392

THE LABORATORY DIAGNOSISOF PREGNANCY

By HERTA SCHWABACHERGroup 9 Laboratory, Watford, Herts.

No pregnancy diagnostic laboratory is in aposition to undertake tests for pregnancy to satisfythe natural desire of every woman to know thetruth as soon as possible. It is desirable that ahealthy married woman be guided by her medicalofficer to await his diagnosis in the consultingroom. The unmarried mother, however, shouldhave the advantage of laboratory diagnosis. Hereearly diagnosis is a definite advantage in makingre-adjustments in social and domestic life.

Medical conditions for which early diagnosis issought are suspected hydatidiform mole, tubulargestation, abortion, abdominal tumours associatedwith amenorrhoea, amenorrhoea associated with ananaemia or endocrine dystrophy, teratoma of thetestes and, lastly, suspected pregnancy in a tubercu-lous patient or a cardiac patient. It is obvious,thatthe mother's life would be jeopardized if pregnancywere allowed to continue in the last two conditions.

Laboratory tests which have been evolved areeither quantitative biochemical estimations orbiological assays.The disadvantages of the chemical tests are two-

fold. Firstly, none of the chemical reactions is asaccurate as the better established biological testsand, secondly, the chemical tests are laboriousand time-consuming in their technique. Voge(1929) introduced the bromine test for the estima-tion of histidine excreted during pregnancy. Thetechnique was modified later by Kapeller-Adler(1934) and by Visscher and Bowman (i934), butthe accuracy rarely exceeded 80 per cent. in anyseries of tests. Colorimetric determination ofoestrogens in the urine have been attempted byGuterman (i945), but his claims of 93 per cent.accuracy have not been confirmed by later workersReinhart and Barnes (1946), who obtained a 25per cent. error.The only reliable pregnancy tests are biological

and are based on the enormous increase in theamount ofgonadotrophic activity during pregnancy.The amount of chorionic gonadotrophin (C.G.)excreted usually reaches a maximum between thesixth and twelfth weeks and then rapidly falls toabout io per cent. of the maximum by about the

sixteenth week and remains at this level till a fewdays after parturition (Fig. i). It.is important toknow the shape of the curve of output of theactivity with each stage of gestation. The cliniciancan then more reliably interpret the result of thetest. A weak positive, for example, around thethree months stage might suggest treatment forthreatened abortion. A very strong positive indilution of i/xo and higher of the extracted prin-ciple from urine would indicate hydatidiform mole.A negative report on urine sent after the thirteenthweek may mean that the patient was excreting lessthan 3,000 I.U. gonadotrophin per litre on the dayof the test (Hobson, I955). The gonadotrophin,when extracted, will be just below the reactingdose for test animal, 70 I.U. for a 70-gm. Xenopus.This can occur anywhere between conception andthe twelfth day of pregnancy and again betweenthe thirteenth week of pregnancy and parturition.The four pregnancy tests which have been

recognized as yielding an adequate standard of testaccuracy are:

I. The Ascheim-Zondek, using immature femalemice.

2. The Friedman, using immature femalerabbits.

3. The Hogben ovulation, using mature femaleXenopus toads.

4. The Galli Mainini spermiation, using maturetoads.

It must be stressed that these tests are not, infact, tests for pregnancy, but tests detecting thepresence of relatively large amounts of gonado-trophin from living chorionic cells. All four testswill give a positive result in normal pregnancy,in cases of hydatidiform moles and chorionic epi-thelioma for periods up to about o days after anabortion if placental tissue is still retained.

i. Ascheim and Zondek (1928) based their teston the discovery that pregnancy urine contains asubstance which is gonadotrophic when injectedinto mice. This test has been modified by differentworkers, but the technique used in the EdinburghPregnancy Diagnostic Laboratory is as follows:Take 30 c.c. from the first sample of urine collected

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from

August 1956 SCHWABACHER : Laboratory Diagnosis of Pregnancy 393

CAteifll,t 6roo trf-t1(cte Cixc- Tonxtk"*isj

.-.

O0

0 0

L -- Y

Wcks. of p,r,nan..railas o,v. o .otl . aAtc. ;

FIG. i.-Gonadotrophin excretion curve in pregnancy.

in the morning. Add four drops of 20 per cent.thymol in alcohol to prevent bacterial growthduring the three days the specimen is kept in arefrigerator.

Four infantile female mice 6 to 8 gm. in weight,three to four weeks old, are injected with the urineto be tested. Each mouse is given 0.5 c.c. each dayfor three days. If the ovaries and uteri are normal,the result is recorded as negative. If not normal,three different structures may be seen (see Plate X,Dukes, I939).

i. Enlarged follicles associated with enlarge-ment of the uterus.

2. Corpora lutea may be recognizable, especiallywith a lens.

3. Haemorrhagic follicles may be large andeasily seen, but often have to be carefullylooked for.

The first reaction of large follicles is not, ifpresent alone, diagnostic of pregnancy, but occurswith urines from patients presenting histories ofearly menopause, severe anaemias and carcinomaof the uterus. It is due to FSH or oestrogens in theurine and is found in the later stages of pregnancy.A further specimen at a later date is requested ifthis reaction is found alone.The presence of corpora lutea, together with

blood spots in one or more mice, denotes a definitepositive result. Such a positive result is obtained incases where chorionic elements are in biological

contact with maternal tissue, also in carcinoma ofthe ovaries and testes. The accuracy of this preg-nancy test estimated on 20,000 tests per annum is99.5 per cent.

2. Friedman (1929) relied on the fact that rabbitsovulate only when their ovaries are specificallystimulated. This occurs after mating or owing tofemale rabbits 'jumping' one another. Femalerabbits must consequently be kept in separatecages one month before use to ensure that at thetime of the test the ovaries do not contain anyruptured follicles and that the animals are notpregnant.To 50 c.c. of urine is added 50 c.c. rectified

spirit and the mixture allowed to stand a fewminutes. A precipitate is formed which consists ofinactive material and this is centrifuged off anddiscarded. Another 150 c.c. of rectified spirit isadded to the mixture and this brings down aprecipitate which contains the active substance.This precipitate is centrifuged off and extractedwith ether to remove toxic substances and theremaining solid matter is dissolved in 5 c.c. ofsaline; 3 c.c. of this solution are injected intra-venously and the animal operated on 24 hourslater. In a few cases the reaction is doubtful in 24hours and then the remaining 2 c.c. of extract areinjected and the animal operated on after a further24 hours.A positive result is returned if one or more

D

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from

394 POSTGRADUATE MEDICAL JOURNAL August 1956ovulated follicles are seen, or if one or morehaemorrhagic follicles are present in one or bothovaries (see Plate XI, Dukes, I939).

In some laboratories 8 to 0o c.c. of crude urineis injected intravenously and the rabbit sacrificedafter 48 hours.

3. The Hogben test. Hogben was interested inthe colour change of animals and while in SouthAfrica in 1927 investigated the chromatic be-haviour of the indigenous toad, Xenopus laevis.This animal is purely aquatic and turns dark ifkept in water in a black container and becomes paleif placed in water in a white container. The eyewas found to be the receptor for this response,the pituitary gland suspected as a mediator in themechanism. When the pituitary gland was re-moved the operated animal remained pale even ina black background and the ovaries atrophied in afew weeks. To prevent their atrophy Hogbeninjected ox pituitary extracts. Not only were theovaries regenerated, but ovulation and extrusionof eggs into the water was observed. Xenopushave cilia the whole length of the oviduct, so thatany eggs ejected from the ovary at ovulation areautomatically extruded from the body by ciliaryaction (Fig. 2). This animal conveniently fails toovulate when kept in captivity. The reliability ofthis animal's response to C.G. depends on thehusbandry it receives. Well-fed animals between60 to 0oo gm. are used. They must respond to70 I.U. ofchorionic gonadotrophin. Toads are usedthree times with an interval of o days and thenrested one month. This means that a single toadis used i8 times a year. The method of keepingthese animals in correct rotation has been describedby Schwabacher (I950) and Hobson (I952).Xenopus is kept in water at 22°C. and fed once aweek with chopped ox liver.By means of the Scott technique (1940) 60 ml.

of urine are concentrated into 5 ml. of extract.One toad only is used for each test and receives2.5 ml. of extract into the dorsal sac. If the toadovulates within 24 hours the result is recorded aspositive. If, however, there is no ovulation in 24hours, a second toad is injected with the remaining2.5 ml. of the same extract. If both toads arenegative 48 hours after the beginning of the testthe result is reported negative. Hence with astrongly reacting urine the result of the test can begiven in 24 hours, but a negative reaction is notreported until 48 hours.We no longer seek confirmation from the doctor

for our tests reported positive. We take it thatan egg denotes the presence of chorionic epi-thelium. It is our routine to send out cardsenquiring whether our negative tests are correcttwo months after the test has been made. Thisinterval allows time for clinical evidenee of preg-

W-... ..NW. $.

":iii...isii: .... ... ...

.......~~~~........

··:I·; ..... .: .T:..ii..:..:·:' .. i9~~~ S'.''@|S;;;..

''::i:5:i:: l | ··'': Si::~Qi :::;- l"'i -I;;;i ez::i:[: ::= :':

FIG. 2.-Positive Hogben reaction.

nancy to be well established. Schwabacher (1950)reported 99.48 per cent. possible accuracy and thisfigure compares favourably with the 24,00ooo testsper annum performed in Edinburgh, where anaccuracy of 99.8 per cent. is achieved (Hobson,1952).

4. The Spermiation test described by GalliMainini (I947) has come into vogue in everycountry where toads are indigenous. Fraser (1950)reviewed the literature which had been producedin 26 species of amphibia. Not all species are suit-able, as some animals spermiate naturally. Thiswas found to be the case in Xenopus laevis(Schwabacher et al., I956). Favourable reports onthe use of Bufo bufo for pregnancy tests have beenpublished in this country by Klopper and Frank(I949), Law (I949), Frazer and Wohlzogen (i949,1950), Ferreira (I954), Haines and Ferreira (1949,1954), Schwabacher, Elkan and Scott (I956).Urinary chorionic gonadotrophin is extracted byScott's technique and o.5 ml. injected into thedorsal sac. The cloacal fluid is examined three tosix hours after injection. It is important that thefinal reading should be at least six hours afterinjection, since positive results were found toappear after the fifth hour and it is not generallypracticable to increase the time still further if the

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from

August 1956 SCHWABACHER : Laboratory Diagnosis of Pregnancy 395

Gt::::

...!'9.- *

.. .:..

*:s:ah··': Xv-~,..,^,..^.Cj

* X ·di ,{ A

FIG. 3.-The normal dark toad together with the palehypophysectomized toad.

urines are to be received, processed and injected onthe same day. The method of housing Bufo isdescribed by Law (I949). We have not beensuccessful in maintaining the Bufo colony on flylarvae and meal worm, but found the animalsmaintain in good condition when fed on liveLucilia (blue-bottle fly). Elkan (1953) published amethod of breeding flies as food for amphibiaduring summer and winter months. Each Bufo isnot subjected to test more than I8 times a year andkept in rotation similar to Xenopus.

Other animals which have been used with lesssuccess for the early diagnosis of pregnancy are therat and the hypophysectomized frog and toad.

The rat ovarian-hyperaemia test. Zondek (I926)observed that after treatment with gonadotrophinthe ovaries of immature rats show a recognizablehyperaemia. Reiprich (I933, 1934) utilized thisreaction as a pregnancy test. The bibliography ofthe evaluation of this test has recently beenrecorded by Elek (I955). The condemning factorfor the rat as a test animal is that false positiveresults are obtained. This is not due to the in-jection of urine; uninoculated control animalsmay yield a positive picture.

The hypophysectomized frog or toad. Hogbenand Winton (I923) described the technique ofremoving the pituitary body and noted that as aresult the skin of the animal changed to a lightercolour (Fig. 3). This lighter hue remained per-manent even when the animal was transferred to adark background. Use was made of this observa-tion by Konsuloff (I934). He found that within amatter of minutes after the injection of pregnancyurine the pale skin of the hypophysectomized toador frog darkened.The colour change is due to the expansion of

melanophore cells, which can be seen easily in theweb of the foot (Hogben and Winton, 1922).Landgrebe and Waring (1941, 1943, I944) showedthat the pars intermedia of the hypophysis elabor-ates the specific melanophore excitant. The in-jection of gonadotrophic extract from pregnancyurine will cause the melanophore cells to expandand the degree of expansion can be compared tothe melanophore index chart given by Hogben andSlome (I931) (Fig. 4). In our experience (I956)the injection of known pregnancy urines yieldedunexpected failures in the Konsuloff and melano-phore expansion reactions in frogs where thepituitary appeared to have been successfullyremoved. Furthermore, when one takes intoaccount the time required to prepare the animalprior to inoculation, this test is no quicker thanthe spermiation reaction.

DiscussionIt has been shown that the reactions in the

hypophysectomized toads and frogs are unsuitedfor pregnancy diagnosis. The occurrence of false

1. 2. 3. 4. 5.

FIG. 4.-The Melanophore index.

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from

396 POSTGRADUATE MEDICAL JOURNAL August 1956

positives and false negatives in the rat test placesit at a disadvantage over the ovulation and sper-miation, Ascheim-Zondek and Friedman tests,where errors are confined to false negatives.The choice of test animal depends on the number

of tests the laboratory has to undertake.The Ascheim-Zondek test performed on four

mice diminishes the chance of error. (Two animalsare used in the Hogben reaction, one toad for thespermiation and one rabbit for the Friedmantest.) As immature mice have to be available andmultiple manipulations are necessary over a periodof four days, this test has been superseded by oneof the following three:The Friedman test is usually performed where a

very occasional pregnancy test is requested. Thecost of the rabbit limits its use.The spermiation reaction is the test of choice in

a laboratory not having to undertake more than afew dozen tests a day. The animals are indigenoushere, can be kept healthy and used repeatedly.The test, therefore, is both cheap, reliable and theresult is reported on the day of receipt of thepregnancy urine.The Edinburgh Pregnancy Diagnostic Labora-

tory established that the Hogben test is more rapid,less expensive and more accurate than the Fried-man and Ascheim-Zondek test (Crew, I939;Landgrebe, I939). The injected toad is placed ona platform in its test jar. The floor of the jar isexamined for eggs macroscopically at 24 and, ifnecessary, at 48 hours. The speed at which a largeseries of reactions can be read makes this animalthe choice for a Central Pregnancy DiagnosticLaboratory. In cases where surgical proceduredepends on the result of the pregnancy test boththe spermiation and ovulation tests are set up.The result of the spermiation reaction is com-municated on the day the specimen is received;the final report of both tests given on the followingday.

The occurrence of false positive reactions due toretained products of conception after parturitionhas already been mentioned. During six years theWatford Pregnancy Diagnostic Laboratory hasperformed some 60,ooo tests. Each year there havebeen about 50 per cent. positive and 50 per cent.negative reactions. On two occasions practitionershave queried the positive result. There is noguarantee that the return of menstruation was notassociated with an abortion or that, despite allcleaning precautions, a Xenopus egg from a positivetest had adhered to the test jar and been carriedover to a negatively reacting toad. The EdinburghPregnancy Diagnostic Laboratory, undertakingtwice this volume of work, have had no falsepositive reactions.A false negative reaction in a fully sensitized toad

is an indication of too few active chorionic cells.False negative results occur :

(a) During the first two weeks of pregnancybefor the peak period of clorionic gonadotrophinexcretion is reached.

(b) From the thirteenth week of pregnancy on-wards, when the urine may contain less than 3,0ooI.U. of chorionic gonadotrophin per litre.

(c) In ectopic gestation or threatened abortionand also in renal disease, where the excretion ofchorionic gonadotrophin may be impaired.

(d) In hydatidiform moles, although the expelledmass may be covered with vesicles, in utero in-sufficient active cells may be embedded in thewall of the uterus to liberate sufficient circulationof chorionic gonadotrophin for a positive reaction.It is of interest here to point out that during thepeak of gonadotrophin excretion, second to thir-teenth week of a normal pregnancy (or six to 18weeks after last menstrual period), it is possible toobtain a positive biological reaction in a i in 10or even I in o00 dilution of extract. Hence not untilafter this period can a reaction in these dilutionsbe diagnostic of molar pregnancy. A positivereaction in i in 200 dilution or higher at any periodof gestation is suggestive of hydatidiform mole orchorionic epithelioma. The gonadotrophin contentof cerebro-spinal fluid is never in greater con-centration than that of urine; it is therefore un-necessary to submit the patient to the discomfortand hazard of a lumbar puncture to obtain fluidfor the diagnosis of molar pregnancy.

(e) During radiation therapy of chorionic epi-thelioma. In three cases where metastases wereconfirmed at autopsy we repeatedly examined theurine during life. Reactions varied from negativeto positive in dilution of i in 200 according to theactivity of the neoplasm.

(f) Due to insufficient chorionic epithelial tissuein teratomatous tissue. Seminoma of the testeshave, in our experience, failed to yield a positiveHogben reaction.

(g) Due to the excretion of subnormal amountsof chorionic gonadotrophin during pregnancy.These cases are rare, but those which have beenfollowed up have had an uneventful pregnancy.Urines from these women have been tested onmultiple occasions on Xenopus known to react to50 I.U. of chorionic gonadotrophin. The speci-mens therefore contained 2,000 I.U. per litre orless.

SummaryI. Biological tests for the diagnosis of early

pregnancy have been described.2. Suggestions have been made for the choice

of the test animal, depending on the number oftests the laboratory expects to undertake.

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from

August 1956 SCHWABACHER : Laboratory Diagnosis of Pregnancy 397

3. Biological reactions in relation to normalpregnancy and pathological conditions have beendiscussed.

BIBLIOGRAPHYASCHEIM, S. and ZONDEK, B. (I928), Klin. Wschr., 7, 8.CREW, F. A. E. (1939), Brit. med. J., 1, 766.DUKES, C. E. (x939), ' Urine,' Oxford University Press.ELEK, S. D. (I955), Brit. med. J., 2, 600.ELKAN, E. (19S3), Brit. J. Herpetology, I, 147.FERREIRA, H. P. (1954), Postgraduate med. J., 30, 355.FRAZER, J. F. D. (I950), Brit. J. Herpetology, x, 84:FRAZER, J. F. D., and WOHLZOGEN, F. X. (x949), Lancet, ii,

306.FRAZER, J. F. D., and WOHLZOGEN, F. X. (1950), Brit. med. J.,

ii, 330.FRIEDMAN, M. H. (I929), Amer. J. Physiol., 90, 617.GALLI MAININI, C. (1947), Semana Med. B. Aires, I, 337.GUTERMAN, H. S. (945), J. clin. Endocrinol., 5, 407.HAINES, M., and FERREIRA, H. P. (I949), Nature, 164, 668.HAINES, M., LAW, J. W., and AUDREY MAY (1954), J. din.

Path., 7, 145.HOBSON, B. M. (1952), J. Obstet. &S Gyn., 59, 352.

HOBSON B. M. (i955), Ibid., 62, 354.HOGBEN, L., and SLOME, D. (1931), Proc. Roy. Soc. Ser B.,

o10 I0o.HOGBEN, L., and WINTON, F. R. (1922), Ibid., 93, 318.HOGBEN, L., and WINTON, F. R. (1923), Ibid., 95, S5.KAPELLER-ADLER, R. (934), Klin. Wschr., 13, 21.KLOPPER, A., and FRANK, H. (1949), Lancet, ii, 9.KONSULOFF, S. (1934), Klin. Wschr., 13, 490, 776.LANDGREBE, F. W. (1939), J. exp. Biol., I6, 89.LANDGREBE, F. W., and WARING, H. (1941), Quart... exp.PkyLA ol.

E ,3II.LANDGREBE, F. W., and WARING, H. (1943), Ibid., 33, I21.

LANDGREBE, F. W., and WARING, H. (i944), Ibid., 33, I.LAW, J. W. (I949), Bull. Inst. Med. Lab. Tech., x4, I75.REINHART, H. L., and BARNES, A. C. (I946), J. din. Endo-

crinol., 6, 664.REIPRICH, W. (I933), Klin. Wschr., 12, I44I.REIPRICH, W. (1934), Z. Geburt, Gynak., 109, 285.SCHWABACHER H. (I950), Brit. med. ., ii, 328.SCHWABACHER, H., ELKAN, E., and SCOTT, F. E. T. (I956),Medical Press 235, 513.SCOTT, L. D. (I940), Brit. J. exp. Path., 21, 320.VISSCHER, J. P., and BOWMAN, D. E. (1934), Proc. Soc. exp.Biol. N. Y., 31, 460.VOGE, C. I. B. (1929), Brit. med. J., ii, 829.ZONDEK, B. (1926), Z. Geburt. Gynak, 90, 372.

PERIPHERAL VASCULAR DISORDERS(Postgraduate Medical Journal)

Price: 3s 10d., post free

THE INVESTIGATION OF PERIPHERAL RAYNAUD PHENOMENONVASCULAR DISORDERS R. P. Jepson, F.R.C.S.Peter Martin, V.R.D., M.Chir., F.R.C.S. (Ed.)

COLLATERAL CIRCULATION IN THELIMBPERIPHERAL ARTERIOGRAPHY AND LMB

AORTOGRAPHY C. J. Longland, M.V.O., M.S., F.R.C.S.Robert E. Steiner, M.B., Ch.B., D.M.R., SYMPATHECIOMY AND PALLIATIVEF.F.R., and David Messent, M.B., F.R.C.S. OPERATIONS FOR OCCLUSIVE

ARTERIAL DISEASETHE MEDICAL MANAGEMENT OF A. J. Slessor, M.V.O., F.R.C.S.OBLITERATIVE ARTERIAL DISEASE OFTHE LEGS BLOOD VESSEL GRAFTINGRobert Semple, M.D., M.R.C.P. C. G. Rob, M.C., M.Chir., F.R.C.S.

Published byTHE FELLOWSHIP OF POSTGRADUATE MEDICINE

60, Portland Place, London, W.1.

RUTHIN CASTLE, NORTH WALESA Clinic for the diagnosis and treatment of Internal Diseases (except Mental or Infectious Diseases). The

Clinic is provided with a staff of doctors, technicians and nurses.The surroundings are beautiful. The climate is mild. There is central heating throughout. The annual

rainfall is 30.5 inches, that is, less than the average for England.The Fees are inclusive and vary according to the room occupied.

For particulars apply to THE SECRETARY, Ruthin Castle, North Wales.Telegrams: Catle, Ruthin. Telphone: Ruthis 66

copyright. on 14 July 2018 by guest. P

rotected byhttp://pm

j.bmj.com

/P

ostgrad Med J: first published as 10.1136/pgm

j.32.370.392 on 1 August 1956. D

ownloaded from