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Background: Pregnancy complications are associated with increased risk of development of cardiometabolic diseases and earlier mortality. However, much of the previous research has been limited to White pregnant participants. We aimed to investigate pregnancy complications in association with total and cause-specific mortality in a racially diverse cohort and evaluate whether associations differ between Black and White pregnant participants. Methods: The Collaborative Perinatal Project was a prospective cohort study of 48 197 pregnant participants at 12 US clinical centers (1959–1966). The Collaborative Perinatal Project Mortality Linkage Study ascertained participants’ vital status through 2016 with linkage to the National Death Index and Social Security Death Master File. Adjusted hazard ratios (aHRs) for underlying all-cause and cause-specific mortality were estimated for preterm delivery (PTD), hypertensive disorders of pregnancy, and gestational diabetes/impaired glucose tolerance (GDM/IGT) using Cox models adjusted for age, prepregnancy body mass index, smoking, race and ethnicity, previous pregnancies, marital status, income, education, previous medical conditions, site, and year. Results: Among 46 551 participants, 45% (21 107 of 46 551) were Black, and 46% (21 502 of 46 551) were White. The median time between the index pregnancy and death/censoring was 52 years (interquartile range, 45–54). Mortality was higher among Black (8714 of 21 107 [41%]) compared with White (8019 of 21 502 [37%]) participants. Overall, 15% (6753 of 43 969) of participants had PTD, 5% (2155 of 45 897) had hypertensive disorders of pregnancy, and 1% (540 of 45 890) had GDM/IGT. PTD incidence was higher in Black (4145 of 20 288 [20%]) compared with White (1941 of 19 963 [10%]) participants. The following were associated with all-cause mortality: preterm spontaneous labor (aHR, 1.07 [95% CI, 1.03–1.1]); preterm premature rupture of membranes (aHR, 1.23 [1.05–1.44]); preterm induced labor (aHR, 1.31 [1.03–1.66]); preterm prelabor cesarean delivery (aHR, 2.09 [1.75–2.48]) compared with full-term delivery; gestational hypertension (aHR, 1.09 [0.97–1.22]); preeclampsia or eclampsia (aHR, 1.14 [0.99–1.32]) and superimposed preeclampsia or eclampsia (aHR, 1.32 [1.20–1.46]) compared with normotensive; and GDM/IGT (aHR, 1.14 [1.00–1.30]) compared with normoglycemic. P values for effect modification between Black and White participants for PTD, hypertensive disorders of pregnancy, and GDM/IGT were 0.009, 0.05, and 0.92, respectively. Preterm induced labor was associated with greater mortality risk among Black (aHR, 1.64 [1.10–2.46]) compared with White (aHR, 1.29 [0.97–1.73]) participants, while preterm prelabor cesarean delivery was higher in White (aHR, 2.34 [1.90–2.90]) compared with Black (aHR, 1.40 [1.00–1.96]) participants. Conclusions: In this large, diverse US cohort, pregnancy complications were associated with higher mortality nearly 50 years later. Higher incidence of some complications in Black individuals and differential associations with mortality risk suggest that disparities in pregnancy health may have life-long implications for earlier mortality.

To evaluate the associations between preconception sleep characteristics and shift work with fecundability and live birth.Secondary analysis of the Effects of Aspirin in Gestation and Reproduction study, a preconception cohort.Four US academic medical centers.Women aged 18-40 with a history of 1-2 pregnancy losses who were attempting to conceive again.Not applicable.We evaluated baseline, self-reported sleep duration, sleep midpoint, social jetlag, and shift work among 1,228 women who were observed for ≤6 cycles of pregnancy attempts to ascertain fecundability. We ascertained live birth at the end of follow up via chart abstraction. We estimated fecundability odds ratios (FORs) using discrete, Cox proportional hazards models and risk ratios (RRs) for live birth using log-Poisson models.Sleep duration ≥9 vs. 7 to8 hours (FOR: 0.81, 95% confidence interval [CI], 0.61; 1.08), later sleep midpoints (3rd tertile vs. 2nd tertile: FOR: 0.85; 95% CI, 0.69, 1.04) and social jetlag (continuous per hour; FOR: 0.93, 95% CI: 0.86, 1.00) were not associated with reduced fecundability. In sensitivity analyses, excluding shift workers, sleep duration ≥9 vs. 7 to8 hours (FOR: 0.62; 95% CI, 0.42; 0.93) was associated with low fecundability. Night shift work was not associated with fecundability (vs. non-night shift work FOR: 1.17, 95% CI, 0.96; 1.42). Preconception sleep was not associated with live birth.Overall, there does not appear to be a strong association between sleep characteristics, fecundability, and live birth. Although these findings may suggest weak and imprecise associations with some sleep characteristics, our findings should be evaluated in larger cohorts of women with extremes of sleep characteristics.Clinicaltrials.gov NCT00467363.

Background: Diet is increasingly recognized as an important determinant of human fertility, with most research focused on specific nutrients or food groups. However, there has been limited assessment of the effect of dietary patterns on fertility. Objectives: We evaluated the association between 4 dietary patterns [the alternative Mediterranean Diet (aMed), the Healthy Eating Index-2010 (HEI-2010), the Danish Dietary Guidelines (DDGI), and the Dietary Inflammatory Index (DII)] and fecundability in 2 preconception cohorts of couples trying to conceive: SF (SnartForaeldre.dk) in Denmark and PRESTO (Pregnancy Study Online) in North America. Methods: Participants completed a baseline questionnaire on sociodemographic, anthropometric, and lifestyle factors and, 10 d later, a validated cohort-specific FFQ. We used data from these respective FFQs to calculate adherence to each dietary pattern. Participants completed bimonthly follow-up questionnaires for ≤12 mo or until pregnancy, whichever came first. We restricted analyses to 3429 SF and 5803 PRESTO participants attempting pregnancy for ≤6 cycles at enrollment. We used proportional probabilities regression models to estimate fecundability ratios (FRs) and 95% CIs, adjusting for potential confounders. Results: Greater DII, indicative of a less anti-inflammatory diet (i.e., poorer diet quality), was associated with reduced fecundability in both SF and PRESTO (DII ≥ -1.5 compared with < -3.3: FR: 0.83; 95% CI: 0.71, 0.97 and FR: 0.82; 95% CI: 0.73, 0.93, respectively). In PRESTO, greater adherence to the aMed or to the HEI-2010 was associated with greater fecundability. In SF, there was no appreciable association between the aMed and fecundability, whereas greater adherence to the DDGI was associated with greater fecundability. Conclusions: In prospective preconception cohort studies from Denmark and North America, higher-quality diets, including diets lower in inflammatory effects, were associated with greater fecundability.

To examine whether semen parameters are associated with live birth among couples seeking infertility treatment after accounting for semen parameter variability.Folic Acid and Zinc Supplementation Trial (FAZST) prospective cohort.Four US reproductive endocrinology and infertility care study centers, 2013-2017.Couples (n = 2,369) seeking fertility consultations at 4 US infertility care study centers.Semen volume, pH, sperm viability, morphology, progressive and total motility, concentration, count, and total and progressive motile count assessed at baseline and at 2, 4, and 6 months after enrollment.Log-binomial models stratified by fertility treatment received (in vitro fertilization [IVF], intrauterine insemination [IUI], ovulation induction [OI], or no treatment) estimated risk differences (RDs) between semen parameter quartiles and live birth and accounted for multiple semen assessments per person. We accounted for abstinence time, the biological interdependence of semen parameters, and potential selection bias because of loss to follow-up.Among couples using OI only or no treatment, 39% had a live birth, and relative to the highest quartile, the lowest quartiles of morphology (RD, -19 [95% CI, -23 to -15] per 100 couples), motility (RD, -13 [95% CI, -17 to -9]), concentration (RD, -22 [95% CI, -26 to -19]), and total motile count (RD, -18 [95% CI, -22 to -14]) were associated with fewer live births. For IUI, 26% had a live birth, and the lowest quartiles of volume (RD, -6 [95% CI, -11 to -0.4]), concentration (RD, -6 [95% CI, -11 to -0.1]), count (RD, -10 [95% CI, -15 to -4]), and total motile count (RD, -7 [95% CI, -13 to -1]) were associated with fewer live births. For IVF, 61% had a live birth, and only morphology (Q1 RD, -7 [95% CI, -14 to 0.2]; Q2 RD, -10 [95% CI, -17 to -2.2]) was associated with live birth.Semen parameters are critical in couples undergoing OI/IUI. Only low morphology was important for live birth after IVF. Although data supporting the use of semen parameters are fragmented across differing populations, current findings are generalizable across the range of male fertility and couple fertility treatments, providing evidence about which semen parameters are most relevant in which settings.NCT#01857310.

BackgroundPremenstrual symptoms, including food cravings, are often a regular complaint among menstruating women. However, existing evidence regarding the biological mechanisms by which these food cravings occur remains unclear. Inflammation may play an essential role in the occurence of these food cravings before menstruation.PurposeThe purpose of the present study was to examine the associations between inflammatory markers and the risk of moderate/severe food cravings while accounting for changes in hormone levels and stress across the menstrual cycle.MethodsThe BioCycle Study followed women (n=259) aged 18-44 for two menstrual cycles. Food cravings (via questionnaire) were assessed up to four times per cycle. Each assessment corresponded to menses and mid-follicular, ovulation, and luteal phases of the menstrual cycle. A wide range of cytokine and chemokine levels (hsCRP, GCSF, GMCSF, IL-4, IL-6, RANTES, MIP1B, etc.) were assessed in blood samples collected at up to 8 visits per cycle, with visits timed using fertility monitors. Cravings for chocolate, sweets, salty, and other foods, and changes in appetite were determined to estimate the odds of moderate or severe cravings. Associations between inflammatory markers and risk of reporting a moderate/severe craving symptom at each cycle visit was determined using weighted generalized linear models (e.g., marginal structural models). Models were adjusted for age, BMI, and race, as well as time-varying covariates such as estradiol, stress, leptin, and total energy intake, and accounted for repeated measures (i.e., multiple cycles per woman). Both inflammatory markers and reports of cravings were modeled to account for variation at each visit.ResultsAn association between higher inflammatory biomarkers such as hsCRP, GCSF, GMCSF, IL-4, IL-6, RANTES, MIP1B, and increased risk of moderate/severe cravings were identified across the menstrual cycle |all risk ratio>0.8, all CIs range>0.7-0.9|. hsCRP retained statistical significance after false discovery rate correction with chocolate, sweet, and salty cravings, while GCSF, GMCSF, IL-6, and RANTES retained significance with chocolate and sweet cravings only.ConclusionThe results suggest a potential role of inflammation in food cravings and appetite changes across the menstrual cycle.

Background Epigenetic mechanisms may underlie associations between maternal caffeine consumption and adverse childhood metabolic outcomes. However, limited studies have examined neonate DNA methylation (DNAm) patterns in the context of preconception or prenatal exposure to caffeine metabolites. Objective We examined preconception and pregnancy caffeine exposure with DNAm alterations in neonate cord blood (n = 378). Design In a secondary analysis of the Effects of Aspirin in Gestation and Reproduction Trial (EAGeR), we measured maternal caffeine, paraxanthine, and theobromine concentrations from stored serum collected preconception (on average 2 months before pregnancy) and at 8 weeks of gestation. In parallel, self-reported caffeinated beverage intake was captured via administration of questionnaires and daily diaries. We profiled DNAm from the cord blood buffy coat of singletons using the MethylationEPIC BeadChip. We assessed associations of maternal caffeine exposure and methylation β-values using multivariable robust linear regression. A false discovery rate (FDR) correction was applied using the Benjamini-Hochberg method. Results In preconception the majority of women reported consuming one or fewer servings/day on average and caffeine and paraxanthine metabolite levels were 88 and 36 µmol/L, respectively. Preconception serum caffeine metabolites were not associated with individual CpG sites (FDR > 5%), though pregnancy theobromine was associated with DNAm at cg09460369 near RAB2A (β = 0.028; SE = 0.005; FDR P = 0.012). Preconception self-reported caffeinated beverage intake compared to no intake was associated with DNAm at cg09002832 near GLIS3 (β = -0.013; SE = 0.002; FDR P = 0.036). No associations with self-reported intake during pregnancy were found. Conclusions Few effects of maternal caffeine exposure on neonate methylation differences in leukocytes were identified in this relatively low caffeine consumption population.Clinical Trial Registry: #NCT00467363.

Objective To determine if 6-month folic acid (5 mg) and zinc (30 mg) supplementation impacts sperm DNA methylation patterns. Design A multicenter, double-blind, block randomized, placebo-controlled trial titled “The Folic Acid and Zinc Supplementation Trial (FAZST).” Setting Infertility care centers. Patient(s) Male partners (18 years and older) from heterosexual couples (female partners aged 18–45 years) seeking fertility treatment were recruited. Intervention(s) Men were randomized 1:1 to receive folic acid (5 mg) and elemental zinc (30 mg) (n = 713) or a matching placebo (n = 757) daily for 6 months. Main Outcome Measure(s) Sperm DNA methylation was analyzed using the EPIC methylation array (Illumina) at 6 months. Differential sperm DNA methylation was assessed at multiple levels (regional, single cytosine phosphate guanine, etc.). We additionally assessed the impact of supplementation on epigenetic age. Result(s) No significant differences were identified between the treatment and placebo groups although some trends appeared to be present. To determine if these trends were noteworthy, we implemented various permutations and found that the patterns we identified were no more than would be expected by random chance. Conclusion(s) The data presented here strongly suggest that this supplementation regimen is not effective at altering sperm DNA methylation. These data comport well with previous findings from the FAZST study that found no impact of supplementation on basic semen analysis parameters or live birth. Clinical Trial Registration Number ClinicalTrials.gov Identifier: NCT01857310.

STUDY QUESTION Is preconception leukocyte telomere length associated with fecundability, pregnancy loss and live birth among women attempting natural conception with a history of 1–2 prior pregnancy losses? SUMMARY ANSWER Preconception leukocyte telomere length is not associated with fecundability, pregnancy loss or live birth. WHAT IS KNOWN ALREADY As women increasingly delay childbearing, accessible preconception biomarkers to predict pregnancy outcomes among women seeking natural conception could improve preconception counseling. Findings of small case–control or cross-sectional studies suggest that telomere attrition is associated with adverse pregnancy outcomes among women undergoing fertility treatment, but prospective studies in non-clinical populations are lacking. STUDY DESIGN, SIZE, DURATION Participants included 1228 women aged 18–40 years with a history of 1–2 prior pregnancy losses who were recruited at four university medical centers (2006–2012). PARTICIPANTS/MATERIALS, SETTING, METHODS Preconception leukocyte telomere length was measured at baseline using PCR and reported as a ratio (T/S) in relation to population-specific standard reference DNA. Women were followed for up to six cycles while attempting to conceive. Associations of telomere length with fecundability, live birth and pregnancy loss were estimated using discrete Cox proportional hazards models and log-binomial models. MAIN RESULTS AND THE ROLE OF CHANCE After adjustment for age, BMI, smoking and other factors, preconception telomere length was not associated with fecundability (Q4 vs Q1 FOR = 1.00; 95% CI = 0.79, 1.27), live birth (Q4 vs Q1 RR = 1.00; 95% CI = 0.85, 1.19), or pregnancy loss (Q4 vs Q1 RR = 1.12; 95% CI = 0.78, 1.62). LIMITATIONS, REASONS FOR CAUTION Telomere length was measured in leukocytes, which is an accessible tissue in women attempting natural conception but may not reflect telomere length in oocytes. Most women were younger than 35 years, limiting our ability to evaluate associations among older women. Participants had a history of 1–2 prior pregnancy losses; therefore, our findings may not be widely generalizable. WIDER IMPLICATIONS OF THE FINDINGS Despite prior research suggesting that telomere length may be associated with pregnancy outcomes among women seeking fertility treatment, our findings suggest that leukocyte telomere length is not a suitable biomarker of pregnancy establishment or maintenance among women attempting natural conception. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (National Institutes of Health, Bethesda, MD, USA; contract numbers HHSN267200603423, HHSN267200603424 and HHSN267200603426). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER The trial was registered with ClinicalTrials.gov, number NCT00467363.

PURPOSE: To examine associations between objectively measured sleep duration and sleep timing with odds of completion of an in vitro fertilization (IVF) cycle. METHODS: This prospective cohort study enrolled 48 women undergoing IVF at a large tertiary medical center between 2015 and 2017. Sleep was assessed by wrist-worn actigraphy, 1–2 weeks prior to initiation of the IVF cycle. Reproductive and IVF cycle data and demographic and health information were obtained from medical charts. Sleep duration, midpoint, and bedtime were examined in relation to IVF cycle completion using logistic regression models, adjusted for age and anti-Müllerian hormone levels. A sub-analysis excluded women who worked non-day shifts to control for circadian misalignment. RESULTS: The median age of all participants was 33 years, with 29% of women >35 years. Ten women had an IVF cycle cancelation prior to embryo transfer. These women had shorter sleep duration, more nocturnal awakenings, lower sleep efficiency, and later sleep timing relative to those who completed their cycle. Longer sleep duration was associated with lower odds of uncompleted IVF cycle (OR = 0.88; 95%CI 0.78, 1.00, per 20-min increment of increased sleep duration). Women with later sleep midpoint and later bedtime had higher odds of uncompleted cycle relative to those with earlier midpoint and earlier bedtime; OR = 1.24; 95%CI 1.09, 1.40 and OR = 1.33; 95%CI 1.17, 1.53 respectively, for 20-min increments. These results were independent of age, anti-Müllerian hormone levels, or sleep duration, and remained significant after exclusion of shift-working women. CONCLUSIONS: Shorter sleep duration and later sleep timing increase the odds of uncompleted cycles prior to embryo transfer.

Shorter gestational age (GA) is a risk factor of developmental delay. GA is usually estimated clinically from last menstrual period and ultrasound. DNA methylation (DNAm) estimates GA using sets of cytosine-guanine-sites coupled with a clock algorithm. Therefore, DNAm-estimated GA may better reflect biological maturation. A DNAm GA greater than clinical GA, known as gestational age acceleration (GAA), may indicate epigenetic maturity and holds potential as an early biomarker for developmental delay risk. We used data from the Upstate KIDS Study to examine associations of DNAm GA and developmental delay within the first 3 years based on the Ages & Stages Questionnaire® (n = 1010). We estimated DNAm GA using two clocks specific to the Illumina Methylation EPIC 850K, the Haftorn clock and one developed from the Effects of Aspirin in Gestation and Reproduction study, in which women were followed to detect pregnancy at the earliest time possible. Among singletons, each week increase in DNAm GA was protective for overall delay (odds ratio:0.74; 95% confidence interval:0.61–0.90) and delay in all domains except for problem-solving skills. Among twins, we observed similar point estimates but lower precision. Results were similar for clinical GA. GAA was largely not associated with developmental delays. In summary, either DNAm GA or clinical GA at birth, but not epigenetic maturity (i.e. GAA), was associated with decreased odds of developmental delay in early childhood. Our study does not support using DNAm GA or GAA as separate risk factors for future risk of developmental delay within the first 3 years of age.

Context Ovarian stimulation (OS) increases pregnancy rates but can cause multiple folliculogenesis and multiple pregnancy. Objective To determine whether the probability of pregnancy differs in OS cycles with mono- vs multifolliculogenesis in women with unexplained infertility (UI). Design Secondary analysis of a multicenter, randomized controlled trial: Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation with 3 treatment arms: gonadotropins, clomiphene, or letrozole, combined with intrauterine insemination. Women were categorized as having either 1 or ≥ 2 mature follicles (≥ 16 mm). Relative risk (RR) and 95% CIs for clinical pregnancy and live birth by number of follicles were estimated using generalized linear models adjusted for age, body mass index, years of infertility, and history of prior live birth. Setting 12 US-based clinical sites. Participants Normally cycling women aged 18 to 40 years with a normal uterine cavity and at least 1 patent fallopian tube. Male partners with ≥ 5 million total motile sperm. Interventions Gonadotropins, clomiphene, or letrozole with insemination Main Outcome Measure(s) Clinical pregnancy rates (CPR) and live birth rates (LBR). Results A single mature follicle > 16 mm resulted in lower CPR (RR, 0.70; 95% CI, 0.54-0.90) and LBR (RR, 0.67; 95% CI, 0.51-0.89) compared with ≥ 2 mature follicles. When stratified by treatment modality, no association of follicle number with CPR or LBR was observed for letrozole or clomiphene, but women using gonadotropins had lower CPR and LBR with monofolliculogenesis. Conclusion In couples undergoing gonadotropin treatment for UI, monofolliculogenesis following OS is related to a lower rate of live birth.

Background Cadmium is an endocrine disrupting chemical that affects the hypothalamic-pituitary-gonadal axis. Though evidence suggests its potential role in altering androgen synthesis and metabolic pathways that are characteristic of polycystic ovary syndrome (PCOS), its relation in healthy women of reproductive age is largely unknown. As women with mild sub-clinical features of PCOS who do not meet the diagnostic criteria of PCOS may still experience reduced fecundability, investigating associations between cadmium and PCOS-phenotypes among healthy women may provide unique insight into the reproductive implications for many on the PCOS spectrum. Therefore, the objective of this study was to evaluate associations between cadmium and androgens, anti-Müllerian hormone (AMH), and metabolic markers in women of reproductive age. Methods This was a prospective cohort study of 251 healthy premenopausal women without self-reported PCOS (mean age 27.3 years and BMI 24.1 kg/m2). Cadmium was measured in blood collected at baseline. Reproductive hormones and metabolic markers were measured in fasting serum 8 times per menstrual cycle for 2 cycles. Linear mixed models and Poisson regression with a robust error variance were used to examine associations between cadmium and reproductive hormones and metabolic markers and anovulation, respectively. Results Median (interquartile range) blood cadmium concentrations at baseline were 0.30 (0.19–0.43) µg/L. Higher levels of testosterone (2.2 %, 95 % confidence interval [CI] 0.4, 4.1), sex hormone-binding globulin (2.9 %, 95 % CI 0.5, 5.5), and AMH (7.7 %, 95 % CI 1.1, 14.9) were observed per 0.1 µg/L increase in cadmium concentrations. An 18 % higher probability of a mild PCOS-phenotype (95 % CI 1.06, 1.31), defined by a menstrual cycle being in the highest quartile of cycle-averaged testosterone and AMH levels, was also found per 0.1 µg/L increase in cadmium levels. No associations were observed for insulin and glucose. These findings were consistent even after analyses were restricted to non-smokers or further adjusted for dietary factors to account for potential sources of exposure. Conclusions Overall, among healthy reproductive-aged women, cadmium was associated with endocrine features central to PCOS, but not with metabolic markers. These suggest its potential role in the hormonal milieu associated with PCOS even at low levels of exposure.

We have recently shown that sperm epigenetic age (SEA), a surrogate measure of biological aging in sperm, is associated with couples' time-to-pregnancy (TTP). Advanced SEA was also observed among smokers, suggesting its susceptibility to environmental exposures. Therefore, we assessed the association between urinary phthalate metabolites and SEA in male partners of couples planning to conceive among the general population.The Longitudinal Investigation of Fertility and the Environment (LIFE) Study was a prospective multi-site and general population cohort study of couples who were interested in becoming pregnant. Among male partners (n = 333), eleven urinary phthalate metabolites were measured and SEA was previously developed using Super Learner ensemble algorithm. Multivariable linear regression was used to evaluate associations of SEA with individual metabolites. Bayesian kernel machine regression (BKMR), quantile g-computation (qgcomp) and weighted quantile sum (WQS) models were used for mixture analyses. Covariates included were BMI, cotinine, race and urinary creatinine.In the single metabolite multivariate analyses, nine (82%) phthalate metabolites displayed positive trends with SEA (range: 0.05-0.47 years). Of these metabolites, advanced SEA was significantly associated with interquartile range increases in exposure of three phthalates [MEHHP (β = 0.23, 95% CI: 0.03, 0.43, p = 0.03), MMP (β = 0.24, 95% CI: 0.01, 0.47, p = 0.04), and MiBP (β = 0.47, 95% CI: 0.14, 0.81, p = 0.01)]. Additionally, in BKMR and qgcomp (p = 0.06), but not WQS models, phthalate mixtures showed an overall positive trend with SEA, with MiBP, MMP and MBzP as major drivers of the mixture effects.This is the first study that combined single exposure and mixture models to associate male phthalate exposures with advanced epigenetic aging of sperm in men planning to conceive among the general population. Our findings suggest that phthalate exposure may contribute to the acceleration of biological aging of sperm.

STUDY QUESTION Are insufficient 25-hydroxyvitamin D (25(OH)D) concentrations, and other markers of vitamin D metabolism, associated with premenstrual symptoms in healthy women with regular menstrual cycles? SUMMARY ANSWER 25(OH)D insufficiency was associated with specific physical premenstrual symptoms, while no associations were observed with psychological symptoms or with other markers of vitamin D metabolism. WHAT IS KNOWN ALREADY Prior studies evaluating vitamin D and premenstrual symptoms have yielded mixed results, and it is unknown whether 25(OH)D insufficiency and other markers of vitamin D metabolism are associated with premenstrual symptoms. STUDY DESIGN, SIZE, DURATION We used two cohorts of women with regular menstrual cycles; 1191 women aged 18–40 years in EAGeR (cross-sectional analysis of a prospective cohort within a randomized trial) and 76 women aged 18–44 years in BioCycle (prospective cohort). In EAGeR, premenstrual symptoms over the previous year were assessed at baseline, whereas in BioCycle, symptoms were assessed prospectively at multiple points over two menstrual cycles with symptoms queried over the previous week. In both cohorts, symptomatology was assessed via questionnaire regarding presence and severity of 14 physical and psychological symptoms the week before and after menses. Both studies measured 25(OH)D in serum. We also evaluated the association of additional markers of vitamin D metabolism and calcium homeostasis, including intact parathyroid hormone (iPTH), calcium (Ca), fibroblast growth factor 23 (FGF23), and 1,25 dihydroxyvitamin D (1,25(OH)2D) with premenstrual symptoms in the BioCycle cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS One cohort of women actively seeking pregnancy (Effects of Aspirin in Gestation and Reproduction (EAGeR)) and one cohort not seeking pregnancy (BioCycle) were evaluated. Log-binomial regression was used to estimate risk ratios (RR) and 95% CIs for associations between insufficient 25(OH)D ( MAIN RESULTS AND THE ROLE OF CHANCE 25(OH)D insufficiency was associated with increased risk of breast fullness/tenderness (EAGeR RR 1.27, 95% CI 1.03, 1.55; BioCycle RR 1.37, 95% CI 0.56, 3.32) and generalized aches and pains (EAGeR RR 1.33, 95% CI 1.01, 1.78; BioCycle 1.36, 95% CI 0.41, 4.45), though results were imprecise in the BioCycle study. No associations were observed between insufficient 25(OH)D and psychological symptoms in either cohort. In BioCycle, iPTH, Ca, FGF23, and 1,25(OH) 2D were not associated with any premenstrual symptoms. LIMITATIONS, REASONS FOR CAUTION Results from the EAGeR study were limited by the study design, which assessed both 25(OH)D at baseline and individual premenstrual symptoms over the past year at the baseline. As such, reverse causality is a potential concern. Though premenstrual symptoms were assessed prospectively in the BioCycle cohort, the power was limited due to small sample size. However, results were fairly consistent across both studies. WIDER IMPLICATIONS OF THE FINDINGS Serum 25(OH)D may be associated with risk and severity of specific physical premenstrual symptoms. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (Contract nos. HHSN267200603423, HHSN267200603424, and HHSN267200603426). JG.R. and D.L.K. have been funded by the NIH Medical Research Scholars Program, a public–private partnership jointly supported by the NIH and generous contributions to the Foundation for the NIH by the Doris Duke Charitable Foundation (Grant #2014194), the American Association for Dental Research, the Colgate Palmolive Company, Genentech, and other private donors. For a complete list, visit the foundation website at http://www.fnih.org. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER Clinicaltrials.gov NCT00467363.

Context Diets high in plant-based protein have gained popularity due to increasing health concerns regarding consumption of animal products. Though links between intakes of certain protein-rich foods and reproductive disorders have been suggested, the relationship of overall animal and vegetable proteins with reproductive hormones among reproductive-aged women is unknown. Objective To evaluate the associations between the intake of dietary protein with reproductive hormones and sporadic anovulation among reproductive-aged women. Design A prospective cohort study, 2005–2007. Setting University at Buffalo, western New York, United States. Participants A total of 259 premenopausal women (18–44 years) without dietary restrictions. Main Outcome Measure(s) Serum reproductive hormones were determined up to 8 times per cycle for 2 cycles. Protein intake was assessed the day prior to hormone assessment at 4 visits/cycle using 24-hour recalls. Results Overall, 84% of participants met the recommended dietary allowance for total protein set for reproductive-aged women. Neither total nor animal protein intake were associated with reproductive hormones or anovulation. However, vegetable protein intake in the lowest tertile was associated with lower luteal phase progesterone (-18.0%, 95% confidence interval [CI] -30.2, -3.6), higher follicle-stimulating hormone (3.8%, 95% CI 0.2, 7.6), and a higher risk of anovulation (risk ratio [RR] 2.53, 95% CI 1.21, 5.26), compared with the middle tertile. Nuts and seeds were the only protein-rich foods associated with an elevated risk of anovulation (RR 2.12, 95% CI 1.17, 3.85). Conclusions Findings suggest that among women who meet the recommended dietary allowance for total protein, low intake of vegetable, but not animal, protein may disturb normal ovulatory function.

Background It has been suggested that premenstrual syndrome (PMS) may derive from either elevated oxidative stress or reduced antioxidant vitamin levels in the body; however, these relationships have been minimally studied in a large cohort of healthy women. Our objective was to estimate the association between serum concentrations of antioxidant vitamins (A, C, and E) and markers of oxidative stress (F2-isoprostane) with symptoms and severity of PMS. Methods The BioCycle study was a prospective cohort study following 259 healthy premenopausal women aged 18–44 years for up to 2 menstrual cycles. Frequency/severity of 20 PMS symptoms were assessed via questionnaires 4 times/cycle, and antioxidant vitamins and oxidative stress biomarkers were measured up to 8 times/cycle to correspond with specific cycle phases. Generalized linear models were used to estimate associations between mean antioxidant concentrations and oxidative stress biomarkers with PMS symptoms and severity; linear mixed models were used to evaluate associations with symptom severity scores within groups (e.g. depression, cravings, pain). Results Higher concentrations of serum antioxidant vitamins were largely not associated with prevalence or severity of PMS symptoms. Though a few associations were observed, only associations between mean γ-tocopherol and decreased odds of swelling of the hands/feet survived adjustment for multiple comparisons (OR 0.33, 95% CI 0.16, 0.65, per ug/dL). However, F2-isoprostanes were associated with prevalence and severity of several symptoms specifically related to depression and cravings (depression score β = 0.07, 95% CI 0.02, 0.12, per 10 ug/dL; cravings score β = 0.16, 95% CI 0.10, 0.22, per 10 ug/dL), as well as with classification of PMS severity (OR 1.07, 95% CI 1.01, 1.14, per 10 pg/dL), with these associations surviving adjustment for false discovery rate. Conclusions F2-isoprostanes, but not antioxidant vitamins, were associated with select PMS symptoms, as well as symptom and severity categories. Specific symptom relationships merit further research.

Anti-müllerian hormone (AMH) is an established marker of ovarian reserve that decreases with age. Though the pool of ovarian follicles is established during fetal development, impacts of in utero exposures on AMH are uncertain. Thus, we sought to evaluate associations of in utero exposures with AMH of adult daughters with a prospective cohort study of adult daughters at university medical centers. Women noted their mother’s reported use of diethylstilbestrol (DES), vitamins, tobacco, alcohol, and caffeine during pregnancy, and their mother’s occupation during pregnancy. All participants were reproductive age women (18–40 years) enrolled in the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial. Serum AMH concentrations were measured at baseline prior to conception and categorized using clinical guidelines. Multinomial regression models estimated associations between each exposure and high (>3.5 ng/mL) and low (

BackgroundVegetarian diets are becoming increasingly popular in the USA. Limited research has examined the health consequences of vegetarian diets during pregnancy. We comprehensively examined associations of vegetarianism during pregnancy with maternal and neonatal outcomes.MethodsWe used data from the Eunice Kennedy Shriver National Institute of Child Health and Human Development’s Fetal Growth Studies–Singletons, a prospective multi-site cohort of 1948 low-risk pregnant women of four races/ethnicities (White, Black, Hispanic, Asian/Pacific Islander) in the USA (2009–2013). Vegetarianism was self-reported and also defined based on dietary patterns measured using a self-administered first-trimester food-frequency questionnaire (full [lacto-ovo and vegan], pesco-, semi- and non-vegetarians). Neonatal outcomes included birthweight and neonatal anthropometric measures, small for gestational age, small for gestational age with neonatal morbidity and preterm delivery. Maternal outcomes included gestational weight gain, gestational diabetes, hypertensive disorders of pregnancy and gestational anaemia.ResultsNinety-nine (6.2%) women self-reported being vegetarian. The diet-based definition identified 32 (2.0%) full vegetarians, 7 (0.6%) pesco-vegetarians and 301 (17.6%) semi-vegetarians. Neonates of diet-based full vegetarians had higher odds of being small for gestational age [adjusted odds ratio (ORadj) = 2.51, 95% confidence interval: 1.01, 6.21], but not of being small for gestational age with a postnatal morbidity. Full vegetarians had marginally increased the odds of inadequate second-trimester gestational weight gain (ORadj = 2.24, 95% confidence interval: 0.95, 5.27).ConclusionVegetarian diets during pregnancy were associated with constitutionally smaller neonatal size, potentially via the mothers’ reduced gestational weight gain. Notably, vegetarianism was not associated with small-for-gestational-age-related morbidities or other adverse maternal outcomes.

To determine the cost-effectiveness of planned oocyte cryopreservation (OC) as a strategy for delayed childbearing to achieve 1 or 2 live births (LB) compared with in vitro fertilization (IVF) and preimplantation genetic testing for aneuploidy (PGT-A) at advanced reproductive age.Decision tree model with sensitivity analyses using data from the Society for Assisted Reproductive Technology Clinical Outcome Reporting System and other clinical sources.Not applicable.A data-driven simulated cohort of patients desiring delayed childbearing with an ideal family size of 1 or 2 LB.Not applicable.Probability of achieving ≥1 or 2 LB, average and maximum cost per patient, cost per percentage point increase in chance of LB, and population-level cost/LB.For those desiring 1 LB, planned OC at age 33 with warming at age 43 decreased the average total cost per patient from $62,308 to $30,333 and increased the likelihood of LB from 50% to 73% when compared with no OC with up to 3 cycles of IVF/PGT-A at age 43. For those desiring 2 LB, 2 cycles of OC at age 33 and warming at age 40 yielded the lowest cost per patient and highest likelihood of achieving 2 LB ($51,250 and 77%, respectively) when compared withpursuing only 1 cycle of OC ($75,373 and 61%, respectively), no OC and IVF/PGT-A with embryo banking ($79,728 and 48%, respectively), or no OC and IVF/PGT-A without embryo banking ($79,057 and 19%, respectively). Sensitivity analyses showed that OC remained cost-effective across a wide range of ages at cryopreservation. For 1 LB, OC achieved the highest likelihood of success when pursued before age 32 and remained more effective than IVF/PGT-A when pursued before age 39, and for 2 LB, 2 cycles of OC achieved the highest likelihood of success when pursued before age 31 and remained more effective than IVF/PGT-A when pursued before age 39.Among patients planning to postpone childbearing, OC is cost-effective and increases the odds of achieving 1 or 2 LB when compared with IVF/PGT-A at a more advanced reproductive age.

We evaluated relationships between preconception adiposity and human offspring sex and sex ratio. Using data from a prospective preconception cohort nested within a randomized controlled trial based at 4 US clinical sites (2006–2012), we used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for male:female sex ratio, and log-identity regression to estimate risk differences (RDs) and 95% CIs for male and female livebirth according to preconception adiposity measures. Inverse-probability weights accounted for potential selection bias. Among 603 women attempting pregnancy, there were meaningful reductions in sex ratio for the highest category of each adiposity measure. The lowest sex ratios were observed for obesity (body mass index of ≥30, calculated as weight (kg)/height (m)2, OR = 0.48, 95% CI: 0.26, 0.88) relative to normal body mass index, and the top tertiles (tertile 3) of serum leptin (OR = 0.50, 95% CI: 0.32, 0.80) and skinfold measurements (OR = 0.50, 95% CI: 0.32, 0.79) relative to the lowest tertiles. Reductions were driven by 11–15 fewer male livebirths per 100 women (for obesity, RD = −15, 95% CI: −23, −6.7; for leptin tertile 3, RD = −11, 95% CI: −20, −3.2; and for skinfolds tertile 3, RD = −11, 95% CI: −19, −3.3). We found that relationships between preconception adiposity measures and reduced sex ratio were driven by a reduction in male births.

BackgroundComplex diseases have multifactorial etiologies making clinically actionable diagnostic markers difficult to identify. Novel tools with higher diagnostic yield and utility in driving personalized care are needed.MethodsWe utilized Illumina methylation array data from over 2400 samples to assess DNA methylation patterns in 20 distinct cell types ranging from sperm to brain as well as various disease states. We generated a simple analysis pipeline for DNA methylation data that focuses on intra-individual methylation variability within gene promoters. The analysis is designed, not to identify single causative gene alterations but instead focuses on any movement away from “healthy” methylation. This approach identifies altered regulation across multiple genes in related pathways thus enabling us to detect shifts in gene regulatory activity associated with distinct tissues and phenotypes. We explored three distinct questions in our assessment. 1) Are patterns of epigenetic variability tissue specific? 2) Do diseased tissues exhibit altered variability patterns compared to normal tissue? 3) Can epigenetic variability be detected in complex disease?ResultsUnsupervised clustering analyses established that patterns of epigenetic variability are tissue specific and that these patterns are at least as predictive of tissue type as differential methylation analysis. We demonstrated the ability to use these patterns to differentiate between healthy and diseased tissue with unsupervised clustering even in cases of complex multifactorial diseases. We applied this method to the clinical use case of male infertility and found that men undergoing intrauterine insemination (IUI) with the lowest number of epigenetically dysregulated promoters in their sperm were almost twice as likely to father a child than men participating in IUI with the highest number of dysregulated promoters (p=0.011). We saw no significant difference in birth rates between groups of men with high and low numbers of dysregulated promoters undergoing in vitro fertilization (IVF), indicating IVF as a better treatment than IUI to achieve live birth in the presence of multi-pathway dysregulation in sperm.ConclusionsThis study demonstrates that patterns of epigenetic variability can differentiate between tissue types. While intuitive, this finding has never been demonstrated previously and suggests that specific epigenetic variability patterns may be used to predict phenotypic changes in disease states as these are, by definition, functional changes to cellular phenotypes. We demonstrate that the variability of gene regulatory marks are distinct between healthy and diseased tissue. This is particularly apparent at genes known to be important to cell function of the tissue of interest. While in some cases these regional alterations can be seen across the entire genome, more often the regulatory alterations that define a pathological phenotype are restricted to genes of known importance to a particular tissue. Importantly, in the case of sperm, we found that these patterns of variability did have utility in predicting infertile patients who would conceive through intrauterine insemination (IUI). We would propose that this discriminatory ability is due to the fact that the signature can be assessed in an n-of-1 context and that the patterns of variability identify any shift away from regulatory normalcy in pathways known to be impactful in the tissue of interest, and not only assessing the presence or absence of rare genetic variants. While the data presented here are encouraging, more work needs to be performed in other tissues to determine when, and in what context, these findings could be clinically actionable.

STUDY QUESTION Do sperm mitochondrial DNA measures predict probability of pregnancy among couples in the general population? SUMMARY ANSWER Those with high sperm mitochondrial DNA copy number (mtDNAcn) had as much as 50% lower odds of cycle-specific pregnancy, and 18% lower probability of pregnancy within 12 months. WHAT IS KNOWN ALREADY Semen parameters have been found to poorly predict reproductive success yet are the most prevalent diagnostic tool for male infertility. Increased sperm mtDNAcn and mitochondrial DNA deletions (mtDNAdel) have been associated with decreased semen quality and lower odds of fertilization in men seeking fertility treatment. STUDY DESIGN, SIZE, DURATION A population-based prospective cohort study of couples discontinuing contraception to become pregnant recruited from 16 US counties from 2005 to 2009 followed for up to 16 months. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm mtDNAcn and mtDNAdel from 384 semen samples were assessed via triplex probe-based quantitative PCR. Probability of pregnancy within 1 year was compared by mitochondrial DNA, and discrete-time proportional hazards models were used to evaluate the relations with time-to-pregnancy (TTP) with adjustment for covariates. MAIN RESULTS AND THE ROLE OF CHANCE Higher sperm mtDNAcn was associated with lower pregnancy probability within 12 months and longer TTP. In unadjusted comparisons by quartile (Q), those in Q4 had a pregnancy probability of 63.5% (95% CI: 53.1% to 73.1%) compared to 82.3% (95% CI: 73.2% to 89.9%) for Q1 (P = 0.002). Similar results were observed in survival analyses adjusting for covariates to estimate fecundability odds ratios (FORs) comparing mtDNAcn in quartiles. Relative to those in Q1 of mtDNAcn, FORs (95% CI) were for Q2 of 0.78 (0.52 to 1.16), Q3 of 0.65 (0.44 to 0.96) and Q4 of 0.55 (0.37 to 0.81), and this trend of decreasing fecundability with increasing mtDNAcn quartile was statistically significant (FOR per log mtDNAcn = 0.37; P LIMITATIONS, REASONS FOR CAUTION This prospective cohort study consisted primarily of Caucasian men and women and thus large diverse cohorts are necessary to confirm the associations between sperm mtDNAcn and couple pregnancy success in other races/ethnicities. WIDER IMPLICATIONS OF THE FINDINGS Our results demonstrate that sperm mtDNAcn has utility as a biomarker of male reproductive health and probability of pregnancy success in the general population. STUDY FUNDING/COMPETING INTEREST(S) This work was funded in part by the National Institute of Environmental Health Sciences, National Institutes of Health (R01-ES028298; PI: J.R.P.) and the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (Contracts N01-HD-3-3355, N01-HD-3-3356 and N01-HD-3-3358). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A

BACKGROUND: Attaining pregnancy is conditional upon a series of complex processes, including adequately timed intercourse, ovulation, fertilization, and implantation. Anovulation is a first line treatment target for couples with difficulty conceiving and is frequently examined in studies of fecundability. OBJECTIVES: To identify whether sporadic anovulation is an important determinant of cumulative pregnancy rates and time to pregnancy among fertile women with regular menstrual cycles. METHODS: We simulated cumulative pregnancy rates and time to pregnancy for 12 consecutive menstrual cycles among 100,000 women based on data-driven probabilities of implantation, fertilization, ovulation, and intercourse occurring in the fertile window. We assumed anovulation probabilities of 1%, 8%, or 14.5% and intercourse averaging once per week, every other day, and daily. The model incorporated reductions in implantation and fertilization rates for successive cycles of non-pregnancy. RESULTS: After 12 cycles, a reduction in the per-cycle incidence of anovulation from 14.5% to 1% resulted in a 4.0% higher cumulative pregnancy rate (86.7 versus 90.7%) and similar time to pregnancy (1-cycle median difference). In contrast, increasing mean unscheduled sexual intercourse frequency from weekly to every other day was associated with a 5-cycle median reduction in time to pregnancy (weekly: 7 cycles; every other day or daily: 2 cycles) and a 28.9% increase in the cumulative pregnancy rate (weekly: 59.9%, every other day: 88.8%; daily: 91.6%). CONCLUSIONS: In presumed fertile women with regular menstrual cycles, routine investigation of anovulation may not be an informative outcome in studies of fecundability, and routine testing to ensure ovulation and treatment of anovulation are unlikely to be medically necessary. While biomarkers or cervical fluid may help time intercourse to the fertile window, time to pregnancy can also be improved through increasing the frequency of unscheduled intercourse. These findings need corroboration in large preconception time to pregnancy studies.

Preeclampsia and gestational hypertension are common complications of pregnancy associated with significant maternal and infant morbidity. Despite extensive research evaluating risk factors during pregnancy, most women who develop a hypertensive disorder of pregnancy are not considered high-risk and strategies for prevention remain elusive. We evaluated preconception blood pressure and its change into early pregnancy as novel risk markers for development of a hypertensive disorder of pregnancy. The EAGeR (Effects of Aspirin in Gestation and Reproduction) trial (2007–2011) randomized 1228 healthy women with a history of pregnancy loss to preconception-initiated low-dose aspirin versus placebo and followed participants for up to 6 menstrual cycles attempting pregnancy and throughout pregnancy if they became pregnant. Blood pressure was measured during preconception and throughout early gestation. The primary outcomes, preterm preeclampsia, term preeclampsia, and gestational hypertension, were abstracted from medical records. Among 586 women with a pregnancy >20 weeks’ gestation, preconception blood pressure levels were higher for preterm preeclampsia (87.3±6.7 mm Hg mean arterial pressure), term preeclampsia (88.3±9.8 mm Hg), and gestational hypertension (87.9±9.1 mm Hg) as compared with no hypertensive disorder of pregnancy (83.9±8.6 mm Hg). Change in blood pressure from preconception into very early pregnancy was associated with development of preeclampsia (relative risk, 1.13 [95% CI, 1.02–1.25] per 2 mm Hg increase in mean arterial pressure at 4 weeks’ gestation), particularly preterm preeclampsia (relative risk, 1.21 [95% CI, 1.01–1.45]). Randomization to aspirin did not alter blood pressure trajectory or risk of hypertension in pregnancy. Preconception blood pressure and longitudinal changes during early pregnancy are underexplored but crucial windows in the detection and prevention of hypertensive disorders of pregnancy. Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00467363.

Background Opioids are commonly prescribed to women of reproductive age, including after delivery and miscarriage. However, to our knowledge, opioid use has not been frequently studied in relation to the common reproductive complications of impaired fecundability and pregnancy. We examined the association of opioid use during the critical window of pregnancy establishment with fecundability and pregnancy loss. Methods We measured opioid use by urine screening and self-report at multiple time points during preconception and early pregnancy in a prospective cohort of women attempting conception (n=1228). The main outcomes included time to hCG-detected pregnancy and incidence of live birth and pregnancy loss. We estimated fecundability odds ratios (FOR) and risk ratios (RR) with 95% confidence intervals (CI) adjusting for sociodemographic characteristics, reproductive characteristics, and use of antidepressants, tobacco, alcohol, and marijuana. Results Prevalence of preconception opioid use was 18% (n=226 of 1228), and in early pregnancy was 5% (n=33 of 685). Opioid use while attempting pregnancy was associated with reduced fecundability (FOR: 0.71; 95% CI: 0.50, 1.0). Risk of pregnancy loss increased as opioid exposure was detected later in gestation, from the beginning of the cycle of conception (RR: 1.5; 95% CI 0.85, 2.6), to week 4 of pregnancy (RR: 2.1; 95% CI: 1.1, 4.1), and to week 4 and 8 of pregnancy (RR: 2.5; 95% CI: 1.3, 5.0). Conclusions Our results are consistent with the hypothesis that opioid exposure while trying to conceive may be harmful, even among healthy, non-opioid-dependent women. Possible risks to fecundability and pregnancy viability are relevant to patients and providers when evaluating pain management approaches.ClinicalTrials.gov registration number: #NCT00467363.

Diet, lifestyle, and psychosocial factors might influence fertility for men and women, although evidence is mixed, and couple-based approaches are needed for assessing associations with reproductive outcomes. The Impact of Diet, Exercise, and Lifestyle (IDEAL) on Fertility Study is a prospective cohort with contemporaneous detailed follow-up of female partners of men enrolled in the Folic Acid and Zinc Supplementation Trial studying couples seeking infertility treatment (2016–2019). Follow-up of men continued for 6 months, while female partners were followed for 9 months while attempting pregnancy and throughout any resulting pregnancy (up to 18 months). Longitudinal data on diet, physical activity (including measurement via wearable device), sleep, and stress were captured at multiple study visits during this follow-up. A subset of women (IDEALplus) also completed daily journals and a body fat assessment via dual-energy x-ray absorptiometry. IDEAL enrolled 920 women, and IDEALPlus enrolled 218. We demonstrated the ability to enroll women in a prospective cohort study contemporaneous to a partner-enrolled randomized trial. In combination with data collected on male partners, IDEAL data facilitates a couple-based approach to understanding associations between lifestyle factors and infertility treatment outcomes. We describe in detail the study design, recruitment, data collection, lessons learned, and baseline characteristics.

Background Riboflavin, vitamin B-6, and vitamin B-12 are key players in one-carbon metabolism as enzymatic cofactors, and deficiency of these nutrients may influence reproductive outcomes possibly through affecting reproductive hormones. Objective The goal was to investigate associations between dietary intakes of riboflavin, vitamin B-6, and vitamin B-12, and menstrual function among premenopausal women. Design This was a secondary analysis of a prospective cohort study conducted at the University at Buffalo during 2005 to 2007. Participants/setting Participants were 259 healthy, regularly menstruating women (aged 18 to 44 years) with self-reported menstrual cycles between 21 and 35 days, who were not trying to conceive, and who had not used hormonal contraception during the past 3 months. Main outcome measures Intakes of B vitamins were assessed via 24-hour dietary recalls four times per menstrual cycle for two cycles. Serum reproductive hormones and plasma homocysteine were measured eight and three times, respectively, per cycle for two cycles. Anovulatory cycles were determined by progesterone concentrations ≤5 ng/mL (15.9 nmol/L) and no observed serum luteinizing hormone peak during the mid or late luteal phase visit. Statistical analysis Weighted linear mixed regressions were used to evaluate associations between cycle-averaged B vitamin intakes and hormones and homocysteine, and generalized linear regressions for associations with anovulation. Models were adjusted for age, race, body mass index, physical activity, alternate Mediterranean diet score, intakes of total energy, protein, fiber, and folate, and percentage of energy intake from fat. Results Higher intakes of riboflavin (per 0.1 mg increase in intake) were inversely correlated with estradiol (−0.87%, 95% CI −1.67 to −0.06) and homocysteine levels (−0.61%, 95% CI −1.10 to −0.12). Higher vitamin B-6 intakes were suggestive of higher follicle-stimulating hormone, although the results were not statistically significant (0.63% difference, 95% CI −0.03 to 1.29, per 0.1 mg increase in intake; P=0.06). Small increases in testosterone and decreases in homocysteine were found with vitamin B-12 intake. No associations were observed between intake of B vitamins and a risk of sporadic anovulation. Conclusions Higher intakes of riboflavin were associated with a small decrease in serum estradiol among healthy, regularly menstruating women. Higher intakes of riboflavin and vitamin B-12 were associated with lower plasma homocysteine concentrations. Overall, riboflavin, vitamin B-6, and vitamin B-12 that are one-carbon nutrients do not appear to influence the ovarian cycle among premenopausal women.

Summary Objective Obesity has become a major, worldwide public health issue and is associated with a greater risk of adverse pregnancy outcomes. Leptin, a hormone produced by adipocytes, is elevated in individuals with obesity and may mediate the association between obesity and pregnancy outcomes. Though leptin levels during pregnancy have been associated with pregnancy outcomes, less is understood regarding preconception levels. Therefore, the objective of this study was to evaluate associations between preconception leptin levels and adverse pregnancy outcomes. Methods This was a prospective cohort study nested within a large randomized controlled trial conducted at four medical centres in the United States. A total of 1078 women completed the parent study; this analysis involved women who became pregnant during that study (n = 776). Patients were healthy women, ages 18 to 40, attempting to conceive, with 1 to 2 prior pregnancy losses. Participants were followed for less than or equal to 6 cycles while trying to conceive and throughout pregnancy if they conceived. Preconception leptin concentrations were measured in serum collected at baseline then categorized by tertiles (using the lowest as reference group). Weighted log‐binomial regression estimated risk ratios (RR) and 95% confidence intervals (CIs) for pregnancy loss, preterm delivery (PTD), gestational diabetes (GDM), and hypertensive disorders in pregnancy, adjusting for age, waist‐to‐hip ratio (WHR), and body mass index (BMI). Results The mean (SD) BMI in this cohort was 25.4 ± 6.0. GDM (RR 18.37; 95% CI, 2.39‐141.55) and hypertensive disorders of pregnancy (RR 2.35; 95% CI, 1.20‐4.61) risks were higher among women in the high tertile after adjusting for age and WHR. The associated risk persisted when adjusting for BMI for GDM but was attenuated for hypertensive disorders in pregnancy. Leptin levels were not associated with risk of pregnancy loss or PTD. Conclusions Women with higher baseline preconception leptin levels had a higher likelihood of experiencing some adverse pregnancy outcomes including GDM and hypertensive disorders of pregnancy. These findings warrant further evaluation, especially in light of the association between leptin and obesity.

STUDY QUESTION How do the calciotropic hormones (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and intact parathyroid hormone (iPTH)) vary across the menstrual cycle and do cyclic patterns of reproductive hormones (estradiol, progesterone, LH, FSH) differ by vitamin D status? SUMMARY ANSWER Calciotropic hormones vary minimally across the menstrual cycle; however, women with 25-hydroxyvitamin D below 30 ng/ml have lower mean estradiol across the menstrual cycle. WHAT IS KNOWN ALREADY Prior human studies suggest that vitamin D status is associated with fecundability, but the mechanism is unknown. Exogenous estrogens and prolonged changes in endogenous estradiol (pregnancy or menopause) influence concentrations of 25-hydroxyvitamin D. In vitro, treatment with 1,25-dihydroxyvitamin D increases steroidogenesis in ovarian granulosa cells. There are little data about changes in calciotropic hormones across the menstrual cycle or cyclic patterns of reproductive hormones by categories of vitamin D status. STUDY DESIGN, SIZE, DURATION A prospective cohort study of 89 self-identified white women aged 18–44, across two menstrual cycles. Participants were a subset of the BioCycle Study, a community-based study conducted at the University of Buffalo, 2005–2007. PARTICIPANTS/MATERIALS, SETTING, METHODS Eligible participants had self-reported regular menstrual cycles between 21 and 35 days and were not using hormonal contraception or vitamins. Early morning fasting blood samples were drawn at up to eight study visits per cycle. Visits were timed to capture information in all cycle phases. Serum samples for 89 women (N = 163 menstrual cycles) were analyzed for estradiol, progesterone, LH, FSH and 25-hydroxyvitamin D (25(OH)D). Variability in calciotropic hormones within and across menstrual cycles was assessed using intraclass correlation coefficients and non-linear mixed models. Given the relative stability of the calciotropic hormones across the menstrual cycle, non-linear mixed models were used to examine differences in the cyclic patterns of estradiol, progesterone, LH and FSH by categories of each calciotropic hormone (split at the median). These models were conducted for all ovulatory cycles (N = 142 ovulatory menstrual cycles) and were adjusted for age, BMI (measured in clinic) and self-reported physical activity. MAIN RESULTS AND THE ROLE OF CHANCE Median 25(OH)D concentration was 29.5 ng/ml (SD 8.4), and only 6% of women had vitamin D deficiency ( LIMITATIONS, REASONS FOR CAUTION Women included in this study had self-reported ‘regular’ menstrual cycles and very few were found to have 25(OH)D deficiency. This limits our ability to examine cycle characteristics, anovulation and the effects of concentrations of the calciotropic hormones found in deficient individuals. Additionally, the results may not be generalizable to women with irregular cycles, other races, or populations with a higher prevalence of vitamin D deficiency. WIDER IMPLICATIONS OF THE FINDINGS These findings support current clinical practice that does not time testing for vitamin D deficiency to the menstrual cycle phase. We find that women with lower vitamin D status (lower 25(OH)D or higher iPTH) have lower mean concentrations of estradiol across the menstrual cycle. Although this study cannot identify a mechanism of action, further in vitro work or clinical trials may help elucidate the biologic mechanisms linking calciotropic and reproductive hormones. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Intramural Research Programs of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (contract numbers: HHSN275200403394C, HHSN275201100002I and Task 1 HHSN27500001) and the National Institute of Environmental Health Sciences. There are no competing interests.

Systematic reviews and meta-analyses are essential for drawing conclusions regarding etiologic associations between exposures or interventions and health outcomes. Observational studies comprise a substantive source of the evidence base. One major threat to their validity is residual confounding, which may occur when component studies adjust for different sets of confounders, fail to control for important confounders, or have classification errors resulting in only partial control of measured confounders. We present the confounder matrix-an approach for defining and summarizing adequate confounding control in systematic reviews of observational studies and incorporating this assessment into meta-analyses. First, an expert group reaches consensus regarding the core confounders that should be controlled and the best available method for their measurement. Second, a matrix graphically depicts how each component study accounted for each confounder. Third, the assessment of control adequacy informs quantitative synthesis. We illustrate the approach with studies of the association between short interpregnancy intervals and preterm birth. Our findings suggest that uncontrolled confounding, notably by reproductive history and sociodemographics, resulted in exaggerated estimates. Moreover, no studies adequately controlled for all core confounders, so we suspect residual confounding is present, even among studies with better control. The confounder matrix serves as an extension of previously published methodological guidance for observational research synthesis, enabling transparent reporting of confounding control and directly informing meta-analysis so that conclusions are drawn from the best available evidence. Widespread application could raise awareness about gaps across a body of work and allow for more valid inference with respect to confounder control.

Salivary steroid immunoassays are widely used in psychoneuroendocrinological studies of menstrual cycle phase, puberty, and menopause. Though manufacturers advertise their assays as suitable, they have not been rigorously validated for these purposes. We collated data from eight menstrual cycle studies across >1,200 women and >9,500 time points. Seven studies collected saliva and one collected serum. All assayed estradiol and progesterone and had an independent measure of cycle phase (LH-surge, menstrual onset). In serum, cycle phase measures strongly predicted steroid concentrations. In saliva, cycle phase poorly predicted estradiol values, which showed an upward bias compared to expectations from serum. For salivary progesterone, predictability from cycle phase was mixed. Widely used enzyme-linked assays performed poorly, while LC-MS/MS performed better. Imputing the population-average serum steroid changes from cycle phase may yield more valid values of hormonal changes for an independent person than directly assessing their hormone levels using salivary immunoassays.

Despite research indicating that sleep disorders influence reproductive health, the effects of sleep on reproductive hormone concentrations are poorly characterized. We prospectively followed 259 regularly menstruating women across one to two menstrual cycles (the BioCycle Study, 2005–2007), measuring fasting serum hormone concentrations up to eight times per cycle. Women provided information about daily sleep in diaries and chronotype and night/shift work on a baseline questionnaire. We evaluated percent differences in mean hormone concentrations, the magnitude of shifts in the timing and amplitude of hormone peaks, and the risk for sporadic anovulation associated with self-reported sleep patterns and night/shift work. We estimated chronotype scores – categorizing women below and above the interquartile range (IQR) as “morning” and “evening” chronotypes, respectively. For every hour increase in daily sleep duration, mean estradiol concentrations increased by 3.9% (95% confidence interval [CI] 2.0, 5.9%) and luteal phase progesterone by 9.4% (CI 4.0, 15.2%). Receiving less than 7 hours of sleep per day was associated with slightly earlier rises in peak levels for several hormones. Women reporting night/shift work (n = 77) had lower testosterone relative to women employed without night/shift work (percent difference: −9.9%, CI −18.4, −0.4%). Women with morning chronotypes (n = 47) had earlier rises in estradiol during their cycles and potentially an earlier rise in luteinizing hormone. Compared to those who had intermediate chronotypes, women with evening chronotypes (n = 42) had a later luteinizing hormone peak of borderline statistical significance. A reduced risk for sporadic anovulation was suggested, but imprecise, for increasing hours of daily sleep leading up to ovulation (risk ratio 0.79, CI 0.59, 1.06), while an imprecise increased risk was observed for women with morning chronotypes (risk ratio 2.50, CI 0.93, 6.77). Sleep-related hormonal changes may not greatly alter ovarian function in healthy women, but have the potential to influence gynecologic health.

Background Although fatty acids are involved in critical reproductive processes, the relationship between specific fatty acids and fertility is uncertain. We investigated the relationship between preconception plasma fatty acids and pregnancy outcomes. Methods We included 1,228 women attempting pregnancy with one to two previous pregnancy losses from the EAGeR trial (2007-2011). Plasma fatty acids were measured at baseline. We used log-binomial regression to assess associations between fatty acids and pregnancy, pregnancy loss, and live birth, adjusting for age, race, smoking, BMI, physical activity, income, parity, treatment arm, and cholesterol. Results Although total saturated fatty acids (SFAs) were not associated with pregnancy outcomes, 14:0 (myristic acid; relative risk [RR] = 1.10, 95% confidence interval [CI] = 1.02, 1.19, per 0.1% increase) and 20:0 (arachidic acid; RR = 1.05, 95% CI = 1.01, 1.08, per 0.1% increase) were positively associated with live birth. Findings suggested a positive association between total monounsaturated fatty acids (MUFAs) and pregnancy and live birth and an inverse association with loss. Total polyunsaturated fatty acids (PUFAs) were associated with lower probability of pregnancy (RR = 0.97, 95% CI = 0.95, 1.00) and live birth (RR = 0.96, 95% CI = 0.94, 0.99), and increased risk of loss (RR = 1.10, 95% CI = 1.00, 1.20), per 1% increase. Trans fatty acids and n-3 fatty acids were not associated with pregnancy outcomes. Conclusions Preconception total plasma MUFAs were positively associated with pregnancy and live birth. PUFAs were inversely associated with pregnancy outcomes. Specific SFAs were associated with a higher probability of live birth. Our results suggest that fatty acids may influence pregnancy outcomes.

Background Women who experience pregnancy loss are especially prone to high stress, though the effects of stress on reproductive outcomes in this vulnerable population are unknown. We assessed relationships between perceived stress and hormones, anovulation, and fecundability among women with prior loss. Methods One thousand two hundred fourteen women with 1-2 prior losses were followed for ≤6 cycles while attempting pregnancy and completed end-of-cycle stress assessments. For cycles 1 and 2, women also collected daily urine and completed daily perceived stress assessments. We assessed anovulation via. an algorithm based on human chorionic gonadotropin (hCG), pregnanediol-3-glucuronide (PdG), luteinizing hormone (LH), and fertility monitor readings. Pregnancy was determined via. hCG. Adjusted weighted linear mixed models estimated the effect of prospective phase-varying (menses, follicular, periovulatory, and luteal) perceived stress quartiles on estrone-1-glucuronide (E1G), PdG, and LH concentrations. Marginal structural models accounted for time-varying confounding by hormones and lifestyle factors affected by prior stress. Poisson and Cox regression estimated risk ratios and fecundability odds ratios of cycle-varying stress quartiles on anovulation and fecundability. Models were adjusted for age, race, body mass index (BMI), parity, and time-varying caffeine, alcohol, smoking, intercourse, and pelvic pain. Results Women in the highest versus lowest stress quartile had lower E1G and PdG concentrations, a marginally higher risk of anovulation [1.28; 95% confidence interval (CI) = 1.00, 1.63], and lower fecundability (0.71; 95% CI = 0.55, 0.90). Conclusion Preconception perceived stress appears to adversely affect sex steroid synthesis and time to pregnancy. Mechanisms likely include the effects of stress on ovulatory function, but additional mechanisms, potentially during implantation, may also exist.