Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (nZ9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all D4 steroids. The 5a-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (D5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus D4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5a-pregnanes) marked the luteo-placental shift, D5 steroids defined fetal gonadal growth and 5a-reduced metabolites of DHP dominated the steroid profile in mid-to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

Pregnane concentrations of ill, neonatal foals remain increased following birth, reflecting a delayed, or interrupted, transition from intra- to extra-uterine life. Serial progesterone and pregnenolone measurement may be useful in aiding diagnosis of NMS.

No abstract

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.ince first crystallized almost eight decades ago, progesterone has remained the only endogenous member of the progestin class of steroids defined by its singular ability to maintain pregnancy (1), acting, in large measure, through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained (2). Birth is thought to be triggered by a decrease in systemic progesterone concentrations (withdrawal) (3), even though this is not evident in mares (4), women, or guinea pigs (5), a disparity that limits the utility of other animal models for preterm labor (6). The vast majority of studies have focused on measuring progesterone, with most using immunoassays that necessarily cross-react with multiple pregnanes (7), the bioactivity of which remain uncharacterized. This is reasonable because, in contrast to androgens, estrogens, and corticoids, for which multiple natural biopotent analogs are known, no other endogenous pregnane has ever been shown to substitute for progesterone in pregnancy in any mammal.However, over five decades ago, Short (8) reported that circulating progesterone concentrations in pregnant mares were surprisingly low at <4 ng/mL, as did Holtan e...

Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3 h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.