Assessing Gestation and Fetal Sex in Wild Assamese Macaques Using Urinary Estrogen Analysis

In mammals, estrogens and progestogens are crucial for gestation, fetal development, and maternal preparation for parturition and lactation. Measuring these hormones allows for the diagnosis of pregnancy, estimation of pregnancy failures, and potentially prenatal sex determination. We evaluated urinary estrogen and progestogen metabolites as biomarkers for gestation detection and for their utility for fetal sex determination in wild Assamese macaques (Macaca assamensis) using 586 samples from 19 females, including 19 successful pregnancies. Four enzyme-immunoassays were tested for suitability in measuring urinary sex steroids using serial dilution: three assays targeting progestogen and one targeting estrogen metabolites (estrone conjugates, E1C). We performed a biological validation by measuring urinary hormone metabolites in one female across pre-, early-, late-, and post-gestation. None of the progestogen measurements reflected gestational status, while E1C levels showed the expected increases during gestation. Next, we measured urinary E1C across gestation in all females and investigated fetal sex effects on maternal E1C levels, expecting differences between females carrying male versus female fetuses. Urinary E1C levels increased as early as 9 days postconception and declined sharply at parturition, mirroring patterns in other primates. During late gestation, females carrying male fetuses had significantly higher E1C levels than those carrying female fetuses, yet overlapping values limit precision for prenatal sex determination. Urinary E1C offers a noninvasive marker for gestation monitoring in Assamese macaques, with application in ecological and conservation research. Additionally, results indicate intra- and inter-species-specific differences in steroid hormone metabolism and excretion, which need to be considered when selecting markers for reproductive monitoring.