Markers of pregnancy loss in Holstein heifers: insights from cervical interferon-stimulated gene mRNA, pregnancy-associated glycoproteins, and progesterone concentrations.

Abstract

Pregnancy loss during the first month of gestation remains a major limitation to reproductive efficiency in dairy cattle, yet the biological stage at which losses occur after the onset of conceptus-derived signaling remains poorly understood in nulliparous heifers. We evaluated cervical interferon-stimulated gene (ISG15, IFI44, OAS1, and RSAD2) expression, circulating progesterone (P4), and pregnancy-associated glycoprotein (PAG) concentrations in grazing Holstein heifers to characterize molecular and endocrine patterns associated with subsequent pregnancy outcomes. Cervical cytobrush samples and blood were collected on d16 after insemination from 103 heifers. Pregnancy status was determined by transrectal ultrasonography on d28 and d33, whereas PAG concentrations were measured on d28. Pregnancy outcomes were subsequently monitored until calving. Based on PAG concentrations, ultrasound determinations and ISG15 expression, heifers were classified as Nonpregnant (n = 10), Early pregnancy loss (EarlyPL; n = 38), Late pregnancy loss (LatePL; n = 7), or Pregnant (n = 48). Evidence of conceptus-derived interferon signaling was detected in 90.3% of inseminated heifers at d16, whereas only 53.4% were pregnant at d28 and 46.6% remained pregnant at d33, indicating that a substantial proportion of losses. All four ISGs were markedly upregulated in EarlyPL, LatePL, and Pregnant heifers relative to Nonpregnant heifers, confirming robust conceptus signaling in most inseminated animals. Among the genes evaluated, ISG15 exhibited the greatest dynamic range and uniquely differentiated all pregnancy outcome categories. P4 concentrations at d16 were similarly elevated in EarlyPL, LatePL, and Pregnant heifers relative to Nonpregnant heifers, indicating that many losses occurred despite adequate luteal support during maternal recognition. In contrast, PAG concentrations at d28 were lower in LatePL than Pregnant heifers, suggesting impaired progression through attachment and early placental development. Collectively, these findings indicate there although embryo signaling is detected and P4 concentrations are elevated on d16, a substantial proportion of losses occur before or shortly after the establishment of early placentation. Cervical ISG15 provided the greatest biological resolution across pregnancy outcomes, whereas PAG concentrations offered complementary insight into progression beyond maternal recognition, supporting a stage-specific model of pregnancy loss in which peri-attachment and early placental processes represent critical determinants of pregnancy maintenance.