Urinary steroids have been studied during early and late pregnancy in domestic horses or sporadic samples at various stages of pregnancy in wild equidae. In our studies, urinary estrone sulfate (E1S) and pregnanediol glucuronide (PdG) were monitored throughout pregnancy in six pony mares by enzyme immunoassay (EIA). Both hormones were corrected by creatinine (Cr) index to compensate for the variation in specific gravity. The mean concentration for ElS, (μg/mg Cr), was .38 ± .03 at d 0, decreased to .17 ± .04 at d 1, and maintained at less than .5 μg/mg Cr until d 30. Although, there was an apparent increase to .80 ± .34 at d 34 (NS, P = .122), the first significant increase was .69 ± .15 at d 46 (P = .0275). Mean concentrations remained relatively stable at this approximate level until d 60. This level was followed by a sustained significant increase observed from d 60 onwards. Mean concentrations of El S increased to 1.11 ± .25, 2.01 ± .45, and 5.48 ± 1.47 at d 64, d 76, and d 86, respectively. Levels of EIS further increased reaching a peak of 143.3 ± 9.51 at d 142 (P = .0006), with maximum for individual mares ranging from d 114 to 170, and also ranging from 115.4 to 286.1 pg/mg. In all cases, maximum concentrations were followed by a gradual decline toward parturition with a more rapid decrease 1 to 3 days before parturition. The first significant decrease following the maximum concentration was 91.40 ± 13.11 (P = .0024) at d 184. Estrone sulfate was 12.1 ± 3.8 one day prepartum and decreased to .4 ± .1 and .1 ± .01 at d 1 and 4 postpartum, respectively. The mean concentrations of PdG (ng/mg Cr) increased from 147 ± 4.3 at d 0 to 50.87 ± .17 (NS, P> .05), 36.8 ± 8.1 (P = .016), and 27.6 ± 7.3 (P = .049) at d 6, 8 and 10, respectively. This increase was followed by a decline and generally the levels fluctuated ranging from 20 to 30 ng/mg Cr until d 80. At d 86, the PdG levels increased to 54.7 ± 11.7 (P = .033). This was followed by a further increase to 141.8 ± 21.4 (P = .0139, compared to d 93) at d 135, then continued to increase to 213.0 ± 25.2 at d 198, and remained at this approximate level until d 303. During the last month of gestation, the mean concentrations of PdG increased from 171.8 ± 9.8 at d 29 prepartum to reach a peak of 388.4 ± 108.6 at d 7 prepartum. Maximum concentrations were followed by a slight decrease to 354.5 ± 84.0 at d 1 prepartum and then decreased to 150.6 ± 23.4 and 39.6 ± 9.3 ng/mg Cr at d 1 and 4 postpartum. In comparing the two hormones, E1S remained baseline followed by a slight increase at d 35, whereas PdG was relatively stable until both hormones increased after d 70 of gestation. This might be related to secretion of both hormones by the fetus and their rapid metabolism by placenta. Estrone sulfate reached a peak at approximately d 142 followed by a decline toward parturition while PdG showed a rapid increase from d 70 to 150, followed by a slow sustained increase to d 300 then increased dramatically again before parturition, while El S continued to decline. The profile of these urinary hormones throughout pregnancy appeared to parallel previously published concentrations in blood. Since the patterns of urinary EIS and PdG are different, their sites and mechanism of metabolism are likely different. The results indicate that the presence of the feto-placental unit is important for the secretion of both estrogens and progestins throughout pregnancy and thus could be utilized as a reliable method for pregnancy determination after three months of pregnancy. / Graduation date: 1993