Modulation of uterine inflammatory response in mares treated with platelet-rich plasma.

Author(s)

Lorenzo Garrido Teixeira Martini Segabinazzi

• Document type: Doctoral Thesis
• Press: Botucatu. 2021-05-06.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária e Zootecnia. Botucatu
• Defense date: 2021-04-15
• Advisor: Marco Antonio Alvarenga

Abstract

Endometritis is the most common cause of subfertility in mares. All mares display a physiological post-breeding endometrial inflammation; however, 15-20% of mares have persistent breeding-induced endometritis (PBIE). Recently, platelet-rich plasma (PRP) is becoming popular in mare practice to mitigate post-breeding induced endometritis and improve fertility. Currently, there is no standardization of methods to prepare PRP for intrauterine use in mares. This study aimed to compare three methods to prepare PRP for intrauterine infusion in mares and to assess the post-breeding inflammatory response, endometrial culture and embryo recovery in mares susceptible to PBIE treated with PRP. In the first study, PRP was produce by three methods. Method 1, blood was collected in blood transfusion bag, transferred to Falcon tubes and double centrifuged; Method 2, blood was collected in vacutainer tubes and centrifuged once; Method 3, blood was collected in a syringe containing anticoagulant, which was kept in an upright-position to sediment for 4h. After processing, cell counts and platelet viability were assessed. In a subset of mares (n=6), PRP was evaluated at 6 and 24h post-cooling at 5°C. In study 2, mares (n=12) susceptible to PBIE had three cycles randomly assigned in a crossover design to receive intrauterine infusions of LRS (control), or autologous PRP or platelet-poor plasma (PPP) pre- (48 and 24h) and post-breeding (6 and 24h). Both PRP and PPP were obtained by Method 1. Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial polymorphonuclear cells (PMNs) were assessed every 24h up to 96h post-breeding. Uterine cytokines (Ilβ, IL6, CXCL8 and IL10) were evaluated before (0h), 6 and 24h post-breeding, and endometrial culture three and nine-days after breed. Embryo flushing was performed 8d post-ovulation. Data were analyzed with mixed model, Tukey’s post-hoc test, and multivariate regression. In study 1, Method 1 resulted in the greatest and method 3 in the fewest platelet concentrations, and the latter had greater WBC than the others (P<0.05). Platelet viability was similar across treatments. Cooling for 24h did not affect platelet counts. However, platelet viability was reduced after cooling PRP produced by method 3. Study 2, PRP treatment reduced endometrial PMNs, post-breeding IUF and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly higher embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). In conclusion, Method 1 resulted in the greatest platelet concentrations, while method 3 resulted had greater WBC counts. Cooling affected platelet viability in PRP obtained with method 3. It remains to be determined whether the different methods and cooling would affect PRP's clinical efficacy. Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property. (AU)

• FAPESP's process: 17/13883-9 - ENDOMETRIAL EXPRESSION OF INFLAMMATORY INTERLEUKINS AND PROTEOMICS OF ENDOMETRIAL FLUID OF MARES SUSCEPTIBLE TO PERSISTENT BREEDING-INDUCED ENDOMETRITIS TREATED WITH PLASMA RICH PLASMA
• Grantee: Lorenzo Garrido Teixeira Martini Segabinazzi
• Support Opportunities: Scholarships in Brazil - Doctorate