, and 0.3 mol/L trehalose [ 13]. After 2 monthsof IVF treatment, the patient became pregnant spontaneously,but this resulted in a biochemical pregnancy. Four months aftervitrification of the embryos, four embryos were warmed for ascheduled embryo transfer, according to the patient ’snaturalmenstrual cycle. Ovulation was confirmed by follicular moni-toringwithtransvaginalultrasonographyonday17ofthecycle.The embryo transfer was planned 5 days after the ovulation.Warmingofmorula-stageembryoswasperformedonthedaybefore the scheduled embryo transfer. Three surviving morula-stage embryos were cultured, a fter warming in Global Medium(LifeGlobal, Gilford, CT, USA) supplemented with 10 % syn-thetic serum substitute (Irvine Scientific, Santa Ana, CA, USA)for 18 h. One embryo showed further development and reachedthe expanded blastocyst stage. The expanded blastocyst wastransferred into the patient’s uterus. Ten days after embryotransfer, her pregnancy test was positive. Two gestational sacswith fetal heart beats in each gestational sac were observed by

Cellular and molecular mechanisms of various types of oocyte aging

It is well established that age-related decline of a womans fertility is related to the poor developmental potential of her gametes. The age-associated decline in female fertility is largely attributable to the oocyte aging caused by ovarian aging. Age-associated oocyte aging results in a decrease in oocyte quality. In contrast to ovarian aging, there is a concept of postovulatory oocyte aging. Postovulatory aging of oocytes, not being fertilized for a prolonged time after ovulation, is known to significantly affect the development of oocytes. Both categories of oocyte aging have similar phenotypes of reproductive failure. However, the mechanisms of the decline in oocyte quality are not necessarily equivalent. An age-dependent increase in aneuploidy is a key determinant of oocyte quality. The reduced expression of molecules regulating cell cycle control during meiosis might be involved in the age-dependent increase in aneuploidy. The mechanism of age-associated oocyte aging might be involved in mitochondrial dysfunction, whose etiologies are still unknown. Alternatively, the mechanism of postovulatory oocyte aging might be involved in reactive oxygen species-induced mitochondrial injury pathways followed by abnormal intracellular Ca2+ regulation of the endoplasmic reticulum. We suggest that future research into the mechanism of oocyte aging will be necessary to develop a method to rescue the poor developmental potential of aged oocytes.

Peroxisome Proliferator-Activated Receptor-γ Agonists Prevent Tumor Necrosis Factor-α-Mediated Inhibition of FSH-Induced Follicle Development and Estradiol Production in a Preantral Follicle Culture System

Abstract: Although 60–80% of the women with polycystic ovary syndrome (PCOS) ovulate with clomiphene citrate (CC), the rest are CC-resistant. Recently, the use of insulin-sensitizing agents such as metformin and pioglitazone have been proposed for inducing ovulation in CC-resistant women with PCOS, and we have reported that administration of bezafibrate, a lipid-lowering fibrate, in addition to CC, successfully induced ovulation in CC-resistant women with PCOS and dyslipidemia. Both pioglitazone and bezafibrate are peroxisome proliferator-activated receptor-&ggr; (PPAR-&ggr;) agonists. This paper reviews the evidence for the direct effects of the drugs, which are PPAR-&ggr; agonists, on follicle development and steroidogenesis, collected using an in vitro mouse preantral follicle culture system. We used the in vitro follicle culture system with the addition of tumor necrosis factor-alpha (TNF-&agr;), which plays a role in insulin resistance, as a model for studying follicle development in women with PCOS. TNF-&agr; inhibited FSH-induced follicle development and steroidogenesis in the follicle culture system. Both pioglitazone and bezafibrate prevented TNF-&agr;mediated inhibition of FSH-induced follicle development and steroidogenesis through the PPAR-&ggr;-stimulating pathway. Our results suggest that insulin-sensitizing drugs, especially PPAR-&ggr; agonists, may directly influence follicle development and steroidogenesis in women with PCOS.

Contents Vol. 76, 2013

M.A. Belfort, Provo, Utah J. Bornstein, Nahariya H.L. Brown, Durham, N.C. C. Chapron, Paris J. de Haan, Maastricht G.A. Dekker, Adelaide, S.A. J.A. Deprest, Leuven K. Hecher, Hamburg S. Kahhale, São Paulo H. Kliman, New Haven, Conn. T.F. Kruger, Tygerberg J.A. Kuller, Raleigh, N.C. M.J. Kupferminc, Tel Aviv H. Minkoff , Brooklyn, N.Y. J. Moodley, Congella J.M. Mwenda, Nairobi H. Odendaal, Tygerberg J.T. Repke, Hershey, Pa. G.R. Saade, Galveston, Tex. B.M. Sibai, Cincinnati, Ohio S.K. Smith, London I.E. Timor-Tritsch, New York, N.Y. S. Uzan, Paris Johan Verhaeghe, Leuven J.J. Walker, Leeds G.D. Wendel, Dallas, Tex. R.A. Wild, Oklahoma City, Okla. G. Zador, Södertälje M.B. Zimmerman, Iowa City, Iowa Gynecological Ultrasound and Imaging George Condous, St. Leonards, N.S.W.