Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels.

PURPOSE: To determine correlations between chemicals in follicular fluid (FF) and follicular reproductive hormone levels. METHODS: The analysis was part of a larger cohort study to determine associations between exposure to EDCs and in vitro fertilization (IVF) outcomes. FF was aspirated from a single leading follicle per participant. Demographics and data on exposure to EDCs were self-reported by the participants using a questionnaire. The concentrations of estradiol (E2), progesterone (PG), anti-Mullerian hormone (AMH), and inhibin B, as well as that of 12 phthalate metabolites and 12 phenolic chemicals were measured in each FF sample. Multivariate linear regression model was used to identify the drivers of hormone levels based on participant's age, BMI, smoking status, and chemical exposure for the monitored chemicals detected in more than 50% of the samples. Benjamini-Hochberg false discovery rate (FDR) correction was applied on the resulting p values (q value). RESULTS: FF samples were obtained from 72 women (mean age 30.9 years). Most of the phthalates and phenolic substances monitored (21/24, 88%) were identified in FF. Ten compounds (7 phthalate metabolites, 3 phenols) were found in more than 50% of samples. In addition, there were positive associations between E2 levels and mono-n-butyl phthalate (MnBP) (beta = 0.01) and mono-isobutyl phthalate (MiBP) (beta = 0.03) levels (q value < 0.05). CONCLUSION: Higher concentrations of several phthalate metabolites, present among others in personal care products, were associated with increased E2 levels in FF. The results emphasize the need to further investigate the mechanisms of action of such EDCs on hormonal cyclicity and fertility in women.

Clinical features of isolated Fallopian tube torsion: evidence from a large series.

We aimed at studying isolated Fallopian tube torsion (IFTT) in a relatively large cohort of women and to evaluate different features of IFTT in comparison to a large cohort of women with non-IFTT adnexal torsion (NIAT). This was a retrospective cohort study. We included women with surgically confirmed ovarian and/or Fallopian tube torsion between March 2011 and June 2020. Fifty-four cases of IFTT were surgically confirmed during the study period and were compared to 422 surgically confirmed NIAT. The rate of controlled ovarian hyperstimulation treatments, current pregnancy, and vomiting was lower in the IFTT group compared with the NIAT group. Cervical tenderness and vaginal discharge were more common in the IFTT group. Oedematous and enlarged ovaries were less common in the IFTT group. In multivariate regression analysis, the following factors were independently associated with IFTT: (i) vaginal discharge [adjusted odds ratio (aOR) 95% CI 8.16, 1.98-33.55]; (ii) cervical motion tenderness (aOR 95% CI 2.71, 1.01-7.29); (iii) fertility treatments (aOR 95% CI 0.26, 0.70-0.77); (iv) previous abdominal surgery (aOR 95% CI 0.46, 0.22-0.96); (v) vomiting (aOR 95% CI 0.38, 0.19-0.76); and (vi) enlarged ovary (aOR 95% CI 0.34, 0.18-0.65). In conclusion, we have identified factors positively and negatively associated with IFTT in a large cohort of women with adnexal torsion.

The effect of coronavirus disease 2019 immunity on frozen-thawed embryo transfer cycles outcome.

OBJECTIVE: To study the effect of patients' immunization after coronavirus disease 2019 (COVID-19) infection or messenger ribonucleic acid (mRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine on frozen-thawed embryo transfer (FET). DESIGN: Cohort retrospective study. SETTING: Tertiary university affiliated medical center. PATIENT(S): All consecutive patients undergoing FET cycles in our center. The study group (immune group) consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) who either recovered from COVID-19 infection or received the mRNA SARS-CoV-2 vaccine. The control groups consisted of patients treated during the COVID-19 pandemic (between January 2021 and August 2021) but were not infected or did not receive the mRNA SARS-CoV-2 vaccine (not-immune2021 group) and those treated between January 2019 and August 2019 (before the pandemic) (not-immune2019 group). INTERVENTION(S): Frozen-thawed embryo transfer cycles. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rates and FET cycles' characteristics. Data on patient age and variables related to infertility treatment were collected from the patient records. RESULT(S): During the study periods, 428 patients underwent 672 FET cycles. The immune group consisted of 141 patients who underwent 264 FET cycles (44 in postinfection and 220 in postvaccination), whereas the not-immune2021 and not-immune2019 groups consisted of 93 and 194 patients undergoing 125 and 283 FET cycles, respectively. Patients' characteristics and the types of endometrial preparations were comparable between the study groups. The implantation rate and clinical and ongoing pregnancy rates per transfer were similar between the study groups (immune group, postinfection and postvaccination; not-immune2021 group; not-immune2019 group). CONCLUSION(S): Coronavirus disease 2019 infection or vaccination did not affect patients' performance or implantation in their subsequent FET cycle.

MRI guided focused ultrasound (MRgFUS) treatment for uterine fibroids among women with and without abdominal scars.

INTRODUCTION: MRI guided focused ultrasound (MRgFUS) is a noninvasive technique for treating uterine fibroids. The presence of abdominal scars can limit the number of women eligible for the procedure, due to absorbance of beam energy. The goals of this study were to assess the number of women that fit the procedure and to compare outcomes among women with or without abdominal scars. MATERIAL AND METHODS: A prospective cohort study of all women that were interested in MRgFUS in a single University-Affiliated Hospital between November 2012 and December 2019. Rates of women that were referred to further screening, fulfilled selection criteria and underwent the procedure were compared between patients with or without abdominal scars. We evaluated the treatment parameters of the two groups and used linear regression model predict non-perfused volume (NPV) at the end of the process. RESULTS: Out of 701 patients, 21.8% were suitable for MRgFUS. Women with scars had significant lower NPV compared with women without scars (60% versus 82.4%, p = 0.021). No serious adverse events were reported in both groups. Linear regression models showed that fibroids' volume, stopping the treatment due to severe pain and the presence of abdominal scars had a statistically significantly negative effect on NPV (betas: -11.51, -6.96, and -6.29, p-values: <0.001, 0.003, and 0.007 respectively), while number of sonication had a statistically significantly positive effect on NPV (beta = 5.98, p = 0.011). CONCLUSION: Regardless of strict inclusion criteria, MRgFUS treatment is less efficient among women with abdominal scars, although still feasible for those who are interested in noninvasive option.

"Add-Ons" for Assisted Reproductive Technology: Do Patients Get Honest Information from Fertility Clinics' Websites?

"Add-on" procedures are actively promoted on some fertility clinic websites as proven means to improve IVF success rates, especially for couples with repeated implantation/IVF failures. However, the actual contribution of these interventions to live birth rates remains inconclusive. At present, little is known about the type and quality of the information provided on the IVF clinics' websites regarding the merits of "add-ons." A systematic evaluation of the quality of information on "add-on" procedures in fertility clinic websites was performed using 10-criteria structured questionnaire. We included English language websites that presented in the Google.com search engine after typing the following key-words:"endometrial scratching"(ES), "intralipid infusions"(ILI), "assisted hatching"(AHA), "PGT-A," or "PGS". In total, 254 websites were evaluated. In most cases, an accurate description of the "add-on" procedures was provided (78.8%). However, only a minority (12%) reported their undetermined effectiveness. The use of PGT-A was more often encouraged (52.8%) than ES (23.6%) and AHA (16%). The cost was infrequently presented (6.9%). Scientific references were only rarely provided for ILI, versus 12.7% for ES, 4.0% for AHA, and 5.6% for PGT-A. The information entry date was often missing. None of the websites reported the clinic's pregnancy-rate following the "add-on" procedures. Information on "add-ons" available to patients from IVF clinic websites is often inaccurate. This could perpetuate false myths among infertile patients about these procedures and raises concern regarding possible commercial bias. It is imperative that IVF clinic websites will better communicate the associated risks and uncertainties of "add-ons" to prospective patients.

Association between follicular fluid phthalate concentrations and extracellular vesicle microRNAs expression.

STUDY QUESTION: Are phthalate metabolite concentrations in follicular fluid (FF) associated with the expression of extracellular vesicle microRNAs (EV-miRNAs)? SUMMARY ANSWER: Phthalate metabolite concentrations are associated with the expression of EV-miRNA and their associated pathways in FFs. WHAT IS KNOWN ALREADY: Phthalate metabolites were recently detected in FF. Urinary phthalate metabolite concentrations alter the expression of EV-miRNAs in FF. STUDY DESIGN, SIZE, DURATION: Prospective study including 105 women recruited between January 2014 and August 2016 in a tertiary university-affiliated hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: We assessed FF concentrations of 12 phthalate metabolites. EV-miRNAs were isolated from aliquots of the same FF, and their expression profiles were measured using a human miRNA panel. Associations between EV-miRNAs that were present in >50% of the samples and phthalate metabolites that were measured in >74% of the FF samples were tested. Genes regulated by EV-miRNAs that were found to be significantly (false discovery rate q-value < 0.1) correlated with FF-phthalates were analyzed for pathways linked with female fertility using miRWalk2.0 Targetscan database, DAVID Bioinformatics Resources and Kyoto Encyclopedia of Genes and Genomes (KEGG). MAIN RESULTS AND THE ROLE OF CHANCE: Of 12 phthalate metabolites, 11 were measured in at least one FF sample. Mono (6-COOH-2-methylheptyl) phthalate (MCOMHP), mono-2-ethyl-5-carboxypentyl phthalate (mECPP), mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP) and mono (7-COOH-2-methyloctyl) phthalate (MCOMOP) were detected in more than 74% of the samples. Of 754 EV-miRNAs tested, 39 were significantly associated either with MEP, MBzP, MCOMOP, MCOMHP and/or with mECPP, after adjusting for multiple testing (P < 0.05). KEGG-based pathway enrichment analysis of the genes regulated by these miRNAs showed that these EV-miRNAs may be involved in pathways related to ovary or oocyte development, maturation and fertilization. LIMITATIONS, REASONS FOR CAUTION: The use of miRNA panel array limits the number of potential relevant miRNAs. Moreover, several of the phthalate metabolites examined may be biased due to internal (enzymatic activity) or external (contamination in medical interventions) causes. WIDER IMPLICATIONS OF THE FINDINGS: Phthalate metabolites may alter follicular EV-miRNAs profile and thus impair pathways that are involved with oocyte development, maturation and fertilization. Our results contribute to understanding of possible mechanism(s) in which endocrine disruptor chemicals interfere with female fertility. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Institutes of Environmental Health Sciences [Grant R21-ES024236]; and Environmental Health Fund, Israel [Grant 1301], no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Pregnancy and neonatal outcomes of twin pregnancies - the role of maternal age.

OBJECTIVES: We aim to study the association of maternal age with maternal and neonatal complications in twin pregnancies. METHODS: A retrospective cohort study of dichorionic-diamniotic twin pregnancies stratified into three groups according to maternal age ("A" <25, "B" 25-34 and "C" 35-44 years old). Outcome measures included pregnancy, delivery and neonatal complications. A sub-analysis of in vitro fertilization pregnancies only was conducted. RESULTS: Compared with younger women (groups A [n=65] and B [n=783]), older women [group C (n=392)] demonstrated significantly higher rates of gestational diabetes mellitus (B 6.6% vs. A 0%, p =0.027, C 10.2% vs. B 6.6%, p =0.032), were more likely to undergo cesarean deliveries (C 66.6%, B 57.6%, A 52.3%, p =0.007), and were at increased risk of having more than 20% difference in weight between the twins (C 24.5%, B 17.4%, A 16.9%, p =0.013). Other outcomes, including preeclampsia, did not differ between the groups. A sub-analysis of the in vitro fertilization only pregnancies was performed. Compared with younger women (groups A [n=18] and B [n=388]), older women (group C [n=230]) underwent more cesarean deliveries (p=0.004), and had more than 20% difference in weight between the twins (p<0.004). Other outcomes, including gestational diabetes mellitus rates and preeclampsia, did not differ between the groups. CONCLUSIONS: Women at advanced maternal age with dichorionic twin pregnancies had significantly higher rates of gestational diabetes mellitus, cesarean deliveries and fetal weight discordancy as compared with younger women. In contrast, the incidence of preeclampsia was not affected by maternal age.

Extracellular vesicles and female reproduction.

Extracellular vesicles (EVs) are nano-sized membrane bound complexes that have been identified as a mean for intercellular communication between cells and tissues both in physiological and pathological conditions. These vesicles contain numerous molecules involved in signal transduction including microRNAs, mRNAs, DNA, proteins, lipids, and cytokines and can affect the behavior of recipient cells. Female reproduction is dependent on extremely fine-tuned endocrine regulation, and EVs may represent an added layer that contributes to this regulation. This narrative review article provides an update on the research of the role of EVs in female reproduction including folliculogenesis, fertilization, embryo quality, and implantation. We also highlight potential pitfalls in typical EV studies and discuss gaps in the current literature.

Obstetric, neonatal and child development outcomes following assisted hatching treatment: a retrospective cohort study.

Assisted hatching (AH) involves artificial disruption of the zona pellucida prior to embryo transfer. The purpose of this study is to examine the safety of AH technique and its effect on obstetrical, perinatal and neonatal outcomes and risk of developmental delay. This is a retrospective cohort of ART cycles using laser AH technique. The study group consisted of 120 cases of AH cycles resulting in singleton pregnancies and live births compared with 113 control cases. A current phone questionnaire was conducted to assess child development in the first year of life. AH was not associated with increased risk for all obstetrical and perinatal outcomes examined including PPROM, gestational diabetes, hypertensive diseases of pregnancy, delivery by cesarean section, gestational age at delivery, low birth weight (LBW), preterm birth and neonatal Apgar score (p>.05). No significant differences were observed between AH and control group in rates and risk of congenital malformations (5.8 vs. 4.4%, respectively, OR 1.33, 95% CI 0.41-4.34) and developmental delay (19.2 vs. 12.8%, respectively, OR 1.62, 95% CI 0.74-3.52). AH did not increase the risk of obstetrical and neonatal complications in singleton pregnancies, including congenital malformations and child developmental delay. AH may therefore be considered a safe method of ART.

Cord blood androgen levels of females from same sex and opposite sex twins - A pilot study.

OBJECTIVE: Opposite-sex twins have shown behavioural and reproductive differences between females and males. These differences may be determined by higher intrauterine levels of androgens among females that were exposed to a male co-twin. The aim of this study was to compare cord blood androgen levels in females from same-sex and opposite-sex twins. DESIGN: A prospective study. In this pilot study, we compared cord blood androgens (DHEA-S, Δ-4 androstenedione, total testosterone-TT) and sex hormone-binding globulin (SHBG) levels in 20 females from same sex and 20 females from opposite-sex dichorionic diamniotic twins. We used generalized estimating equation (GEE) modelling to assess differences in cord blood androgens between females from same-sex twin pregnancies and females from opposite-sex twin pregnancies. PATIENTS: Twenty opposite-sex twin pairs (female-male twins) and 20 same-sex twin pairs (female-female). MEASUREMENTS: Cord blood total testosterone, Δ-4 androstenedione, DHEA-S and sex hormone-binding globulin (SHBG) levels. RESULTS: No difference in the levels of androgens as Δ-4 androstenedione, total testosterone and SHBG was identified between females that were exposed to a female co-twin compared with females that were exposed to a male co-twin. DHEA-S levels were significantly lower among females from opposite-sex twins compared with females from same-sex twins. CONCLUSIONS: Our preliminary data do not support the hypothesis that females exposed to male co-twins are exposed to higher levels of androgens in utero compared with females exposed to female co-twins. Further studies are needed to explain the reported behavioural and reproductive differences among opposite-sex twins.

Machine learning vs. classic statistics for the prediction of IVF outcomes.

PURPOSE: To assess whether machine learning methods provide advantage over classic statistical modeling for the prediction of IVF outcomes. METHODS: The study population consisted of 136 women undergoing a fresh IVF cycle from January 2014 to August 2016 at a tertiary, university-affiliated medical center. We tested the ability of two machine learning algorithms, support vector machine (SVM) and artificial neural network (NN), vs. classic statistics (logistic regression) to predict IVF outcomes (number of oocytes retrieved, mature oocytes, top-quality embryos, positive beta-hCG, clinical pregnancies, and live births) based on age and BMI, with or without clinical data. RESULTS: Machine learning algorithms (SVM and NN) based on age, BMI, and clinical features yielded better performances in predicting number of oocytes retrieved, mature oocytes, fertilized oocytes, top-quality embryos, positive beta-hCG, clinical pregnancies, and live births, compared with logistic regression models. While accuracies were 0.69 to 0.9 and 0.45 to 0.77 for NN and SVM, respectively, they were 0.34 to 0.74 using logistic regression models. CONCLUSIONS: Our findings suggest that machine learning algorithms based on age, BMI, and clinical data have an advantage over logistic regression for the prediction of IVF outcomes and therefore can assist fertility specialists' counselling and their patients in adjusting the appropriate treatment strategy.

Outcome of two sequential singleton pregnancies and twin pregnancies among primiparous women at advanced age undergoing IVF.

PURPOSE: To compare the obstetrical and detailed neonatal outcomes of primipara of advanced maternal age conceiving two sequential singleton pregnancies by IVF with those of primipara conceiving twins by IVF. METHODS: A retrospective study of all primiparous women aged ≤ 38 years and conceived by IVF who delivered sequential singletons or delivered twins at a single tertiary university affiliated medical center between 2011 and 2019. We performed two main comparisons: 1. First vs. second singleton pregnancies. 2. Two singleton pregnancies vs. twin pregnancies. RESULTS: Overall, there were 63 women with consecutive singleton IVF pregnancies. The median age was 40.0 at first pregnancy and 42.0 in the second pregnancy. Pregnancy and delivery complications rates did not differ significantly between the first and the second singleton pregnancies, including gestational hypertensive disorders (7 (11.1%) vs. 4 (6.3%), p = 0.530), gestational diabetes mellitus (13 (20.6%) vs 18 (28.5%), p = 0.410), intrauterine growth restriction (6 (9.5%) vs. 4 (6.3%), p = 0.744), or cesarean delivery (25 (39.7%) vs. 29 (46%), p = 0.589). Rates of delivery before 32 weeks gestation were similar for both first and second singleton pregnancies (1.6%, p > 0.999). The proportion of neonatal adverse outcome in both first and second singleton pregnancies groups was low and did not differ between the groups. Compared with women who delivered sequential singletons, women with twin pregnancies had significantly higher cesarean delivery rates (113 (83.7%) vs. 29 (46%), p < 0.001) and lower gestational ages at delivery (36.2 vs. 38.4, p < 0.001) than women with two singleton deliveries. Adverse neonatal outcomes were significantly higher for twin pregnancies, including birthweight < 1500 g (17 (12.6%) vs. 2 (3.2%), p = 0.036), neonatal intensive care unit admission (57 (42.2%) vs. 4 (6.3%), p < 0.001), neonatal hypoglycemia (23 (17%) vs. 3 (4.8%), p = 0.017), and respiratory distress syndrome (14 (10.4%) vs. 1 (1.6%), p = 0.040). Length of neonatal hospitalization was significantly longer for twins (9 vs. 5 days, p < 0.001). The rate of gestational hypertensive disorders (preeclampsia and gestational hypertension) was similar between the groups, but the rates of severe preeclampsia trended higher among women who carried twins (8 (5.9%) vs. 0, p = 0.057). CONCLUSIONS: Sequential singleton pregnancies at primipara women of advanced maternal age have an overall very good outcome, with no clinically significant difference between the pregnancies. In addition, their outcome is much better compared with twins.

Impact of the mode of conception on gestational hypertensive disorders at very advanced maternal age.

RESEARCH QUESTION: To study gestational hypertensive disorders in oocyte donation pregnancies compared with other modes of conception at very advanced maternal age. DESIGN: A historical cohort study of all women aged 45-47 years who gave birth to singletons at a tertiary medical centre between March 2011 and May 2018, at 24 weeks' gestation or later. Pregnancy outcomes were compared between donor oocyte (IVF-OD), IVF using autologous oocytes (IVF-A) and naturally conceived pregnancies. A multivariate logistic regression was used to evaluate the association between the mode of conception and gestational hypertensive disorders. RESULTS: The final analysis included 159, 68 and 73 patients in the IVF-OD, IVF-A and natural conception groups, respectively. The rate of gestational hypertensive disorders was significantly higher among those who conceived by IVF compared with those who conceived naturally but did not differ between the two IVF groups (27.0% for IVF-OD, 19.1% for IVF-A, P = 0.204; 5.5% for natural conception, P < 0.001 and P = 0.013 compared with IVF-OD and IVF-A, respectively). The results remained similar in a multivariate logistic regression analysis. The rate of Caesarean deliveries was significantly higher in the IVF-OD and IVF-A groups compared with the natural conception group (83.6%, 70.6% and 37.0%, respectively, P < 0.001), but other pregnancy outcomes did not differ between the groups. CONCLUSIONS: IVF pregnancies in the late fifth decade of life were associated with significantly higher rates of gestational hypertensive disorders compared with naturally conceived pregnancies. No difference existed between the two IVF groups. These results may highlight the impact of IVF itself on gestational hypertensive disorders at very advanced maternal age.

Body mass index in relation to extracellular vesicle-linked microRNAs in human follicular fluid.

OBJECTIVE: To study whether increased body mass index is associated with altered expression of extracellular vesicle microRNAs (EV-linked miRNAs) in human follicular fluid. DESIGN: Cross-sectional study. SETTING: Tertiary-care university-affiliated center. PATIENT(S): One hundred thirty-three women undergoing in vitro fertilization (IVF) were recruited from January 2014 to August 2016. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): EV-linked miRNAs were isolated from follicular fluid and their expression profiles were measured with the use of the Taqman Open Array Human miRNA panel. EV-linked miRNAs were globally normalized and inverse-normal transformed. Associations between body mass index (BMI) and EV-linked miRNA outcomes were analyzed by means of multivariate linear regression and principal component analysis. RESULT(S): Eighteen EV-linked miRNAs were associated with an increase in BMI after adjusting for age, ethnicity, smoking status, and batch effects. Hsa-miR-328 remained significant after false discovery rate adjustments. Principal component analyses identified the first principal component to account for 40% of the variation in our EV-linked miRNA dataset, and adjusted linear regression found that the first principal component was significantly associated with BMI after multiple testing adjustments. Using Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we predicted gene targets of EV-linked miRNA in silico and identified PI3K-Akt signaling, ECM-receptor interaction, focal adhesion, FoxO signaling, and oocyte meiosis pathways. CONCLUSION(S): These results show that a 1-unit increase in BMI is associated with altered follicular fluid expression of EV-linked miRNAs that may influence follicular and oocyte developmental pathways. Our findings provide potential insight into a mechanistic explanation for the reduced fertility rates associated with increased BMI.

Supraphysiological Concentrations of Bisphenol A Alter the Expression of Extracellular Vesicle-Enriched miRNAs From Human Primary Granulosa Cells.

Bisphenol A (BPA) is a widely used chemical that has been detected in follicular fluid and associated with adverse reproductive effects. Granulosa cells have an important role in follicular growth and oocyte maturation, however, little is known about the biological mechanisms of BPA toxicity on human granulosa cells. In this study, we exposed primary granulosa cells to different concentrations of BPA (0, 20, 200, 2000, and 20 000 ng/ml) and used quantitative polymerase chain reaction to measure the expression levels of miRNAs enriched in extracellular vesicles (EV-enriched miRNAs), and cellular levels of selected target genes of differentially expressed EV-enriched miRNAs. We found that exposure to 20 000 ng/ml BPA was associated with decreased levels of EV-miR-27b-3p (FC = 0.58, p = .04) and increased levels of its biologically relevant target genes FADD (FC = 1.22, p = .01), IGF1 (FC = 1.59, p = .06), and PPARG (FC = 1.73, p = .001) as compared with the control. In addition, we observed that under the same exposure conditions, the expression levels of miR-27b-3p in granulosa cells were also downregulated (FC = 0.65, p = .03) as compared with the control. Our findings suggest that both cellular and extracellular changes in gene expression may mediate BPA toxicity in granulosa cells.

An optimized model for hCG stimulation of human mural granulosa cell culture.

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6-8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.

Maternal Phthalate and Personal Care Products Exposure Alters Extracellular Placental miRNA Profile in Twin Pregnancies.

Prenatal exposure to endocrine-disrupting chemicals (EDCs) exerts both short- and long-term adverse effects on the developing fetus. However, the mechanisms underlying these effects have yet to be uncovered. Maternal-fetal signaling is mediated in part by signaling molecules (eg, microRNAs [miRNAs]) contained in extracellular vesicles (EVs) that are released by the placenta into the maternal circulation. We investigated whether maternal exposure to the EDCs phthalates and personal care products alters the miRNA profile of placental-derived EVs circulating in maternal blood. Blood and urine samples from pregnant women with uncomplicated term dichorionic, diamniotic twin pregnancies were analyzed as part of a larger study investigating correlations between exposure of phthalate and personal care products and epigenetic alterations in twin pregnancies. We explored correlations between maternal urinary levels of 13 phthalate and 12 personal care products metabolites and the miRNA profile of placental EVs (EV-miRNAs) circulating in maternal blood. The expression of miR-518e was highest among women with high urinary levels of monobenzyl phthalate and methyl paraben. miR-373-3p was the least expressed in women exposed to high levels of methyl paraben, and miR-543 was significantly downregulated in women exposed to high levels of paraben metabolites, dichlorophenol metabolites, and triclosan. In conclusion, this pilot study reveals that prenatal exposure to EDCs is associated with altered profile of circulating placenta-derived EV-miRNAs. Further studies are needed to generalize these results to singleton pregnancies and to assess whether these alterations are associated with pregnancy complications.

Urinary concentrations of phenols and phthalate metabolites reflect extracellular vesicle microRNA expression in follicular fluid.

BACKGROUND: Phenols and phthalates are potential endocrine disrupting chemicals (EDCs) that are associated with adverse health outcomes. These EDCs dysregulate a number of biomolecules and pathways, including microRNAs. MicroRNAs can be carried in transport systems called extracellular vesicles (EVs) that are present in most biofluids. EVs in the follicular fluid, which fills the ovarian follicle and influences oocyte developmental competency, carry microRNAs (EV-miRNAs) that have been associated with In Vitro Fertilization (IVF) outcomes. However, it remains unclear whether EDCs affect EV-miRNAs in follicular fluid. OBJECTIVES: This study sought to determine whether urinary concentrations of phenols and phthalates biomarkers are associated with EV-miRNAs expression in follicular fluid collected from women undergoing IVF treatment. METHODS: This cross-sectional study included 130 women recruited between January 2014 and August 2016 in a tertiary university-affiliated hospital. Participants provided urine samples during ovarian stimulation and on the day of oocyte retrieval. We assessed urinary concentrations of five phenols, eight phthalate metabolites, and one phthalate alternative metabolite. EV-miRNAs were isolated from follicular fluid and their expression profiles were measured using the TaqMan Open Array® Human microRNA panel. We fitted multivariable linear regression models and principal component analysis to examine associations between individual and molar sums of exposure biomarkers and EV-miRNAs. RESULTS: Of 754 miRNAs tested, we detected 133 EV-miRNAs in the microRNA array which expressed in at least 50% of the follicular fluid samples. After adjusting for multiple testing, we identified eight EV-miRNAs associated with individual phenols and phthalate metabolites, as well as molar ΣDEHP that met a q < 0.10 false-discovery rate (FDR) threshold. Hsa-miR-125b, hsa-miR-106b, hsa-miR-374a, and hsa-miR15b was associated with mono(2-ethylhexyl) phthalate concentrations, hsa-let-7c with concentrations mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), and the sum of metabolites of di(2-ethylhexyl) phthalate, hsa-miR-24 with mono-n-butyl phthalate concentrations, hsa-miR-19a with cyclohexane-1,2-dicarboxylic acid monohydroxy isononyl ester (MHiNCH), and hsa-miR-375 with ethyl paraben concentrations. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, gene targets and pathways of these EV-miRNAs were predicted in silico and 17 KEGG FDR-significant pathways related to follicular development and oocyte competence were identified. CONCLUSIONS: Our results show that urinary concentrations of select phenol and phthalate metabolites are correlated with altered EV-miRNAs expression in follicular fluid. These findings may provide insight regarding the molecular mechanisms underlying adverse effects of phenol and phthalate exposure on female fertility.

Outcomes of singleton versus twin pregnancies in the fifth and sixth decades.

BACKGROUND: Pregnancies in the fifth and sixth decades of life have been increasing, but current data are limited regarding the outcomes of twin compared with singleton pregnancies in this age group and to twin pregnancies at younger age. OBJECTIVE: To compare obstetrical and neonatal outcomes of IVF conceived pregnancies, in twin gestations of women who were ≥45 years old at delivery to singletons at similar age and twin gestations at the age of <35 years, and to assess if the complications are mainly influenced by the very advanced maternal age or by the multifetal pregnancy. STUDY DESIGN: A retrospective cohort study from a single tertiary medical center of women aged ≥45 at delivery between March 2011 and January 2018 and women aged <35 at delivery with twin pregnancies that conceived by IVF. Exclusion criteria were spontaneous pregnancies or pregnancies after ovulation induction, monochorionic twin pregnancies, higher order multiple gestations, or women that underwent fetal reduction. Pregnancy, delivery, postpartum and neonatal outcomes were compared between singleton and twin pregnancies. Multivariate logistic regression was used to evaluate the association between twin pregnancies and adverse outcomes. RESULTS: Out of 67,355 deliveries, 612 were of women ≥45 years old, of whom 492 women conceived via IVF (395 singleton and 97 twin pregnancies). Of those, 60 women were ≥50 years old, 49 of them carried singleton and 11 carried twin pregnancies. Baseline characteristics were similar between groups. Women at the fifth and sixth decades with twins had significantly higher rates of preeclampsia (32.0% vs. 10.9%, p < 0.001), gestational diabetes mellitus (35.4% vs. 23.8%, p = 0.020), preterm deliveries <32 weeks of gestation (8.2% vs. 1.3%, p = 0.001), and fetal growth restriction (18.6% vs. 7.6%, p = 0.001) compared with singleton pregnancies at similar maternal age. After multivariate analysis adjusting for confounders, the odds ratio for the composite of preeclampsia, gestational hypertension, intrauterine fetal growth restriction, and placental abruption was 3.19 for twin compared with singleton pregnancies and 1.73 for gestational diabetes mellitus. Pregnancy complications among older women with twins were also significantly higher when compared with younger women with twins (<35 years old). Women in the fifth and sixth decades with twins had higher rates of cesarean deliveries (91.8% vs. 56.4%, p<0.001), gestational hypertension, preeclampsia, and gestational diabetes mellitus (10.3% vs. 4.2%., p=0.016; 32.0% vs. 6.2%, p<0.001; 35.1% vs. 8.1%, p<0.001, respectively) than the younger group carrying twins. CONCLUSIONS: Twin pregnancies in the fifth and sixth decades carry significantly higher complications rate compared with singleton pregnancies at the same age and twin pregnancies at younger age.