Higher cortisol:cortisone ratios in the preovulatory follicle of completely unstimulated IVF cycles indicate oocytes with increased pregnancy potential.

BACKGROUND: Conception following gonadotrophin-stimulated IVF and embryo transfer has been associated with a higher intrafollicular cortisol:cortisone ratio and decreased metabolism of cortisol to cortisone. The role of glucocorticoids in human oocyte maturation is not fully understood, but active glucocorticoid (cortisol) may be important. This study relates intrafollicular cortisol and cortisone concentrations to oocyte fertilization and embryo implantation in unstimulated cycles. METHODS: Patients aged <40 years with favourable sperm underwent unstimulated IVF-embryo transfer. Study 1 related intrafollicular cortisol levels to oocyte and IVF outcome: (i) fertilized, pregnant (n = 9); (ii) fertilized, not pregnant (n = 21); and (iii) unfertilized (n = 12). Study 2 was a case-control study of 27 patients (same outcome groups of equal size) which measured intrafollicular cortisol, cortisone and the cortisol:cortisone ratio. RESULTS: Conception cycles demonstrated higher cortisol concentrations compared with the fertilized group (study 1) [median (95% confidence interval): 299 (249-330) versus 227 nmol/l (185-261); P < 0.05] and higher cortisol:cortisone ratios when compared with the unfertilized group (study 2) [7.38 (5.23-9.19) versus 3.56 (1.75-7.46) respectively; P = 0.02]. Of the women with cortisol:cortisone ratios greater than the outcome independent mean of 5.90, 58% conceived compared with only 13% with ratios <5.90 (P < 0.02). CONCLUSION: Higher cortisol:cortisone ratios in conception cycles suggest that active glucocorticoid may be important for final oocyte maturation and embryo implantation in unstimulated cycles.

Connective tissue growth factor in the ovarian paracrine system.

The endocrine actions of follicle stimulating hormone and luteinising hormone on ovarian cells are transduced by locally produced paracrine factors that regulate the formation of extracellular matrix, proteolytic enzymes and protease inhibitors, which continuously remodel the parenchymal environment in which follicles develop. We recently identified connective tissue growth factor (CTGF) as a gene expressed during the predifferentiated stage of granulosa cell development in rat ovary. The CTGF gene encodes a protein that is implicated in the regulation of connective tissue synthesis, mototaxis, angiogenesis and cellular interaction with ECM at various sites in the body. Stimulation of granulosa cells by FSH in vitro and in vivo induces follicular maturation associated with down-regulation of granulosa cell CTGF mRNA expression. The gene remains expressed in cells of the innermost (antrally located) granulosa compartment up to and after the point of ovulation. Based on the inferred biological properties of CTGF protein and the spatiotemporal pattern of CTGF mRNA expression in the ovary, we postulate roles for ovarian CTGF during early stages of follicular development and after ovulation in the formation of the corpus luteum.

Total cortisol levels are reduced in the periovulatory follicle of infertile women with minimal-mild endometriosis.

PROBLEM: To measure and compare concentrations of total and free glucocorticoids with oocyte fertilizing capacity in the follicular fluid (FF) of women with minimal-mild endometriosis and tubal damage. METHOD OF STUDY: Follicular fluid was collected from individual periovulatory follicles during oocyte retrieval for in vitro fertilization (IVF) in natural cycles. Total and free levels of cortisol and cortisone were measured using specific radioimmunoassays after chloroform extraction. RESULTS: Cortisol concentrations in women with minimal-mild endometriosis were significantly lower compared with controls (women with tubal infective damage) (258 versus 328 nmol/L, P < 0.02). There was no correlation between total or free concentrations of cortisol or cortisone and the fertilization capacity of the oocyte. CONCLUSIONS: Total cortisol levels are lower in the follicles of women with endometriosis. Our findings provide further evidence of follicular dysfunction contributing to the subfertility associated with minimal-mild endometriosis.

Differentiation-dependent expression of connective tissue growth factor and lysyl oxidase messenger ribonucleic acids in rat granulosa cells.

Searching for novel genes involved in tissue remodeling during ovarian folliculogenesis, we carried out differential display RT-PCR (DDRT-PCR) on RNA from gonadotropin-stimulated rat granulosa cells (GC). GC from preantral and early antral follicles in immature rat ovaries were cultured in serum-free medium containing no hormone (control), recombinant human FSH (10 ng/ml), 5alpha-dihydrotestosterone (DHT; 10(-6) M), or FSH plus DHT. Total cellular RNA was extracted from cells at 6, 12, 24, and 48 h of treatment for DDRT-PCR analysis, corresponding to an estimated 60% saturation of the messenger RNA (mRNA) population. Six distinct complementary DNA clones were obtained that reproduced the DDRT-PCR profile on a Northern blot of the corresponding RNA samples. Two of these clones detected transcripts that were strongly down-regulated by FSH. One corresponded to connective tissue growth factor (CTGF), a cysteine-rich secreted protein related to platelet-derived growth factor that is implicated in mitogenesis and angiogenesis, and a second was identical to lysyl oxidase (LO), a key participant in extracellular matrix deposition. In detailed expression studies, Northern analysis revealed a single, approximately 2.5-kb CTGF transcript maximally suppressed within 3 h of exposure to FSH with or without DHT and two LO transcripts ( approximately 3.8 and approximately 5.2 kb) maximally suppressed at 6 h. DHT alone did not affect CTGF mRNA, but strongly enhanced LO mRNA relative to the control value. In vivo, CTGF and LO transcripts were significantly suppressed in GC 48 h after equine CG injection (10 IU, ip) compared with untreated controls and were further reduced 12 h after administration of additional 10 IU hCG to induce luteinization. In situ hybridization confirmed GC in preantral/early antral follicles as principal sites of CTGF and LO mRNA expression. We conclude that expression of CTGF and LO mRNAs is inversely related to GC differentiation. The encoded proteins probably have roles in the regulation of tissue remodeling and extracellular matrix formation during early follicular development.

Expected contribution to serum oestradiol from individual ovarian follicles in unstimulated cycles.

Data relating serum oestradiol concentration to follicle size in unstimulated cycles are lacking. We provide precise data on serum concentrations expected for any follicle diameter (FD) in the mid- to late follicular phase. Infertile women (n = 35) with apparently normal ovulatory cycles were studied in detail in 128 unstimulated monofollicular cycles leading to IVF. Using mathematical modelling to account for repeated cycles in the same woman, the relationship between serum oestradiol and FD was explored and reference ranges for serum oestradiol at individual FD were calculated. Serum oestradiol concentrations [number of patients, geometric mean, 95% confidence interval (CI)] at the onset of the LH surge were higher in 'fertilized' cycles (73, 1279, 1180-1378 pmol/l) compared with 'unfertilized' cycles (31, 1055, 929-1197 pmol/l, P: = 0.008) and 'no oocyte' cycles (24, 1064, 922-1227 pmol/l, P: = 0.03) respectively. In 'fertilized' cycles, oestradiol concentrations rose exponentially with FD and for each size of follicle the oestradiol distribution was skewed. Functional oocyte competence varied in apparently normal ovulatory cycles and was correlated with pre-ovulatory serum oestradiol but not FD. Serum oestradiol varies within wide limits for maturing follicles of any given diameter prior to the onset of the LH surge.

Relationship between midcycle luteinizing hormone surge quality and oocyte fertilization.

OBJECTIVE: To determine whether alterations in preovulatory follicular fluid (FF) levels of LH, FSH, and steroids are associated with the probability of fertilization. DESIGN: Retrospective analysis of prospective study results. SETTING: Reproductive medicine clinic of a university teaching hospital. PATIENT(S): Infertile women, with unstimulated, apparently regular cycles in an IVF research program. INTERVENTION(S): Measurement of preovulatory FF levels of LH, FSH, E2, and P and serum LH levels by fluoroimmunometry. MAIN OUTCOME MEASURE: Oocyte fertilization. RESULT(S): There were 84 transferable embryos (rate of normal fertilization and cleavage, 67%), and 41 oocytes (33%) failed to fertilize. Analysis of the matched FF showed that the median concentration of FF LH was significantly higher for cleaving embryos than for unfertilized oocytes (14.6 vs. 10.4 IU/L). Serum LH concentrations were similarly higher in cycles with cleaving embryos. There were no statistically significant differences in FF concentrations of FSH, E2, or P in the two groups. CONCLUSION: Reduced preovulatory FF LH levels are associated with impaired fertilization of oocytes in vitro, despite normal FF FSH and steroid levels.

Onset of the preovulatory luteinizing hormone surge: diurnal timing and critical follicular prerequisites.

OBJECTIVE: To determine the diurnal variation in the onset of the preovulatory LH surge in women. DESIGN: Prospective open cohort study. SETTING: University hospital research program. PATIENT(S): Thirty-five women with infertility resulting from tubal damage that was associated with minor endometriosis or with infertility of prolonged unexplained etiology. INTERVENTION(S): Women underwent transvaginal ultrasonography and serum E2 estimation daily during monitored cycles before unstimulated natural cycle IVF: exogenous gonadotropins were not administered. MAIN OUTCOME MEASURE(S): Serum E2 concentration, follicle diameter, and endometrial thickness. RESULTS: Of 169 cycles. 155 progressed to an ovulatory LH surge, of which 146 occurred within 8 hours of assessment of the outcome measures. The relationship between follicle diameter and E2 was weak, but an abnormal value for one always was countered by a normal value for the other. CONCLUSIONS: Most women begin the preovulatory LH surge between midnight and 8:00 A.M., but with no particular variation by day of the week. The relationship between follicle size and serum E2 is not sufficiently strong to predict the LH surge confidently on the basis of only one variable, but the LH surge is unlikely to occur before either the follicle diameter has reached 15 mm and/or the serum E2 level has reached 600 pmol/L.

The detection and confirmation of 11 beta-hydroxysteroid dehydrogenase type 1 transcripts in human luteinized granulosa cells using RT-PCR and plasmid pUC18.

The activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in human granulosa cells has been shown to be associated with the outcome of treatment following in-vitro fertilization and embryo transfer. There are two known isoforms of 11 beta-HSD which differ significantly in their actions and co-factor requirements. The net activity of 11 beta-HSD which differ significantly in their actions and co-factor requirements. The net activity of 11 beta-HSD within the human ovary is unclear, but may be of particular importance within the ovarian follicle in regulating possible glucocorticoid influences on the oocyte. This study presents preliminary information regarding establishment of techniques to identify transcripts of the 11 beta-HSD isoforms within human granulosa cells and human cumulus cells using reverse transcription-polymerase chain reaction. In view of the high expression of the type 1 11 beta-HSD isoform and the possibility of other 11 beta-HSD isoforms in the ovary, plasmid technology was used to confirm the technique specifically identifying the known isoforms.

Increased follicular fluid total and free cortisol levels during the luteinizing hormone surge.

OBJECTIVE: To determine the changes in follicular fluid (FF) total and free cortisol during the LH surge in naturally ovulating women. PATIENT(S): Twenty-six women having diagnostic laparoscopy during the follicular phase of normal menstrual cycles were selected. INTERVENTION(S): Blood samples were collected 1 day before, the day of, and 1 day after surgery and the results of serum E2 and LH were used to divide the cycles retrospectively into pre- and post-LH surge groups. Follicular fluid was collected during laparoscopy. MAIN OUTCOME MEASURE(S): Serum P, total and free cortisol, and FF volume, E2, P, total cortisol, and free cortisol were measured on the day of surgery. RESULT(S): Median serum and FF P levels were significantly higher in the post-LH surge group compared with the pre-LH surge group (0.54 versus 1.54 ng/mL [1.7 versus 4.85 nmol/L] and 5.03 versus 28.0 micrograms/mL [15.8 versus 88.0 mumol/L], respectively). Follicular fluid volume also increased significantly after the surge (2.5 versus 4.5 mL). Median serum total and free and percent free cortisol were higher after the surge, although not significantly. In contrast, FF total, free, and percent free levels increased dramatically between pre- and post-LH surge samples (4.41 versus 43.6 ng/mL [16.0 versus 158 nmol/L], 0.138 versus 6.68 ng/mL [0.5 versus 24.2 nmol/L], and 3.3% versus 15.0%, respectively; P < 0.05). CONCLUSION(S): An increase in total and free cortisol occurs in the follicle during the LH surge. Cortisol and its regulation by 11 beta-hydroxysteroid dehydrogenase therefore may exert a physiologic role in oocyte maturation or ovulation.

Ovarian dysfunction in endometriosis-associated and unexplained infertility.

PURPOSE: The impact of endometriosis and unexplained infertility on follicular function and fertilization of oocytes in cycles totally unperturbed by exogenous gonadotrophins, when compared with controls with tubal damage, were examined. METHODS: In natural cycles, without any exogenous gonadotropins, endocrine and ultrasonographic studies of follicular maturation in 18 women with minor endometriosis (41 cycles), 15 women with unexplained infertility (31 cycles), and 34 women with tubal damage (88 cycles) were performed. RESULTS: The endometriosis group had a significantly longer follicular phase (median: 15, 13, and 13 days). Both endometriosis and unexplained infertility had significantly reduced LH concentrations in follicular fluid compared with tubal damage (median: 12.1, 11.5, and 15.9 IU/L, respectively). Endometriosis was associated with a significantly reduced fertilization rate compared with unexplained infertility or tubal damage (46, 65, and 69%, respectively). CONCLUSIONS: These data show continuing evidence of ovulatory dysfunction leading to reduced fertilization rates in women with minor endometriosis.

Relative bioactivity of endogenous luteinizing hormone during superovulation therapy for in vitro fertilization in normal-cycling women.

PURPOSE: Impaired outcome of in vitro fertilization treatment has been related to abnormal luteining hormone (LH) secretion and to the occurrence of a premature LH surge. The purpose of this study was to compare LH serum concentrations, measured by bioassay (LH-b) and immunoassay (LH-i), during early and late follicular phases in normal-ovulatory women who were stimulated with clomiphene, human menopausal gonadotropin (HMG), and human chorionic gonadotropin (hCG), while undergoing in vitro fertilization (IVF) treatment for infertility, due mainly to tubal damage. RESULTS: Of 22 patients, 15 had an LH surge (surge group) before receiving hCG and 7 did not (nonsurge group). LH-b and LH-i concentrations were higher in the late follicular phase than before clomiphene treatment, but the LH-b/LH-i (B/I) ratios remained unchanged and there were no significant differences between the LH surge and the nonsurge groups. In the surge group, the B/I ratio appeared to fall during the LH surge [presurge values, 5.4 +/- 0.5 (SD) and 5.2 +/- 0.5; surge values, 4.9 +/- 0.6 and later 4.0 +/- 0.6], but the differences were not significant, and in some individuals, the B/I ratio later rose as the LH surge progressed. CONCLUSIONS: Our findings suggest that interference with IVF outcome by a spontaneous LH surge is due to quantitative and/or exposure time effects on the oocyte, rather than to any qualitative variation in bioactivity of LH.

Effect of progestogen therapy on follicular development, related hormone concentrations and fertilization in vitro in unstimulated cycles and unexplained and endometriosis-associated infertility.

Evidence of pituitary-ovarian dysfunction in unexplained and endometriosis-associated infertility has been reported previously. Hormone-suppressive therapy is often used in an attempt to improve fertility, although benefits have not been proven. Our study examines the effect of progestogen (medroxyprogesterone acetate) treatment on women with endometriosis-associated and unexplained infertility, compared with women with tubal damage as functional controls. Pre-ovulatory follicular size and serum and follicular fluid hormone concentrations were measured, and oocyte collection and in-vitro fertilization were attempted, in natural cycles totally unperturbed by exogenous gonadotrophins, for two cycles before and two cycles following treatment with medroxyprogesterone acetate for 2 months. In the endometriosis and unexplained infertility groups, compared with the tubal group, the treatment led to significant reductions in the integrated luteinizing hormone (LH) values (483 versus 664, 559 versus 762 and 864 versus 820 notional IU/l respectively). There were no changes in serum oestradiol or follicular fluid oestradiol, progesterone, follicle stimulating hormone or LH concentrations after treatment. The results suggest that progestogen therapy has no beneficial effect on the pituitary-ovarian dysfunction which contributes to endometriosis-associated and unexplained infertility.

Stress and stress-related hormones during in-vitro fertilization treatment.

Whether stress and infertility are linked as cause or consequence is unclear, and there is no consensus on the most appropriate methods for measuring stress in infertile women. To address this question, we measured changes in biochemical and questionnaire-based assessments of stress in infertile women. Median baseline, follicular phase and pre-operative serum prolactin (229, 311 and 457 mIU/l) cortisol (278, 369 and 496 nmol/l) and state anxiety score (38, 40 and 49) respectively all increased during stimulated in-vitro fertilization (IVF) treatment. There was no such increase in a control group having similar laparoscopic surgery unrelated to infertility, or in women having unstimulated IVF without laparoscopy, suggesting that anxiety levels are greatest in stimulated IVF, increase as a result of the treatment, and are adequately reflected by state anxiety scores. Baseline serum prolactin in unstimulated IVF (384 mIU/l) was significantly higher than control (177 mIU/l), although this was not reflected in serum cortisol or state anxiety score. Trait anxiety was constant within and between groups, suggesting that stress is not contributing greatly to the infertility. Women who achieved a pregnancy had similar state anxiety scores to those who failed, suggesting that the degree of anxiety observed during IVF treatment is unlikely to influence the chance of pregnancy. There was a trend towards lower trait anxiety in women who became pregnant, but the numbers were small.

Reduced preovulatory granulosa cell steroidogenesis in women with endometriosis.

To examine the cause of altered follicular fluid steroid levels and lower in vitro fertilization rate observed in infertile women with minor endometriosis, we have compared the production of estradiol (aromatase activity) and progesterone of freshly isolated granulosa cells (3h. incubation) from such women and a control group with tubal or unexplained infertility, having IVF during unstimulated or gonadotropin-stimulated cycles. As previously observed, mature oocytes from women with endometriosis had a reduced fertilization and cleavage rate in vitro in unstimulated cycles (19/37[51%] vs. 69/94[73%], p < 0.05) and stimulated cycles (20/37[57%] vs. 32/39[82%], p < 0.01). Median [95%CI] basal aromatase activity was lower in endometriosis compared with control in unstimulated cycles (2.84[2.03-3.49] pmol E2/10(3) cells/3h, n = 31 vs. 3.63[2.72-3.49], n = 55, p = 0.057) and stimulated cycles (0.31[0.16-0.50], n = 14 vs. 0.99[0.70-1.52], n = 20, p < 0.001). Progesterone production followed a similar pattern in unstimulated (0.56[0.50-0.89] pmol/10(3) cells/3h, n = 29 vs. 1.23[0.69-1.54], n = 52,) and stimulated (0.37[0.20-0.73], n = 16 vs. 0.95[0.72-1.17], n = 21) cycles (p < 0.05). Addition of FSH, LH or hCG (30ng/mL) to the incubation medium enhanced progesterone production 2 to 3-fold, but had no effect on aromatase activity. Our results indicate a defect in granulosa cell steroidogenesis associated with endometriosis, which could affect oocyte function and explain the reduction in fertilizing capacity and subsequent competence of the corpus luteum, and the associated subfertility.

Pituitary-ovarian dysfunction as a cause for endometriosis-associated and unexplained infertility.

This study examines circulating and follicular hormone concentrations and fertilization of oocytes in cycles totally unperturbed by exogenous gonadotrophins in 10 women (25 cycles) with untreated minimal-mild endometriosis and nine women (23 cycles) with prolonged unexplained infertility compared with 16 women (50 cycles) with tubal damage as functional controls. Endometriosis was associated with a significantly longer follicular phase (median 15, 12, 13 days respectively) and reduced oestrogen secretion (median index area under the curve 3063, 3842, 3805 units respectively) compared with controls. Both endometriosis and unexplained infertility had significantly reduced serum luteinizing hormone (LH) surges [median peak serum (LH) 43, 39, 55 IU/l respectively and median area under the curve 661, 687, 823 units respectively] and reduced LH concentrations in follicular fluid (median 19.6, 10.6, 9.2 IU/l respectively). These findings suggest that infertility associated with minor endometriosis and of apparently unexplained aetiology share a common pathophysiology in impaired LH surge secretion. Whether that represents a primary pituitary disorder or is secondary to a defective ovarian signal is discussed.

Time-dependent effects of transforming growth factor alpha on aromatase activity in human granulosa cells.

Transforming growth factor alpha (TGF alpha) is implicated as a paracrine growth factor in the regulation of human granulosa cell function. To investigate this further, we have examined the actions of TGF alpha on the basal and follicle-stimulating hormone (FSH)-stimulated aromatase activity of human granulosa cells to determine how this growth factor influences oestrogen biosynthesis in the follicle. Granulosa cells from women having in-vitro fertilization during untreated or gonadotrophin-stimulated cycles were cultured for 1-6 days in the presence or absence of FSH or TGF alpha at a range of doses. Aromatase activity, expressed as oestradiol production, was determined after culture during a 3 h test period. After 2 days, TGF alpha (1-300 ng/ml) decreased basal and FSH-stimulated aromatase activity in a dose-dependent manner (ED50 = 3 ng/ml). In contrast, after 4 days, TGF alpha enhanced both basal and FSH-stimulated aromatase activity. Repeated experiments revealed a consistent pattern of inhibition on day 2, which was more marked in the presence of FSH (reduction by 30.6 +/- 9.1%, mean +/- SEM; n = 14; P < 0.01), and stimulation on day 4 in both the absence (increased by 61.4 +/- 20.6%, mean +/- SEM; n = 6; P < 0.05) and presence of FSH (increased by 36.0 +/- 15.2%, mean +/- SEM; n = 8; P < 0.05). The results provide further evidence that TGF alpha is a paracrine factor in the control of oestrogen biosynthesis, but the actions can be either inhibitory or stimulatory depending on the duration of exposure.

Multiple follicular development associated with herbal medicine.

After three endocrinologically normal cycles while undergoing unstimulated in-vitro fertilization treatment, a woman took a herbal medicine (Vitex agnus castus) at the beginning of a fourth unstimulated IVF treatment cycle. In this fourth cycle, her serum gonadotrophin and ovarian hormone measurements were disordered. One embryo resulted from the three eggs collected but a pregnancy did not ensue. She had symptoms suggestive of mild ovarian hyperstimulation syndrome in the luteal phase. Two subsequent cycles were endocrinologically normal. We do not advocate the use of this herbal medicine to promote normal ovarian function.

Factors influencing follicle-stimulating hormone-responsive steroidogenesis in marmoset granulosa cells: effects of androgens and the stage of follicular maturity.

Preovulatory changes in the steroidogenic function of primate granulosa cells were studied using the cyclic marmoset (Callithrix jacchus) as a model. Antral follicles (greater than or equal to 0.5 mm diameter) were dissected from mid-late follicular phase ovaries (7 days after prostaglandin-induced luteolysis) and classified by diameter as small (0.5-1.0 mm), medium (1.1-1.9 mm) or large (greater than or equal to 2.0 mm). Granulosa cells from follicles in each size category were isolated and pooled to assess steroid biosynthesis. The aromatase activity of freshly isolated granulosa cells from large follicles was 200 times greater than that of small follicles, confirming their relatively advanced preovulatory status. Granulosa cells were cultured for 48 h in the presence and absence of human (h) FSH (0.1 ng/ml), with and without 0.1 microM androgen (testosterone or 5 alpha-dihydrotestosterone), to assess basal and hormone-responsive steroidogenesis (progesterone accumulation in culture medium and aromatase activity in washed granulosa cell monolayers). Basal granulosa cell steroidogenesis increased with follicular size, and there was a development-related pattern of response to hFSH and androgen. hFSH responsiveness (maximum fold-stimulation induced by hFSH) declined with follicular size, being 2-6 times greater for granulosa cells from small vs. large follicles. On the other hand, hFSH sensitivity increased with follicular size; the dose of hFSH giving 50% of the maximum response (ED50) for cells from large follicles being 10-20 times less than that of cells from small follicles. For granulosa cells from small follicles, treatment with 0.1 microM androgen in the presence of hFSH led to dramatic (up to 16-fold) enhancement of steroidogenic responses to hFSH. In contrast, for granulosa cells from large follicles, the presence of androgen substantially inhibited aromatase activity stimulated by hFSH and had weak inhibitory effects on progesterone accumulation. These results show that granulosa cell steroidogenesis becomes increasingly sensitive to hFSH during preovulatory follicular development in marmosets. The marked ability of androgen to directly augment hFSH-responsive steroidogenesis in vitro is lost during preovulatory development, such that androgen acts in mature granulosa cells to suppress hFSH-stimulated aromatase activity. These observations are evidence of development-dependent changes in granulosa cell responses to FSH and androgens which may contribute to the control of preovulatory follicular development in primates.

Cellular aspects of pre-ovulatory folliculogenesis in primate ovaries.

According to current concepts of pre-ovulatory folliculogenesis in primate ovaries, each growing follicle has a 'threshold' requirement for stimulation by FSH which must be met if it is to enter the oestrogen-secretory phase of pre-ovulatory development. Until recently, our understanding of the intra-follicular mechanisms underlying FSH action on granulosa cells was based largely on information from non-primate laboratory animals, mainly polyovulators such as rats. The present paper describes studies on FSH-regulated granulosa cell function in relation to pre-ovulatory development in vivo and in vitro using a laboratory primate, the common marmoset (Callithrix jacchus). Measurement of aromatase activity is used as an index of granulosa cell cytodifferentiation to verify three major tenets of the 'threshold' hypothesis: (i) that granulosa cells acquire increased responsiveness to FSH and LH during pre-ovulatory growth; (ii) that these developmentally related changes are directly induced by FSH; and (iii) that intrafollicular steroids modify FSH action and thereby contribute to the establishment of follicular FSH thresholds. The results obtained highlight the value of this experimental animal model for studies of cellular and molecular aspects of pre-ovulatory folliculogenesis which are relevant to human ovaries.