Hamatani Toshio

Abstract Objective To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Design Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Setting Clinical and experimental research. Patients Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. Intervention(s) None. Main outcome measure(s) The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Results Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. Conclusion We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model.

Aim: Women undergoing infertility treatment often need to balance work and fertility treatment. Therefore, we evaluated the quality of life (QOL) and impact of infertility treatment on Japanese working women and their careers. Methods: We conducted an online questionnaire at 18 clinics in Japan. Responses were collected from 835 women, 713 of whom were working. The participants were divided into three groups based on treatment stage. Data were collected using the FertiQoL and an original questionnaire created by the authors. The Mann–Whitney U test and a multinomial logistic analysis were used. Results: Approximately 90% of the participants felt that treatment could hinder their work and 8% had quit their jobs. Low QOL was associated with sadness and despair due to infertility and mood disorders, disruptions to life and work, and the complicated medications and procedures involved in treatment. Social isolation and the effect of fertility treatment on daily life and work strongly hindered the careers of working women in the third stage of treatment (in vitro fertilization and intracytoplasmic sperm injection). Approximately 70% of the participants required support to subsidize treatment costs and sought shorter working hours and flextime systems. Only 55% informed their workplaces about the fertility treatment, but about 70% easily gained understanding by informing them. Conclusions: For many working women, infertility treatment posed barriers to their careers, which could explain the low QOL. Urgent introduction of a support system is necessary in Japan, and understanding and social acceptance of infertility appears to be important.

© 2019 The Authors Zygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early-stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of γ-H2AX despite increased expression of the DNA repair genes Rad51l3 and Bard. Re-expression of Zscan5b reduced γ-H2AX content, implying a role for Zscan5b in DNA damage repair processes. A co-immunoprecipitation analysis showed that Zscan5b bound to the linker histone H1, suggesting that Zscan5b may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis. In this article, Yamada and colleagues show that Zscan5b deficiency increases DNA stress, compromises chromosomal structure during mitosis, and leads to the development of early-stage cancers. Zscan5b deficiency may offer a murine model of human chromosomal breakage syndromes.

© 2018, United States & Canadian Academy of Pathology. Tetraspanin CD9 is essential for sperm–egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Reproductive capacity in women starts to decline beyond their mid-30s and pregnancies in older women result in higher rates of miscarriage with aneuploidy. Age-related decline in fertility is strongly attributed to ovarian aging, diminished ovarian reserves, and decreased developmental competence of oocytes. In this review, we discuss the underlying mechanisms of age-related decline in oocyte quality, focusing on oxidative stress (OS) in oocytes. The primary cause is the accumulation of spontaneous damage to the mitochondria arising from increased reactive oxygen species (ROS) in oocytes, generated by the mitochondria themselves during daily biological metabolism. Mitochondrial dysfunction reduces ATP synthesis and influences the meiotic spindle assembly responsible for chromosomal segregation. Moreover, reproductively aged oocytes produce a decline in the fidelity of the protective mechanisms against ROS, namely the ROS-scavenging metabolism, repair of ROS-damaged DNA, and the proteasome and autophagy system for ROS-damaged proteins. Accordingly, increased ROS and increased vulnerability of oocytes to ROS lead to spindle instability, chromosomal abnormalities, telomere shortening, and reduced developmental competence of aged oocytes.

To investigate pregnancy outcomes in women after abdominal radical trachelectomy (RT) for early-stage cervical cancer.|The patients' background, fertility, and pregnancy outcomes were reviewed in a total of 61 pregnancies in 48 of 172 women who underwent abdominal RT at Keio University Hospital between September 2002 and December 2013.|There were 5 women with stage IA1, 2 with stage IA2, and 41 with stage IB1. Histological types were as follows: squamous cell carcinoma (n = 36), adenocarcinoma (n = 10), and adenosquamous cell carcinoma (n = 2). The pregnancy rate of women attempting to conceive after abdominal RT was 44% (48/109). The mean ± SD duration from abdominal RT to conception was 3.1 ± 1.9 years. Of 61 pregnancies, 42 pregnancies were achieved by fertility treatment (in vitro fertilization-embryo transfer, 39; intrauterine insemination, 3). After excluding one pregnancy without detailed clinical information, there were 42 live births (5 in 22-27 weeks, 11 in 28-33weeks, 20 in 34-36 weeks, and 6 in 37-38 weeks), 13 miscarriages, and 5 ongoing pregnancies. While there were 10 first trimester miscarriages, 3 pregnancies ended in the second trimester owing to chorioamnioniti

Background: Patients hoping to preserve their fertility receive conservative treatment with high-dose medroxyprogesterone acetate (MPA) for well-differentiated endometrioid adenocarcinoma (EC) or atypical endometrial hyperplasia (AEH). Such treatment generally involves frequent intrauterine operations, including dilation and curettage (D&C) and endometrial biopsy (EMB), which could result in endometritis, endometrial thinning, or intrauterine adhesion. In turn, any of these outcomes could adversely affect implantation and pregnancy development. The current study thus aimed to identify factors that might affect pregnancy following conservative treatment by MPA. Methods: We compared a pregnancy group (45 patients) with a non-pregnancy group (53 patients) of MPA-treated patients to evaluate the factors affecting clinical pregnancy establishment. We undertook a multivariate logistic regression analysis based on factors shown by univariate analysis to be significantly different between the groups. Univariate analysis identified number of D&C, endometrial thickness, duration of MPA administration, age of pregnancy permission (the age at which a patient was first allowed to attempt pregnancy after disappearance of the lesion), period of disappearance of lesions, and recurrence as independent variables. Results: The odds ratios (95 % confidence interval) of multivariate analysis for disease recurrence, endometrial thickness during ovulation, and age of pregnancy permission were 0.283 (0.102-0.785), 1.677 (1.251-2.248), and 0.889 (0.792-0.998), respectively. There was no significant difference in the other independent variables between groups. Conclusions: We identified three factors considered to affect pregnancy establishment following conservative treatment with MPA: recurrence, endometrial thickness during ovulation, and the age of the pregnancy permission. Introduction of infertility treatment including assisted reproductive technology (ART) soon after achieving tumor disappearance by MPA would therefore be beneficial for patients with disease recurrence, thin endometrium, or a higher age of pregnancy permission.

Recently, infertility treatment-related psychological effects are receiving increased attention. However, whether sexual satisfaction is reduced amongst infertile couples remains to be elucidated. In this study, sexual satisfaction of Japanese infertile couples was assessed using a validated questionnaire designed to assess the male and female partner individually, and the couple as a whole for the first time. This study randomly included 170 infertile couples seen at the outpatient clinic and 170 couples that had recently achieved spontaneous pregnancy. All couples were given the Japanese version of the Golombok-Rust Inventory of Sexual Satisfaction (GRISS). In couples aged 35 years or older, the male partners showed significantly worse sexual satisfaction scores than the female partners. Sexual satisfaction also deteriorated with therapeutic interventions, with mental factors affected more than physical factors. Therapeutic interventions such as timed sexual intercourse and assisted reproductive technology were considered emotionally stressful for infertile couples, with sexual satisfaction accordingly lower in this group than in couples achieving spontaneous pregnancy. GRISS successfully evaluated lower sexual satisfaction associated with infertility, and hence is a useful tool for identifying couples whose sexual satisfaction could be enhanced by counselling or other stress-reduction modalities.

We induced differentiation of human amnion-derived mesenchymal stem cells (AMCs) and menstrual blood-derived mesenchymal stem cells (MMCs) into endometrial stroma-like cells, which could be useful for cell therapy to support embryo implantation. Interestingly, the expression patterns of surface markers were similar among AMCs, MMCs, and endometrial stromal cells. In addition, whereas treatment with estrogen and progesterone was not very effective for decidualizing AMCs and MMCs, treatment with 8-Br-cAMP prompted remarkable morphological changes in these cells as well as increased expression of decidualization markers (prolactin and insulin-like growth factor binding protein-1) and attenuated expression of surface markers unique to mesenchymal stem cells. These results demonstrated that bone marrow-derived stem cells, which are considered a potential source of endometrial progenitor cells, as well as AMCs and MMCs show in vitro decidualization potential, which is characteristic of endometrial stromal cells.

In mammals, uterine epithelium is remodeled cyclically throughout adult life for pregnancy. Despite the expression of CD9 in the uterine epithelium, its role in maternal reproduction is unclear. Here, we addressed this issue by examining uterine secretions collected from patients undergoing fertility treatment and fertilization-competent Cd9(-/-) mice expressing CD9-GFP in their eggs (Cd9(-/-)TG). CD9 in uterine secretions was observed as extracellular matrix-like feature, and its amount of the secretions associated with repeated pregnancy failures. We also found that the litter size of Cd9(-/-)TG female mice was significantly reduced after their first birth. Severely delayed re-epithelialization of the endometrium was then occurred. Concomitantly, vascular endothelial growth factor (VEGF) was remarkably reduced in the uterine secretions of Cd9(-/-)TG female mice. These results provide the first evidence that CD9-mediated VEGF secretion plays a role in re-epithelialization of the uterus.

Background: Oocytes may undergo two types of aging. The first is induced by exposure to an aged ovarian microenvironment before being ovulated, known as 'reproductive or maternal aging', and the second by either a prolonged stay in the oviduct before fertilization or in vitro aging prior to insemination, known as 'postovulatory aging'. However, the molecular mechanisms underlying these aging processes remain to be elucidated. As telomere shortening in cultured somatic cells triggers replicative senescence, telomere shortening in oocytes during reproductive and postovulatory aging may predict developmental competence. This study aimed to ascertain the mechanisms underlying altered telomere biology in mouse oocytes during reproductive and postovulatory aging.Methods: We studied Tert expression patterns, telomerase activity, cytosolic reactive oxygen species (ROS) production, and telomere length in fresh oocytes from young versus reproductively-aged female mice retrieved from oviducts at 14 h post-human chorionic gonadotropin (hCG), in vivo or in vitro postovulatory-aged mouse oocytes at 23 h post-hCG. Oocytes were collected from super-ovulated C57BL/6 J mice of 6-8 weeks or 42-48 weeks of age. mRNA and protein expressions of the Tert gene were quantified using real-time quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and immunochemistry. Telomerase activity was measured by a telomeric repeat amplification protocol assay, while telomere length was measured by Q-PCR and quantitative fluorescence in situ hybridization analyses.Results: The abundance of Tert expression in oocytes significantly decreased during reproductive and postovulatory aging. Immunofluorescent staining clearly demonstrated an altered pattern and intensity of TERT protein expression in oocytes during reproductive aging. Furthermore, relative telomerase activity (RTA) in oocytes from reproductively-aged females was significantly lower than that in oocytes from young females. In contrast, RTA in postovulatory-aged oocytes was similar to that in fresh oocytes. Oocytes from reproductively-aged females and postovulatory-aged oocytes showed higher ROS levels than oocytes from young females. Relative telomere length (RTL) was remarkably shorter in oocytes from reproductively-aged females compared to oocytes from young females. However, postovulatory aging had no significant effect on RTL of oocytes.Conclusions: Long-term adverse effects of low telomerase activity and increased ROS exposure are likely associated with telomere shortening in oocytes from reproductively-aged female mice. © 2013 Yamada-Fukunaga et al. licensee BioMed Central Ltd.

What are the reproductive and obstetric outcomes in patients undergoing radical abdominal trachelectomy (RAT) for early-stage cervical cancer? When RAT was performed before a pregnancy achieved with fertility treatments, pregnancy rate of 36.2 was obtained and 71.4 of these women gave birth at 32 weeks of gestation. Reproductive and obstetric outcomes after radical vaginal trachelectomy (RVT) are well documented; however, these outcomes after RAT have not been well studied. This is a retrospective cohort study of patients at a single institution who underwent RAT and became pregnant. Reproductive and obstetric outcomes of 114 patients who had undergone RAT from September 2002 to December 2010 were investigated. Women of reproductive age with early-stage cervical cancer who wished to preserve their fertility were documented. Patients median age was 33 years (2540 years). A total of 31 pregnancies were achieved in 25 patients and 6 patients had 2 pregnancies. Eighteen of 25 patients (72.0) had infertility problems; 17 patients conceived with IVF-embryo transfer and 1 patient with intrauterine insemination. The pregnancy rate among patients who wished to conceive was 36.2 (25/69). Among 31 pregnancies in 25 patients, 4 patients had first trimester miscarriage and 1 patient had second trimester miscarriage. Excluding the five patients who miscarried and the five ongoing pregnancies, all the 21 patients had deliveries by Cesarean section. Four patients had a preterm birth in the second trimester and 17 patients delivered in the third trimester. Of the 17 pregnancies that reached the third trimester, 2 (11.8) were preterm births between 29 and 32 weeks, 11 (64.7) were delivered between 32 and 37 weeks and 4 (23.5) at 37 weeks of gestation. Because of the retrospective data collection, not all pregnancies may have been recorded. Prospective multicenter studies are needed to determine if the results shown in this retrospective cohort can be generalized to all patients with early-stage cervical cancer who wish to undergo the fertility-sparing RAT procedure. There was no funding for this study. No conflicts of interest.

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for beta-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-C-13(8)] octanoate added to the culture media into intermediate metabolites of the TCA cycle via beta-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

Objective: To provide a focused review of the scientific literature pertaining to spermatozoal RNA. Design: Review of the literature and appraisal of relevant articles. Setting: Not applicable. Patient(s): Infertile male. Intervention(s): None. Main Outcome Measure(s): Spermatozoal RNAs as potential epigenetic modifiers in early embryo development and as clinical markers of male infertility. Result(s): The nucleus of mature spermatozoa contains a complex population of mRNAs and miRNAs despite its transcriptionally inert state. Conclusion(s): A specific set of functional RNAs are delivered into oocytes during fertilization and are thought to contribute extragenomically to early embryonic development. Even if spermatozoal RNAs is merely residual, it still has the potential to greatly improve the investigative and diagnostic potential of male infertility. (Fertil Steril (R) 2012;97:275-81. (C)2012 by American Society for Reproductive Medicine.)

Background: This study aims to gather information either supporting or rejecting the hypothesis that acute stress may induce ovulation in women. The formulation of this hypothesis is based on 2 facts: 1) estrogen-primed postmenopausal or ovariectomized women display an adrenal-progesterone-induced ovulatory-like luteinizing hormone (LH) surge in response to exogenous adrenocorticotropic hormone (ACTH) administration; and 2) women display multiple follicular waves during an interovulatory interval, and likely during pregnancy and lactation. Thus, acute stress may induce ovulation in women displaying appropriate serum levels of estradiol and one or more follicles large enough to respond to a non-midcycle LH surge. Methods: A literature search using the PubMed database was performed to identify articles up to January 2010 focusing mainly on women as well as on rats and rhesus monkeys as animal models of interaction between the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes. Results: Whereas the HPA axis exhibits positive responses in practically all phases of the ovarian cycle, acute-stress-induced release of LH is found under relatively high plasma levels of estradiol. However, there are studies suggesting that several types of acute stress may exert different effects on pituitary LH release and the steroid environment may modulate in a different way (inhibiting or stimulating) the pattern of response of the HPG axis elicited by acute stressors. Conclusion: Women may be induced to ovulate at any point of the menstrual cycle or even during periods of amenorrhea associated with pregnancy and lactation if exposed to an appropriate acute stressor under a right estradiol environment.

Mining gene-expression-profiling data identified a novel gene that is specifically expressed in preimplantation embryos. Hmgpi, a putative chromosomal protein with two high-mobility-group boxes, is zygotically transcribed during zygotic genome activation, but is not transcribed postimplantation. The Hmgpi-encoded protein (HMGPI), first detected at the 4-cell stage, remains highly expressed in pre-implantation embryos. Interestingly, HMGPI is expressed in both the inner cell mass (ICM) and the trophectoderm, and translocated from cytoplasm to nuclei at the blastocyst stage, indicating differential spatial requirements before and after the blastocyst stage. siRNA (siHmgpi)-induced reduction of Hmgpi transcript levels caused developmental loss of preimplantation embryos and implantation failures. Furthermore, reduction of Hmgpi prevented blastocyst outgrowth leading to generation of embryonic stem cells. The siHmgpi-injected embryos also lost ICM and trophectoderm integrity, demarcated by reduced expressions of Oct4, Nanog and Cdx2. The findings implicated an important role for Hmgpi at the earliest stages of mammalian embryonic development. © The Author 2009. Published by Oxford University Press.

Delayed implantation (embryonic diapause) occurs when the embryo at the blastocyst stage achieves a state of suspended animation. During this period, blastocyst growth is very slow, with minimal or no cell division. Nearly 100 mammals in seven different orders undergo delayed implantation, but the underlying molecular mechanisms that direct this process remain largely unknown. In mice, ovariectomy before preimplantation ovarian estrogen secretion on day 4 of pregnancy initiates blastocyst dormancy, which normally lasts for 1-2 weeks by continued progesterone treatment, although blastocyst survival decreases with time. An estrogen injection rapidly activates blastocysts and initiates their implantation in the progesterone-primed uterus. Using this model, here we show that among -20,000 genes examined, only 229 are differentially expressed between dormant and activated blastocysts. The major functional categories of altered genes include the cell cycle, cell signaling, and energy metabolic pathways, particularly highlighting the importance of heparin-binding epidermal growth factor-like signaling in blastocyst- uterine crosstalk in implantation. The results provide evidence that the two different physiological states of the blastocyst, dormancy and activation, are molecularly distinguishable in a global perspective and underscore the importance of specific molecular pathways in these processes. This study has identified candidate genes that provide a scope for in-depth analysis of their functions and an opportunity for examining their relevance to blastocyst dormancy and activation in numerous other species for which microarray analysis is not available or possible due to very limited availability of blastocysts.

SP-10 is a sperm intra-acrosomal protein, specific to the testis, that is believed to play an important role in egg-sperm binding. While the molecular characterization of the SP-10 protein has been clarified, little is yet known of its functional role in fertilization. We therefore established a monoclonal antibody (mAb pep-SP10) against a peptide (pep-SP10) that included the most hydrophilic portion of human SP-10 between the 135th and 149th amino acids. Human SP-10 was found to be localized in the equatorial region of acrosome-reacted sperm by immunofluorescent staining using our mAb pep-SP10. Monoclonal Ab pep-SP10 inhibited sperm-oolemma binding in the zona-free hamster egg penetration test, but it did not inhibit sperm-zona binding in the hemizona assay. Furthermore, we demonstrated that the oolemmal ligands of human SP-10 did not include beta(1) integrins, the most promising candidates for oocyte ligands involved in sperm-oolemma binding, based on the findings of a human sperm-cultured cell binding assay using F9 mouse embryonal carcinoma cells and F9-transformed cells lacking beta(1) integrins. In conclusion, our present data suggest that human SP-10, expressed on the equatorial region of acrosome-reacted sperm, indeed mediates sperm-oolemma binding in a beta(1) integrin-independent manner, but not sperm-zona binding.