Pferdeheilkunde15 (1999)6 (November/Dezember)
614-617
Transvaginalultrasound-guidedreductionof twin
conceptusesin the mare
LeeH.A. Morrisl,R.E.S.Greenwood2
and W.R.Allenl
1University
of Carnbddge,
Department
Veterinary
N.4edicine,
of Clinical
Equine
Fertility
Unit,Mertoun
Paddocks,
Cambridge
, Reynolds
House,
HighStreet,
Newrnarket,
Suffolk
Summary
It is nowpossible
to treatmarescarrying
twinconceptuses
betlveendays35 and55 of gestation
by transvaginal
ultrasound-guided
aspiration of the allantoic
fluidof oneof the conceptuses.
In thisreviewof sixmarestreatedin thismanner,
two successfully
carrieda singleton
to
term,onemarecontinued
to carrylivetwinsto term,anothorsubsequently
lostthe remaining
singleton
conceptus
after100daysof gestatiprocedure.
on andtwo lost bothconceptuses
within3 daysof the aspiration
Theseresuitsrevealthe potentialvalueof the techniquebut
highlights
the needfor furtherresearch
to determine
the optimumstageof gestation
at whichto carryit out in orderto maximize
the chan
cesof survival
of the remainino
fetus.
Keywords:
twin,transvaginal
ultrasound,mare,allantois,aspiration
Transvaginale, ultrasonographiegeführteRedulition von Zwillingsanlagen bei der Stute
Es ist möglich geworden,Zwillingsanlagen
bei der Stute zwischenTrächtigkeitstag35 und 55 mittels transvaginaler,ultrasonographiegestützter selektiverAspirationvon Ailantoisflüssigkeit
auf einen Einlingzu reduzieren.In der vorliegendenuberslcht von sechs Stuten, die in
dieserWeise behandeltwurden, trugen zwei Stuten einen Einlingbis zum normalenGeburtsterminaus, eine Stute trug weiterhinZwillinge
aus, eine weitereStute verloreinen Einlingnach ca 100 Tagen Gravidität.Zwd Stuten verlorenbeide Fruchtanlageninnerhalbvon 3 Tagen
nach der Asplration.Die Untersuchungsergebnisse
belegengrunds.itzlichdie Brauchbarkeitder Methode,sie zeigenaber auch deutlichauf,
dass weitererForschungsbedarf
auf diesemGebietbesteht.,nsbesonderedürftedie Terminierungdes Trächtigkeitszeitpunktes
zu definieren
sein, der optimalfür die Aspirationist und die Uberlebenschancen
des verbliebenenEinlingswesentlicherhöht.
Schlüsselwörter: Zwillinge,transvaginaleUltraschalluntersuchung,
Stute,Allantois,Aspiration
Introduction
Sincethe adventof transrectalultrasoundscanningfor early accuratediagnosisof pregnancyin the mare in the early
19BO's (Palmer and Driancourt, 1980; Simpson et al.,
1982) the overall twin conception rate in Thoroughbred
mares in the United Kingdom has risen from 2.2o/oin 1983
(Sandersonand Allen, 1987)to around 7.2% in 1998 (authors, unpublisheddata), This increaseis almost certainly
due to a combinationof the increasedaccuracyof diagnosis of twins using modern high resolutionscannerswith 5,0
and 7.5 MHz transducersand the high rate of success
(> 95%) when manuallycrushingone conceptusper rectum
before day 20 of gestation;increasedsurvivalof femaleembryos to term will inevitablyhasten the spread of twinning
geneswithinthe population,
Of 95 twin conceptionsdiagnosedbetween days 13 and
22 of gestationin Thoroughbredmares in the Newmarket
regionof Englandduringthe 1998 breedingseason,B were
not treated.The twin conceotusesin 4 of these mares had
reducedspontaneouslyto a singleton,and both conceptuses had disappearedin the other 4 mares, by the time of
their subsequentpregnancyexaminations(authors,unpublisheddata).Pascoe (1983)noted that only some 13o/oof
mares left carrying untreated twin conceptuses gave birth
to a single livefoal and those that aborted twins sufferedan
increasedrisk of dystocia and reduced fertilityin the follo-
614
wing breeding season. Furthermore,twin foals that do
surviveto term are usuallyundersizedand they show increased perinatal mortality (Jeffcott and Whitwell, 1973;
Merkt et al., 1982; Pascoe, 1983). The study by Jeffcott
and Whitwell(7973) indicatedIhat 22% of the spontaneous
abortionsin Thoroughbredmaresin the U.K. duringthe period between 1967 and 1970 were caused by twinning
whereas nowadays, with early ultrasound scanning and
successfulcrushingof one conceptus,the equivalentfigure
has fallen Io 7o/o(4, Blunden, personal communication).
Nonetheless,undiagnosedtwin pregnanciesstill represent
an economicloss in time and fedilitywithin the Thoroughbred breedingindustry,especiallyin those mares in which
persistenceof the eCG-secretingfetal endometrialcups in
the maternal endometriumafter removal of the twins suppresses oestrus and ovulation for many months (Allen et
al., 1973).
Treatment options for managing undiagnosed twins
beyondday 35 of gestationhave includedserialdailyinjections of prostaglandinF, analoguesto cause lutoelysisand
abortion(Daelset al. 1989/,attemptedmanualcrushingof
one conceptus (Roberis, 1982), surgical removal of one
conceptus (Pascoe and Stover, 19Bg), blind transvaginal
aspirationof allantoic fluid from one conceptus (Pascoe,
1979),dietary restrictionof the mare (Merkt et al., 1982)or
Pferdeheilkunde
15
LeeH.A.MorriseI al.
transabdominalultrasound-guided
injectionof concentrated
KCI into the heart (Rantanenand Kincaid, 19BB)or intrathoracic injectionof procainepenicillin(McKinnonand Rantanen, 1998),after day 100 of gestation.A transvaginalultrasound-guidedtechnique for aspiratingfetal fluids from
singletonor twin conceptusesbetweendays 20 and 65 of
gestation has been described by Bracher et al. (1993),
Macpherson et al. (1995) and Squr,reset al. (1994)and this
paper discussesthe use of this transvaginalapproach to
treat 6 cases of twins between davs 35 and 55.
Materials and methods
The procedurewas carriedout on 6 mares carryingtwins,
one at day 35, two at day 45, one at day 48, one at day 50
and one at day 55, of gestation.In addition,6 experimental
mares carryingsingletonconceptusesunderwenttransvaginal ultrasound-guided
aspirationof allantoicfluid on day
32 of gestationfollowed immediatelyby videoendoscopic
examinationof the ounctured conceotus in the uterus as
describedby Bracherand Allen (1992).
With the mare restrainedin stocks,the sizesand locationof
the twin conceptuseswithin the uteruswas determinedby
transrectal linear arruy ultrasonographyand the choice
made as to which would be easiestto grasp and penetrate
with the needle,Around 20 ml of localanaesthetic(2% LignocaineHydrochloride;
C-VetVeterinaryProducts,Leyland,
UK) was infusedinto the mare's rectumto achieverelaxation for improvedmanioulationof the uterus.A nose twitch
was appliedto the mare for additionalrestraintif and when
necessarybut pharmacological
sedationwas avoidedin order to maintaingood uterinetone. The 6.5 MHz curvilinear
ultrasoundprobe (Dynamiclmaging Ltd, Lothian,UK) was
envelopedin a plasticrectalglovewith the fingersremoved,
lubricatedwith sterilegel (K-Y jelly;Johnson and Johnson
Ltd, Maidenhead,UK) and guided into the vagina using a
sterilegloved hand. The operator's hand was then withdrawn from the vaginaand insertedinto the rectumin order
to pull the gravidhorn backwardsinto the pelviccavityand
hold it there. Once the uterus was fixed in a suitableposition,the ultrasoundprobe was advancedto make contact
with the cranialwall of the vaginaand the conceptusselected for eliminationwas identifiedon the ultrasoundmonitor.
A 60 cm, 16 gauge, single lumen needle(Cook Veterinary
Products,Brisbane,Australia)was insertedinto the needle
guide attachedto the ultrasoundprobe by a second operator. The needle was advanced in the guide until, with a
sharp definedthrust,it piercedthe vaginaland uterinewalls
and the conceptusmembranes.The progressof the needle
was visibleon the ultrasoundmonitorand when it entered
the allantoiccavity between 60 and 180 ml of clear fluid
was aspirated using a 60 ml syringe.Subsequently,the
needlewas retractedand the orobe withdrawnfrom the vag rn a .
In one mare, which underwentthe procedureon day 48 of
gestation,the orallyactiveprogestagen,altrenogest(Regumate; Hoechst RousselVet, Bucks, UK) and the prostaPferdeheilkunde
15
gl andi nsynthetasei nhi bi tor,fl uni xi nmegl umi ne(Finadyne;
ScheringPlough, Middlesex,UK) were administeredonce
dailyfrom one day before,to three days after,the manipul ati on.
Results
The resultsof the transvaginal
aspirationattemptsare summarized in Table 1. Only two of the 6 treated mares (33%o)
producedlivesingletonfoalsat term. One other mare,aspirated at day 35 of gestation,carried a singletonconceptus
until at least day 100, but thereafterunderwentan unobserved spontaneousabortion. Another mare, treated on
day 45 of gestation,subsequentlycarried both conceptuses to term and the continuationof the twins was not recognised by the referringveterinarysurgeon when carrying
out 6 once-weeklypost-treatmentultrasoundexaminations.
Both foals were born live from a spontaneousparturition
but the smallerof the two survivedfor only 48 hours, In the
two mares,treated on days 45 and 48 days of gestationrespectively,both conceptuses had died within 3 days after
the aspirationprocedureand the mares were given serial
daily injectionsof the prostaglandinFr. analogue,cloprostenol (Estrumate,Schering-Plough,
Middlesex,UK) to induce luteolysisand expulsionof the fetal remnants.There
was no evidenceof intrauterineinfectionassociatedwith
eitherof thesefailures.
Hysteroscopicexaminationof the 6 mares that underwent
transvaginalaspirationof allantoicfluid from a singleton
conceptusrevealeda singleentrancehole in eitherthe chorioallantoicor choriovitellinemembranes.The conceotus
had collapsed and there was evidence of haemorrhage
from chorionic blood vessels. However, there were no
obvious accumulationsof allantoicfluid within the uterine
l umen.
The placentawas obtainedfrom the 17 y.o. maiden mare
carryingtwins that underwentallantoicaspirationon day 55
of gestation and carried the remainingsingleton foal to
term. The olacentawas oassed intactwithin 10 minutesafter birth of the foal, which was healthyand vigorousand
Tab.1: Transvaginal
ultrasound-guided
aspirationof allantoic
fluidfromoneof twinconceptuses
in the mare
Transvag
inale,ultraschallgeführte
AspirationderAllantoisflüssigkeit
einerFruchteinerZwillingsträchtigkeit
Mare Stageof gestation Distributionof the Numberof foals
number
at time of
:onceptuseswithin
born alive
aspiration
the uterus
(days)
1
;
z
;
)
5
;O
);
4,5
4C
4,g
;;
cc
Unilateral
n
üÄiiäiäiär
äiiäiäiäi
O
:.;
.
öilaIeral
äiiäiä'äl
:,,
öilaIeral
I
;
;
\,,
z
1
615
Transvaginal
ultrasound-guided
reduction
of twinconceptuses
inthemare
had an estimatedbodyweightof 40 kg. The allantochorion
weighed 3.02 kg and it exhibiteda star shaped avillous
area near the junction between the body and the gravid
horn regionsof the placenta,representingthe area of appositionof the two placentasat the time of aspiration(Figu re 1 a ) .A m um m if iedc o n c e p tu sme a s u ri n g2 .5 x 1 .5 cm
was attachedto this avillousregionby a fibrouspedicleand
directly opposite it a large pedunculatedallantochorionic
pouch protrudedinto the allantoiccavity(Figure1b).
endometrialcups. However,for a variety of reasons this
does not always occur and a few pairs of twins persist
beyond the crucialstage of day 34-36 after ovulationwhen
the progenitorchorionicgirdle invadesthe maternalendometriumand the endometrialcuo secretionis set in motion
(Allenet al., 1973).The present use of transvaginalultrasound-guidedaspirationto eliminateone of the twin conceptuses after day 35 offers an alternativeto aborting both
conceptusesand trying,usuallyin vain,to inducethe mare
to returnto oestrusso she can be mated again.
The techniqueis very dependentupon adherenceto strict
hygiene to avoid introducingbacteria into the progesterone-dominated,and hence very susceptible,uterus. The
speed and ease of the manipulationmay also influencethe
results.Technically,the uterus is easier to grasp and fix
withinthe pelvisbeforeit beginsto extend beyond the pelvic bnm aroundday 40. However,the viabilityof the remaining conceptusmay be improvedif the aspirationis carried
out at laterstagesof gestation(i.e.> day 50) when the developingchorionicvilli have achieveda more definiteand
stable interdigitationwith the endometrialepithelium,but
the graviduterusis stillnot too pendulousto manipulateinto the oel vi s.
Success of the techniqueis obviouslythe productionof a
singleviablefoal at term. Examinationof the placentaand
foal from one of the survivorsin this study showed both to
be at the lower end of the normal range for an aged, maiden
Thoroughbred mare (Sandra Wilsherand W.R. Allen,
Fig. 1a: Mummifiedfetus attachedto the chorionof the surviving
unpublisheddata).lt is also clearthat two extremesof failure
twin
are possible.ln one case, the productionof twins at term
Am Chorion des überlebendenZwillingspartners
haftenrevealedthe resilienceof the conceptusesto manipulation
der mumifizierlterFetus
and localizedtrauma,which can be assumedto have been
induced by the techniquebased upon hysteroscopicobservationsmade in this study.On the other hand,the risk of
losing both conceptusesalso remainsquite high. In both
cases in this study the two conceptusesfailedwithin2 days
of the orocedure,which is consistentwith the resultsof the
earlierstudies on transvaginalultrasound-guidedallantoic
aspiration of singleton pregnancies (Macpherson et al.,
7995; Squires et al., 7994). Bracher et al. (1993) defined
successas the presenceof viablesingletonat 10 days after
the aspirationof the co-twrnand they thereforefailedto account for possiblelossesat laterstagesof gestation.However, in one such case in the presentstudy it was not possibleto determineif the abortionwas due to the asoiration
orocedureundertakensome weeks oreviouslvor was from
an unrelatedcause.
The absenceof significantaccumulationsof fluid in the uterine lumentwo days afterthe procedurein our 6 clinicalcases of twins and the 6 experimentalcases of singletons,
pouch apposingthe mummifiedfetus
Fig. 1b: Allantochorionic
differsfrom the resultsof the previousstudies performedon
Mumifizierter
Fetuseinqeoasst
in eineAllantochoriontasche singleton
conceptuses(Macphersonet al., 1995;Squireset
al., 1994)and the study performedon twins (Bracheret al.,
Di scu ss ion
1993). The former authors administeredprogestagento
their asoiratedmares and demonstratedlower concentratildeally,twin conceptuses are nowadays diagnosed and
ons of prostaglandinmetabolitesin the blood of the mares
treated successfullywithin 13-17 days after ovulationand
that retaineda viablefetus comoaredto those in which the
well before the formation of the gonadotrophin-secreting fetus died. Squireset al. (1994)noted that, despitean initial
616
Pferdeheilkunde
15
Lee H.A. Morns eI al.
fall in serum progesterone concentrations 2-4 days after
the procedure, the corpus luteum did not regress completely and progesterone levels rose again. lt is most likely that
the eCG secreted at this later stage of gestation helps to
maintain and protect the corpora lutea from the potentially
luteolytic actions of any prostaglandin released as a consequence of the aspiration procedure.
At this preliminary stage in the development and application
of the technique, it is not possible to draw conclusions regarding any effects of the treatment on the birthsize and
growth rate of the surviving foal. However, with further refinement of the method and investigation into the optimum
stage of gestation at which to carry out aspiration of the allantois, the technique may hopefully become a reliable method of reducing twin conceptuses to a viable singleton in
the mare.
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Acknowledgements
Two of the aspirationswere performedat the Universityof Guelph,
Canadain associationwith Drs WalterJohnsonand ClairePlanteand
one was pedormed in associationwith Mr Andrew McGladderyof
Rossdaleand Partners,Veterinary
Surgeonsin Newmarket.
Lee H.A. Morris
Universityof Cambridge
Departmentof ClinicalVeterinaryMedicine
EquineFertilttyUnrt
Merloun Paddocks
Wooditton Rd
Newmarket
Suffolk,CBB9BH
U.K.
tel.:0044- 1638 - 66 24 91
fax:0044- 16 38 - 66 72 07
GreenwoodEllisand partners
ReynoldsHouse
High Street,Newmarket
Suffolk CBB gAH
UK
617