Current knowledge in equine reproduction stipulates that systemic changes associated with pregnancy only occur following maternal recognition of pregnancy (MRP) and subsequent maintenance of the corpus luteum (CL), initiated around day 10 of pregnancy. The earliest known immune responses to pregnancy begin around day 35, when MHC-expressing trophoblast cells of the chorionic girdle invade the endometrium. We hypothesised that systemic changes, independent of classical MRP, might occur as early as day 7.
A series of investigations comparing the systemic proteomic, lipidomic and metabolomic profiles of pregnant and non-pregnant mares (n=99) supported this hypothesis, revealing distinct pregnancy profiles in all three studies. These changes were independent of progesterone levels. Functional pathways typically associated with virus recognition and immune response evasion were enriched on gene ontology analysis of the proteomics data. Specifically, abundances of immunoglobulin components responsible for antigen specificity (light chain variable regions) were consistently altered in pregnant mares at day 7 post-ovulation (p<0.001) and persisted to day 14. To further probe the difference in antigen specificity of these profiles, we isolated immunoglobulins and their targets by cross-linking and co-immunoprecipitation of blood plasma proteins from pregnant (n=3) and non-pregnant (n=3) mares and analysed the resulting pull-down lysates using mass spectrometry. Over 600 target proteins were identified in the lysates. Immunoglobulin abundances in the pull-down lysates were stable across groups but two proteins consistently and uniquely co-precipitated with immunoglobulins in the non-pregnant mares. Both of these proteins (versican [VCAN] and dynein light chain roadblock-type 2 [DYNLRB2]) have roles in immune modulation and viral entry, and high expression in placental tissue. Their role in the equine pregnancy is yet to be established.
In conclusion, we present the first evidence for a possible systemic process of immune recognition of early pregnancy in the mare, which is underway by day 7 and precedes classical MRP.