Equine endometrial Th1 and Th2 evaluation during early pregnancy

Abstract

As in all mammals, continuous feto-maternal dialogue is essential for successful pregnancy in mares. Nevertheless, several features of pregnancy in the mare are unusual and differ markedly from equivalent events in other species. The nature of the signal by which the equine embryo biochemically indicates its presence in the mare´s uterus for pregnancy maintenance remains a mystery to the present day, but the equine conceptus must begin to release this factor resulting in maternal recognition of pregnancy around day 10 to day 13 after ovulation. In humans and some animal species, the Th1/Th2 balance is essential for immunological tolerance in pregnancy. This immunohistochemical study aimed to assess the T-bet (Th1) and GATA3 (Th2) lymphocyte populations in the endometrium on days 7, 10, and 13 after ovulation in cyclic and pregnant mares. Mares were randomly assigned to the day of sample collection in both groups. In the first cycle, endometrial biopsies from 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10), and 13 (n = 10) post-ovulation (Cyclic group). In the second cycle, the same mares were bred to a fertile stallion, and endometrial samples were collected on days 7, 10, and 13 post ovulation. Immediately after biopsies, uteri were flushed, and those with an embryo recovered were allotted to the Pregnant group. Of the 30 mares flushed, embryos were recovered from6 mares on day 7, 6 on day 10, and 6 on day 13. Samples from mares without embryo recovery were excluded from both groups. Immunohistochemistry was used to detect Th1 (T-bet PA5-28881, Invitrogen) or Th2 (GATA3 L50-823, Thermo Fisher) proteins in the endometrium. Only stained lymphocytes from the stratum compactum (SC) were counted. Five fields were analyzed at 400x magnification. A two-way ANOVA was performed to evaluate the absolute number of T-bet/GATA3 cells and their ratio as a dependent factor and experimental groups, day, and their interactions as independent variables. T-bet-stained cells decreased from days 7 to 13 in both the Cyclic (day 7=73 vs 13=48) and Pregnant groups (day 7=83 vs 13=39) (P=0.0179); no difference was observed between groups. GATA3 stained cells remained constant over the days, with more stained cells in the Pregnant (day 7=37; 10=35; 13=34) than in the Cyclic group (day 7=23; 10=12; 13=12) (P=0.0001). The Th1/Th2 ratio did not show a difference (P>0.2222). Th2 cells are recruited into the SC of the endometrium of the pregnant mare. These cells could be secreting interleukins and establishing an anti-inflammatory microenvironment in early pregnancy. In conclusion, the presence of Th2 cells in pregnant mares could be important for embryonic development and maternal recognition of pregnancy regarding immunological tolerance.