Kanako Hayashi

Endometriosis (EuE) is a chronic, incurable disease. Due to limited efficacy, high recurrence rates, and serious side effects of current treatments, development of new, targeted, nonhormonal therapies is urgently needed. We previously reported that niclosamide, an FDA‐approved anthelmintic drug, attenuates endometriotic lesion growth. We further identified folate receptor‐β (FRβ)‐positive macrophages as contributors to disease progression. Significantly, niclosamide inhibits FRβ ⁺ macrophages and reduces inflammation, innervation, and angiogenesis. To develop niclosamide as a nonhormonal and selective immune cell‐targeted therapy for EuE, we engineered a folic acid (FA)‐conjugated 2‐deoxyglucose dendrimer (FA‐2DG‐D) using click chemistry to enable selective FRβ‐mediated uptake. Conjugation of niclosamide to FA‐2DG‐D yielded a targeted nanotherapeutic (FA‐2DG‐D‐Niclo) with enhanced aqueous solubility, controlled intracellular release, and excellent batch‐to‐batch reproducibility. In a mouse model of EuE, FA‐2DG‐D demonstrated lesion‐specific accumulation and selective internalization by FRβ ⁺ macrophages with minimal off‐target organ retention. A single intraperitoneal dose of FA‐2DG‐D‐Niclo (25 or 50 mg/kg/bw of niclosamide) significantly reduced FRβ ⁺ macrophage burden, suppressed lesion number and volume, and markedly improved EuE‐associated hyperalgesia at 2 weeks post‐treatment. Together, these findings establish FRβ ⁺ macrophages as a potential target in EuE and present FA‐2DG‐D‐Niclo as a nonhormonal, macrophage‐focused nanomedicine for precise and effective endometriosis treatment.

Endocrine-disrupting chemicals (EDCs) are a global concern for human health and the environment. EDCs include plasticizers, pharmaceutical agents, industrial chemicals, fungicides, and pesticides. Thus, EDC exposure is a manmade consequence in our current lives. More than 1000 chemicals are classified as EDCs, which can interfere with any aspect of the endocrine system, resulting in reproductive disorders, metabolic dysfunctions, cardiovascular diseases, neurological and immune defects, and cancers. Because EDCs affect steroid hormone regulation, there are crucial associations between EDCs and gynecological diseases. This review focuses on the inroads by which faulty signaling mechanisms and cellular responses to major EDCs, such as bisphenols, phthalates, per- and polyfluoroalkyl substances, and parabens, lead to the initiation or progression of uterine disorders, including endometriosis, leiomyoma (uterine fibroids), adenomyosis, and endometrial hyperplasia. We also summarize the features, risks, and functions of EDCs in each disease.

This study investigated how chronic pelvic pain (CPP) develops using rhesus macaques with naturally occurring endometriosis and a multiple lesion induction mouse model (MIM), as repeated retrograde menstruation is considered an underlying mechanism of endometriosis pathogenesis. MIM increased lesion numbers and elevated hypersensitivity. Elevated persistent glial cell activation was observed across multiple brain regions or spinal cords in MIM and rhesus macaques. Elevated TRPV1, SP, and CGRP expressions in the dorsal root ganglia (DRG) were persistent in MIM. MIM induced the severe disappearance of TIM4 ^hi MHCII ^lo residential macrophages and an influx of increased pro-inflammatory TIM4 ^lo MHCII ^hi macrophages in the peritoneal cavity. Cytokine levels were persistently elevated in MIM. Furthermore, dienogest (a synthetic progestin) and fingolimod (a selective immunosuppressor) reduced hyperalgesia and neuroinflammation. Our results indicate that recurrent retrograde menstruation can be a peripheral stimulus that induces nociceptive pain and creates a composite chronic inflammatory stimulus, leading to neuroinflammation and sensitization of the central nervous system. The circuits of neuroplasticity and stimulation of peripheral organs via a feedback loop of neuroinflammation may mediate widespread endometriosis-associated CPP. These findings in mice were further supported by results from the spontaneously developed advanced endometriosis in rhesus macaques via recurrent retrograde menstruation. Repeatedly occurring retrograde menstruation intensifies central sensitization driven by neuroinflammation in rhesus macaques and a mouse model of endometriosis.

Endometriosis is a chronic, incurable disease. Due to limited efficacy, high recurrence rates, and serious side effects of current treatments, development of new, targeted, non-hormonal therapies is urgently needed. We previously reported that niclosamide, an FDA-approved anthelmintic drug, attenuates endometriotic lesion growth. We further identified folate receptor-β (FRβ)-positive macrophages as contributors to disease progression. Significantly, niclosamide inhibits FRβ ⁺ macrophages and reduces inflammation, innervation, and angiogenesis. To develop niclosamide as a non-hormonal and selective immune cell-targeted therapy for endometriosis, we engineered a folic acid-conjugated 2-deoxyglucose dendrimer (FA-2DG-D) using click chemistry to enable selective FRβ-mediated uptake. Conjugation of niclosamide to FA-2DG-D yielded a targeted nanotherapeutic (FA-2DG-D-Niclo) with enhanced aqueous solubility, controlled intracellular release, and excellent batch-to-batch reproducibility. In a mouse model of endometriosis, FA-2DG-D demonstrated lesion-specific accumulation and selective internalization by FRβ⁺ macrophages with minimal off-target organ retention. A single intraperitoneal dose of FA-2DG-D-Niclo (25 or 50 mg/kg/bw of niclosamide) significantly reduced FRβ⁺ macrophage burden, suppressed lesion number and volume, and markedly improved endometriosis-associated hyperalgesia at two weeks post-treatment. Together, these findings establish FRβ⁺ macrophages as a potential target in endometriosis and present FA-2DG-D-Niclo as a non-hormonal, macrophage-focused nanomedicine for precise and effective endometriosis treatment. Graphic

Conditional ablation of Igf1r in early folliculogenesis has demonstrated the necessity of insulin signaling to progress to the antral stage, whereas ablation of both Insr and Igfr1 in the periovulatory window allows the formation of the antrum but reduces the efficiency of ovulation and subsequent luteinization. For this study, we examined the independent and shared actions in single and double knockouts (DKO) for Insr and Ifg1r using Esr2-Cre. As this recombinase is active during neonatal ovarian development and the initial wave of folliculogenesis, we hypothesized that abnormalities in ovary formation and establishment of the initial follicle pool would occur, which could alter female reproductive lifespan. We found that ablation of both receptors led to a delay in puberty, altered mating frequency, and ultimately infertility for Igf1rd/d and DKO females. Quantitation of germ cell cyst breakdown, and formation of primordial and primary follicles were normal in the neonatal window and at puberty, suggesting insulin signaling was not essential for establishment of ovarian reserve. However, the loss of IGF1R signaling impaired transition from primary to secondary follicles, which was worsened when IGF ligand cross-reactivity from INSR signaling was lost in DKO mice. DKO mice also exhibited abnormal follicle activation in the absence of hormone stimulation, but no subsequent proliferation of granulosa cells or antrum formation occurred. In adult mice, loss of either receptor disrupted estrous cyclicity, with DKO mice rarely leaving metestrus indicating abnormal regulation of the HPG axis contributing to subfertility and infertility observed in single and double receptor knockouts.

Exposure to endocrine-disrupting chemicals (EDCs), such as bisphenol (BP) A, disrupts reproduction across generations. Germ cell epigenetic alterations are proposed to mediate these transgenerational defects. Previously, we have shown that prenatal exposure to environmentally relevant doses of BPA or its substitute, BPS, caused transgenerationally maintained reproductive impairments associated with neonatal spermatogonial epigenetic changes in male mice. However, the mechanisms sustaining these changes across generations remain unclear. This study aimed to systematically elucidate the mechanism of transgenerational inherence by prenatal BPA and BPS exposure in the murine germline from F1 to F3 generations at both transcriptomic and epigenetic levels. Pregnant CD-1 females (F0) were orally administered BPA or BPS at doses of 0 (vehicle control), 0.5, 50, or 1000 µg/kg/b.w./day from gestational day 7 to birth. Sperm counts and motility were examined in F1, F2, and F3 adult males. THY1⁺ germ cells on postnatal day 6 from F1, F2, and F3 males at a dose of 50 µg/kg/b.w./day were used for analysis by single-nucleus (sn) multi-omics (paired snRNA-seq and snATAC-seq on the same nucleus). Prenatal exposure to BPA and BPS with 0.5, 50, and 1000 µg/kg/b.w./day reduced sperm counts in mice across F1 to F3 generations. In the F1 generation, BPA or BPS exposure with 50 µg/kg/b.w./day disrupted the balance between maintaining the undifferentiated and differentiating spermatogonial populations. Differentially accessible peaks (DAPs) by snATAC-seq were primarily located in the promoter regions, with elevated activity of key transcription factors, including SP1, SP4, and DMRT1. Notably, similar gene expression and chromatin changes were observed in directly exposed F1 and F2 generations but differed in the indirectly exposed F3 generation. Approximately 80% of DAPs in F1 and F2 spermatogonia overlapped with histone post-translational modifications linked to transcription activation (e.g., H3K4me1/2/3 and H3K27ac). Across F1 to F3 generations, although BPA exerted more potent effects on gene expression in F1 spermatogonia, BPS induced longer-lasting effects. Interestingly, DMRT1 motif activity was persistently elevated in all three generations following ancestral BPA or BPS exposure. Our work provides the first systematic analyses of transgenerational gene and chromatin dynamics following prenatal exposure to BPA or BPS. These results suggest that prenatal exposure to environmentally relevant doses of BPA or BPS alters chromatin accessibility and transcription factor motif activities, consequently contributing to disrupted transcriptional levels in neonatal spermatogonia, and some are sustained to F3 generations, ultimately leading to the reduction of sperm counts in adults. https://doi.org/10.1289/EHP16981.

The use of cannabis during pregnancy and nursing is a growing public health concern, and the multigenerational impacts of perinatal cannabis exposure remain largely unknown. To address this knowledge gap, we sought to examine the long-term consequences of perinatal cannabis use on reproductive function and how it might impact subsequent generations. Pregnant female mice were exposed to control vehicle or cannabis extract [25, 100, or 200 mg/ml Δ9-tetrahydrocannabinol (THC) in the cannabis extract] from gestational day 1 to postnatal day 21 (twice/day), encompassing the duration of pregnancy through weaning. Based on plasma THC concentrations in F0 females, we chose 100 and 200 mg/ml THC in the cannabis extract for subsequent studies. The selected doses and exposure conditions did not disrupt pregnancy or nursing in F0 females. Pregnancy and neonatal outcomes, including gestational length, litter size, and sexual ratio, were not affected by cannabis exposure. However, cannabis-exposed neonatal F1 pups were smaller. Cannabis exposure delayed vaginal opening as a sign of puberty onset and disrupted estrous cyclicity in F1 females. However, its effects were minor in F2 and F3 females. F1–F3 females showed no abnormal ovarian and uterine histology or plasma estradiol-17β levels and could produce normal offspring without pregnancy issues. These results suggest that the developmental stages of the hypothalamus and pituitary are likely perturbed by gestational and nursing cannabis exposure in F1 females. However, they are not sufficient to compromise adult reproductive function. The present results indicate limited transgenerational effects of perinatal cannabis exposure on female reproductive parameters.

The Rhox family of homeobox transcription factors comprises established regulators of gonad function, but their downstream targets have been relatively elusive, particularly in the female reproductive tract. Here, we characterize Ins2 as a downstream target of the two granulosa cell-specific factors, Rhox5 and Rhox8, in the ovary. While INS2 is classically produced by islet cells in the pancreas, we found that Ins2 gene expression is present in the mural granulosa cell layer of large antral follicles, and it was not significantly reduced in Rhox5-null mice. This was a surprising finding as we previously validated Ins2 as a direct target of RHOX5 in Sertoli cells, the male counterpart to granulosa cells that serves the germ cell nurse function in the testis. In the ovary, RHOX8 appears to be the major driver of Ins2 expression, as evidenced from the maximal activity of Ins2 promoter reporter plasmids when RHOX8 protein was active within granulosa cells in vitro and the downregulation of endogenous Ins2 in mice with the granulosa cell-specific knockdown of RHOX8 in vivo. RHOX5 induces Rhox8 expression in pre-antral granulosa cells and then becomes relatively silent in peri-ovulatory follicles. However, Rhox8 does not peak until after the ovulatory LH surge. The induction of Rhox8 by progesterone, after the normal window of RHOX5 has passed, may explain why Rhox5-null female mice display apparently normal fertility, if RHOX8 is capable of the redundant stimulation of target genes that are essential for ovulation.

The use of cannabis during pregnancy and nursing is a growing public health concern, and the multigenerational impacts of perinatal cannabis exposure remain largely unknown. To address this knowledge gap, we sought to examine the long-term consequences of perinatal cannabis use on reproductive function and how it might impact subsequent generations. Pregnant female mice were exposed to control vehicle or cannabis extract [25, 100, or 200 mg/ml Δ9-tetrahydrocannabinol (THC) in the cannabis extract] from gestational day 1 to postnatal day 21 (twice/day), encompassing the duration of pregnancy through weaning. Based on plasma THC concentrations in F0 females, we chose 100 and 200 mg/ml THC in the cannabis extract for subsequent studies. The selected doses and exposure conditions did not disrupt pregnancy or nursing in F0 females. Pregnancy and neonatal outcomes, including gestational length, litter size, and sexual ratio, were not affected by cannabis exposure. However, cannabis-exposed neonatal F1 pups were smaller. Cannabis exposure delayed vaginal opening as a sign of puberty onset and disrupted estrous cyclicity in F1 females. However, its effects were minor in F2 and F3 females. F1-F3 females showed no abnormal ovarian and uterine histology or plasma estradiol-17β levels and could produce normal offspring without pregnancy issues. These results suggest that the hypothalamus and pituitary are likely perturbed by perinatal cannabis exposure, and the early hypothalamus-pituitary-ovarian axis is disrupted in F1 females. However, they are not sufficient to compromise adult reproductive function. The present results indicate limited transgenerational effects of perinatal cannabis exposure on female reproductive parameters.