To evaluate the relative importance of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in follicular development and oocyte fertility in the human species, the use of recombinant human FSH, human menopausal gonadotrophin (HMG), and very highly purified urinary human FSH (FSH-HP) plus oestradiol valerate for ovarian stimulation and in-vitro fertilization (IVF) were compared in three cycles in a woman with isolated congenital gonadotrophin deficiency who had never been treated with ovarian stimulating agents. The total number of ampoules of gonadotrophins used was lower in the HMG treatment cycle. Ovarian response and IVF outcome in the three treatment cycles were as follows: (i) HMG cycle: normal follicular growth, normal pattern of oestradiol and inhibin through the menstrual cycle, high fertilization rate (93%); (ii) recombinant FSH cycle: normal follicular growth, low oestradiol and abnormal inhibin, finally poor rate of fertilization (28%); (iii) FSH-HP plus oestradiol valerate cycle: normal follicular growth, normal pattern of inhibin and poor fertilization rate (27%). Luteal plasma progesterone concentrations were much higher in the HMG treatment cycle. This case shows that FSH is the only factor required in order to induce follicular growth in the human, although LH or a product derived from its action may assist in order to achieve full follicular maturity and oocytes capable of fertilization. Though oestradiol might have a mediatory role in the process of follicular maturation, our results favour a direct primary role of LH in complete maturation of the follicle.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.

FSH stimulates follicular growth through inducing granulosa cell proliferation. Our working hypothesis is that mitogenesis is facilitated by a locally produced growth/differentiation factor(s) that modulates FSH action in developing granulosa cells. The present study was undertaken to examine the effect of the related gonadal peptides activin and inhibin on granulosa cell proliferation. Monolayer cultures of granulosa cells isolated from preantral/early antral follicles in immature rat ovaries were established by incubation overnight in serum-free medium 199. Treatment was initiated with serum-free medium containing recombinant human (rh) activin and/or rh-inhibin in the presence or absence of FSH. After incubation for 18 h, medium was collected for progesterone determination (as a marker of cell differentiation), and the cell monolayers were incubated for 2 h in the presence of [3H]thymidine to measure DNA synthesis. Activin dose dependently (1-100 ng/ml) stimulated DNA synthesis (minimal effective dose, 1 ng/ml), whereas inhibin or FSH alone was without effect. When activin (1 ng/ml), but not inhibin, was present with FSH, the gonadotropin caused dose-dependent increases in [3H]DNA synthesis over a wide range of FSH concentrations (1-100 ng/ml). This property of activin was unaltered by the additional presence of inhibin (1-100 ng/ml). To analyze the role of cAMP in mediating the mitogenic action of FSH in the presence of activin, experiments were repeated substituting a membrane-permeable cAMP agonist, 8-bromo-cAMP (8br-cAMP; 0.1-3 mM). Similar to FSH, 8br-cAMP had no effect on granulosa cell DNA synthesis in the absence of activin. However, in the presence of activin (1 ng/ml) 8br-cAMP was stimulatory. The dose response to 8br-cAMP revealed a biphasic effect on DNA synthesis and differentiation: DNA synthesis rose to a maximum in the presence of 0.5 mM 8br-cAMP and declined thereafter. Progesterone synthesis only started to increase in the presence of 0.1 mM 8br-cAMP, rising to a maximum at 3 mM 8br-cAMP, at which concentration DNA synthesis was fully suppressed. We conclude that activin induces DNA replication in rat granulosa cells. In the presence of activin, FSH and 8br-cAMP are mitogens. These actions of FSH and 8br-cAMP occur at doses too low to stimulate steroidogenesis, emphasizing the role of intracellular cAMP tone in granulosa cell proliferation and differentiation.

The physiological significance of these results will not become clear until patterns of activin and inhibin protein production and the expression of their receptors have been more thoroughly characterized in relation to follicular development. Meanwhile, in situ hybridization studies on rat and monkey ovaries suggest that inhibin/activin beta-subunit mRNA (favoring synthesis of activin) is relatively abundant in granulosa cells of immature antral follicles, whereas alpha-subunit mRNA (favoring synthesis of inhibin) predominates in Graafian follicles. The increased production of follistatin associated with advanced preovulatory development would serve to further reduce the activin "tone" relative to inhibin (Fig. 1). At the level of protein action in vitro, the pattern emerging is that inhibin minimally affects granulosa cell steroidogenesis at any stage of follicular development, whereas activin has pronounced modulatory effects that alter with follicular maturity. As suggested previously,60 the ability of activin to enhance gonadotropin-responsive aromatase activity and simultaneously suppress progesterone production by mature granulosa cells has physiological implications in that it hints at a mechanism for promoting estrogen synthesis and simultaneously suppressing progesterone synthesis, which is precisely what occurs in the preovulatory follicle. The effects of inhibin and activin on human thecal androgen synthesis observed in vitro suggest how these proteins might act locally to modulate preovulatory follicular growth and estrogen synthesis in vivo (Fig. 2).57 In essence, we propose that activin acting at early stages of antral follicular development plays a role in follicular recruitment through sensitizing immature granulosa cells to the cytodifferentiative action of FSH. On the other hand, inhibin is more likely to play a role in preovulatory follicular selection and maintenance of follicular dominance. Studies of follicular fluid levels of androgen and estrogen in relation to granulosa cell aromatase activity indicate that the capacity of the theca interna to generate aromatase substrate (androstenedione) increases hand in hand with aromatase activity in the human preovulatory follicle. It has therefore been suggested that a positive feedback loop (granulosa on theca) exists that promotes thecal androgen synthesis and hence estrogen synthesis in this follicle.64 The discovery that inhibin production in vitro is greatest by granulosa cells isolated from preovulatory follicles strongly implicates inhibin as a component of this feedback loop.

The menopausal transition is characterized by the appearance of elongated cycles, which become longer and more frequent as menopause approaches. Several endocrine abnormalities have been attributed to these cycles; however, no quantitative studies of their causes and consequences exist to date. This study is based on sequential daily urinary concentrations of FSH, LH, estrone 3-glucuronide (E1G), and pregnanediol 3-glucuronide (PdG) from 34 women with perimenopausal menstrual irregularity (total of 289 cycles). The timing of ovarian response was determined as the day of E1G take-off (ETO). Other parameters measured were the mean FSH concentration before ETO (FSH(ETO)) and the midluteal levels of PdG, E1G, and LH. There was a strong parallelism between ETO and cycle length variability. FSH(ETO) levels increased gradually with ETO. Both ETO and FSH(ETO) were inversely related to luteal PdG and directly related to E1G. PdG and LH levels were inversely related. All comparisons were highly significant (P < 0.0001). We conclude that delayed ovarian response underlies the elongation of the menstrual cycle in the menopausal transition, which is likely to be caused by a temporary lack of ovarian responsiveness to FSH. A progressive decline in luteal PdG with increased E1G occurs in association with these trends.

Continuous administration of gonadotrophin-releasing hormone analogues (GnRHa) in patients stimulated for the purpose of IVF might have a direct effect on the ovary. We have evaluated the IVF outcome of patients treated with Buserelin and subsequently with a combination of follicle-stimulating hormone (FSH) and human menopausal gonadotrophin (HMG). Patients were divided into three groups according to the number of oocytes obtained by transvaginal ultrasound-guided follicular aspiration: group 1 (n = 35), in which 1-5 oocytes were retrieved; group 2 (n = 30), in whom 6-10 oocytes were obtained; group 3 (n = 32), in whom greater than or equal to 11 oocytes were collected. Only couples with normal semen samples at oocyte retrieval were included in this study. The dose of Buserelin employed was not different between groups. However, the amount of FSH/HMG necessary to reach an optimal response significantly (P less than 0.01) decreased as follicular development increased. The quality of the oocytes obtained was evaluated based on the appearance of the oocyte-corona-cumulus complex, fertilization rate, morphological appearance of the embryos, and implantation rate. The fertilization rate was significantly (P less than 0.01) decreased in group 3 (57.2%) in comparison with groups 1 (77.1%) and 2 (74.2%). There was no significant difference between the groups in the quality of the embryos obtained or the quality of those replaced into the uterus. The implantation rate per embryo transferred was significantly (P less than 0.05) higher in group 1 (16.5%) in comparison with groups 2 (6.6%) and 3 (8.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

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Measurement of reproductive hormones in urine is a practical way of obtaining large amounts of information; however, there is still controversy on how to overcome problems derived from volume fluctuations between samples. Creatinine adjustment is a widely accepted solution, however, it introduces an extra cost, and large studies involving multiple sequential determinations would benefit from more economical solutions. We determined the value of creatinine adjustment, and compared it with a mathematical method that uses the smoothed profile of pregnanediol (PdG) as a reference to adjust other hormonal markers. To do this, we investigated the effects on three major urinary reproductive hormonal markers (luteinizing hormone (LH), estrone 3-glucuronide (E1G) and PdG) in 17 complete menstrual cycles. Detection of the day of LH peak did not differ between raw and adjusted data. Creatinine adjustment reduced variation in pre-ovulatory E1G levels between individuals, though the effect was negligible within individuals. No significant differences were found regarding post-ovulatory PdG rise. Although creatinine adjustment significantly reduces variability, producing smoother profiles, an equivalent degree of smoothness is obtained using the PdG adjustment. We conclude that under the current technology, for the retrospective study of urinary hormonal profiles in the human menstrual cycle, PdG adjustment is a valid alternative to creatinine.