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Home / Equine Research Bank / Equine Vet J / Concentrations of sulphated estrone, estradiol and dehydroep...
Equine veterinary journal2019; 51(6); 802-808; doi: 10.1111/evj.13109

Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares.

Abstract: Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation. Results: Estrone sulphate concentrations peaked by week 26 at almost 60 μg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed. Conclusions: Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards. Conclusions: Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation.
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Publication Date: 2019-04-10 PubMed ID: 30891816DOI: 10.1111/evj.13109Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on a detailed study of the concentrations of certain hormones in pregnant mares, with the data gathered through a method called liquid chromatography tandem mass spectrometry (LC-MS/MS), which is more accurate than previous methods.

Experiment and Methodology

  • This study conducts a longitudinal analysis, meaning it measures the changes over a continuous period, of hormone concentrations during the pregnancy of mares (female horses). The researchers focus particularly on conjugated oestrogens and androgens, which are types of steroids.
  • The researchers have utilized a method called liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides more accurate measurements compared to methods used in previous such studies, which relied on immunoassays.
  • They developed an analytical method using LC-MS/MS for the measurement of sulphated estrone, estradiol, testosterone, and dehydroepiandrosterone (DHEAS) during equine gestation.
  • A group of mares were treated with a substance called letrozole in the late stages of their pregnancies to inhibit oestrogen synthesis, and their hormone concentrations were compared to the control group (mares given a placebo).

Results

  • The study found that the concentration of a hormone called estrone sulphate peaked by the 26th week of pregnancy, at levels much higher than previously reported in similar studies using immunoassays.
  • The hormone, DHEAS increased from week 7 to 9 of gestation, but was consistently low for the remainder of the gestation period and testosterone sulphate was undetectable at any stage.
  • Estradiol sulphate concentrations were found to be highly correlated with those of estrone sulphate, but at much lower levels.
  • Both oestrogen sulphate concentrations visibly decreased from their peak to the time of giving birth.
  • Letrozole did inhibit the concentrations of estrone and estradiol sulphates at particular stages of late gestation, but no increase in DHEAS was observed.

Conclusions

  • One cited limitation of the study is the small number of mares that were sampled and available for analysis. Another limitation is the lack of analysis of 5α-reduced and B-ring unsaturated steroids due to the lack of available standards.
  • From the findings, the researchers conclude that depending on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares, and may detect androgen conjugates that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation.

Cite This Article

APA
Legacki EL, Scholtz EL, Ball BA, Esteller-Vico A, Stanley SD, Conley AJ. (2019). Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares. Equine Vet J, 51(6), 802-808. https://doi.org/10.1111/evj.13109

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 51
Issue: 6
Pages: 802-808

Researcher Affiliations

Legacki, E L
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Scholtz, E L
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Ball, B A
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.
Esteller-Vico, A
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.
Stanley, S D
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Conley, A J
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.

MeSH Terms

  • Animals
  • Dehydroepiandrosterone / blood
  • Dehydroepiandrosterone / metabolism
  • Estradiol / blood
  • Estradiol / metabolism
  • Estrone / analogs & derivatives
  • Estrone / blood
  • Estrone / metabolism
  • Female
  • Horses / blood
  • Mass Spectrometry / methods
  • Mass Spectrometry / veterinary
  • Pregnancy
  • Pregnancy, Animal / blood

References

This article includes 66 references
  1. Cole HH, Hart GH. The potency of blood serum of mares in progressive stages of pregnancy in effecting the sexual maturity of the immature rat.. Am. J. Physiol. 93, 57-68.
  2. Cole HH, Hart GH. Sex hormones in the blood serum of mares. II The sera of mares from the 222nd day of pregnancy to the first heat period post-partum.. Am. J. Physiol. 94, 597-603.
  3. Cole HH, Saunders FJ. The concentration of gonad-stimulating hormone in blood serum and of oestrin in the urine throughout pregnancy in the mare.. Endocrinology 19, 199-208.
  4. Glen WL, Barber R, McConkey HM, Grant GA. Isolation of beta-dihydroequilin and alpha-dihydroequilenin from the urine of pregnant mares.. Nature 177, 753.
  5. Savard K. The estrogens of the pregnant mare.. Endocrinology 68, 411-416.
  6. Nett TM, Holtan DW, Liné Estergreen V. Plasma estrogens in pregnant and postpartum mares.. J. Anim. Sci. 37, 962-970.
  7. Rance TA, Park BK. The measurement of oestrone, equilin and dehydroepiandrosterone in the peripheral plasma of pregnant pony mares by radioimmunoassay.. J. Steroid Biochem. 9, 1065-1069.
  8. Terqui M, Palmer E. Oestrogen pattern during early pregnancy in the mare.. J. Reprod. Fertil. Suppl. 27, 441-446.
  9. Levy MJ, Boyne MT 2nd, Rogstad S, Skanchy DJ, Jiang X, Geerlof-Vidavsky I. Marketplace analysis of conjugated estrogens: determining the consistently present steroidal content with LC-MS.. AAPS J 17, 1438-1445.
  10. Kindahl H, Knudsen O, Madej A, Edqvist LE. Progesterone, prostaglandin F2alpha, PMSG and oestrone sulphate during early pregnancy in the mare.. J. Reprod. Fertil. Suppl. 32, 353-359.
  11. Hyland JH, Wright PJ, Manning SJ. An investigation of the use of plasma oestrone sulphate concentrations for the diagnosis of pregnancy in mares.. Aust. Vet. J. 61, 123.
  12. Hoffmann B, Gentz F, Failing K. Investigations into the course of progesterone, oestrogen and eCG concentrations during normal and impaired pregnancy in the mare.. Reprod. Domest. Anim. 31, 717-723.
  13. Kasman LH, Hughes JP, Stabenfeldt GH, Starr MD, Lasley BL. Estrone sulfate concentrations as an indicator of fetal demise in horses.. Am. J. Vet. Res. 49, 184-187.
  14. Stabenfeldt GH, Daels PF, Munro CJ, Kindahl H, Hughes JP, Lasley B. An oestrogen conjugate enzyme immunoassay for monitoring pregnancy in the mare: limitations of the assay between days 40 and 70 of gestation.. J. Reprod. Fertil. Suppl. 44, 37-44.
  15. Henderson K, Stevens S, Bailey C, Hall G, Stewart J, Wards R. Comparison of the merits of measuring equine chorionic gonadotrophin (eCG) and blood and faecal concentrations of oestrone sulphate for determining the pregnancy status of miniature horses.. Reprod. Fertil. Dev. 10, 441-444.
  16. Legacki EL, Scholtz EL, Ball BA, Stanley SD, Berger T, Conley AJ. The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry.. Reproduction 151, 421-430.
  17. Bhavnani BR. The saga of the ring B unsaturated equine estrogens.. Endocr. Rev. 9, 396-416.
  18. Tate J, Ward G. Interferences in immunoassay.. Clin. Biochem. Rev. 25, 105-120.
  19. Krasowski MD, Drees D, Morris CS, Maakestad J, Blau JL, Ekins S. Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction.. BMC Clin. Pathol. 14, 1-13.
  20. Wudy SA, Schuler G, Sanchez-Guijo A, Hartmann MF. The art of measuring steroids: principles and practice of current hormonal steroid analysis.. J. Steroid Biochem. Mol. Biol. 179, 88-103.
  21. Scholtz EL, Krishnan S, Ball BA, Corbin CJ, Moeller BC, Stanley SD, McDowell KJ, Hughes AL, McDonnell DP, Conley AJ. Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5alpha-dihydroprogesterone.. Proc. Natl Acad. Sci. USA 111, 3365-3370.
  22. Esteller-Vico A, Ball BA, Troedsson MHT, Squires EL. Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.. Biol. Reprod. 96, 414-423.
  23. Wesson JA, Ginther OJ. Fetal and maternal gonads and gonadotropins in the pony.. Biol. Reprod. 22, 735-743.
  24. Mostl E. The horse feto-placental unit.. Exp. Clin. Endocrinol. 102, 166-168.
  25. Raeside JI. A brief account of the discovery of the fetal/placental unit for estrogen production in equine and human pregnancies: relation to human medicine.. Yale J. Biol. Med. 90, 449-461.
  26. Raeside JI, Liptrap RM, McDonell WN, Milne FJ. A precursor role for DHA in a feto-placental unit for oestrogen formation in the mare.. J. Reprod. Fertil. Suppl. 27, 493-497.
  27. Conley AJ. Review of the reproductive endocrinology of the pregnant and parturient mare.. Theriogenology 86, 355-365.
  28. Legacki EL, Ball BA, Corbin CJ, Loux SC, Scoggin KE, Stanley SD, Conley AJ. Equine fetal adrenal, gonadal and placental steroidogenesis.. Reproduction 54, 445-454.
  29. Raeside JI. Dehydroepiandrosterone in the fetal gonads of the horse.. J. Reprod. Fertil. 46, 423-425.
  30. Raeside JI, Gofton N, Liptrap RM, Milne FJ. Isolation and identification of steroids from gonadal vein blood of the fetal horse.. J. Reprod. Fertil. Suppl. 32, 383-387.
  31. Raeside JI, Liptrap RM, Milne FJ. Relationship of fetal gonads to urinary estrogen excretion by the pregnant mare.. Am. J. Vet. Res. 34, 843-845.
  32. Pashen RL, Allen WR. The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare.. J. Reprod. Fertil. Suppl. 27, 499-509.
  33. Pashen RL, Sheldrick EL, Allen WR, Flint AP. Dehydroepiandrosterone synthesis by the fetal foal and its importance as an oestrogen precursor.. J. Reprod. Fertil. Suppl. 32, 389-397.
  34. Hyland JH, Langstrom B. Changes in urinary and plasma oestrone sulphate concentrations after induction of foetal death in mares at 45 days of gestation.. Aust. Vet. J. 67, 349-351.
  35. Walters KW, Corbin CJ, Anderson GB, Roser JF, Conley AJ. Tissue-specific localization of cytochrome P450 aromatase in the equine embryo by in situ hybridization and immunocytochemistry.. Biol. Reprod. 62, 1141-1145.
  36. Heap RB, Hamon M, Allen WR. Studies on oestrogen synthesis by the preimplantation equine conceptus.. J. Reprod. Fertil. Suppl. 32, 343-352.
  37. Legacki EL, Corbin CJ, Ball BA, Scoggin KE, Stanley SD, Conley AJ. Steroidogenic enzyme activities in the pre- and post-parturient equine placenta.. Reproduction 155, 51-59.
  38. Ainsworth L, Ryan KJ. Steroid hormone transformations by endocrine organs from pregnant mammals. I. Estrogen biosynthesis by mammalian placental preparations in vitro.. Endocrinology 79, 875-883.
  39. Marshall DE, Dumasia MC, Wooding P, Gower DB, Houghton E. Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry.. J. Steroid Biochem. Mol. Biol. 59, 281-296.
  40. Abd-Elnaeim MM, Derar IR, Wilsher S, Allen WR, Leiser R, Schuler G. Immunohistochemical localization of oestrogen receptors alpha and beta, progesterone receptor and aromatase in the equine placenta.. Reprod. Domest. Anim. 44, 312-319.
  41. Daels PF, Albrecht BA, Mohammed HO. Equine chorionic gonadotropin regulates luteal steroidogenesis in pregnant mares.. Biol. Reprod. 59, 1062-1068.
  42. Hoffmann B, Falter K, Vielemeier A, Failing K, Schuler G. Investigations on the activity of bovine placental oestrogen sulfotransferase and -sulfatase from midgestation to parturition.. Exp. Clin. Endocrinol. Diabetes 109, 294-301.
  43. Polei M, Viergutz T, Tomek W, Schuler G, Furbass R. Estrogen-specific sulfotransferase (SULT1E1) in bovine placentomes: inverse levels of mRNA and protein in uninucleated trophoblast cells and trophoblast giant cells.. Biol. Reprod. 91, 48.
  44. Cox JE. Oestrone and equilin in the plasma of the pregnant mare.. J. Reprod. Fertil. Suppl. 23, 463-468.
  45. Raeside JI, Liptrap RM. Patterns of urinary oestrogen excretion in individual pregnant mares.. J. Reprod. Fertil. Suppl. 23, 649-675.
  46. Pashen RL, Allen WR. Maternal and foetal endocrinology during late pregnancy and parturition in the mare.. Equine Vet. J. 16, 233-238.
  47. Allen WR, Wilsher S, Stewart F, Ousey J, Fowden A. The influence of maternal size on placental, fetal and postnatal growth in the horse. II. Endocrinology of pregnancy.. J. Endocrinol. 172, 237-246.
  48. Corvol P, Bardin CW. Species distribution of testosterone-binding globulin.. Biol. Reprod. 8, 277-282.
  49. Silberzahn P, Zwain I, Martin B. Concentration increase of unbound testosterone in plasma of the mare throughout pregnancy.. Endocrinology 115, 416-419.
  50. Canisso IF, Ball BA, Scoggin KE, Squires EL, Williams NM, Troedsson MH. Alpha-fetoprotein is present in the fetal fluids and is increased in plasma of mares with experimentally induced ascending placentitis.. Anim. Reprod. Sci. 154, 48-55.
  51. Conley AJ, Plant TM, Abbott DH, Moeller BC, Stanley SD. Adrenal androgen concentrations increase during infancy in male rhesus macaques (Macaca mulatta).. Am. J. Physiol. Endocrinol. Metab. 301, 1229-1235.
  52. Geyer J, Bakhaus K, Bernhardt R, Blaschka C, Dezhkam Y, Fietz D, Grosser G, Hartmann K, Hartmann MF, Neunzig J, Papadopoulos D, Sanchez-Guijo A, Scheiner-Bobis G, Schuler G, Shihan M, Wrenzycki C, Wudy SA, Bergmann M. The role of sulfated steroid hormones in reproductive processes.. J. Steroid Biochem. Mol. Biol. 172, 207-221.
  53. McLachlan JA, Newbold RR. Estrogens and development.. Environ. Health Perspect. 75, 25-27.
  54. Tong MH, Jiang H, Liu P, Lawson JA, Brass LF, Song WC. Spontaneous fetal loss caused by placental thrombosis in estrogen sulfotransferase-deficient mice.. Nat. Med. 11, 153-159.
  55. Corona G, Elia C, Casetta B, Da Ponte A, Del Pup L, Ottavian E, Toffoli G. Liquid chromatography tandem mass spectrometry assay for fast and sensitive quantification of estrone-sulfate.. Clin. Chim. Acta 411, 574-580.
  56. Tournier M, Pouech C, Quignot N, Lafay F, Wiest L, Lemazurier E, Cren-Olive C, Vulliet E. Determination of endocrine disruptors and endogenic androgens and estrogens in rat serum by high-performance liquid chromatography-tandem mass spectrometry.. Steroids 104, 252-262.
  57. Schuler G, Sanchez-Guijo A, Hartmann MF, Wudy SA. Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS.. J. Steroid Biochem. Mol. Biol. 179, 55-63.
  58. Martins-Junior HA, Pinaffi FL, Simas RC, Tarouco AK, Ferreira CR, Silva LA, Nogueira GP, Meirelles FV, Eberlin MN, Perecin F. Plasma steroid dynamics in late- and near-term naturally and artificially conceived bovine pregnancies as elucidated by multihormone high-resolution LC-MS/MS.. Endocrinology 155, 5011-5023.
  59. Blaschka C, Schuler G, Sanchez-Guijo A, Zimmer B, Feller S, Kotarski F, Wudy SA, Wrenzycki C. Occurrence of sulfonated steroids and ovarian expression of steroid sulfatase and SULT1E1 in cyclic cows.. J. Steroid Biochem. Mol. Biol. 179, 79-87.
  60. Canisso IF, Ball BA, Esteller-Vico A, Williams NM, Squires EL, Troedsson MH. Changes in maternal androgens and oestrogens in mares with experimentally-induced ascending placentitis.. Equine Vet. J. 49, 244-249.
  61. Chavatte PM, Rossdale PD, Tait AD. Modulation of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.. Equine Vet. J. 27, 342-347.
  62. Brooks RV, Klyne W. Steroids of pregnant mare's urine. V. Identification of two androstane-3:16-diols; the structure of uranolone.. Biochem. J. 65, 663-668.
  63. Tait AD, Hodge LC, Allen WR. The biosynthesis of 3beta-hydroxy-5,7-androstadien-17-one by the horse fetal gonad.. FEBS Lett. 182, 107-110.
  64. Tait AD, Santikarn S, Allen WR. Identification of 3beta-hydroxy-5,7-pregnadien-20-one and 3beta-hydroxy-5,7-androstadien-17-one as endogenous steroids in the fetal horse gonad.. J. Endocrinol. 99, 87-92.
  65. Wynn M, Ball BA, Legacki EL, Conley A, Loux S, May J, Esteller-Vico A, Stanley SD, Scoggin K, Squires EL, Troedsson MH. Inhibition of 5alpha-reductase alters pregnane metabolism in the late pregnant mare.. Reproduction 155, 251-258.
  66. Krone N, Hughes BA, Lavery GG, Stewart PM, Arlt W, Shackleton CH. Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS).. J. Steroid Biochem. Mol. Biol. 121, 496-504.

Citations

This article has been cited 6 times.
  1. Arbogast DM, Metrione LC, Jones MK, Donelan EM, Roth TL, Freeman EW, Rispoli LA. Pregnancy- and age-associated variation in serum dehydroepiandrosterone concentrations in black and white rhinoceroses. Conserv Physiol 2026;14(1):coag007.
    doi: 10.1093/conphys/coag007pubmed: 41695197google scholar: lookup
  2. Scoggin KE, Adlan F, Fedorka CE, Rakha SI, Stout TAE, Troedsson MHT, Ali HE. Gestation-Stage Related Changes in the IGF System Components in the Equine Placenta. Biomolecules 2025 Aug 6;15(8).
    doi: 10.3390/biom15081135pubmed: 40867581google scholar: lookup
  3. Gokulakrishnan K, Mohan V, Srikumar BN. Neurosteroid Levels in Pregnancy and Its Implications for Mental Health: A Literature Review. Ann Neurosci 2025 Jul 19;:09727531251351075.
    doi: 10.1177/09727531251351075pubmed: 40693244google scholar: lookup
  4. Scarlet D, Schuler G, Malama E, Bollwein H, Bocci C, Colleoni S, Lazzari G, Galli C, Kowalewski MP. Endocrine profile and OPU-ICSI outcomes in mares: a comparative study. Reprod Fertil 2025 Jul 1;6(3).
    doi: 10.1530/RAF-25-0027pubmed: 40539920google scholar: lookup
  5. Klöppner L, Harps LC, Parr MK. Sample Preparation Techniques for Growth-Promoting Agents in Various Mammalian Specimen Preceding MS-Analytics. Molecules 2024 Jan 9;29(2).
    doi: 10.3390/molecules29020330pubmed: 38257243google scholar: lookup
  6. Gabai G, Mongillo P, Giaretta E, Marinelli L. Do Dehydroepiandrosterone (DHEA) and Its Sulfate (DHEAS) Play a Role in the Stress Response in Domestic Animals?. Front Vet Sci 2020;7:588835.
    doi: 10.3389/fvets.2020.588835pubmed: 33195624google scholar: lookup
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