The effects of prenatal azithromycin exposure on offspring ovarian development at different stages, doses, and courses.

Azithromycin, a commonly used macrolide antibiotic for treating chlamydial infections during pregnancy, has sparked investigations into its potential effects on offspring development. Despite these inquiries, there remains uncertainty about the specific impact of prenatal azithromycin exposure (PAzE) on offspring ovarian development and the precise "effect window". Pregnant mice, following clinical guidelines for azithromycin dosing, were orally administered azithromycin at different gestational stages [(gestational day, GD) 10-12 or GD 15-17], doses (50, 100, or 200 mg/kg·d), and courses (single or multiple). On GD 18, we collected offspring blood and ovaries to examine changes in fetal serum estradiol (E2) levels, fetal ovarian morphology, pre-granulosa cell function, and oocyte development. Multiple courses of PAzE resulted in abnormal fetal ovarian morphological development, disorganized germ cell nests, enhanced ovarian cell proliferation, and reduced apoptosis. Simultaneously, multiple courses of PAzE significantly increased fetal serum E2 levels, elevated ovarian steroidogenic function (indicated by Star, 3ß-hsd, and Cyp19 expression), disrupted oocyte development (indicated by Figlα and Nobox expression), and led to alterations in the MAPK signal pathway in fetal ovaries, particularly in the high-dose treatment group. In contrast, a single course of PAzE reduced fetal ovarian cell proliferation, decreased steroidogenic function, and inhibited oocyte development, particularly through the downregulation of Mek2 expression in the MAPK signal pathway. These findings suggest that PAzE can influence various aspects of fetal mouse ovarian cell development. Multiple courses enhance pre-granulosa cell estrogen synthesis function and advance germ cell development, while a single terminal gestation dose inhibits germ cell development. These differential effects may be associated with changes in the MAPK signal pathway.

Effects of atosiban on clinical outcome in frozen-thawed embryo transfer: a propensity score matching study.

PURPOSE: To evaluate the effects of atosiban on clinical outcomes in patients undergoing frozen-thawed embryo transfer. METHODS: The clinical data of 1093 infertile patients who underwent frozen-thawed embryo transfer in our center from January 2019 to December 2020 were retrospectively analyzed (control, 418; atosiban, 675). Propensity score matching (PSM) analysis identified 400 matched pairs of patients. The implantation rate, clinical pregnancy rate, live birth rate, biochemical pregnancy rate, abortion rate, multiple pregnancy rate, and ectopic pregnancy rate between the two groups were compared. RESULTS: Before PSM, patients differed by infertility factors, number of transferred embryos, and endometrial preparation protocol (P < 0.05). After PSM, characteristics were similar in corresponding patients of the atosiban and control groups. After propensity score matching, we found that there was no significant difference in the implantation rate, clinical pregnancy rate, live birth rate, biochemical pregnancy rate, abortion rate, multiple pregnancy rate, and ectopic pregnancy rate in atosiban and control group (P > 0.05). CONCLUSION: Atosiban did not improve the clinical outcomes of infertile patients with frozen-thawed embryo transfer.

Differential expression of placental 11ß-HSD2 induced by high maternal glucocorticoid exposure mediates sex differences in placental and fetal development.

A variety of adverse environmental factors during pregnancy cause maternal chronic stress. Caffeine is a common stressor, and its consumption during pregnancy is widespread. Our previous study showed that prenatal caffeine exposure (PCE) increased maternal blood glucocorticoid levels and caused abnormal development of offspring. However, the placental mechanism for fetal development inhibition caused by PCE-induced high maternal glucocorticoid has not been reported. This study investigated the effects of PCE-induced high maternal glucocorticoid level on placental and fetal development by regulating placental 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) expression and its underlying mechanism. First, human placenta and umbilical cord blood samples were collected from women without prenatal use of synthetic glucocorticoids. We found that placental 11ß-HSD2 expression was significantly correlated with umbilical cord blood cortisol level and birth weight in male newborns but not in females. Furthermore, we established a rat model of high maternal glucocorticoids induced by PCE (caffeine, 60 mg/kg·d, ig), and found that the expression of 11ß-HSD2 in male PCE placenta was decreased and negatively correlated with the maternal/fetal/placental corticosterone levels. Meanwhile, we found abnormal placental structure and nutrient transporter expression. In vitro, BeWo cells were used and confirm that 11ß-HSD2 mediated inhibition of placental nutrient transporter expression induced by high levels of glucocorticoid. Finally, combined with the animal and cell experiments, we further confirmed that high maternal glucocorticoid could activate the GR-C/EBPα-Egr1 signaling pathway, leading to decreased expression of 11ß-HSD2 in males. However, there was no significant inhibition of placental 11ß-HSD2 expression, placental and fetal development in females. In summary, we confirmed that high maternal glucocorticoids could regulate placental 11ß-HSD2 expression in a sex-specific manner, leading to differences in placental and fetal development. This study provides the theoretical and experimental basis for analyzing the inhibition of fetoplacental development and its sex difference caused by maternal stress.

Gestational exposure to PCB-118 impairs placental angiogenesis and fetal growth.

Maternal exposure to polychlorinated biphenyls (PCBs) results in adverse effects on fetal development. However, the underlying mechanism has not been sufficiently explored in respect to particular PCBs. Placental angiogenesis plays a crucial role in feto-maternal substances transportation and fetal development. The present study was conducted to investigate the effects of prenatal PCB118 exposure on placental angiogenesis and fetal growth. The pregnant dam received PCB118 at environmentally relevant doses (0, 20, or 100 µg/kg/day) intragastrically from gestational day (GD) 7.5-18.5 to establish an in vivo model. Compared with the control group, the fetal body and placental weights of the PCB118 (100 µg/kg/day) group were significantly decreased and the intrauterine growth retardation (IUGR) rates were increased both in the female and male fetus. Furthermore, we found that placental histology was significantly impaired and the number of blood vessels was decreased in the PCB118 group. Additionally, gestational exposure to PCB118 caused anomalous mRNA expression of the genes in the placenta regarding angiogenesis. These findings indicate that PCB118 may contribute to the occurrence of IUGR by provoking placental angiogenesis dysfunction. This study clarified the adverse effects and potential mechanism of prenatal PCBs exposure on fetal growth, providing a new theoretical and experimental basis for future treatment and prevention.

Inducible factors and interaction of pulmonary fibrosis induced by prenatal dexamethasone exposure in offspring rats.

This study aimed to investigate the correlation between prenatal dexamethasone exposure (PDE) and susceptibility to pulmonary fibrosis in offspring. Healthy female Wistar rats were given dexamethasone (0.2 mg/kg.d) or an equal volume of normal saline subcutaneously from 9 to 20 days after conception. Some of their female offspring underwent ovariectomy (OV) at 22 weeks after birth. All animals were euthanized at 28 weeks after birth. The morphological changes related to pulmonary fibrosis and extracellular matrix-related gene expression were detected, and Two-way ANOVA analyzed the interaction between PDE and OV. The results showed that adult offspring rats in FD group (female rats with PDE treatment) had early pulmonary fibrosis changes, such as pulmonary interstitial thickening, and increased expression of type IV collagen (COL4), α -smooth muscle actin (α-SMA) and fibronectin (FN) in lung tissues compared with those in FC group (female rats with saline treatment). In addition, adult offspring rats in FDO group (female rats with PDE and OV treatment) showed signs of pulmonary fibrosis, including apparent extracellular matrix deposition, increased lung injury scores (P＜0.01, P＜0.05), and extracellular matrix related gene expression (P＜0.01, P＜0.05), compared with rats in FDS (female rats with PDE treatment alone) or rats in FCO group (female rats with OV treatment alone). Moreover, PDE and OV had an interactive effect on the development of pulmonary fibrosis in female adult offspring. This study first reported the correlation between PDE and susceptibility to pulmonary fibrosis in female offspring rats, as well as the synergistic effect of PDE and OV in this pathological event, which provided a basis for further understanding of the pathogenesis of fetal originated pulmonary fibrosis.

P-gp expression inhibition mediates placental glucocorticoid barrier opening and fetal weight loss.

BACKGROUND: Prenatal adverse environments can cause fetal intrauterine growth retardation (IUGR) and higher susceptibility to multiple diseases after birth, related to multi-organ development programming changes mediated by intrauterine overexposure to maternal glucocorticoids. As a glucocorticoid barrier, P-glycoprotein (P-gp) is highly expressed in placental syncytiotrophoblasts; however, the effect of P-gp on the occurrence of IUGR remains unclear. METHODS: Human placenta and fetal cord blood samples of IUGR fetuses were collected, and the related indexes were detected. Pregnant Wistar rats were administered with 30 mg/kg·d (low dose) and 120 mg/kg·d (high dose) caffeine from gestational day (GD) 9 to 20 to construct the rat IUGR model. Pregnant mice were administered with caffeine (120 mg/kg·d) separately or combined with sodium ferulate (50 mg/kg·d) from gestational day GD 9 to 18 to confirm the intervention target on fetal weight loss caused by prenatal caffeine exposure (PCE). The fetal serum/placental corticosterone level, placental P-gp expression, and related indicator changes were analyzed. In vitro, primary human trophoblasts and BeWo cells were used to confirm the effect of caffeine on P-gp and its mechanism. RESULTS: The placental P-gp expression was significantly reduced, but the umbilical cord blood cortisol level was increased in clinical samples of the IUGR neonates, which were positively and negatively correlated with the neonatal birth weight, respectively. Meanwhile, in the PCE-induced IUGR rat model, the placental P-gp expression of IUGR rats was decreased while the corticosterone levels of the placentas/fetal blood were increased, which were positively and negatively correlated with the decreased placental/fetal weights, respectively. Combined with the PCE-induced IUGR rat model, in vitro caffeine-treated placental trophoblasts, we confirmed that caffeine decreased the histone acetylation and expression of P-gp via RYR/JNK/YB-1/P300 pathway, which inhibited placental and fetal development. We further demonstrated that P-gp inducer sodium ferulate could reverse the inhibitory effect of caffeine on the fetal body/placental weight. Finally, clinical specimens and other animal models of IUGR also confirmed that the JNK/YB-1 pathway is a co-regulatory mechanism of P-gp expression inhibition, among which the expression of YB-1 is the most stable. Therefore, we proposed that YB-1 could be used as the potential early warning target for the opening of the placental glucocorticoid barrier, the occurrence of IUGR, and the susceptibility of a variety of diseases. CONCLUSIONS: This study, for the first time, clarified the critical role and epigenetic regulation mechanism of P-gp in mediating the opening mechanism of the placental glucocorticoid barrier, providing a novel idea for exploring the early warning, prevention, and treatment strategies of IUGR.

Sexual dimorphism in placental development and its contribution to health and diseases.

According to the Developmental Origin of Health and Disease (DOHaD), intrauterine exposure to adverse environments can affect fetus and birth outcomes and lead to long-term disease susceptibility. Evidence has shown that neonatal outcomes and the timing and severity of adult diseases are sexually dimorphic. As the link between mother and fetus, the placenta is an essential regulator of fetal development programming. It is found that the physiological development trajectory of the placenta has sexual dimorphism. Furthermore, under pathological conditions, the placental function undergoes sex-specific adaptation to ensure fetal survival. Therefore, the placenta may be an important mediator of sexual dimorphism in neonatal outcomes and adult disease susceptibility. Few systematic reviews have been conducted on sexual dimorphism in placental development and its underlying mechanisms. In this review, sex chromosomes and sex hormones, as the main reasons for sexual differentiation of the placenta, will be discussed. Besides, in the etiology of fetal-originated adult diseases, overexposure to glucocorticoids is closely related to adverse neonatal outcomes and long-term disease susceptibility. Studies have found that prenatal glucocorticoid overexposure leads to sexually dimorphic expression of placental glucocorticoid receptor isoforms, resulting in different sensitivity of the placenta to glucocorticoids, and may further affect fetal development. The present review examines what is currently known about sex differences in placental development and the underlying regulatory mechanisms of this sex bias. This review highlights the importance of placental contributions to the origins of sexual dimorphism in health and diseases. It may help develop personalized diagnosis and treatment strategies for fetal development in pathological pregnancies.

Selection of reliable reference genes for analysis of gene expression in the rat placenta.

The selection of suitable reference genes (RGs), especially the identification of the proper combination of RGs is the key to obtain reliable results of gene expression for quantitative real-time polymerase chain reaction (qRT-PCR). To date, there is no relevant study dealing with the stability of RGs in rat placenta. In this study, the geNorm, NormFinder, and BestKeeper software were used to analyze the expression stability of the candidate RGs in placenta under physiological and prenatal caffeine exposure (PCE) conditions. The expression of Tbp, Gapdh and Ywhaz in female and Polr2a, Gapdh and Ywhaz in male placenta were highly stable under physiological conditions, and there was no obvious gender difference. We further found that two RGs were sufficient for reliable normalization in female and male placenta and the combination of Ywhaz and Gapdh was the most suitable compound RGs under physiological conditions. Under PCE conditions, Ywhaz, Gapdh and Polr2a were the most stable genes in both female and male placenta. Among them, Ywhaz and Gapdh were chosen as the best paring. Finally, selected RGs were employed for normalization of the expression of a clear target gene and the results of standardization supported our choice. In conclusion, our study confirmed that Ywhaz/Gapdh combination was the most suitable RGs in rat placenta under physiological and PCE pathological conditions and provided a theoretical and experimental basis for physiological and pathological research of the rat placenta.

miRNA320a-3p/RUNX2 signal programming mediates the transgenerational inheritance of inhibited ovarian estrogen synthesis in female offspring rats induced by prenatal dexamethasone exposure.

Our previous studies found that prenatal dexamethasone exposure could cause abnormal follicular development in fetal rats. This study intends to observe the transgenerational inheritance effects of ovarian estrogen inhibition in offspring exposed to dexamethasone (0.2 mg/kg • d) from gestational day 9 (GD9) to GD20 in Wistar rats, and explore the intrauterine programming mechanisms. Prenatal dexamethasone exposure reduced the expression of ovarian cytochrome P450 aromatase (P450arom), the level of serum estradiol (E2) and the number of primordial follicles, while increased the number of atresia follicles before and after birth in F1 offspring rats. At the same time, the expression of miRNA320a-3p in F1 ovaries was down-regulated, and RUNX2 expression increased significantly. These changes were continued to F2 and F3 generations, accompanied by consistently down-regulated miRNA320a-3p expression in oocyte of F1 and F2 adult offspring. In vitro, fetal rat ovaries and KGN human ovarian granulosa cells were treated with dexamethasone. It showed that dexamethasone decreased miRNA320a-3p and P450arom expression, as well as E2 synthesis, and increased RUNX2 expression. All these effects could be reversed by the GR antagonist RU486. The overexpression of miRNA320a-3p in vitro could also reverse the effects of dexamethasone on RUNX2, P450arom, and E2 levels. The dual-luciferase reporter gene experiment further confirmed the direct targeted regulation of miRNA320a-3p on RUNX2. These results indicate that prenatal dexamethasone exposure induces ovarian E2 synthesis inhibition mediated by the GR/miRNA320a-3p/RUNX2/P450arom cascade signal in fetal rat ovary, which has transgenerational inheritance effects and may related to the inhibited miRNA320a-3p expression in oocyte.

The effect of ICSI in infertility couples with non-male factor: a systematic review and meta-analysis.

PURPOSE: We performed a systematic review and meta-analysis of available literature to investigate the efficacy of the intracytoplasmic sperm injection (ICSI) in couples with non-male factor with respect to the clinical outcomes. METHODS: The literature search was based on EMBASE, PubMed, and the Cochrane Library. All studies published after 1992 until February 2020 and written in English addressing patients in the presence of normal semen parameters subjected to ICSI and in vitro fertilization (IVF) were eligible. Reference lists of retrieved articles were hand-searched for additional studies. The primary outcomes were fertilization rate, clinical pregnancy rate, and implantation rate; the secondary outcomes were good-quality embryo rate, miscarriage rate, and live birth rate. RESULTS: Four RCTs and twenty-two cohort studies fulfilling the inclusion criteria were included. Collectively, a meta-analysis of the outcomes in RCTs showed that compared to IVF, ICSI has no obvious advantage in fertilization rate (RR = 1.16, 95% CI: 0.83-1.62), clinical pregnancy rate (RR = 1.04, 95% CI: 0.66-1.64), implantation rate (RR = 1.12, 95% CI: 0.67-1.86), and live birth rate (RR = 1.17, 95% CI: 0.43-3.15). Pooled results of cohort studies demonstrated a statistically significant higher fertilization rate (RR = 1.16, 95% CI: 1.03-1.31) and miscarriage rate (RR = 1.04, 95% CI: 1.01-1.06) in the ICSI group; furthermore, higher clinical pregnancy rate (RR = 0.85, 95% CI: 0.77-0.94), implantation rate (RR = 0.78, 95% CI: 0.65-0.95), and live birth rate (RR = 0.86, 95% CI: 0.79-0.94) was founded in the IVF group; no statistically significant difference was observed in good-quality embryo rate (RR = 0.98, 95% CI: 0.93-1.04). CONCLUSION: ICSI has no obvious advantage in patients with normal semen parameters. Enough information is still not available to prove the efficacy of ICSI in couples with non-male factor infertility comparing to IVF.

The effects, underlying mechanism and interactions of dexamethasone exposure during pregnancy on maternal bile acid metabolism.

As important members in steroids related signal pathways, bile acids are very important in regulating substance metabolism and immune homeostasis. However, bile acids are highly cytotoxic, and the excessive accumulation can induce several abnormalities such as cholestatic liver injury. It is known that the bile acid metabolism alters during pregnancy and mostly will not result in pathologies. However, the effect of dexamethasone exposure during pregnancy on bile acid metabolism is still unknown. In this study, pregnant Wistar rats were subcutaneously administered dexamethasone (0.2 mg/kg.d) or saline from gestation day 9-21, while virgin rats were given the same treatment for 13 days. We found that, physiological pregnancy or dexamethasone exposure during non-pregnancy did not affect maternal serum TBA level and liver function. Nevertheless, dexamethasone exposure during pregnancy increased serum TBA level and accompanied with liver injury. Furthermore, we discovered that the conservation of bile acid homeostasis under pregnancy or dexamethasone exposure was maintained through compensatory pathways. However, dexamethasone exposure during pregnancy tipped the balance of liver bile acid homeostasis by increasing classical synthesis and decreasing efflux and uptake. In addition, dexamethasone exposure during pregnancy also increased serum estrogen level and nuclear receptors mRNA expression levels. Finally, two-way ANOVA analysis showed that dexamethasone exposure during pregnancy could induce or facilitate maternal cholestasis and liver injury by up-regulating ERα and CYP7A1 expression. This study confirmed that dexamethasone exposure during pregnancy was related to maternal intrahepatic cholestasis of pregnancy and should be carefully monitored in clinical settings.

Prevalence and type-specific distribution of human papillomavirus infection among women in mid-western rural, Nepal- A population-based study.

BACKGROUND: Cervical cancer is the most common cancer among women in Nepal. The prevalence of human papillomavirus (HPV) 16 and or HPV 18 among women with cervical pre-cancer and cancer is higher than the incidence of HPV in the world population. The population-based epidemiological data of HPV in the general population in most parts of the country remains unknown. The objective of this study was to assess the prevalence and type distribution of HPV infection and association of abnormal cytology with high risk HPV infection among women in mid-western rural, Nepal. METHODS: A population-based cross sectional study was conducted in Jumla, one of the most remote districts in Nepal. A total of 1050 cervical samples were collected from married and non- pregnant women aged 20-65 years during mobile Cervical Cancer Screening Clinics conducted from May 2016 to January 2017. The presence of HPV DNA was firstly confirmed by HPV consensus PCR using PGMY09/PGMY11 designed primers, then HPV positive samples were further genotyped by the membrane hybridization method to detect the 21 high-risk HPV (HR-HPV) and low-risk HPV types. The prevalence of HR-HPV among women with normal and abnormal cytology was calculated. Data were analyzed using SPSS software for Windows. P < 0.05 was considered statistically significant. RESULTS: A total of 998 women were eligible for this study with the mean age 32.6 ± 8.6 years, and the mean marital age was 16.7 ± 3.8 years. The overall prevalence of HPV infections was 19.7%. HR-HPV and low-risk HPV were 11.7 and 8.7% respectively. The six most common HR-HPV types were HPV16, 39, 58, 33, 51 and 18. HR-HPV infection among the women with abnormal and normal cytology was of 27.3 and 10.8% respectively. CONCLUSIONS: There was a higher prevalence of HR-HPV infection among women living in Jumla than other parts of Nepal. This study provides preliminary information on overall HPV and type-specific HR-HPV prevalence, HR-HPV 16, 39, 58, 33, 51, and 18 are the most prevalent genotypes in this region. The data contribute to the epidemiological knowledge about HPV and type-specific HR-HPV genotypes prevalence in mid-Western Nepal.

Inactivation of Vibrio parahaemolyticus by Aqueous Ozone.

Vibrio parahaemolyticus contamination causes serious foodborne illness and has become a global health problem. As a disinfectant, aqueous ozone can effectively kill a number of bacteria, viruses, parasites, and other microorganisms. In this study, three factors, namely, the aqueous ozone concentration, the exposure time, and the bacterial density, were analyzed by response surface methodology, and the aqueous ozone concentration was the most influential factor in the sterilization ratio. Under low aqueous ozone concentrations (less than 0.125 mg/l), the bacterial cell membranes remained intact, and the ozone was detoxified by intracellular antioxidant enzymes (e.g., superoxide dismutase and catalase). Under high aqueous ozone concentrations (more than 1 mg/l), cell membranes were damaged by the degree of peripheral electronegativity at the cell surface and the concentration of lactate dehydrogenase released into the extracellular space, and the ultrastructures of the cells were confirmed by transmission electron microscopy. Aqueous ozone penetrated the cells through leaking membranes, inactivated the enzymes, inhibited almost all the genes, and degraded the genetic materials of gDNA and total RNA, which eventually led to cell death.

Burden of cervical neoplasia in mid-western rural Nepal: a population-based study.

OBJECTIVE: To assess the burden of cervical neoplasia in mid-western rural, Nepal using cytology, visual inspection with acetic acid (VIA) and visual inspection with Lugol's iodine (VILI). METHOD: A cross-sectional, population-based study was conducted. Total of 2,279 married, non-pregnant women aged 20-65 years participated in a screening clinic from May 2016 to January 2017. All eligible women completed self-report of socio-demographic and reproductive health data followed by screening tests. Biopsies were obtained from areas on the cervix assessed by VIA and or VILI to be abnormal. Final disease was confirmed by biopsy report. RESULTS: A total of 96.09% (n=2,190) women were eligible for this study with mean age 32.78±9.33 years. The overall rate of positive cytology, VIA, and VILI were 3.69%, 12.45%, and 16.89%, respectively. Sixty-two cases were biopsy proven cervical neoplasia. Altogether 78 (3.69%) cases were cytologically abnormal: 25 (1.18%) were atypical squamous cells of undetermined significance, 33 (1.56%) were low-grade squamous intraepithelial lesion, 11 (0.52%) were high-grade squamous intraepithelial lesion, and 9 (0.42%) were squamous cell carcinoma. Illiterate women appeared to be at higher risk for cervical neoplasia (p<0.001). Similarly, age ≥46 years (p<0.013), participant's multiple marriages or sexual partners (p<0.005), and positive human immunodeficiency virus status (p<0.001) were significantly associated with abnormal cytology. CONCLUSION: Based on cytology report, there is 3.69% prevalence of cervical neoplasia among women in a rural region of mid-western, Nepal. A "screen and treat" approach would be more attractive in low resource settings.