Ovarian reserve and ageing: 2 sides of the same coin? Insights from humans and gastropods

Background

A decline in ovarian reserve is associated with the ageing process. The Klotho gene, named after the goddess of ageing, was first described in 1997 and associated with premature ageing in mice. However, its potential as a biomarker of oocyte quality and ovarian ageing has recently attracted considerable attention. The Klotho protein exists in three forms — α- (with membrane-bound and secreted isoforms), β- and γ-Klotho — and can be detected in organs such as the kidney and brain, as well as in bodily fluids including blood, urine, and cerebrospinal fluid. We hypothesise that Klotho may reflect the progressive decline in reproductive capacity that accompanies the transition from pre- to postmenopause. Therefore, our aim is to analyse possible correlations between Klotho and other biomarkers of ovarian reserve and ageing, in patients with and without hormone therapy (HT) as well as controls. Furthermore, we want to establish an animal model to deepen our knowledge on Klotho with regard to its role in stress regulation.

Methods

An ongoing cross-sectional study has been performed since April 2024 at the University Clinic of Gyn. Endocrinology and Reproductive Medicine, including (peri-)menopausal patients and controls. Currently, patients with PCOS are also being recruited. Blood samples were processed and stored at –80 °C. Secreted and soluble α-, β-Klotho and FGF-23 were analysed using enzyme-linked immunosorbent assays (ELISA). Serum levels of anti-Müllerian hormone (AMH), leptin, follicle-stimulating (FSH) and luteinising hormone (LH), inhibin B, as well as cytokine concentrations of IL-1β, IL-10, IL-6, MCP-1 (CCL2) and TNF-α, were measured in patients and controls. Transcriptome-derived Klotho sequences from various gastropod species were validated in vivo. Basal mRNA expression of Klotho and stress related proteins as well as the response to Cd exposure were assessed by qRT-PCR. Statistical analyses were performed using GraphPad Prism (version 10.1.2, p<0.05 significant).

Results

So far, 109 menopausal patients (MP) and 50 healthy controls (Co) have been included, of which 38 MP and 0 Co underwent HT. The mean age differed significantly: 53 ± 8 years (MP) vs. 30 ± 6 years (Co). BMI did not differ significantly between the two groups. Both secreted and soluble α-Klotho were higher in Co. In patients with HT, secreted α-Klotho was higher than in patients without HT, but this difference was not significant (p>0.05). FGF-23, leptin, LH and FSH were higher in MP, whereas AMH, inhibin and oestradiol were higher in Co. The first results showed no significant correlations or differences with regard to immune parameters. Gastropods displayed variable basal expression levels of Klotho and response patterns.

Discussion

Within our study population, α-Klotho levels were higher in Co, which is related to the significant age difference. Menopausal patients with HT exhibited a trend towards higher Klotho levels than without HT, suggesting a potential beneficial effect of HT on ageing. The multifunctional Klotho protein may perform various functions within gastropods, which are a promising animal model.