論文

The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period (Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 (Bmal1). By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock’s circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte–erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(−) CD34+ cells, the CD36 expression was more evident in CD109− MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Abstract: Assisted reproductive technology (ART) led to the birth of 60,381 infants in 2020 in Japan. This number is set to increase as the future interest in ART is anticipated to rise. Couples receiving ART are monitoring the outcomes of these treatments to see whether any differences exist between babies conceived naturally and those conceived via ART. This study investigated the relationship between the long-term outcome of children born from ART with a focus on physical and psychomotor developments. A large volume of data concerning each relationship with ART was collected from various observational studies. Several findings indicate that, over time, the physical characteristics of babies born by ART, and those born naturally are comparable. However, some reports indicate that, until they reach school age, there may be a small difference in growth. ART and naturally conceived children do not vary in academic achievement or attention deficit hyperactivity disorder. Taken together, it is difficult to conclude with certainty that ART is the source of these differences since they may arise from the child’s genetic factors or their environment.

Background: Skipping breakfast is associated with dysmenorrhea in young women. This suggests that the delay of food intake in the active phase impairs uterine functions by interfering with circadian rhythms. Objectives: To examine the relation between the delay of feeding and uterine circadian rhythms, we investigated the effects of the first meal occasion in the active phase on the uterine clock. Methods: Zeitgeber time (ZT) was defined as ZT0 (08:45) with lights on and ZT12 (20:45) with lights off. Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum consumption), group II (time-restricted feeding during ZT12-16, initial 4 h of the active period), and group III (time-restricted feeding during ZT20-24, last 4 h of the active period, a breakfast-skipping model). After 2 wk of dietary restriction, mice in each group were killed at 4-h intervals and the expression profiles of uterine clock genes, Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1), Per1 (period circadian clock 1), Per2, and Cry1 (cryptochrome 1), were examined. Results: qPCR and western blot analyses demonstrated synchronized circadian clock gene expression within the uterus. Immunohistochemical analysis confirmed that BMAL1 protein expression was synchronized among the endometrium and myometrium. In groups I and II, mRNA expression of Bmal1 was elevated after ZT12 at the start of the active phase. In contrast, Bmal1 expression was elevated just after ZT20 in group III, showing that the uterine clock rhythm had shifted 8 h backward. The changes in BMAL1 protein expression were confirmed by western blot analysis. Conclusions: This study is the first to indicate that time-restricted feeding regulates a circadian rhythm of the uterine clock that is synchronized throughout the uterine body. These findings suggest that the uterine clock system is a new candidate to explain the etiology of breakfast skipping-induced uterine dysfunction.

Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. Previously, we found decreased SERPINA1 (alpha-1 antitrypsin) expression in endometriosis-like lesions in a mouse model of endometriosis, suggesting that it exacerbated inflammation in these lesions. However, the molecular mechanism(s) by which SERPINA1 affects expression of inflammatory factors and development of endometriotic lesions have not been fully characterized. To investigate the role of intracellular SERPINA1 in endometrial stromal cells (ESCs), we performed RNA sequence analysis using RNA extracted from ESCs in which SERPINA1 was knocked down. The analysis identified several toll-like receptor (TLR)-related factors as being upregulated. Silencing of SERPINA1 increased expression of TLR3 and TLR4 in ESCs, as well as several TLR signaling pathway components, including MYD88, IRAK1/4, interleukin (IL)-1β, and interferon (IFN)-β. TLR3 or TLR4 agonists increased expression of inflammatory factors in SERPINA1-knockdown ESCs, whereas TLR3 or TLR4 inhibitors decreased expression. In addition, treatment with recombinant IL-1β or IFN-β increased expression of MYD88 and inflammatory factors in ESCs. Immunohistochemical analysis of endometriotic tissues showed that TLR3, TLR4, and MYD88 were localized in endometriosis lesions. Taken together, the data suggest that reduced expression of SERPINA1 induces expression of inflammatory factors by ESCs, which in turn are associated with TLR3/4, IL-1β, and IFN-β signaling. Regulation of intracellular SERPINA1 levels in ESCs may be a strategy to inhibit inflammatory responses in endometriotic lesions.

CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-alpha, progesterone receptor, and alpha-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

Purpose: This descriptive analysis evaluated the 2022 assisted reproductive technology (ART) data collected by the Japan Society of Obstetrics and Gynecology registry. Methods and Results: In 2022 (cutoff date 30 November 2023), 634 of 635 registered ART facilities participated; 602 implemented ART treatment, with 543 630 registered cycles and 77 206 neonates (9.1% and 10.6% increases from the previous year). For fresh cycles, freeze-all in vitro fertilization and intracytoplasmic sperm injection cycles increased, resulting in 2183 and 2822 neonates, respectively. In total, 275 296 cycles resulted in oocyte retrieval, with 158 247 (57.5%) freeze-all cycles. Total single embryo transfer (ET) and singleton pregnancy rates were 82.4% and 97.2%, respectively. The singleton live birth rate was 97.4%. The number of frozen–thawed ET (FET) cycles was 264 412, with 98 348 pregnancies and 72 201 neonates. The single ET rate was 85.3%. The rate of singleton pregnancies was 96.9%; that of singleton live births was 96.9%. Per registered cycle, women had a mean age of 37.6 (standard deviation: 4.8) years; 210 322 cycles (38.7%) were conducted for women aged ≥40 years. Conclusions: Significant growth in ART cycles and outcomes reflects the impact of recent expanded insurance coverage.

Purpose: To summarize assisted reproductive technology (ART) data for 2023 collected through the Japan Society of Obstetrics and Gynecology registry. Methods and Results: 625 out of 638 registered ART facilities took part in the study, documenting 561 664 treatment cycles and 85 048 newborns (+3.3% and +10.2% increases from 2022). The average age of women undergoing treatment was 37.3 years (standard deviation: 4.8); 205 986 cycles (36.7%) involved women aged ≥ 40 years. Among fresh cycles, oocyte retrieval was performed in 281 665 cycles, including 168 343 freeze-all cycles (59.8%). A total of 2329 pregnancies and 3375 newborns resulted from in vitro fertilization and intracytoplasmic sperm injection cycles, yielding 1772 and 2502 newborns, respectively. The overall rates for single embryo transfer (SET) and singleton delivery were 80.5% and 96.5%. Frozen–thawed embryo transfer accounted for 271 361 cycles, resulting in 109 850 pregnancies and 80 774 newborns, with a SET rate of 84.7% and a singleton delivery rate of 96.3%. Conclusions: In 2023, the second year of insurance coverage, the registry recorded the highest numbers of treatment cycles and newborns. Effective registry systems planned for 2026 will enable comprehensive evaluation of emerging trends in Japanese ART practice.

Infertility and reproductive issues are commonly observed in animals with clock abnormalities. Substantial rodent data is available; however, relatively few studies have investigated the connection between fertility and clock abnormalities in humans. Therefore, this study aimed to analyze the expression of circadian clock genes and their impact on genes involved in collagen production in the human myometrium and leiomyomas (LM). The relationship between the expression of brain and muscle aryl-hydrocarbon-receptor-nuclear-translocator (Arnt)-like protein-1 (BMAL1) and the genes responsible for collagen synthesis in the human MM and LMs were investigated. Human MM and LM tissues were collected for analysis from patients who underwent hysterectomy analysis. Immunohistochemical analysis, cell culturing, immunofluorescence, small interfering RNA transfection, reverse transcription quantitative real-time polymerase chain reaction, scratch wound assays, and transwell assays were employed to gain a comprehensive understanding of the cellular and molecular processes. A correlation was found between BMAL1 expression and genes regulating collagen synthesis in primary cultures of human MM and LM cells. Moreover, the inhibition of BMAL1 differentially increased the migration and invasion of MM and LM cells. This work discloses the role of BMAL1 in collagen production in primary cultures of human MM and LM cells, offering insight into clock gene involvement in both normal and pathological uterine conditions. Furthermore, this study highlights the crucial role of BMAL1 in collagen synthesis in human MM and LM cells, underscoring the significance of BMAL1 in the regulation of reproductive physiology. These results suggest that BMAL1 might be a useful target molecule for anti-LM therapy.

Objective: Rare disease Background: Intraplacental choriocarcinoma (IC) is a rare subtype of gestational choriocarcinoma that can be associated with placental hemorrhage and metastases to the mother and fetus. Chemotherapy can be effective in cases of placental localization and may not adversely affect fertility. This report describes a 32-year-old woman who conceived 10 months after chemotherapy for IC and achieved successful term delivery of that pregnancy. Case Report: A 32-year-old woman delivered her first child by vacuum extraction due to weak labor and fetal distress. The newborn presented with severe anemia, as well as elevated alpha-fetoprotein and fetal hemoglobin levels, suggesting fetomaternal hemorrhage. Placental histopathology revealed IC. Postpartum elevation of human chorionic gonadotropin levels prompted EMA-CO (etoposide, methotrexate, actinomycin D/cyclophosphamide, vincristine) chemotherapy; 7 treatment courses were completed without side effects or metastasis. Ten months after completion of chemotherapy, the patient spontaneously conceived. She subsequently delivered a healthy boy at 38+5 weeks of gestation; placental examination did not reveal malignancy. During the 5-year follow-up period, both children developed normally, and no maternal recurrence or metastasis was observed. Conclusions: This report describes a case of postnatal diagnosis of IC via placental histopathology, followed by successful treatment and subsequent pregnancy within 1 year after chemotherapy completion. The findings highlight the importance of accurate diagnosis and appropriate management of IC; they support previous observations that chemotherapy does not preclude future successful pregnancies. Additionally, the report underscores the clinical challenges of IC, its implications for future pregnancies, and the need for long-term follow-up concerning both mother and child.

Aim: To examine the cumulative ongoing pregnancy/live birth rate per patient by encompassing all embryo transfer (ET) cycles associated with a single oocyte retrieval cycle among women with diminished ovarian reserve (DOR), and to investigate whether serum anti-Müllerian hormone (AMH) levels can predict a live birth success. Methods: This multi-institutional retrospective cohort study included infertile women with DOR (AMH <1.1 ng/mL) who began their first cycle of in vitro fertilization and/or intracytoplasmic sperm injection between January and December 2021. Outcomes using the retrieved embryos were evaluated for up to four ET cycles. Results: Of 606 patients, 113 did not undergo ET. A total of 605 ET cycles were analyzed. Clinical pregnancy and ongoing pregnancy/live birth rates per ET cycle were significantly dependent on age, not AMH levels. Ongoing pregnancy/live birth rates per patient increased significantly with higher AMH levels. A significant difference in the cumulative ongoing pregnancy/live birth rate was observed depending on whether cases with no remaining embryos were included among the censored cases. Conclusion: Assisted reproductive technology (ART) outcomes per ET cycle in women with DOR were similar to those reported in the Japanese ART registry without distinguishing DOR. The greater the number of good-quality embryos that can be frozen, the higher the expected cumulative live birth rate.

Accumulating evidence indicates that disruption of the circadian clock contributes to the development of lifestyle-related diseases. We have previously shown that exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, can strongly affect the molecular clocks in the peripheral tissues. This study aimed to investigate the effects of its dosing time and the central nervous system-specific GLP-1 receptor knockdown (GLP1RKD) on the hepatic clock in mice treated with exenatide. Male C57BL/6J and GLP1RKD mice were housed under a 12-h/12-h light/dark cycle, and feeding was restricted to either the light period (L-TRF) or the first 4 h in the dark period (D-TRF). In parallel, exenatide was administered 4–5 times, once daily either at the beginning of the dark (ZT 12) or light period (ZT 0), and we assessed the mRNA expression rhythms of clock genes in the liver thereafter. Exenatide administration at ZT 12 counteracted the phase shift effect of the L-TRF on the hepatic clock of wild-type mice, whereas the dosing at ZT 0 enhanced its effect. However, exenatide did not influence the phase of the hepatic clock under D-TRF regardless of the dosing time. The effect of exenatide in wild-type mice weakened in GLP1RKD mice. These results showed that exenatide dosing time-dependently affects the hepatic circadian clock through the central GLP-1 system. Exenatide administration at the beginning of the active period (i.e., in the morning for humans) might prevent disruption of the peripheral clocks caused by irregular eating habits.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4-positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1-derived HPV 16-positive W12 cells via an ELF3-dependent pathway. In organotypic raft cultures, FOXP4-downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte-derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3-dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

In humans, the placenta provides the only fetomaternal connection and is essential for establishing a pregnancy as well as fetal well-being. Additionally, it allows maternal physiological adaptation and embryonic immunological acceptance, support, and nutrition. The placenta is derived from extra-embryonic tissues that develop rapidly and dynamically in the first weeks of pregnancy. It is primarily composed of trophoblasts that differentiate into villi, stromal cells, macrophages, and fetal endothelial cells (FEC). Placental differentiation may be closely related to perinatal diseases, including fetal growth retardation (FGR) and hypertensive disorders of pregnancy (HDP), and miscarriage. There are limited findings regarding human chorionic villous differentiation and placental development because conducting in vivo studies is extremely difficult. Placental tissue varies widely among species. Thus, experimental animal findings are difficult to apply to humans. Early villous differentiation is difficult to study due to the small tissue size; however, a detailed analysis can potentially elucidate perinatal disease causes or help develop novel therapies. Artificial induction of early villous differentiation using human embryonic stem (ES) cells/induced pluripotent stem (iPS) cells was attempted, producing normally differentiated villi that can be used for interventional/invasive research. Here, we summarized and correlated early villous differentiation findings and discussed clinical diseases.

Objective: Hypoxia, which occurs during the development of cervical cancer, confers chemotherapy resistance. MicroRNA expression is regulated by hypoxia and is associated with the onset and progression of certain types of cancer. MicroRNA-100 (miR-100) is a microRNA, associated with nasopharyngeal and oral squamous cell carcinomas, whose expression is decreased in cervical cancer. This study aims to ascertain the effect of hypoxia on expression levels of both miR-100 and its target genes, as well as exploring the sensitivity to paclitaxel under hypoxic conditions. Methods: We investigated the effect of hypoxia on miR-100 expression. We also explored the regulators of paclitaxel response under hypoxic conditions of cervical cancer. Results: Using RT-qPCR, we found that expression of miR-100 in cervical cancer cell lines SiHa and HeLa is significantly higher under hypoxic conditions (1% O2). We also confirmed that human ubiquitin-specific protease 15 (USP15) is the one of the target proteins of miR-100. Hypoxia and overexpression of miR-100 both reduced the activity of the luciferase reporter containing the 3′-untranslated region of USP15, which contains the miR-100 binding site. Furthermore, a western blot analysis showed that hypoxia suppresses the expression of the USP15 protein, while RT-qPCR showed hypoxia-induced downregulation of USP15 mRNA levels. We also discovered that overexpression of miR-100 induces paclitaxel resistance, thereby reducing the drug's therapeutic effect on cell death. Conclusions: Our results are consistent with the hypothesis that cervical cancer cells overexpress miR-100 in response to hypoxia and that miR-100 is a facilitator of USP15 downregulation and inactivation.

Objective: Imeglimin is a novel antidiabetic drug structurally related to metformin. Metformin has been shown to modulate the circadian clock in rat fibroblasts. Accordingly, in the present study, we aimed to determine whether imeglimin can impact the circadian oscillator in mouse embryonic fibroblasts (MEFs). Methods: MEFs carrying a Bmal1-Emerald luciferase (Bmal1-ELuc) reporter were exposed to imeglimin (0.1 or 1 mM), metformin (0.1 or 1 mM), a nicotinamide phosphoribosyltransferase inhibitor FK866, and/or vehicle. Subsequently, Bmal1-ELuc expression and clock gene mRNA expression levels were measured at 10-min intervals for 55 h and 4-h intervals for 32 h, respectively. Results: Imeglimin significantly prolonged the period (from 26.3 to 30.0 h at 0.1 mM) and dose-dependently increased the amplitude (9.6-fold at 1 mM) of the Bmal1-ELuc expression rhythm; however, metformin exhibited minimal effects on these parameters. Moreover, imeglimin notably impacted the rhythmic mRNA expression of clock genes (Bmal1, Per1, and Cry1). The concurrent addition of FK866 partly inhibited the effects of imeglimin on both Bmal1-ELuc expression and clock gene mRNA expression. Conclusion: Collectively, these results reveal that imeglimin profoundly affects the circadian clock in MEFs. Further studies are needed to evaluate whether imeglimin treatment could exert similar effects in vivo.

Objective: To assess the relationship between meal consumption frequency and assisted reproductive technology (ART) outcomes among female patients with infertility. Research Methods & Procedures: This cohort study was conducted from February 2022 to January 2024 at Tokyo Medical University Hospital. Overall, 101 female patients with infertility issues and without a history of stroke, heart disease, cancer, or type 1 or type 2 diabetes were enrolled in this study. The factors extracted from the questionnaire included demographic information, meal consumption frequency before ART and at 20 years of age, smoking status, and alcohol consumption status. Data on other factors, including age, body mass index, anti-Müllerian hormone level, and parity history, were collected from medical records. The assessed clinical outcomes included number of transplanted embryos, clinical pregnancies, ongoing pregnancies, live births, and miscarriages. Results: After adjusting for potential confounding factors, including age, smoking status, alcohol consumption status, body mass index, anti-Müllerian hormone level, and parity history, a multivariate analysis of ART outcomes was performed. Patients were categorized into groups based on the frequency of weekly consumption of breakfast, lunch, and dinner. Patients who consumed breakfast 6–7 times a week were significantly more likely to have higher rates of live birth and lower rates of miscarriage in pregnancies conceived through ART. Conclusions: Consumption of breakfast 6–7 times a week before ART was associated with increased success rates following ART. This highlights the potential importance of regular breakfast consumption for optimizing ART outcomes.

In recent years, local governments in Japan have established a public financial support system for fertility preservation in pediatric, adolescent, and young adult cancer patients. Fertility preservation has become popular for patients with cancers included in the gonadal toxicity risk classification of the 2017 edition of the Guideline for Fertility Preservation in Children, Adolescents and Young Adult Cancer Patients from the Japan Society of Clinical Oncology. However, patients with cancer and non-cancer diseases that are not included in the Guideline’s gonadal toxicity risk classification also often receive treatment that may affect fertility, but they are often denied the opportunity of fertility preservation because no public financial support is available for diseases not listed in the Guideline. The national research project proposes including these diseases in the indications and treatment for fertility preservation. Therefore, we cooperated with the Japan Society for Fertility Preservation and the Ministry of Health, Labour and Welfare research group to solicit opinions from experts in each therapeutic area and reviewed the literature and overseas guidelines. This paper summarizes the findings of the project. We believe that it will be an important source of information for clinicians treating patients who need fertility preservation but note that the appropriateness of fertility preservation for the disorders listed in this report needs to be continuously reviewed as medical care advances.