Impact of DEHP on mitochondria-associated endoplasmic reticulum membranes and reproductive toxicity in ovary.

Di(2-ethylhexyl) phthalate (DEHP) is a widely recognized environmental endocrine disruptor that potentially impacts female reproductive function, although the specific mechanisms leading to such impairment remain unclear. A growing body of research has revealed that the endoplasmic reticulum and mitochondrial function significantly influence oocyte quality. The structure of mitochondria-associated endoplasmic reticulum membranes (MAMs) is crucial for facilitating the exchange of Ca2+, lipids, and metabolites. This study aimed to investigate the alterations in the composition and function of MAMs after DEHP exposure and to elucidate the underlying mechanisms of ovarian toxicity. The female mice were exposed to DEHP at doses of 5 and 500 mg/kg/day for one month. The results revealed that DEHP exposure led to reduced serum anti-Müllerian hormone levels and increased atretic follicles in mice. DEHP induced endoplasmic reticulum stress and disrupted calcium homeostasis in oocytes. Furthermore, DEHP impaired the mitochondrial function of oocytes and reduced their membrane potential, and promoting apoptosis. Similar results were observed in human granulosa cells after exposure to mono-(2-ethylhexyl) phthalate (MEHP, metabolites of DEHP) in vitro. Proteomic analysis and transmission electron microscopy revealed modifications in the functional proteins and structure of the MAMs, and the suppression of oxidative phosphorylation pathways. The findings of this investigation provide a new perspective on the mechanism underlying the reproductive toxicity of DEHP in females.

Associations between phthalate metabolites and cytokines in the follicular fluid of women undergoing in vitro fertilization.

Many studies have showed that phthalates have reproductive and embryonic toxicity, while the potential mechanisms are mostly unknown. Inflammation may play a mediating part in phthalate exposure and adverse reproductive endpoints. A cross-sectional survey was conducted to investigate the associations of phthalate metabolites with inflammatory cytokines in the follicular fluid (FF) of women undergoing in vitro fertilization (IVF). We determined the levels of eight phthalate metabolites and five cytokines in the FF of 76 women, including interleukin (IL)- 6, IL-8, IL-10, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). The associations of individual phthalate exposure with cytokines in FF samples were explored by multiple linear regression. We further evaluated the combined effects of multiple phthalate exposures on FF levels of cytokines by using Bayesian kernel machine regression (BKMR) models. We found that there was a positive relationship between mono-ethyl phthalate (MEP) and IL-6 in the FF (percent change:12.4%; 95% CI: 1.3%, 24.9%). In contrast, elevated mono-benzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP) and %MEHP levels were associated with decreased MCP-1. In the BKMR models, phthalate metabolite mixtures were positively associated with TNF-α when the mixtures were lower than 65th percentile compared with their medians. In the stratified analyses, MEHP was inversely associated with MCP-1 among women with BMI ≥ 23 kg/m2 (test for interaction <0.05). Our results suggest that certain phthalate metabolites or their mixtures may alter levels of inflammatory cytokines in the FF, and further research is necessary to elucidate the mechanisms underlying the relationship between phthalates exposure, ovarian dysfunction and adverse pregnancy outcomes.

Associations of sleep characteristics with outcomes of IVF/ICSI treatment: a prospective cohort study.

STUDY QUESTION: Are sleep characteristics associated with outcomes of IVF/ICSI treatment? SUMMARY ANSWER: Nocturnal sleep <7 h/night and disturbed sleep are related to impaired oocyte and embryo yield, while longer nocturnal sleep might reduce the chance of a successful pregnancy, and the associations between nocturnal sleep duration and IVF/ICSI outcomes are modified by maternal age and subjective sleep quality. WHAT IS KNOWN ALREADY: Disturbed sleep and circadian rhythm contribute to impaired fecundity in the general population, but the effects of sleep characteristics on IVF/ICSI outcomes are largely unknown. STUDY DESIGN, SIZE, DURATION: This study was conducted among 1276 women undergoing IVF/ICSI treatment between December 2018 and September 2019 based on the Tongji Reproductive and Environmental cohort. Owing to the limited number of multiple cycles, we included only the outcomes of their first IVF/ICSI cycle in the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data on sleep characteristics were collected via questionnaires on the day of oocyte retrieval. IVF/ICSI outcomes were abstracted from medical records. Quasi-Poisson, quasi-binomial or logistic regression models were used to assess the relations between sleep characteristics and reproductive outcomes after adjusting for relevant confounders. We also performed stratified analyses by subjective sleep quality (good versus poor) and maternal age (≤30 versus >30 years). MAIN RESULTS AND THE ROLE OF CHANCE: Compared with women who slept 7 to <8 h/night, those who slept <7 h/night exhibited decreases in the number of retrieved and mature oocytes of 11.5% (95% CI: -21.3%, -0.48%) and 11.9% (95% CI: -22.4%, -0.03%), respectively. A mid-sleep time (MST) earlier than 2:21 a.m. (<2:21 a.m.) or later than 3:00 a.m. (≥3:00 a.m.) and poor subjective sleep quality were inversely associated with the fertilization rate. Women who had trouble falling asleep more than three times per week had a lower number of mature oocytes (-10.5%, 95% CI: -18.6%, -1.6%), normal fertilized oocytes (-14.8%, 95% CI: -23.7%, -4.8%) and good-quality embryos (-15.1%, 95% CI: -25.4%, -3.5%) than those who had no such trouble. In addition, women who slept 9 to <10 h/night had a lower chance of clinical pregnancy compared to women who slept 7 to <8 h/night (odds ratio = 0.65, 95% CI: 0.44, 0.98). In the stratified analyses, the positive associations of nocturnal sleep duration with the number of good-quality embryos and fertilization rate existed only among the women with poor subjective sleep quality (P for interaction = 0.02 and 0.03, respectively). Additionally, we found that the positive associations of nocturnal sleep duration with implantation or clinical pregnancy only existed among women aged over 30 years (P for interaction = 0.04 and 0.01, respectively). LIMITATIONS, REASONS FOR CAUTION: Sleep characteristics are self-reported, which may lead to misclassification. MST serves as a proxy of chronotype and may be non-differentially misclassified resulting in an underestimate of the association of interest. Measuring sleep characteristics on the day of oocyte retrieval may lead to bias. Chance findings cannot be excluded because of the limited number of women with <7 h or ≥10 h nocturnal sleep and multiple testing. Our results may be biased by other confounders and may not be generalizable to women of other ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: Unhealthy sleep characteristics, including short nocturnal sleep, inappropriate sleep time, poor subjective sleep quality and having trouble falling asleep, may impair oocyte quantity and its potential to mature or be fertilized. Long nocturnal sleep might reduce the chance of clinical pregnancy among infertile females, especially women younger than 30 years old. Prolonged nocturnal sleep duration may be a potential beneficial behavior for improving IVF/ICSI outcomes for women aged over 30 years and women with poor subjective sleep quality, which requires further investigation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81771654) and the National Key R&D Program of China (No. 2018YFC1004201). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Associations between medication use and phthalate metabolites in urine and follicular fluid among women undergoing in vitro fertilization.

BACKGROUND: Phthalates, which are used as excipients of drugs, have been related to adverse reproductive outcomes. However, the relationships between medication use and phthalate exposure among women undergoing in vitro fertilization (IVF) have not been studied. OBJECTIVE: To investigate the associations between the medication intake and phthalate metabolites in urine and follicular fluid (FF). METHOD: Eight phthalate metabolites were measured in urine and FF samples from 274 women undergoing IVF using liquid chromatography-tandem mass spectrometry. Information on recent medication intake was obtained via interview by trained staff. We constructed generalized linear regression models to examine the associations of medication intake with phthalate metabolite concentrations and dose-response relationships between the number of medicines used and metabolite concentrations in two matrices. RESULTS: Four of 10 drugs were used by more than 10% of the participants, including vitamins (23.0%), traditional Chinese medicine (TCM, 22.3%), antioxidants (12.4%) and amoxicillin (10.2%). Participants who had used TCM had 26.0% (95% CI: 0.0, 58.8%), 32.6% (95% CI: 4.2, 68.8%) and 32.3% (95% CI: 2.6, 70.6%) higher urinary mono-n-butyl phthalate (MBP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) concentrations, respectively, than those who had not. Antioxidant intake was associated with a 30.6% (95% CI: -48.5, -6.6%) decrease in the urinary MBP concentration. Compared with non-users, women who reported the use of medicines had 53.2% (95% CI: 2.7, 128.5%) higher concentrations of MMP and a 37.7% (95% CI: -60.7, -1.5%) lower level of MBP in FF, respectively. CONCLUSION: Our data suggest that the intake of some medications may increase phthalate exposure among women undergoing IVF.

Predictors of phthalate metabolites in urine and follicular fluid and correlations between urine and follicular fluid phthalate metabolite concentrations among women undergoing in vitro fertilization.

BACKGROUND: Phthalate metabolites in follicular fluid (FF) may negatively affect normal folliculogenesis; however, the predictors of phthalate metabolite concentrations in urine and FF and relationships between urine and FF phthalate metabolite concentrations among women undergoing in vitro fertilization (IVF) are poorly understood. OBJECTIVE: To investigate predictors of phthalate metabolites in urine and FF and correlations between urine and FF phthalate metabolite concentrations among women undergoing IVF. METHOD: We recruited 305 women seeking infertility treatment at a reproductive center in Wuhan, China, from October to November 2016. Information regarding demographic characteristics, personal care product use and plastic material contact was obtained through direct interviews. Concentrations of 8 phthalate metabolites in urine and FF samples were measured using high-performance liquid chromatography and tandem mass spectrometry. Associations regarding metabolite concentrations in urine and FF samples were analysed by Spearman's correlation and linear regression. Generalized linear regression was used to examine potential predictors of phthalate metabolite concentrations in urine and FF. RESULTS: Weak to moderate associations between urine and FF samples were found for monoethyl phthalate (MEP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) (correlation coefficient: MEP, 0.350; MEOHP, 0.377); no associations were observed for other metabolites. The predictive powers of urinary metabolite concentrations in determining FF metabolite concentrations were uniformly low, with R2 ≤ 0.113. Body mass index (BMI) and educational level were inversely associated with the urinary concentrations of certain metabolites. Higher household income, intake of bottled drinks within 48 h, and use of shower gel and soap were frequently associated with higher urinary metabolite concentrations. BMI, higher household income and use of disposable plastic cups within 48 h were associated with higher metabolite concentrations in FF. CONCLUSION: Phthalate metabolite concentrations in urine and FF vary according to sociodemographic characteristics and lifestyle factors. Phthalate metabolite concentrations in urine may not be appropriate for estimating ovary phthalate exposure.

The associations of urinary phthalate metabolites with the intermediate and pregnancy outcomes of women receiving IVF/ICSI treatments: A prospective single-center study.

BACKGROUND: Phthalate exposure was reported to induce defects in ovarian function, and further influence embryo development and pregnancy outcomes. However, the data about the associations of phthalates with intermediate and pregnancy outcomes of in vitro fertilization (IVF) cycles are scarce in the Chinese population. METHODS: A total of 663 women receiving IVF/intracytoplasmic sperm injection (ICSI) treatments in our center were enrolled in this analysis. They provided one urine sample on the day of oocyte retrieval. We measured urinary concentrations of eight phthalate metabolites. Generalized linear models were used to analyze the associations of urinary phthalate metabolites with ovarian response, fertilization, early embryo development, and pregnancy outcomes. RESULTS: Among all the phthalate metabolites, mono-n-butyl phthalate (MBP) had the highest urinary concentration with a median level of 101.51 µg/g creatinine (Cr). MBP concentration was inversely associated with normal fertilization odds (overall P-trend < 0.01). There was a significant correlation of monoethyl phthalate (MEP) with decreased odds of normal fertilization in medium-concentration group compared to low-concentration group (overall P-trend = 0.02). No significant associations of metabolite concentrations with the odds of good-quality embryos on day 3 or blastocyst formation were found. Monomethyl phthalate (MMP) and MEP in medium-concentration group reduced 22.4% (95% CI: 0.64-0.94, overall P-trend = 0.04) and 21.9% (95% CI: 0.64-0.95, overall P-trend = 0.05) of the odds to gain good-quality blastocyst compared to low-concentration group. The eight phthalate metabolites were not correlated to clinical pregnancy rate, live birth rate, or early miscarriage rate. There was no significant association of di (2-ethylhexyl) phthalate (DEHP) metabolites observed with any clinical outcomes in the total population. After excluding male infertility, mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) in medium-concentration group turned to be associated with a higher number of retrieved oocytes (overall P-trend = 0.04), whereas mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) in medium-concentration group was associated with a lower odds of normal fertilization compared to low-concentration group (overall P-trend = 0.02). CONCLUSIONS: Urinary MBP concentration was much higher compared to other phthalate metabolites in this cohort of Chinese IVF/ICSI women, and also higher than it was reported by studies in other countries. MBP showed adverse impacts on fertilization. MMP and MEP could affect blastocyst quality, but not embryo quality on day 3. DEHP metabolites didn't show consistent reproductive toxicities as demonstrated in previous studies.

Association between blastocyst morphology and live birth rate following frozen-thawed single blastocyst transfer: Results from a 5-year retrospective analysis of 2593 cryopreserved blastocysts.

AIM: Our aim was to investigate associations between blastocyst morphology parameters and live birth outcome and to make possible additional recommendations for existing embryo selection strategies. METHODS: This retrospective cohort study included 2593 frozen-thawed single blastocyst transfers (SBT) cycles from 2012 to 2016. Multivariable logistic regression model was used to analyze the independent predictive effectiveness of blastocyst parameters for live birth rate (LBR). RESULTS: The participants enrolled in the present study were 32 (28-35) years old with a median body mass index of 21.20 (19.60-23.40) kg/m2 , among whom 1058 (40.8%) women had live births. Among the three blastocyst morphology parameters, we found only inner cell mass grade and trophectoderm cell grade had significant effects on LBR (P < 0.001). When adjusting for potential confounders in a multivariable logistic regression model, the expansion and hatching (EH) stage of blastocoel also showed obvious correlation with LBR. Blastocysts at EH stage 4-5 had a significantly higher LBR than that at stage 3 (P < 0.05). Additionally, the timing of blastulation was also an important predictor of LBR. Blastocysts vitrified on day 6 and day 7 yielded a lower LBR than that vitrified on day 5 (P < 0.001). CONCLUSION: The timing of blastulation and all blastocyst morphology parameters were associated with LBR independently. Although the most important parameter for predicting clinical outcomes remains undetermined, the timing of blastulation was a stable predictor of live birth for frozen-thawed SBT.

Intrauterine transplantation of autologous menstrual blood stem cells increases endometrial thickness and pregnancy potential in patients with refractory intrauterine adhesion.

AIM: This study was designed to evaluate the effects of intrauterine transplantation of menstrual blood stem cells (MenSCs) on endometrial thickness and pregnancy outcomes in patients with refractory intrauterine adhesion (IUA). METHODS: This study included a group of infertile women (n = 12, age 22-40 years), with refractory IUA. Autologous MenSCs isolated from the women's menstrual blood were expanded in vitro and transplanted into their uteruses, followed by hormone replacement therapy. Transvaginal ultrasound examination was performed to assess the endometrial thickness. Transabdominal ultrasound was conducted to detect pregnancy outcome. RESULTS: Autologous MenSCs were successfully isolated and expanded from menstrual blood and transplanted into the uterus of each patient. A significant improvement of the endometrial thickness was observed from 3.9 ± 0.9 to 7.5 ± 0.6 mm (P < 0.001). No adverse reaction was observed. The duration of menstruation was increased from 2.4 ± 0.7 to 5.3 ± 0.6 days (P < 0.001). Five out of 12 patients achieved clinical pregnancy and the pregnancy rate was 41.7%. CONCLUSIONS: Intrauterine transplantation of autologous MenSCs results in regeneration of endometrium, a prolongation of menstrual duration and an increase rate of pregnancy in patients with refractory IUA.

Blocking TRPA1 and TNF-α Signal Improves Bortezomib-Induced Neuropathic Pain.

BACKGROUND/AIMS: Bortezomib (BTZ) is largely used as a chemotherapeutic agent for the treatment of multiple myeloma. However, one of the significant limiting complications of BTZ is painful peripheral neuropathy during BTZ therapy. The purpose of this study was to examine the underlying mechanisms leading to neuropathic pain induced by BTZ. METHODS: ELISA and western blot analysis were used to examine the levels of tumor necrosis factor alpha (TNF-α) and its receptor, transient receptor potential ankyrin 1 (TRPA1) and intracellular p38-MAPK and JNK signal in the lumbar dorsal root ganglion. Behavioral test was performed to determine mechanical pain and cold sensitivity in a rat model. RESULTS: Systemic injection of BTZ significantly increased mechanical pain and cold sensitivity as compared with control animals (P< 0.05 vs. control rats). Our data also showed that protein expression of TRPA1 was upregulated in the dorsal root ganglion of BTZ rats and blocking TRPA1 attenuated mechanical pain and cold sensitivity in control rats and BTZ rats (P< 0.05 vs. vehicle control). Notably, the inhibitory effect of blocking TRPA1 on mechanical pain and cold sensitivity was smaller in BTZ rats than that in control rats. In addition, a blockade of TNF-α attenuated intracellular p38-MAPK and JNK signal in the dorsal root ganglion. This also decreased TRPA1 expression and alleviated mechanical hyperalgesia and cold hypersensitivity in BTZ rats. CONCLUSION: We revealed specific signaling pathways leading to neuropathic pain induced by chemotherapeutic BTZ. The data also suggest that blocking TRPA1 and tumor necrosis factor alpha is beneficial to alleviate neuropathic pain during BTZ intervention.

Elevated Serum Exosomal miR-125b Level as a Potential Marker for Poor Prognosis in Intermediate-Risk Acute Myeloid Leukemia.

BACKGROUND: The prognostic significance of miR-125b in intermediate-risk acute myeloid leukemia has not been well investigated. The aim of the study was to reveal the relationship between the elevated exosomal miR-125b level and the poor prognosis in adult patients with this disease. METHODS: A total of 154 consecutive patients with intermediate-risk acute myeloid leukemia were enrolled. Exosomes were isolated from blood specimens. The exosomal miR-125b level was determined using quantitative real-time polymerase chain reaction. Patients received standardized therapy and were followed up for 1-24 months. Details about relapse and overall death were recorded. RESULTS: Patients were divided into the high miR-125b level group (n = 77) and the low miR-125b level group (n = 77). In the multivariate Cox proportional hazard regression model, the high miR-125b level group was separately associated with increased risks of relapse and overall death in 2 years (hazard ratio [HR] 2.84, 95% CI 1.81-4.33 and HR 2.69, 95% CI 1.87-4.52). Kaplan-Meier analysis also revealed that a high miR-125b level was related to a higher cumulative relapse and overall death rates (p < 0.001 and p < 0.001, respectively). CONCLUSION: Circulating exosomal miR-125b concentration might be an independent prognostic indicator in intermediate-risk acute myeloid leukemia patients. An elevated miR-125b level indicated higher risks of relapse and overall death.

Urinary phthalate metabolites in relation to serum anti-Müllerian hormone and inhibin B levels among women from a fertility center: a retrospective analysis.

BACKGROUND: Phthalates, a class of endocrine disruptors, have been demonstrated to accelerate loss of ovarian follicle pool via disrupting folliculogenesis, and lead to diminished ovarian reserve. However, human data are limited. Here, we aimed to examine whether urinary phthalate metabolites are correlated with markers of ovarian reserve among women attending a fertility clinic. METHODS: We measured eight phthalate metabolites in urine samples collected from 415 women seeking infertility treatment at the Reproductive Medicine Center of Tongji Hospital, Wuhan, China. Data on measures of ovarian reserve, as indicated by serum anti-Müllerian hormone (AMH) and inhibin B (INHB) levels, were retrieved retrospectively through electronic medical charts. Multivariate linear models were performed to estimate the associations of urinary phthalate metabolites and serum AMH and INHB. We further explored the potential nonlinearity of the relationships with restricted cubic spline analysis. RESULTS: Overall, we found largely null associations between urinary phthalate metabolites and serum AMH. The multivariable adjusted differences in serum INHB levels comparing the highest quartile of urinary MEHP to the lowest were - 18.29% (95% CI: - 31.89%, - 1.98%; P-trend = 0.04). Women in the second to fourth quartiles of MEOHP had a significant decrease of - 23.74% (95% CI: -35.85%, - 9.24%), - 19.91% (95% CI: -33.30%, - 3.82%) and - 20.23% (95% CI: -34.43%, - 2.96%), respectively, in INHB levels compared to the first quartile. In the spline analysis, we identified a nonlinear relationship between MEOHP exposure and serum INHB. CONCLUSIONS: We provided evidence for a negative association between urinary concentrations of certain phthalate metabolites and serum INHB levels, suggesting an adverse effect of phthalates exposure on growing antral follicles. Whether phthalates exposure at environmentally level will pose a risk for ovarian reserve needs further investigation.

Lipopolysaccharide-induced toll-like receptor 4 signaling in esophageal squamous cell carcinoma promotes tumor proliferation and regulates inflammatory cytokines expression.

Emerging evidence suggests toll-like receptor 4 (TLR4) signaling contributes to cancer development and progression. However, the consequences of signaling via the TLR4 pathway in esophageal squamous cell carcinoma (ESCC) are still unclear. Here, we investigated the impact of Lipopolysaccharide (LPS)-induced TLR4 signaling on ESCC cell proliferation, inflammatory cytokines expression, and downstream molecular mechanisms. Seventy-eight ESCC and 26 normal esophageal specimens were analyzed by immunohistochemistry, and two cell lines (Eca-109 and TE-1) were used for in vitro studies. LPS, a natural agonist of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell proliferation and inflammatory cytokines regulation were examined. Specific inhibitors of mitogen-activated protein kinase (MAPK) (extracellular regulated protein kinase [ERK] and p38) signaling pathways were used to investigate the role of each pathway in LPS-TLR4 signaling. TLR4 protein was increased in ESCC tumor tissues compared with the adjacent normal tissues. TLR4 over-expression was significantly correlated with tumor differentiation grade, lymph node metastasis, and UICC stage. LPS-induced activation of TLR4 signaling promoted cancer cell proliferation, increased production of proinflammatory or immunosuppressive cytokines TNF-α, TGF-ß and inhibited the anti-inflammatory cytokine IL-10. LPS-TLR4 signaling was associated with the activation of ERK and p38 MAPK signaling pathways. Further inactivation of the two pathways by specific inhibitors attenuated cell proliferation and inflammatory cytokines expression induced by LPS. Our results indicate that LPS-TLR4 signaling in cancer cells contributes to the progression of human ESCC.

Differential expression of bone morphogenetic protein 5 in human lung squamous cell carcinoma and adenocarcinoma.

Bone morphogenetic proteins (BMPs) play important roles in tumor cell proliferation, metastasis, and invasion. However, the expression patterns of BMPs in patients with non-small-cell lung cancer (NSCLC) and their correlations with NSCLC pathogenesis have not been examined yet. In this study, the mRNA levels of BMP family members in NSCLC tissues were analyzed and results showed that the mRNA levels of BMP5 and BMP7 were significantly down-regulated and up-regulated, respectively, in tumor tissues compared with those in the corresponding noncancerous tissues. Interestingly, the mRNA level of BMP5 was significantly higher in lung adenocarcinoma tissues than that in lung squamous cell carcinoma tissues. Furthermore, results from immunohistochemistry analysis confirmed stronger expression of BMP5 protein in lung adenocarcinoma than in lung squamous cell carcinoma. Our findings suggested that BMP5 might be a potential prognostic biomarker or therapeutic target for patients with NSCLC.

Monomethyl Phthalate Causes Early Embryo Development Delay, Apoptosis, and Energy Metabolism Disruptions Through Inducing Redox Imbalance.

Phthalates are a class of environmental endocrine disrupting chemicals which can cause reproductive system damages. However, data about reproductive toxicity spectrum of phthalate metabolites among Chinese women undergoing in vitro fertilization (IVF) treatments are scarce yet. Previous studies regarding underlying embryo toxicities focused on oxidative stress and apoptosis, while energy metabolism abnormality might be another key cause for embryo developmental disruptions. Here, we found that among the measured eight phthalate metabolites, monomethyl phthalate (MMP) had the second highest urinary concentration in women receiving IVF. Compare to the lowest exposure level group, MMP in tertile 3 was associated with fewer counts of oocyte retrieved and good-quality embryos, and MMP in tertile 2 was correlated with reduced good-quality embryo rate. The direct embryo toxicities of MMP were studied using mouse 2-cell embryos. Consistent to results found in human populations, exposure to MMP induced mouse early embryo developmental delay. Furthermore, MMP exposure led to excessive reactive oxygen species production in early embryos, and antioxidant can partially rescue the early embryo development slow down. Embryo apoptosis could also be caused by oxidative stress. To be noted, elevated apoptosis level was not found in live "slow" embryos but dead embryos, which suggested that apoptosis was not related to early embryo developmental delay. Additionally, MMP exposure depleted adenosine triphosphate (ATP) synthesis of early embryos, which could be reversed by antioxidant. In conclusion, MMP, as the newly found embryonic toxicant in Chinese women, resulted in early embryo development delay, apoptosis, and energy metabolism disruptions via inducing redox imbalance.

Phthalate metabolites in urine and follicular fluid in relation to menstrual cycle characteristics in women seeking fertility assistance.

BACKGROUND: Phthalates have been shown to disrupt the estrous cycle in animal studies. However, epidemiological research investigating their associations with menstrual cycle characteristics is limited. OBJECTIVE: To explore the relationships between phthalate exposure and menstrual cycle characteristics among women seeking fertility assistance. METHODS: We determined the levels of eight phthalate metabolites in both follicular fluid (FF) and urine specimens collected from 441 women in the Tongji Reproductive and Environmental (TREE) cohort, using high-performance liquid chromatography and tandem mass spectrometry. Information about menstrual cycle parameters was obtained through a questionnaire. The impacts of individual and joint exposure to phthalates on menstrual cycle characteristics were assessed using multivariable linear regression, Poisson regression, and quantile g-computation approaches. RESULTS: After adjusting for relevant covariates, we found that per log10-unit increase in mono(2-ethylhexyl) phthalate (MEHP) level in urine specimens was associated with a decrease of 0.20 days (95 % CI: -0.37, -0.03) in bleeding duration. We also observed that mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and the sum of di(2-ethylhexyl) phthalate (DEHP) metabolites (∑DEHP) concentrations in FF samples were inversely related to cycle length [ß = -1.92 (95 % CI: -3.10, -0.75) and -1.87 (95 % CI: -3.56, -0.19), respectively]. However, we generally observed null associations between phthalate metabolites and irregular cycle, dysmenorrhea, hypomenorrhea, or cycle length variation. Furthermore, we also found that phthalate metabolite mixtures in FF and urine were generally unrelated to menstrual cycle characteristics. CONCLUSION: Our findings suggest that some DEHP metabolites in FF and urine are inversely associated with menstrual cycle length and menstrual bleeding duration in women attending a fertility center.

DBP Exposure Affects Oocyte Fertilization Via Extracellular Vesicles-Derived miR-116-5p in Ovarian Granulosa Cells Through Downregulating FOXO3a Expression.

Mono-butyl phthalate (MBP), the metabolite of dibutyl phthalate (DBP), is the most abundant phthalate metabolite found in Chinese women. Extracellular vesicles (EVs) are nanoscale lipid bilayer particles produced by extensive kinds of cells, serving a key role in intercellular communication. Extracellular vesicle miRNAs (EV-miRNAs) in follicular fluid (FF) have been evidenced to be associated with female reproductive health. The objective of this study was to investigate the associations of EV-miRNAs expressed profile with DBP exposure in FF of female participants and expose its potential mechanism in impaired oocyte development. Based on participants' FF MBP concentrations and fertilization status, we compared the miRNA expression between the FF-EVs of group A (high DBP exposure and impaired fertilization) and group B (low DBP exposure and normal fertilization). Compared with group B, miR-1246, miR-3679-5p, miR-423-5p, miR-5585-3p, miR-116-5p, miR-172-5p were upregulated, while miR-34b-3p was downregulated in group A. Target genes of the differently expressed miRNAs were predicted, and the functional analysis was performed. Furthermore, we exposed human ovarian granulosa tumor cell line (KGN) to MBP (4ug/L) to isolate the EVs from the culture medium and validated the expression levels of different miRNAs. We found that MBP exposure was significantly associated with increased levels of miR-116-5p (P = 0.01). In addition, we demonstrated that the most different miRNA, miR-116-5p regulated oocyte fertilization by inhibiting FOXO3a. Our findings suggested that EV-miRNAs in the FF might mediate MBP toxicity in oocytes.

Clusterin-carrying extracellular vesicles derived from human umbilical cord mesenchymal stem cells restore the ovarian function of premature ovarian failure mice through activating the PI3K/AKT pathway.

BACKGROUND: Emerging evidence has highlighted the therapeutic potential of human umbilical cord mesenchymal stem cells (UC-MSCs) in chemotherapy-induced premature ovarian failure (POF). This study was designed to investigate the appropriate timing and molecular mechanism of UC-MSCs treatment for chemotherapy-induced POF. METHODS: Ovarian structure and function of mice were assessed every 3 days after injections with cyclophosphamide (CTX) and busulfan (BUS). UC-MSCs and UC-MSCs-derived extracellular vesicles (EVs) were infused into mice via the tail vein, respectively. Ovarian function was analyzed by follicle counts, the serum levels of hormones and ovarian morphology. The apoptosis and proliferation of ovarian granulosa cells were analyzed in vitro and in vivo. Label-free quantitative proteomics was used to detect the differentially expressed proteins in UC-MSC-derived EVs. RESULTS: After CTX/BUS injection, we observed that the ovarian function of POF mice was significantly deteriorated on day 9 after CTX/BUS infusion. TUNEL assay indicated that the number of apoptotic cells in the ovaries of POF mice was significantly higher than that in normal mice on day 3 after CTX/BUS injection. Transplantation of UC-MSCs on day 6 after CTX/BUS injection significantly improved ovarian function, enhanced proliferation and inhibited apoptosis of ovarian granulosa cells, whereas the therapeutic effect of UC-MSCs transplantation decreased on day 9, or day 12 after CTX/BUS injection. Moreover, EVs derived from UC-MSCs exerted similar therapeutic effects on POF. UC-MSCs-derived EVs could activate the PI3K/AKT signaling pathway and reduce ovarian granulosa cell apoptosis. Quantitative proteomics analysis revealed that clusterin (CLU) was highly expressed in the EVs of UC-MSCs. The supplementation of CLU proteins prevented ovarian granulosa cells from chemotherapy-induced apoptosis. Further mechanistic analysis showed that CLU-knockdown blocked the PI3K/AKT signaling and reversed the protective effects of UC-MSCs-derived EVs. CONCLUSIONS: Administration of UC-MSCs and UC-MSCs-derived EVs on day 6 of CTX/BUS injection could effectively improve the ovarian function of POF mice. UC-MSCs-derived EVs carrying CLU promoted proliferation and inhibited apoptosis of ovarian granulosa cells through activating the PI3K/AKT pathway. This study identifies a previously unrecognized molecular mechanism of UC-MSCs-mediated protective effects on POF, which pave the way for the use of cell-free therapeutic approach for POF.

Associations between exposure to phthalates and liver function among women undergoing assisted reproductive technology.

Phthalates can induce hepatotoxicity in animal studies. We aimed to assess the associations of individual and mixture of urinary phthalate metabolites with serum liver function indicators among 764 women undergoing assisted reproductive technology (ART). In linear models, we observed inverse correlations between urinary mono-benzyl phthalate and serum total protein (TP) as well as globulin (ß=-0.27 and -0.23, respectively, P<0.05). Additionally, negative associations were identified between mono-isobutyl phthalate and mono-butyl phthalate (MBP) and aspartate aminotransferase-to-alanine transaminase ratio (AST/ALT) (P<0.05). MBP and the sum of all phthalate metabolites (∑all.phth.m) were positively associated with bilirubin, with ß ranging from 0.14 to 0.47. Most phthalate metabolites were also positively related to gamma-glutamyl transferase (GGT) (all P<0.05). In Bayesian kernel machine regression models, phthalate mixture was positively associated with bilirubin and GGT, whereas inversely associated with AST/ALT and TP. Our results suggest that phthalate exposure may impair liver function among women undergoing ART.

Associations between urinary phthalate concentrations and antral follicle count among women undergoing in vitro fertilization.

Background: Phthalates are ubiquitously used in a variety of products and have an adverse effect on folliculogenesis. However, previous epidemiological studies on the associations between phthalate exposure and antral follicle count (AFC) produced conflicting results. The present study aimed to evaluate the associations between urinary phthalate metabolite concentrations and AFC among women undergoing in vitro fertilization (IVF). Methods: We collected 525 urine samples and measured 8 phthalate metabolites from IVF patients. Poisson regression models were conducted to evaluate the associations between phthalate metabolite concentrations and AFC. In addition, participants were stratified into a younger group (< 35 years) and an older group (≥ 35 years) to explore the potential effect modification by age. We also performed sensitivity analyses by restricting our analyses to only infertile women diagnosed with tubal factor infertility to test the robustness of the results. Results: Significant positive associations were observed among urinary MBP, MEOHP and ∑PAEs concentrations and AFC after adjusting for age, BMI, year of study and infertility diagnosis. Compared with women in the first tertile, women in the third tertile of MBP and MEOHP had 7.02% (95% CI: 1.18%, 12.9%) and 8.84% (95% CI: 2.83%, 14.9%) higher AFC, respectively, and women in the second and third tertiles of ∑PAEs had 6.19% (95% CI: 0.37%, 12.0%) and 9.09% (95% CI: 3.22%, 15.0%) higher AFC, respectively. In addition, MBP, MEOHP and ∑PAEs also had significant positive associations with AFC in trend tests for dose-response. In the age-stratified analysis, we found a stronger relationship between phthalate metabolite concentrations and AFC among older women and an inverse association among younger women. We observed similar results in the sensitivity analyses. Conclusion: We found positive associations between phthalate exposure and AFC, which support the idea that phthalate exposure may accelerate primordial follicle recruitment and lead to higher AFC in women undergoing IVF. More studies are needed to better understand their relationships.

Associations of personal care products use with reproductive outcomes of IVF/ICSI treatment.

Introduction: Personal care products (PCPs) contain a number of endocrine-disrupting chemicals (EDCs) that could potentially affect the reproductive function in women of childbearing age. However, studies focused on the effects of PCPs use on reproductive outcomes are very limited. The current study aimed to explore the relationships between PCPs use patterns and reproductive outcomes in women undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment. Methods: A total of 1500 women from the Tongji Reproductive and Environmental (TREE) study between December 2018 and January 2020 were included in this study. Participants provided characteristics of PCPs use within the previous three months. Retrieved oocyte number, mature oocyte number, two distinct pronuclei (2PN) zygote number, fertilization rate, cleavage rate, blastocyst formation rate, implantation, clinical pregnancy, miscarriage, and live birth were followed up as reproductive endpoints. Generalized linear regression model was utilized to assess the associations between various categories of PCPs use and reproductive endpoints of IVF/ICSI. Results: After adjusting for relevant covariates, women who used skin care products ≥14 times per week had a reduction of 22.4% in the maturation rate (95% CI: -39.2%, -1.6%) compared to participants who did not use skin care products. After transferring fresh embryos, women who used cosmetics 1-2 times per week (adjusted OR = 2.2, 95% CI: 1.0, 4.8) or 3-7 times per week (adjusted OR = 2.5, 95% CI: 1.2, 5.2) had a higher possibility of miscarriage than those who did not use cosmetics. There was negative association between the use of gel or soap and the cleavage rate among women aged < 30 years old (P for interaction = 0.01). Among women with BMI ≥ 24 kg/m2, the use of gel or soap was negatively associated with the blastocyst formation rate (P for interaction = 0.04), while cosmetics use was negatively associated with the maturation rate (P for interaction = 0.001). Conclusion: Our findings suggest that the use of PCPs in women of reproductive age have a potential adverse impact on IVF/ICSI outcomes, particularly skin care and cosmetic products.