Ronit Machtinger

Metabolism disrupting chemicals and metabolic disorders

The recent epidemics of metabolic diseases, obesity, type 2 diabetes(T2D), liver lipid disorders and metabolic syndrome have largely been attributed to genetic background and changes in diet, exercise and aging. However, there is now considerable evidence that other environmental factors may contribute to the rapid increase in the incidence of these metabolic diseases. This review will examine changes to the incidence of obesity, T2D and non-alcoholic fatty liver disease (NAFLD), the contribution of genetics to these disorders and describe the role of the endocrine system in these metabolic disorders. It will then specifically focus on the role of endocrine disrupting chemicals (EDCs) in the etiology of obesity, T2D and NAFLD while finally integrating the information on EDCs on multiple metabolic disorders that could lead to metabolic syndrome. We will specifically examine evidence linking EDC exposures during critical periods of development with metabolic diseases that manifest later in life and across generations.

Parma consensus statement on metabolic disruptors

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16–18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as “metabolic disruptors”, in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome.

Morphological systems of human embryo assessment and clinical evidence

Success rates with IVF have improved remarkably since the procedure was first established for clinical use with the first successful birth in 1978. The main goals today are to perform single-embryo transfer in order to prevent multiple pregnancies and achieve higher overall pregnancy rates. However, the ability to identify the most viable embryo in a cohort remains a challenge despite the numerous scoring systems currently in use. Clinicians still depend on developmental rate and morphological assessment using light microscopy as the first-line approach for embryo selection. Active research in the field involves developing non-invasive methods for scoring embryos and ranking them according to their ability to implant and give rise to a healthy birth. Current attention is particularly being focused on time-lapse evaluation. Available data from preliminary studies indicate that these systems are safe;prospective data now need to be collected to determine whether these methods do improve implantation rates. This review gives brief consideration to the use of morphological evaluations in assisted reproduction treatment, discusses the types of embryo scoring,digital imaging and biometric approaches currently in use and comments on future developments for embryo evaluation.

The association between severe obesity and characteristics of failed fertilized oocytes

STUDY QUESTION Is the cytoskeletal and chromosomal organization of failed fertilized oocytes from severely obese patients (BMI ≥ 35 kg/m²) altered compared with that in patients with normal BMI (BMI 18.5-24.9 kg/m²)? SUMMARY ANSWER Compared with normal BMI patients, severe obesity was associated with a greater prevalence of spindle anomalies and non-aligned chromosomes in failed fertilized oocytes. WHAT IS KNOWN AND WHAT THIS PAPER ADDS Obesity is associated with poor reproductive outcomes, but little is known regarding the underlying mechanisms. To address potential mechanisms, our study compared the cytoskeletal and chromosome organization in failed fertilized oocytes from severely obese and normal BMI patients. DESIGN The study population was drawn from IVF patients treated in a hospital-based infertility clinic between February 2010 and July 2011. The prevalence of meiotic spindle and chromosome alignment anomalies in failed fertilized oocytes from patients with severe obesity (i.e. Class II and III; BMI 35.0-50.1 kg/m²) was compared with those from patients with normal BMI (BMI 18.5-24.9 kg/m²). Oocytes were fixed and then labeled for tubulin, actin and chromatin. Spindle number and integrity, as well as chromosome alignment, were assessed using immunofluorescence microscopy and, in some cases, confocal microscopy. Generalized estimating equations were applied, which account for the correlation among oocytes from the same patient to estimate odds ratio (OR), 95% confidence intervals (CIs) and two-sided Wald P-values. Models were adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and polycystic ovarian syndrome (PCOS) a priori. PARTICIPANTS AND SETTING University-affiliated infertility clinic. A total of 276 oocytes that failed to fertilize from 137 patients were evaluated: 105 oocytes from severely obese women (n = 47) and 171 oocytes from normal BMI patients (n = 90). MAIN RESULTS AND THE ROLE OF CHANCE (i) Significantly more oocytes from the severely obese group exhibited two spindles compared with those from the normal BMI group (58.9 versus 35.1%; OR = 2.68, CI = 1.39-5.15, P-value = 0.003). (ii) Among oocytes with a single spindle, those from severely obese patients showed a significantly higher prevalence of disarranged spindles with non-aligned chromosomes compared with those from normal BMI patients (28.6 versus 8.6%; OR = 4.58, CI = 1.05-19.86, P-value = 0.04). BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION Inclusion of only failed fertilized oocytes, small sample size, unknown factors such as non-PCOS comorbidity. GENERALIZABILITY TO OTHER POPULATIONS For this study, by design, it is unclear whether the findings are generalizable to successfully fertilized oocytes, and whether this oocyte-level influence of obesity is generalizable to infertile women who do not undergo stimulation or, more broadly, to spontaneous conceptions in fertile women. STUDY FUNDING/COMPETING INTEREST(S) none. TRIAL REGISTRATION NUMBER n/a.

Bisphenol-A and human oocyte maturation in vitro

STUDY QUESTION Does exposure to bisphenol-A (BPA) affect the maturation of human oocytes in vitro? SUMMARY ANSWER There was a dose-response association of BPA exposure with altered human oocyte maturation in vitro. WHAT IS KNOWN ALREADY There is widespread exposure of the general population to BPA. BPA has been detected in the human follicular fluid. Animal studies have shown that BPA exposure is associated with maturation arrest and spindle abnormalities in maturing oocytes. STUDY DESIGN, SIZE, DURATION A randomized trial, using 352 clinically discarded oocytes from 121 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS The study population was drawn from patients undergoing IVF/ICSI cycles in our program at Brigham and Womens Hospital from March 2011 to April 2012. Oocytes from only one cycle for each patient were included in the study. Cycles with at least two germinal vesicle stage oocytes were included with random allocation of one oocyte to culture for 30 h without BPA and remaining sibling oocytes to medium-containing BPA (20, 200 ng/ml or 20 µg/ml). Oocytes were fixed and labeled for tubulin, actin and chromatin and examined with immunofluorescence and confocal microscopy. Oocytes were assessed for meiotic stage (n = 292), and those at metaphase II (MII, n = 175) were further classified according to their spindle configurations and patterns of chromosome alignment. McNemars test was used to compare dichotomized maturation status. Generalized estimating equations were used to account for the correlation between oocytes from the same woman and for the spindle analysis. MAIN RESULTS AND THE ROLE OF CHANCE As the BPA dose increased, there was a decrease in the percentage of oocytes that progressed to MII (P = 0.002) and increases in the percentage of oocytes that were degenerated (P = 0.01) or that had undergone spontaneous activation (P = 0.007). Among MII oocytes, as the BPA dose increased, there was a significant trend (by test for trend) for a decreased incidence of bipolar spindles (P < 0.0001) and aligned chromosomes (P = 0.02). LIMITATIONS, REASONS FOR CAUTION Although we used sibling oocytes to overcome potential confounders, such as infertility diagnosis and maternal age, additional studies with a larger number of oocytes are required to confirm the present results. Having access only to clinically discarded oocytes, we were limited to evaluating only those oocytes that failed to mature in vivo despite having been exposed to gonadotrophin stimulation and the ovulatory trigger of HCG. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study investigating the effect of BPA on oocyte meiotic maturation, spindle morphology and chromosome alignment in human oocytes. Together with prior animal studies, the data support the negative influences of BPA on cell cycle progression, spindle architecture and chromosome organization during oocyte maturation. Furthermore, the increased rates of abnormal maturation in oocytes exposed to BPA may be relevant to our understanding of the decrease in fertility reported in the last decades. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the NIEHS Center Grant Pilot Project (P30-ES000002). R.M. was sponsored by a fellowship from the Environmental Health Fund, Israel and by the Frederick L. Hisaw Endowment, Harvard School of Public Health. There are no conflicts of interest. TRIAL REGISTRATION NUMBER n/a.

Does local injury to the endometrium before IVF cycle really affect treatment outcome? Results of a randomized placebo controlled trial

Aim: To evaluate the effect of local injury to the endometrium during spontaneous menstrual cycles before in vitro fertilization (IVF) treatment on implantation and pregnancy rates in women with recurrent implantation failure (RIF). Methods: In a prospective randomized controlled trial (RCT), a total of 36 patients, with RIF undergoing IVF, were randomized to two groups. In 18 patients, endometrial biopsies were performed using a pipelle curette on days 9–12 and 21–24 of the menstrual cycle preceding IVF treatment. In 18 control patients, a cervical pipelle was performed. Results: The implantation rate (2.08% versus 11.11%; p = 0.1), clinical (0% versus 31.25%; p < 0.05) and live births rates (0% versus 25%; p = 0.1) were lower in the experimental group compared with controls. Conclusion: Our RCT did not find any benefit from local injury to the endometrium in women with a high number of RIFs. Further studies are warranted to better define the target population of patients who may benefit from this procedure.

Ibuprofen and paracetamol for pain relief during medical abortion: a double-blind randomized controlled study

OBJECTIVE To determine the efficacy of a nonsteroidal anti-inflammatory drug vs. paracetamol in pain relief during medical abortion and to evaluate whether nonsteroidal anti-inflammatory drugs interfere with the action of misoprostol. DESIGN A prospective double-blind controlled study. SETTING University-affiliated tertiary hospital. PATIENT(S) One hundred twenty women who underwent first-trimester termination of pregnancy. INTERVENTION(S) Patients received 600 mg mifepristone orally, followed by 400 microg of oral misoprostol 2 days later. They were randomized to receive ibuprofen or paracetamol when pain relief was necessary. Patients completed a questionnaire about side effects and pain score and returned for an ultrasound follow-up examination 10-14 days after medical abortion. MAIN OUTCOME MEASURE(S) Success rates, as defined by no surgical intervention, and pain scores were assessed. RESULT(S) Ibuprofen was found to be statistically significantly more effective for pain relief after medical abortion compared with paracetamol. There was no difference in the failure rate of medical abortion, and the frequency of surgical intervention was slightly higher in the group that received paracetamol (16.3% vs. 8.5%). CONCLUSION(S) Ibuprofen was found to be more effective than paracetamol for pain reduction during medical abortion. A history of surgical or medical abortion was predictive for high pain scores. Despite its anti-prostaglandin effects, ibuprofen use did not interfere with the action of misoprostol.

Bisphenol A, oocyte maturation, implantation, and IVF outcome: review of animal and human data

Recent data have raised concerns about the detrimental effect of chronic exposure to environmental chemicals. Some chemicals affect the endocrine system (endocrine disruptors) and have been linked to several diseases, including infertility. One such endocrine disruptor is bisphenol A (BPA), a monomer widely used in the plastic industry, with nearly ubiquitous exposure. In this review, data on the effects of BPA on female fertility are summarized. Specifically, its effect is considered on folliculogenesis, oocyte maturation, embryo quality, and implantation, both in animal and human models. Animal studies have shown that BPA might impair prophase I, follicular growth, and implantation, and may be associated with spindle abnormalities. In humans, while in-vitro studies have suggested an association between BPA exposure and impaired oocyte meiosis, clinical evidence indicate possible adverse effects of BPA exposure on IVF outcomes. As human clinical data are still scarce, larger studies are required to further elucidate the effects of BPA exposure on female fertility.

Does BPA alter steroid hormone synthesis in human granulosa cells in vitro

STUDY QUESTION Does Bisphenol A (BPA) impair steroid hormone production in human luteinized granulosa cells in vitro? SUMMARY ANSWER At supra-physiological concentrations, BPA alters progesterone and estradiol synthesis in vitro and significantly reduces the mRNA and protein expression levels of three genes encoding steroidogenesis enzymes. WHAT IS KNOWN ALREADY In IVF patients, the effects of BPA exposure on cycle outcome are controversial. Previous animal studies have shown that granulosa cell steroid hormone synthesis is compromised after BPA exposure, but their findings have been difficult to replicate in humans due, in part, to the low availability of discarded biological material. STUDY DESIGN, SIZE, DURATION Luteinized granulosa cells obtained from 44 fertile and infertile patients undergoing in vitro fertilization were cultured for 48 h with different concentrations of BPA (0, 0.2, 0.02, 2.0, 20 µg/ml). PARTICIPANTS/MATERIALS, SETTING, METHODS Culture medium and total RNA extracted from the luteinized granulosa cells were examined for estradiol and progesterone levels as well as mRNA and protein expression of steroidogenesis enzymes, using enzyme immunoassays, real-time PCR and western blots. MAIN RESULTS AND THE ROLE OF CHANCE Treatment of granulosa cells with 2 or 20 µg/ml BPA for 48 h resulted in significantly lower progesterone biosynthesis (P < 0.005 and <0.001, respectively). Estradiol production was significantly altered only after incubation with 20 µg/ml of BPA (P < 0.001). These concentrations also significantly reduced the mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP11A1 and CYP19A1 without affecting StAR and 17β-hydroxysteroid dehydrogenase mRNA expression. Similarly, 3β-HSD, CYP11A1 and CYP19A1 protein levels were reduced after administration of 20 µg/ml BPA. Lower BPA concentrations similar to, and up to 100 times, the concentrations measured in human follicular fluid, serum and urine did not alter steroidogenesis in primary granulosa cell cultures. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study investigating the effects of acute exposure (48 h) of BPA on discarded material. As such, the model may not accurately reflect the effect of BPA on the physiological events of follicular steroid hormone synthesis in vivo. WIDER IMPLICATIONS OF THE FINDINGS Our results show that in vitro exposure to BPA at low doses does not affect granulosa cells steroidogenesis. Combined with recent in vivo studies, these data can be reassuring but further studies are needed to assess the effects of chronic exposure to BPA on ovarian steroidogenesis. STUDY FUNDING AND COMPETING INTERESTS This study was supported by grant number 1936/12 from the Israeli Science Foundation (ISF). The authors have no conflict of interest.

In vitro maturation for patients with repeated in vitro fertilization failure due to “oocyte maturation abnormalities”

OBJECTIVE To investigate the efficacy of in vitro oocyte maturation (IVM) in women with repeated failure of IVF treatments due to follicular developmental abnormalities. DESIGN Prospective longitudinal study. SETTING The IVF unit of a university-affiliated medical center. PATIENT(S) Seven women with three or more IVF failures due to abnormal oocyte development resulting in no egg retrieval (empty follicle syndrome [EFS]), oocyte maturation arrest, or failure of fertilization. INTERVENTION(S) Immature oocyte collection, with or without minimal ovarian stimulation, IVM of the oocytes, insemination by intracytoplasmic sperm injection (ICSI), and embryo transfer. MAIN OUTCOME MEASURE(S) Number of aspirated oocytes, maturation, fertilization and cleavage rates, pregnancy, and ongoing pregnancy. RESULT(S) Seven women underwent seven IVM cycles. Four of them received a minimal stimulation of 75 IU FSH for 3-4 days. Oocytes were retrieved from all subjects (mean 7.1 +/- 3.3 oocytes/cycle). The in vitro maturation rate was 39.6% (mean 2.7 +/- 2.9 matured oocytes available for fertilization in four patients). The mean fertilization rate was 45.8% +/- 31.6%. Four women had embryo transfer. Two patients with previous genuine EFS conceived and delivered. CONCLUSION(S) IVM should be considered for women with previous genuine EFS. The role of IVM in other indications of oocyte maturation deficits warrants further investigation.