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Background: During hormone replacement therapy (HRT) to optimize endometrial growth for thawed-frozen embryo transfer (FET), patients normally undergo estrogen administration for 14 days (day 14 group). However, if optimal endometrial thickness is not achieved in 14 days, estrogen treatment is continued for seven more days at a higher dose (day 21 group). This study aimed to determine the effects of this extended estrogen administration regimen on pregnancy outcomes. Methods: A retrospective analysis was performed using our patient database from November 2016 to March 2018. We analyzed 303 cycles of blastocyst-FET and compared patient background characteristics and pregnancy outcomes between day 14 and day 21 groups. Results: In day 21 group, the pregnancy rate tended to be lower while the miscarriage rate and ectopic pregnancy rate were higher than those in day 14 group. However, the differences between the groups were not statistically significant. Conclusions: Delayed endometrial growth resulted in poorer pregnancy outcomes in FET, but the differences were not statistically significant. Thus, it could be meaningful to add estrogen administration for 7 days to avoid the poor outcomes caused by a thin endometrium.

Endoplasmic reticulum (ER) stress is associated with several aging-related diseases; however, the mechanism underlying age-related deterioration of oocyte quality is unclear. Here, we used post-ovulatory, in vivo aged mouse oocytes as a model. Super-ovulated oocytes harvested from the oviduct at 14 h and 20 h post-hCG injection were designated as ‘fresh’ and ‘aged’, respectively. Embryo development following IVF was compared between fresh, aged and ER stress-induced oocytes. Expression of the ER stress marker GRP78 was examined at each stage. To evaluate the effect of salubrinal, an ER stress suppressor, on embryo development following IVF, expression levels of GRP78 and phospho-eukaryotic initiation factor 2 alpha were compared between aged and salubrinal-treated aged oocytes. Embryo transfer of salubrinal-treated aged oocytes was performed to examine the safety of salubrinal. Similar to aged oocytes, ER stress-induced oocytes showed lower fertilization rates and poor embryo development. Following IVF, expression of GRP78 decreased with embryo development. GRP78 expression was significantly higher in aged oocytes than in fresh oocytes. Salubrinal lowered GRP78 levels and improved embryo development. No adverse effect of salubrinal treatment was found on the birth weight of pups or on organogenesis in mice. The limitation of this study was that protein kinase-like ER kinase was the only ER stress pathway examined; the role of IRE1 and ATF6 pathways was not considered. Nevertheless, salubrinal can significantly improve embryo development in in vivo aged oocytes undergoing ER stress. Hence, regulation of ER stress might represent a promising therapeutic strategy to overcome poor oocyte quality.

The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients’ ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients’ burden for easy to use FP by a government subsidy and medical insurance coverage. This model has been termed the Danish model (“the woman stays - the tissue moves”). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

Aim This study aimed to assess the efficacy of the endometrial receptivity array (ERA) as a diagnostic tool and the impact of personalized embryo transfer (pET) for the treatment of patients with recurrent implantation failure (RIF) in Japan. Methods Fifty patients with a history of RIF with frozen-thawed blastocyst transfers were recruited from July, 2015 to April, 2016. Endometrial sampling for the ERA and histological dating and a pET according to the ERA were performed. The receptive (R) or non-receptive (NR) status of the endometrium as a result of the first ERA, endometrial dating, and pregnancy rates after the pET were analyzed. Results Of the patients with RIF, 12 (24%) were NR. Among them, eight (66.7%) were prereceptive. A clinical follow-up was possible in 44 patients who underwent the pET. The pregnancy rates were 58.8% per patient and 35.3% per first pET in the R patients and 50.0% per patient and 50.0% per first pET in the NR patients. Discrepancies between the ERA results and histological dating were seen more in the NR patients than in the R patients. Conclusions For patients with unexplained RIF, there is a significance in searching for their personal window of implantation (WOI) using the ERA, considering the percentage of those who were NR and the pregnancy rates that resulted from the pET. By transferring euploid embryos in a personal WOI, much better pregnancy rates are expected.

We examined the prognostic factors for pregnancy in 210 vitrified-warmed embryo transfer (ET) cycles in 121 patients. The univariate analysis showed that age, gravida, the number of cycles associated with infertility caused by endometriosis, the number of previous assisted reproductive technology (ART) treatment cycles, and the number of ICSI procedures were significantly lower in pregnant cycles compared with non-pregnant cycles. The percentages of ET using at least one intact embryo and of ET using at least one embryo that had developed further after warming were significantly higher in pregnant cycles compared with non-pregnant cycles. Multivariate logistic regression analysis showed that previous ART treatment cycles, ET with at least one intact embryo, and ET using at least one embryo that had developed further were independent prognostic factors for pregnancy in vitrified-warmed ET cycles. We conclude that fewer previous ART treatment cycles, ET using at least one intact embryo, and ET with embryos that have developed further after warming might be favourable prognostic factors for pregnancy in vitrified-warmed ET cycles.

Study question Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? Summary answer The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. What is already known To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. Study design, size, duration This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. Participants/materials, setting, methods We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. Main results and the role of chance The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Limitations, reasons for caution Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Wider implications of the findings Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. Study funding/competing interests This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Trial registration number Not applicable.

STUDY QUESTION Does in vivo aging of mouse oocytes affect mitochondrial function? SUMMARY ANSWER Mitochondrial function was impaired in post-ovulatory in vivo-aged mouse oocytes and microinjection of somatic cell mitochondria did not rescue poor fertilization and embryonic development rates. WHAT IS KNOWN ALREADY The mechanisms underlying the decline in oocyte quality associated with oocyte aging remain unknown, although studies have suggested that the decline is regulated by mitochondrial dysfunction. However, only a limited number of studies have provided direct evidence implicating mitochondrial dysfunction in oocyte quality during the aging of oocytes. STUDY DESIGN, SIZE, DURATION We used post-ovulatory, in vivo-aged mouse oocytes as a model for studying low-quality oocytes in oocyte aging. PARTICIPANTS/MATERIALS, SETTING, METHOD Superovulated oocytes released from the oviduct at 14 h and 20-24 h post-hCG injection were designated as 'fresh' and 'aged' oocytes, respectively. Membrane potentials and oxygen consumption in single oocytes were evaluated as measures of mitochondrial function in fresh and aged oocytes. Mitochondrial transcriptional factor A (TFAM) expression levels were examined by western blotting, and colocalization of mitochondria and TFAM was analyzed by measuring immunofluorescence in fresh and aged oocytes. IVF and blastocyst formation rates were calculated after oocyte microinjection with mitochondria derived from liver cells. MAIN RESULTS AND THE ROLE OF CHANCE The average mitochondrial membrane potential in fresh oocytes was significantly higher than that in aged oocytes (P

We examined how clomiphene citrate (CC) reduces estrogen receptor-α (ERα) in a human endometrial cancer cell line. Ishikawa human endometrial cancer cells were treated with ERα ligands such as 17β-estradiol (E2), CC, and the pure antiestrogen, ICI 182,780 (ICI). Thereafter, the expression levels of ERα protein and mRNA were analyzed by western blot and real-time quantitative PCR, respectively, and those of ubiquitinated ERα were analyzed by immunoprecipitation of ERα followed by immunoblotting with an anti-ubiquitin antibody. The expression levels of ERα protein after treatment with E2, CC, and ICI were significantly decreased compared to pre-treatment levels without a corresponding decrease in ERα mRNA. These ligands significantly increased the levels of ubiquitinated ERα compared to vehicle treatment. Co-treatment with the proteasome inhibitor, MG132, abrogated the decrease in ERα levels caused by treatment with the ligands only. We demonstrated, for the first time, a CC-induced decrease in ERα mediated by the ubiquitin-proteasome pathway in human endometrial cancer cells.

The azoospermia factor (AZF) region is important for spermatogenesis, and deletions within these regions are a common cause of oligozoospermia and azoospermia. Although several studies have reported this cause, the present research, to the best of our knowledge, is the first large-scale study assessing this factor in Japan. In this study, 1030 male patients with infertility who were examined for Y chromosome microdeletion using the polymerase chain reaction-reverse sequence-specific oligonucleotide (PCR-rSSO) method, a newly developed method for Y chromosome microdeletion screening, were included. The study enrolled 250 patients with severe oligospermia and 717 patients with azoospermia. Among the 1030 patients, 4, 4, 10, and 52 had AZFa, AZFb, AZFb+c, and AZFc deletions, respectively. The sperm recovery rate (SRR) of microdissection testicular sperm extraction in patients with AZFc deletions was significantly higher than that in those without AZF deletions (60.0% vs 28.7%, P = 0.04). In patients with gr/gr deletion, SRR was 18.7%, which was lower than that in those without gr/gr deletion, but was not statistically significant. In conclusion, our study showed that the frequency of Y chromosome microdeletion in male patients in Japan was similar to that reported in patients from other countries, and SRR was higher in patients with AZFc deletion.

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-γ (PPAR-γ) agonists. This paper reviews the evidence for the direct effects of the drugs, which are PPAR-γ 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-α), which plays a role in insulin resistance, as a model for studying follicle development in women with PCOS. TNF-α inhibited...

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 Ca(2+) oscillations at fertilization as a result of impaired Ca(2+) 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 Ca(2+) 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 Ca(2+) regulation in the endoplasmic reticulum.

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 M trehalose. We compared the 0.5 and 1.0 M 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 M trehalose group (96.5%) was significantly higher than that in the 0.5 M trehalose group (57.0%). The percentage of embryo transfers after warming in the 1.0 M trehalose group (94.3%) was significantly higher than that in the 0.5 M trehalose group (83.7%). The clinical pregnancy rate in the 1.0 M trehalose group (25.0%) was significantly higher than that in the 0.5 M trehalose group (11.1%). Conclusion: Embryo survival and clinical pregnancy rates were higher when a 1.0 M trehalose solution was used than when a 0.5 M trehalose solution was used during the embryo warming process.

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.

Prostaglandins (PGs) are implicated in various physiological and pathological functions because of their vasoactive, mitogenic, and differentiating properties. PGs have long been known to participate in various female reproductive functions. The cyclooxygenase (COX) isozymes, COX-1 and COX2, are the rate-limiting enzymes of PGs. Gene targeting studies have revealed that COX-2, but not COX-1, derived PGs are essential for ovulation, fertilization, implantation, and decidualization. However, the roles of PGs in oocyte maturation remain controversial. We have clarified that COX-2-deficient mice have defective oocyte meiotic and cytoplasmic maturation associated with defective cumulus expansion in vivo and in vitro. Lack of COX-2-derived PGE2 leads to impaired cumulus cell-oocyte interactions, which are critical for the production of fertilization-competent oocytes. This review summarizes the published data regarding the roles of PGs in oocyte maturation especially in gene targeting studies.

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.

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. Bec...

A unicornate uterus with a rudimentary horn is a rare Mullerian duct abnormality. This uterine anomaly may cause variable gynecologic complications, including hematometra, endometriosis leading to acute pelvic pain, infertility, and obstetric complications, including miscarriage, preterm delivery, and rupture of the uterus, especially when the pregnancy implants in the rudimentary horn. To date, the standard treatment for the latter condition has been laparotomy, as patients usually experience massive abdominal hemorrhage or pregnancies too large to attempt laparoscopic removal. We report a case of a non-communicating rudimentary uterine horn pregnancy which was preoperatively diagnosed by ultrasonography, hysterofiberscopy, and magnetic resonance imaging, and then subsequently resected laparoscopically.

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca2+ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCzeta in generating changes in egg intracellular Ca2+ despite the fact that PLCzeta constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca2+ oscillations has not been examined. Reducing egg PLCbeta1 by a transgenic RNAi approach resulted in a significant decrease in Ca2+ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCbeta1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca2+ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCzeta in egg activation.

Mayer-Rokitansky-Küster-Hauser syndrome is a rare congenital anomaly characterized by congenital aplasia or hypoplasia of the uterus and vagina. We report a case of Mayer-Rokitansky-Küster-Hauser syndrome with multiple large pelvic masses diagnosed by three-dimensional computed tomography (CT) angiography.A 40-year-old woman with Mayer-Rokitansky-Küster-Hauser syndrome presented with an abdominal mass that had grown for 3 months. Magnetic resonance imaging (MRI) confirmed several solid masses, and normal bilateral ovaries were detected; three-dimensional CT revealed that these tumors were supplied from the right ovarian and uterine arteries, suggesting that they arose from the uterus. Accordingly, leiomyoma was suspected. Laparoscopic surgery was contraindicated, and the patient therefore underwent laparotomy. The masses were resected with the bilateral rudimentary uteri and fallopian tubes, and pathologic evaluation confirmed leiomyoma.Combined MRI and three-dimensional CT angiography can accurately evaluate the origin and anatomic properties of leiomyomas in patients with Mayer-Rokitansky-Küster-Hauser syndrome.

In recent years, postponement of marriage and childbearing in women of reproductive age has led to an increase in the incidence of age-related infertility. The reproductive aging process in women is assumed to occur due to a decrease in both the quantity and quality of the oocytes, with the ultimate result being a decline in fecundity. This age-related decline in fecundity is strongly dependent on oocyte quality, which is critical for fertilization and subsequent embryo development. Aged oocytes display increased chromosomal abnormality and dysfunction of cellular organelles, both of which factor into oocyte quality. In particular, mitochondrial dysfunction has been suggested as a major contributor to the reduction in oocyte quality as well as to chromosomal abnormalities in aged oocytes and embryos. Participation of oxidative stress in the oocyte aging process has been proposed because oxidative stress has the capacity to induce mitochondrial dysfunction and directly damage many intracellular components of the oocytes such as lipids, protein, and DNA. In an attempt to improve mitochondrial function in aged oocytes, several therapeutic strategies have been investigated using both animal models and assisted reproductive technology. Here, we review the biological mechanisms and present status of therapeutic strategies in the female reproductive aging field and indicate possible future therapeutic strategies.

Postovulatory aging of oocytes significantly affects embryonic development. Also, altered Ca2+ oscillation patterns can be observed in fertilized, aged mouse oocytes. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum, and the latter relies on ATP availability, we simultaneously measured changes in intracellular ATP concentration ([ATP]i) and Ca2+ oscillations in fresh and aged mouse oocytes. We continuously assessed changes in [ATP]i from intracellular free Mg2+ concentration measured by fluorescent dye Magnesium Green (MgG) while intracellular Ca2+ concentration ([Ca2+]i) was monitored by Fura-PE3. At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation in [Ca2+]i, indicating a relative decrease in [ATP]i. In fresh oocytes, it was quickly followed by a significant decrease below baseline, indicating a relative increase in [ATP]i. In contrast, in aged oocytes, such a decrease in MgG fluorescence was not observed. In a separate experiment, ATP content in fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in unfertilized fresh and aged oocytes. Intracellular ATP content at 5 h after fertilization was increased in both oocytes, where fresh oocytes showed a significantly higher intracellular value than aged oocytes. These findings suggest that aged mouse oocytes fail to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization might lead to an altered Ca2+ oscillation pattern and poor developmental potency, which is commonly noted in aged oocytes.

Although estrogen is known to protect against β-amyloid (Aβ)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Aβ-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Aβ-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) α gene (PCER). Raloxifene, like 17β-estradiol (E2), significantly inhibited Aβ-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Aβ, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2− and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor κB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer’s disease.

The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17beta-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFkappaB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFkappaB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFkappaB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFkappaB.

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

Background Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age and is characterized by chronic anovulation. Insulin resistance may be a key component of the pathogenesis of this disorder. Pioglitazone is a thiazolidinedione derivative that acts by improving insulin resistance via the peroxisome proliferator-activated receptor-γ (PPAR-γ) pathway. Reportedly, pioglitazone improves the anovulation status in patients with PCOS. In the present study, we examined whether pioglitazone directly affects ovarian follicular development and steroidogenesis using in vitro mouse preantral follicle culture system. Methods An isolated individual in vitro mouse preantral follicle culture was used to test the effects of pioglitazone on the follicle development and steroidogenesis. Tumor necrosis factor-α (TNF-α), which plays a role in insulin resistance, has been reported to inhibit the follicle stimulating hormone (FSH)-induced follicular development and steroidogenesis in an in vitro mouse preantral follicle culture system. Therefore, we examined whether pioglitazone counteracts these effects by TNF-α. We assessed the follicle diameter and follicle survival and antral-like cavity formation rates, the 17β-estradiol (E2) levels in the culture medium, and the ovulation rate using the in vitro preantral follicle culture. Results Pioglitazone treatment counteracted the inhibition of TNF-α in FSH-induced follicle development in a dose-dependent manner. Pioglitazone, at a concentration of 5 μM, which was the minimum effective concentration, significantly counteracted the inhibition of TNF-α in FSH-induced follicle survival (29 versus 56%, P P P P Conclusions Pioglitazone counteracted the inhibition by TNF-α on FSH-induced follicle development and steroidogenesis in the in vitro mouse preantral follicle culture. The results suggest that pioglitazone may directly affect the follicular development and steroidogenesis.

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 Ca(2+) oscillations at fertilization as a result of impaired Ca(2+) 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 Ca(2+) 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 Ca(2+) regulation in the endoplasmic reticulum.

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.

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.

We examined whether impairment of intracellular Ca(2+) 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 Ca(2+) oscillations at fertilization: these Ca(2+) oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca(2+) store was decreased in in vitro-aged oocytes. A decrease in the Ca(2+) store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca(2+)-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca(2+) oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca(2+) homeostasis (such as a decrease in the ER Ca(2+) store) caused an alteration in Ca(2+) 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 H(2)O(2) or thapsigargin. Acting together, alteration in Ca(2+) oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.

Postovulatory aging of oocytes significantly affects embryonic development. Also, altered Ca2+ oscillation patterns can be observed in fertilized, aged mouse oocytes. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum, and the latter relies on ATP availability, we simultaneously measured changes in intracellular ATP concentration ([ATP]i) and Ca2+ oscillations in fresh and aged mouse oocytes. We continuously assessed changes in [ATP]i from intracellular free Mg2+ concentration measured by fluorescent dye Magnesium Green (MgG) while intracellular Ca2+ concentration ([Ca2+]i) was monitored by Fura-PE3. At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation in [Ca2+]i, indicating a relative decrease in [ATP]i. In fresh oocytes, it was quickly followed by a significant decrease below baseline, indicating a relative increase in [ATP]i. In contrast, in aged oocytes, such a decrease in MgG fluorescence was not observed. In a separate experiment, ATP content in fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in unfertilized fresh and aged oocytes. Intracellular ATP content at 5 h after fertilization was increased in both oocytes, where fresh oocytes showed a significantly higher intracellular value than aged oocytes. These findings suggest that aged mouse oocytes fail to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization might lead to an altered Ca2+ oscillation pattern and poor developmental potency, which is commonly noted in aged oocytes.

In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca(2+) oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca(2+) homeostasis mainly through direct effects on the machinery involved in intracellular Ca(2+) storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca(2+)-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 microM hydrogen peroxide (H(2)O(2)) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H(2)O(2)-treated fresh oocytes. The frequency of Ca(2+) oscillations was significantly increased whereas the amplitude of individual Ca(2+) transients was lowered in the aged and H(2)O(2)-treated fresh oocytes. The rates of rise and decline in individual Ca(2+) transients were decreased in these oocytes, indicating impaired Ca(2+) handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO(2) in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca(2+) oscillations play a key role in the development of fertilized aged oocytes.

A 46 year-old man with hypertrophic obstructive cardiomyopathy (HOCM) was referred to this clinic complaining of shortness of breath. His symptoms remained even after he had been treated with bisoprolol, lisinopril, and verapamil. We started treating him with cibenzoline, instead of verapamil. Continuous-wave Doppler examination revealed a marked decrease from 95.9 mm Hg to 8.8 mmHg in the left ventricular outflow tract pressure gradient after administration of cibenzoline, and the symptom disappeared. The electrical axis of the electrocardiogram deviated from +53 to +95 degrees, and the QRS interval was increased from 0.095 to 0.114 seconds. By the M-mode echocardiogram, the interventricular septum was observed moved paradoxically in the early, systolic phase. We suggest the possibility that cibenzoline modifies intraventricular conduction and causes asynchronous contraction of the left ventricle, resulting in decrease in the left ventricular outflow tract pressure gradient.