In vitro effects of platelet-rich plasma on bovine endometrial-derived cells

Endometritis is one of the most important causes of reduced fertility in cows as it might affect endometrial function and impair future pregnancy rates. Regenerative medicine by platelet rich plasma (PRP) treatment may offer a new therapy strategy. PRP is defined as the plasma portion with the highest platelet concentration. It contains several growth factors (i.e PDGF, TGF- , PDEGF, PDAF, IGF-1) that have been proven to have beneficial effects on tissue regeneration and anti-inflammatory properties (Marx, 2001). Despite its broad-range applications, few papers have been focusing on the biological effects and the action mechanisms of PRP at cellular and molecular levels. In this pre-clinical study, in view of its in vivo application, the effect of PRP on the expression of genes involved in the regulation of oestrous cycles and fetal-maternal interaction, such as cycloxygenase 2 (COX2), tumor protein p53 (TP53), oestrogen receptors (ER and ER ) and progesterone receptor (PR) was evaluated on bovine endometrial cells. The expression of the transcription factor c-Myc was also investigated to evaluate the potential mitogenic effect of PRP. To produce PRP, whole blood from the mammary vein of different cows were collected into bags containing CPDA-1. The whole blood was centrifuged at 100xg for 30 min and then at 1500xg for 10 min. The resulting platelet pellet was diluted to obtain a concentration of 1x109 platelet/ml. The PRP was frozen at -70°C and thawed at room temperature for three times to promote the release of platelet-derived growth factors. Endometrial cells were obtained from endometrium tissue of normal-cycling cows by digestion with 1mg/ml of collagenase type I for 3h. Endometrial-derived cells were cultured in vitro until passage (P) 10 with two different concentrations of PRP (5% and 10%) and mRNA levels of examined genes were quantified by qPCR. The results were compared to those of control cells, cultured with 10% fetal calf serum (FCS) only. This in vitro study showed a significant increases in the expression of all studied genes in cells at P5 cultured in presence of 5% PRP compared to 10% PRP or 10% FBS. In particular, PR expression increased 5.44-fold and ER expression increased 250-fold. At P10, decrease in the expression of all the evaluated genes was observed, with the only exception of TP53, whose expression remained constant. These data indicate that PRP determines an up-regulation of genes that play an important role in reproduction. It is conceivable that PRP derived growth factors are involved in this mechanism. Indeed, c-Myc, that was up-regulated in this study and is involved in cell proliferation and growth, is activated by EGF that is a component of PRP (Anitua et al., 2004). In future, it will be interesting explore the anti-inflammatory properties of PRP by in vivo studies. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent 2001; 10:225-228. Anitua E et al. Autologous platelets as a source of proteins for healing and tissue regeneration. hromb Haemost 2004; 91:4-15.