Department
of Obstetrics andGynecology,
Ohio StateUniversity,
Columbus, Ohio,
USAFERTILITY
CONTROL
THROUGH ACTIVE IMMUNIZATION
USING PLACENTA PROTEINS
By
Vernon C. Stevens
ABSTRACT
Numerous studies have demonstrated that antibodies to
placental proteins
in avariety
ofspecies
arecapable
ofpreventing
ordisrupting gestation.
Early
work in this area wasprimarily
directed towards thepassive
im-munization of rodents with
heterologous
antisera to wholeplacental
ex-tracts.Toxicity
and renaldamage
oftenaccompanied fertility
inhibition. More recent studiesreported
lesstoxicity
and ahigher
specificity
ofantibodies to
reproductive
function whenanti-placental
antibodies wereabsorbed withserumand extracts of
non-reproductive
organs.Few studieshave been
reported
in which active immunization withplacental proteins
was
employed.
The most detailed studies of active immunization haveemployed highly purified placental
hormones. Immunizations of rats and rabbits with humanplacental
lactogen
have resultedin marked reduction inreproductive
function. Immunization ofhuman females withchemically
altered(hapten-coupled)
HCG resulted in theproduction
of antibodiesreacting
with unaltered HCG andpituitary
LH. These antibodies werecapable
ofreducing
the level ofendogenous
serum LH in pre- andpost-menopausal
women.They
also altered the events of the menstrualcycle
in
premenopausal
women. Morespecific
inhibition of chorionicgonado-trophin
has been obtainedby
immunization of baboons with thebeta sub-unit of HCG.Antifertility
effects without alterations in the menstrualcycle
of female baboons immunized with the beta subunit of HCG have beenreported.
The antibodiesproduced
in these animals reactedsigni-ficantly
with human LH in vitro. Thepossibility
ofusing
hormonal and non-hormonalplacental proteins
asantigens
for thespecific
Antibodies
raised toplacental proteins
have been shown todisrupt
pregnancy inlaboratory
animals in several studies. Fewreports
have demonstratedthat
the action
of
such antibodies werespecific
to theproducts
of
conception
anddid not react to
antigens
of
non-reproductive
organs.Further,
noapplication
of
passive
immunizationprocedures
has beensuggested
for
application
tohuman
fertility
control because ofproblems
associated withsensitivity
reactions torepeated injections
of
foreign
serumproteins.
In recent years, progresshas
been made toward the isolation of
highly purified
proteins
fromplacental
tissue andpreliminary experiments
have been conductedusing
active im¬ munizationtechniques
toprevent
ordisrupt gestation.
These
latterexperiments
have
provided
data
which now indicate that a method ofhuman
fertility
control maybe
developed by
immunization withplacental proteins.
Although
at our current stateof
knowledge
there is noopportunity
toapply
passive
immunizationmethods
tohuman
fertility
control,
many data sug¬gesting
feasibility
of
methods and mechanisms of action have been obtained inpassive
immunization studies. In order toprovide background
information,
some
of
the moresignificant
studies in this area will be reviewed inthis
report.
Passive immunization studies
Disruption
of pregnancyby
antisera toplacental
antigens
wasdemonstrated
early
in thiscentury
(Dobrowski
1903);
however,
few
further studies werereported
untilmuch
later.Antisera
towhole
ratplacental
extracts weread¬
ministered to normal
pregnant
rats ondays
11 and 12of
gestation
(Seegal
Se Loeb1940).
These
workersfound
that the treatmentproduced
résorption
of
66 per cent of
foetuses; however,
equal
orgreater
damage
to pregnancy couldbe
produced by
injections
of
antisera to ratblood.
Therefore,
noconclusions
could be reached on the basis
of this
study regarding specific
action of theantibodies.
Later,
the sameauthors
(Loeb
et al.1949)
demonstrated that abortion was induced in 100 per centof
pregnant
ratsinjected
with antiserum to ratplacenta together
withdeoxycorticosterone
acetate.Although
theanti-serum
used
inthese
latterexperiments
was moreeffective
inproducing
abortion,
alltreated
rats showed renalhypertrophy
andnephritis.
Anotherreport
(Cohen
SeNedzel
1940)
described theabortion-inducing
actionof
anti-sera to
guinea pig
placenta
when administered toguinea pigs.
Although
anti¬bodies
to several organs werepresent
inthe
antisera,
in vitroabsorption
re¬moved
non-placenta specific
antibodies andfive of
sixpregnant
animals abortedafter
theadministration of
absorbed antiserum.Similar studies
as above were conducted in mice(Koren
et al.1968).
An
antiserum raisedto
whole
mouseplacentae
inrabbits
wasinjected
intopregnant
and liver
damage
was observed in treated animals andapproximately
10 percentdied
following
immunization. A control groupof animalsreceiving
normal rabbit serum did not suffer total loss ofpregnancies
and renal lesions werenot correlated to the
disruption
of
pregnancy in the treated animals. These studiessuggested
that at least someof
theabortions
wereproduced
by specific
antibodies to
placentae.
Another
study using
rabbit anti-mouseplacenta
serum in mice showedabortion and
toxicity despite prior
absorption
ofthe
antisera(Nemirovsky
1970).
However,
in 1971 Behrman andco-workers
reported
studies of passive immunizationof
mice with rabbit anti-mouseplacenta
serum inwhich
muchless
toxicity
wasobserved(Kometani
8c Behrman1971).
Antisera wereabsorbed
repeatedly
with normal mouse serum andtoxicity
levels
were establishedprior
to the
antifertility
studies. Abortion could beproduced
in all animalsif
sera were administered onday
11 or later ingestation.
Fewer abortions
were ob¬served when immunizations were
performed
during
early
pregnancy. Evidence waspresented
tosuggest
thatcytotoxic
effects
produced by
antiserawere com¬plement
dependent.
Immunodiffusion
tests ofeffective
antisera indicated anti¬ bodies to at least four differentplacental proteins.
Perhaps
the mostcarefully
conductedstudy
ofthe effects of
passive
im¬
munization in rodents
using
antisera toplacental proteins
wasreported
by
Beer et al.
(1972).
Ratplacenta
at 12-14days
ofgestation
werehomogenized
and
partially
fractionated
by centrifugation.
Theresulting trophoblast
cellswere used to immunize
rabbits
and the antiseraobtained
were used to pas¬sively
immunizepregnant
rats at 7-18days
of
gestation.
The unabsorbed seraproduced
abortion in 100 per centof the
animals but 2 of 12 rats diedfrom
this
treatment. In the samestudy,
rabbit
anti-ratlymphocyte
serum causedabortion in 25 per cent
of
the treated rats but 67 per cent of the animals died.Subsequently,
rabbit anti-ratplacenta
serum was absorbed with ratlympho¬
cytes;
thetoxicity
of
the antiseradisappeared
but itsabortifacient
activity
wasretained.
Conversely,
when
the anti-ratplacenta
serum was absorbedwith
rattrophoblast
tissue,
theabortifacient
activity
was nolonger
present.
Histological
examination
of
uteri, decidua, ovaries,
kidneys, lungs
and livers offemale
ratsaborted with
absorbed
antiplacenta
serum revealed no detectabledamage.
The
abortion-inducing
actionof
anti-ratplacental
antisera was noteffective
in
disrupting
pregnancy inhamsters
or mice. Noeffects
upon pregnancy infemale
rats wereobserved
when antisera to rattrophoblast
produced
in malerats were used.
Studies
have also been conductedusing
antibodies
topurified placental
proteins.
El Tomi et al.(1971 )
demonstrated that abortion could beproduced
in ratsby
administering
antisera tohighly purified
human
placental lactogen
(HPL)
raised
inrabbits.
Antisera were shown to cross-react with rat LH and ratprolactin
and the numberof
implantations
were reduced inanimals
in-jected
with antiseraearly
ingestation.
Animals treated later ingestation
had normalimplantation
numbers butsignificant
foetal
résorption
occurred. Thisstudy suggested
that sera to HPL reacted with ratplacental
antigen(s)
neces¬sary
for
normalgestation.
Whether
suchsubstance(s)
had anyfunctional
similarity
toplacental
lactogen
was notapparent
from
thisstudy.
A similar
study
to the above wasreported
thefollowing
year(Gusdon
1972).
In thisstudy,
totalfoetal
résorption
wasobserved
inpregnant
ratsafter
the
injection
of
rabbit anti-HPL sera.No effects
werefound
innon-treated
controls or rats
injected
with normal rabbit serum. Thestriking
observation
from
thisreport
was that thosefemales
receiving
anti-HPL serum returned tonormal oestrous
cycles
after
abortionbut failed
to conceive uponrepeated
matings
for
11 monthsafter
the initialimmunization.
Noexplanation
for this unusual observation wasprovided.
Similar abortifacient
activity
of anti-HPLsera was also
observed
in mice(Yamini
et al.1972).
These authors demon¬strated a
complement
dependent
cytotoxic
actionof
anti-HPL andsuggested
that
pregnancydisruption
maybe
causedby
the
anti-lymphocyte
antibodies contained in the anti-HPL sera. Stevens et al.(1971) reported
that abortionswere
produced
inpregnant
baboonsusing
baboon
anti-HPL sera. No detailedcharacterization of the
antisera used wasmade
although
cross-reactionwith
baboon
placental
extract was demonstrated.The
studiesreported using
anti¬bodies to HPL leave many
questions
unanswered and thestudy
of the effectsof antibodies
specific
for
placental lactogen
in aprimate
species
wouldprovide
moremeaningful
data.Antisera
tohumanchorionic
gonadotrophin (HCG)
have shown
thecapability
of
interfering
withreproduction
innon-human
species.
Highly
cross-linkedHCG
injected
into rabbits resulted in theproduction
of
anti-HCG
sera whichcross-reacted with
endogenous
mousegonadotrophin (Schlumbcrger
SeAnderer
1969).
In thesestudies,
injections
of anti-HCG
serainto female
mice inducedprolonged
oestrousperiods
for
18-20days
and
disrupted fertility
for
periods
of
37-100days.
No antisera were administered topregnant
mice and the rela¬tionship
of anti-HCG
sera and mouseplacental proteins
was uncertain. Al¬though
the effects of HCG
antiserauponfertility
were notdetermined,
Moug-dal
et al.(1971)
demonstrated
that such sera werecapable
of
reducing
thesecretion of
progesterone
from the
corpusluteum
innon-pregnant
rhesus mon¬keys.
These seraapparently
cross-reactedwith
endogenous monkey
LH,
butreactivity
withchorionic
gonadotrophin
from
pregnant
monkeys
was notstudied.
A cross-reaction
of
antiserato the subunitsof HCG
withnon-human
primate
chorionic
gonadotrophins
has,
however,
beenshown
by
other workers
(Hodgen
et
al.
1973).
Arelatively
low
cross-reactionof
antisera to the betasubunit of
HCG
with human orbaboon LHhas
alsobeen observed
(Stevens, unpublished).
Fig.
1.Induction of abortion in a
pregnant
baboon withsheep
anti-beta HCG serum(Stevens,
unpublished).
HCG
which cross-reacts with baboonCG
wasadministered
to apregnant
baboon on
day
20of
gestation.
This
resulted in an immediate fall in endo¬genous baboon chorionic
gonadotrophin
and ovarian steroid levels.Heavy
vaginal bleeding
wasobserved
approximately
36 hoursafter
the intravenousinjection
of the antisera(Fig. 1).
This workclearly
confirmed
the classicalconcept
that chorionicgonadotrophin
is necessary formaintaining
the steroid secretionfrom
theprimate
corpus luteum of pregnancy.In recent years, extensive
efforts
have been made to evaluate theeffects
upon
reproduction
of
antisera to non-hormonalprimate placental proteins.
Inan extension of earlier work
using
antisera towhole
placental
extracts,
Behr¬man and co-workers
recently reported antifertility
effects of
antisera directedto
specific
placental proteins
of rhesus andsquirrel
monkeys (Behrman
etal.
1974).
Theseworkers isolated
a fractionfrom
monkey
placental
extracts thatwith normal serum.
Although
no antisera wereproduced
to thisfraction,
the antiserareacting
to itproduced
abortions
in 63-80 per centof the rhesus
andsquirrel monkeys
studied. Whilethis
report suggests
that antibodiesproducing
abortions may bespecifically
directed to aplacental protein,
the criteriaof
purity
of
thereacting
fraction
were not documented and an exhaustive charac¬terization
of
the abortifacient antisera was notpresented.
Two
"placenta-specific"
proteins
have been isolatedfrom
human termpla¬
centaeby
Bohn
(1972).
One
of theseproteins
ispresent
inlarge
amounts in pregnancy sera(SPj)
while the other is not(PP:->)·
Antisera raised inrabbits
to human
SPi
have beenshown
to react with extractsof
placentae
and preg¬ nancy sera fromrhesus
monkeys,
Cynomolgus monkeys
and baboons(Bohn,
unpublished),
but not with pregnancy seraof
rats andguinea pigs
(Bohn
8cRonneberger
1973).
Antibodies to humanSPi
wereeffective
inproducing
abortions in 8 of 10
Cynomolgus monkeys
at 19-55days
of
gestation
following
a
single injection
(Bohn,
unpublished).
Thus it appearsthat
antibodies toSPi
will cross-react
readily
withantigens
from
primate
placentae
but
not withplacental proteins
from
animalsof
lowerphylogenetic
levels. Further studiesarenecessarytodetermine the
specificity
and mechanism ofabortifacient
actionof antibodies
toSPj.
Active immunization studies
Relatively
few
studies have been conductedemploying
active immunizationtechniques
with
homologous
orheterologous
antigens.
Evenfewer
reports
haveappeared
in whichhighly purified
proteins
were used to elicitantibody
respon¬ ses. In 1966 Okunda 8cGroilman
reported
studies in whichfemale
rats wereimmunized with whole rat
placental
extracts.Although
8of
12 immunized ratsconceived
following
mating,
only
one rat had anyliving
foetuses
by
day
20of
gestation.
The antiseraproduced
in these animals reactedstrongly
withkidney
extracts,
and upon examination allanimals showed
nephritis.
Whether
disruption
of
fertility
in these animals was related toantibodies
toplacental
proteins
could
notbe
ascertained.Similar
procedures
were used and similarresults were obtained in another
study,
in whichrabbits
were immunizedwith
total rabbit
conceptus
from
the ninthday
of
gestation
(Menge 1968).
Another
study
employed
the
guinea pig
as thespecies
immunized with wholeplacental
extracts(Paine
8cKennedy
1968).
Five animals
were immunizedusing
Freund's
complete
adjuvant.
The numberof
oestrusperiods required
for
con¬ception
wasgreater
than
normal in the treated animals. Reducedfertility
wasobserved in two
animals,
one died and theother
two had normalpregnancies.
A somewhat more detailedstudy
was conductedby
Boss
8c Neken(1970)
inwhich
females
were immunized withfractions
of
ratplacental
homogenates.
fractions obtained
by centrifugation,
andinjections
of
mostfractions
causedantibody
responses in 50 per cent or moreof the
rats in each group.Only
about 30 per cent of the immunized rats became
pregnant,
but in those that did conceive there was no detectabledisruption
ofgestation.
A similartype
of
experiment
wasperformed
by Menge
(1968)
in cattle. Tenheifers
were in¬jected
withhomogenates
of whole bovineconceptus
(32-38
days
gestation)
in Freund'scomplete adjuvant
and were then bredby
artificial insemination.Immunized
heifers
required
a mean of 3.8 inseminations perconception
while control animals neededonly
anaverageof
1.2. Inpregnant
immunized animals, nosignificant
difference in the numberof viable foetuses
atday
16of
gesta¬
tion was observed when
compared
to controlanimals.
In
yet
anotherstudy,
noeffects
onconception
orgestation
were observedwhen extracts of
homologous placentae
wereinjected
intorabbits;
however,
the
numberof
pregnancies
was reducedby
one-half if theinjections
of
ex¬tracts were
followed
by
chronic administration ofHCG
(Goldzieher
etal.
1970).
Noeffects
uponconception
or on the courseof
pregnancy were notedfrom
injections
of HCG
alone inthis
study.
Similarly,
Glass Se Mroueh(1967)
found
that rabbits immunized withHCG
readily
became
pregnant
and ex¬hibited normal
gestation.
Significant disruptions
of the oestrouscycle
andgestation
wereobserved
infemale
ratsfollowing
active immunization withpurified
human
placental
lactogen (El
Tomi et al.1970).
The meancycle length
wasnearly
doubledinHPL
injected
ratsoverthe control animalswhich
were treated with Freund'sadjuvant.
Theconception
rates were alsoslightly
reduced in the immunizedanimals;
however,
there was nosignificant
reduction
inimplantation
rates inthose
animalsconceiving.
However,
thenumber
of
foetuses
surviving
untilterm was
markedly
reduced in HPL immunizedrats,
and in the animals im¬munized at 90
days
of
age, no young were bornfrom
any of 28females
ofthis
group. In another group, immunized at 21days
of
age and mated3monthslater,
24of
37 animals conceived and about 60 per centof the
foetuses
sur¬vived. These authors
speculated
that a ratplacental
protein
cross-reacted with antibodiesgenerated
to HPLwhich
resulted in adiminished
fertility.
Another
report
from thesamelaboratory
suggested
that HPLimmunizations
exert an
effect
onreproduction
also in the rabbit(El
Tomi et al.197lo).
Female animalsimmunized
withpurified
HPL andFreund's
adjuvant
readily
produced
antibodies to HPTHowever,
nodisruption
ofgestation
was appa¬rent. No
difference
was found in the number of corpora lutea orimplantation
sites
between
control and immunized rabbits.On
the otherhand,
all animalsshowed
somefoetal
résorption
and the numberof
young born insurviving
litters was
only
about
15 per cent of controllitters.
Both the rat andrabbit
studies discussed
above
suggested
immunological similarity
of
ahuman
placen¬
Since
noneof
the studies discussed above describedeffects of
active im¬munization with a
highly
purified
protein
to ahomologous
species,
few
datawere
available
tosuggest
feasibility
of
using
such methods as a meansof
human
fertility
control. Thedesign
of
studiesfor
potential
humanapplication
of
active immunization wasplagued
by
twomajor problems.
The firstof
these is thatisoantigens
arerarely
antigenic
and the second is theunacceptability
of
Freund's
complete
adjuvant
for
human use. In anattempt
to circumvent theseproblems,
Stevens
(1973)
conducted studies in whichhomologous antigens
(pituitary gonadotrophins)
werehapten-coupled
with a diazonium salt andinjected
into femalebaboons
in anoil
vehicle.The
findings
from this
study
indicated that such
antigen
alteration could result in theproduction
of anti¬ bodiescross-reacting
andneutralizing biological
effectsof
endogenous
un¬altered
antigens
without the aid of
conventionaladjuvants.
The dataobtained
in thisstudy
stimulated anotherstudy
inwhich
postmenopausal
women wereimmunized with
hapten-coupled
FICG
in saline(Stevens,
unpublished).
Low levelsof
antibodies toHCG
and human LH were detected 5-7 weeksafter
thefirst of four
injections.
The concentration ofendogenous
LH wasreduced
in100 0- 60-40 20 200
|
100 I 11 il II II¡1
!¡
"
!!
? ni
;
· , w . .11 '
\!
Subject: . . Age 57 FSH »-« LH ---Immunization ( IO mg) ANTHOOYu
~l I I I I I I I I I I I III r~i 2 9 16 23 30 7 14 21 2« 4 II le 26 I » 16 22 2» ß 13 20 27 3 IO 17 24June July Aug Sept. Oct. Nov
Fig.
2.Effects of immunization with
hapten-coupled
HCG upon FSH and LH levels in apostmenopausal
woman(Stevens,
unpublished).
100- 80-60 40-20 200 100-I
U
i
í
'
Î1
l'Ii
' I I M ii II ' ' II II -I * > ;i I! ''!
II · 4-4-*-MIN SES 27J »NTI90DY TITIR Subject: CS Age: 45 FSH >-« LH Immunization (IOmg) m EZ2 zaUX
- — — — — — — — — ~ — — -1— — — — — ~- — -~ — —rT 2 9 W 13 90 7 « 21 2» « II 16 28 t ß 22 29 S4^r
13 90 27 9 IO 17 24July Aug. Sept. Oct. Nov.
Fig.
3.Effects of immunization with
haptcn-coupled
HCG upon FSH and LH levels in apremenopausal
woman(Stevens, unpublished).
8
of
10 womenduring
theperiod
ofsignificantly
elevated
antibody
levels.Antibodies were
abundant for
only
1-2 months(Fig. 2).
In the samestudy,
4
previously
sterilizedpremenopausal
women weresimilarly
immunized and
ahigher
levelof antibodies
wasobserved.
Likewise,
endogenous
LH levels werereduced
for
alonger period
of
time(Fig. 3).
Subsequent
detailed studies showedthat
these
premenopausal
women wereanovulatory,
butunfortunately,
no con¬trol
studies
wereperformed
to determine theirovulatory
statusprior
to theimmunization.
In order to demonstrate the
effectiveness of
immunological procedures
for
disrupting
hormonal
levels inendocrinologically
normal women, anotherstudy
was conducted in which control data
showed
normal hormonalpatterns
prior
to the start
of
immunization(Stevens
SeCrystle 1973).
In this latterstudy,
ninepremenopausal
women,previously
sterilizedby
bilateralsalpingectomy,
werestudied
through
a controlmenstrualcycle by assessing
serum levels ofpituitary
and ovarian hormones.
Only
sixof
these exhibited normalpatterns.
Sub¬
haptenic
groups perHCG
molecule. Antibodiesreacting
withHCG
and LHwere
detected
in allsubjects
at 3-5weeks of
immunization.During
a secondmenstrual
cycle,
theeffect of
the immunization upon hormonalpatterns
wasassessed.
Midcycle
LHpeaks
were reduced orabolished
in all sixsubjects
andbaseline
LH values were reduced infour
women. Patterns of serumprogeste¬
roneindicated
that 4of
the 6 did not ovulatebut
the other twoshowed
ele¬ vatedluteal
phase
progesterone
levelsdespite
reduced
LHpeaks
atmidcycle.
The data obtained
from
one of thesesubjects
are shown inFig.
4.Four
of
these sixsubjects
were studiedduring
anothercycle
six monthsafter
the first immunization.
Significantly
elevatedantibody
levels werefound
in 3of the 4
cycles. Midcycle
LHpeaks
were absent in 3 of the 4cycles
but
theprogesterone
patterns
suggested
that two of thecycles
wereovulatory.
Levelsof
serumoestrogens
weregenerally
reduced inall four
cycles
and oneof
thoseshowed
only slight
elevation atmidcycle (Fig.
5). Throughout
the entirestudy
ofHCG
immunization ofpremenopausal
women, menses andcycle length
re¬mained normal in all
subjects. Lymphocyte
transformation
testsfor
delayed
hypersensitivity
revealed normalfindings.
SUBJECT 3 ANTIBOCW TITER
^LLi
-I-H-J
II
li
íJliU
26 23 29CYCLE LENGTH (day·)
7 4 6 ß 10 12 14 16
DAY OF CYCLE
20 22 24
Fig.
4.Normal hormonal patterns and effects of immunization with
hapten-coupled
HCG uponhormonalpattern
inapremenopausal
woman(Stevens
SeCrystle 1973).
SUBJECT 2 6 MONTH FOLLOW-UP : 100 ANTIBODY TITER
I
50+
0 +-I FSH 40i
LHI
30 E 20 IO 300 --200 --100 -ESTRONE 600 4O0 '-200 -8 12 6 ESTRADIOL PROGESTERONE « O 2 4 6 IO 12 14 16 18 20 22 24 DAYOF CYCLEFig-
ó.Hormonal patterns in a
premenopausal
woman six months after immunizationwith
hapten-coupled
HCG(Stevens
SeCrystle 1973).
Although
thehuman studies described
above illustrated thefeasibility
of
reducing
thecirculating
levelsof
a human hormoneby
isoimmunization,
itshould be borne in mind that
HCG
antibodiesnormally
do not discriminate betweenHCG
and LH neitherbiologically
norimmunologically.
Therefore,
the presence
of
persistent
antibodies
toHCG
in young women may notonly
render
them
anovulatory
but islikely
to result(upon prolonged treatment)
inamenorrhoea
and clinicalsymptoms
of
ovariandeficiency.
Thus,
while these studiesweresignificant
as amodelfor
immunological investigations
inhumans,
no directapplication
to humanfertility
control wasprovided.
Recent
studies have
indicated that antibodiesproduced
tothe
beta subunitSince the
subunit
preparations
used were notcompletely
pure, it washoped
that
a"pure"
beta subunit ofHCG
might
elicitantibody
response toHCG,
but
not to LH.Consequently,
ahighly purified
preparation
of the betasubunit
of HCG
was used to immunizesheep,
rabbits
and baboons(Stevens,
unpublished).
Theantibodies
produced
reactedwith
HCG,
baboonCG
and humanLH,
but not with baboon LH. Thesefindings discouraged
the useof
beta
HCG
in humans as a meansof
fertility
control
butpermitted antifertility
evaluation
of HCG
immunization in baboons.During
thisstudy,
10fertile
baboons
were immunized and weresubsequently
mated
with males of provenfertility. Preliminary
evaluationof
theantifertility
effect of the
immunization revealed that no pregnancy lastedpast
expected
timeof
mensesfollowing
30
matings
(Table 1).
Theprevious conception
rate in non-immunized baboons in thecolony
was about 90 per cent.These
studies indicate that in case anantigen
could
beprepared
that elicitedantibodies
specific
for
HCG
and notreacting
withhuman
LH,
animmunological
method for
humanfertility
con¬trol maybecome feasible.
Table 1.
Effects of immunization of female baboons with beta HCG on
fertility.
Baboon No. No. of
matings
OvulationsPregnancy*
1 3 4 5 7 9 10 12 14 16 3 3 3 3 3 3 3 3 3 3 Total 10 30 28
Pregnancy
delined as any animal nothaving
menses at or beforeexpected
time forREFERENCES
Beer . E.,
Billingham
R. E. SeYoung
I. S. L.:J.
exp. Med. 735(1972)
1177. Behrman S.J.,
YoshidaT.,
Amano Y. SePaine P.: Amer.J.
Obstet.Gynec.
77S(1974)
616.
Bohn H.: Arch.
Gynäk.
272(1972)
165.Bohn H. Se
Ronneberger
H.: Arch.Gynäk.
275(1973)
277. BossJ.
H. Se Nelken D.: Fértil, and Steril. 27(1970)
508.Cohen H. R. Se Nedzel R. A.
J.:
Proc. Soc. exp. Biol.(N. Y.)
43(1940)
249. Dobrowski M. D.: Bull. Int. Cracovie 5(1903)
526.El Tomi A. E.
F.,
Boots L. Se Stevens V. C:Endocrinology
87(1970)
1181. El Tomi A. E. F., Boots L. Se Stevens V. C:Endocrinology
88(1971 )
805.El Tomi A. E.
F.,
Crystle
C. D. SeStevens V. C: Amer.J.
Obstet.Gynec.
709(19716)
74.Glass R. H. & Mroueh .: Amer.
J.
Obstet.Gynec.
97(1967)
1082.Goldzieher
J.
W.,Matthijasen
C. Se MandelJ.
E.:Contraception
2(1970)
323. GusdonJ.
P.,Jr.:
Amer.J.
Obstet.Gynec.
772(1972)
472.Hodgen
G.D.,
Nixon W.E.,
VaitukaitisJ.
L.,
Tullner W. W. Se Ross G. T.: Endo¬crinology
92(1973)
705.Kometani K. Se Behrman S.
J.:
Int.J.
Fértil. 16(1971)
193.Koren
.,
Abrams G. Se Behrman R. S.].:
Amer.J.
Obstet.Gynec.
702(1968)
340. Loeb E. N., Knowlton A. L, Stoerk M. G. SeSeegal
B. C:J.
exp. Med. 89(1949)
287.Menge
A. C: Proc. Soc. exp. Biol.(N. Y.)
727(196S)
1271.Menge
A. C:J. Reprod.
Fértil. 18(1969)
67.Moudgal
N. R., MacDonald G.J.
SeGreep
R. O.:J.
clin. Endocr. 32(1971)
579.Nemirovsky
M. S.:Experientia (Basel)
26(1970)
1138.Okunda T. Se Grollman .: Arch. Path.
(Chic.)
82(1966)
246. Paine P.J.
SeKennedy
S. .: Int.J.
Fértil. 73(1968)
198.Ross G.
T.,
VaitukaitisJ.
L. Se RobbinsJ.
B. In:Margoulies
M. and Greenwood F.C,
Eds. Structure
Activity
Relationships
or Protein andPolypeptide
Hormone,
Excerpta
Med.
(Amst.)
247(1972)
153.Schlumberger
H. D. Se Anderer F. .: Acta endocr.(Kbh.)
60(1969)
681.Seegal
B. C. Se Loeb E. N.: Soc. exp. Biol. Med. 45(1940)
248.Stevens V. C: Obstet, and
Gynec.
42(1973)
496.Stevens V. C. Se
Crystle
C. D.: Obstet, andGynec.
42(1973)
485. Stevens V.C,
PowellJ.
E. SeSparks
S.].:
Biol.Reprod.
5(1971)
97. Yamini G., Chard T. Se Blake M. E.:J.
Endocr. 55(1972)
29.DISCUSSION
Cinader: The strategy of immunization with
chemically
modifiedautologous
antigens
(Cinader 1963),
nowbeing
applied
to hormones and toplacental antigens,
is based onmodel
experiments
inwhich animals withnconatally
induced tolerancewereimmunizedwith
para-azo-sulphonic
acid derivatives of thetolerogen
(Cinader
Se Dubert 1955,1956).
Theensuing
response results inantibody
which can combine withtolerogen.
This
reactivity
is attributable topeptide
determinants which areconformationally
Se Cinader
1967).
Theproportions
ofhapten/tolerogen
must be determined as a func¬tion of the number of
responding
individuals and of thehighest antibody quantity
directedagainst peptide
determinants. Thishapten/tolerogen proportion
is lower than that which induces maximalantibody
response, directedagainst
hapten.
In this con¬text, it should be noted that the
capacity
torespond
with tolerance breakdown is undergenetic
control(St.
Rose 8c Cinader1973).
Individuals which are resistant totolerance circumvention can be made
responsive by adjuvant
administration. Somethought might
begiven
to thepossible
use ofadjuvants
which do not have harmful side effects.Tulwar: About two years ago, a WHO Research and
Training
Centre(RTC)
wasestablished in New
Delhi,
and one of themajor projects
of that RTC is to try todevelop antiplacental approaches
tofertility
control.Our
approach
isslightly
different from the one that Dr. Stevens hadadopted.
Let meemphasize
that in none of ourexperiments
have we used whole HCG. We haveonly
worked with the beta subunit. For thenon-endocrinologists
in thisaudience I may mention that thealpha
subunit of HCG is identicalwith that of three other hormones,TSH,
FSH andLH,
andconsequently
there is adanger
of crossreaction if one useswhole HCG. The beta
subunit,
even in thepurest
form available, whenanalysed by
electrophoresis
onpolyacrylamide
gels
showsmicroheterogeneity.
It resolves in about8 bands. We have studied the
antigenic properties
of each of these 8 bands, and thereactivity
of each of these bandsalong
thepolyacrylamide
gel
has beenanalyzed
with the anti-HCG serum and anti-HLH serum. These arecompetitive
type
experiments
where the
binding
of labelled HCG to anti-betaHCG,
or of labelled LH to anti-LH is determined. When theantigenic
determinants for beta HCG areanalyzed,
the HLHreactivity partly
lies outside thespectrum,
partly
within thespectrum.
The outside part can be removedchemically,
but to remove the oneoverlapping
thereactivity
against
HCG weused animmunosorbentagainst
aheterologous
LHpreparation.
Withgradient absorption
one can remove thehigh
affinity
absorbing
sites to arrive at apreparation
whichgives
almostnegligible
crossreaction,practically
no crossreactionwith HLH. On the other
hand,
it preserves the determinantsagainst
HCG. At thisstage,
it isessentially
oneband,
though
athigh
concentrations one may see twobands. Another
major
difference is that thishighly
purified
beta HCG isconjugated
to a
proteinic
carrier rather than modifiedby haptenic
groups. Thecomplex
of betaHCG with tetanus toxoid in
appropriate proportions
isimmunogcnic
in alarge
variety
ofspecies, ranging
frommouse, rabbit, goat,monkey
to human. Theimmunity
is of along-lasting
type; there is a rise in theantibody
titergradually reaching
aplateau
around the 30th week in thegoat
andslightly
earlier in themonkey.
Later there is atendency
todecay,
but with anotherinjection
apowerful
booster responseis obtained. The antiserum does not react with any of the hormones we
usually
comeacross, i.<?.,
HGH,
HPL,FSH,
TSH,
even HLH atphysiological
concentrations. Onthe other
hand,
the antiserum reactsimmunologically
with the total HCG molecule.Immunological reactivity
does notnecessarily
mean that the antiserum would alsoneutralize the
biological
effects. Thebiological
effectiveness has been tested in at least two or three systems, such as the ventral prostate assay and theproduction
of pro¬gesterone
by
isolated corpus luteum slices. The antiserum is able to neutralize the HCG-induced effects in these systems.In
short,
the main differences betweenDr. Stevens'approach
and ours is that we aredealing
with achemically
andimmunochemically purified
preparation
of betathat we are
studying
is the subunit of the hormoneconjugated
in discreteproportions
with aproteinic
carrier, atype
of vaccineapproved
for human use, i.e., the tetanustoxoid,
which I thinkishighly immunogenic
andhas desirable immunizationproperties.
Stevens: I think this is very nice work that Dr. Talwar has done and I would agree with him that antisera to beta subunit of HCG react very little with LH. Even withour
preparations,
which were notchemically
characterized soextensively,
we haveproduced
antisera in animals with as low as less than one per cent of the reaction with HLH asthey
do with HCG.However,
the ReviewGroup
of WHO decided thatthis was still too much crossreaction to attempt studies in
humans, and, therefore,
didnot recommend to follow this line of
experimentation
in human females.Simons: You said that
lymphocyte
transformation tests revealednegative
or normalUndings
in your active immunization studies. Could youclarify
what you meanplease?
Secondly,
you referred to thedesirability
ofavoiding adjuvants,
such asFreund's,
in
immunizing procedures
which haveapplication
to humans. It may bepossible
toachieve an
adjuvant
effect in humansby combining antigen
immunization with a BCGimmunization programme. Such an
approach
would have the additionaladvantage
ofassociation with a
widely practised public
healthprocedure.
Do you know ofa reasonwhich
might preclude
such anapproach?
Stevens:
First,
lymphocyte
transformation tests were notperformed
directly
undermysupervision.
As I understand it, theprocedure
was the blast formationtype
of test in which the modified HCG was introduced with theperipheral
lymphocytes
of thepatients
and observation of the number of cells that had been transformed. There was no difference between the control transformation and thesamples
taken at variousperiods
during
andfollowing
immunization.As far as the use of
adjuvants
is concerned,what I meant to say was that one couldnot
preclude
the use of alladjuvants
but should not useadjuvants
of the Freund'scomplete
type.
Certainly,
there aremany types ofadjuvants acceptable
for human use,such as the ones you have
suggested,
and we arecurrently
performing
an extensiveevaluation of many
adjuvants
that could be used with various kinds of altered orchemically
modifiedproteins
andpeptides.
Fudcnberg:
Withregard
to the useofBCG,
ifBCG is to begiven repeatedly,
this canresult in humoral
immunity.
Some cancerpatients
so treatedclearly
havedeveloped
immune
complex
disease. There have also been a widevariety
of other very un¬desirable side
effects,
so I thinkrepeated
use of BCG isprecluded.
In this context,have you looked at your immunized baboons to see if
they
had anyurinary
abnor¬mality
findings
or renal function that aresuggestive
of latentantigen-antibody
com¬plex
renal disease?Stevens: The
only
studies we have donewere to look for the excretion of excess albu¬min in urine. The results were
negative.
Talwar: With the
preparation
that we use we havenot observed any untoward effectat any level. Last
September,
our NationalAgency
gave uspermission
for some con¬trolled human trials. With the
help
of our Obstetrics &Gynecology Department,
a few cases have been studied and verycarefully
monitored for avariety
of systems, such askidney,
liver,
thyroid,
adrenal andpituitary
functions,
and up till now there isAmino acid
Sequence
of/?-hCG
a^-hLH
CG S«lTHrÏGki-Pro-Lau-Arg-Proj^Cy<IÂr}Pro-lll-Ain-Aki-Thr-Uu-Akl-Vol-Glu-L7l-6li>-6l)f-Cyi-Pro-Vol-LH S«f4Afa|-Glx-Pro-L«u-Ar9-ProJTr^Cy»4si«JPro-ll«-Ai»-Alo-Tllr-L«u-Akl-V<il-GI«-L>«-GI«-Cly-Cyi-Pra-vol-CG LH CG LH CG LH CG LH Cyt-lle-Thr-Vol-A«\-Thr-Thr-ll«-C»»-Ata-G(y-Tyr-Cyt-Pro-Tlir-M«tmr-Anj-VoHC>·-lk- »·-V<ll-A·x· n»-Thr-ll·-Cy·-Alo-Gly-Ty -Cy»-P O-Thr-M*·A |-M<-L«I|L«l-ßlI^
Vol-Lou-Pro-Alo-Leu-Pro-Glny^Vol-Cyt-U«nrTyr-Arg-Aip-Vol-Arg-Ph·
-Gki- Sor-Ila-Arg-Uu-Pro-ay-Cyt-Pro-An-ei»-
Pro-Vol-Pro-GI»^PjO}vol-Cy»4n^Tyr-Ara^U»-Vol-Arj-Pht-Gbi-S«f-llt-Arg-Lou-Pro-Gry<y«-Pl»-Ar9-«y-Val-Ain-Pro-Vol-VOI-S«rVol-Aix-Pro-Vol-Vol-S«rPho-ProVol-Alo-Lto-Sor-Cy^ArgfCy«
Tyr-Alo
Vol-Alo-Lou-SW-Cyi-jGiiifCy·
Alo-I^C^-Arg-Aig-S«r-TrrflI3-A»e-Cj«-Gly-ProC)ri-Arg-Arg-Sor-Thr-gerj-A«i-Cy·-G^-Gr»-Pro-Lv»-A»-HI«-Pro-L«u-Tnr-Cy«lAiptA«pHr^-Ars-Pr»^-Aio-SW-S«r-S«c-S«r-Ly«-Ala-PTO-ProH|
Gly-Gr/-Pro-Ly«-A»x-H(t-Pro-L*u-Thr-Cy»kax4A«i·S*r-Ly>-Gly|Fyo-S^-Lou-Pro-S>r-Pro-Sor-Arg-Uu-Pro-Gry-Pro-S<r-A«p-Thr-r>o-M<-lH»u-Pro-G<n|
Morgón,«01.(1973)Fig,
.The amino acid sequence of beta subunits of human LH and HCG as
reported
by Morgan
et al.(1973).
* indicates location of
carbohydrate
attachment onnative hormone.Amino acid
Sequence
of
aC-Terminal
Peptide
of/?-hCG
CG- I CG-2 PtM -Gki -Asp -Sw -S*r -S«t -S«r Ly«-Ale-Pro-Pro-Pro-S«r-L«u-Pro-S«f-Prx)-S»r- Ph«-Gln-A»p-S»r-S«-S»r-Ly»-Alo-Pro-Pro-Pro-Ser-Uu-Pro-S«i-Pro-S»r-CG-I CG-2 Arg-Leu-Pro-Gly-Pro Arg-Lou-Pro-Grj-Pro Asp-Thr-Pro-II« -L»u -Pro-Gin K7
An-Thr-Pro-II«-L«u-Pro-Gin-fs«r-L«u-Prol
CG-1 Morgan,·*al.(1973) CG-2 Carl·«,·,al.(1973)Fig-
B.The amino acid sequence of a C-terminal
peptide
of the beta subunit of HCG that is notrepresented
in the beta subunit of LH. The two structuresrepresent
sequencesreported by
two different groups of workers.*indicates location of
carbohydrate
attachment on native hormone.(CG-1
Mitchison: Dr.
Stevens,
could you tell us about the C-terminalpeptide,
which seemsto be
unique
to HCG?Stevens: This is a
point
that Ihoped
to discuss. Thatlong
tail that you saw earlier on the beta subunit of HCG whencomparing
the structures of beta subunits of HCG and LH(Fig. A)
isrepresented by
its amino acid structure on this slide(Fig. B).
Slightly
different sequences have beenreported by
two different groups of workers. There was a deletion of a serine atposition
121by
one worker and there is a dif¬ference in three amino acids at the C-terminal
end;
however, these structures areessentially
the same. We have madepreparations
ofthis,
and aslightly larger peptide
from natural HCG
by
chymotrypsin
digestion.
We have alsoprepared
asynthetic
peptide using
this structurewith 4 more amino acidsat the N-terminal end. Antibodies have beenproduced
to thesepeptides
in rabbits. We havenot had sufficientquantities
to immunize baboons
yet,
buthopefully,
we can start very soon. The antibodies pro¬duced to these
peptides
react with the nativeHCG,
the beta subunit ofHCG,
but not with human LH. We have found that thesepeptides
are notimmunogenic
under all circumstances.They
need to beconjugated
with alarger
molecule orhaptenic
group to render themantigenic.
Weplan
to prepare avariety
of thesesynthetic
peptides
ofdifferent
lengths
and with differentconjugations
and to test with many types ofadjuvants
to try to find the most desirable immunization reagents and schedule that willproduce long
termantibody
levels with asingle injection.
Fox: Have you made any
histological
studies on target organs for thegonadotrophins
of immunized animals?
Stevens:
Yes,
in the animals that were immunized withthe HCG beta subunit we havedone some studies. Fluorescent-labelled antibodies were reacted with
pituitary
tissueand with tissues of a host of other organs. In the animals that had been immunized
for a
long period
oftime,
we have donehistopathological
examinations on someorgans,
including
thepituitary.
We have not found any evidence of tissuedamage.
However,
one must remember thatwe did not show any evidence ofcrossreacting
anti¬ bodies to baboonpituitary
hormones,
so ournegative findings
are notterribly
sur¬prising.
Wehope
that we can obtain someguidelines
for the kinds ofsafety
studiesthat should be done in this area in order to conduct a critical evaluation of this
method.
Rowe: Dr.
Stevens,
you assess immune responseby antibody
measurement and then you observe an effect on hormones and onfertility.
Do you have direct evidence thatthese effects are due to
antibody?
Can you, for instance, transferantibody
passively
and see thesame effect?Stevens: I could show you another series of slides
showing
data obtained when normal pregnant baboons were immunized with hormones raised in the baboons as well asantibodies raised in other animals. We have shown that we can abort baboons with
anti-HCG sera.
Using placental lactogen,
we have immunized pregnant baboons withbaboon
antiplacental
lactogen
sera and observed similar effects. I am notwilling
toconclude that this action is
directly
due to the neutralization of thecirculating
orintracellular hormone. The
antigen-antibody
reaction with the hormonemight
have caused somesecondary
damage, resulting
in abortion. The answer to thesequestions
must await more detailed studies.
you did not mention either in your written or in your oral
presentation.
How didyoumeasure LH in thepresence of LH
crossreacting
antisera, andviceversa, how didyoumeasure antiserum levels in the presence of
endogenous crossreacting
hormone?Stevens: To make a very short answer, I cannot
explain
it.However,
you must realize that the antibodies wereproduced
to alteredantigen
that did not have identical determinants as the native molecule. Theonly explanation
I cangive
is thatperhaps
some of the antibodies were not reactive with native hormone, but were with the 125I-]abelledantigen
used to detect them. The antibodies to nativeantigen
used in theradioimmunoassay
would reactwith thenativeendogenous
hormone,
thusreflecting
a measurable level of hormone in the same serum
sample
in which antibodies werefound.
Talwar:
May
I ask Dr. Stevens whether in the six cases where whole HCG wasgiven,
you have seen the effect onthyroid
or TSH?Stevens:
No,
we have not studied the effects onthyroid
or TSH.Voisin: What we are
aiming
at in thismeeting
is the induction of immune reactions directedagainst antigens
involved in thereproduction
mechanisms with the idea that these reactions must counteract these processes. We must,however,
keep
in mind thata
significant
part of the immune reaction acts in theopposite
direction and that thisseems to be of great use in
physiological
situations. In theexperiments
I shall referto, which were donein our
laboratory
with GerardChaouat. we studied non-hormonalplacental
antigens,
butantigens
that arelinked to the surface oftrophoblastic
cells,
asDr. Talwar alluded to. We made use of two strains of
mice,
C57 BL/KS andA/J,
differing
both atthe H-2 and non-H-2 loci. KS females weremade pregnantby
eitherKS or
A/J
males for 1-5pregnancies.
Theirplacentae
were examined for thepresenceand
specificity
of maternalimmunoglobulins
bound to theplacenta.
Whatwas found,in brief, was as follows: First, maternal
immunoglobulins
werestrongly
fixed on and not washable off the cell membrane oftrophoblast.
This was much moreprominent
in
allogeneic
thaninsyngeneic pregnancies
and even more so inplacentae
frommulti-Table A.
Facilitation
(enhancement)
action ofplacental
eluates on Sarcoma 1(A/Jax)
tumours"'grafted
on C57BlKs mice**.Origin
ofplacental
eluatesFather Mother
Number of
grafted
mice Lethal takesRejections
A/Jax
C57Ks C57Ks C57Ks 10 15 1 152 IO6
Sa¡
cellsgrafted
on either maleor female C57Ks.pai'ous females. Immunofluorescence studies showed that maternal
immunoglobulins
are
strongly
fixed at the surface ofplacental,
trophoblastic
cells,
even after along
perfusion
followedby
severalwashings
of the slides. These antibodies weremainly
of the
IgGj
type(mouse
IgG|
likeguinea pig
IgGj
are theanaphylactic,
non-com¬plement fixing antibodies).
These antibodies can be eluted.They
arespecific
of thepaternal transplantation antigens,
andthey
have theproperties
offacilitating
anti¬bodies,
that is, wheninjected
into normal KS micethey
render them able to accept thegraft
ofpaternal
strainA/J
sarcoma cells. As shownby
the data of TableA,
out of 10 mice there were 9 lethal takes of the sarcoma
graft,
while,
when the eluates
-prepared
in the same way
-came from
isogenic
placentae,
there was no one take out of 15grafted
mice. Thesimple
messagebrought by
theseexperiments
is that immunization may go two ways, facilitation reaction as well asrejection
reaction, theformer
counteracting
the latter, and this is as true inreproduction immunology
as itis true in cancer
immunology
and intransplantation
immunology.
Stevens: I think it is
important
topoint
outthat there areseveralpossible
mechanismswhich may be
operative
for theantifertility
effects of HCG antibodies. Onepossibility
is that the antibodiesmight
becytotoxic
to the cells of thetrophoblast.
This has beencarefully
describedby
Morisada et al.(1972).
There is also thepossibility
that when the zonapellucida
comesoff,
there may be acoating
of theblastocyst
with antibodies,thereby preventing implantation
in much the same way that antibodies to the zonapellucida
may act.Furthermore,
you must remember that chorionicgonadotrophin
is necessaryendocrinologically
forextending
the life of the corpus luteum of pregnancyso thatthe uterine environmentwill be maintained
during
theearly
stages ofgestation.
Antibody
neutralization could prevent this maintenance.Lymphocyte suppressing
ac¬tivity
of HCG has also beensuggested.
Should antibodies remove HCG fromtropho¬
blast cell
surfaces,
normalimmunological
surveillancemay terminate the pregnancy. Weplan
now to prepare several differentsynthetic
peptides,
allrepresenting
part
orparts
of the C-terminalportion
of the HCG beta subunit that is exclusive to the chemical structure of HCG. We furtherplan
toconjugate
thesepeptides
withvarying
types
ofacceptable
conjugates
that could be used in humans, and then evaluate each of these types withvarying
vehicles(adjuvants)
and time schedules to obtain the ideal method of immunization. When theseexperiments
arecompleted,
we will im¬munize baboons and
perform
critical studies ofsafety
andefficacy.
Then,hopefully,
we will get
permission
from our Ethics Committee toperform
human trials.References:
Cansen R.
B.,
Bah! 0. P. & Swaminathan N.:J.
biol. Chem. 248(1973)
6810. Cinader B.: Canad. Cancel Conf. 5(1963)
279.Cinader B. 8c Dubert
J.
M.: Brit.J.
exp. Path. 36(1955)
515. Ciuadcr . 8- DubertJ.
M.: Proc. roy. Soc. 146(1956)
18.CinaderB.. St.Rose