We request permission to use available funds to complete assays of Anti-Mullerian hormone (AMH) on the serum residuals that are currently held in Glasgow from samples taken at the 15+ clinic. An NIH funded grant (PI: DA Lawlor) provided funds for fasting glucose, insulin and lipids to be completed on a predicted 7000 samples at the 15+ clinic. Blood samples from this clinic were only available on ~3500 samples and relevant assays are now near complete on these. Because a smaller number of samples were assayed funds are available to complete AMH assays on these 3500; and sufficient serum for these assays is currently available in Professor Sattar's laboratory in Glasgow. AMH assays would be relevant to the NIH grant application since we will use these to examine developmental origins of ovarian and testicular function which are in turn related to vascular and metabolic health outcomes.

We would like to address the following objectives in relation to AMH:

a. Describe the distribution of AMH in contemporary males and females at mean age 15

b. Determine the association of parental smoking in pregnancy; maternal weight gain in pregnancy; blood pressure change in pregnancy; gestational diabetes/glycosuria in pregnancy and parental smoking whilst breast feeding (in those offspring who were breastfed) with offspring AMH levels at mean age 15

c. Determine the prospective associations of offspring smoking, fat mass and change in fat mass, growth trajectories from birth to age 15, age at menarche (females) with AMH levels at mean age 15

d. With available data (current 3000 or larger if grants funded) complete a genome-wide association study with AMH (In collaboration with David Evans & Nic Timpson) of AMH levels

e. Examine the cross-sectional associations of AMH with glucose, insulin and lipids at age 15.

Background

AMH and ovarian and testicular function

Anti-Mullerian hormone (AMH, Mullerian-inhibiting substance) is a member of the transforming-growth factor-beta family. AMH has the primary role of regression of the Mullerian duct in the male fetus during early testis differentiation. However, expression of AMH persists after completion of the reproductive duct system in males, and furthermore commences expression in females at this time, in whom it is produced by ovarian granulosa cells from early fetal life[1]. Although AMH is initially observed in granulosa cells of primary follicles, maximal expression occurs in preantral and small antral follicles[2, 3]. AMH expression declines as antral follicles increase in size, with nominal expression restricted to the granulosa cells of the cumulus[3]. This loss of AMH expression during the follicle stimulating hormone (FSH)-dependent final stages of follicular growth, and the lack of expression by atretic follicles[4], suggests that basal levels of AMH may more accurately reflect the total developing follicular cohort and consequently potential ovarian response to FSH. The clinical utility of this, and a demonstration of the likely causal signficance of AMH as a measure of ovarian function, is that AMH is strongly associated with oocyte yield, clinical pregnancy and live birth in IVF cycles[4-7]. It is also a sensitive measure of the gonadotoxic effect of differential chemotherapy regimens and falls rapidly after toxic stimuli[8-12]. It is elevated in polycystic ovarian syndrome, a condition associated with increased preantral follicles[13-18]. Lastly, it can indicate the timing of the menopause transition approximately 5 years prior to the sentinel event as determined by amenorrhoea and circulating follicle stimulating hormone levels[19, 20]. Importantly, AMH has also been shown to be relatively consistent across the menstrual cycle[21-23], consistent with its role reflecting the continuous, non-cyclic growth of small follicles in the ovary. AMH has therefore overtaken other markers and is now recognised as the optimal measure of follicular reserve in females[7, 24, 25].

AMH is also produced in males, and the ontogeny of AMH is similar across species[26, 27], in that circulating AMH produced by Sertoli cells remains high until the onset of puberty, when they progressively decrease, correlating with the stage of pubertal development. This decline in AMH is principally due to the inhibitory effect of intratesticular testosterone and meiotic cells on Sertoli cell AMH expression[28], and male AMH values decrease to female levels[29]. Male AMH therefore provides a unique handle on Sertoli cell number and function - the principal determinant of testicular germ cell number. Consistent with this subfertile men have a significantly lower AMH than controls[30], and that even the relatively mild insult of a varicocele is associated with a lower AMH in prepubertal, pubertal and adult males[30, 31].

Prenatal/developmental determinants of ovarian and testicular function

Gestational cigarette smoking is plausibly a strong determinant of ovariant and testicular function and likely to be related to AMH via intrauterine and lactation exposure. In female fetuses, smoking may have a direct toxic effect on the primordial follicle, leading to premature exhaustion of the follicular germ pool[32, 33]. In animal models impairment of fertility in the offspring following prenatal exposure to polycyclic aromatic hydrocarbons (in cigarette smoke) via the mother during pregnancy has been demonstrated. Histological analysis of ovarian tissue from the exposed offspring mice demonstrate a markedly reduced number of primordial follicles[34, 35], suggesting that a detrimental impact on ovarian reserve and follicular dynamics underlies this phenomenon. Notably, in mice models, the combination of pre-pregnancy and lactational exposure to polycyclic hydrocarbons was associated with a 70% reduction in primordial follicle number[36]. This loss of primordial follicles and primary follicles if applicable to humans has profound biological consequences, as it is generally accepted that mammals are born with a finite number of primordial follicles that are incapable of proliferating and replenishing, and it is this dogma which underlies the chronological decline in the fecundity of both natural[37-39] and stimulated ovarian cycles[40, 41] and the relatively static onset of the menopause.

Human studies examining the impact of maternal smoking on the ovarian reserve of the offspring have been limited[42, 43]. A small epidemiological study of 230 women with offspring recall of maternal smoking status during the index pregnancy demonstrated a reduced cumulative conception rate[42]. This association was robust to adjustment with frequency of intercourse, the offspring's age and own smoking status and childhood exposure. Prenatal exposure to maternal smoking and reduced fecundability in the offspring was also observed in a recall study of 663 women from Minnesota[43]. Analysis of time to pregnancy in 1653 female twins also demonstrated a reduced fecundability in the exposed female offspring[44]. In contrast for offspring exposed during childhood to parental smoking an increased fecundability in the offspring was observed in both of these studies[43]. Importantly this apparently conflicting data regarding the timing of exposure is dependent on offspring recall of parental smoking status and has not examined differential smoking status across gestation, lactation and childhood and has no information regarding dose-dependent effects. With the prospective data in ALSPAC we will be able to examine the association of smoking in pregnancy, during infancy (and lactation where relevant) and childhood on AMH levels an index of ovarian reserve (see above) and we will be able to compare associations with paternal smoking to establish whether maternal associations are likely to be acting through intrauterine mechanisms.

With respect to males maternal cigarette smoking during gestation has been increasingly associated with rising incidences of cryptorchidism and hypospadias and reductions in testis size, sperm counts/quality, and fertility[45-47]. Thus, we hypothesise that maternal smoking during pregnancy and lactation will also be related to reduced AMH levels in males as well as females.

In both males and females there is epidemiological evidence of an association between obesity, metabolic parameters and fertility and other reproductive outcomes. These characteristics also cluster within families, with intergenerational associations. Whilst AMH levels have been assessed in prepubertal offspring of mothers with PCOS (and shown to be elevated in comparison to similar aged offspring of women without PCOS[14], to our knowledge no one has previously examined the association of maternal obesity, weight gain and metabolic/ vascular characteristics during pregnany with offspring AMH levels

Genetic determinants of AMH

Many reproductive characteristics and diseases have high levels of heritability including age at menarche (50-70%; [48]), age at menopause (~ 50%;[49, 50]) and PCOS (~60% [51]). Analysis of 359 women with PCOS, demonstrated an association between circulating AMH levels and three SNPs of the ACVR1 gene which encodes the common ALK2 component of the heteromeric AMH receptor complex in an allele dose manner[52]. In contrast AMH was not associated with SNPs in either the AMH gene or the specific AMH type II receptor part of the heteromer, despite biological effects when expressed in cell lines[53]. We hypothesise that in a general population a number of common variants will have modest associations with AMH levels. In particular given that AMH and follicular function are linked to metabolic derangements, we will examine common variants of genes, regulating metabolic function and adiposity.

Associations of AMH with vascular and metabolic traits

The relationship reproductive health with vascular and metabolic traits, and specifically the association of PCOS with insulin resistance and reduced insulin secretion would predict associations of AMH with glucose, insulin and lipid levels.

Methods

AMH will be assayed on existing ALSPAC samples from 15+ at Professor Naveed Sattar's laboratory. The AMH assay used will be the commercial ELISA kit provided by DSL (Webster, Texas, USA). This kit is in routine use in our laboratories[5, 7]. Current inter and intra-assay CVs in our laboratory are 3.0% and 2.6% respectively. The Glasgow laboratory adheres to UK external quality control for all parameters and is Clinical Pathology Accreditation (CPA) accredited.

For objectives a-c and e relevant datasets will be compiled by DA Lawlor and standard linear / logistic regression models used in analyses. Sensitivity tests of possible non-paternity will be used when comparing maternal and paternal exposures with offspring AMH levels.

For objective d, data management and analyses will be undertaken by Dave Evans & Nic Timpson from MRC CAiTE, University of Bristol.

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