The anterior pituitary-like hormone in late pregnancy toxemia

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nullified a possible progestin effect. In all cases bleeding occurred during administration or after cessation of medication. The hot flushes and other symptoms characteristic of the menopausal syndrome were completely relieved during the period of treatment, but recurred in all cases some time after cessation of medication. kind

I wish to express assistance and

my grateful appreciation to Dr. Walter efforts in making this work possible.

T. Dannreuther

for

his

REFERENCES

(1) Newtaedter,

T.: AK J. OBST. & GYNEC. 29: 680, 1935. (2) Frar&, R. T.: 97: 1852, 1931. (3) To be published. (4) Fran& B. T., ami Gold(5) iVla.rer, C., and G&l.stein, L.: Clinical berger, iK. A. : J. A. M. A. 90: 376, 1928. (6) Endocrinology of the Female, Philadelphia, 1932, W. B. Saunders Co., p. 165. Neustaedter, T. : Endocrinology 20: 639, 1936. (7) FZ&mmm, C. F. : Western J. Surg. 45: 61, 1937. (8) Hisaw, F. L., a,nd Leonard, S. L.: Am. J. Phyaiol. 92: 574, 1930. (9) Hkaw, F. L.: AM. J. OBST. & GYNEC. 29: 638, 1935. (10) H&&w, F. L., Greep, R. O., and Fe&d, H. L.: Proc. Sot. Exper. Biol. & Med. 36: 640, 1937. (11) Kwfmmnn, C. : Zentralbl. f. Gynak. 56: 2058, 1932. (12) Idem: Ibid. 57: 42, 1933. (13) Clazcberg, C.: Ibid. 56: 2460, 1932. (14) Elden, G. A.: Endocrinology 20: 47, 1936. (15) Werner, A. -4.. and Collier, W. D.: J. A. M. A. 100: 633, 1933. (16) Idem: 5. A. Id. A. 101: 1466, 1933. (17) Werner, A. A., Jones, Grey, Roberts, J., Brom, G. O., Neikm, C. H., and Bothermich, N. 0.: J. A. M. A. 109: 1027, 1937. (18) RooJG, J.: Endocrinology 19: 269, 1935. (19) Kaufmann,, C.: J. Obst. & Gyuaec. Brit. Emp. 42: 409, 1935. J.

A.

M.

A.

27 EAST

93m

STREET

THE ANTERIOR A GEORAJE VAN

PITUITARY-LIKE HORMONE PR.EGNANCY TOXEMIA” SUMMARY

S.

OF

M.D.,

SMITH,

(From

the Fearitig

Research

SINCE 1932

RESULTS AND

BROOKLINE,

Laboratory,

IN LATE

0.

WATKINS

SMITH,

PH.D.,

MASS. Free

Hospital

for

Women)

T

HE presence of abnormally high levels of the anterior pituitarylike hormone+ in the serum and urine of patients with late pregnancy toxemia and eclampsia was first reported in 1933.2 This finding has since been confirmed.3-6 Quantitative assays of the anterior pituitary-like hormone in the serum and urine of patients during normal and pathologic pregnancies are still being carried on in this laboratory and at the present writing (March, 1939) 560 serums and 390 urines from 173 pregnant women have been titered. Results concerning 127 of these pregnancies have been previously reported in

*The Mrs. William Lowell Putnam investigation of the toxemias of pregnancy. tCommonly referred to as A.P.L. B~monylras: P.U. (pregnancy urinary hormone). pregnancy prolan, antuitrin S, follutein, antophysin (now labslled korotron). ‘A.P.L..” etc., etc. Since this gonadotropic factor of human pregnancy has been shown to be a product of chorionic t&sue,’ the logical appellation for it would be “chorionic gonadotropic hormone.” Analyses of pladental tissue and tests upon hypophysectomized rats by us@ have demonstrated that the excessive gonad-stimulating power of the serum of patients with late pregnancy toxemia is due to a factor which does not differ qualitatively from that found in normal pregnancy. Anselmino and Hoffmain+ have reached the same conclusion concerning the gonadotropic hormone of late pregnancy toxemia.

detail.2,

i-10

The total data which have accumulated during the seven years since the inception of this investigation seem sufficient to warrant separate summary. Although urines and placentas from a large number of the cases included in this report have been assayed for the anterior pituitarylike hormone, the results of serum analyses only will be summarized. In the main, the findings in urines and placentas have been eonfirmatory of the serum titers, but the method for extracting the anterior pituitary-like hormone from the serum (see below) is more reliable than are those for urine or placental tissue. Moreover, the quantitative determination of any urinary constituent depends upon the accurate collection of a twenty-four-hour specimen. This can be accomplished only under the most rigidly supervised conditions, whereas the collection of blood involves no such chance for error. We have, therefore, in recent years come to rely almost entirely upon serum values. Furthermore, in any attempt to predict late pregnancy toxemia one must necessarily assay serum, since the urinary values for the anterior pituitary-like hormone do not rise to abnormal levels until toxemia has become clinically manifest, whereas excessive amounts in the serum are demonstrable four to six weeks prior to the development of toxic signs.iO Our criteria for normal levels of the gonadotropic factor in pregnancy are based upon the analyses of 210 serums from 64 patients who were normally pregnant and continued so to delivery. In 36 of these cases, serum assays were performed at intervals from as early as the sixth week of gestation to delivery. The rest were studied by single specimens during the last trimester. A peak in the curve of the a.nterior pituitary-like hormone in both the serum and urine has been found to occur at about the time of the second missed period,1°-13 the values at this time being frequently as high as or higher than those observed in cases of chorionepithelioma or hydatidiform mole. This high level, however, is maintained for only a comparatively short, time, one to three weeks, a fact which supplies an important means of differentiating between early pregnancy and mole. By the beginning of the fourth month the anterior pituitary-like hormone of serum and urine has reached a constant low level and in no instance of normal pregnancy between the fifth and eighth months have the serum values been higher than 100 R.U. per 100 cc. During the last four weeks of gestation in a number of cases the serum anterior pituitary-like hormone rose somewhat above this amount. Values above 100 R.IJ. per 100 cc. prior to the fifth missed period or within four weeks of term are therefore not considered abnormal. In Table I the 173 cases studied in late pregnancy have been divided into those with normal and those with high levels of anterior pituitary-like hormone. None of the 11 with a clinical diagnosis of nephritic toxemia or essential hypertension had elevated serum anterior pituitary-like hormone-that these cases were probably diagnosed correctly is supported by the fact that signs were present by the second trimester. A total of 13 women had premature deliveries, 5 of these with normal and S with excessive amounts of anterior pituitary-like hormone. Of the 85 patients

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TABLE SUMXARY

I.

JOURNAJl

SERUM OF TOTAL

OF

ANTERIOR RESULTS.

oHSTE~T~~ICS

AND

GYNECOLOGY

PITUITARY-LIKE E~ORAIONE 1932 TO PEBRUARY, 1939,

-

IN LATE PREGNANCY. INCLUSIVE. 173 CASES

---L.~.

('LISICAL

DIAGNOSIS DELIVERY

AT

TIAIE

PATLENTS ANTERIOR

OF

ormal pregnancy G-4, 71.1 per Pre-eclampsia or edampsia 10,” 11.1 per Nephritic toxemia 9,t 10 per Essential hypertension 2.2 per 2,t Premature delivery at 6 to S 5, 5.6 per months I___.---_---__ Totals 90 *Two of these patients died of eclampsia (1 known to have gone iSymptoms began

through a subsequent early in pregnancy.

cent cent, cent rent cent

--I i75,

Table

II

summarizes

month

by

82 of

repeated

the

serum

above

173

with

to

83 autopsy).

with

no

came

DIAGNOSIS DELIVERY

Normal pregnancy Pre-eclampsia Nephritic toxemia Essential hypertension Premature delivery

,ZT TIAIE

at

7

OF

to

months

35, 2, G,” 1,” S 5.

72 4 12 2 JO

began

early

in

9.7 per

cent

patients

the

serum no

who

of

per per

cent cent

are

pituitarypregnancies

90 per cent having other 10 per cent followed

from

determining

the

whether

FOR SERUM ANTERIOR SEVENTH MONTHS OF

PATIENTS ABNORMAL

percent. per cent cent

anterior

were

purpose

others

normal

hormone, and the

WITH NORNAL PITuITARY-LIKE HOII;LIONE

per

Two

for

___--50

Totals *Symptoms

PATIENTS ANTERIOR

cent

toxemia.

were

TABLE II. Sum~s~ OF RESULTS OF RI%,a:aTEn ANALYSES PITI!ITAT;Y-LIKE HORA~ONE DURING THE FIFTH, SIXTH, AND PRE~XAXCY. s:! (1.2SES CLINIC,\L

per

-

pregnancy

analyses

90.3

8,

classed as pre-eclamptic or eclamptic, 75 had high values like hormone and 10 had normal levels. Thus there among the 83 patients with high anterior pituitary-like been diagnosed as having pre-eclampsia or eclampsia having delivered prematurely. fifth

PATIENTS WITH HIGH ANTERIOR PITUITARY-LIKE HORMONFJ

WITH XORAIAL PITUITARY-LIKE HORXONE

WITH RISE

25,

78

per

cent

7,

22

per

cent

32

-

pregnancy.

or not late pregnancy toxemia could be predicted prior to its clinical manifestation on the basis of serum anterior pituitary-like hormone values. In none of the 7 patients diagnosed as having nephritic toxemia or essential hypertension were the values at any time above the limits of normal. Twelve women had premature deliveries, 5 with normal and 7 with high levels of serum anterior pituitary-like hormone. Of the $7 women who developed pre-eclan@a after the seventh modh, 25 had had aa wnmistakable rise in se-rzlm anterior pU&ary-like hmmone fear to sti weeks Fe&&y. Two women, in whom repeated serum analyses failed to reveal any abnormality, later developed toxemia. Thus none of the 32 women whose serum values exceeded normal levels during the fifth, sixth, or seventh month experienced a continued uneventful gestation. Twenty-five developed pre-eclampsia” and 7 delivered prematurely at about the time when toxic signs might have been expected to appear. CONCLUSIONS

1. Pre-eelamptic toxemia and eclampsia are usually (88 per cent in this series) characterized by the finding of excessive amounts of an*It should be emphasized that, althouxh the highest pituitary-like hormone have been encountered in cases excess of this factor does not consistently bear a direct of symptoms and signs.

of

values

for

eclampsia.

relationship

serum

the

to

the

anterior

degree

severity

of

terior pituitary-like hormone in the serum. This excess has betit found to precede clinical signs by four to six weeks. 2. In a small pewentage of cases with a clinical diagnosis of pweclampsia or eclampsia, the serum anterior pituitary-like hormone tlors not exceed normal levels. 3. In patient,s quite definitely diagnosetl as nephritic or hypertensivca (symptoms beginning earlier in pregnancdy), the serum auterior pituitary-like hormone is normal. 4. Premature delivery may or may not be associated with exc?essivr anterior pituitary-like hormone. 5. The finding of high levels of this substancJe in the serum during the fifth, sixth, or seventh month warrants the prediction of impentling pre-eclampsia or premature delivery. Quantification of serum anterior pituitary-like hormone during the fifth, sixth, and seventh months of pregnancy has become an accepted procedure both in this laboratory and at the New England Deaconess Hospital (Boston), where diabetic patients (in whom the incidence of prema.ture delivery and pre-eclamptic toxemia is high) are being studied.* It has been found of wnsiderable value as a means of predicting later+ trouble and governing the management of patients. For this reason it seems advisable to include in this report, an exacat description of horn these tests are performed. At two- to three-week intervals between the fifth and eighth mont,hs of pregnanq, IO C.C. of venous blood are collected without any anticoagulant and the cl@ allowe to flmn. The wrum is separated, cleared of any cells by centrifugation and measured accurately into four 15 cx. centrifuge tubes, each tube to contain 1.0 c.G., 0.5 f.c., 0.5 fx., ad 0.:: cx., respectively. About 10 C.C. of 95 per raent ethyl alwhc~lt are added to each tube, the contents mixed and the tubes place11 in the refrigerator for twelve to 18 hours. After centrifuging and pouring off the supernatant alcohol, the precipitate is ma&et1 (stirring rod and shaking) tmce with ether, approximatclg Ir! c.?., and again centrifuged. The ether is then poured off and the tubes alloTvet to tlrnin for a few minutes, after which 2 C.C. of normal saline is atltled and rriiswl *Of the 173 patients followed, 31 were diabetic patients under the care of Ifrs. E. P. Joslin, Priscilla White, and R. S. Titus, of the George F. Baker Clinic of th? New England Deaconess Hospital, Boston. In 25 of these, repeated analyses were performed. They are not considered separately, since the conclusions reached for’ There were 10 the whole group apply equally well to diabetic pregnancies alone. normal pregnancies, all with normal levels of anterior pituitary-like hormone in the serum. In the one case of nephritic toxemia, the values for serum anterior pituitarySeven diabetic patients delivered prematurely, 3 like hormone never exceeded normal. All with normal and 4 with high levels of serum anterior pituitary-like hormone. 13 of the diabetic patients who had symptoms diagnosed as pre-eclampsia had excesThus, there were no sive amounts of anterior pituitary-like hormone in the ser;um. normal pregnancies among the 17 cases with high antenor pituitary-like hormone. Of the 1.5 patients in whom an abnormal rise was detected during the flfth, sixth, and seventh months, 11 developed pre-eclampsia some weeks after the flrst abnormal As in nondiabetic patients. premature rise, and the other 4 delivered prematurely. delivery may or may not be associated with high anterior pituitary-like hormone, but the incidence of premature delivery (rather than prc-eclampsia) in the high anterior pituitary-like hormone group was considerably greater among the ,liab&t, patients (23.5 per cent) than among the nondiabetic patients (6 per cent). In all of our studies of pregnant diabetic women we have received the indispensable cooperation of Dr. Priscilla White in the collection of specimens and supplying of clinical data. Of the 31 diabetic patients included in this series. 27 are being with especial reference to the clinical appliseparately reported by her and others,‘” cation of the quantification of serum anterior pituitar>--like hormone in diabetic preannncies. .:The serum is concentrated by alcohol precipitation for two leasons: first, to I’?move estrogens which when injected simultaneously enhance the anterior pituits;;; second, to reduce the toxicity. 1 like hormone reaction in immature rats, and, method describe<1 gives quantitative recovery of anterior pituitary-like hormone :ril
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with the precipitate, forming a smooth emulsion. The ether held back by the precipitate and saline is removed by stirring the contents up onto the sides of the centrifuge tubes while rotating, warming with the hand and blowing into the tubes. (No more heat than this should be applied--to avoid the possibility of any destruction of ‘the hormone.) The stirring rods are washed down with 3 C.C. of saline, making the total content of each tube 6 c.c., this being the final test solution. Each extract is administered subcutaneously into a nineteento twenty-one-day-old female rat, the injections being given twice a day for three days, 1 C.C. per injection. On the morning of the fifth day (ninety-six hours from the first injection) the rats are sacrificed and the ovaries examined for the appearance of grossly visible discrete corpora l&tea. The anterior pituitary-like hormone of the serum being tested is considered normal provided none of the test animals or only the animal receiving the extract of 1.0 C.C. (test for 100 R.U. per 100 CC.) gives a positive result. If all the animals sho!v discrete corpora, the serum may be said to contain more than 333 R.U. of anterior pituitary-like hormone per 100 CA., a definitely elevated level. If the animal receiving the extract of 1.0 C.C. and the 2 animals receiving the extract of 0.5 CC. are positive, whereas the fourth animal has no corpora lutea, the serum may be said to contain 200 R.U. of anterior pituitary-like hormone per 106 c.c., an amount exceeding normal. For clinical purposes only 4 such tests arc required with any I serum, unless the test for 333 R.U. per 100 C.C. is negative and the 2 tests for 200 R.U. per 100 C.C. fail to give In this event the test for 200 R.U. per 100 C.C. is repeated on 2 check results. more immature rats, and if more than I of the 4 animals receiving an extract of 0.5 cc. show definite corpora, the anterior pituitary-liko hormone may be considered to exceed normal levels. Inasmuch as the reading of the end point requires considerable experience and even then is not always clear-cut, the importance of making check determinations with further specimens from the same patient and of using a series of at least 4 No patient should be reported as tests for each serum cannot be over-emphasized. having excessive anterior pituitary-like hormone unless high values are acquired in at least 2 specimens of serum. DISC’C’SYlON

It is logical to assume that the abnormal rise of serum anterior pituitary-like hormone, being the earliest indicator both of an hormonal imbalance and of impending pre-eolampsia or premature delivery, is most closely associated with the p+aary etiology. This, however, is not the only hormonal abnormality which has been found to characterize determination of the estrogens has revealed the disease. Quantitative that these are relatively low in the urine, serum, and placentas of (the excretion product of toxemic patients.‘, ?-lo Urinary pregnandiol progestin) has also been found to be below normal levels.14, 15, I9 A detailed study of urinary estrogens,*4, lo in which 3 different estrogenic factors have been assayed separately, indicates that progestin deficiency results in a marked change in the metabolism of the estrogens and in their more rapid destruction. Evidence for such a progestin-deficient metabolism of the estrogens has been found thus far in 8 pre-eclamptic patients at the time of the development of toxic signs.17 The hypothesis has been proposed that some change associated with a faulty metabolism of the estrogens due to progestin deficiency (such as a toxic estrogen breakdown product) may be directly responsible for the, generalized vascular derangement of pre-eclampsia and eclampsia.14 Whether or not this hypothesis proves to be correct, our quantitative

S3IITH

AND

SMITH:

L,ATE

PREGNANCY

TOXEMIA

fxl

studies of pre-eclampsia point toward the following sequence of events : (1) Excessive amounts of circulating anterior pituitary-like hormone. (2) a decrease of pr0gest.m and “total’? estrogen at a time wheu they normally increase, and (3) a strikin, tp change in the metabolism of estrogens due to reduced progestin, this last abnormality being demonstrable at the time of the onset of clinically recognizable toxemia. The role of anterior pituitary-like hormone, which distinguishes human pregnancy from that of practically all other mammals, in the physiology of normal gestation has not been ascertained, but one naturally supposes that this gonadotropic factor plays an important part in the placenta’s elaboration of progestin and estrogen. since it ii: such a potent stimulator of these factors from the ovaries of experimental animals. Of course the placenta has not been proved the source of progestin and estrogen, but the circumstantial evidence pointing toward it as the source is overwhelming. There come to mind 3 possible explanations for the apparent paradox introduced by the finding of high levels of anterior pituitary-like hormone together with a deficiency of progestin and estrogen in pre-eelampsia. 1’1) The increase of the gonadotropic substance map be a prot&ive measure, an attempt to counteract a failing production of progestin and estrogen. If this explanation were correct, one would expect the increase to follow rather than precede the drop in lurinary estrogens and pregnandiol. (2) Excessive amounts of anterior pituitary-like hormone, due to an imbalance of placental function. over a sufficiently long period may have, in certain patients, an inhibitory iiifluence upon the secretion of progestin and estrogen just as its prolonged administra tion experimentally is known to result in ovarialr atrophy due to t,he stimulation of antibodies. If this were the ease. the presence of antibodies in the material being assayed would inhibit the reaction and depress the titer. (3) T o us, the most plausible explanation lies in a failing utilization of anterior pituitary-like hormone in t,he produci.ion of these steroids and consequent building up of the l.evel of anterior pituitary-like hormone in the blood. This third hypothesis is supported by the following considerations. In normal pregnancy a ra.pid decline in the level of anterior pituitary-like hormone takes place during t,he third and fourth months. This decline coincides both with a rapidly increasing elaboration of estrogens and progestin (based 011 urinary estrogen and pregnandiol) and with the “taking over,” presumably by the placenta, of the functions of the regressing corpus luteum. These events are in accord wit,h the idea t,hat anterior pituitary-like hormone is utilized normally for the secretion of estrogen and progestin by the placenta, and that, in being so ut,ilized, its level in the circulation is kept low. Since the low level of serum anterior pituitary-like hormone is maintained during the fifth, sixth, and sevent,h months of normal gestation, during which time t.he production of estrogens and progest,in steadily increases, an abnormal rise would be interpreted as failure of utilization. On this basis one would place the primary et,iology of pre-eelampsia in whatevw I~~~SPS Ihe

624

AYE,KICAiX

JOUKNAL

OF

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AND

(I’YNECOLOt3Y

placenta to be deficient in the utilization of anterior lntuitary-like hormotre for production of estrogen and progestin. It is entirely conceivable that a failing utilization of anterior pituitary-like hormone might, be accompanied by decreased formation in which event no abnormality would be revealed by its quantificatiorl. It is further conceivable that decreased format,ion might result from damage to the placenta by toxic separation, pre-eelampsia, or eclampsia. Such situations could exp.lain the normal values for serum anterior pituitary-like hormone fomid in 12 per cent of the pre-eelamptic aud rclaniptic patients as reported above. The fact that 22 per cent of the lmtients in whom an abnormal rise of serum anterior pituitary-like hormone was detected during the fifth, sixth, and seventh months delivered prematurely deserves comment. All the factors involved in the initiation of labor have not been ascert,ained but quantitative studies in wonlet hare revealed a marked drop in estrogens Pjust.before deliver,v,lG. Ii. lo alid a decrease in progestin has also been indicated by the changed metabolism of the estrogens which accompanies normal labor.“! ” The same hormonal changes which precede am1 accompany the onset of pre-eclampsia, therefore, appear to pertail rather suddenly at the time of normal delivery. Under certain conditions these changes might be associated with premature delivery rather than toxemia. REFEREROES ( I j Gey, G. O., Setynr, 8. E., trd TlflEmnrt, L. M,: Science 88: 306 1938. (i’) Smith, G. T., n?bd Smith, 0. IV.: Pro<>. Hoc. Exper. Biol. & Med. 30: 918, 19:3X (3) Heim, K.: Klin. Wchnschr. 13: 1614, 1934. (4) Ba~rg, R., anil Le Grand, G. : Arch. internat. de m&l. expC. 10: 551, 193.5. (5) Anselmino, E. J., and Hqffmann, F. : Ztschr. f. Geburtsh. u. Gynak. 114: 52> 1936, (6) Tmwy, B., and Parker, P. : Endoc~rinology 21: 687, 1937. (7) Bmith, G. T., oad IYWLWL, 0. TV.: Am. J. Physiol. 107: 128, 1934. (6) Idem: Surg. Gynrc. Obst. 61: 27, 1935. (9) I&m: Ibid. 61: 175, 1935. (10) Idem: AK J. OUST. & GYNEC. 33: 365, 1937. (71) Idem.: New England J. Med. 215: 9OS, 1936. (12) Brcwnf, J. 8. L., mnnd Venning, E. M.: Am. J. Physiol. 116: 18, 1936. (In) Eum. c, IT., Eoh,ls, C. I,., and 7Vonder, L?. H. : J. A. 1%. A. 108: 287, 1937. (14) Smith, G. P.. rend Bntiith, 0. TV.: AM. J. OBST. & GYNEC. 36: 769, 793s. (15) 7t’eiZ. Paul G.: Science 87: 72, 1938. (16) Cohen, R. I,., XayriGdlL, G. F., aord Watscnt, X. : T,ancet 1: 674, 1935. (17) Rmith, G. V., Smith, 0. JT., and l%nuus, CT.: Am. J. Physiol. 121: 98, 1935. (18) White, P., T&m, R. R., dodin. E. P., md Hunt. H.: Am. J. N. Rr. In press. (19) Rmith. G. r.. mn,d ,%~ith, 0. TV.: .isr. J. OBST. & Civzsir’. (Tn press.)

Ucko,

H.

H.:

Staining

of

Vaginal

Smears,

Lancet

1:

1113,

1!1RS.

The author believes that the practical value of repeated vaginal smears is not He sugrecognized sufficiently because of technical difficulties in staining them. gests fixing in equal parts of alcohol and ether, in which the slides can be kept indefinitely. Before staining, the slide is covered with methyl alcohol for two minutes, and then stained with diluted Giemsa prepared by adding 10 drops of the standard solution of 5 c.c. of distilled water. Staining requires twenty minutes. Cornified cells are stained red, partially cornified ones violet, and noncornified cells blue. CARL

P.

HUHEEL