To study the relationship between FK506-binding protein 51 (FKBP51) and ovarian aging and/or diminished ovarian reserve (DOR) in human ovaries by comparing FKBP51 levels in granulosa (GC) and cumulus cells (CC), collected during controlled ovarian stimulation (COS) from women of advanced reproductive age and/or with a diagnosis of DOR with that of young women with normal ovarian reserve. To explore the association between increased FKBP51 expression and human ovarian aging further, expression of FKBP51 was compared in ovarian stroma of post-menopausal versus pre-menopausal women. Lastly, this relation was further queried by comparing ovarian expression of several collagen genes as markers of ovarian fibrosis in 14-month-old wild type (Fkbp5^+/+) and Fkbp5 knockout (Fkbp5^-/-) mice. Laboratory based experimental study. Academic-affiliated assisted reproductive technology unit/laboratory SUBJECTS: (1) Samples collected included follicular fluid (FF), CC, GC and serum from group 1: Young women with normal ovarian reserve (<35 years; n=12); group 2: DOR (anti-Mullerian hormone (AMH) 37 years; n=8). (2) Ovarian stromal tissues obtained from surgical specimen of post-menopausal (50-65 years; n=6) and pre-menopausal (18-30 years; n=6). (3) 14-month-old ovarian tissues from Fkbp5^+/+and Fkbp5^-/- mice. Comparison of FKBP51 expression in GC and CC from women undergoing COS, ovarian stromal tissue from pre- and post-menopausal women, and ovarian tissue from aged Fkbp5^+/+and Fkbp5^-/- mice. (1) Level of FKBP51 in human GC and CC, collected during COS by performing RT-quantitative PCR (RT-qPCR). (2) Immunohistochemistry (IHC) to detect FKBP51 levels and Picrosirius Red Staining (PSR) to detect collagen deposition in human ovarian stromal tissue. (3) RT-qPCR to compare expression levels of several collagen genes in Fkbp5^+/+ and Fkbp5^-/- old mice ovaries. (4) Serum and FF levels of TGF-β1, and soluble Endoglin measured by ELISA. IHC revealed that FKBP51 HSCORE levels in ovarian stromal tissue were significantly higher in post- vs. pre-menopausal women (Mean± SEM; 160.52±17.75 vs. 120.67±14.33, P= 0.002). Stronger Picrosirius Red staining, suggestive of fibrosis, was seen in post- vs. pre-menopausal women (54.06± 6.94 vs. 37.50± 14.29, P=0.02). Analysis of qPCR revelated that 1) Col1a1, Col1a2, Col3a1 levels were significantly lower in ovaries obtained from 14-month-old Fkbp5^-/-vs. Fkbp5^+/+ mice; 2) FKBP5 levels significantly increased in cumulus cells of advanced age women vs. younger women (1.71± 0.22 vs. 1.11± 0.15, P= 0.03); and 3) FKBP5 levels were ∼3-fold higher in granulosa cells of women with DOR vs. age-matched control (3.22± 1.11 vs. 1.30± 0.54 P= 0.03). This study for the first time demonstrates expression profile of FKBP51 in human ovary and its potential role in ovarian aging. Our results indicate that the upregulation of FKBP51 is associated with ovarian aging. Moreover, in women undergoing IVF treatment, enhanced FKBP51 expression is seen in those with DOR or women of advanced maternal reproductive age, who have poor prognosis. Therefore, drugs targeting inhibition of FKBP51 expression and/or activity may delay ovarian aging or treat premature ovarian aging.

Prenatal stress (PNS) impairs offspring ovarian development by exerting negative long-term effects on postnatal ovarian function and folliculogenesis. FKBP51 is a stress-responsive protein that inhibits glucocorticoid and progesterone receptors. We hypothesize that FKBP51 contributes to impaired ovarian development and folliculogenesis induced by PNS. Timed-pregnant Fkbp5^+/+ (wild-type) and Fkbp5^−/− (knockout) mice were randomly assigned to either the undisturbed (nonstress) or PNS group, with exposure to maternal restraint stress from embryonic days 8 to 18. Ovaries from the offspring were harvested and stained, and follicles were counted according to their stages. Ovarian expressions of FKBP51 were evaluated by immunohistochemistry and Fkbp5 and steroidogenic enzymes were evaluated by qPCR. Compared to controls, Fkbp5^+/+ PNS offspring had increased peripubertal primordial follicle atresia and fewer total follicles in the adult and middle-aged groups. In adult Fkbp5^+/+ offspring, PNS elevated FKBP51 levels in granulosa cells of primary to tertiary follicles. Our results suggest that PNS administration increased FKBP51 levels, depleted the ovarian reserve, and dysregulated ovarian steroid synthesis. However, these PNS effects were tolerated in Fkbp5^−/− mice, supporting the conclusion that FKBP51 contributes to reduced ovarian reserve induced by PNS.