Post-insemination intrauterine infusion of Platelet-Rich Plasma (PRP) in sub-fertile mares: preliminary results

Purpose of the work. A post-mating-induced endometritis is a physiological self-limited and necessary reaction to clear the uterus after insemination.1 Recently, platelet rich-plasma (PRP) was shown effective in the treatment of chronic endometritis2 and in modulating inflammatory uterine response3 by production of growth factors, chemokines and cytochines. Aim of this study was to evaluate the effect of intrauterine autologous PRP infusion on the post- insemination inflammatory endometrial response and on the pregnancy rate in sub-fertile mares inseminated with cooled-semen. Materials and methods. PRP treatment was performed in 11 sub-fertile mares [median age 11; range 7-19] unresponsive to conventional therapies. Artificial inseminations (AI) were performed 8 or 24 hours after induction of ovulation with hCG (2000UI). A dose of 800-1200 x 106of cooled semen diluted (50 x 106 sperms/ml)with a milk based commercial extender was deposited in the uterine body. At this moment, 300 ml of blood were collected from the jugular vein in blood bags containing CPDA-1. PRP was prepared with the method of double centrifugation2-4: blood was centrifugated at 120 g x 20 min and the all the plasma was pipetted in new tubes to further centrifugation at 240x g for 10 min. Subsequently, the supernatant was discarded, and the remaining fraction was used as PRP. Low volume flushes (LVF) with 120 ml of sterile saline were performed 6-8 hours after AI and immediately after 20 ml of autologous PRP containing on average of 196000 platelets/μL, were infused into the uterus. At 36h from the hCG administration, mares were checked for ovulation and a second LVF was performed. The turbidity of LVF samples were scored with a 0-5 scale and then divided in two aliquots, one of which was immediately frozen at -20°C for further evaluation of inflammation markers and the other one was centrifuged at 240xg for 10 min. The pellet was smeared and the number of neutrophils (PMN) and epithelial cells (EP) were counted in 5 fields at 400x magnification. Outcomes. Data were checked for normality with Shapiro-Wilk test. EP and PMN cells and the ratios PMN/EP pre and post PRP infusions were compared by Wilcoxon signed-rank test. The turbidity scores were compared by Mood median test. Differences were considered significant at p<0.05. Turbidity of the LVF fluid was significantly lower post PRP infusion (p=0,0002), scored 4 (median; range 3-5) and 2 (range 1-3) respectively pre and post. PMN were 171.07 ±276.53 (median ± dev st) and 12.57±35.02, EP were 6.67±4.2 and 79.71±115.34 and PMN/EP were 21.29±29.75 and 0.39 ±1.03 respectively pre and post PRP infusion. All the differences were highly significant (p=0.0006; p=0.0022; p=0.0002). Pregnancy rate at the first cycle post treatment was 63%. 318 23° CONGRESSO INTERNAZIONALE SIVE Conclusion. Preliminary results show that PRP infusions post insemination could be effective in modulating uterine inflammatory response to semen and improving pregnancy rate in subfertile mares.