Delayed conception in women with low-urinary iodine concentrations: a population-based prospective cohort study.

STUDY QUESTION: Is iodine deficiency associated with decreased fecundability? SUMMARY ANSWER: Moderate to severe iodine deficiency is associated with a 46% decrease in fecundability. WHAT IS KNOWN ALREADY: Iodine deficiency is common in women of childbearing age but its effect on fecundability has not been investigated. STUDY DESIGN, SIZE, DURATION: The LIFE Study, a population-based prospective cohort study, enrolled 501 women who had discontinued contraception within 2 months to become pregnant between 2005 and 2009. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women reported on risk factors for infertility by interview then kept daily journals of relevant information. Women used fertility monitors to time intercourse relative to ovulation then used home digital pregnancy tests to identify pregnancies on the day of expected menstruation. Urine samples for iodine analysis were collected on enrollment. MAIN RESULTS AND THE ROLE OF CHANCE: Samples were in the deficiency range in 44.3% of participants. The group whose iodine-creatinine ratios were below 50 µg/g (moderate to severe deficiency) had a 46% reduction in fecundity (P = 0.028) compared with the group whose iodine-creatinine ratios were in the adequate range: adjusted fecundability odds ratio of becoming pregnant per cycle, 0.54 (95% confidence interval 0.31-0.94). LIMITATIONS, REASONS FOR CAUTION: Iodine concentrations vary within individuals over time, so the data must be interpreted by group as we have done; residual confounding is possible. WIDER IMPLICATIONS OF THE FINDINGS: Significant delays in becoming pregnant occur at iodine concentrations that are common in women in the USA and parts of Europe. Replicating these findings will be important to determine whether improving iodine status could be beneficial in improving fecundability. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, USA. Contracts N01-HD-3-3355; N01-HD-3-3356; N01-HD-3-3358 and HHSN275201100001l/HHSN27500007. None of the authors has any conflict of interest to declare.

Biological variability in serum anti-Müllerian hormone throughout the menstrual cycle in ovulatory and sporadic anovulatory cycles in eumenorrheic women.

STUDY QUESTION: Does serum anti-Müllerian hormone (AMH) vary significantly throughout both ovulatory and sporadic anovulatory menstrual cycles in healthy premenopausal women? SUMMARY ANSWER: Serum AMH levels vary statistically significantly across the menstrual cycle in both ovulatory and sporadic anovulatory cycles of healthy eumenorrheic women. WHAT IS KNOWN ALREADY: Studies to date evaluating serum AMH levels throughout the menstrual cycle have conflicting results regarding intra-woman cyclicity. No previous studies have evaluated an association between AMH and sporadic anovulation. STUDY DESIGN, SIZE, DURATION: We conducted a prospective cohort study of 259 regularly menstruating women recruited between 2005 and 2007. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women aged 18-44 years were followed for one (n = 9) or two (n = 250) menstrual cycles. Anovulatory cycles were defined as any cycle with peak progesterone concentration ≤5 ng/ml and no serum LH peak on the mid or late luteal visits. Serum AMH was measured at up to eight-time points throughout each cycle. MAIN RESULTS AND THE ROLE OF CHANCE: Geometric mean AMH levels were observed to vary across the menstrual cycle (P < 0.01) with the highest levels observed during the mid-follicular phase at 2.06 ng/ml, decreasing around the time of ovulation to 1.79 ng/ml and increasing thereafter to 1.93 (mid-follicular versus ovulation, P < 0.01; ovulation versus late luteal, P = 0.01; mid-follicular versus late luteal, P = 0.05). Patterns were similar across all age groups and during ovulatory and anovulatory cycles, with higher levels of AMH observed among women with one or more anovulatory cycles (P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Ovulatory status was not verified by direct visualization. AMH was analyzed using the original Generation II enzymatically amplified two-site immunoassay, which has been shown to be susceptible to assay interference. Thus, absolute levels should be interpreted with caution, however, patterns and associations remain consistent and any potential bias would be non-differential. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates a significant variation in serum AMH levels across the menstrual cycle regardless of ovulatory status. This variability, although statistically significant, is not large enough to warrant a change in current clinical practice to time AMH measurements to cycle day/phase. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD (Contracts # HHSN275200403394C, HHSN275201100002I Task 1 HHSN27500001). The authors have no conflicts of interest to declare.

Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes.

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Synergistic activation of the inhibin alpha-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate.

The inhibin alpha-subunit gene is expressed in the ovary, testis, adrenal, and pituitary. Because this pattern of expression corresponds to that of the orphan nuclear receptor, steroidogenic factor-1 (SF-1), we hypothesized that the inhibin alpha promoter might be regulated by SF-1. Expression of exogenous SF-1, in an SF-1 deficient cell line, caused modest stimulation of the inhibin alpha promoter. However, activation of the cAMP pathway, which is known to regulate inhibin alpha expression, greatly enhanced the actions of SF-1. Coexpression of SF-1 with the catalytic subunit of cAMP-dependent protein kinase A caused greater than 250-fold stimulation, whereas only 4- or 7-fold stimulation was seen by the SF-1 or protein kinase A pathway alone. Synergistic stimulation by SF-1 and the cAMP pathway was also seen in GRMO2 granulosa cells, which express endogenous SF-1. Deletion and site-directed mutagenesis localized a novel SF-1 regulatory element (TCA GGGCCA; -137 to -129) adjacent to a variant cAMP-response element (CRE; -120 to -114). The synergistic property of SF-1 and cAMP stimulation was inherent within this composite inhibin alpha fragment (-146 and -112), as it was transferable to heterologous promoters. Mutations in either the CRE or the SF-1 regulatory element completely eliminated synergistic activation by these pathways. The binding of SF-1 and CRE binding protein (CREB) to the inhibin alpha regulatory elements was relatively weak in gel mobility shift assays, consistent with their deviation from consensus binding sites. However, SF-1 was found to interact with CREB using an assay in which epitope-tagged SF-1 was expressed in cells and used to pull down in vitro translated CREB. Expression of CREB binding protein (CBP), a coactivator that interacts with SF-1 and CREB, further enhanced transcription by these pathways. Stimulation by the SF-1 and cAMP pathways was associated with increased histone H4 acetylation, suggesting that chromatin remodeling accompanies their actions. We propose a model in which direct interactions of SF-1, CREB, and associated coactivators like CBP induce strongly cooperative transactivation by pathways that individually have relatively weak effects on transcription.

Differential gonadotropin-releasing hormone stimulation of rat luteinizing hormone subunit gene transcription by calcium influx and mitogen-activated protein kinase-signaling pathways.

Gonadotropin secretion and gene expression are differentially regulated by hypothalamic GnRH pulses by unknown mechanisms. GnRH stimulates calcium influx through L-type voltage-gated channels and activates phospholipase C, leading to increased protein kinase C (PKC) and mitogen-activated protein kinase activity. We found differential contributions of these pathways to GnRH-stimulated rat LH subunit transcription in pituitary gonadotropes and cell lines. Endogenous transcription of the alpha- and LHbeta-subunits in rat pituitary cells was stimulated by GnRH. Independent PKC activation by phorbol myristate acid stimulated only the alpha-subunit gene. In contrast, an L-channel antagonist (nimodipine) inhibited only LHbeta stimulation by GnRH, and an L-channel agonist (BayK 8644) stimulated only basal LHbeta transcription. GnRH induction of a rat alpha-subunit promoter construct in alphaT3 cells was unaffected by nimodipine or elimination of external calcium, while both treatments eliminated the LHbeta response. Application of a mitogen-activated kinase kinase (MEK) inhibitor (PD098059) decreased basal and GnRH-stimulated alpha-subunit promoter activity and had no effect on LHbeta promoter activity. In pituitary cells from mice bearing an LHbeta promoter-luciferase reporter transgene, GnRH stimulation was inhibited by nimodipine but not by PD098059. Thus, GnRH induction and basal control of the alpha-subunit gene seem to occur through the PKC/mitogen-activated protein kinase pathway, while induction of the LHbeta gene is dependent on calcium influx. Differential signaling from the same receptor may be a mechanism for preferential regulation of transcription.

Thyrotropin (TSH)-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C.

TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.