Ovulation induction in clomiphene-resistant anovulatory women: differential follicular response to purified urinary follicle-stimulating hormone (FSH) versus purified urinary FSH and luteinizing hormone

We studied 15 anovulatory women undergoing ovulation induction with purified human urinary FSH or purified human urinary FSH and LH [human menopausal gonadotropins (hMG)]. All patients had either sporadic or no vaginal bleeding after progesterone therapy and failed to ovulate after receiving clomiphene (250 mg for 5 days) plus hCG. Other causes of infertility were ruled out. Sixteen cycles of FSH and 12 cycles of hMG were administered according to a standard protocol. Estradiol, progesterone, androstenedione, testosterone, LH, and FSH concentrations were quantitated by RIA. Follicular diameter was determined using ultrasound. There was no significant difference in the amount of FSH or hMG used per patient, in the duration of therapy before hCG administration, or in the length of the luteal phase in any patient. There was a difference in the number of follicles greater than 1000 mm3 per cycle in those patients receiving FSH compared to the number in those receiving hMG [2.8 +/- 1.3 (+/- SEM) vs. 4.4 +/- 1.5 follicles; P = 0.026). The maximum follicular phase serum estradiol (18.3 vs. 34.8 ng/ml) and maximum luteal phase progesterone concentrations (1289 vs. 2808 pg/ml; P = 0.026) were also different between the FSH and hMG groups. Linear regression analysis revealed a significant correlation between the peripheral serum estradiol levels and the total follicular volume of follicles in the hMG-treated group which was not apparent in the FSH-treated group. These findings suggest that exogenous LH may not be required to induce folliculogenesis in anovulatory patients.     

Stimulation of ovarian follicular maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency

According to the 2-cell theory, ovarian steroidogenesis requires the coordinate action of both FSH and LH. To evaluate the relative importance of these hormones in follicular maturation, a randomized cross-over study was performed in 10 women with complete gonadotropin deficiency (absence of pulsatile LH secretion and no LH response to LHRH). Five women were treated with highly purified FSH (LH bioactivity, 0.09%) and 3 months later with human menopausal gonadotropin (hMG; LH bioactivity, 65%), each given for 10 days at a daily dose of 225 IU FSH, im. The sequence was reversed in the other 5 women. hCG (5000 IU) was administered im 24 h after the last injection of FSH or hMG. Plasma estradiol (E2), estrone (E1), androstenedione (A), testosterone, LH, and FSH concentrations and urinary LH and FSH were measured daily by RIA. Ultrasonography was performed during each treatment and 2 days after each hCG injection. After FSH treatment, mean plasma and urinary FSH levels increased, mean plasma LH did not change, and urinary LH increased slightly but not significantly from 91 +/- 32 (SE) to 164 +/- 55 mIU/24 h (10(-3) IU/24 h). After hMG treatment, mean plasma and urinary LH and FSH levels increased accordingly. The mean basal plasma E2 [11 +/- 1 pg/mL (40 +/- 4 pmol/L)] and E1 [14 +/- 4 pg/mL (52 +/- 15 pmol/L)] levels increased after FSH treatment to 207 +/- 69 pg/mL (760 +/- 253 pmol/L) and 82 +/- 21 pg/mL (303 +/- 78 pmol/L), respectively (P less than 0.01), but plasma A did not change. In response to hMG, the mean plasma E2, E1, A, and testosterone levels increased more than during FSH treatment. Ultrasonography revealed multiple preovulatory follicles (greater than or equal to 16 mm) in 2 women after hMG and 1 woman after FSH treatment; therefore, hCG was not administered. In 3 women given FSH, hCG did not induce ovulation. hCG induced ovulation in 8 women given hMG and in 6 women given FSH, based on ultrasonography and plasma progesterone levels. Thus, in the presence of profound gonadotropin deficiency pharmacological doses of FSH, with minute LH contamination, are capable of stimulating ovarian follicular maturation, underlining the key role of FSH in folliculogenesis.     

The association between granulosa cell aromatase activity and oocyte-corona-cumulus-complex maturity from individual human follicles

The steroidogenic capability of granulosa cells isolated from 12 preovulatory human follicles was correlated with the stage of maturation of the corresponding oocyte-corona-cumulus-complex ( OCCC ). Individual follicles from human menopausal gonadotropin (hMG) stimulated cycles were aspirated 36 h after administration of hCG. Granulosa cells were cultured for 150 min and corresponding OCCC were evaluated for maturity before fertilization with human sperm. Granulosa cell aromatase activity was measured using 1 beta-3H-testosterone as substrate by quantitating the amount of 3H2O produced. Progesterone production by the granulosa cells was measured as was follicular fluid levels of combined hCG and LH activity and FSH and PRL. Follicular fluid concentrations of combined hCG plus LH activity decreased somewhat while FSH levels increased as OCCC matured. PRL levels did not vary. Granulosa cell progesterone production did not change with maturity of OCCC . However, aromatase activity decreased as OCCC matured with levels from granulosa cells with immature OCCC vs. intermediate and mature OCCC of 260 +/- 148 vs. 129 +/- 53 (SE) pg E2/10(5) cells, respectively (P less than 0.07). Although granulosa cells responded variably to hMG stimulation from individual to individual, and the response was not predictable from peripheral serum estradiol levels, follicles isolated from the same patient had a definite diminution in aromatase activity with OCCC maturation. From these preliminary results, aromatase activity in immediately preovulatory granulosa cells declined as OCCC matured in hMG/hCG stimulated cycles.     

The effect of leuprolide acetate on ovulation induction with human menopausal gonadotropins in polycystic ovary syndrome

The use of exogenous gonadotropins for treatment of clomiphene-resistant chronic anovulation in women with the polycystic ovary syndrome (PCO) is hazardous and often ineffective, possibly because of the abnormal endogenous gonadotropin secretion characteristic of PCO. We evaluated the effect of leuprolide acetate, a long-acting GnRH agonist, on serum gonadotropin and sex steroid concentrations before and during human menopausal gonadotropin (hMG) induction of ovulation in women with PCO. In this controlled prospective randomized study, leuprolide was administered daily for 4 weeks, followed by concomitant hMG administration. Gonadotropin and steroid hormone concentrations were compared with those during ovulation induction cycles in women with PCO receiving hMG only. Daily administration of leuprolide for 4 weeks resulted in significantly decreased serum LH, estradiol, and testosterone concentrations, but no change in serum progesterone, FSH, and dehydroepiandrosterone sulfate. Compared to ovulation induction using hMG alone, leuprolide administration before and during hMG treatment prevented preovulatory rises in serum LH and P concentrations, while having no effect on serum FSH, testosterone, estradiol, and dehydroepiandrosterone sulfate. We conclude that leuprolide administered to women with PCO decreases gonadal steroid production and is capable of preventing premature luteinization during hMG induction of ovulation.     

Gonadotropin-releasing hormone (GnRH) analog suppression renders polycystic ovarian disease patients more susceptible to ovulation induction with pulsatile GnRH

Pulsatile GnRH administration consistently restores normal reproductive hormone levels and ovulation in women with hypogonadotropic hypogonadism, but is less effective in those with polycystic ovarian disease (PCOD). We pharmacologically created a hypogonadotropic condition with a GnRH analog (GnRH-A) in six women with PCOD to investigate the role of deranged gonadotropin secretion in PCOD and to improve the response to pulsatile GnRH ovulation induction. Before GnRH and GnRH-A treatment the women with PCOD had increased LH pulse frequency [one pulse every 55 +/- 2 (+/- SE) min; P less than 0.05] and LH pulse amplitude (10.9 +/- 1.4 U/L; P less than 0.05) compared to normal women in the follicular phase of their menstrual cycle. Each PCOD woman completed one cycle of pulsatile GnRH administration for ovulation induction before (pre-A cycles; n = 6) and one or two cycles after (post-A cycles; n = 9) GnRH-A administration [D-Ser(tBu)6-Des,Gly10-GnRH; 300 micrograms, sc, twice daily for 8 weeks]. Pulsatile GnRH (5 micrograms/bolus) was given at 60-min intervals using a Zyklomat pump. Daily blood samples were drawn during the pulsatile GnRH ovulation induction cycles for the determination of serum LH, FSH, estradiol (E2), progesterone, and testosterone, and pelvic ultrasonography was done at 1- to 4-day intervals. Mean (+/- SE) serum LH levels were elevated during the pre-A cycle (49.2 +/- 3.1 IU/L) and decreased to normal levels during the post-A cycles (19.6 +/- 1.4 IU/L; P less than 0.0001). Mean testosterone concentrations were lower during the post-A cycles [88 +/- 2 ng/dL (3.1 +/- 0.1 nmol/L)] than during the pre-A cycles [122 +/- 3 ng/dL (4.2 +/- 0.1 nmol/L); P less than 0.0001]. In the follicular phase of the post-A cycles E2 levels were significantly lower [81 +/- 5 pg/mL (300 +/- 20 pmol/L) vs. 133 +/- 14 pg/mL (490 +/- 50 pmol/L); P less than 0.0001], preovulatory ovarian volume was smaller (24.6 +/- 2.0 vs. 31.4 +/- 2.4 cm3; P less than 0.01), and the FSH to LH ratio was higher (0.56 +/- 0.03 vs. 0.16 +/- 0.01) than in the pre-A cycle, suggesting more appropriate function of the pituitary-gonadal axis. Excessive LH and E2 responses to pulsatile GnRH administration in the early follicular phase of the pre-A cycle were abolished in the post-A cycles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation of human follicular fluid inhibin activity with spontaneous and induced follicle maturation

We sought to correlate the inhibin activity of individual ovarian follicles (greater than 16 mm in diameter) from untreated (7 patients; 7 follicles), clomiphene-stimulated (150 mg/day; menstrual cycle days 5-9; 9 patients, 14 follicles), and human menopausal gonadotropin (hMG)-stimulated (150 IU/day; menstrual cycle days 3-11; 8 patients; 23 follicles) ovarian cycles and to correlate these results with the follicular fluid (FF) steroid concentration. Follicular aspirates were obtained via laparoscopy from 24 regularly menstruating patients when the diameter of the largest follicle reached 20 mm, as determined by serial ultrasonography. FF concentrations of estradiol, progesterone, testosterone, 17-hydroxyprogesterone, and androstenedione were determined by RIA. Inhibin activity was determined using the inhibition of basal 24-h FSH secretion by dispersed rat anterior pituitary cells. Inhibin values were highest among the follicles aspirated from those patients who received hMG [277 +/- 31 (+/- SE) U/ml] compared to untreated subjects (51 +/- 13 U/ml) or those who received clomiphene (96 +/- 14 U/ml). Estradiol was highest in FF from untreated patients (2295 +/- 1155 ng/ml) compared to levels in patients who received hMG (368 +/- 1.76 micrograms/ml) or clomiphene (1049 +/- 174 ng/ml). FF progesterone values were highest in untreated patients (9.4 +/- 2.59 micrograms/ml) compared to those in hMG-treated (5.04 +/- 1.76 micrograms/ml) and clomiphene-treated patients (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values were similarly higher in the untreated (1.55 +/- 0.21 micrograms/ml) and clomiphene-treated (2.54 +/- 0.27 micrograms/ml) patients than in the hMG-treated group (0.73 +/- 0.09 micrograms/ml). FF androstenedione (untreated, 50.7 +/- 30 ng/ml; clomiphene-treated, 73.4 +/- 23.4 ng/ml; hMG-treated, 60.2 +/- 19.8 ng/ml) and testosterone (6.66 +/- 2.45, 5.98 +/- 1.46, and 6.39 +/- 2.16 ng/ml, respectively) concentrations in all three patient groups were similar. In untreated patients, there was a highly significant positive correlation between intrafollicular inhibin activity and FF estradiol, testosterone, and androstenedione concentrations and a statistically significant negative correlation between intrafollicular inhibin activity and FF progesterone concentrations. Patients receiving clomiphene therapy demonstrated at least two different response patterns, one with a positive and one a negative correlation between intrafollicular inhibin activity and FF steroid concentrations. The patients receiving hMG therapy had no statistically significant correlation between intrafollicular inhibin     

Vaginal progesterone administration before ovulation induction with exogenous gonadotropins in polycystic ovarian syndrome

We studied the value of vaginal progesterone (P4) in suppressing serum LH concentrations and restoring normal luteal phase serum LH concentrations before administration of exogenous gonadotropins in anovulatory women with the polycystic ovarian syndrome (PCOS). P4 (50 mg every 12 h) was administered by vaginal suppository to 9 women (18 cycles) for 14 days before ovulation induction with human menopausal gonadotropin (hMG) and hCG. Serum LH, FSH, estradiol, P4, and PRL levels were measured daily. A biphasic effect on LH secretion occurred during P4 administration. Peak serum LH levels occurred on day 5 (125% of basal levels; P less than 0.05) of vaginal P4 suppository use, followed by a progressive fall (P less than 0.05) to 79% of basal levels, but serum LH levels were still higher than those in normal women despite achieving physiological luteal phase P4 concentrations. Ovulation occurred in 56% of cycles after P4 and hMG/hCG treatment and in 65% of control cycles after hMG/hCG alone. In 7 women, serum LH was measured at 10-min intervals for 6 h before and after vaginal P4 administration for 10 days. LH pulse frequency decreased from 7.4 +/- 1.1 to 4.4 +/- 1.2 pulses/6 h (P less than 0.01), and LH pulse amplitude increased from 3.8 +/- 1.8 to 6.1 +/- 2.9 IU/L (P less than 0.01) after P4 administration. We conclude that vaginal P4 (50 mg every 12 h) 1) produces serum P4 concentrations within the normal range for the luteal phase of the menstrual cycle; 2) elevates serum LH, but not FSH, within 5 days; 3) decreases LH pulse frequency and increases LH pulse amplitude after 10 days, but does not normalize serum LH values; and 5) fails to improve the results of subsequent ovulation induction with exogenous gonadotropins in patients with PCOS.     

Periovulatory follicular fluid hormone levels in spontaneous human cycles

We measured follicular fluid hormone levels in 48 normally cycling infertile women who underwent follicle puncture and oocyte retrieval during diagnostic laparoscopy at time-bracketed intervals after an endogenous LH surge. Follicular fluid LH, FSH, PRL, estrone (E1), estradiol (E2), progesterone (P), androstenedione (A), and testosterone (T) concentrations and P/E2 and A/E2 ratios were determined. Oocytes were classified as germinal vesicle (gv), metaphase I (mI), metaphase II (mII), or degenerating (dg). Follicular fluid (ff) hormone levels then were correlated with the stage of oocyte maturation. There were no differences in ff E1 or E2 levels at any stage of oocyte maturation, except that the mean ff E2 concentration was significantly (P less than 0.05) lower in ff containing dg oocytes [2,474 +/- 1,435 (+/- SE) nmol/L] than in those containing the other oocyte stages. The mean P levels were significantly (P less than 0.0001) higher in ff containing mI (48,781 +/- 10,240 nmol/L) and mII (41,801 +/- 11,098 nmol/L) oocytes than in ff containing gv oocytes (1371 +/- 696 nmol/L). The mean A level was highest (P less than 0.01) in dg-associated ff. Similarly, T was highest (P less than 0.05) in ff containing dg (52 +/- 14 nmol/L) oocytes than in ff containing mI (10.7 +/- 10.1 nmol/L) or mII (10.1 +/- 4 nmol/L) oocytes, and it was also elevated (P less than 0.05) in gv ff (72 +/- 33 nmol/L) compared to mII ff. The above differences also were reflected in the P/E2 ratio, which was significantly higher (P less than 0.05) in mI and mII ff, as well as in the A/E2 ratio, which was higher (P less than 0.05) in ff containing mI and mII oocytes compared to ff containing gv or dg oocytes. These data define the evolving changes in the microenvironment of the follicular fluid of preovulatory follicles of normally cycling women. They also provide reference points for analysis of ff obtained from women during stimulated cycles intended for in vitro fertilization.     

THE EFFECT OF PULSATILE FOLLICLE STIMULATING HORMONE ON THE. ENDOGENOUS LUTEINIZING HORMONE SURGE IN WOMEN

The effect of pulsatile administration of 'pure' FSH on the endogenous LH surge was investigated in 10 infertile but otherwise normal women. In each woman the LH surge in the spontaneous cycle preceding the treatment cycle was characterized in blood samples taken every 6 h. FSH was injected s.c. via a pump (28 IU every 3 h) starting on cycle day 2. Only five of the FSH-treated women displayed an endogenous LH surge, and this was markedly attenuated in four of them. The LH surge occurred significantly earlier in the FSH-treated than in the corresponding spontaneous cycle (cycle day 10.2 +/- 0.5 vs 13.6 +/- 0.8 mean- +/- SEM, P less than 0.05), although it tended to occur later in the FSH-treated cycles with a higher total follicular fluid volume of follicles 12-15 mm in diameter. This volume was even greater in the FSH-treated cycles without an endogenous LH surge. Serum progesterone levels increased significantly in all five FSH-treated cycles after the onset of the LH surge and ovulation was confirmed by ultrasound in four of them. These results suggest that the LH surge during superovulation induction with pulsatile FSH in normally cycling women is a variable event. We postulate that unknown inhibitory substances secreted be small growing follicles antagonize the positive feedback effect of E2 on LH secretion.     

Luteinzing hormone activity in menotropins optimizes folliculogenesis and treatment in controlled ovarian stimulation

Although the role that LH plays in folliculogenesis is still controversial, recent evidence points toward facilitatory actions of LH activity in ovulation induction. Thus, we compared the response to either highly purified FSH (75 IU FSH/ampoule; group A, 25 subjects) or human menopausal gonadotropin (75 IU FSH and 75 IU LH/ampoule; group B, 25 subjects) in normoovulatory GnRH agonist-suppressed women, candidates for intrauterine insemination. A fixed regimen of 2 daily ampoules of highly purified FSH or human menopausal gonadotropin was administered in the initial 14 days of treatment; menotropin dose adjustments were allowed thereafter. Treatment was monitored with daily blood samples for the measurement of LH, FSH, 17beta-estradiol (E(2)), progesterone, testosterone, hCG, inhibin A, and inhibin B, and transvaginal pelvic ultrasound was performed at 2-day intervals. Although preovulatory E(2) levels were similar, both the duration of treatment (16.1 +/- 0.8 vs. 12.6 +/- 0.5 days; P< 0.005) and the per cycle menotropin dose (33.6 +/- 2.4 vs. 23.6 +/- 1.1 ampoules; P < 0.005) were lower in group B. In the initial 14 treatment days the area under the curve of FSH, progesterone, testosterone, inhibin A, and inhibin B did not differ between the 2 groups, whereas LH, hCG, and E(2) areas under the curve were higher in group B. The occurrence of small follicles (<10 mm) and the inhibin B/A ratio in the late follicular phase were significantly reduced in group B. A nonsignificant trend toward a higher multiple gestation rate was present in group A (60% vs. 17%). We conclude that ovulation induction with LH activity-containing menotropins is associated with 1) shorter treatment duration, 2) lower menotropin consumption, and 3) reduced development of small ovarian follicles. These features can be exploited to develop regimens that optimize treatment outcome, lower costs, and reduce occurrence of complications such as multiple gestation and ovarian hyperstimulation.     

Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells as a function of chronological age and ovulation induction regimen.

We examined whether the proliferative index of granulosa cells as determined by flow cytometry varied with a women's age or ovulation induction regimen that included leuprolide acetate (LA). This prospective cohort study included three groups of patients undergoing assisted reproductive technologies. Group I consisted of 9 women age less than or equal to 30 yr, who received LA plus human menopausal gonadotropin (hMG). Group II included 9 women age more than or equal to 40 yr, who received LA plus hMG. Group III consisted of 6 women age less than or equal to 30 yr who received hMG alone. A total of 79 preovulatory follicles containing greater than 10(4) granulosa cells were obtained from these 24 women and examined by flow cytometry. Group I was compared to group II to match for ovulation induction regimen and to examine proliferative index as a function of age. Group I was compared to group III to match for age and to examine proliferative index as a function of ovulation induction regimen. Outcome measures included proliferative index of granulosa cells as a function of age, ovulation induction regimen, ampules of hMG, estradiol on day of hCG, and serum FSH. Group I demonstrated a greater proliferative index than group II: 23.4% +/- 1.4 vs. 18.4% +/- 0.96 (P less than 0.01). Group I had a greater proliferative index than group III: 23.4% +/- 1.4 vs. 11.9 +/- 0.61 (P less than 0.001). Although both age and the presence of LA appeared to affect the PI, multiple linear regression demonstrated that only the addition of LA and not age, per se, had an independent effect upon granulosa cells undergoing proliferation (P less than 0.0005). We conclude that LA followed by hMG leads to an increase in the percentage of granulosa cells undergoing proliferation when compared to ovulation induction regimens that include hMG alone. Chronological age does not appear to have a significant independent influence upon the proliferative index.     

Ovulation induction with pulsatile gonadotropin-releasing hormone and continuous human menopausal gonadotropin in polycystic ovarian disease

Various treatments have been applied to polycystic ovarian (PCO) type of anovulation. However, none of them was definitive in terms of the efficacy and side effects. Six anovulatory women of PCO type were treated with pulsatile gonadotropin-releasing hormone (GnRH) of various pulse intervals and continuous human menopausal gonadotropin (hMG). The efficacy and rationale of the treatments were discussed. The subjects were diagnosed PCO by GnRH test and/or laparoscopy. They did not ovulate with clomiphene, clomiphene-hCG and hMG-hCG therapies. Their pretreatment serum FSH and LH levels and FSH/LH ratios were 6.9 +/- 1.2 mIU/ml, 15.7 +/- 5.1 mIU/ml, and 0.54 +/- 0.19 (Mean +/- SD), respectively. The treatment consisted of 3 protocols: 1) pulsatile GnRH (5-10 micrograms/pulse) of 90 min interval, 2) pulsatile GnRH (5-10 micrograms/pulse) of 120 min interval and 3) continuous hMG (150 IU/day) through subcutaneous route. Follicular growth was monitored sonographically and an intramuscular bolus of 10,000 IU hCG was given when the dominant follicle reached 20 mm in diameter. During both GnRH treatments serum FSH levels and FSH/LH ratios did not elevate substantially. Serum LH, E2 and PRL levels elevated acutely and transiently during the initial phase of GnRH treatments. Follicular growth was observed in a small fraction of the cases, but none of them ovulated. In contrast, continuous hMG treatment induced significant elevation in serum FSH levels (8.2 +/- 1.7 mIU/ml; p less than 0.01) and FSH/LH ratios (1.73 +/- 0.57; p less than 0.001). Transient hyperprolactinemia was accompanied with the preovulatory E2 rise. All the cases ovulated and 3 singleton pregnancies followed. These findings draw conclusions as follows. Pulsatile GnRH administration may desensitize the pituitary presumably due to increased GnRH pulse frequency as a consequence of two independent pulse generators, intrinsic and exogeneous. It may induce transient hyperprolactinemia through a paracrine system between gonadotrophs and lactotrophs. As a due course pulsatile GnRH therapy is questionable for ovulation induction in cases with functioning hypothalamic-pituitary axis. The fact that continuous hMG effectively induced follicle maturation with elevated FSH/LH ratios suggested that FSH dominance might be a prerequisite for folliculogenesis. The fluctuating nature of gonadotropins might not be mandatory for folliculogenesis.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purified FSH stimulates production of inhibin by the human ovary

Ovarian inhibin production is stimulated by the administration of human menopausal gonadotrophins or following a rise in endogenous LH and FSH. In order to determine whether FSH specifically stimulates inhibin secretion in vivo, immunoassayable serum inhibin levels were measured following the administration of a highly purified preparation of urinary FSH free of significant contamination with LH. Ten anovulatory women underwent a protocol of induction of ovulation with purified FSH and human chorionic gonadotrophin (hCG). During the induction of ovulation, blood samples were taken for radioimmunoassay of FSH, LH, oestradiol, progesterone and inhibin. During the administration of FSH there were increases in plasma concentrations of FSH, oestradiol and inhibin (P less than 0.01) but no significant change in the concentration of LH. Oestradiol and inhibin concentrations rose in parallel and were closely correlated (tau = 0.920, n = 110, P less than 0.001). There was also a direct correlation between the measured level of FSH and inhibin (tau = 0.512, n = 110, P less than 0.05), but there was no correlation between LH and oestradiol, inhibin or FSH. Inhibin (tau- = 0.702, n = 10, P less than 0.01) and oestradiol (tau- = 0.691, n = 10, P less than 0.01) were correlated with the number of follicles seen on ovarian ultrasound. Levels of oestradiol and inhibin reached a peak on the day of hCG administration or on the following day. Inhibin levels then fell over the next 2 days in all cycles. In an ovulatory cycle resulting in conception, inhibin and oestradiol then rose in parallel with progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

A common single nucleotide polymorphism in exon 10 of the human follicle stimulating hormone receptor is a major determinant of length and hormonal dynamics of the menstrual cycle

CONTEXT: FSH is essential for follicular maturation. Data from ovarian hyperstimulation cycles suggest that FSH action is attenuated by a frequent single nucleotide polymorphism of the FSH receptor gene exchanging Asn for Ser at codon 680. OBJECTIVE: We hypothesized that the FSH receptor genotype influences menstrual cycle dynamics. DESIGN: Menstrual cycle was monitored from the midluteal phase through ovulation until the consecutive menstruation. SETTING: The study was conducted at the University research center. SUBJECTS: Women homozygous for the Asn680 (n = 12) and Ser680 (n = 9) variants with normal menstrual cycles volunteered for the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASUREMENTS: Follicular growth, serum LH, FSH, estradiol, progesterone, inhibin A, inhibin B and antimullerian hormone were measured. RESULTS: During the luteo-follicular transition, serum levels of estradiol, progesterone, and inhibin A were significantly lower, and FSH started to rise earlier in the Ser680/Ser680 group. FSH levels were steadily and significantly higher, and the mean area under the FSH curve was 31% greater in this group (P < 0.002). No differences were observed in estradiol, inhibin B, and growth velocities of dominant follicles. The time from luteolysis to ovulation was significantly longer in women with the Ser680/Ser680 (13.6 +/- 1.01 d) compared with Asn680/Asn680 (11.3 +/- 0.61 d, P < 0.05) genotype with a significant difference in total menstrual cycle length (29.3 vs. 27.0 d, respectively; P < 0.05). CONCLUSIONS: The FSH receptor Ser680/Ser680 genotype is associated with higher ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the intercycle transition, and longer menstrual cycles.     

Urinary luteinizing hormone testing and prediction of ovulation in spontaneous, clomiphene citrate and human menopausal gonadotropin-stimulated cycles. A clinical evaluation

A urinary luteinizing hormone test was utilized to predict ovulation in 99 spontaneous, 122 clomiphene citrate, and 82 human menopausal gonadotropin stimulated cycles. Tests were performed in early morning and evening specimens and follicular development was monitored by daily ultrasonography. A positive detection rate of 98, 97, and 94%, respectively, was obtained. Evidence of luteinized unruptured follicles was seen more frequently in stimulated cycles, concurring with negative test results. In 2 spontaneous, 1 clomiphene citrate and 5 hMG induced cycles two distinct LH surges were detected concomitant with a pattern of follicular atresia and subsequent new follicular development. Most ovulations occurred between 16 and 28 h after LH detection, significantly earlier in spontaneous than in clomiphene citrate stimulated cycles (p less than 0.02), whereas pre-ovulatory follicles were larger in the clomiphene citrate group (p less than 0.001). The mean duration of the follicular and luteal phases, as calculated from the LH peak, was substantially shorter in the hMG cycles than in the other two groups (p less than 0.001).     

Gonadotropic stimulation of inhibin secretion by the human ovary during the follicular and early luteal phase of the cycle

We studied the pattern of secretion of inhibin bioactivity from the ovary into peripheral blood during the follicular and early luteal phase of the menstrual cycle in women receiving gonadotropin therapy. Multiple follicular development was stimulated in 5 women undergoing in vitro fertilization and embryo transfer for tubal infertility using three different treatments designed to vary the concentration of FSH and LH (14 cycles). The women received clomiphene citrate (150 mg/day) from days 2-6 alone or supplemented with either exogenous human menopausal gonadotropin (28 IU/3 h) or pure FSH (28 IU/3 h) from day 6 until the day of follicle aspiration. Inhibin concentrations increased 10-fold in parallel with those of estradiol, from 0.2-0.3 U/mL on day 2 (before the onset of treatment) to 4-5 U/mL on day 14 of the cycle (time of the peak LH level). Coincidental to the LH surge, the inhibin concentration declined 2- to 3-fold before increasing again early in the luteal phase. The concentration of inhibin was higher in the gonadotropin-treated group (clomiphene plus human menopausal gonadotropin/FSH) than in the group treated with only clomiphene during the follicular phase. The number of follicles stimulated was significantly higher (P less than 0.001) in the group given exogenous gonadotropins [4.8 +/- 0.4 (SE)] than in the clomiphene alone group (2.2 +/- 0.4). These data strongly suggest that both the Graafian follicles and the corpus luteum secrete inhibin, which together with estradiol and progesterone may play a role in the regulation of FSH secretion during the luteal phase.     

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.     

Longitudinal evaluation of the different gonadotropin pulsatile patterns in anovulatory cycles of young girls.

We studied 13 adolescents (mean gynecological age 29.2 +/- 14.1 months) with anovulatory cycles and 7 women with ovulatory cycles (mean gynecological age 33.1 +/- 15.3 months) as a control group. Adolescents with anovulatory cycles were grouped on the basis of mean plasma LH values: group 1 (n = 7) with high LH values, and group 2 (n = 6) with normal LH values. In all women plasma gonadotropin concentrations were measured at 10-min intervals for 8 h on day 4 of the cycle. Pulsatile gonadotropin secretion was also studied in each subject a second time 40 months later, to establish the outcome of the different pulsatile patterns. Group 1 had more frequent and greater LH pulses than the other two groups (which were similar) and had the highest plasma 17 beta estradiol, testosterone, androstenedione, and 17 hydroxyprogesterone concentrations. Longitudinal control showed that: in group 1, three subjects out of seven acquired ovulatory cycles and there was a fall in mean LH plasma levels (30 +/- 5 vs. 9 +/- 4 IU/L; P less than 0.01), number of pulses (8.3 +/- 1.5 vs. 5 +/- 0; P less than 0.025), mean amplitude (13 +/- 3 vs. 5 +/- 2 IU/L; P less than 0.02) and an increase in interpulse interval (56 +/- 10 vs. 91 +/- 6 min; P less than 0.01). In four subjects anovulatory cycles persisted and the LH pulsatile profile remained unchanged. In group 2, five subjects out of six acquired ovulatory cycles, but there were no significant changes in the number of pulses (6 +/- 1 vs. 6 +/- 2; P = NS), interpulse interval (97 +/- 30 vs. 85 +/- 30 min; P = NS), or amplitude (5 +/- 2 vs. 4 +/- 2 IU/L; P = NS). The results indicate that: 1) anovulatory young women with early normal plasma LH values have an adequate GnRh pulsatile pattern which will easily lead to ovulation; 2) anovulatory young women with high LH plasma values may have a reproductive system blocked in a pathological condition, similar to that observed in polycystic ovary syndrome; 3) only few subjects with high plasma LH values are able to achieve ovulation and normalize LH pulsatile pattern as a consequence of a new mode of GnRh release.     

Does metformin modify ovarian responsiveness during exogenous FSH ovulation induction in normogonadotrophic anovulation? A placebo-controlled double-blind assessment

OBJECTIVE: To assess whether the addition of metformin to gonadotrophin ovulation induction in insulin-resistant, normogonadotrophic, anovulatory women alters ovarian responsiveness to exogenous FSH. DESIGN: Placebo-controlled double-blind assessment in an academic hospital. RESULTS: After a progestagen withdrawal bleeding, patients were randomised for either metformin (n = 11) or placebo (n = 9) treatment. In cases of absent ovulation, exogenous FSH was subsequently administered to induce ovulation. Only during metformin treatment did body mass index and androgen (androstenedione and testosterone) levels decrease, whereas FSH and LH levels increased significantly. In the metformin group, a single patient ovulated before the initiation of exogenous FSH. Significantly more monofollicular cycles and lower preovulatory oestradiol concentrations were observed in women receiving FSH with metformin compared with FSH alone. CONCLUSIONS: Metformin co-treatment in a group of insulin-resistant, normogonadotrophic, anovulatory patients resulted in normalization of the endocrine profile and facilitated monofollicular development during the FSH induction of ovulation.