Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Meeting the demand for fertility services: the present and future of reproductive endocrinology and infertility in the United States.

The field of reproductive endocrinology and infertility (REI) is at a crossroads; there is a mismatch between demand for reproductive endocrinology, infertility and assisted reproductive technology (ART) services, and availability of care. This document's focus is to provide data justifying the critical need for increased provision of fertility services in the United States now and into the future, offer approaches to rectify the developing physician shortage problem, and suggest a framework for the discussion on how to meet that increase in demand. The Society of REI recommend the following: 1. Our field should aggressively explore and implement courses of action to increase the number of qualified, highly trained REI physicians trained annually. We recommend efforts to increase the number of REI fellowships and the size complement of existing fellowships be prioritized where possible. These courses of action include: a. Increase the number of REI fellowship training programs. b. Increase the number of fellows trained at current REI fellowship programs. c. The pros and cons of a 2-year focused clinical fellowship track for fellows interested primarily in ART practice were extensively explored. We do not recommend shortening the REI fellowship to 2 years at this time, because efforts should be focused on increasing the number of fellowship training slots (1a and b). 2. It is recommended that the field aggressively implements courses of action to increase the number of and appropriate usage of non-REI providers to increase clinical efficiency under appropriate board-certified REI physician supervision. 3. Automating processes through technologic improvements can free providers at all levels to practice at the top of their license.

Evaluation of a New Model for Human Chorionic Gonadotropin Rise in Pregnancies of Unknown Viability.

OBJECTIVE: To evaluate the performance of a new human chorionic gonadotropin (hCG) threshold model to classify pregnancies as viable or nonviable using a longitudinal cohort of individuals with pregnancy of unknown viability. The secondary objective was to compare the new model with three established models. METHODS: This is a single-center, retrospective cohort study of individuals seen at the University of Missouri from January 1, 2015, until March 1, 2020, who had at least two consecutive quantitative hCG serum levels with an initial level greater than 2 milli-international units/mL and 5,000 milli-international units/mL or less, with the first interval between laboratory draws no greater than 7 days. Prevalence of correct classification of viable intrauterine pregnancies, ectopic pregnancies, and early pregnancy losses was evaluated with a new proposed hCG threshold model and compared with three established models describing minimum expected rates of hCG rise for a viable intrauterine pregnancy. RESULTS: Of an initial cohort of 1,295 individuals, 688 patients met inclusion criteria. One hundred sixty-seven individuals (24.3%) had a viable intrauterine pregnancy; 463 (67.3%) had an early pregnancy loss; and 58 (8.4%) had an ectopic pregnancy. A new model based on the total additive percent rise of hCG at 4 and 6 days after initial hCG (70% or greater and 200% or greater rise, respectively) was created. The new model was able to correctly identify 100% of viable intrauterine pregnancies while minimizing incorrect classification of early pregnancy losses and ectopic pregnancies as normal pregnancies. At 4 days after initial hCG, 14 ectopic pregnancies (24.1%) and 44 early pregnancy losses (9.5%) were incorrectly classified as potentially normal pregnancies. At 6 days after initial hCG, only seven ectopic pregnancies (12.1%) and 25 early pregnancy losses (5.6%) were incorrectly classified as potentially normal pregnancies. In established models, up to nine intrauterine pregnancies (5.4%) were misclassified as abnormal pregnancies and up to 26 ectopic pregnancies (44.8%) and 58 early pregnancy losses (12.5%) were incorrectly classified as potentially normal pregnancies. CONCLUSION: The proposed new hCG threshold model optimizes a balance between identifying potentially viable intrauterine pregnancies and minimizing misdiagnosis of ectopic pregnancies and early pregnancy losses. External validation in other cohorts is needed before widespread clinical use.

Disrupted methylation patterns at birth persist in early childhood: a prospective cohort analysis.

BACKGROUND: Alterations in the epigenome are a risk factor in multiple disease states. We have demonstrated in the past that disruption of the epigenome during early pregnancy or periconception, as demonstrated by altered methylation, may be associated with both assisted reproductive technology and undesirable clinical outcomes at birth, such as low birth weight. We have previously defined this altered methylation, calculated based on statistical upper and lower limits of outlier CpGs compared to the population, as an 'outlier methylation phenotype' (OMP). Our aim in this study was to determine whether children thus identified as possessing an OMP at birth by DNA methylation in cord blood persist as outliers in early childhood based on salivary DNA methylation. RESULTS: A total of 31 children were included in the analysis. Among 24 children for whom both cord blood DNA and salivary DNA were available, DNA methylation patterns, analyzed using the Illumina Infinium MethylationEPIC BeadChip (850 K), between cord blood at birth and saliva in childhood at age 6-12 years remain stable (R2 range 0.89-0.97). At birth, three out of 28 children demonstrated an OMP in multiple cord blood datasets and hierarchical clustering. Overall DNA methylation among all three OMP children identified as outliers at birth was remarkably stable (individual R2 0.908, 0.92, 0.915), even when only outlier CpG sites were considered (R2 0.694, 0.738, 0.828). CONCLUSIONS: DNA methylation signatures in cord blood remain stable over time as demonstrated by a strong correlation with epigenetic salivary signatures in childhood. Future work is planned to identify whether a clinical phenotype is associated with OMP and, if so, could undesirable clinical outcomes in childhood and adulthood be predicted at birth.

Embryo cryopreservation leads to sex-specific DNA methylation perturbations in both human and mouse placentas.

In vitro fertilization (IVF) is associated with DNA methylation abnormalities and a higher incidence of adverse pregnancy outcomes. However, which exposure(s), among the many IVF interventions, contributes to these outcomes remains unknown. Frozen embryo transfer (ET) is increasingly utilized as an alternative to fresh ET, but reports suggest a higher incidence of pre-eclampsia and large for gestational age infants. This study examines DNA methylation in human placentas using the 850K Infinium MethylationEPIC BeadChip array obtained after 65 programmed frozen ET cycles, 82 fresh ET cycles and 45 unassisted conceptions. Nine patients provided placentas following frozen and fresh ET from consecutive pregnancies for a paired subgroup analysis. In parallel, eight mouse placentas from fresh and frozen ET were analyzed using the Infinium Mouse Methylation BeadChip array. Human and mouse placentas were significantly hypermethylated after frozen ET compared with fresh. Paired analysis showed similar trends. Sex-specific analysis revealed that these changes were driven by male placentas in humans and mice. Frozen and fresh ET placentas were significantly different from controls, with frozen samples hypermethylated compared with controls driven by males and fresh samples being hypomethylated compared with controls, driven by females. Sexually dimorphic epigenetic changes could indicate differential susceptibility to IVF-associated perturbations, which highlights the importance of sex-specific evaluation of adverse outcomes. Similarities between changes in mice and humans underscore the suitability of the mouse model in evaluating how IVF impacts the epigenetic landscape, which is valuable given limited access to human tissue and the ability to isolate specific interventions in mice.

Impact of mode of conception on early pregnancy human chorionic gonadotropin rise and birth weight.

Objective: To assess whether the mode of conception and embryo biopsy impact first-trimester human chorionic gonadotropin (hCG) dynamics and subsequent risk of small for gestational age (SGA) or large for gestational age (LGA). Design: Retrospective cohort study. Setting: University fertility center. Patients: Six hundred-two pregnant patients with singleton live births. Interventions: Serial serum hCG measurements were obtained between 10 and 28 days postconception to determine the within-woman rate of change in hCG (slope) by mode of conception (unassisted pregnancy, fresh embryo transfer (ET), frozen ET, and frozen ET following preimplantation genetic testing for aneuploidy (PGT-A). Main Outcome Measures: Primary outcomes included birth weight, SGA, and LGA. Results: Mode of conception is not independently associated with birth weight, SGA, or LGA. Mediation analysis revealed an expected one-day increase in log-transformed hCG varied by mode of conception: unassisted (0.41), fresh ET (0.39), frozen ET (0.42), PGT-A (0.44). Human chorionic gonadotropin rise has a positive effect on birth weight (55 g per SD increase in hCG slope) and is associated with SGA (odds ratio, 0.65), but not with LGA (odds ratio, 1.18). Conclusions: Human chorionic gonadotropin rise is an important mediator of the mode of conception/birth weight relationship. Preimplantation genetic testing for aneuploidy has the highest rate of hCG rise, followed by frozen ET, unassisted, and fresh ET. Faster rise is associated with higher birth weight and lower risk of SGA but does not impact LGA risk. Importantly, PGT-A does not increase the risk of extreme birth weight relative to other modes of conception evaluated.

Effects of preconception lifestyle intervention in infertile women with obesity: The FIT-PLESE randomized controlled trial.

BACKGROUND: Women with obesity and infertility are counseled to lose weight prior to conception and infertility treatment to improve pregnancy rates and birth outcomes, although confirmatory evidence from randomized trials is lacking. We assessed whether a preconception intensive lifestyle intervention with acute weight loss is superior to a weight neutral intervention at achieving a healthy live birth. METHODS AND FINDINGS: In this open-label, randomized controlled study (FIT-PLESE), 379 women with obesity (BMI ≥ 30 kg/m2) and unexplained infertility were randomly assigned in a 1:1 ratio to 2 preconception lifestyle modification groups lasting 16 weeks, between July 2015 and July 2018 (final follow-up September 2019) followed by infertility therapy. The primary outcome was the healthy live birth (term infant of normal weight without major anomalies) incidence. This was conducted at 9 academic health centers across the United States. The intensive group underwent increased physical activity and weight loss (target 7%) through meal replacements and medication (Orlistat) compared to a standard group with increased physical activity alone without weight loss. This was followed by standardized empiric infertility treatment consisting of 3 cycles of ovarian stimulation/intrauterine insemination. Outcomes of any resulting pregnancy were tracked. Among 191 women randomized to standard lifestyle group, 40 dropped out of the study before conception; among 188 women randomized to intensive lifestyle group, 31 dropped out of the study before conception. All the randomized women were included in the intent-to-treat analysis for primary outcome of a healthy live birth. There were no significant differences in the incidence of healthy live births [standard 29/191(15.2%), intensive 23/188(12.2%), rate ratio 0.81 (0.48 to 1.34), P = 0.40]. Intensive had significant weight loss compared to standard (-6.6 ± 5.4% versus -0.3 ± 3.2%, P < 0.001). There were improvements in metabolic health, including a marked decrease in incidence of the metabolic syndrome (baseline to 16 weeks: standard: 53.6% to 49.4%, intensive 52.8% to 32.2%, P = 0.003). Gastrointestinal side effects were significantly more common in intensive. There was a higher, but nonsignificant, first trimester pregnancy loss in the intensive group (33.3% versus 23.7% in standard, 95% rate ratio 1.40, 95% confidence interval [CI]: 0.79 to 2.50). The main limitations of the study are the limited power of the study to detect rare complications and the design difficulty in finding an adequate time matched control intervention, as the standard exercise intervention may have potentially been helpful or harmful. CONCLUSIONS: A preconception intensive lifestyle intervention for weight loss did not improve fertility or birth outcomes compared to an exercise intervention without targeted weight loss. Improvement in metabolic health may not translate into improved female fecundity. TRIAL REGISTRATION: ClinicalTrials.gov NCT02432209.

Effect of an Active vs Expectant Management Strategy on Successful Resolution of Pregnancy Among Patients With a Persisting Pregnancy of Unknown Location: The ACT or NOT Randomized Clinical Trial.

Importance: Women with an early nonviable pregnancy of unknown location are at high risk of ectopic pregnancy and its inherent morbidity and mortality. Successful and timely resolution of the gestation, while minimizing unscheduled interventions, are important priorities. Objective: To determine if active management is more effective in achieving pregnancy resolution than expectant management and whether the use of empirical methotrexate is noninferior to uterine evacuation followed by methotrexate if needed. Design, Setting, and Participants: This multicenter randomized clinical trial recruited 255 hemodynamically stable women with a diagnosed persisting pregnancy of unknown location between July 25, 2014, and June 4, 2019, in 12 medical centers in the United States (final follow up, August 19, 2019). Interventions: Eligible patients were randomized in a 1:1:1 ratio to expectant management (n = 86), active management with uterine evacuation followed by methotrexate if needed (n = 87), or active management with empirical methotrexate using a 2-dose protocol (n = 82). Main Outcomes and Measures: The primary outcome was successful resolution of the pregnancy without change from initial strategy. The primary hypothesis tested for superiority of the active groups combined vs expectant management, and a secondary hypothesis tested for noninferiority of empirical methotrexate compared with uterine evacuation with methotrexate as needed using a noninferiority margin of -12%. Results: Among 255 patients who were randomized (median age, 31 years; interquartile range, 27-36 years), 253 (99.2%) completed the trial. Ninety-nine patients (39%) declined their randomized allocation (26.7% declined expectant management, 48.3% declined uterine evacuation, and 41.5% declined empirical methotrexate) and crossed over to a different group. Compared with patients randomized to receive expectant management (n = 86), women randomized to receive active management (n = 169) were significantly more likely to experience successful pregnancy resolution without change in their initial management strategy (51.5% vs 36.0%; difference, 15.4% [95% CI, 2.8% to 28.1%]; rate ratio, 1.43 [95% CI, 1.04 to 1.96]). Among active management strategies, empirical methotrexate was noninferior to uterine evacuation followed by methotrexate if needed with regard to successful pregnancy resolution without change in management strategy (54.9% vs 48.3%; difference, 6.6% [1-sided 97.5% CI, -8.4% to ∞]). The most common adverse event was vaginal bleeding for all of the 3 management groups (44.2%-52.9%). Conclusions and Relevance: Among patients with a persisting pregnancy of unknown location, patients randomized to receive active management, compared with those randomized to receive expectant management, more frequently achieved successful pregnancy resolution without change from the initial management strategy. The substantial crossover between groups should be considered when interpreting the results. Trial Registration: ClinicalTrials.gov Identifier: NCT02152696.

Sleep Habits of Women With Infertility.

CONTEXT: Sleep plays important roles in metabolic and reproductive function, and polycystic ovary syndrome (PCOS) is associated with sleep disturbances, including increased prevalence of obstructive sleep apnea. OBJECTIVE: We sought to evaluate sleep parameters in infertile women with PCOS compared with women with unexplained infertility (UI) and identify risk factors for disturbed sleep. METHODS: At private and academic ambulatory gynecology and infertility practices, we evaluated a prospective cohort of women diagnosed with PCOS or UI from 2 randomized clinical trials. We included 1603 infertile women enrolled in 2 concurrent randomized clinical trials. The main outcome measures were self-reported sleep measures. RESULTS: Sleep duration <6 hours (6.1% vs 2.7%; P < .001), habitual snoring (37.8% vs 19.0%; P < .001), and clinical sleepiness (12.0% vs 8.6%; P < .026) were more common in women with PCOS than those with UI. After adjusting for covariates, PCOS and elevated fasting insulin were associated (P = .010) with clinical symptoms of obstructive sleep apnea (OSA) diagnosis, whereas PCOS, elevated insulin (P = .003), WC >88 cm (P = .003), and current smoking (P = .012) were associated with habitual snoring. Clinical depression score (P < .001) and PCOS diagnosis (P = .002) were associated with perceived daytime sleepiness. Short sleep duration and clinical symptoms of OSA were not associated with conception and live birth rates. CONCLUSION: Infertile women with PCOS more commonly report sleep disturbances than those with UI. Markers of insulin resistance are associated with previous diagnosis of OSA, habitual snoring, and short sleep duration. The presence of clinical symptoms of OSA or short sleep duration does not affect fertility treatment response.

Lifestyle modifications alone or combined with hormonal contraceptives improve sexual dysfunction in women with polycystic ovary syndrome.

OBJECTIVE: To describe the prevalence of female sexual dysfunction in a well-defined polycystic ovary syndrome (PCOS) population, and to assess the impact of common PCOS treatments on sexual function. DESIGN: Secondary analysis of a randomized controlled trial, oral contraceptive pills and weight loss in PCOS. SETTING: Two academic medical centers. PATIENTS: Women with PCOS (N = 114) defined by the Rotterdam criteria. INTERVENTIONS: Continuous oral contraceptive pill (OCP) or intensive lifestyle modification (Lifestyle) or the combination (Combined) for 16 weeks. MAIN OUTCOME MEASURES: Change in Female Sexual Function Index (FSFI) and Female Sexual Distress Scale-Revised (FSDS-R) scores after 16 weeks. RESULTS: There was no change in total FSFI or FSDS-R score in any treatment group; however, an increase in the FSFI desire domain subscore was observed in the Lifestyle and Combined treatments, indicating improved sexual desire over the 16-week period. Overall, 33 participants (28.9%) met criteria for sexual dysfunction by FSFI criteria (baseline score ≤26.55). Among this group, FSFI score improved after 16 weeks of Lifestyle and Combined treatments. There was no change in prevalence of sexual dysfunction in treatment groups at 16 weeks. Use of OCPs did not alter FSFI scores. CONCLUSION(S): Female sexual dysfunction is highly prevalent among women with PCOS. Our findings suggest that common treatments for PCOS, including intensive lifestyle modification and the combination of intensive lifestyle modification and OCPs, have the potential to improve sexual function in these women; the mechanism for these improvements is likely multifactorial. CLINICAL TRIAL REGISTRATION NUMBER: NCT00704912.

Effects of Oral Contraception and Lifestyle Modification on Incretins and TGF-ß Superfamily Hormones in PCOS.

OBJECTIVE: To examine the effects of common treatments for polycystic ovary syndrome (PCOS) on a panel of hormones (reproductive/metabolic). DESIGN: Secondary analysis of blood from a randomized controlled trial of three 16-week preconception interventions designed to improve PCOS-related abnormalities: continuous oral contraceptive pills (OCPs, N = 34 subjects), intensive lifestyle modification (Lifestyle, N = 31), or a combination of both (Combined, N = 29). MATERIALS AND METHODS: Post-treatment levels of activin A and B, inhibin B, and follistatin (FST), as well as Insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 2 (IGFBP-2), glucagon, glucagon-like peptide 1 (GLP-1) and 2, and oxyntomodulin were compared to baseline, and the change from baseline in these parameters were correlated with outcomes. RESULTS: Oral contraceptive pill use was associated with a significant suppression in activin A, inhibin A, and anti-mullerian hormone (AMH), but a significant increase in FST. IGF-1, IGFBP-2, glucagon, and GLP-2 levels were significantly decreased. Oxyntomodulin was profoundly suppressed by OCPs (ratio of geometric means: 0.09, 95% confidence interval [CI]: 0.05, 0.18, P < 0.001). None of the analytes were significantly affected by Lifestyle, whereas the effects of Combined were similar to OCPs alone, although attenuated. Oxyntomodulin was significantly positively associated with the change in total ovarian volume (rs = 0.27; 95% CI: 0.03, 0.48; P = 0.03) and insulin sensitivity index (rs = 0.48; 95% CI: 0.27, 0.64; P < 0.001), and it was inversely correlated with change in area under the curve (AUC) glucose [rs = -0.38; 95% CI: -0.57, -0.16; P = 0.001]. None of the hormonal changes were associated with live birth, only Activin A was associated with ovulation (risk ratio per 1 ng/mL increase in change in Activin A: 6.0 [2.2, 16.2]; P < 0.001). CONCLUSIONS: In women with PCOS, OCPs (and not Lifestyle) affect a wide variety of reproductive/metabolic hormones, but their treatment response does not correlate with live birth.

Timing of exposure to gonadotropins has differential effects on the conceptus: evidence from a mouse model†.

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications.

Intrauterine insemination performance characteristics and post-processing total motile sperm count in relation to live birth for couples with unexplained infertility in a randomised, multicentre clinical trial.

STUDY QUESTION: Are intrauterine insemination (IUI) performance characteristics and post-processing total motile sperm count (TMC) related to live birth rate in couples with unexplained infertility? SUMMARY ANSWER: Patient discomfort with IUI and lower inseminate TMC were associated with a reduced live birth rate, while time from hCG injection to IUI, sperm preparation method and ultrasound guidance for IUI were not associated with live birth success. WHAT IS ALREADY KNOWN: We previously determined that some baseline characteristics of couples with unexplained infertility, including female age, duration of infertility, history of prior loss and income, were related to live birth rate across a course of ovarian stimulation and IUI treatment. However, the relationship between treatment outcomes and per-cycle characteristics, including ultrasound guidance for IUI, timing of IUI relative to hCG injection, difficult or painful IUI and inseminate TMC, are controversial, and most prior investigations have not evaluated live birth outcome. STUDY DESIGN, SIZE, DURATION: This was a secondary analyses of 2462 cycles from the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) clinical trial. This prospective, randomised, multicentre clinical trial determined live birth rates following IUI after ovarian stimulation with clomiphene citrate, letrozole or gonadotropins in 854 couples with unexplained infertility. It was conducted between 2011 and 2014, and couples could undergo up to four consecutive treatment cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMIGOS was an NIH-sponsored Reproductive Medicine Network trial conducted at 12 clinical sites. Participants were women with unexplained infertility who were between 18 and 40 years of age. Cluster-weighted generalised estimating equations (GEE), which account for informative clustering of multiple IUI treatment cycles within the same patient, were used to determine associations between IUI performance characteristics, including inseminate TMC, and live birth rate. Efficiency curves were also generated to examine the relationship between inseminate TMC and live birth rate. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for treatment group and baseline factors previously associated with live birth across a course of OS-IUI treatment, patient discomfort during the IUI procedure was associated with a reduction in live birth rate (aRR 0.40 (0.16-0.96)). Time from hCG trigger injection to IUI was not significantly associated with outcome. Higher TMC was associated with greater live birth rate (TMC 15.1-20.0 million (14.8%) compared to ≤5 million (5.5%)) (aRR 2.09 (1.31-3.33)). However, live births did occur with TMC ≤ 1 million (5.1%). LIMITATIONS, REASONS FOR CAUTION: This investigation is a secondary analysis, and AMIGOS was not designed to address the present question. Since timed intercourse was allowed as part of the AMIGOS trial, we cannot rule out the possibility that any given pregnancy resulted from intercourse rather than IUI. WIDER IMPLICATIONS OF THE FINDINGS: Most factors associated with the performance of IUI were not significantly related to obtaining live birth. Our findings suggest that higher TMC inseminated leads to an increase in live birth rate up to TMC ~20 million. However, there may be no reasonable threshold below which live birth is not possible with IUI. STUDY FUNDING/COMPETING INTEREST(S): Funding was received through grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): U10 HD077680, U10 HD39005, U10 HD38992, U10 HD27049, U10 HD38998, U10 HD055942, HD055944, U10 HD055936 and U10 HD055925. This research was made possible by funding by the American Recovery and Reinvestment Act. Dr Hansen reports grants from NIH/NICHD and Yale University during the conduct of the study, grants from Roche Diagnostics and grants from Ferring International Pharmascience Center US outside the submitted work. Dr Peck reports support from Ferring Pharmaceuticals outside the submitted work. Dr Coward has nothing to disclose. Dr Wild reports grants from NICHD during the conduct of the study. Dr Trussell has nothing to disclose. Dr Krawetz reports grants from NICHD during the conduct of the study, grants from Merck and support from Taylor and Frances and from Springer, outside the submitted work. Dr Diamond reports grants from NIH/NICHD, Yale University, during the conduct of the study and support from Advanced Reproductive Care AbbVie, Bayer and ObsEva, outside the submitted work. Dr Legro reports support from Bayer, Kindex, Odega, Millendo and AbbVie and grants and support from Ferring, outside the submitted work. Dr Coutifaris reports grants from NICHD/NIH and personal fees from American Society for Reproductive Medicine, outside the submitted work. Dr Alvero has nothing to disclose. Dr Robinson reports grants from NIH during the conduct of the study. Dr Casson has nothing to disclose. Dr Christman reports grants from NICHD during the conduct of the study. Dr Santoro reports grants from NIH during the conduct of the study. Dr Zhang reports grants from NIH during the conduct of the study and support from Shangdong University outside the submitted work. TRIAL REGISTRATION NUMBER: n/a.

Pregnancy registry: three-year follow-up of children conceived from letrozole, clomiphene, or gonadotropins.

OBJECTIVE: To study the development of children conceived from non-IVF infertility treatments consisting of gonadotropins, clomiphene, or letrozole. DESIGN: Prospective cohort study. SETTING: U.S. academic health centers. PATIENT(S): Children of women with polycystic ovary syndrome who conceived with letrozole (LTZ) or clomiphene (CC) in the PPCOS II study or women with unexplained infertility (AMIGOS study) who conceived with LTZ, CC, or gonadotropin (GN). INTERVENTION(S): Longitudinal annual follow-up from birth to age 3. MAIN OUTCOME MEASURE(S): Scores from Ages and Stages Developmental Questionnaire (ASQ), MacArthur-Bates Communicative Development Inventory (MCDI), and annual growth. RESULT(S): One hundred eighty-five children from 160 families participated in at least one follow-up evaluation from the two infertility trials. Most multiple gestations in the follow-up study resulted from GN treatment (n = 14) followed by CC (n = 6) and LTZ (n = 3). There were no significant differences among the three groups at any time point with respect to abnormal scores on the ASQ. On the MCDI Words and Gestures, the LTZ group scored significantly higher than the GN group for most items (phrases, early gestures, later gestures, and total gestures). Children in the CC group scored significantly higher than the GN group for the later gestures and total gestures items. CONCLUSION(S): Differences in growth and cognitive developmental rates among children conceived with first-line infertility therapies, including LTZ, are relatively minor and likely due to differences in multiple pregnancy rates.

Families with children resulting from ART: psychosocial and financial implications.

STUDY QUESTION: What are the psychosocial and financial issues experienced among families with children 2-12 years of age conceived by ART? SUMMARY ANSWER: Our results suggest that families with children, 2-12 years of age, conceived via ART are doing well, although impacts were identified on parents of twins and higher-order multiples. WHAT IS KNOWN ALREADY: Multiple births have been associated with higher morbidity and mortality of children, as well as financial costs to families and society. STUDY DESIGN SIZE DURATION: This study was an assessment of familial response to birth of singletons, twins and higher order multiples at child's ages of 2-12. PARTICIPANTS/MATERIALS SETTING METHODS: Semi-structured interviews and surveys were conducted with mothers (n = 348) and fathers (n = 338) of singletons, twins and higher-order multiple gestations who received fertility services. MAIN RESULTS AND THE ROLE OF CHANCE: No significant differences were observed between the groups in domains of primary caregiving or parental separation/divorce. Impacts were identified on parent's ability to maintain employment. The revised 15-item scores of the Impact on Family Scale were significantly lower, reflecting more negative impacts, among families with twins (beta = -2.6, 95% confidence interval (CI), -4.7, -0.5, P = 0.014) and multiples (beta = -7.4, 95% CI, -10.4, -4.5, P < 0.001) than among families with singletons. Similarly, the Parenting Stress Index total scores were significantly lower among families with twins and multiples, indicating greater levels of stress, when compared to those with singletons. In addition, the Beck Depression Inventory total score were significantly higher for twins and multiples, and the Child Behaviour Checklist for ages 1.5-5 total problem score was significantly higher for twins when compared to singletons. LIMITATIONS REASONS FOR CAUTION: The study was limited to families who received fertility treatment and constitutes a population that was well educated and had higher incomes. Additionally, interview data was self-reported. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Institutes of Health (NIH)/Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U10 HD39005 (to M.P.D.), U10 HD077680 (to K.R.H.), U10 HD077844 (to A.Z.), U10 HD077841 (to M.C.), U10 HD38992 (to R.S.L.), U10 HD27049 (to C.C.), U10 HD055925 (to H.Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NICHD or NIH.Dr Virginia Miller-no conflicts; Dr Michael P. Diamond-NIH Funding, AbbVie, Bayer and ObsEva Funding; Board of Directors and Stockholder for Advanced Reproductive Care; Dr Karl R. Hansen-Yale University/Reproductive Medicine Network/NICHD, Roche Diagnostics and Ferring International Pharmascience Center US funding; Dr Anne Steiner-NIH Funding; Dr Marcelle I. Cedars-no conflicts; Dr Richard Legro-consultant for Ogeda, Millendo, Kindex and Bayer; Ferring and Astra Zeneca funding; Dr Stephen A. Krawetz-no conflicts; Dr Christos Coutifaris-NIH Funding; Dr Hao Huang-no conflicts; Dr Nanette Santoro-no conflicts; Dr Heping Zhang-NIH Funding. TRIAL REGISTRATION NUMBER: N/A.

Androgenicity and fertility treatment in women with unexplained infertility.

OBJECTIVE: To determine whether biochemical or clinical markers of androgenic activity predict live birth rate with ovarian stimulation in the unexplained infertility population. DESIGN: Secondary analysis of the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) clinical trial. SETTING: Multicenter university-based clinical practices. PATIENT(S): Nine hundred couples with unexplained infertility were included. Women were 18-40 years old with regular menses, a normal uterine cavity, at least one patent fallopian tube, and a male partner with ≥5 million motile sperm. Women were randomized to receive gonadotropin, clomiphene, or letrozole with IUI for four or fewer four treatment cycles. Women were evaluated for biochemical (total testosterone, DHEAS, and free androgen index) and clinical markers of androgenic activity (sebum, acne, and hirsutism). Multivariable logistic regression models adjusting for treatment group, maternal age, and body mass index were performed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcome was live birth. Secondary outcomes included conception, clinical pregnancy, and pregnancy loss. RESULT(S): When comparing 900 women in the AMIGOS trial based on quartiles of serum TT, women were of younger age, higher body mass index, and higher waist circumference with increasing TT. Increasing quartiles of TT also showed increasing DHEAS and free androgen index values. Serum androgens were not associated with outcomes of live birth, conception, clinical pregnancy, or pregnancy loss. Clinical androgen markers were not associated with pregnancy outcomes. CONCLUSION(S): In a randomized cohort of women with unexplained infertility, biochemical and clinical measures of androgens did not predict live birth rate after ovarian stimulation treatment. CLINICAL TRIAL REGISTRATION NUMBER: NCT 01044862.

Epigenetic changes and assisted reproductive technologies.

Children conceived by Assisted Reproductive Technologies (ART) are at moderately increased risk for a number of undesirable outcomes, including low birth weight. Whether the additional risk is associated with specific procedures used in ART or biological factors that are intrinsic to infertility has been the subject of much debate, as has the mechanism by which ART or infertility might influence this risk. The potential effect of ART clinical and laboratory procedures on the gamete and embryo epigenomes heads the list of mechanistic candidates that might explain the association between ART and undesirable clinical outcomes. The reason for this focus is that the developmental time points at which ART clinical and laboratory procedures are implemented are precisely the time points at which large-scale reorganization of the epigenome takes place during normal development. In this manuscript, we review the many human studies comparing the epigenomes of ART children with children conceived in vivo, as well as assess the potential of individual ART clinical and laboratory procedures to alter the epigenome.

Human chromatin remodeler cofactor, RNA interactor, eraser and writer sperm RNAs responding to obesity.

In the United States almost 33% of adults are considered obese (BMI > 30 kg/m2). Both animal models and to a lesser extent human studies, have associated BMI, a measure of obesity, with alterations in sperm DNA methylation and RNAs. Sperm RNAs from the Assessment of Multiple Gestations from Ovarian Stimulation trial, were isolated and sequenced. A Generalized Linear Model identified 487 BMI associated human sperm RNA elements (short exon-sized sequences). They partitioned into four patterns; a continual increase with BMI, increase once obese (BMI>30 kg/m2); a steady decrease with BMI; and decrease once overweight (BMI 25 - 30 kg/m2). Gene Ontology revealed a unique relationship between BMI and transcripts associated with chromosome organization, adipogenesis, cellular stress and obesity-related inflammation. Coregulatory networks linked by Chromatin remodeler cofactors, RNA interactors, Erasers and Writers (CREWs) were uncovered to reveal a hierarchical epigenetic response pathway.