Development of a preclinical screening pipeline to test the efficacy of anti-fibrotic and anti-inflammatory drugs on ovarian aging

Abstract Study question Can either systemic or targeted ovarian delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs have a therapeutic effect on ovarian aging in mice? Summary answer We have established an in vitro and in vivo pre-clinical pipeline in the mouse to evaluate the effect of drugs on ovarian aging. What is known already The female reproductive system is the first to age in humans, beginning when women reach their mid-thirties. Reproductive aging results in infertility and hormonal perturbations which have general health consequences. Most research on reproductive aging has focused on the age-associated reduction in oocyte quantity and quality. However, our lab discovered that the ovarian microenvironment (stroma) in which the gamete develops changes with age. Specifically, a pro-inflammatory milieu develops with a significant increase in pro-inflammatory cytokine production including interleukin-6. Concurrently, collagen I/III accumulates resulting in fibrosis. This fibrotic and inflammatory milieu likely impacts oocyte quality contributing to reproductive aging. Study design, size, duration Etanercept was selected as an anti-inflammatory drug because it binds and inhibits TNF-a/b. Pirfenidone was selected as anti-fibrotic drug because it targets collagen I/III and TGF-b1. In vitro studies were performed using tissue explant-cultures to evaluate the toxicity of the drugs on the ovary. In vivo studies were based on the delivery of these drugs either systemically or targeted to the ovary using mini-osmotic pumps to evaluate the impact of these drugs on ovarian aging. Participants/materials, setting, methods In vitro studies: Ovaries from 7-month (N = 6) and 14-month (N = 6) old CD1 mice were isolated, dissected into equal pieces, and incubated in Pirfenidone (0.05, 0.5, 5mM), Etanercept (1, 10, 100g/ml), or the drug diluent (controls). After 24h the explants were collected and tissue architecture (H&E), collagen content (Picrosirius red/PSR), and cell death (Caspase-3) was analyzed using histochemical approaches. In vivo studies: Alzet mini-osmotic pumps were implanted subcutaneously and 2-delivery approaches tested: systemic (N = 2) and ovarian-targeted(N = 2). Main results and the role of chance We first evaluated the toxicity of Etanercept and Pirfenidone on the ovary using our in vitro system. H&E staining revealed that the structure of the Etanercept and Pirfenidone-treated ovaries were similar to controls. The collagen content was also not significantly different in all groups (p > 0.05, One-way ANOVA, Tukey’s test). However, 5mM Pirfenidone and 10 and 100g/ml Etanercept treatments resulted in a trend of decreased collagen relative to the control ovaries. Cleaved Caspase-3 fluorescent intensity/area was also similar in all conditions (p > 0.05, One-way ANOVA, Tukey’s test). Therefore, our in vitro studies show that Pirfenidone and Etanercept are not toxic demonstrating the potential use of these drugs in the ovary. Next, we tested the use of these drugs in vivo with osmotic pumps in 7-month old mice, when ovarian fibrosis is first observed. As a proof-of-concept, the pump was filled with Alcian Blue (dye). Pumps were implanted subcutaneously for systemic and targeted delivery. For the targeted delivery, the pump was connected to a catheter which was inserted underneath the bursa. The pumps were well tolerated by the mice. Two weeks after surgery the catheters were still attached to the ovaries and none of the ovaries showed structural abnormalities when analyzed with H&E. Limitations, reasons for caution We have established a preclinical pipeline to screen the potential of drugs to extend reproductive longevity. Studies are ongoing to determine the efficacy of Pirfenidone and Etanercept in delaying fibrosis and inflammation in the ovary to improve reproductive outcomes. Experiments to determine the side effects of these drugs are required. Wider implications of the findings Our in vitro studies show that Pirfenidone and Etanercept are not toxic to the ovary suggesting a therapeutic potential of anti-fibrotic and anti-inflammatory drugs in delaying reproductive aging. Our in vivo preclinical pipeline represents a novel system to evaluate the therapeutic effect of these and other drugs on ovarian aging. Trial registration number N/A