Abstract :
[en] Since 1976, estrogen concentrations in pregnant mares have been thought to be unrelated to fetal
sex, but these studies were based on immunoassays. Despite their widespread use, these assays
have limited specificity due to cross-reactivity and are rarely validated in equine, compromising
their accuracy. Liquid chromatography tandem mass spectrometry (LC-MS/MS) provides precise
steroid quantification, allowing deeper insights into steroid metabolism and potential fetal gender
differences. The current standard for fetal sexing, transrectal ultrasonography, requires technical
expertise, has a limited gestational window, and may yield inconsistent results. This study
hypothesizes influence of fetal sex on maternal sulfonated estrogen levels and aims to develop a
non-invasive method for fetal gender determination in mares. From 2020-2024, 68 mares from
Belgian stud farms, managed under standardized conditions regarding diet, housing, and
reproductive practices, were included, resulting in 115 pregnancies. Blood samples (n = 596,
median per gestation: 5 (Q1–Q3: 4–6)) were collected from 4-11 months to quantify estrone-sulfate
(E1S) and estradiol-sulfate (E2S) using a validated LC-MS/MS method for equine. Mares with
placentitis were excluded. Statistical analyses (SAS 9.4, p < 0.05) used logistic regression to assess
fetal sex effects, accounting for maternal age, breed, and parity, and estimate associations between
fetal sex and maternal hormone concentrations. Most mares were Warmbloods (45.6%) or Spanish
purebred horses (44.1%). Male foals accounted for 51.7% of the births, with a sex ratio of 1:1.07.
Parity and breed did not affect the sex ratio, while age tended to be significant (p = 0.06). Estrone
and estradiol-sulfate concentration followed a quadratic trajectory (p = 0.0003), peaking at 5
months for females and 6 months for males. Fetal gender influenced hormone concentrations for
E2S (p < 0.0001) and E1S (p = 0.012). Males exhibited higher E2S from 169-308 days, with the
most significant differences at 169-196 days (p = 0.0003), 197-224 days (p = 0.0019), and 225-
252 days (p = 0.0031). Females had higher E2S at 113–140 days (p = 0.032). These results contrast
with previous reports, where no fetal sex-related differences in maternal estrogen concentrations
were observed. Fetal gonads secrete androgens that drive placental estrogen production, and their
bioavailability influences maternal estrogen concentrations. At 5 months, female fetuses reach
peak E2S concentration, exhibiting higher concentrations than males. Around six months, male
fetuses surpass females in E2S concentrations as they reach their peak. This shift is due to
differential steroidogenic activity. Although male fetal gonads contain fewer interstitial cells at this
stage, their enzymatic machinery is more developed, leading to greater androgen production. These
are aromatized in the placenta into estradiol by cytochrome P450 19A1, whose transcript
expression also peaks around 6 months, before placental sulfotransferases responsible for estrogen
sulfonation increase E2S excretion in maternal circulation, particularly with male fetuses. These
findings suggest maternal E2S concentrations differ by fetal sex, indicating LC-MS/MS could
serve as a reliable, non-invasive alternative to ultrasound for fetal monitoring. Expanding the
cohort earlier in pregnancy could improve predictive accuracy and enable fetal sex determination
as early as ultrasound, optimizing breeding management.
