Determination of conjugated oestrogens in maternal blood plasma

Determination of conjugated oestrogens in maternal blood plasma

G. Schulerand B. Hoffmann
Pferdeheilkunde15 (1999)6 (November/Dezember)
627-629
Determinationof conjugatedoestrogensin maternal
blood plasmaand urinefor pregnancydiagnosisand
monitoringof foetal well-beingin the mare
G. Schu/erand 8. Hoffmann
Klinik
fürGeburtshilfe,
Gynäkologie
undAndrologie
derGroß-undKlentiere
mlttierärztlicher
Ambulanz,
nivers
JustusLiebig-U
tätGressen
Summary
Dueto the distinctdifferences
of oestrogen
concentrations
in pregnant
and nonpregnantanimalsthe radioimmunological
determination
of
conjugated
oestrogens
in maternalbloodplasmaor serumjs a highJy
reliable
methodfor pregnancy
in maresduringthe interval
diagnosis
production
corresponding
to the phaseof placental
oestrogen
As
betweenarounddays70 80 aftermating/insemination
and parturition.
placental
providedby thefoetalgonads,anylossof foetalvitalityis strictlyassociated
oestrogen
biosynthesis
witha
dependson precursors
placentaloestrogenproduction.
decreased
Therefore,
oestrogen
of foetalwellconcentrations
are a usefulparameter
for the monitoring
production
beingin casesof unclearsymptomsof impairedpregnancy.
Priorto the onsetof placental
oestrogen
determination
of ovarian
pregnancy
estronesulfatecan alsobe applied(> day40).However,
diagnosis
maybe problematic
valuesbedue to slightlyoverlapping
tween1 ng/mlto 2.5 ng/mllikewise
occurring
in maresat oestrusanda minorfractionof pregnant
mares.Thedetermination
of urinaryconcanbe usedas a non-invasive
alternative
to the measurement
in bloodplasmawithinthe interval
to the
correspondlng
lugatedoestrogens
phaseof placental
(> day80).Prelimlnary
productionbetweendays40
oestrogenproduction
resultsalsosuggestthat ovarianoestrogen
and 80 aftermating(insemination)
maybe monitored
for pregnancy
diagnosis,
but the preliminary
cut-offlevelat 175 ng/mlneedsfurther
confirmation.
Keywords:
horse,pregnancydiagnosis,oestrogens,blood,urine
Messung konjugierter Blut- und Harnöstrogene zur Graviditätsdiagnoseund Überwachung der Fetusvitalität beim Pferd
Die kächtigkeitsspezifische
Ostrogenproduktion
der Stute kann entsprechendder Ostrogenquellenin zwei Phaseneingeteiltwerden. In der
ersten Phasefindet sich im peripherenmaternalenPlasmanach einem erstenAnstieg um den 40. Graviditätstageine leichteErhöhungder
Estronsulfatkonzentrationen
von Basalniveau(< 0,5 nglml)auf 1,5-10 ng/ml bis zum ca. 70.-80. Graviditätstag.Als Ostrogenquellefungiert
hierbeidas Ovar. In der folgendenplazentarenPhase kommt es im maternalenBlutplasmazu einem steilenAnstieg der Konzentrationen
konjugierterOstrogeneauf Maximalwerteum 700 ng/ml in der Graviditätsmitte,
gefolgtvon einemallmählichenAbfaltauf Werte um 50-100
ng/ml unmittelbarvor der Geburt. Unter der Geburtfallendie Ostrogenkonzentrationen
weiter steil auf Basalnjveauab. Aufgrundder außerordentlichhohen Ostrogenkonzentrationen
in der plazentarenPhase stellt die radioimmunologische
BestimmungkonjugierterOstrogenein
Blutplasmaoder -serumab dem 80. Tag nach der Bedeckung/Besamung
eine äußerstzuverlässigeN4ethode
zur hormonellenTrächtgkeitsdiagnosedar. Da die Präkursorender plazentarenOstrogeneden fetalenGonadenentstammen,ist eine Beeinträchtigungder Vitalitätdes
Fohlensstets mit einem Abfallder plazentarenÖstrogeneverbunden.Dahereignetsich die Östrogenbestimmung
auch zur lrächtigkeitsü
berwachungin FällenunklarerSymptomeeinesdrohendenAbortes.Auch zwischendem 40.-80. Graviditätstagist in den allermeistenFällen
eine Trächtigkeitsdiagnose
anhand der BestimmungkonjugierterOstrogenemöglich.Schwierigkeitenkönnen sich hier in einzelnenFälen
durch die geringfügigüberlappendenl.4eRwerte
rossigerbzw. frühgraviderStuten im Bereichzwischen1,0 und 2,5 ng/ml ergeben.Neben
der Bestimmungim Blutplasmakann in dem der plazentarenPhaseentsprechendenZeitraumauch die lvlessungkonjugierterÖstrogeneim
Urin als nicfrtinvasive
alternativeMethodeangewandtwerden. VorläufigeErgebnissedeuten an, daß auch in der der ovariellenPhase entsprechendenZeitspanneOag 40 80) anhandder Ostrogenkonzentrationen
im UrineineAussagehinsichtlichdes BestehenseinerGravidität
möglichist. Der vorläufigeGrenzwertvon 175 ng/ml bedarfjedocheinerweiterenBestätigungdurch einegrößereAnzahLvonTieren.
Schlüsselwörter:
Pferd,Trächtigkeitsdiagnose,
Ostrogene,Blut, Urin
Introduction
Due to the limitedbreedingseasonan accuratepregnancy
diagnosisas soon as possibleafterthe last matingor inseminationis of outstandingimportancefor horse breeders.
Ultrasonography-aided
rectal palpation has become the
standardmethod which allowspreciseand earlyconfirmation of singletonand twin pregnanciesfrom day 1B onward
concomitantwith the orovisionof informationabout the viability of the foetus (McKinnonet al., 1993; Shideler, 1993).
However,in spite of this situationthere is stillroom for hormonal pregnancydiagnosiswhich may be even more advantageousin ceftainsituationssuch as pregnancydiagno'15
Pferdeheilkunde
sis in vicious mares or if the investigationmust be performed under otherwise unsuitableconditions, in small
racesand donkeysand in animalswith injuredrectum,Furthermore,hormonalpregnancydiagnosiscan be used to
verifythe diagnosisin cases of uncertainrectal diagnosis,
especiallyat later stages of pregnancywhen the enlarged
uterusis not sufficiently
accessibleto examination.Hormonal pregnancydiagnosismay also be an alternativefor less
experienced, non-specialised practitioners in doubtful
cases. Moreover,there is an increasinginterestof horse
ownersin non-invasive
and thereforemore economicalme-
627
Determinatton
of conjugatedoestrogensin maternalbloodplasmaand urinefor pregnancydiagnosis
thods. Non-invasivemethods are also of soecialinterestin
wild equids or zoo animals.Severalmethods of hormonal
pregnancydiagnosisin horsesare availablesuch as progesterone measurementin blood at the time of the next oestrus in case of unsuccessfulbreeding,the detection of
equine chorionicgonadotrophinin blood (Hoffmannet al.,
1996),the measurementof pregnancy-associated
oestrogens in blood, urine (Schuler,1998)or faeces (Möstl et al.,
1983; Palme et al., 19Bg) or the determinationof faecal
concentrationsof pregnancy-specificgestagens (Schwarzenbergeret al., 19Bg).Inour laboratory,hormonalpregnancy diagnosisis routinelyperformedby radioimmunological
determinationof total estrone in blood plasma or serum,
- in urine.
and - as a non-invasive
alternative
In the mare largeamountsof oestrogensare producedduring pregnancywith sulfoconjugatedforms dominatingby
far over free ones (Hoffmannet al., 1996).Equinepregnancy associated oestrogen production can be divided into
two phases accordingto the source of oestrogens.A first
rise of ovarianoriginfrom basal levelsbelow 0.5 ng/ml up
to plasmaconcentrationsbetween 1 .5-10 ng/ml commences around day 40 of gestation (Daelset al., 1990; Hoffmann et al., 1996),followedby a plateauuntil around day
BO,when placentaloestrogenproductronincreasessharply
providingplasma concentrationsup to values around 700
ng/ml at midgestation,Thereafteroestrogenconcentrations
reachingvaluesof about 100 ng/ml in
declineprogressively
the last week of gestation.The final drop occurs immediately prior to parturitionand baselinelevelsare reached1-2
days post partum (Hoffmannet al., 1996).Since in the horse
oestrogensare predominantlyexcretedvia the urine (Palme
et al., 7996),the urinaryoestrogenprofileis basicallyidenti ca l to t he one in blo o d o l a s m a b u t o n a 1 0 0 -1 0 00-fol d
higherlevel(Evanset al., 1984; Monfort et al., 1991).
tion and adjustmentto RIA conditionswere achievedby the
additionof 20 pl 0.8 N HCI and 20 prlconcentratedphosphate buffer (8.356 g NarHPOo,2.686 g KH2PO4,and
0.325 g NaN.).Dependingon the stage of gestationthe redissolvedsamoles were then further diluted orior to RlA.
W hen taki ng1.0 ml as a starti ngposi ti onthe fi naldilut ions
vari ed betw een 1:5 and 1:1000. In order to obtai n exact
values in case of low oestrogenconcentrations,the 1:5dilution was measured of each sample, irrespectiveof the
assumed stage of gestation.Paired dilutionsof 1:5 and
1:5OOwere chosen for samples provided without any
anamnesticinformation.The antiserumused was directed
againstestrone(Hoffmannet al., 1994)andexhibitedthe followingcross reactions:estrone:1OOyo,
equilenine23.83o/o,
equi l i ne: 7 .21o/o,estradi ol -17B :1.94o/o,estradiol- 17a:
1.O2yo,estriol:0.06%, Concentrationsof estrone equivalentswere calculatedby comparisonwith a standardcurve
consistingof eight standardsbetween 20 fmol and 3200
fmol estrone/tube.Intra- and interassaycoefficientsof variationvaried between 9.4% and 12.5%. Classificationof
estroneconcentrationsas indicativefor pregnancywas based on the data of Hoffmannet al. fi99d.
Determinationof pregnancy-specificoestrogens in urine
The procedurefor the measurementof pregnancyassociated oestrogensin urine was identicalto the method described above for the determinationin blood olasma and
serum. Due to the considerablyhigher oestrogenconcentrati onsi n uri ne,di l uti onsof 1: 500 and 1:50000w er e chosen for all urinesamoles.
Results and discussion
Due to the distinct differencesof oestrogen concentrations
in pregnantand non-pregnantmares the determinationof
conjugatedoestrogensin maternalblood plasmais a highly
Determination of pregnancy-specific oestrogens in blood
reliablemethod for pregnancydiagnosisduring the phase
pnsma or serum
of placentaloestrogenproductionlastingfrom around day
(RlA)used for the determinationof
The radioimmunoassay
70-80 of gestationuntil parturition.As placentaloestrogen
pregnancyassociatedoestrogenswas adopted from Gentz
biosynthesisdependson precursorsprovidedby the foetal
(1994)and modifiedfor routineperformance.Since in the
gonads (reviewedby Möstl, 1994),any loss of foetal vitality
mare sulfoconjugatedforms dominate by far over the free
strictlycoincideswith a decreasedplacentaloestrogenproones, no separate estimationof free and conjugatedoestduction (Kasmanet al., 19BB;Hoffmannet al., 1996).Thus,
rogens was intended and they were determinedas total
oestrogen concentrationsare a useful parameter for the
estrone equivalentsafter hydrolysisof the conjugates.For
monitoringof foetal well-beingin cases of unclear symphydrolysisof conjugatedoestrogens,160 pl 625 mM acetoms and suspected impaired pregnancies.Accordingly,
tate buffer pH 4.8 and 50 pl dilutedB-glucuronidase-aryl- with the exceptionof one case, all maresexhibitingestrone
sulfatase from helix pomatia (Serva Feinbiochemika concentrationsclearly below the respective normal range
Gmb H& Co,D- 69155 H e i d e l b e rgd; i l u te d 1 :2 5 i n 0 .15 M
aborted within one to five days. The exceptionrefersto one
NaCl),were added to plasma or serum samples (0.a ml).
mare which gave birthto an abnormallysmallbut viablefoal
After an overnightincubationat 37'C oestrogenswere exafter a prolongedgestationof 420 days. There subnormal
tracted with toluene.To ensureredissolution
of the extracconcentrationsof estrone equivalentsaround 3-6 ng/ml
ted oestrogens,160 pl 0.1 N NaOH with 0,1%oraI serumal(normalrange:50-200 ng/ml)were observedin the lasttwo
bumin were added to the evaporatedsampleswhich were
weeks of gestation.On the other hand, mares with threathen incubatedfor 20 min at room temoerature.Neutralisa- tened aborlionsshowinq normal oestrooenconcentrations
Material and methods
628
P ferdehei l k unde15
G. Schulerand B. Hoffmann
maintainedpregnancythroughoutthe time of observationin
the clinic,Apaft from subnormaloestrogenconcentrations
also extraor{rnarilyhigh values can be indicativefor impaired gestation(Hoffmannet al., 1996),Furthermore,twin
pregnanciesare associatedwith oestrogenlevelsin the upper range.
Priorto the onset of placentaloestrogenproductiondeterminationof estronesulfateof ovarianorigincan also be applied, However,pregnancydiagnosismay be problematic
due to slightlyoverlappingvalues between 1 to 2.5 ng/ml
likewise occurring in mares at oestrus (Makawiti et al.,
1983; Koskinenet al., 19Bg)and a minor fractionof eCGpositivemares (Schuler, 1998). However, since the detection of eCG and not the positiveoutcome of gestationwas
used to classifyanimalsas pregnant,no informationis available if pregnancywas still intact at the time of testing as
eCG productioncan persistfor a relativelylong time after
foetal death (Hoffmannet al,, 1996). Hence the ,,true"cutoff level might be somewhat higher.This is indicatedby
preliminaryresultsfrom the additionalprogesteronedeterminationin cases of doubtfuloestrogenconcentrations;
all
samplestested for progesteronebecauseof oestrogenlevels within the overlappingrange exhibitedconcentrations
whichwere not consistentwith earlypregnancy(< 1.5 ng/ml;
Hoffmannet al., 1996).
The same radioimmunological
method is applicablefor the
determinationof urinaryoestrogenswhich allows the replacement of the overcome chemical assays of Cuboni
(1934)or Rommel (1964).Due to the extremelyhigh concentrationsexceeding56 pg/ml at midgestationthe determinationof urinaryoestrogenlevelsis a very reliablemepregnancydiagnosis.
thod for non-invasive
Firstresultsof urinaryoestrogendetermination
also suggest
that ovarianoestrogenproduction between days 50 and BO
may be monitoredfor pregnancy
aftermating(insemination)
preliminary
however,
diagnosis;
the
cut-off level at 175
ng/ml needsfurtherconfirmation.
ring normaland impairedpregnancyin the mare. Reprod.Dom.
A n i m .3 1 , 7 1 7 - 7 2 3
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P f erdeh e i l k u n d e1 5
G. Schuler
B. Hoffmann
Klinikfür Geburtshilfe
Gynäkologieund Andrologie
der Groß- und Kleintieremit tierärztlicherAmbulanz
J ustus -Liebig - Universitäl Giessen
FrankfurterStraße 106
35392 Gießen
Tel.:06 41 - 993 87 04
Fax:0641 - 993 87 09
629