Objective

To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs).

Design

Ex vivo and in vitro experimental study.

Setting

University.

Animal(s)

Immature female Sprague Dawley rats.

Intervention(s)

Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or β-glycan antibody.

Main outcome measure(s)

As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription-polymerase chain reaction.

Result(s)

After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, β-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent.

Conclusion(s)

These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs by virtue of FSH seem to be required.

Ovarian testosterone increases the response of antral follicles to stimulation, declines with age, and has effects mediated or potentiated by insulin-like growth hormone I (IGF-I). Increased circulating insulin and IGF-I, exogenous testosterone, and increased local ovarian testosterone concentrations due to aromatase inhibition or exogenous luteinizing hormone/human chorionic gonadotropin are all associated with an increased ovarian response to gonadotropins. These factors should be further investigated alone or in combination for enhancing oocyte yield with fertility treatments, particularly in older reproductive-age women.

Context

In women with polycystic ovary syndrome (PCOS), 17-hydroxyprogesterone (17-OHP) responses to gonadotropin stimulation vary from increased to indistinguishable compared with normal controls.

Objective

To determine whether 17-OHP responses to recombinant-human chorionic gonadotropin (r-hCG) are individually correlated to the size of antral follicles among women with PCOS.

Design setting and participants

A prospective study conducted in 19 women with PCOS and 20 normal controls at an academic medical center.

Interventions

Blood samples were obtained before and 24 hours after administration of 25 μg of r-hCG. Ovarian imaging was conducted with three-dimensional pelvic ultrasonography. Each subject underwent a 2-hour oral glucose tolerance test.

Main outcome measures

Basal and stimulated levels of 17-OHP, androgens, estradiol, progesterone, anti-Mullerian hormone (AMH), insulin, glucose, follicle number, and size.

Results

In women with PCOS, mean antral follicle count (AFC) was greater than that of controls, although the size of cohort follicles within individual subjects was not correlated to 17-OHP responses. The numbers of 2- to 3-mm and 3- to 4-mm follicles in PCOS were significantly greater than in controls, whereas differences between larger follicles were not observed. Increased AMH in PCOS was correlated to AFC, but not 17-OHP responses. Insulin sensitivity did not correlate to r-hCG‒stimulated 17-OHP after adjustment for body mass index.

Conclusions

17-OHP responses to hCG in individuals with PCOS were not correlated to the distribution of antral follicles. Greater numbers of small antral follicles in women with PCOS than in controls suggest an extension of accelerated growth from the preantral stage.

Purpose

Factors that differentially regulate oocyte and granulosa cell growth within the early preantral follicle and how these factors differ at each stage of follicle growth remain poorly understood. The aim of this study was to isolate and evaluate the effect of recombinant growth and differentiation factor 9 (GDF9) on oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle growth during in vitro culture.

Methods

Primary stage follicles (diameters of 50-89 μm) and early secondary stage follicles (diameters of 90-120 μm) were isolated from immature mice, and individual, intact follicles were cultured in vitro in the presence and absence of recombinant GDF9. The effects of GDF9 on follicle growth were determined by the assessment of changes in the follicle volume during culture. The growth of the granulosa cell and oocyte compartments of the follicles was evaluated separately at each stage.

Results

GDF9 significantly increased the growth of isolated follicles at both the primary and early secondary follicle stages. Independent evaluation of the granulosa cell and oocyte compartments revealed that, while GDF9 promoted granulosa cell growth at both stages of folliculogenesis, oocyte growth was stage specific. GDF9 promoted growth of the oocyte at the primary, but not the early secondary, follicle stage.

Conclusions

These findings demonstrate a stage-specific role for GDF9 in the regulation of oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle development.

Paracrine interactions between ovarian theca-interstitial cells (TICs) and granulosa cells (GCs) play an important role in the regulation of follicular steroidogenesis. Androgens serve as substrates for aromatization as well as affect GC function. This study evaluated the effects of co-culture of GC with TICs and the role of testosterone (T) and 5-alpha-dihydrotestosterone (DHT), and estradiol (E2) in modulation of GC expression of genes involved in the production of progesterone: 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase (Hsd3b) and cholesterol side-chain cleavage (Cyp11). GCs obtained from immature Sprague-Dawley rats and were cultured in chemically defined media without or with TICs, DHT, or T. Hsd3b and Cyp11 transcripts were analyzed by qt-PCR. Co-culture of GCs with TICs stimulated Hsd3b and CYP11 expression in GCs. DHT and T induced a concentration-dependent upregulation of Hsd3b and CYP11 expression, as well as increased progesterone concentrations in spent media. E2 also increased expression of Hsd3b, and Cyp11. Effects of androgens were abrogated in the presence of an anti-androgen bicalutamide and the antiestrogen ICI 182780 (ICI). In conclusion, present findings demonstrate that androgens upregulate production of progesterone in GCs; these effects are likely due to a combination of direct action on androgen receptors and effects mediated by estrogen receptors.

Background

The influence of estradiol (E₂) on granulosa cell (GC) function has not been tested clinically in women with polycystic ovary syndrome (PCOS). The objective of this study is to determine if E₂ influences GC responses to FSH in women with PCOS.

Methods

This is a two phase, single cohort study conducted over a 2-year period at a single academic center. Nine women with PCOS according to NIH criteria. In Phase 1, FSH stimulation of GC responses as measured by E₂ and Inhibin B (Inh B) were assessed before and at 5 and 6 weeks after GnRH agonist administration. In Phase 2, the same protocol was employed with the addition of an aromatase inhibitor (letrozole, LET) administered daily beginning at week 4 for 2 weeks.

Results

In Phase 1, recovery of FSH, E₂ and Inh B from ovarian suppression occurred at 5 and 6 weeks after GnRH agonist injection and preceded resumption of LH and androgen secretion. In Phase 2, hormone recovery after GnRH agonist was characterized by elevated FSH and suppressed E₂ levels whereas recovery of LH and androgen levels were unchanged. In Phase 1, FSH stimulated E₂ and Inh B responses were unaltered during recovery from ovarian suppression. In Phase 2, E₂ and Inh B fold changes after FSH were significantly reduced at weeks 5 (p < 0.04) and 6 (p < 0.01), respectively.

Conclusion

In anovulatory women with PCOS, chronic, unopposed E₂ secretion may contribute, at least in part, to enhanced ovarian responsiveness to FSH.

Trial registration

NCT02389088.

Objective

To compare androgen responses during ACTH infusion among women with polycystic ovary syndrome (PCOS) and healthy women.

Design

Cross-sectional study.

Setting

Academic medical center.

Patient(s)

Women with PCOS (n = 13) and healthy controls (n = 15).

Intervention(s)

Blood samples were obtained frequently during a 6-hour dose-response ACTH infusion.

Main outcome measure(s)

Comparison of basal and stimulated levels of 17α-hydroxyprogesterone (17-OHP), androgens, and cortisol (F) during ACTH infusion with those after hCG injection within individual subjects.

Result(s)

In women with PCOS increased 17-OHP, androstenedione (A), and DHEA responses during ACTH infusion were comparable to those observed in healthy controls. The magnitude of responses was highly variable among women with PCOS. Within individual women with PCOS adrenal responses to ACTH and ovarian responses to hCG were significantly correlated. Cortisol responses to ACTH were similar in women with PCOS and healthy controls.

Conclusion(s)

Within individual women with PCOS, enhanced androgen responses to ACTH are accompanied by comparable androgen responsiveness to hCG. These findings suggest that dysregulated steroidogenesis leading to hyperandrogenemia in this disorder is likely present in both adrenal and ovarian tissues.

Clinical trial registration number

NCT00747617.

Objective

To study the effects of ibuprofen on androgen production, gene expression, and cell viability in rat theca-interstitial cells exposed to the proinflammatory stimuli interleukin-1β (IL-1β) and lipopolysaccharide (LPS).

Design

Animal study.

Setting

University-based research laboratory.

Patients/animals

Theca-interstitial cells were isolated from 30 day old female Sprague Dawley rats.

Interventions

Theca cells were cultured with pro-inflammatory media containing IL-1β and LPS and compared with cells cultured in control media.

Main outcome measures

Androstenedione quantification was performed on conditioned cell culture medium using liquid chromatography-mass spectrometry. Theca cell viability was assessed using PrestoBlue cell viability assay. The gene expression of Cyp17a1, Cyp11a1, and Hsd3b was analyzed using quantitative polymerase chain reaction.

Results

Both proinflammatory stimuli IL-1β and LPS increased androstenedione concentration in cell culture medium, and these effects were mitigated with ibuprofen. Both inflammatory agents in addition increased the expression of key genes involved in androgen synthesis: Cyp17a1, Cyp11a1, and Hsd3b; the addition of ibuprofen to the culture medium inhibited these effects. Theca cell viability increased with IL-1β and LPS. Ibuprofen inhibited the IL-1β-mediated increase in cell viability but did not reverse the effects of LPS.

Conclusions

In conclusion, our findings support the hypothesis that many of the alterations induced by inflammatory stimuli in theca-interstitial cells are abrogated by the addition of ibuprofen.

Objective

To determine whether granulosa cells contribute to excess androgen production, by assessing inhibin B (Inh B) responses to hCG in women with polycystic ovary syndrome (PCOS) and in normal women.

Design

Prospective study.

Setting

Academic medical center.

Patient(s)

Twenty women with PCOS and 16 normal women.

Intervention(s)

Blood samples obtained before and 24 hours after injection of 25 μg recombinant hCG (r-hCG).

Main outcome measure(s)

Basal and stimulated Inh B, E2, androstenedione (A), and T responses after r-hCG administration.

Result(s)

In normal and PCOS women, r-hCG induced a significant reduction of Inh B levels. Lowered Inh B responses were not related to body mass index, PCOS status, or age by multivariate regression. Recombinant hCG significantly increased serum A and E2 in both normal and PCOS women.

Conclusion(s)

In normal and PCOS women, Inh B production was decreased following r-hCG administration. These findings strongly suggest that in PCOS women androgen excess is not enhanced by LH-stimulated Inh B production.

Clinical trial registration number

NCT00747617.

Recent studies report the involvement of intra-ovarian factors, such as inflammation and oxidative stress, in the pathophysiology of polycystic ovary syndrome (PCOS), the most common endocrine disorder of reproductive age women. Endoplasmic reticulum (ER) stress is a local factor that affects various cellular events during a broad spectrum of physiological and pathological conditions. It may also be an important determinant of pro-fibrotic remodeling during tissue fibrosis. In the present study, we showed that ER stress was activated in granulosa cells of PCOS patients as well as in a well-established PCOS mouse model. Pharmacological inducers of ER stress, tunicamycin and thapsigargin, were found to increase the expression of pro-fibrotic growth factors, including transforming growth factor (TGF)-β1, in human granulosa cells, and their expression also increased in granulosa cells of PCOS patients. By contrast, treatment of PCOS mice with an ER stress inhibitor, tauroursodeoxycholic acid or BGP-15, decreased interstitial fibrosis and collagen deposition in ovaries, accompanied by a reduction in TGF-β1 expression in granulosa cells. These findings suggest that ER stress in granulosa cells of women with PCOS contributes to the induction of pro-fibrotic growth factors during ovarian fibrosis, and that ER stress may serve as a therapeutic target in PCOS.