Sex-dependent associations of maternal androgen levels with offspring BMI and weight trajectory from birth to early childhood.

CONTEXT: In preclinical studies, high androgen levels during pregnancy are associated with low birth weight and rapid postnatal weight gain in the offspring. However, human data linking prenatal androgens with birth weight and early life weight gain in the offspring are scarce. DESIGN: We evaluated 516 mother-child pairs enrolled in the New England birth cohorts of the Collaborative Perinatal Project (1959-1966). We assayed androgen bioactivity in maternal sera during third-trimester using a receptor-mediated luciferase expression bioassay. Age and sex-specific BMI Z-scores (BMIz), defined using established standards, were assessed at birth, 4 months, 1 year, 4 years, and 7 years. We used linear mixed models to evaluate the relation of maternal androgens with childhood BMIz overall and by sex. We examined the association of maternal androgens with fetal growth restriction. The association of weight trajectories with maternal androgens was examined using multinomial logistic regression. RESULTS: Higher maternal androgen levels associated with lower BMIz at birth (ß = - 0.39, 95% CI: - 0.73, - 0.06); this relation was sex-dependent, such that maternal androgens significantly associated with BMIz at birth in girls alone (ß = - 0.72, 95% CI: - 1.40, - 0.04). The relation of maternal androgens with fetal growth restriction revealed dose threshold effects that differed by sex. There was no significant association between maternal androgens and weight trajectory overall. However, we found a significant sex interaction (p = 0.01); higher maternal androgen levels associated with accelerated catch-up growth in boys (aOR = 2.14, 95% CI: 1.14, 4.03). CONCLUSION: Our findings provide evidence that maternal androgens may have differential effects on the programming of intrauterine growth and postnatal weight gain depending on fetal sex.

The use of long acting subcutaneous levonorgestrel (LNG) gel depot as an effective contraceptive option for cotton-top tamarins (Saguinus oedipus).

Cotton-top tamarins (Saguinus oedipus) are a critically endangered species that have been bred successfully in captivity for many years. For two decades, the Cotton-top Tamarin SSP(©) has been challenged with a high rate of reproduction combined with a history of contraceptive failures and nonrecommended births using the current Depo Provera(®) (medroxyprogesterone acetate) injection followed by MGA (melengestrol acetate) implant contraception combination. To address these issues we have developed and tested the use of levonorgestrel (LNG) as an effective contraception option for cotton-top tamarins. LNG was delivered in an injectable, gel matrix consisting of polylactic-co-glycolic acid, triethyl citrate and N-methylpyrrolidone. This gel matrix forms a biodegradable depot at the subcutaneous injection site providing slow release of the active ingredient. Gel matrix composition and LNG concentration were adjusted in four gel formulations to maximize the duration of contraceptive efficacy while minimizing immediate post-injection increases in fecal LNG concentration. LNG treatment (68.44 ± 8.61 mg/kg) successfully eliminated ovarian cycles (fecal pregnanediol-3-glucuronide (PdG) and estrone conjugates (E(1) C)) for 198.8 ± 70.3 days (formulation four; range 19-50 weeks). It was demonstrated that subcutaneous LNG depot injection was an effective, reversible contraceptive option for the management of cotton-top tamarins in captivity.

Effect of porcine zonae pellucidae immunisation on ovarian follicular development and endocrine function in domestic ewes (Ovis aries).

Domestic ewes (Ovis aries) were immunised with porcine zonae pellucidae (pZP) or pZP conjugated to keyhole limpet haemocyanin (KLH) in adjuvant(s) to examine the feasibility of the species to serve as a model for further development of pZP-based vaccines in ungulates. Two immunisation groups were employed, with a third group receiving only adjuvant (n = 5 per group). Early in the study, oestrous activity was monitored by the use of a vasectomised ram fitted with a marking harness. Eventually, ewes were exposed to an intact ram for breeding. In addition, weekly serum and every-other-day faecal samples were collected to measure pZP antibodies and progesterone metabolite concentrations respectively. At the conclusion of the study, fecundity was established, and ovarian tissue was examined. Ewes immunised against pZP : KLH with adjuvant produced minimal antibody absorbance levels, displayed normal oestrous cycles, became pregnant upon introduction of the intact ram and exhibited normal ovarian histopathology. Ewes immunised against pZP with adjuvant produced high antibody absorbance levels, were acyclic following primary immunisation and were infertile. Examination of the ovarian tissue revealed atrophic changes that included: (1) the absence of growing follicles; (2) significant reduction in the number of primordial follicles; and (3) the presence of abnormal granulosa cell clusters lacking oocytes. Antisera displayed immunoreactivity to the major components of pZP, and immunohistochemical labelling of ovarian tissue showed specificity to the ZP. These data are the first generated in an ungulate species showing deleterious effects of pZP immunisation on folliculogenesis and oestrous cyclicity.

In vivo effects of delta 1-testololactone on peripheral aromatization.

To evaluate the in vivo effect of delta 1-testololactone on peripheral aromatization, studies were performed on seven postmenopausal women with metastatic breast cancer. Analysis of variance indicated that there were significant increases of circulating androstenedione (p less than 0.05) and estradiol (p less than 0.001) during administration of different doses of testololactone. Androstenedione levels were increased with all doses of testololactone tested (50, 100, 250, and 500 mg every 6 hr for 14 days each), while estradiol rose with only the 250- and 500-mg dosages. With administration, there was a significant decrease of estrone (p less than 0.001) with the mean level falling from 26 +/- 3 (S.E.) to 11 +/- 2 pg/ml. The addition of adrenal suppression (dexamethasone, 1 mg nightly at 11 p.m.) significantly lowered androstenedione (p less than 0.05) but had no effect on estrone or estradiol levels. Long-term therapy (up to 6 months) with the 250-mg dosage showed continual suppression of estrone with no escape being observed. Studies to determine the reason for the increase of estradiol with testololactone suggested cross-reactivity of the antibody with in vivo metabolites of the drug. However, these possible metabolites did not bind to uterine cytosol estrogen receptors. The decrease in estrone with testololactone administration presumably explains its antitumor properties.

Molecular target of endocrine disruption in human luteinizing granulosa cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin: inhibition of estradiol secretion due to decreased 17alpha-hydroxylase/17,20-lyase cytochrome P450 expression.

Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.

Measurement of periimplantational relaxin concentrations in the macaque using a homologous assay.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Absence of regular pulsatile gonadotropin secretion during implantation in the rhesus macaque.

The secretory response of the primate corpus luteum (CL) to CG after implantation suggests that gonadotropin receptors are not depleted despite increasing CG production and continuous elevated tropic stimulation. Such continuous stimulation is known to cause down-regulation of receptors in other tissues. To determine if CG secretion is intermittent during the initial stages of CL rescue, we assessed the secretory pattern of CG during the periimplantation period by collecting frequent (4/h) blood samples in two studies (for 4 h on 3 separate days between days 8-13, or for 2 separate 13-h sequences between days 10-15 postovulation) in 13 chair-adapted females. Day 0 of gestation was defined as the day of ovulation, as estimated by peak urinary estrone conjugate excretion in females mated on days 9, 11, and 13 of the menstrual cycle. Hormone concentrations were measured by either RIA [irFSH; estradiol and progesterone (P)] or Leydig cell bioassay (bioLH or bioCG). In the first study, 4 of 6 females conceived, and the mean for bioLH was not significantly elevated until days 12-13. In the second study, 5 of 7 females conceived, and the episodic secretory pattern of circulating pituitary bioLH typically observed in cycling females (2.7 +/- 0.3 peaks/13 h) was replaced by a relatively nonpulsatile, but steadily increasing profile during days 12-15 of gestation (1.5 +/- 0.4 peaks/13 h). Although occasional large fluctuations in bioLH/CG and P were noted, the bioLH/P peaks were less congruent than those in nonfertile cycles, and there was no diurnal pattern in the secretion of either hormone. In contrast, irFSH concentrations did not fluctuate and were similar in the two groups of females [2.93 +/- 0.28 vs. 2.34 +/- 1.7 ng/ml (mean +/- SEM)]. These data demonstrate that 1) a steady, gradually increasing secretory pattern of CG is associated with rescue of the CL; 2) the circulating profile of CG during the periimplantation interval is not consistently episodic and does not support the hypothesis that intermittent CG release prevents LH/CG receptor down-regulation in the CL during early pregnancy; 3) increased bioLH/CG levels during conceptive cycles in rhesus monkeys are not detected until days 12-13 after the midcycle bioLH peak; 4) irFSH patterns on pooled aliquots appear to be uninformative with regard to gonadotropin dynamics in early pregnancy; and 5) urinary estrone conjugate measurements provide a practical method for the precise timing of infrequent events, such as implantation, in the laboratory macaque.

Testicular androgen and estrogen secretion and benign prostatic hyperplasia in the beagle.

The natural history of benign prostatic hyperplasia (BPH) in the dog is characterized by a slow progression through two phases. The early phase of the disease process is characterized by glandular hyperplasia and occurs as early as 2.5 yr of age. The late phase of the disease is characterized by cystic hyperplasia and occurs after 4 yr. The results of the present study are the first to compare Leydig cell structure and steroidogenic function in dogs with BPH with those in age-matched controls. It was discovered that glandular BPH can occur in young dogs (2.5 yr) in which Leydig cell mass and ultrastructure and maximally stimulated androgen secretion are indistinguishable from those in age-matched controls. These results support the concept that the early phase of BPH (glandular hyperplasia) is not related temporally to some defect in the Leydig cell. In contrast, the late phase of BPH (cystic hyperplasia) in beagles 6 yr of age is associated with diminished smooth endoplasmic reticulum in the Leydig cell and with diminished production of androgens by perfused testes in vitro. During the course of these studies, we discovered that the testes of young beagles with BPH, but not age-matched controls or old beagles with BPH, secrete an unidentified molecule (putative estrogen). This molecule was characterized partially in that it is extractable from testicular venous effluent with diethyl ether, elutes in a discrete fraction in several different high performance liquid chromatographic systems, reacts with an antibody that recognizes 17 beta-estradiol, estrone, and estriol, and competes with 17 beta-[3H]estradiol for the rat uterine estrogen receptor. Based on the elution volume from high performance liquid chromatography, the unknown molecule (putative estrogen) is not estriol, estradiol, or estrone.

The functional relationship between priming and releasing actions of luteinizing hormone-releasing hormone.

To examine the relationship between the priming and releasing actions of LRF on LH secretion, 14 normal cycling women received 4 different rates of LRF infusion (0.005, 0.01, 0.05, and 0.1 microgram/m2.min for 4 h). The releasing action of the infusion was measured as the area under the curve and the priming effect was assessed by the acute LH increment in response to a test pulse of LRF (10 microgram) at the end of infusion. At the lower 2 infusion rates, there were only minor changes in releasing function, but it increased exponentially (r = 0.986) with higher rates of infusion. In contrast, the priming effect of the lower 2 doses of infusion increased markedly as a function of infusion rate, but no additional priming was found with the higher rates of infusion. Thus, over the range of infusion rates employed, the releasing and priming functions of LRF appear to be dose dependent. These results indicate that the interdependent releasing and priming actions of LRF on LH secretion are functionally dissociable and that large elevations of LRF tend to favor release, while small LRF increments seem to promote priming preferentially.

The effects of estrogen and progesterone on the functional capacity of the gonadotrophs.

The functional capacity of the gonadotrophs under the influence of exogenous estrogen and progesterone was assessed by repeated stimulation with submaximal doses of LH-releasing factor (LRF) (10 mug at 2-h intervals) of subjects during the early follicular phase of the cycle and of hypogonadal women. The initial increment of peak serum LH and FSH concentrations after the first administration of LRF, was used to describe the pituitary sensitivity, and the integrated release during the 10 hours of LRF-stimulated pulses was utilized to approximate the pituitary gonadotropin reserve. During the early follicular phase, response to LRF stimulations was relatively stable with corresponding release of LH and FSH. An augmentation of sensitivity, as well as the reserve, for both LH and FSH was elicited by an incremental change in circulating estradiol levels, a change imposed by daily administration of estradiol benzoate for 4 days during the early follicular phase. Under the same conditions, the addition of progesterone (10 mg, im) at the end of the estradiol benzoate treatment induced a marked amplification of the estrogen-augmented pituitary gonadotropin sensitivity and reserve. The pituitary sensitivity, relatively higher than the reserve in hypogonadal subjects, was reversed by the administration of ethinyl estradiol (20-50 mug/day) for 7 days. These data indicate that the functional capacity of the gondotrophs is profoundly modulated by estrogen through relative changes in pituitary sensitivity and reserve, and that progesterone in low doses exhibited an amplifying effect on estrogen-primed gonadotrophs in both the pituitary sensitivity and the reserve.

PIP: Low doses of luteinizing hormone-releasing factor (LH-RF) were injected at 2-hour intervals into 6 women in the early follicular phase and to hypogonadal women, to estimate acute LH and follicle stimulating hormone (FSH) pool size and gonadotropin reserve. 5 injections of 10 mcg LH-RF iv every 2 hours on Cycle Days 2-6 produced LH peaks of constant height (about 40 mIU/ml) within 30 minutes, a small gradual elevation in FSH, and a doubling of basal estradiol levels. The increment in LH seen in the 1st peak was defined as the pituitary "sensitivity" and the integrated area of release over the 10 hours was d efined as the pituitary gonadotropin "reserve." Estradiol benzoate 2, 4, 6, and 8 mcg/kg twice daily for 4 days increased sensitivity (110-160 mIU LH/ml) and reserve. Progesterone 10 mg im given with the last estradiol dose amplified markedly both sensitivity and reserve, and changed the form of the LH curves, such that the 2 subjects with lowest basal LH and highest peak levels had spontaneous spikes after the induced LH peaks. In comparison, LH and FSH increments were higher in h ypogonadal women than in normal subjects, so that separte FSH peaks were discernible. The 1st LH and FSH peaks after estradiol treatment were much lower than the 4 subsequent peaks. These data were interpreted to mean that pituitary sensitivity was greater in hypogonadal subjects, but lowered by estrogen. Progesterone apparently amplified both sensitivity and reserve stimulated by estrogen.

The role of estrogen in the modulation of pituitary sensitivity of LRF (luteinizing hormone-releasing factor) in men.

The role of estradiol in modulating pituitary gonadotropin release in the human male was studied by evaluating the effects of clomiphene on the pituitary gonadotropin response to synthetic LRF (150 mug) in 6 eugonadal men. Administration of clomiphene (100 mg single daily dose time 5 days) induced a significant elevation of the basal levels of LH, FSH, estradiol and testosterone. However, the pituitary release of LH and FSH in response to LRF was markedly diminished by the clomiphene treatment. This finding suggests that in men, as in women, endogenous estradiol provides feedback regulation of gonadotropin output by the pituitary.

PIP: The effects of clomiphene on the pituitary response to LRF in eugonadal men were studied to investigate the role of estradiol in the feedback modulation of gonadotropin secretion in men. 150 mcg LRF was given to 6 healthy men (20-28 years) and luteinizing hormone (LH) and follicle stimulating hormone (FSH) evaluated. A 5-day course of clomiphene (100 mg daily) was then given and the LRF test repeated. Serum FSH, LH, estradiol, and testosterone (T) were determined. Clomiphene caused a significant elevation in the basal levels of LH, FSH, Estradiol, and T. Pituitary release of LH and FSH in response to LRF was markedly reduced by clomiphene treatment indicating that estradiol provides feedback regulation of gonadotropin output by the pituitary.

Gonadotropin secretion in response to low and high doses of LRF in normal and hypogonadal women (functional disparity of the gonadotrophs).

A comparison was made of gonadotropin release after an iv bolus of large (150 mug) and small (10 mug) doses of LRF during various phases of the menstrual cycle and in primary hypogonadal women. Gonadotropin release was quantitatively similar to small and large doses of LRF in hypogonadal women with low estradiol levels. In contrast, normal women from the early to late follicular phase of the cycle, the gonadotropin response to a small dose of LRF (10 mug) was not as great as that observed with a large dose of LRF (150 mug) and did not elicit the augmented gonadotropin response of the late follicular phase. During the mid-luteal phase, a significant enhancement of responses to small doses of LRF was observed. The sustained gonadotropin release induced by the large dose of LRF in the late follicular phase was no longer apparent during the mid-luteal phase, although the dose-related discrimination of LH release was maintained. These dose-related disparities in the gonadotropin release to LRF might represent functional expressions of the two-pool dynamics of the gonadotrophs induced by ovarian steroids.

Assessments of the functional capacity of the gonadotrophs in men: effects of estrogen and clomiphene.

Serum LH and FSH responses to serial injections of LRF with small (10 mug x 5), large (150 mug x 5), decremental (300 to 10 mug), and incremental (10 to 300 mug) doses at 2-hour intervals were assessed in eugonadal men. At constant doses, pulses of LRF induced pulsatile LH release which was qualitatively similar but quantitatively greater for the large than for the small dose of LRF. There were no periods of refractoriness or augmentation of subsequent responses from prior exposure to LRF when administered at 2-hour intervals. LH responses to incremental and decremental doses of LRF resulted in corresponding measurable changes in the magnitude of pituitary LH release. The FSH responses to pulses of LRF at all doses tested were uniformly small. These data suggest that analyses of the initial and integrated release (10 h experiment) to pulses of LRF may disclose the functional capacity of the gonadotrophs and that small variations in the endogenous LRF delivered may represent a significant factor in the control of LH release. The small dose (10 mug x 5) of pulses of LRF was utilized in the assessment of estrogen and clomiphene treatments on the functional capacity of the gonadotrophs in normal men. Compared with the pretreatment results, both constant doses of ethinyl estradiol (50 mug/day x 7 days) and incremental doses of estradiol benzoate (100 to 400 mug, twice daily injections x 4 d) induced an attenuation of the initial release as well as of the integrated response 10 h) to pulses of LRF. Clomiphene treatment (100 mg/day x 5 d), likewise, resulted in a reduction of gonadotropin release to all pulses of LRF. These data suggest that circulating estrogen in intact men may have both a negative and a positive feedback effect on the gonadotrophs and that the testicular estrogen secretion as well as the extraglandular sources of estrogen, may play a critical role in the regulations of gonadotropin secretion in man.

The functional changes of the pituitary gonadotrophs during the menstrual cycle.

Submaximal doses of LRF, administered over a period of several hours, either by repeated pulses (10 mug at 2 h intervals X 5) or by constant infusion (0.2 mug/min X 4 h), have permitted the assessment of changes in the releasable gonadotropin during the menstrual cycle. Quantitations in the acute releasable and ultimately releasable gonadotropins were made which represent, respectively, the sensitivity and reserve of the gonadotrophs. The functional expression of these two components of gonadotropin release exhibited profound changes during the menstrual cycle and were in synchrony with the cyclicity of ovarian steroid levels; during the early follicular phase, both sensitivity and reserve were at a minimum, but with increasing levels of E2, a preferential increase in reserve over sensitivity (P less than 0.005) was found. Although both sensitivity and reserve increased dramatically near the midcycle, the relative change in these two components was reversed from the late follicular phase to the midcycle surge. The presence of this phenomenon may be causally related to the development of a self-priming effect of LRF at this time, as evidenced by an augmentation of gonadotropin release to the 2nd pulse of LRF. Thus, a build-up in pituitary store consequent to the greater increase in reserve than in sensitivity, together with the appearance of the self-priming effect of LRF induced by progressively rising levels of E2, may constitute the essential dynamics required for the development of the midcycle gonadotropin surge. Pituitary sensitivity and reserve continued to be high during the early luteal phase but reduced progressively thereafter. In all studied, FSH responses were less obvious but showed remarkable parallelism to the pattern of LH responses. We have concluded that the functional capacity of the gonadotrophs exhibits a remarkable cyclic change and that the adenohypophysis represents a critical feedback site in the development of pre-ovulatory gonadotropin surge.

Enhanced ovarian steroid secretion before implantation in early human pregnancy.

Previous studies have compared ovarian steroid production in the luteal phase of nonconceptive and conceptive cycles. Some investigators reported higher preimplantational levels of progesterone in conceptive cycles vs. nonconceptive cycles, but other studies have found no differences. Many of these results were difficult to interpret because the studies included infertile women and/or women who received exogenous hormones. In this study we have characterized the profiles of gonadotropin secretion and ovarian steroid response during early pregnancy in a population of spontaneously ovulating women and compared them to those in nonconceptive cycles of recently fertile women. Blood samples were collected daily during the luteal phase from 24 women during 51 cycles of artificial insemination with donor semen. Cycles were segregated to those from women who had a successful term pregnancy (normal group) and those having an early spontaneous abortion (SAB group) and were also classified as nonconceptive or conceptive based on measurements of hCG. Serum LH and FSH did not show marked differences between nonconceptive and conceptive cycles in the periimplantation period in either the normal or SAB group. In the normal group, estradiol concentrations were significantly higher in conceptive cycles than in nonconceptive cycles beginning 6 days after the LH peak and continuing through the end of the cycle, while differences in progesterone concentrations bordered on or exceeded significance during the same time period. In the SAB group, preimplantation differences in pituitary gonadotropin and ovarian steroid secretion were not observed, whereas the postimplantation hCG concentrations in the SAB group were significantly lower than those in the normal group. It is reasoned that embryos with defective post-implantation hCG secretion may have had this defect before detection of hCG in serum, thus accounting for the lack of stimulation of steroid secretion in these pregnancies. These findings suggest that the enhanced ovarian steroid secretion in conceptive cycles may be due to a gonadotropic stimulus from the preimplantation embryo.

The two pools of pituitary gonadotropin: regulation during the menstrual cycle.

Information on the relative activity and on the functional relationships between the acutely releasable (1st) and reserve (2nd) pools of pituitary LH during the course of the normal menstrual cycle was obtained via a 4 h LRF infusion (0.2 mug/min X 4 h). This was immediately followed by 3 pulses of LRF (10 mug at 2 h intervals) to assess further the size of the acutely releasable pool after activation of the reserve pool by the infusion. Our observations indicate that two functional pools of LH are present in all phases of the menstrual cycle and that comparative pool size or activity is influenced profoundly by ovarian steroid feedback as well as by the pattern of input of hypothalamic LRF. From the early to the late follicular phase, in synchrony with the rising levels of E2, the size of the 2nd pool is preferentially augmented. A small increase in the 1st pool activity is not apparent until the late follicular phase when a 5-fold increase in the size of the 2nd pool is also attained. During the mid-luteal phase and in association with relatively high progesterone (P) and E2, th large 2nd pool is maintained as in the late follicular phase but the 1st pool is strikingly smaller. Activation of the 2nd pool of LH by the LRF infusion (priming) increases the acutely releasable LH (1st pool) in all three phases of the cycle, as evidenced by an enhanced response to the 1st but not subsequent pulses of LRF at the end of the infusion as compared with non-infused controls. This priming effect is likely a reflection of activation or "shifting" of LH from the larger 2nd pool to the smaller 1st pool. It is found that this priming effect is greatest during the mid-luteal phase as compared to other phases of the cycle. During the days of mid-cycle LH surge, a dramatic reversal of the relative activity of the two pools in observed and this is manifested by an enormous increase in the activity of the 1st relative to the 2nd pool. In contrast to other phases of the cycle, the release of LH from the 2nd pool is not sustained and this premature decline in LH release despite continuous LRF infusion appears to be due to pituitary depletion of LH as evidenced by the failure of the pituitary response to pulses of LRF immediately following the infusion...

Bone health is not affected by luteal phase abnormalities and decreased ovarian progesterone production in female runners.

The primary purpose of this study was to determine whether decreased ovarian progesterone production, associated with short and inadequate luteal phases in exercising women, was associated with decreased bone mineral density (BMD) and altered bone metabolism. Thirty-three eumenorrheic menstruating women participated in this study for 3 months. Subjects were required to collect daily urine samples for three consecutive menstrual cycles and have blood and urine collected weekly. Daily urine samples were analyzed for free LH, estrone conjugates (E1C), and pregnanediol 3-glucuronide (PdG), adjusted for creatinine, whereas weekly blood and urine samples were analyzed for bone markers, estradiol, progesterone, FSH, and LH. Based on the analyses of these samples, subjects were divided into three groups: sedentary ovulatory (SedOvul; n = 9), exercising ovulatory (ExOvul; n = 14), and exercising luteal phase defects (ExLPD; n = 10). The three groups were matched for age (27.6 +/- 1.0 yr), weight (60.6 +/- 1.9 cm), and reproductive maturity (14.5 +/- 1.0 yr), PdG production during the luteal phase was lower (P = 0.004) in the ExLPD women compared to that in the SedOvul group (2.4 +/- 0.4 vs. 5.1 +/- 0.6 ng/mL creatinine, respectively). The ExOvul group also had less (P < 0.01) PdG production during the luteal phase (3.5 +/- 0.3 ng/mL creatinine) compared to the SedOvul group. The total production of PdG, as assessed by area under the curve analysis, was also lower (P < 0.001) in the ExOvul and ExLPD groups compared to that in the SedOvul group. E1C production, however, was not different (P > 0.05) among the groups, except for E1C during the early follicular phase, which was lower (P = 0.043) in the ExLPD group than that in the SedOvul group. BMD and biochemical markers of bone metabolism were unaffected by and not associated with the compromised progesterone environment, but BMD values at the proximal femur (r = 0.354; P = 0.061) and total body (r = 0.359; P = 0.056) were associated with decreased early follicular E1C production. We conclude the following. 1) Luteal phase disturbances occur independent of training volume, and volume of training does not have to be severe to result in menstrual disturbances. 2) As a result of exercise, disturbance in progesterone production is not associated with decreased bone mass. 3) Long follicular phases are associated with reduced estrogen production during the early follicular phase, which are both associated with decreased bone mass. 4) Provided the estradiol status is adequately maintained, BMD is unaffected by decreased progesterone production associated with short and inadequte luteal phases in exercising women.

High frequency of luteal phase deficiency and anovulation in recreational women runners: blunted elevation in follicle-stimulating hormone observed during luteal-follicular transition.

The purposes of this investigation were to evaluate the characteristics of three consecutive menstrual cycles and to determine the frequency ofluteal phase deficiency (LPD) and anovulation in a sample of sedentary and moderately exercising, regularly menstruating women. For three consecutive menstrual cycles, subjects collected daily urine samples for analysis of FSH, estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and creatinine (Cr). Sedentary (n=11) and exercising (n=24) groups were similar in age (27.0+/-1.3 yr), weight (60.3+/-3.1 kg), gynecological age (13.8+/-1.2 yr), and menstrual cycle length (28.3+/-0.8 days). Menstrual cycles were classified by endocrine data as ovulatory, LPD, or anovulatory. No sedentary women (0%) had inconsistent menstrual cycle classifications from cycle to cycle, but 46% of the exercising women were inconsistent. The sample prevalence of LPD in the exercising women was 48%, and the 3-month sample incidence was 79%. In the sedentary women, 90% of all menstrual cycles were ovulatory (SedOvul; n=28), whereas in the exercising women only 45% were ovulatory (ExOvul; n=30); 43% were LPD (ExLPD; n=28), and 12% were anovulatory (ExAnov; n=8). In ExLPD cycles, the follicular phase was significantly longer (17.9+/-0.7 days), and the luteal phase was significantly shorter (8.2+/-0.5 days) compared to ExOvul (14.8+/-0.9 and 12.9+/-0.3 days) and SedOvul (15.9+/-0.6 and 12.9+/-0.4 days) cycles. Luteal phase PdG excretion was lower (P < 0.001) in ExLPD (2.9+/-0.3 microg/mg Cr) and ExAnov (0.8+/-0.1 microg/mg Cr) cycles compared to SedOvul cycles (5.0+/-0.4 microg/mg Cr). ExOvul cycles also had less (P < 0.01) PdG excretion during the luteal phase (3.7+/-0.3 microg/mg Cr) than the SedOvul cycles. E1C excretion during follicular phase days 2-5 was lower (P=0.05) in ExOvul, ExLPD, and ExAnov cycles compared to SedOvul cycles and remained lower (P < 0.02) in the ExLPD and ExAnov cycles during days 6-12. The elevation in FSH during the luteal-follicular transition was lower (P < 0.007) in ExLPD (0.7+/-0.1 ng/mg Cr) cycles compared to SedOvul and ExOvul cycles (1.0+/-0.1 and 1.1+/-0.1 ng/mg Cr, respectively). Energy balance and energy availability were lower (P < 0.05) in ExAnov cycles than in other menstrual cycle categories. The blunted elevation in FSH during the luteal-follicular transition in exercising women with LPD may explain their lower follicular estradiol levels. These alterations in FSH may act in concert with disrupted LH pulsatility as a primary and proximate factor in the high frequency of luteal phase and ovulatory disturbances in regularly menstruating, exercising women.

Bone mass and subtle abnormalities in ovulatory function in healthy women.

Women with occasional anovulatory or short luteal phase menstrual cycles have been reported to lose bone mineral density (BMD) at a greatly accelerated rate compared to women without such abnormalities. To investigate this association, we performed a longitudinal study of BMD in a group of healthy premenopausal women enrolled in a comprehensive study of ovulatory function. Subjects had collected daily urine samples that were analyzed for estrone and progesterone metabolites by enzyme-linked immunoassay. The 53 participants collected urine for an average of 4.1 cycles. Computer algorithms identified 7 (13.2%) women with luteal phase abnormalities (> 1 anovulatory cycle or cycle with luteal phase length < or = 10 days) and 17 (32.1%) women with other menstrual abnormalities. Areal BMD (grams per cm2) was measured at the lumbar spine, hip, and whole body using dual energy x-ray absorptiometry; BMD was measured 2-3 times over an average observation period of 17.5 months. At baseline, women with luteal abnormalities had mean BMD similar to those of the 29 women with no abnormal cycles: lumbar spine, 1.06 vs. 1.09 g/cm2; total hip, 0.95 vs. 0.94 g/cm2; whole body, 1.15 vs. 1.11 g/cm2 (P > 0.10; adjusted for age and weight at baseline, parity, physical activity level, and calcium intake). When compared at follow-up to women with no abnormal cycles, women with luteal abnormalities tended to gain BMD at the spine and hip (P > 0.10). On whole body measurement, women with luteal abnormalities tended to lose BMD compared to women with no abnormal cycles (-1.1%/yr vs. 0%/yr; P = 0.08); however, the magnitude of loss was not unusual for women in this age range and was within the coefficient of variation for replicate measurements. Neither mean luteal phase length, percent time in luteal phase, nor average daily excretion of progesterone metabolites was associated with baseline BMD or percent annual change in BMD at any measurement site. Thus, we did not confirm a relationship between luteal abnormalities and accelerated bone loss in this population of healthy premenopausal women.

Relaxin in the peri-implantation period.

The time of appearance of relaxin in peripheral blood was determined in conceptive and non-conceptive cycles using a sensitive and specific double-antibody enzyme-linked immunoassay for human relaxin. For study of relaxin in early pregnancy, daily plasma samples were collected from women receiving artificial insemination of donor semen. The day of ovulation was determined by daily LH monitoring and ultrasound observation. In three conceptive cycles, relaxin was significantly elevated over baseline 9-10 days following the LH peak. Relaxin concentrations quickly rose over the next 15 days of observation to over 800 pg/ml. Relaxin was observed to increase 1 to 2 days prior to the first detectable increase in plasma hCG as measured by enzyme-linked immunosorbent assay. To compare the relaxin profile in conceptive cycles with normal luteal phase concentrations, relaxin was also measured in daily plasma samples collected from women contracepting with barrier methods, bilateral tubal ligation, or abstinence. A small but consistent rise in relaxin in the late luteal phase was observed in nine of eleven women, which began 6-9 days after the LH peak, averaged approximately 50 pg/ml, and was declining by the next menses. It is concluded that a small but measurable rise in plasma relaxin is associated with the normal luteal phase and that relaxin secretion is accelerated around the time that hCG is first detected in conceptive cycles. This acceleration of relaxin secretion which is associated with the onset of hCG may provide additional evidence for identification of transient early pregnancy.