Ibuprofen inhibits key genes involved in androgen production in theca-interstitial cells.

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-1ß (IL-1ß) 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-1ß 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-1ß 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-1ß and LPS. Ibuprofen inhibited the IL-1ß-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.

Evidence of profound ovarian suppression on combined hormonal contraception resulting in dramatically different ovarian reserve testing and oocyte retrieval outcomes: case report and review of the literature.

OBJECTIVE: To describe a case report and demonstrate that degree of ovarian suppression with continuous combined hormonal contraception (CHC) may be more profound than previously described and may present similarly as decreased ovarian reserve. DESIGN: Case report and review of the literature. SETTING: Private practice in vitro fertilization center. PATIENTS: A 36-year-old single gravida 0 presenting for oocyte cryopreservation on CHC. INTERVENTIONS: Discontinuation of vaginal ring combined hormonal contraceptive for 6 months. MAIN OUTCOME MEASURES: Antral follicle count, antimüllerian hormone, day 3 follicle-stimulating hormone, total oocytes, and mature oocytes retrieved before and after discontinuation of CHC. RESULTS: After a 6-month break from CHC, our patient's antimüllerian hormone level increased from undetectable levels to 3.45 ng/mL, day 3 follicle-stimulating hormone level decreased from 14.9 IU/mL-6.17 IU/mL, and antral follicle count improved from 0-28. In addition, the number of oocytes retrieved after a 4-month CHC break and 6-month break increased from 8 to 29, respectively. CONCLUSIONS: In patients on long-term combined continuous hormonal contraception, profound ovarian suppression can result in a clinical picture of diminished ovarian reserve and extremely poor response to high-dose stimulation, which may be reversed by more time off from suppression.

Unexplained recurrent pregnancy loss and unexplained infertility: twins in disguise.

STUDY QUESTION: Is B-cell CLL/lymphoma 6 (BCL6) endometrial expression, a surrogate biomarker of endometriosis, elevated in women with unexplained recurrent pregnancy loss (uRPL) and unexplained infertility (UI) compared to fertile subjects? SUMMARY ANSWER: Endometrial BCL6 expression is elevated to a similar degree in women with uRPL and UI compared to fertile controls. WHAT IS KNOWN ALREADY: Endometriosis has been linked to the genesis of endometrial progesterone resistance and to specific nuclear proteins, including endometrial BCL6. BCL6 overexpression (immune histologic score > 1.4) has been strongly associated with poor reproductive outcomes in IVF cycles in women with UI. Our previous data have demonstrated an accuracy of 94% for diagnosing endometriosis, and BCL6 protein is elevated in the decidua of women with uRPL. STUDY DESIGN SIZE DURATION: In this case-control study, at a tertiary university teaching hospital, 110 samples (control n = 28; uRPL n = 29; UI n = 53) from pathological archives were analyzed. Timed endometrial biopsies were obtained between 2 January 2002 and 31 December 2016. PARTICIPANTS/MATERIALS SETTING METHOD: LH-timed endometrial biopsies were obtained from women with UI, uRPL (two or more consecutive losses) and normal fertile subjects during the mid-secretory phase of the menstrual cycle. Endometrial BCL6 protein levels were compared in women with UI and uRPL and fertile controls using western blot analysis and immunohistochemistry (HSCORE). MAIN RESULTS AND THE ROLE OF CHANCE: The mean age of the uRPL group was significantly higher than the others [mean (SD)] control = 32.7 (2.6); uRPL = 35.8 (3.7); UI = 32.7 (4.4); P = 0.002, ANOVA]. Seventy-nine percent of women in both subfertile groups (uRPL and UI, 65 out of 82) displayed elevated BCL6 protein levels. From these, a subset of cases with abnormal BCL6 went to laparoscopy and endometriosis was found in 9 out of 11 cases of uRPL and in 20 out of 21 cases of UI. Median BCL6 HSCORE for controls versus uRPL and UI was significantly different [median (interquartile); control = 0.3 (0.02 to 0.5); uRPL = 3 (1.9 to 3.6); UI = 2.9 (1.6 to 3.1); P < 0.0001, Kruskal-Wallis]. A significant trend in the association between the degree of infertility (fertile, uRPL and UI) and the HSCORE level (negative, medium and high) was found (P < 0.001; x 2 for trend). Western blot of representative samples from each group demonstrated similar findings based on protein levels in the whole endometrium. After running ANCOVA analysis for age difference, the BCL6 difference among groups was still significant (P-value < 0.0001). LIMITATIONS REASONS FOR CAUTION: We studied subjects with two consecutive pregnancy losses rather than the definition adopted in Europe of three losses. The findings may lack external validity in other clinical settings (e.g. low prevalence of endometriosis). WIDER IMPLICATIONS OF THE FINDINGS: Based on the data presented here, we postulate that the degree of BCL6 expression may represent a continuum of progesterone resistance and response to inflammation that occurs in women with endometriosis, yielding different degrees of infertility, from uRPL to UI. STUDY FUNDING/COMPETING INTERESTS: This study was supported by NICHD/NIH R01 HD067721 (SLY and BAL), by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior: Grant 99999.003035/2015-08 (BAL) and by CAPES/PROAP (RFS). Two authors (BAL, SLY) have licensed intellectual property for the detection of endometriosis. Dr Bruce Lessey is an unpaid scientific Advisor for CiceroDx. The other authors report no conflict of interest.

Inflammatory Stimuli Trigger Increased Androgen Production and Shifts in Gene Expression in Theca-Interstitial Cells.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder characterized by theca cell hyperplasia and excessive androgen production. An increasing body of evidence has pointed to a close association between PCOS and low-grade chronic systemic inflammation. However, the mechanistic basis for this linkage is unknown. Therefore, we evaluated the effects of the inflammatory agents lipopolysaccharide (LPS) and IL-1ß on rat theca-interstitial cells (TICs). We found that incubation with either LPS or IL-1ß elicited a dose-dependent increase in both TIC viability and androgen production. Using RNA sequencing analysis, we found that both of these inflammatory agents also triggered profound and widespread shifts in gene expression. Using a stringent statistical cutoff, LPS and IL-1ß elicited differential expression of 5201 and 5953 genes, respectively. Among the genes upregulated by both LPS and IL-1ß were key regulatory genes involved in the cholesterol and androgen biosynthesis pathways, including Cyp17a1, Cyp11a1, Hsd3b, and Hmgcr. This provides a molecular explanation for the mechanism of action of inflammatory agents leading to increased androgen production. Gene ontology and pathway analysis revealed that both LPS and IL-1ß regulated genes highly enriched for many common functions, including the immune response and apoptosis. However, a large number of genes (n = 2222) were also uniquely regulated by LPS and IL-1ß, indicating that these inflammatory mediators have substantial differences in their mechanism of action. Together, these findings highlight the potential molecular mechanisms through which chronic low-grade inflammation contributes to the pathogenesis of androgen excess in PCOS.