Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

How is the production of progesterone (P₄) and 17-hydroxy-P₄ (17-OH-P₄) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. In the early follicular phase, theca cells primarily synthesized 17-OH-P₄ while granulosa cells produced limited P₄, maintaining the P₄:17-OH-P₄ ratio 1. The concentration of 17-OH-P₄ in the luteal phase was similar to that in the follicular phase, but P₄ production increased in the luteal phase, yielding a P₄:17-OH-P₄ ratio significantly >1. The P₄:17-OH-P₄ ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P₄ during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100–130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E₂), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E₂ and FSH to the culture medium.

Testicular Germ Cell Tumour (TGCT) is one of the most common tumours in young men. Increasing evidence shows that the extracellular matrix has a key role in the prognosis and metastasis of various human cancers. This study analysed the relationship between the matrix protein ameloblastin (AMBN) and potential biological markers associated with TGCT diagnosis and prognosis. The relationship between AMBN and TGCT prognosis was determined by bioinformatic analysis using the expression profiles of three RNAs (long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs) from The Cancer Genome Atlas (TCGA) database, and available clinical information of the corresponding patients. Prediction and validation of competitive endogenous RNA (ceRNA) regulatory networks related to AMBN was performed. AMBN and its associated ceRNA regulatory network were found to be related to the recurrence of TGCT, and LINC02701 may be used as a diagnostic factor in TGCT. Furthermore, we identified PELATON (Plaque Enriched LncRNA In Atherosclerotic And Inflammatory Bowel Macrophage Regulation) as an independent prognostic factor for TGCT progression-free interval.

STUDY QUESTION: Does the human ovary possess the enzymatic machinery required for local corticosteroid synthesis during follicular development? SUMMARY ANSWER: The human ovary exhibits dynamic corticosteroid metabolism during follicular development and expresses enzymes consistent with local corticosteroid synthesis. WHAT IS KNOWN ALREADY: Cortisol is a systemic glucocorticoid that binds to the glucocorticoid receptor (NR3C1), which is expressed in ovarian tissues. While excess systemic cortisol impairs follicular development, a surge in local cortisol activity around ovulation appears necessary for limiting the impact of the inflammatory process taking place. The preovulatory upregulation of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) suggests local conversion of cortisone to active cortisol. However, whether the ovary can synthesize corticosteroids de novo remains uncertain. STUDY DESIGN, SIZE, DURATION: This observational study included samples from 34 women undergoing ovarian cryopreservation and samples from 50 women undergoing IVF/ICSI according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women donated follicular fluid (FF), plasma, and granulosa cells. Steroid concentrations in FF and plasma were quantified using LC-MS/MS. Granulosa cell mRNA expression of steroidogenic enzymes (CYP11B1, CYP11B2, CYP21A2, HSD11B1, HSD11B2) was assessed by RT-qPCR. In addition, immunofluorescence (IF) was used to localize CYP21A2 in preantral and small antral follicles. FF samples were stratified according to follicle size (2–18 mm) and ovulatory status. Hormone dynamics and FF/plasma ratio were evaluated using linear mixed-effects models and non-parametric tests. MAIN RESULTS AND THE ROLE OF CHANCE: Intrafollicular cortisol levels increased with follicle size up to 8–10 mm, then declined until close to ovulation, where a sharp increase was observed (from 10), suggesting local synthesis. This is supported by the expression of CYP21A2 and low-level CYP11B1 mRNA in GCs, and by IF localization of CYP21A2 in preantral and small antral follicles. In contrast, FF cortisol concentrations were consistently lower than in plasma (FF/plasma ratio <0.1), indicating active inactivation by HSD11B2, which was abundantly expressed. HSD11B1 expression was low but detectable in periovulatory follicles, consistent with local reactivation of cortisone to cortisol. Among mineralocorticoids, corticosterone was only detectable at ovulation, while aldosterone was undetectable. LIMITATIONS, REASONS FOR CAUTION: We were unable to localize CYP11B1 at the protein level due to limitations in available antibodies. Additionally, FF and plasma samples were obtained from different individuals, precluding direct paired comparisons. Some variability in plasma sample collection timing throughout the day also limits conclusions regarding diurnal fluctuations. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that the human ovary possesses the capacity for partial corticosteroid synthesis, utilizing locally abundant precursors. Cortisol bioactivity appears to be temporally regulated through both inactivation and reactivation mechanisms, enabling a controlled, pro-resolving inflammatory response at ovulation. This mechanism may be essential for timely corpus luteum formation and maintenance of luteal phase integrity. Dysregulation of local cortisol dynamics may contribute to luteal phase insufficiency in a subset of women. The concept of a follicular “inflammatome” and its role in ovulatory success warrants further exploration. FUNDING: This research was supported by the University Hospital of Copenhagen (Rigshospitalet), The University of Copenhagen, and Novo Nordisk Foundation grant number NNF21OC00700556, as well as founded by the Interreg ÔKS V through ReproUnion (www.reprounion.eu) and a grant from Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the manuscript, or the decision to submit it for publication. DISCLOSURES: During the preparation of this work, the author(s) used ChatGPT 4o for language assistance. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article. Furthermore, CYA has received consultant fees and honoraria for lectures from Ferring and IBSA. TRIAL REGISTRATION NUMBER: N/A.