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High‐mobility group box 1 (HMGB1) is critical for inflammatory homeostasis and successful pregnancy, and there is a strong association among elevated levels of HMGB1, polycystic ovary syndrome (PCOS), chronic inflammation and pregnancy loss. However, the mechanisms responsible for PCOS‐driven regulation of uterine HMGB1 and its candidate receptors [toll‐like receptor (TLR) 2 and 4] and inflammatory responses during pregnancy remain unclear. In this study, we found a gestational stage‐dependent decrease in uterine HMGB1 and TLR4 protein abundance in rats during normal pregnancy. We demonstrated that increased expression of HMGB1, TLR2 and TLR4 proteins was associated with activation of inflammation‐related signalling pathways in the gravid uterus exposed to 5α‐dihydrotestosterone and insulin, mimicking the clinical features (hyperandrogenism and insulin resistance) of PCOS and this elevation was completely inhibited by treatment with the androgen receptor (AR) antagonist flutamide. Interestingly, acute exposure to lipopolysaccharide suppressed HMGB1, TLR4 and inflammation‐related protein abundance but did not affect androgen levels or AR expression in the gravid uterus with viable fetuses. Furthermore, immunohistochemical analysis revealed that, in addition to being localized predominately in the nuclear compartment, HMGB1 immunoreactivity was also detected in the cytoplasm in the PCOS‐like rat uterus, PCOS endometrium and pregnant rat uterus with haemorrhagic and resorbed fetuses, possibly via activation of nuclear factor κB signalling. These results suggest that both AR‐dependent and AR‐independent mechanisms contribute to the modulation of HMGB1/TLR2/TLR4‐mediated uterine inflammation. We propose that the elevation of HMGB1 and its receptors and disruption of the pro‐/anti‐inflammatory balance in the gravid uterus may participate in the pathophysiology of PCOS‐associated pregnancy loss. [ABSTRACT FROM AUTHOR]

The mechanisms through which the androgen-dependent activation of the androgen receptor (AR) regulates gravid uterine ferroptosis remain unknown. We show that while co-exposure of pregnant rats to the androgen 5adihydrotestosterone (DHT) and insulin (INS) triggered uterine ferroptotic signaling cascades, additional treatment with the anti-androgen flutamide increased expression of the key ferroptosis-inhibitory proteins SLC7A11, GSH, and GPX4; reduced iron content; normalized levels of ferroptosisassociated Tfrc, Fpn1, and Ho1 mRNAs; reduced levels of proteins modified by 4-HNE (a marker of ferroptosis); and restored protein levels of NRF2, a key transcription factor regulating antioxidant defense signaling, in the gravid uterus. Furthermore, exposure to DHT alone increased uterine ferroptosis, and NRF2 abundance was negatively correlated with AR status. Coimmunoprecipitation and Western blot assays revealed that the AR physically interacted with endogenous NRF2, and this interaction was increased by DHT exposure in vivo. Our results suggest that AR overactivation and NRF2 suppression cooperate in the regulation of NRF2-targets in uterine ferroptosis. [ABSTRACT FROM AUTHOR]

Abstract Background Several peripheral proteins that might be useful for detecting the presence of ectopic pregnancy (EP) have been evaluated, but none have been proven entirely useful in the clinic. We investigated the presence and the possible changes in circulating molecules that distinguish between normal intrauterine pregnancy (IUP) and tubal ectopic pregnancy. Methods Non-pregnant women during the menstrual cycle, women with IUP, and women with tubal EP after informed consent. Serum levels of 17β-estradiol (E2), progesterone (P4), testosterone (T), beta-human chorionic gonadotropin (β-hCG), vascular endothelial growth factor-A (VEGF-A), placental growth factor (PIGF), and a distintegrin and metalloprotease protein 12 (ADAM12) were analyzed. Receiver operating characteristic analysis was used to assess the diagnostic discrimination of EP and gestational age-matched IUP. Results E2, P4, PIGF, and ADAM12 levels increased and β-hCG decreased throughout IUP. E2 and VEGF-A levels were significantly different between women with tubal EP and IUP. However, using a serum β-hCG cut-off of less than 1000 mIU/mL, P4 was significantly lower in women with tubal EP compared to IUP. Although E2 was inversely correlated with VEGF-A in women in the early stages of IUP, E2 was not correlated with VEGF-A in women with EP prior to tubal surgery. There were no significant differences in either PIGF or ADAM12 alone between women with tubal EP or IUP. Although no significant correlations were seen between E2 and PIGF or P4 and ADAM12 in women in the early stages of IUP, E2 was positively correlated with PIGF and P4 was positively correlated with ADAM12 in women with EP prior to tubal surgery. Our studies defined associations but not causality. Conclusions Individual measurements of serum E2 or VEGF-A levels are strongly related to early pregnancy outcomes for women with IUP and EP, and pregnancy-associated E2 and VEGF-A levels provide diagnostic accuracy for the presence of tubal EP. This study demonstrates that correlation analysis of E2/VEGF-A and E2/PIGF serum levels may be able to distinguish a tubal EP from a normal IUP.

Context: Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium.Objective: This work aimed to increase the limited understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients.Methods: Endometrial biopsy samples were collected from non-PCOS (n = 17) and PCOS (n = 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and underwent pharmacological manipulation in vitro. The expression and localization of toll-like receptor 2 (TLR2)/4, key elements of innate immune signal transduction and nuclear factor κB (NFκB) signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues.Results: We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of interferon regulatory factor-7 (IRF-7) and NFκB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that 5-dihydrotestosterone (DHT) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased interferon α (IFNα) and IFNɣ abundance. The effects of DHT on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an antiandrogen. Although 17β-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed interleukin-1 receptor-associated kinase 4 (IRAK4), p-IRF-7, IRF-7, IκB kinase α (IKKα), p-NFκB p65, IFNɣ, and tumor necrosis factor α protein expression.Conclusion: Cytokine synthesis and increased endometrial inflammation in PCOS patients are coupled to androgen-induced TLR4/IRF-7/NFκB signaling, which is inhibited by metformin treatment. [ABSTRACT FROM AUTHOR]

Abstract Background The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations. Methods A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone. Results The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls. Conclusions The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

Abstract Background The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma. Methods Western blot and immunohistochemistry with specific antibodies were used to characterize the expression and cellular localization of the mPRs in mouse and human tissues. Taqman (Quantitative Polymerase Chain Reaction) assays were used to quantify mRNA levels in the fallopian tubes of two different mouse models after injections with different hormones and specific antagonists. Results In the fallopian tubes of both mouse and human, the expression of mPR beta and mPR gamma proteins was exclusively found in the ciliated cells. Whereas mPR beta was found on the cilia, mPR gamma was localized at the base of the same ciliated cells, as previously reported. In gonadotropin-primed mice, both mPRs genes were down-regulated after an injection with progesterone. Treatment with estradiol rapidly down-regulated the level of mPR beta mRNA and protein in immature mice. The mPR gamma protein was down-regulated around the time of ovulation in cycling women, similar to the regulation observed in mice stimulated to ovulate via gonadotropin injections. Conclusion Our findings show the presence and hormonal regulation of two distinct mPRs associated with the cilia of the fallopian tubes in both mice and women. It is hypothesized that these receptors are involved in the control of ciliary movement and, thus, gamete transport in the fallopian tubes of mammals.

The estrogen signaling pathway via nuclear estrogen receptors (ER) α and β is considered to be the master regulator of the cellular glucose metabolism in the uterus. While in vivo animal studies have demonstrated that 17β-estradiol (E2) treatment increases the expression levels and activities of several glycolytic enzymes in the uterus, the specific ER subtype-dependent regulation of key glycolytic enzymes in the uterus has not been experimentally verified. In this study, the localization of ERα and ERβ in human and mouse endometria were evaluated using immunohistology. Given that ERα and ERβ are not functionally equivalent, ERα, ERβ and ERαβ knockout (ERα-/-, ERβ-/- and ERαβ-/-) mice were utilized to determine the expression pattern of glycolytic enzymes in the uterus. It was found that the level of ERα was higher than that of ERβ in the human and mouse endometrial epithelial and stromal cells, and both receptors were downregulated by E2 treatment in the mouse uterus. The expression of the hexokinase 1 and GAPDH was increased in ERα-/- and ERβ-/- mice compared with wild-type controls. Increased phosphofructokinase expression was observed in ERα-/- and ERαβ-/- mice, whereas increased pyruvate kinase isozyme M2 and pyruvate dehydrogenase expression was observed in ERβ-/- and ERαβ-/- mice. The findings indicated for the first time that while estrogen regulates ERα and ERβ expression in the uterus, ERα and ERβ selectively regulate uterine glycolytic enzyme expression during glycolysis. Additionally, the link between endometrial ER subtypes and glycolysis in women with polycystic ovary syndrome (PCOS) is discussed. The findings suggested that the E2-dependent ER-mediated regulation of glycolysis may be involved in the disturbance of the glucose metabolism in patients with PCOS with endometrial dysfunction. [ABSTRACT FROM AUTHOR]