First and second trimester urinary metabolic profiles and fetal growth restriction: an exploratory nested case-control study within the infant development and environment study.

BACKGROUND: Routine prenatal care fails to identify a large proportion of women at risk of fetal growth restriction (FGR). Metabolomics, the comprehensive analysis of low molecular weight molecules (metabolites) in biological samples, can provide new and earlier biomarkers of prenatal health. Recent research has suggested possible predictive first trimester urine metabolites correlating to fetal growth restriction in the third trimester. Our objective in this current study was to examine urinary metabolic profiles in the first and second trimester of pregnancy in relation to third trimester FGR in a US population from a large, multi-center cohort study of healthy pregnant women. METHODS: We conducted a nested case-control study within The Infant Development and the Environment Study (TIDES), a population-based multi-center pregnancy cohort study. We identified 53 cases of FGR based on the AUDIPOG [Neonatal growth - AUDIPOG [Internet]. [cited 29 Nov 2016]. Available from: http://www.audipog.net/courbes_morpho.php?langue=en ] formula for birthweight percentile considering maternal height, age, and prenatal weight, as well as infant sex, gestational age, and birth rank. Cases were matched to 106 controls based on study site, maternal age (± 2 years), parity, and infant sex. NMR spectroscopy was used to assess concentrations of four urinary metabolites that have been previously associated with FGR (tyrosine, acetate, formate, and trimethylamine) in first and second trimester urine samples. We fit multivariate conditional logistic regression models to estimate the odds of FGR in relation to urinary concentrations of these individual metabolites in the first and second trimesters. Exploratory analyses of custom binned spectroscopy results were run to consider other potentially related metabolites. RESULTS: We found no significant association between the relative concentrations of each of the four metabolites and odds of FGR. Exploratory analyses did not reveal any significant differences in urinary metabolic profiles. Compared with controls, cases delivered earlier (38.6 vs 39.8, p < 0.001), and had lower birthweights (2527 g vs 3471 g, p < 0.001). Maternal BMI was similar between cases and controls. CONCLUSIONS: First and second trimester concentrations of urinary metabolites (acetate, formate, trimethylamine and tyrosine) did not predict FGR. This inconsistency with previous studies highlights the need for more rigorous investigation and data collection in this area before metabolomics can be clinically applied to obstetrics.

Polymorphisms in the estrogen receptor alpha gene (ESR1), daily cycling estrogen and mammographic density phenotypes.

BACKGROUND: Single nucleotide polymorphisms (SNPs) involved in the estrogen pathway and SNPs in the estrogen receptor alpha gene (ESR1 6q25) have been linked to breast cancer development, and mammographic density is an established breast cancer risk factor. Whether there is an association between daily estradiol levels, SNPs in ESR1 and premenopausal mammographic density phenotypes is unknown. METHODS: We assessed estradiol in daily saliva samples throughout an entire menstrual cycle in 202 healthy premenopausal women in the Norwegian Energy Balance and Breast Cancer Aspects I study. DNA was genotyped using the Illumina Golden Gate platform. Mammograms were taken between days 7 and 12 of the menstrual cycle, and digitized mammographic density was assessed using a computer-assisted method (Madena). Multivariable regression models were used to study the association between SNPs in ESR1, premenopausal mammographic density phenotypes and daily cycling estradiol. RESULTS: We observed inverse linear associations between the minor alleles of eight measured SNPs (rs3020364, rs2474148, rs12154178, rs2347867, rs6927072, rs2982712, rs3020407, rs9322335) and percent mammographic density (p-values: 0.002-0.026), these associations were strongest in lean women (BMI, ≤23.6 kg/m2.). The odds of above-median percent mammographic density (>28.5 %) among women with major homozygous genotypes were 3-6 times higher than those of women with minor homozygous genotypes in seven SNPs. Women with rs3020364 major homozygous genotype had an OR of 6.46 for above-median percent mammographic density (OR: 6.46; 95 % Confidence Interval 1.61, 25.94) when compared to women with the minor homozygous genotype. These associations were not observed in relation to absolute mammographic density. No associations between SNPs and daily cycling estradiol were observed. However, we suggest, based on results of borderline significance (p values: 0.025-0.079) that the level of 17ß-estradiol for women with the minor genotype for rs3020364, rs24744148 and rs2982712 were lower throughout the cycle in women with low (<28.5 %) percent mammographic density and higher in women with high (>28.5 %) percent mammographic density, when compared to women with the major genotype. CONCLUSION: Our results support an association between eight selected SNPs in the ESR1 gene and percent mammographic density. The results need to be confirmed in larger studies.

First trimester phthalate exposure and anogenital distance in newborns.

STUDY QUESTION: Is first trimester phthalate exposure associated with anogenital distance (AGD), a biomarker of prenatal androgen exposure, in newborns? SUMMARY ANSWER: Concentrations of diethylhexyl phthalate (DEHP) metabolites in first trimester maternal urine samples are inversely associated with AGD in male, but not female, newborns. WHAT IS KNOWN ALREADY: AGD is a sexually dimorphic measure reflecting prenatal androgen exposure. Prenatal phthalate exposure has been associated with shorter male AGD in multiple animal studies. Prior human studies, which have been limited by small sample size and imprecise timing of exposure and/or outcome, have reported conflicting results. STUDY DESIGN, SIZE, DURATION: The Infant Development and the Environment Study (TIDES) is a prospective cohort study of pregnant women recruited in prenatal clinics in San Francisco, CA, Minneapolis, MN, Rochester, NY and Seattle, WA in 2010-2012. Participants delivered 787 infants; 753 with complete data are included in this analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Any woman over 18 years old who was able to read and write English (or Spanish in CA), who was <13 weeks pregnant, whose pregnancy was not medically threatened and who planned to deliver in a study hospital was eligible to participate. Analyses include all infants whose mothers provided a first trimester urine sample and who were examined at or shortly after birth. Specific gravity (SpG) adjusted concentrations of phthalate metabolites in first trimester urine samples were examined in relation to genital measurements. In boys (N = 366), we obtained two measures of anogenital distance (AGD) (anoscrotal distance, or AGDAS and anopenile distance, AGDAP) as well as penile width (PW). In girls (N = 373), we measured anofourchette distance (AGDAF) and anoclitoral distance (AGDAC). We used multivariable regression models that adjusted for the infant's age at exam, gestational age, weight-for-length Z-score, time of day of urine collection, maternal age and study center. MAIN RESULTS AND THE ROLE OF CHANCE: Three metabolites of DEHP were significantly and inversely associated with both measures of boys' AGD. Associations (ß, 95% confidence interval (CI)) between AGDAS and (log10) SpG-adjusted phthalate concentrations were: -1.12 (-2.16, -0.07) for mono-2-ethylhexyl phthalate (MEHP), -1.43, (-2.49, -0.38) for mono-2-ethyl-5-oxohexyl phthalate (MEOHP), and -1.28 (-2.29, -0.27) for mono-2-ethyl-5-hydroxyhexyl (MEHHP). Associations were of similar magnitude for AGDAP. Associations were weaker and not statistically significant for PW. No other phthalate metabolites were associated with any genital measurement in boys. No phthalate metabolites were associated with either AGD measure in girls. LIMITATIONS, REASONS FOR CAUTION: Exposure assessment was based on a single first trimester urine sample, which may have introduced exposure misclassification. In addition, significant between-center differences suggest that this measurement is difficult to standardize. WIDER IMPLICATIONS OF THE FINDINGS: Our findings are consistent with multiple rodent studies and most human studies which were far smaller. The data we report here suggest that even at current low levels, environmental exposure to DEHP can adversely affect male genital development resulting in reproductive tract changes that may impact reproductive health later in life. These findings have important implications for public policy since most pregnant women are exposed to this ubiquitous chemical. STUDY FUNDING/COMPETING INTERESTS: Funding for TIDES was provided by the following grants from the National Institute of Environmental Health Sciences: R01ES016863-04 and R01 ES016863-02S4. The authors report no conflict of interest.

A factor analysis approach to examining relationships among ovarian steroid concentrations, gonadotrophin concentrations and menstrual cycle length characteristics in healthy, cycling women.

STUDY QUESTION: How are ovarian steroid concentrations, gonadotrophins and menstrual cycle characteristics inter-related within normal menstrual cycles? SUMMARY ANSWER: Within cycles, measures of estradiol production are highly related to one another, as are measures of progesterone production; however, the two hormones also show some independence from one another, and measures of cycle length and gonadotrophin concentrations show even greater independence, indicating minimal integration within cycles. WHAT IS KNOWN ALREADY: The menstrual cycle is typically conceptualized as a cohesive unit, with hormone levels, follicular development and ovulation all closely inter-related within a single cycle. Empirical support for this idea is limited, however, and to our knowledge, no analysis has examined the relationships among all of these components simultaneously. STUDY DESIGN, SIZE, DURATION: A total of 206 healthy, cycling Norwegian women participated in a prospective cohort study (EBBA-I) over the duration of a single menstrual cycle. Of these, 192 contributed hormonal and cycle data to the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects provided daily saliva samples throughout the menstrual cycle from which estradiol and progesterone concentrations were measured. FSH and LH concentrations were measured in serum samples from three points in the same menstrual cycle and cycle length characteristics were calculated based on hormonal data and menstrual records. A factor analysis was conducted to examine the underlying relationships among 22 variables derived from the hormonal data and menstrual cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Six rotated factors emerged, explaining 80% of the variance in the data. Of these, factors representing estradiol and progesterone concentrations accounted for 37 and 13% of the variance, respectively. There was some association between measures of estradiol and progesterone production within cycles; however, cycle length characteristics and gonadotrophin concentrations showed little association with any measure of ovarian hormone concentrations. LIMITATIONS, REASONS FOR CAUTION: Our summary measures of ovarian hormones may be imprecise in women with extremely long or short cycles, which could affect the patterns emerging in the factor analysis. Given that we only had data from one cycle on each woman, we cannot address how cycle characteristics may covary within individual women across multiple cycles. WIDER IMPLICATIONS OF THE FINDINGS: Our findings are generalizable to other healthy populations with typical cycles, however, may not be applicable to cycles that are anovulatory, extreme in length or otherwise atypical. The results support previous findings that measures of estradiol production are highly correlated across the cycle, as are measures of progesterone production. Estradiol and progesterone concentrations are associated with one another, furthermore. However factor analysis also revealed more complex underlying patterns in the menstrual cycle, highlighting the fact that gonadotrophin concentrations and cycle length characteristics are virtually independent of ovarian hormones. These results suggest that despite integration of follicular and luteal ovarian steroid production across the cycle, cycle quality is a multi-faceted construct, rather than a single dimension. STUDY FUNDING/COMPETING INTEREST(S): The EBBA-I study was supported by a grant from the Norwegian Cancer Society (49 258, 05087); Foundation for the Norwegian Health and Rehabilitation Organizations (59010-2000/2001/2002); Aakre Foundation (5695-2000, 5754-2002) and Health Region East. The current analyses were completed under funding from the National Institutes of Health (K12 ES019852). No competing interests declared.

Anogenital distance in newborn daughters of women with polycystic ovary syndrome indicates fetal testosterone exposure.

Polycystic ovary syndrome (PCOS) affects ~7% of reproductive age women. Although its etiology is unknown, in animals, excess prenatal testosterone (T) exposure induces PCOS-like phenotypes. While measuring fetal T in humans is infeasible, demonstrating in utero androgen exposure using a reliable newborn biomarker, anogenital distance (AGD), would provide evidence for a fetal origin of PCOS and potentially identify girls at risk. Using data from a pregnancy cohort (The Infant Development and Environment Study), we tested the novel hypothesis that infant girls born to women with PCOS have longer AGD, suggesting higher fetal T exposure, than girls born to women without PCOS. During pregnancy, women reported whether they ever had a PCOS diagnosis. After birth, infant girls underwent two AGD measurements: anofourchette distance (AGD-AF) and anoclitoral distance (AGD-AC). We fit adjusted linear regression models to examine the association between maternal PCOS and girls' AGD. In total, 300 mother-daughter dyads had complete data and 23 mothers reported PCOS. AGD was longer in the daughters of women with a PCOS diagnosis compared with daughters of women with no diagnosis (AGD-AF: ß=1.21, P=0.05; AGD-AC: ß=1.05, P=0.18). Results were stronger in analyses limited to term births (AGD-AF: ß=1.65, P=0.02; AGD-AC: ß=1.43, P=0.09). Our study is the first to examine AGD in offspring of women with PCOS. Our results are consistent with findings that women with PCOS have longer AGD and suggest that during PCOS pregnancies, daughters may experience elevated T exposure. Identifying the underlying causes of PCOS may facilitate early identification and intervention for those at risk.

Timing of prenatal phthalate exposure in relation to genital endpoints in male newborns.

Prior studies report that penile size and male anogenital distance (AGD), sensitive markers of androgen action in utero, may be shortened by prenatal exposure to certain phthalates, including diethylhexyl phthalate (DEHP), but no human study has investigated the importance of exposure timing in these associations. The aim of this study was to examine the significance of exposure timing on the action of prenatal phthalates in particular DEHP, on male infant penile size and AGD. In The Infant Development and the Environment Study (TIDES) we measured penile width (PW) as well as anoscrotal distance (AGDAS ) and anopenile distance (AGPAP ) in newborn males. We modeled these endpoints in relation to phthalate metabolite concentrations in maternal urine samples collected in each trimester (T1, T2, and T3) in a subset of TIDES mothers (N = 168). PW was inversely associated with T2 oxidized DEHP metabolites, mono-2-ethyl-5-oxohexyl (MEOHP, ß=-0.48; 95% confidence interval, -0.93, -0.02), MEHHP (-0.48; -0.92, -0.05), mono-2-ethyl-5-carboxypentyl (MECPP, -0.51; -1.01, -0.004), although no appreciable associations were seen between PW and T1 and T3 DEHP metabolite concentrations in this subset. Concentrations of DEHP metabolites in T1 urine samples were inversely related to male AGD. For example, in T1 samples in this subset of women mono-2-ethyl-5-hydroxyhexyl (MEHHP) was inversely associated with male AGDAP (ß = -1.73; 95% confidence interval, -3.45, 0.0004). However, no appreciable associations were seen between AGD measures and any DEHP metabolite in T2 and T3 samples. These data suggest that DEHP exposure is inversely associated with AGD and PW, with PW primarily associated with T2 exposure and AGD associations seen only for T1 exposure, but no associations were found between T3 DEHP metabolites and any of these genital endpoints. These findings are consistent with data on critical windows in rodent studies, supporting the biological plausibility of these associations in humans.

Stability of proposed biomarkers of prenatal androgen exposure over the menstrual cycle.

The prenatal hormonal milieu is widely believed to shape health later in life; however, there are considerable methodological challenges associated with measuring the in utero hormonal environment. Two potential biomarkers of prenatal androgen exposure that can be measured postnatally have been proposed: anogenital distance (AGD) and the ratio of the second to fourth digits of the hand (2D:4D). Although both measures are widely used research tools, their use in adult women may be complicated by the dramatic fluctuations in reproductive hormones across the menstrual cycle. To determine whether there is cyclical variation in these biomarkers, we conducted a longitudinal study of 12 naturally cycling, nulliparous adult women. Trained examiners assessed two measures of AGD [anus to clitoris (AGD-AC) and anus to fourchette (AGD-AF)] and 2D:4D in both hands for the duration of three menstrual cycles, taking measurements during the follicular, peri-ovulatory and luteal phases of each cycle. Despite the small sample size, longer (more masculine) AGD was associated with lower (more masculine) digit ratios, as predicted by the literature. Using multi-level linear regression models, we found that AGD and 2D:4D measurements did not differ significantly across cycle phases. AGD-AF and digit ratios in both hands were associated with age at menarche, suggesting a possible common developmental trajectory. These results demonstrate that AGD and 2D:4D are stable across the menstrual cycle. In addition, research is needed to determine how reliably these measures reflect the in utero hormonal milieu.

Thymic microenvironment and lymphoid responses to sublethal irradiation.

Sublethal irradiation of the murine thymus has been a useful tool for depleting the thymus of dividing immature thymocyte subsets, to sequence thymocyte differentiation events occurring from radiation-resistant precursors. This massive reduction in thymocytes also represents a model in which the bidirectional interplay between the thymic stromal cells and lymphocytes can be investigated. The purpose of this study was thus twofold: to precisely map the initiation of thymopoiesis as a prelude to assessing the effects of injected mAb to novel thymic antigens; and to use a panel of mAbs to determine the alterations in the thymic stroma during the T-cell depletion and reconstitution phases. The striking finding from this study was that following T-cell depletion, there was a marked upregulation of specific stromal antigens, which retracted with the reappearance of T cells. Thus, following sublethal irradiation, there are modifications in the thymic microenvironment that may by necessary to support renewed thymopoiesis and the complete restoration of the thymus involved the synchronous development of both the stromal and lymphocytic components.

Thymic shared antigen-2: a novel cell surface marker associated with T cell differentiation and activation.

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3-CD4-CD8-, CD4+CD8+, and CD3highCD4-CD8+ thymocytes but is down-regulated on approximately 40% of CD3highCD4+CD8- thymocytes. Expression on peripheral TCR-alphabeta+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2-/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8- thymocytes and TCR-gamma delta+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.