Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.

Statistical evaluation of coincident prolactin and luteinizing hormone pulses during the normal menstrual cycle.

The purpose of this work was 2-fold. First, we sought to develop statistical criteria by which it could be established that the coincident occurrence of pulses of two different hormones exceeds that which would occur by chance alone, thereby suggesting that secretion of the two hormones is either coupled or controlled from a single source generator. Using computer simulations of uncoupled pulse generators operating at different frequencies, we were able to derive the appropriate statistical criteria and to apply them to achieve our second objective, to determine whether the occasional coincidence of plasma LH and serum PRL pulses that occurs throughout the menstrual cycle in normal women exceeds that which would happen by chance. The results of the computer simulations indicated that pulses emanating from two completely independent oscillators will occur coincidently at a predictable rate, despite the fact that the generator sources are not coupled; moreover, the rate of coincidence is increased when the pulse frequency of one of the source generators is increased. Using this knowledge and the statistical criteria we derived, we analyzed the coincidence of LH and PRL pulses in five normal women during their early follicular, late follicular, and midluteal phases and in another five women during their late luteal phase. We found that the number of PRL pulses that occurred coincidently with LH pulses consistently exceeded that which would be predicted if the two pulse generators were operating completely independently of one another; however, only during the late follicular and late luteal phases was the coincidence level between LH and PRL pulses sufficiently high in a sufficient number of women to conclude that there was coupling between the pulse sources. These studies suggest, first, that stringent and rigorous statistical criteria must be applied to the analysis of spontaneously coincident secretory phenomena before it can be deduced that two pulse generators are indeed coupled, and second, that the pulse generators governing the secretion of PRL and LH are probably coupled, at least during certain phases of the menstrual cycle.

Nocturnal slowing of pulsatile luteinizing hormone secretion in women during the follicular phase of the menstrual cycle.

The gonadotropin secretion pattern in normal reproductive age women (n = 5) was evaluated for the presence of a circadian rhythm. The women volunteered for a series of 24-h admissions in different phases of their menstrual cycles (early follicular, late follicular, and midluteal). Plasma LH and FSH levels were determined by RIA in blood samples drawn through indwelling venous catheters at 20-min intervals throughout a normal 24-h sleep-wake cycle. The gonadotropin secretory pattern was subjected to cosine analysis for identifying rhythmicity. The LH interpulse interval fluctuated with a significant 24-h rhythm during the early follicular phase in four of the five women. The maximum interpulse intervals occurred during the early morning between 0100 and 0500 h (mean, 0250 h), with a corresponding increase in LH pulse amplitude occurring within the same time interval (mean, 0320 h). We found no consistent 24-h rhythms in overall mean plasma LH levels during any phase of the menstrual cycle, nor did we find a significant rhythmicity in either LH interpulse interval or LH pulse amplitude during the late follicular or luteal phase. These results demonstrate that the LH pulse-generating system is frequency modulated on a circadian basis during the early follicular phase of the human menstrual cycle.

Corpus luteum insufficiency induced by a rapid gonadotropin-releasing hormone-induced gonadotropin secretion pattern in the follicular phase.

The pulse frequency of LH and FSH (and by inference, GnRH) is a major determinant of the relative baseline plasma levels of LH and FSH. Luteal phase deficiency has been reported to be associated with increased gonadotropin pulse frequency and inadequate preovulatory follicular development. In this study we induced in normal women a supraphysiological gonadotropin pulse frequency in the follicular phase to determine its effect on follicular development and corpus luteum function. Specifically, we tested the hypothesis that a supraphysiological GnRH pulse frequency would result in deficient luteal phase production of progesterone. The subjects were six normal ovulatory women (age range, 23-35 yr). They were initially studied during a control cycle (cycle 1). Then, 25 ng/kg GnRH was administered iv every 30 min from the early follicular phase of the next cycle (cycle 2) until ovulation occurred. GnRH administration resulted in increased follicular phase plasma LH and FSH levels and LH to FSH ratios, multiple preovulatory follicles (mean, 2.8) with increased mean integrated estradiol [1302 (pg/mL)day (cycle 1) vs. 2550 (pg/mL)day (cycle 2); P less than 0.05; 4780 vs. 9360 (pmol/L)day, Systeme International units], spontaneous ovulation, decreased luteal phase plasma immunoreactive and bioactive LH levels, decreased luteal phase length [13.5 days (cycle 1) vs. 8.8 days (cycle 2); P less than 0.05], and decreased mean integrated progesterone secretion [152 (ng/mL)day (cycle 1) vs. 66 (ng/mL)day (cycle 2); P less than 0.01; 482 vs. 209 (nmol/L)day, Systeme International units]. We conclude that high frequency LH and FSH secretion during the follicular phase can induce inadequate progesterone secretion during the subsequent luteal phase, and we infer that the pathophysiological basis for this induced luteal phase deficiency is decreased LH support of corpus luteum function.

Luteal phase deficiency: abnormal gonadotropin and progesterone secretion patterns.

Luteal phase deficiency (LPD) is a reproductive disorder associated with infertility and spontaneous abortion. This study was undertaken to determine whether LPD might be related to an abnormal pattern of gonadotropin secretion. We tested this hypothesis by evaluating the pattern of pulsatile LH secretion in both the follicular and luteal phases of the menstrual cycle in normal women (n = 21) and women with LPD (n = 20), which was diagnosed on the basis of two out of phase endometrial biopsies. In addition, we sought to determine whether changes in progesterone (P) pulse patterns could account for the decrease in average serum P levels in women with LPD. To this end, we examined the pulse patterns of P and compared these patterns between normal women and those with LPD. Frequent blood sampling was performed in both groups to determine their respective hormone secretion patterns. In the follicular phase, blood samples were obtained every 10 min for 12 h; in the luteal phase the samples were obtained every 10 min for 12 h; in the luteal LH, FSH, and P were assayed in each sample. Pulse detection was performed by an adaptive threshold method of pulse analysis. The LH pulse frequency was significantly higher in the women with LPD than in the normal women in the early follicular phase [P less than 0.05; LPD, 12.8 +/- 1.4 (+/- SE); normal, 8.2 +/- 0.7 pulses/12 h]. LH pulse frequency was similar in the early and late follicular phases in the women with LPD, whereas it was higher in the late follicular phase in normal women. Mean serum FSH levels were not different between groups in both the early and late follicular phases. In the luteal phase the P pulse amplitude and mean serum P level were significantly lower in the LPD group than in the normal women (P less than 0.01). We conclude that 1) a too rapid LH pulse pattern in the early follicular phase may lead to inadequate LH support of the corpus luteum and become manifest as LPD; 2) the mechanism for inadequate P secretion in LPD is decreased P pulse amplitude; 3) the finding of similar serum FSH levels in the two groups in both the early and late follicular phases did not support compromised folliculogenesis as an etiological factor for LPD.

Prolactin secretion and corpus luteum function in women with luteal phase deficiency.

Luteal phase deficiency (LPD) as a clinical infertility problem is considered to have a heterogeneous etiology. Hyperprolactinemia has long been considered a causative factor of LPD. In this context we investigated PRL secretion in 18 women with LPD. All of the subjects were infertile with 2 out of phase (greater than 2 days) endometrial biopsies; 10 of the women also had daily blood samples, this latter subgroup had significantly decreased integrated luteal phase progesterone (P) levels compared to normal women with in-phase biopsies. PRL secretion was investigated as follows: 1) daily blood levels; 2) pulsatile secretion patterns in 3 cycle phase [early follicular (12 h); late follicular (12 h); midluteal (24 h)], 3) LH-PRL coupling, and 4) nocturnal patterns. Results were compared to findings in 36 normal women. The mean daily levels of PRL over the menstrual cycle were not different between the two groups (LPD, 12.1 +/- 1.5; normal, 13.8 +/- 0.8 microgram/L; P = 0.3). There was no correlation between luteal phase integrated P and PRL levels for either group. There was a small difference in the PRL pulse amplitude in the early follicular phase between the LPD and normal women (2.6 +/- 0.3 vs. 5.5 +/- 1.3 micrograms/L; P less than 0.05). There were no significant differences between groups in PRL pulse frequency or mean level during the 12 or 24 h in any cycle phase. There was an equivalent amount of LH-PRL pulse coupling in both groups in all three cycle phases. Diurnal and nocturnal PRL secretion was studied by breaking the 24 h data (midluteal) into day (0700-2300 h) and night (2300-0700) segments. Mean PRL levels were higher at night in both groups (LPD, 15.9 vs. 12.6; normal, 15.4 vs. 9.3 micrograms/L; P less than 0.05), as expected. There were no differences in nocturnal PRL secretory patterns between the two groups. In summary, we have serious reservations whether abnormalities in PRL secretion are a common or integral part of the pathophysiology of LPD. From previous work we know these subtle abnormalities in PRL secretion in LPD are associated with definite abnormalities in gonadotropin secretion. We believe these gonadotropin abnormalities are probably more significant in terms of decreased P secretion.

The importance of signal pattern in the transmission of endocrine information: pituitary gonadotropin responses to continuous and pulsatile gonadotropin-releasing hormone.

We tested the hypothesis that pulsatile GnRH stimulation of the pituitary is required for normal gonadotropin secretion in humans. We administered GnRH in pulsatile and continuous regimens in varying order to each of five women with hypothalamic amenorrhea and presumed endogenous GnRH deficiency. Mean serum levels of GnRH were similar during the pulsatile and continuous regimens. All women ovulated during the pulsatile regimen (progesterone, greater than 31.8 nmol/L (10 ng/mL); none ovulated during the continuous regimen. Compared to pretreatment levels, FSH and estradiol, as measured by RIA, and LH, as measured by bioassay, increased significantly during the pulsatile GnRH regimen, but not during the continuous regimen. However, LH and alpha-subunit, as measured by RIA, increased significantly during both continuous and pulsatile GnRH administration. We conclude that a pulsatile pattern of GnRH is essential to normal functioning of the human female reproductive axis. Continuous administration of GnRH, producing mean serum levels of the peptide indistinguishable from those found during pulsatile administration, stimulates some rise in a nonbioactive form of radioimmunoassayable LH-like material and alpha-subunit, but does not stimulate bioactive LH, FSH, estradiol, or progesterone and does not lead to ovulation.

Onset and characteristics of the midcycle surge in bioactive and immunoactive luteinizing hormone secretion in normal women: influence of physiological variations in periovulatory ovarian steroid hormone secretion.

Limited studies in nonhuman primates suggest that the midcycle LH surge is characterized by distinctly different patterns of bioactive (LH-BIO) and immunoactive (LH-RIA) LH secretion. To further examine the patterns of midcycle LH-BIO and LH-RIA secretion and explore the influence of physiological variations in steroid hormone feedback on LH surge dimensions we studied seven normal ovulatory women over the periovulatory interval. In each, blood samples were obtained every 3 h and transvaginal ultrasonography was performed every 12 h over a 5-7 day interval at midcycle. Serum levels of LH-RIA, FSH, estradiol (E2), progesterone (P4), and 17-hydroxyprogesterone were determined by RIA; LH-BIO was estimated using a mouse leydig cell bioassay. Hormone data were standardized to the time of surge onset in LH-RIA (time zero), defined as a 100% increase above a 6-point running mean baseline value; surge cessation was defined as a decline to below baseline concentration. Mean LH-RIA surge duration was 54.0 +/- 4.0 h. LH-BIO surge onset was simultaneous with that of LH-RIA and coincident with the peak in E2 levels (mean data). Mean P4 and 17-hydroxyprogesterone rose in a parallel, phasic manner, an abrupt increase in slope occurred between -6 h and +30 h but an acute rise in P4 was not consistently observed among individuals. The surge onset to follicle rupture interval (mean 37.6 +/- 4.2 h) positively correlated with peak LH-RIA (r = 0.76, P less than 0.05), surge amplitude (r = 0.74, P less than 0.05) and surge onset to peak interval (r = 0.87, P less than 0.02), but not surge duration. There were no significant relationships between E2 or P4 (mean, peak, integrated, slope) and surge amplitude or duration (LH-RIA, FSH), peak value, or surge onset to peak interval (LH-RIA, LH-BIO, FSH). These data suggest that in women, 1) onset of the midcycle surge in LH-RIA and LH-BIO is simultaneous, and 2) surge characteristics are not influenced by physiological variations in steroid hormone secretion that occur beyond the thresholds required for surge initiation.

Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor in spontaneous menstrual cycles of normal women of advanced reproductive age.

Previous studies indicate that the menstrual cycles of older reproductive age women are characterized by a selective elevation of FSH associated with early development and ovulation of a dominant follicle. Several intraovarian hormones and growth factors have been identified that appear to serve important paracrine roles. The purpose of this study was to examine follicular fluid (FF) hormones and growth factors in the dominant follicle of unstimulated cycles of older, ovulatory women. We aspirated FF from the preovulatory dominant follicle in natural menstrual cycles of older subjects (age, 40-45 yr; n = 20) and younger controls (age, 20-25 yr; n = 19). FF was analyzed for estradiol, progesterone, testosterone, androstenedione, inhibin A and B, total activin A, total follistatin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor (VEGF) concentrations. We found that the dominant follicles from older women contain normal concentrations of steroids, inhibin A and B, IGF-II, IGFBP-2, and IGFBP-3; increased concentrations of follistatin, activin A, and VEGF; and decreased concentrations of IGF-I. Therefore, under the influence of elevated FSH, the dominant follicle in older women is highly competent in terms of hormone and growth factor secretion. We postulate that elevated FF activin may be related to the early ovulation observed in older women, whereas elevated VEGF may be related to the meiotic spindle abnormalities observed in the oocytes of older reproductive age women.

The importance of luteinizing hormone in the control of inhibin and progesterone secretion by the human corpus luteum.

Serum inhibin levels rise markedly during the luteal phase of the human menstrual cycle and are closely correlated with serum progesterone (P) levels, suggesting that the corpus luteum (CL) secretes inhibin. While FSH is the major regulator of inhibin secretion by the granulosa cells, the control of CL inhibin secretion is unclear. We hypothesized that, like P, CL inhibin secretion would be LH dependent. To examine this possibility, normal women were given the GnRH antagonist [Ac-D2Nal1, D4CL Phe2, D3Pal3, Arg5, DGlu6 (AA), DAla10]GnRH (Nal-Glu antagonist) for 3 consecutive days commencing on day 6-8 of the luteal phase. The daily doses were 2.5 (n = 3), 10 (n = 4), and 25 micrograms/kg (n = 5), sc. Serum LH levels fell 2 h after injection, and the fall was maximal (70-74%) at 6 h; the degree of suppression was not dose dependent. The duration of suppression was dose related, being less than 12 h, between 12, and 24 h, and more than 24 h for the 2.5, 10, and 25 micrograms/kg doses, respectively. Serum FSH levels declined by 22-43%, but the effect was not dose related. Serum P levels fell by 42-45% 8 h after each dose of antagonist. They returned to baseline 24 h after the 2.5 micrograms/kg dose, but after both the 10 and 25 micrograms/kg doses serum P levels continued to fall, and menstrual bleeding commenced within 48-72 h after the first antagonist injection. Serum inhibin levels were not altered relative to normal cycles by the 2.5 micrograms/kg dose, but fell by 48% and 58%, and 62% and 73% respectively, 48 and 72 h after the 10 and 25 micrograms/kg doses, respectively. Serum P and inhibin levels correlated closely in all women. To examine the relative roles of FSH and LH in the control of CL function, Nal-Glu antagonist (25 micrograms/kg, sc) was administered at 0 and 24 h commencing on day 6-8 of the luteal phase, in combination with either human menopausal gonadotropin (hMG; 150 IU, im, every 12 h) or hCG (1500 IU, im, once), both commencing at 0 h. hMG administration led to a rapid (by 2 h) and marked (3- to 9-fold) rise in serum FSH levels, whereas serum LH remained low, similar to antagonist alone treatment cycless.(ABSTRACT TRUNCATED AT 400 WORDS)

Luteal phase deficiency: characterization of reproductive hormones over the menstrual cycle.

The recurrent deficiency of progesterone (P) secretion by the corpus luteum has been associated with infertility and habitual abortion and given the clinical diagnosis of luteal phase deficiency (LPD). There is evidence that both follicular and luteal phase abnormalities can result in LPD cycles. In this study we have examined reproductive hormone levels and preovulatory follicular size in women with LPD (n = 10). For the purposes of this study, LPD was determined by an endometrial biopsy in the studied cycle that was more than 2 days out of phase. These biopsies were performed in women with infertility or habitual abortion who exhibited an out of phase biopsy in a prior cycle. The control group consisted of 28 normal women. Daily serum levels of the following hormones were determined in each subject: LH and FSH [immuno- and bioactive (LH-immuno and LH-bio)], P, estradiol (E2), and inhibin. The LPD women exhibited significant decreases in integrated luteal phase levels of inhibin [10,615 +/- 898 vs. 13,560 +/- 662 (U/L).days; P less than 0.02] and E2 [5,015 +/- 275 vs. 6,435 +/- 393 (pmol/L).days (1366 vs. 1753 (pg/mL).days); P less than 0.05] in addition to the expected decrease in P [280 +/- 23 vs. 420 +/- 23 (nmol/L).days (88 vs. 132 (ng/mL).days); P less than 0.01]. On days 6-11 after the LH surge (day 0), there was a significant (P less than 0.05) decrease in mean LH-bio levels in LPD compared with those in normal women (146 +/- 26 vs. 212 +/- 24 micrograms/L). The midcycle LH surge was deficient in LPD when both LH-immuno [482 +/- 30 vs. 672 +/- 43 (micrograms/L).days; P less than 0.01] and LH-bio [1711 +/- 179 vs. 2248 +/- 226 (micrograms/L).days; P less than 0.05] levels were compared with normal values. When comparing the follicular phase in LPD with that in normal women, similar follicle size, peak and integrated E2 levels, and mean LH and FSH (immuno and bio) levels were found. The only follicular phase abnormality noted in this study was decreased mean levels of serum inhibin in the early and midfollicular phases (221 +/- 19 vs. 308 +/- 25 U/L; P less than 0.01). In this group of women with LPD, low levels of inhibin in the follicular phase were consistent with the concept of a defect in function of the preovulatory follicle, possibly as a result of previously described defects in gonadotropin secretion in this condition.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels.

The factors responsible for the variability in plasma leptin levels observed among individuals with similar body compositions remain unclear. To examine the impact of dietary variables, we compared the changes in leptin levels induced by fasting and dietary fat restriction with the expected decrease following a significant loss in adipose mass. A 21.4 +/- 3.7% weight loss led to a 76.3 +/- 8.1% decrease in mean plasma leptin level (25.2 +/- 9.3 to 6.1 +/- 3.4 ng/mL, P = 0.0001) in a group of 9 obese males. Despite a weight loss of only 2.6 +/- 0.8%, mean plasma leptin levels fell by 61.9 +/- 25.2% (8.5 +/- 4.5 to 2.4 +/- 0.5 ng/mL, P < 0.01) in 7 nonobese females subjected to 3 days of fasting. Leptin levels in fasted subjects returned to baseline within 12 h of refeeding. Individual high- and low-fat meals given to 19 subjects after an overnight fast had no effect on plasma leptin levels. Reduction in dietary fat content from 37-10% of total calories for 7 weeks was also without effect on plasma leptin levels in these subjects. We conclude that plasma leptin levels primarily reflect total adipose mass, rather than meal consumption or dietary energy source, but that the reduction in leptin levels with ongoing fasting is disproportionate to the reduction in adipose mass. The ability of fasting to deactivate this presumed physiological satiety system may have been advantageous in environments characterized by rapid changes in food availability.

Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women.

Women experience a decline in fertility that precedes the menopause by several years. Previous studies have demonstrated a monotropic rise in FSH associated with reproductive aging: however, the mechanism of this rise and its role in the aging process are poorly understood. The purpose of this study was to characterize ovarian follicular development and ovarian hormone secretion in older reproductive age women. Sixteen women, aged 40-45 yr, with regular ovulatory cycles were studied. The control group consisted of 12 ovulatory women, aged 20-25 yr. Serum obtained by daily blood sampling was analyzed for FSH, LH, estradiol (E), progesterone, and inhibin (Monash polyclonal assay). Follicle growth and ovulation were documented by transvaginal ultrasound. Older women had significantly higher levels of FSH throughout the menstrual cycle. E, progesterone, LH, and inhibin levels did not differ between the two age groups when compared relative to the day of the LH surge. Ultrasound revealed normal growth, size, and collapse of a dominant follicle in all subjects. Older women had significantly shorter follicular phase length associated with an early acute rise in follicular phase E, reflecting accelerated development of a dominant follicle. We conclude that older reproductive age women have accelerated development of a dominant follicle in the presence of the monotropic FSH rise. This is manifested as a shortened follicular phase and elevated follicular phase E. The fact that ovarian steroid and inhibin secretion were similar to those in the younger women suggests that elevated FSH in women of advanced reproductive age may represent a primary neuroendocrine change associated with reproductive aging.

Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age.

Reproductive aging in women (a physiological decline in the function of the hypothalamic-pituitary-ovarian axis) is an infrequently investigated and poorly understood biological phenomenon. Although menstrual irregularity and anovulation are known to precede the menopause, normal women in their fifth decade experience a profound decrease in fertility while still experiencing regular menstrual cycles. To further our understanding of the physiological changes associated with reproductive aging, this study examined the spontaneous development and function of ovarian follicles in normal women, aged 40-45 yr. The subjects were women (n = 21), aged 40-45 yr, who had regular 25- to 35-day ovulatory menstrual cycles, were not infertile, had no medical problems, and met specific criteria for weight, diet, and exercise. The controls were normal women (n = 20), age 20-25 yr, who met the same criteria. The subjects were monitored with daily hormone measurements [LH, FSH, estradiol (E), progesterone (P), and inhibin] and pelvic sonograms from day 1 of their study cycle until the dominant ovarian follicle reached a mean diameter of 15 mm and/or a serum E level of 550 pmol/L or higher was attained. At that time, 10,000 U hCG were given, and a transvaginal sonographic follicle aspiration was performed 32 h later. The follicular fluid (FF) was collected, stored frozen at -70 C, and later analyzed for E, P, testosterone (T), androstenedione, inhibin, insulin-like growth factor I (IGF-I), and IGF-II. The number of cycle days to aspiration was lower (11.6 vs. 15.6 days; P < 0.001) and the early follicular phase mean FSH and mean E levels were higher (9.3 vs. 6.6 mIU/mL and 305 vs. 160 pmol/L; P < 0.01) in the older (O) group compared to the younger group. There was a strong trend toward higher FF mean E (2280 vs. 1931 nmol/L) and lower FF mean T (978 vs. 2361 pmol/L) levels in group O. The E/T ratio was significantly higher (5253 vs. 2408; P < 0.03) in group O. In group O, the mean FF P levels were increased as well (25.1 vs. 18.8 micromol/L; P < 0.01). The serum mean IGF-I (153 vs. 226 ng/mL; P < 0.001) and FF mean IGF-I (113 vs. 158 ng/mL; P < 0.02) levels were significantly decreased in group O. There were no differences between groups in serum or FF IGF-II or inhibin levels. Whether reproductive aging is an intrinsic ovarian process or the ovary is simply responding to exogenous influences, the ovary in general and its follicles in particular are the primary site of the effects of aging. Ovarian follicles in older ovulatory women have some unique features: 1) the follicles are the same size as those in younger women, but form more rapidly; 2) secretion of E and inhibin is not compromised; 3) the concentrations of steroids in the FF are indicative of a healthier follicle, i.e. increased P levels and higher estrogen to androgen ratio; and 4) serum and FF levels of IGF-I are decreased, but there are no differences in IGF-II levels.

Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles.

UNLABELLED: This study sought to compare circulating and follicular fluid (FF) concentrations of dimeric inhibin A and B utilizing specific two-site ELISAs for these hormones in normal older and younger ovulatory women. METHODS. Normally ovulating women age 40-45 (n = 10) and 20-25 (n = 13) were studied throughout the follicular phase with daily blood sampling, transvaginal ultrasound examinations, and dominant follicle aspiration. When the dominant follicle reached a mean diameter of 16 mm or serum estradiol (E2) was > or = 550 pmol/L, 10,000 IU of hCG was administered intramuscularly followed 32 hours later by transvaginal follicle aspiration. Serum and FF samples were analyzed for E2, FSH, and inhibin A and B. Daily hormone levels were compared by ANOVA, and mean results were compared using t-tests. RESULTS: Older women developed a dominant follicle sooner, meeting criteria for hCG cycle day 10.6 +/- 0.4 vs. 14.5 +/- 1.0 p < 0.001. As expected, the older group had higher maximal serum FSH concentrations compared to the younger women (11.4 +/- 0.5 vs. 8.0 +/- 0.4 IU/L, p < 0.001). We compared hormone concentrations from days-1 to 3 (where day 0 = day of maximal FSH concentration). E2 concentration was higher in the older women (p = 0.002), and there was no significant difference in inhibin A secretion (p = 0.61). In contrast, mean inhibin B concentration was significantly lower in the older women (p = 0.04). On the day of aspiration of the dominant follicle, serum inhibin B was decreased in the older subjects (42.6 +/- 6.5 vs. 153.1 +/- 53 pg/ml, p = 0.02), whereas older subjects had higher levels of inhibin A (106 +/- 16 vs. 60.4 +/- 9.4 pg/ml, p = 0.04) and similar E2 levels (665 +/- 35.2 vs. 687 +/- 92 pmol/L, p = 0.83). There were no differences in FF concentrations of inhibin B (164 +/- 31 vs. 174 +/- 37 ng/ml, p = 0.85), inhibin A (317.7 +/- 38 vs. 248 +/- 57 ng/ml, p = 0.16), or E2 (2074 +/- 294 vs. 2474 +/- 338 nmol/L, p = 0.82) in the older and younger women. CONCLUSION. Follicular phase inhibin B secretion is decreased in older ovulatory women who demonstrate a monotropic FSH rise, whereas inhibin A secretion is similar to that in younger women. The dominant follicle in these older women appears to be normal in terms of FF E2 and inhibin content. We speculate that decreased inhibin B secretion most likely reflects a diminished follicular pool in older women and may be an important regulator of the monotropic FSH rise.

Progesterone modulation of pulsatile luteinizing hormone secretion in normal women.

Recent studies show that the frequency and amplitude of pulsatile LH secretion change during the normal human menstrual cycle; however, the neuroendocrine mechanisms underlying these changes are poorly understood. To assess the role of progesterone (P) in regulating LH secretion patterns, we treated normal women (n = 5) with im P in oil during the follicular phase of their cycle and compared LH pulse frequency, amplitude, and mean plasma level during treatment to those in normal cycling women. Normal women were studied five times in five menstrual cycles. Each study lasted 24 h, with a sampling interval of 20 min. The cycle phases studied were early follicular (twice), late follicular (LF), midluteal, and LF with P therapy to simulate luteal phase plasma P levels. LH pulse frequency was slower (P less than or equal to 0.001) in the midluteal phase than in either the early follicular phase or LF, and furthermore, P, administered in the normal follicular phase, slowed LH pulse frequency, augmented pulse amplitude, and reduced mean plasma LH levels compared to those in untreated women studied at the same cycle phase (P less than or equal to 0.02). We infer that P secretion by the ovary mediates the change in the LH secretory pattern during the luteal phase of the normal menstrual cycle, and that at least part of this effect is mediated by the central nervous system.

Polysymptomatic complaints and Briquet's syndrome in polycystic ovary disease.

To test the hypothesis that there is an association between polycystic ovary disease and Briquet's syndrome, the authors administered a health questionnaire to infertile women with polycystic ovary disease, infertile women with tubal disease, and normal women. The patients with polycystic ovary disease endorsed significantly more physical and psychological complaints than either control group. Structured interviews revealed that five of the 39 (13%) met diagnostic criteria for definite or probable Briquet's syndrome. This study gives support to an association between polysymptomatic complaints, Briquet's syndrome, and polycystic ovary disease.

The usefulness of a urinary LH kit for ovulation prediction during menstrual cycles of normal women.

OBJECTIVE: To assess the predictive value of monitoring urine LH at home using a rapid, colorimetric enzyme immunoassay test (Ovuquick) once every evening. METHODS: Twenty-six strictly defined normal women with no history of infertility were enrolled in studies involving urine LH tests. Each subject had transvaginal sonography and serum LH tests performed two times per day beginning in the middle of a normal menstrual cycle. All subjects performed urine LH testing at home every evening. The time of the peak serum LH measurement was considered the surge. Ovulation was determined using sonographic criteria with confirmation by normal luteal-phase progesterone levels (3 ng/mL or greater). Two clinically relevant intervals were determined: interval I, time from peak serum LH to positive urine LH, and interval II, time from positive urine LH to follicular collapse by ultrasonography. RESULTS: All 26 cycles examined were ovulatory, based on sonographic and progesterone level criteria. The mean time (+/- standard error of the mean [SEM]) for interval I was 2 +/- 2 hours (95% confidence interval [CI] -2 to 6). The mean time (+/- SEM) for interval II was 20 +/- 3 hours (95% CI 14-26). Positive predictive values for follicular collapse within 24 or 48 hours after positive urine LH testing were 73 and 92%, respectively. CONCLUSION: Urine LH testing every evening is a reliable method of predicting ovulation within the ensuing 48 hours.

The steroid profile of a virilizing ovarian tumor.

A case report of a 25-year-old female with a sex cord stromal virilizing ovarian tumor is presented. The pathway of ovarian steroid secretion in this tumor is elucidated with the dominant elements being pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The tumor primarily made testosterone (T) with lesser elevations of androstenedione (A), dehydroepiandrosterone (DHEA), and dihydrotestosterone (DHT). Expert pathologic opinions differed whether this neoplasm was a Sertoli-Leydig tumor or a virilizing granulosa tumor; therefore, it was probably a gynandroblastoma. A unilateral salpingo-oophorectomy was performed and the patient promptly resumed normal ovarian function with ovulation.

An in vitro fertilization program using satellite physicians.

In order to provide an in vitro fertilization (IVF) service for a large geographic region, we developed the concept of Satellite IVF Centers. The goals of this program were as follows: 1) to facilitate patient participation by decreasing travel expenditures, time for screening appointments, and IVF cycle cancellations, and 2) to involve community physicians in a regionalized program. We established centers in nine cities within the Washington, Alaska, Montana, and Idaho region serviced by the University of Washington, and in Alberta, Canada. A 2-day training session was held to provide participating physicians with a basic knowledge of IVF. We considered the roles of the satellite IVF physician to include identification of couples for IVF, initiation of ovulation induction cycles, and determination of appropriate induction cycles for oocyte recovery. The staff of the University Department of Laboratory Medicine standardized the methodologies for estradiol measurements and maintained a quality control analysis for all participating community laboratories. Satellite activity was coordinated through the IVF office at the University, which monitored physicians' decisions based on hormone and ultrasound data from days 7 and 8 of the stimulation cycle. Patients meeting specific ovarian response criteria arrived at the University on day 9 of the stimulation cycle and completed IVF. During 18 months, 72 patients were screened and initiated cycles at the nine participating centers. Forty-four of these patients were sent to the University for oocyte recovery, which resulted in 39 embryo transfers and eight pregnancies. This service has reached a large geographic community, dramatically reduced the cost of IVF for this community, and met with both patient and physician acceptance.