Premature ovarian insufficiency (POI), also known as premature ovarian failure (POF), is characterized by abnormal ovarian function in females under the age of 40 [
1]. POI is marked by elevated levels of follicle-stimulating hormone (> 40 IU/mL), decreased levels of estradiol (< 30 pg/mL), and anti-Müllerian hormone (< 1 ng/mL) [
2]. The prevalence of POI cases varies between 0.9% and 1.2% in different societies [
3]. Several factors such as physiological conditions, genetic traits, autoimmune diseases, infections, surgical procedures, environmental effects, and idiopathic reasons have been linked to POI occurrence [
4].
Histological and molecular studies have indicated that POI is characterized by fibrotic changes in ovarian tissue via the engagement of several signaling pathways [
5,
6]. Among these, the TGF-β/Smads signaling axis regulates the growth of ovarian follicles at different developmental stages. Dysregulated TGF-β/Smads signaling pathway accounts for follicular atresia and inhibition of follicles, increasing the possibility of POI conditions [
7,
8]. Local production of important inflammatory cytokines such as TNF-α and IL-10 has been shown to promote POI consequences [
9,
10]. For example, TNF-α induces primary follicle apoptosis, vascular endothelial damage, and reduction of sex-related hormones [
7].
Hormone replacement therapy (HRT) is the most common treatment for POI patients, but it lacks complete recovery of ovarian tissue activity and accounts for the occurrence of some female-related malignancies [
11‐
13]. Oocyte and embryo cryopreservation and donation [
14], as well as gonadotropin-releasing hormone (GnRH) therapy [
15], have also been suggested for POI patients. Unfortunately, these modalities cannot restore normal ovarian tissue activity. Recently, new therapeutic strategies based on stem cells [
16‐
18], and their secretome have been proposed as an effective treatment option for various complications such as infertility [
19]. Numerous studies have shown that different cell types can produce and release nano-sized vesicles such as exosomes (Exos), microvesicles, and apoptotic bodies into the extracellular vesicles [
20,
21]. Exos, with an average size of 40 to 150 nm, are abundant in several biofluids such as plasma, urine, and amniotic fluid [
22,
23]. Exos harbor several signaling molecules that act in a paracrine manner to target cells, resulting in the regulation of specific molecular cascades [
24‐
26]. Amniotic fluid (AF) is an easily accessible biological fluid with significant levels of Exos [
27,
28]. AF-derived Exos (AF-Exos) act as mediators of paracrine communication within the intrauterine environment through the transfer of metabolic substances, including proteins, ions, carbohydrates, lipids, enzymes, and hormones, as well as genomic content (dsDNA, ssDNA, miRNA, mRNA), growth factors, and cytokines that support the growth of the placenta and fetus during pregnancy [
29‐
33]. Emerging data have pointed to the fact that Exo-based treatments yield promising therapeutic outcomes for infertility, cancers, infectious diseases, on-target drug delivery, etc. [
33‐
37]. Unlike whole-cell-based therapy, the application of Exos has several advantages such as low immunogenicity, non-toxicity, and ease of access to target cells [
38‐
41]. In the current investigation, we studied the regenerative potential of AF-Exos in a rat model of POI concerning fibrotic changes and the TGF-β/Smads signaling.