Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry

Ledeck, Joy; Dubrowski, Thomas; Schoumacher, Matthieu; Peeters, Stéphanie; Le Goff, Caroline; Egyptien, Sophie; Deleuze, Stefan; Cavalier, Etienne; Ponthier, Jérôme

doi:10.1016/j.anireprosci.2026.108098

Article (Scientific journals)

Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry

Ledeck, Joy; Dubrowski, Thomas; Schoumacher, Matthieu et al.

2026 • In Animal Reproduction Science, 286, p. 108098

Peer Reviewed verified by ORBi

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https://hdl.handle.net/2268/341171

DOI
10.1016/j.anireprosci.2026.108098

PubMed
41512440

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Keywords :

mare; gestation; pregnancy; equine; conjugated steroids; steroids; fetal sex; serum; Liquid chromatography-tandem mass spectrometry; LC-MS/MS; horse; foal; fetal gender determination; conjugated; dehydroepiandrosterone 3-sulfate; estrone 3-Sulfate; 17β-Estradiol 3-β-D-Glucuronide; estrone β-D-Glucuronide; 17β-Estradiol 3-O-Sulfate; equilin 3-O-β-D-Glucuronide; 17β-Dihydro-Equilin 3-Sulfate

Abstract :

[en] Refined profiling of conjugated estrogens and androgens during equine pregnancy using liquid chromatography-tandem mass spectrometry (LC-MS/MS) could provide accurate fetal-sex determination. Current methods for fetal-sex prediction remain limited by timing, accuracy, and operator expertise. This study investigated sex-specific differences in maternal conjugated steroid profiles to develop a reliable, non-invasive predictive method. Samples were collected from 141 mares of various breeds starting at sixteen weeks of pregnancy. The samples were pooled according to gestational stage, divided into 28-day blocks, and analysed using a validated LC-MS/MS method. Quantified analytes included estrone, estradiol, equilin sulfates, glucuronides, and dehydroepiandrosterone sulfate. Method validation encompassed linearity, trueness, precision, accuracy, uncertainty, quantification limits, recovery, matrix effects, carryover, sensitivity, and stability. The effects of fetal sex, breed, parity, and maternal age on steroid concentrations were investigated. Data were split into a Test group for model development and a Validation group for performance assessment. Fetal sex was the principal factor influencing conjugated estrogen and dehydroepiandrosterone profiles (p =0.01–0.0001). Female pregnancies exhibited higher classical conjugated steroid concentrations at block 5, whereas male pregnancies showed delayed elevations persisting until term. Equilin derivatives were consistently lower in males. Estrone sulfate (E1S) showed reliable univariate performance for fetal-sex prediction (area under the curve (AUC) 0.710–0.865, blocks 5–10). Multivariate models using all conjugated steroids achieved superior accuracy (AUC 0.747–0.912, blocks 5–11), providing an alternative method to univariate model and ultrasonography. The validated LC-MS/MS method improves understanding of equine feto-placental endocrinology and offers a non-invasive tool for practical fetal sexing.

Research Center/Unit :

FARAH. Médecine vétérinaire comparée - ULiège

Disciplines :

Veterinary medicine & animal health

Author, co-author :

Ledeck, Joy ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Médecine vétérinaire comparée

Dubrowski, Thomas ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale

Schoumacher, Matthieu ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale

Peeters, Stéphanie ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale

Le Goff, Caroline ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale

Egyptien, Sophie ; Université de Liège - ULiège > Département d'Enseignement et de Clinique des animaux de Compagnie (DCC) > Reproduction des animaux de compagnie

Deleuze, Stefan ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Médecine vétérinaire comparée

Cavalier, Etienne ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale

Ponthier, Jérôme ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Médecine vétérinaire comparée

Language :

English

Title :

Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry

Publication date :

March 2026

Journal title :

Animal Reproduction Science

ISSN :

0378-4320

eISSN :

1873-2232

Publisher :

Elsevier BV

Volume :

286

Pages :

108098

Peer reviewed :

Peer Reviewed verified by ORBi

Additional URL :

https://api.elsevier.com/content/article/PII:S0378432026000011?httpAccept=text/xml

Name of the research project :

PE2C2

Funders :

FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture
FWB - Fédération Wallonie-Bruxelles

Available on ORBi :

since 09 February 2026

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