The stereological investigation of conditioned medium originated from human adipose tissue-derived mesenchymal stem cells and platelet-rich plasma effects on polycystic ovary syndrome in a rat model

Abstract Background: Platelet-rich plasma (PRP) and conditioned medium (CM) originating from human adipose tissue mesenchymal stem cells (ATMSCs) as two enriched sources of growth factors have a potential impact on tissue repair. Objective: This investigation aimed to investigate the effects of PRP and CM on ovarian structures in letrozole-induced polycystic ovarian syndrome (PCOS) in female rats through the stereological methods. Materials and Methods: To obtain PRP, blood samples from Wistar rats were collected in citrate tubes, centrifuged (400g, 10 min) to separate components. Plasma+Buffy coat was recentrifuged (800g, 10 min); Platelet-poor-plasma (PPP) discarded, PRP (lower layer) obtained and stored at − 20°C. To attain the mentioned mesenchymal CM, first, adipose tissue was collected from liposuction samples by collagenase digestion and cultured in DMEM/FBS. To approve the isolation of ATMSCs, the flow cytometry method, based on the expression status of CD44, CD90, CD34 and CD45 markers, was carried out. ATMSCs were then grown in serum-free DMEM, and supernatant was centrifuged and stored. Forty female Wistar rats were allocated into five groups (Control, negative control (letrozole [LTZ]), letrozole and PRP [LTZ-PRP], letrozole and CM [LTZ-CM] and letrozole and PRP + CM [LTZ-PRP + CM] groups). The Control group received normal saline (0.9% NaCl, 200 μl) orally. In the negative control, PCOS was induced by letrozole (1 mg/kg dissolved in normal saline, daily). Animals were treated by PRP, CM and PRP + CM (200 μL, IP, 1 and 14 days after PCOS induction). At the end of the experiment, body mass index (BMI) and body weight were evaluated, and then, blood samples were taken for the evaluation of serum testosterone level. The animals were dissected, and their ovaries were excised, fixed, sectioned and stained by H&E. Stereological approaches were utilized for estimation of the volume of the ovary, ovarian cortex and medulla, corpus luteum, oocytes, ovarian cysts and the number of different ovarian follicles and granulosa cells. Results: In the LTZ-PRP and LTZ-CM groups, ovarian weight (OW) was significantly increased compared to the LTZ group ( P < 0.001 and P < 0.05, respectively). The cortex volume (CV) and ovarian volume (OV) of the LTZ-PRP group were dramatically elevated compared to the LTZ group ( P < 0.0001). A remarkable elevation of the preantral follicles number (PFN) in the mentioned group compared to the LTZ group was also observed ( P < 0.05). The estimation of the antral follicle number (AFN), atretic follicle number (AtFN) and granulosa cell number (GCN) in the LTZ-PRP group showed a significant increment compared to the LTZ group ( P < 0.0001). The PFN and AFN in the LTZ-CM group compared to the LTZ group were significantly elevated ( P < 0.01). In this group, AtFN and GCN were also increased significantly in comparison with the LTZ group ( P < 0.001). In terms of serum testosterone level (STL), there was a significant elevation in the LTZ-PRP group compared to the LTZ group ( P < 0.0001). Regarding the LTZ-PRP + CM group, the body weight (BW) and body mass index (BMI) were considerably higher than the LTZ-CM group ( P < 0.05 and P < 0.01, respectively). CV and OV were also increased significantly compared to the LTZ-CM group ( P < 0.001). The AtFN in this group was considerably elevated in comparison with the LTZ-CM group ( P < 0.0001). Moreover, STL in the mentioned combinatorial group was conspicuously increased compared to the LTZ-CM group ( P < 0.0001). Conclusion: PRP, alone and combined with CM, promoted parameters related to the weight and volume of the cortex and the ovary, and the number of granulosa cells and follicles in different phases compared with the PCOS group. Thus, it seems that the PRP has a more prominent role regarding these parameters than CM. However, more research is recommended to validate these results.