Fig. 1. Pirfenidone reduces ovarian collagen and promotes ovulation in reproductively aged mice.

(A) Experimental design to test the effects of candidate antifibrosis drugs on ovarian collagen deposition, ovulation, and oocyte developmental competence. (B to D) Female mice (15 months old) were treated with pirfenidone via injection (250 mg/kg, ip; 4 days) or orally (500 mg/kg, po; 2 weeks) or with nintedinab (50 mg/kg, ip; 4 days) and compared to those treated with vehicle (Veh) and young (Y; 2 months old) controls. Following gonadotropin stimulation, ovaries and oviducts were dissected and ovulation was assessed in each female (B, left) (n = 3 to 9 females per group as indicated) and ovulated oocytes were counted (B, right). Ovarian fibrosis was measured by PSR staining of collagen (C and D). Scale bars, 200 μm. (E to G) Female mice (12 months old) were treated with pirfenidone orally (500 mg/kg; 2 weeks; n = 12) or vehicle (n = 16) alongside young controls (n = 8), and ovarian fibrosis was assessed by PSR strain (E). Ovulated oocytes were counted (F) and subjected to in vitro fertilization, and embryo development was monitored to the two-cell (2C) and blastocyst stage (G). Means ± SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way analysis of variance (ANOVA) (D, E, and G) or two-tailed t test (F) compared to vehicle-treated old mice.