PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Androgen-mediated Perturbation of the Hepatic Circadian System Through Epigenetic Modulation Promotes NAFLD in PCOS Mice.

In women, excess androgen causes polycystic ovary syndrome (PCOS), a common fertility disorder with comorbid metabolic dysfunctions including diabetes, obesity, and nonalcoholic fatty liver disease. Using a PCOS mouse model, this study shows that chronic high androgen levels cause hepatic steatosis while hepatocyte-specific androgen receptor (AR)-knockout rescues this phenotype. Moreover, through RNA-sequencing and metabolomic studies, we have identified key metabolic genes and pathways affected by hyperandrogenism. Our studies reveal that a large number of metabolic genes are directly regulated by androgens through AR binding to androgen response element sequences on the promoter region of these genes. Interestingly, a number of circadian genes are also differentially regulated by androgens. In vivo and in vitro studies using a circadian reporter [Period2::Luciferase (Per2::LUC)] mouse model demonstrate that androgens can directly disrupt the hepatic timing system, which is a key regulator of liver metabolism. Consequently, studies show that androgens decrease H3K27me3, a gene silencing mark on the promoter of core clock genes, by inhibiting the expression of histone methyltransferase, Ezh2, while inducing the expression of the histone demethylase, JMJD3, which is responsible for adding and removing the H3K27me3 mark, respectively. Finally, we report that under hyperandrogenic conditions, some of the same circadian/metabolic genes that are upregulated in the mouse liver are also elevated in nonhuman primate livers. In summary, these studies not only provide an overall understanding of how hyperandrogenism associated with PCOS affects liver gene expression and metabolism but also offer insight into the underlying mechanisms leading to hepatic steatosis in PCOS.

Anti-Müllerian hormone treatment enhances oocyte quality, embryonic development and live birth rate†.

The anti-Müllerian hormone (AMH) produced by the granulosa cells of growing follicles is critical for folliculogenesis and is clinically used as a diagnostic and prognostic marker of female fertility. Previous studies report that AMH-pretreatment in mice creates a pool of quiescent follicles that are released following superovulation, resulting in an increased number of ovulated oocytes. However, the quality and developmental competency of oocytes derived from AMH-induced accumulated follicles as well as the effect of AMH treatment on live birth are not known. This study reports that AMH priming positively affects oocyte maturation and early embryonic development culminating in higher number of live births. Our results show that AMH treatment results in good-quality oocytes with greater developmental competence that enhances embryonic development resulting in blastocysts with higher gene expression. The transcriptomic analysis of oocytes from AMH-primed mice compared with those of control mice reveal that AMH upregulates a large number of genes and pathways associated with oocyte quality and embryonic development. Mitochondrial function is the most affected pathway by AMH priming, which is supported by more abundant active mitochondria, mitochondrial DNA content and adenosine triphosphate levels in oocytes and embryos isolated from AMH-primed animals compared with control animals. These studies for the first time provide an insight into the overall impact of AMH on female fertility and highlight the critical knowledge necessary to develop AMH as a therapeutic option to improve female fertility.

Jumonji Domain-containing Protein-3 (JMJD3/Kdm6b) Is Critical for Normal Ovarian Function and Female Fertility.

In females, reproductive success is dependent on the expression of a number of genes regulated at different levels, one of which is through epigenetic modulation. How a specific epigenetic modification regulates gene expression and their downstream effect on ovarian function are important for understanding the female reproductive process. The trimethylation of histone3 at lysine27 (H3K27me3) is associated with gene repression. JMJD3 (or KDM6b), a jumonji domain-containing histone demethylase specifically catalyzes the demethylation of H3K27me3, that positively influences gene expression. This study reports that the expression of JMJD3 specifically in the ovarian granulosa cells (GCs) is critical for maintaining normal female fertility. Conditional deletion of Jmjd3 in the GCs results in a decreased number of total healthy follicles, disrupted estrous cycle, and increased follicular atresia culminating in subfertility and premature ovarian failure. At the molecular level, the depletion of Jmjd3 and RNA-seq analysis reveal that JMJD3 is essential for mitochondrial function. JMJD3-mediated reduction of H3K27me3 induces the expression of Lif (Leukemia inhibitory factor) and Ctnnb1 (ß-catenin), that in turn regulate the expression of key mitochondrial genes critical for the electron transport chain. Moreover, mitochondrial DNA content is also significantly decreased in Jmjd3 null GCs. Additionally, we have uncovered that the expression of Jmjd3 in GCs decreases with age, both in mice and in humans. Thus, in summary, our studies highlight the critical role of JMJD3 in nuclear-mitochondrial genome coordination that is essential for maintaining normal ovarian function and female fertility and underscore a potential role of JMJD3 in female reproductive aging.

Lipopolysaccharide induces lipolysis and insulin resistance in adipose tissue from dairy cows.

Intense and protracted adipose tissue (AT) fat mobilization increases the risk of metabolic and inflammatory periparturient diseases in dairy cows. This vulnerability increases when cows have endotoxemia-common during periparturient diseases such as mastitis, metritis, and pneumonia-but the mechanisms are unknown. Fat mobilization intensity is determined by the balance between lipolysis and lipogenesis. Around parturition, the rate of lipolysis surpasses that of lipogenesis, leading to enhanced free fatty acid release into the circulation. We hypothesized that exposure to endotoxin (ET) increases AT lipolysis by activation of classic and inflammatory lipolytic pathways and reduction of insulin sensitivity. In experiment 1, subcutaneous AT (SCAT) explants were collected from periparturient (n = 12) Holstein cows at 11 ± 3.6 d (mean ± SE) before calving, and 6 ± 1 d and 13 ± 1.4 d after parturition. Explants were treated with the endotoxin lipopolysaccharide (LPS; 20 µg/mL; basal = 0 µg/mL) for 3 h. The effect of LPS on lipolysis was assessed in the presence of the ß-adrenergic agonist and promoter of lipolysis isoproterenol (ISO; 1 µM; LPS+ISO). In experiment 2, SCAT explants were harvested from 24 nonlactating, nongestating multiparous Holstein dairy cows and exposed to the same treatments as in experiment 1 for 3 and 7 h. The effect of LPS on the antilipolytic responses induced by insulin (INS = 1 µL/L, LPS+INS) was established during ISO stimulation [ISO+INS, LPS+ISO+INS]. The characterization of lipolysis included the quantification of glycerol release and the assessment of markers of lipase activity [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and phosphorylated HSL Ser563 (pHSL)], and insulin pathway activation (AKT, pAKT) using capillary electrophoresis. Inflammatory gene networks were evaluated by real-time quantitative PCR. In periparturient cows, LPS increased AT lipolysis by 67 ± 12% at 3 h across all time points compared with basal. In nonlactating cows, LPS was an effective lipolytic agent at 3 h and 7 h, increasing glycerol release by 115 ± 18% and 68.7 ± 16%, respectively, relative to basal. In experiment 2, LPS enhanced ATGL activity with minimal HSL activation at 3 h. In contrast, at 7 h, LPS increased HSL phosphorylation (i.e., HSL activity) by 123 ± 11%. The LPS-induced HSL lipolytic activity at 7 h coincided with the activation of the MEK/ERK inflammatory pathway. In experiment 2, INS reduced the lipolytic effect of ISO (ISO+INS: -63 ± 18%) and LPS (LPS+INS: -45.2 ± 18%) at 3 h. However, the antilipolytic effect of INS was lost in the presence of LPS at 7 h (LPS+INS: -16.3 ± 16%) and LPS+ISO+INS at 3 and 7 h (-3.84 ± 23.6% and -21.2 ± 14.6%). Accordingly, LPS reduced pAKT:AKT (0.11 ± 0.07) compared with basal (0.18 ± 0.05) at 7 h. Our results indicated that exposure to LPS activated the classic and inflammatory lipolytic pathways and reduced insulin sensitivity in SCAT. These data provide evidence that during endotoxemia, dairy cows may be more susceptible to lipolysis dysregulation and loss of adipocyte sensitivity to the antilipolytic action of insulin.

Looking at the Future Through the Mother's Womb: Gestational Diabetes and Offspring Fertility.

Altered nutrition or intrauterine exposure to various adverse conditions during fetal development or earlier in a mother's life can lead to epigenetic changes in fetal tissues, predisposing those tissues to diseases that manifest when offspring become adults. An example is a maternal obesity associated with gestational diabetes (GDM), where fetal exposure to a hyperglycemic, hyperinsulinemic, and/or hyperlipidemic gestational environment can provoke epigenetic changes that predispose offspring to various diseased conditions later in life. While it is now well established that offspring exposed to GDM have an increased risk of developing obesity, metabolic disorders, and/or cardiovascular disease in adult life, there are limited studies assessing the reproductive health of these offspring. This mini-review discusses the long-term effect of in utero exposure to GDM-associated adverse prenatal environment on the reproductive health of the offspring. Moreover, using evidence from various animal models and human epidemiological studies, this review offers molecular insight and understanding of how epigenetic reprogramming of genes culminates in reproductive dysfunction and the development of subfertility or infertility later in adult life.

Role of fibulin-5 insufficiency and prolapse progression on murine vaginal biomechanical function.

The vagina plays a critical role in supporting the pelvic organs and loss of support leads to pelvic organ prolapse. It is unknown what microstructural changes influence prolapse progression nor how decreased elastic fibers contributes to vaginal remodeling and smooth muscle contractility. The objective for this study was to evaluate the effect of fibulin-5 haploinsufficiency, and deficiency with progressive prolapse on the biaxial contractile and biomechanical function of the murine vagina. Vaginas from wildtype (n = 13), haploinsufficient (n = 13), and deficient mice with grade 1 (n = 9) and grade 2 or 3 (n = 9) prolapse were explanted for biaxial contractile and biomechanical testing. Multiaxial histology (n = 3/group) evaluated elastic and collagen fiber microstructure. Western blotting quantified protein expression (n = 6/group). A one-way ANOVA or Kruskal-Wallis test evaluated statistical significance. Pearson's or Spearman's test determined correlations with prolapse grade. Axial contractility decreased with fibulin-5 deficiency and POP (p < 0.001), negatively correlated with prolapse grade (ρ = - 0.80; p < 0.001), and positively correlated with muscularis elastin area fraction (ρ = - 0.78; p = 0.004). Circumferential (ρ = 0.71; p < 0.001) and axial (ρ = 0.69; p < 0.001) vaginal wall stresses positively correlated with prolapse grade. These findings demonstrated that fibulin-5 deficiency and prolapse progression decreased vaginal contractility and increased vaginal wall stress. Future work is needed to better understand the processes that contribute to prolapse progression in order to guide diagnostic, preventative, and treatment strategies.

Androgens regulate ovarian gene expression by balancing Ezh2-Jmjd3 mediated H3K27me3 dynamics.

Conventionally viewed as male hormone, androgens play a critical role in female fertility. Although androgen receptors (AR) are transcription factors, to date very few direct transcriptional targets of ARs have been identified in the ovary. Using mouse models, this study provides three critical insights about androgen-induced gene regulation in the ovary and its impact on female fertility. First, RNA-sequencing reveals a number of genes and biological processes that were previously not known to be directly regulated by androgens in the ovary. Second, androgens can also influence gene expression by decreasing the tri-methyl mark on lysine 27 of histone3 (H3K27me3), a gene silencing epigenetic mark. ChIP-seq analyses highlight that androgen-induced modulation of H3K27me3 mark within gene bodies, promoters or distal enhancers have a much broader impact on ovarian function than the direct genomic effects of androgens. Third, androgen-induced decrease of H3K27me3 is mediated through (a) inhibiting the expression and activity of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase that promotes tri-methylation of K27 and (b) by inducing the expression of a histone demethylase called Jumonji domain containing protein-3 (JMJD3/KDM6B), responsible for removing the H3K27me3 mark. Androgens through the PI3K/Akt pathway, in a transcription-independent fashion, increase hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. Furthermore, proof of concept studies involving in vivo knockdown of Ar in the ovary and ovarian (granulosa) cell-specific Ar knockout mouse model show that ARs regulate the expression of key ovarian genes through modulation of H3K27me3.

Androgen-induced epigenetic modulations in the ovary.

In recent years, androgens have emerged as critical regulators of female reproduction and women's health in general. While high levels of androgens in women are associated with polycystic ovary syndrome (PCOS), recent evidence suggests that a certain amount of direct androgen action through androgen receptor is also essential for normal ovarian function. Moreover, prenatal androgen exposure has been reported to cause developmental reprogramming of the fetus that manifests into adult pathologies, supporting the Developmental Origins of Health and Disease (DOHaD) hypothesis. Therefore, it has become imperative to understand the underlying mechanism of androgen actions and its downstream effects under normal and pathophysiological conditions. Over the years, there has been a lot of studies on androgen receptor function as a transcriptional regulator in the nucleus as well as androgen-induced rapid extra-nuclear signaling. Conversely, new evidence suggests that androgen actions may also be mediated through epigenetic modulation involving both the nuclear and extra-nuclear androgen signaling. This review focuses on androgen-induced epigenetic modifications in female reproduction, specifically in the ovary, and discusses emerging concepts, latest perceptions, and highlight the areas that need further investigation.

Developmental programming: prenatal testosterone-induced epigenetic modulation and its effect on gene expression in sheep ovary†.

Maternal perturbations or sub-optimal conditions during fetal development can predispose the offspring to diseases in adult life. Animal and human studies show that prenatal androgen excess may be an underlying cause of polycystic ovary syndrome (PCOS) later in life. In women, PCOS is a common fertility disorder with comorbid metabolic dysfunction. Here, using a sheep model of PCOS phenotype, we elucidate the epigenetic changes induced by prenatal (30-90 day) testosterone (T) treatment and its effect on gene expression in fetal day 90 (D90) and adult year 2 (Y2) ovaries. RNA-seq study shows 65 and 99 differentially regulated genes in prenatal T-treated fetal and adult ovaries, respectively. Interestingly, there were no differences in gene inducing histone marks H3K27ac, H3K9ac, and H3K4me3 or in gene silencing marks, H3K27me3 and H3K9me3 in the fetal D90 ovaries of control and excess T-exposed fetuses. In contrast, except for H3K4me3 and H3K27me3, all the other histone marks were upregulated in the prenatal T-treated adult Y2 ovary. Chromatin immunoprecipitation (ChIP) studies in adult Y2 ovaries established a direct relationship between the epigenetic modifications with the upregulated and downregulated genes obtained from RNA-seq. Results show increased gene inducing marks, H3K27ac and H3K9ac, on the promoter region of upregulated genes while gene silencing mark, H3K9me3, was also significantly increased on the downregulated genes. This study provides a mechanistic insight into prenatal T-induced developmental programming and its effect on ovarian gene expression that may contribute to reproductive dysfunction and development of PCOS in adult life.

Role of core circadian clock genes in hormone release and target tissue sensitivity in the reproductive axis.

Precise timing in hormone release from the hypothalamus, the pituitary and ovary is critical for fertility. Hormonal release patterns of the reproductive axis are regulated by a feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. The timing and rhythmicity of hormone release and tissue sensitivity in the HPG axis is regulated by circadian clocks located in the hypothalamus (suprachiasmatic nucleus, kisspeptin and GnRH neurons), the pituitary (gonadotrophs), the ovary (theca and granulosa cells), the testis (Leydig cells), as well as the uterus (endometrium and myometrium). The circadian clocks integrate environmental and physiological signals to produce cell endogenous rhythms generated by a transcriptional-translational feedback loop of transcription factors that are collectively called the "molecular clock". This review specifically focuses on the contribution of molecular clock transcription factors in regulating hormone release patterns in the reproductive axis, with an emphasis on the female reproductive system. Specifically, we discuss the contributions of circadian rhythms in distinct neuronal populations of the female hypothalamus, the molecular clock in the pituitary and its overall impact on female and male fertility.

Gestational Diabetes Epigenetically Reprograms the Cart Promoter in Fetal Ovary, Causing Subfertility in Adult Life.

Intrauterine exposure to various adverse conditions during fetal development can lead to epigenetic changes in fetal tissues, predisposing those tissues to disease conditions later in life. An example is gestational diabetes (GD), where the offspring has a higher risk of developing obesity, metabolic disorders, or cardiovascular disease in adult life. In this study, using two well-established GD (streptozotocin- and high-fat and high-sugar-induced) mouse models, we report that female offspring from GD dams are predisposed toward fertility problems later in life. This predisposition to fertility problems is due to altered ovarian expression of a peptide called cocaine- and amphetamine-regulated transcript (CART), which is known to negatively affect folliculogenesis and is induced by elevated leptin levels. Results show that the underlying cause of this altered expression is due to fetal epigenetic modifications involving glucose- and insulin-induced miRNA, miR-101, and the phosphatidylinositol 3-kinase/Akt pathway. These signaling events regulate Ezh2, a histone methyltransferase that promotes H3K27me3, a gene-repressive mark, and CBP/p300, a histone acetyltransferase that promotes H3K27ac, a transcription activation mark, in the fetal ovary. Moreover, the CART promoter has depleted 5-methylcytosine (5mC) and enriched 5-hydroxymethylcytosine (5hmC) levels. The depletion of H3K27me3 and 5mC repressive marks and subsequent increase in H3K27ac and 5hmC gene-activating marks convert the Cartpt promoter to a "superpromoter." This makes the Cartpt promoter more sensitive to leptin levels that predispose the GD offspring to fertility problems. Therefore, this study provides a mechanistic insight about fetal epigenome reprogramming that manifests to ovarian dysfunction and subfertility later in adult life.

Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells.

Anti-Müllerian hormone (AMH) produced by ovarian granulosa cells (GCs) plays a crucial role in ovarian function. It is used as a diagnostic and/or prognostic marker of fertility as well as for pathophysiological conditions in women. In this study, we investigated the underlying mechanism for regulation of AMH expression in GCs using primary mouse GCs and a human GC tumor-derived KGN cell line. We find that growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) together (GDF9 + BMP15), but not when tested separately, significantly induce AMH expression in vitro and in vivo (serum AMH). Our results show that GDF9 + BMP15 through the PI3K/Akt and Smad2/3 pathways synergistically recruit the coactivator p300 on the AMH promoter region that promotes acetylation of histone 3 lysine 27 (H3K27ac), facilitating AMH/Amh expression. Intriguingly, we also find that FSH inhibits GDF9 + BMP15-induced increase of AMH/Amh expression. This inhibition occurs through FSH-induced protein kinase A/SF1-mediated expression of gonadotropin inducible ovarian transcription factor 1, a transcriptional repressor, that recruits histone deacetylase 2 to deacetylate H3K27ac, resulting in the suppression of AMH/Amh expression. Furthermore, we report that ovarian Amh mRNA levels are significantly higher in Fshß-null mice (Fshß-/-) compared with those in wild-type (WT) mice. In addition, ovarian Amh mRNA levels are restored in Fshß-null mice expressing a human WT FSHß transgene (FSHß-/-hFSHßWT). Our study provides a mechanistic insight into the regulation of AMH expression that has many implications in female reproduction/fertility.

Androgens Regulate Ovarian Gene Expression Through Modulation of Ezh2 Expression and Activity.

A substantial amount of evidence suggests that androgen signaling through classical androgen receptors is critical for both normal and pathologic ovarian physiology. Specifically, we and others have shown that, in mouse granulosa cells, androgen actions through both extranuclear and nuclear androgen receptor signaling are critical for normal follicle development and ovulation. Here, we show that androgens through the PI3K/Akt pathway rapidly (within minutes) phosphorylate and inhibit activity of the Polycomb group protein enhancer of zeste homolog 2 (Ezh2). Over the course of 24 to 48 hours, androgens then induce expression of the microRNA miR-101, which targets Ezh2 messenger RNA (mRNA), leading to a nearly complete loss of Ezh2 protein expression. This long-term androgen-induced loss of Ezh2 actions ultimately results in sustained reduction of the H3K27me3-repressive mark in the promoter region of the Runt-related transcription factor-1 (Runx1) gene, a luteinizing hormone (LH)-induced transcription factor essential for ovulation, leading to increased Runx1 mRNA expression. Accordingly, blocking androgen-induced inhibition of Ezh2 in vivo adversely affects LH-induced Runx1 mRNA expression and subsequent ovulation. Importantly, although estrogen treatment of granulosa cells similarly causes rapid activation of the PI3K/Akt pathway and short-term phosphorylation of Ezh2, it does not induce miR-101 expression and thereby does not reduce overall Ezh2 expression, demonstrating the androgen specificity of long-term Ezh2 suppression. Thus, this study provides insight regarding how androgen-induced extranuclear kinase signaling and intranuclear transcription through Ezh2 modifications may influence the expression pattern of genes, ultimately affecting various downstream physiological processes.

Potential therapeutic applications of human anti-Müllerian hormone (AMH) analogues in reproductive medicine.

Members of the transforming growth factor-beta (TGF-beta) superfamily are key regulators of various physiological processes. Anti-Müllerian hormone (AMH) which is also commonly known as Müllerian-inhibiting substance (MIS) is a member of the TGF-beta superfamily and an important regulator of reproductive organ differentiation and ovarian follicular development. While AMH has been used for diagnostic purposes as a biomarker for over 15 years, new potential therapeutic applications of recombinant human AMH analogues are now emerging as pharmacologic agents in reproductive medicine. Therapeutic uses of AMH in gonadal tissue may provide a unique opportunity to address a broad range of reproductive themes, like contraception, ovulation induction, onset of menopause, and fertility preservation, as well as specific disease conditions, such as polycystic ovarian syndrome (PCOS) and cancers of the reproductive tract. This review explores the most promising therapeutic applications for a novel class of drugs known as AMH analogues with agonist and antagonist functions.

Infiltrating Myeloid Cells Exert Protumorigenic Actions via Neutrophil Elastase.

Tissue infiltration and elevated peripheral circulation of granulocytic myeloid-derived cells is associated with poor outcomes in prostate cancer and other malignancies. Although myeloid-derived cells have the ability to suppress T-cell function, little is known about the direct impact of these innate cells on prostate tumor growth. Here, it is reported that granulocytic myeloid-derived suppressor cells (MDSC) are the predominant tumor-infiltrating cells in prostate cancer xenografts established in athymic nude mice. MDSCs significantly increased in number in the peripheral circulation as a function of xenograft growth and were successfully depleted in vivo by Gr-1 antibody treatment. Importantly, MDSC depletion significantly decreased xenograft growth. We hypothesized that granulocytic MDSCs might exert their protumorigenic actions in part through neutrophil elastase (ELANE), a serine protease released upon granulocyte activation. Indeed, it was determined that NE is expressed by infiltrating immune cells and is enzymatically active in prostate cancer xenografts and in prostate tumors of prostate-specific Pten-null mice. Importantly, treatment with sivelestat, a small-molecule inhibitor specific for NE, significantly decreased xenograft growth, recapitulating the phenotype of Gr-1 MDSC depletion. Mechanistically, NE activated MAPK signaling and induced MAPK-dependent transcription of the proliferative gene cFOS in prostate cancer cells. Functionally, NE stimulated proliferation, migration, and invasion of prostate cancer cells in vitro IHC on human prostate cancer clinical biopsies revealed coexpression of NE and infiltrating CD33+ MDSCs.Implications: This report suggests that MDSCs and NE are physiologically important mediators of prostate cancer progression and may serve as potential biomarkers and therapeutic targets. Mol Cancer Res; 15(9); 1138-52. ©2017 AACR.

The Circadian Timing System and Environmental Circadian Disruption: From Follicles to Fertility.

The internal or circadian timing system is deeply integrated in female reproductive physiology. Considerable details of rheostatic timing function in the neuroendocrine control of pituitary hormone secretion, adenohypophyseal hormone gene expression and secretion, gonadal steroid hormone biosynthesis and secretion, ovulation, implantation, and parturition have been reported. The molecular clock, an autonomous feedback loop oscillator of interacting transcriptional regulators, dictates the timing and amplitude of gene expression in each tissue of the female hypothalamic-pituitary-gonadal (HPG) axis. Although multiple targets of the molecular clock have been identified, many associated with critical physiological functions in the HPG axis, the full extent of clock-driven gene expression and physiology in this critical system remains unknown. Environmental circadian disruption (ECD), the disturbance of temporal relationships within and between internal clocks (brain and periphery), and external timing cues (eg, light, nutrients, social cues) due to rotating/night shift work or transmeridian travel have been linked to reproductive dysfunction and subfertility. Moreover, ECD resulting from exposure to endocrine disrupting chemicals, environmental toxins, and/or irregular hormone levels during sexual development can also reduce fertility. Thus, perturbations that disturb clock function at the molecular, cellular or systemic level correlate with significant declines in female reproductive function. Here we briefly review the evidence for molecular clock function in each tissue of the female HPG axis (GnRH neuron, pituitary, uterus, oviduct, and ovary), describe the human epidemiological and animal data supporting the negative effects of ECD on fertility, and explore the potential for novel chronotherapeutics in women's health and fertility.

Definition by FSH, AMH and embryo numbers of good-, intermediate- and poor-prognosis patients suggests previously unknown IVF outcome-determining factor associated with AMH.

BACKGROUND: Though outcome models have been proposed previously, it is unknown whether cutoffs in clinical pregnancy and live birth rates at all ages are able to classify in vitro fertilization (IVF) patients into good-, intermediate- and poor prognosis. METHODS: We here in 3 infertile patient cohorts, involving 1247, 1514 and 632 women, built logistic regression models based on 3 functional ovarian reserve (FOR) parameters, including (1) number of good quality embryos, (2) follicle stimulating hormone (FSH, mIU/mL) and (3) anti-Müllerian hormone (AMH, ng/mL), determining whether clinical pregnancy and live birth rates can discriminate between good, intermediate and poor prognosis patients. RESULTS: All models, indeed, allowed at all ages for separation by prognosis, though cut offs changed with age. In the embryo model, increasing embryo production resulted in linear improvement of IVF outcomes despite transfer of similar embryo numbers; in the FSH model outcomes and FSH levels related inversely, while the association of AMH followed a bell-shaped polynomial pattern, demonstrating "best" outcomes at mid-ranges. All 3 models demonstrated increasingly poor outcomes with advancing ages, though "best" AMH even above age 43 was still associated with unexpectedly good pregnancy and delivery outcomes. Excessively high AMH, in contrast, was at all ages associated with spiking miscarriage rates. CONCLUSIONS: At varying peripheral serum concentrations, AMH, thus, demonstrates hithero unknown and contradictory effects on IVF outcomes, deserving at different concentrations investigation as a potential therapeutic agent, with pregnancy-supporting and pregnancy-interrupting properties.

Intra-cellular mechanism of Anti-Müllerian hormone (AMH) in regulation of follicular development.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-ß superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent.