328

TABLE III-CALCULATED AND OBSERVED INCIDENCE OF MAXIMUM

SATELLITED CHROMOSOMES IN MONGOLS

The resulting distribution is compared with that actuallyfound in table ill.The correspondence between calculated and observed

figures is not so good as that shown in table 11. This is notsurprising since the figures for the expected frequencydistribution in that table were calculated so as to corres-

pond closely to the observed figures; and the number ofmongols in the series is comparatively small. However,the calculated figures do suggest two things which appearto be so; that most mongols will have either 3 or 4satellited G-group chromosomes; and that less than3 satellited chromosomes will be uncommon in them.

DiscussionTwo suggestions have been put forward as a result of

the findings: (1) that one of the two chromosome pairs21/22 is almost always satellited, the other in less than50% of instances; and (2) that the mongol chromosome isalmost always satellited.

It is still uncertain whether the chromosome pairs 21and 22 can be distinguished from each other. Size is anunreliable criterion (Patau 1965) and autoradiography hasshown no conclusive difference (Schmid 1963), althoughthere are suggestions that one pair is late replicating, asalso is the mongol chromosome (Patau 1965). It is,perhaps, futile to speculate on the identity of the mongolchromosome until pairs 21 and 22 have been rigidlydistinguished and defined, although our findings dosomewhat curiously echo the conception in the originalDenver Report (1960) that one of the two pairs (21) is

always satellited. The findings, however, suggest thatmongolism must in all cases be associated with trisomy ofeither 21 or 22 and that if it be established that one ofthese chromosomes is involved in mongolism, the other isautomatically absolved.Of greater interest perhaps is the light which the

findings may throw on the mechanism of non-disjunction.The secondary constrictions related to the satellites havebeen widely considered to be concerned with the nucleolus;this has not been convincingly demonstrated in mitoticchromosomes (although satellite association may be

secondarily due to the nucleolar association of theacrocentric chromosomes) but has been demonstrated inmeiotic prophase (Ferguson-Smith 1964). It may well bethat a large secondary constriction on an acrocentric

chromosome, which is closely associated with the nucleolus(and with other acrocentrics) is a contributory cause (oreven a necessary precondition) for non-disjunction of thisparticular chromosome to happen.We thank Prof. J. D. Boyd for his encouragement in this work,

which was supported by a grant for equipment from the WellcomeFoundation; Mrs. V. Harrison and Miss M. Kitson for technicalassistance and performing the satellite-counts; Mr. J. F. Crane andMr. G. Oakes for the photographic work; Dr. G. E. Roberts, Dr.S. B. Roberts, and Dr. A. M. Myers for giving us the blood fromtheir patients at the Ida Darwin Hospital, Fulbourn; and the 50undergraduates and others who allowed us to take blood-samples.

References at foot of next column

DR. FORD, DR. WOOLLAM: REFERENCES

Denver Report (1960) Lancet, i, 1063.Ferguson-Smith, M. A. (1964) Cytogenetics, 3, 124.Ford, E. H. R., Woollam, D. H. M. (1967) Lancet, ii, 26.Engmann, F. R. (1967) ibid. p. 1114.Miller, O. J., Mukherjee, B. B., Breg, W. R. (1962) Trans. N.Y. Acad. Sci.

24, 372.Patau, K. (1965) in Human Chromosome Methodology (edited by J. J.

Yunis); p. 155. New York and London.Schmid, W. (1963) Cytogenetics, 2, 175.

URINARY ŒSTROGEN IN LATE PREGNANCY

ŒSTRIOL EXCRETION AS A GUIDETO IMPENDING FŒTAL DEATH BEFORE TERM

R. F. HEYS*M.B. Manc., M.R.C.O.G.

J. S. SCOTTM.D. Glasg., F.R.C.S.E., F.R.C.O.G.

OF THE DEPARTMENT OF OBSTETRICS AND GYNÆCOLOGY

R. E. OAKEYPh.D. Durh., A.R.I.C.

S. R. STITCHPh.D. Brist., F.R.I.C.

OF THE STEROID LABORATORY, DEPARTMENT OF CHEMICAL PATHOLOGY

MEDICAL SCHOOL, LEEDS 2

Summary A new, rapid and simple method wasassessed for the determination of urinary

œstriol as a means of predicting the likelihood of intra-uterine death of the foetus, before term, in abnormalpregnancy. Assays were carried out in 403 pregnancieswhich were considered to be associated with increasedfoetal risk. Subnormal œstriol excretions were recordedin 59. Intrauterine death occurred in 18 pregnancies,including 2 cases of erythroblastosis fœtalis. In 12

(75%) of the remaining 16, the œstriol excretion wassubnormal when last measured before intrauterine death;in 3 others it was in the " borderline " range. The casesin which œstriol levels were not subnormal at the last

assay before intrauterine foetal death are discussed indetail. There were 38 premature deliveries followingsubnormal œstriol levels. 4 (11%) babies died in theneonatal period. It is concluded that a subnormalœstriol level carries a high risk of subsequent foetal death,and that this assay, if acted upon in the light of the clinicalsituation, can contribute to a reduction in perinatalmortality.

Introduction

A recurring need in clinical obstetrics is a reliablemeans of forecasting the likelihood of fcetal death in uteroduring the last two months of gestation-the stage at

which the foetus would stand a reasonable chance ofsurvival if it were delivered. Although no infallible meansof predicting festal death is yet available, it has beenclaimed that the determination of maternal urinarysteroids does give some indication of the likelihood ofintrauterine death (Zondek and Goldberg 1957, Greeneand Touchstone 1963, Wray and Russell 1964). Of thecurrent methods, those estimating the major urinaryoestrogens (aestriol) seem to be the most helpful in provid-ing an indication of foetal and placental function and thelikelihood of intrauterine death (Macnaughton 1967).This follows from the fact that the foetus provides themajor portion of the steroid precursors for oestriol bio-synthesis in the placenta (Frandsen and Stakemann 1961,Easterling et al. 1966). By contrast, progesterone pro-duction, reflected by the excretion of pregnanediol, occursin the placenta with little or no participation by thefoetus (Cassmer 1959, Klopper et al. 1966). (Estriolexcretion therefore measures both foetal and placental

* Present address: Halifax General Hospital, Halifax, Yorkshire.

329

function, whereas pregnanediol excretion is an index ofplacental activity only.Methods for the estimation of urinary oestriol, however,

have tended to be slow and expensive, and their clinicalvalue has been disputed. Thus, although Greene andTouchstone (1963), Banerjea (1962) and MacLeod et al.(1967) believe that such assays can provide helpfulinformation in the management of complicated preg-nancies, Booth et al. (1965) considered that routinedeterminations of oestriol in pregnancy by the methodsthen available were unlikely to save foetal life. The

delay in obtaining results contributed to their conclusion.The situation obviously requires re-evaluation in the

light of the simpler and more rapid assay techniqueswhich are now available.

Method

A method has been developed in these laboratories (Oakeyet al. 1967) for the estimation of total urinary Kober chromo-gens (mainly oestriol) by means of a colorimetric techniquewhich is more rapid than those previously described (Ittrich1960, Cartlidge et al. 1961, Frandsen 1963, Montagu 1964).With this procedure it is possible to obtain a duplicate resultwithin three and a half hours of completion of the twenty-four-hour urine collection, and for one technician to assay15 urines in duplicate with internal standards in one workingday with simple apparatus available in most hospitallaboratories.The present paper is an assessment of the clinical value of

the method as a guide to the prediction of impending foetaldeath during twenty-eight to forty weeks of pregnancy.(Estriol estimations were carried out in 403 complicatedpregnancies at Leeds Maternity Hospital on 1229 twenty-four-hour urine specimens obtained after twenty-six weeks’gestation from patients with living foetuses whose pregnancies

Fig. 1-Results of cestriol estimations before intrauterine death ofthe foetus.

Upper and lower limits of normal 24-hr. urinary oestriol excretionin late pregnancy and

" borderline " range are shown.

TABLE I-PREGNANCIES WITH EVIDENCE OF INCREASED RISK OF FCETAI.

DEATH

TABLE II—CESTRIOL EXCRETION BEFORE INTRAUTERINE DEATH

* See text. I.U.D. =intrauterine death of foetus.

progressed at least to the stage of viability (twenty-eight weeks)." Complicated " was defined as the presence of any featurewhich suggested the possibility of an increased risk of intra-uterine festal death. The urine was assayed at least twiceweekly when values were between the lower limit of the normalrange and the lower limit plus 2-5 mg. (according to gestationperiod) (fig. l)-referred to as the range of

" borderline "

excretion. This practice was followed as a result of experiencegained during this investigation.

Results(Estriol Excretion in Normal PregnancyThe upper and lower limits of twenty-four-hour

urinary oestriol excretion in the last ten weeks of normalpregnancy (fig. 1) were determined from an assay of 431twenty-four-hour urine specimens from 168 patientswith normal pregnancies (Oakey et al. 1967).(Estriol Excretion before Fcetal Death In UteroUrinary oestriol was assayed in 403 pregnancies when

clinical evidence suggested a risk of foetal death (table I).Intrauterine death occurred in 18 of these; 2 of thesedeaths were due to erythroblastosis foetalis.The findings in the remaining 16 pregnancies, none of

which were associated with gross anatomical abnormality,are summarised in table 11 and fig. 1. In 12 (75%) of thesethe excretion of oestriol was subnormal when last measuredbefore the fcetal death, and in 3 it was within the " border-line " range. Out of the 403 pregnancies, subnormaloestriol excretions were recorded in 59. Of these, 14were followed by intrauterine foetal deaths, 5 by signs offoetal asphyxia, and 15 by retarded fcetal growth (Gruen-wald and Minh 1961), and in 10 there was only a singlelow reading.Premature Delivery and Infant Survival after Subnormal(Estriol Excretion

There were 38 live births after premature deliveryof patients who had shown subnormal oestriol excretion.Of the 38 babies, 4 ( 11 ° ° ) died in the neonatal period.Out of 21 other pregnancies in which restriol excretionwas below normal but the baby was not delivered pre-maturely, there were 14 (67S) intrauterine deaths.2 of the deaths occurred between thirty-two and thirty-four weeks’ gestation and 7 after thirty-four weeks-i.e.,

330

at a stage when extrauterine survival was a reasonable

possibility.PaetaZ Deaths with Normal (Estriol Excretion

Of the 3 pregnancies with oestriol excretion within the" borderline " range at the last assay before intrauterine

death, 2 were associated with previous subnormal valuesand, in 1 of these, no assay was carried out for nine daysbefore death of the foetus. In the 3rd pregnancy therehad been a steady fall in the oestriol excretion before thelast estimation, which was carried out six days before thedeath of the foetus. Had present policy on assays in the" borderline " zone been in operation at that time, furtherassays would have been carried out before foetal death in2 of these pregnancies.The mother of the 1 baby that died four days after the

last assay with an oestriol excretion above the " border-line " range had a history of 2 unexplained stillbirths; thefirst of these infants weighed 4 lb. 8 oz. (2043 g.) at term,the second 5 lb. 14 oz. (2668 g.) at thirty-nine weeks.Between these pregnancies, she had 2 surviving infants,the 1st weighing 6 lb. 10 oz. (3008 g.) delivered bycxsarean section on account of unexplained foetal asphyxiaat the onset of labour, and the 2nd weighing 8 lb. 3 oz.(3713 g.) delivered spontaneously at forty-one weeks.The pregnancy which resulted in the death of the fcetus

reported here was, apparently, clinically uncomplicatedbefore intrauterine death in early labour at thirty-eightweeks’ gestation. The failure to sustain the rise inoestriol excretion should, however, be noted (fig. 1). It is

probably significant that the weight of this infant wassatisfactory (6 lb. 10 oz.; 3008 g.) for its period of gesta-tion. This suggests that foeto-placental function was goodto within a short time of foetal death as, indeed, the resultsof the oestriol assays indicated.

Discussion

From the findings in this last patient, it seems that

foeto-placental function may sometimes fail too rapidly tobe detected by oestriol assays at weekly intervals, but onour data this is uncommon. Certainly, where the historysuggests the possibility of rapid failure of a previouslynormally-functioning placenta, oestriol assays should becarried out frequently. In many other cases, however,intrauterine death in a previous pregnancy had evidentlybeen preceded by chronic placental insufficiency as

indicated by retarded foetal growth. Associated with thisthere was usually an unsatisfactory oestriol excretion. In

patients with such a history we found that a normalrising oestriol excretion throughout pregnancy indicateda good foetal prognosis and that it was of considerablevalue in avoiding unnecessary premature delivery. Thecontrast in oestriol values obtained in successive preg-nancies in such a case is shown in fig. 2.Our assessment of the data obtained from this method

of assay is that a subnormal or falling oestriol excretionindicates a high risk of imminent intrauterine death.

Subsequent foetal death in utero occurred in 24% of allpatients with subnormal oestriol levels. Furthermore,death occurred within four days of the first subnormalassay in two-thirds of these patients.These figures undoubtedly underestimate the risk of

early intrauterine death in such patients. Half of thewomen who had a liveborn infant after subnormaloestriol excretion were delivered within 7 days of the firstlow reading, after induction of labour or cassarean section.In most cases the decision to intervene was influenced by

Fig. 2-24-hour urinary oestriol excretion in successive pregnanciesin the same patient.

the results of the assay. It is likely that a number offoetal deaths were thereby avoided.With erythroblastosis foetalis, foetal death in utero is not

a consequence of inadequate placental function. Further-

more, for a period of some days or weeks before death,the foetus is in the virtually incurable state of hydropsfcetalis. These considerations and our data from cases oferythroblastosis foetalis (Heys et al. 1968) led us to,conclude that oestriol assays were unlikely to be of valuein improving foetal salvage in erythroblastosis foetalis.

Conclusion

Rapid oestriol assays, interpreted in the light of theclinical situation, can make a practical contribution toprevention of foetal death in utero. The value of themethod is principally in the thirty-two to thirty-eightweeks’ period of gestation if there is cause for concernabout the state of the foetus. If oestriol levels suggestserious foetal jeopardy, the likelihood of survival will inmany instances be greater if the baby is delivered, than ifit is allowed to remain in utero.

We are grateful to all our colleagues who in different waysfacilitated this investigation. Financial support (to R. E. 0.) fromthe Medical Research Council, and gifts of steroids from ScheringA.G. (Berlin) are gratefully acknowledged.

Requests for reprints should be addressed to R. E. 0.

REFERENCES

Banerjea, S. K. (1962) J. Obstet. Gynœc. Br. Commonw. 69, 963.Booth, R. T., Stern, M. I., Wood, C., Sharples, M. J. H., Pinkerton,

J. H. M. (1965) ibid. 72, 229.Cartlidge, V. K., Spencer, C. M., Swyer, G. I. M., Woolf, A. J. (1961)

J. Endocr. 22, xvi.Cassmer, O. (1959) Acta endocr., Copenh. suppl. 45.Easterling, W. E., Simmer, H. H., Dignam, W. J., Frankland, M. V.,

Naftolin, F. (1966) Steroids, 8, 157.Frandsen, V. A. (1963) The Excretion of Œstriol in Normal Human

Pregnancy. Copenhagen.— Stakemann, G. (1961) Acta endocr., Copenh. 38, 383.

Greene, J. W., Touchstone, J. C. (1963) Am. J. Obstet. Gynec. 85, 1.Gruenwald, P., Minh, H. N. (1961) ibid. 82, 312.Heys, R. F., Scott, J. S., Oakey, R. E., Stitch, S. R. (1968) Unpublished.Ittrich, G. (1960) Acta endocr., Copenh. 35, 34.

References continued at foot of next column

331

DR. HEYS AND OTHERS: REFERENCES—continued

Klopper, A. I., Turnbull, A. C., Anderson, A. B. M. (1966) J. Obstet.Gynœc. Br. Commonw. 73, 390.

MacLeod, S. C., Brown, J. B., Beischer, N. A., Smith, M. A. (1967) Aust.N.Z. J. Obstet. Gynœc. 7, 25.

Macnaughton, M. C. (1967) Am. J. Obstet. Gynœc. 97, 998.Montagu, K. (1964) J. Obstet. Gynœc. Br. Commonw. 71, 92.Oakey, R. E., Bradshaw, L. R. A., Eccles, S. S., Stitch, S. R., Heys, R. F.

(1967) Clin. chim. Acta, 15, 35.Wray, P. M., Russell, C. S. (1964) J. Obstet. Gynœc. Br. Commonw. 71, 97.Zondek, B., Goldberg, S. (1957) J. Obstet. Gynœc. Br. Emp. 64, 1.

PRACTICABILITY AND COST OF

ŒSTRIOL ASSAYS FOR SAVING BABIESIN A MATERNITY HOSPITAL

R. E. OAKEYPh.D. Durh., A.R.I.C.

S. R. STITCHPh.D. Brist., F.R.I.C.

OF THE STEROID LABORATORY, DEPARTMENT OF CHEMICAL PATHOLOGY

R. F. HEYS *

M.B. Manc., M.R.C.O.G.J. S. SCOTT

M.D. Glasg., F.R.C.S.E., F.R.C.O.G.OF THE DEPARTMENT OF OBSTETRICS AND GYNÆCOLOGY

MEDICAL SCHOOL, LEEDS 2

Summary After scrutiny of the relevant records fora three-year period, it was concluded that

15 out of 244 stillborn babies would have survived if thematernal œstriol excretion had been known. The

requisite daily assays by a simple and rapid method (1100per annum) would have cost £800 per annum, or £160 perbaby saved. Since larger numbers could be handled withthe same resources in a central service, the cost could bereduced to £50 per baby saved. It was concluded that evenwhen the resources for chemical pathology are limited aservice for the determination of œstriol of the typedescribed should have high priority.

Introduction

ASSESSMENT of the value of a laboratory investigation inmedical practice must relate resources to benefits.Resources are relatively simple to determine; less simpleto determine are the benefits. In the case of oestriolassays to prevent perinatal death, however, an approxi-mate balance sheet can be drawn.We have elsewhere recorded our results with a simple

and rapid method for the determination of urinaryoestrogen (Oakey et al. 1967, Heys et al. 1968 a, b).

Potential of Method

In the years 1960-62 inclusive, when oestriol assayswere not carried out, there were 6636 births at Leeds

Maternity Hospital. We have scrutinised the records ofthe 244 stillbirths in this period which were recorded afterthirty-two weeks’ gestation. In 84 of these, oestriol

assays would have had nothing to offer (e.g., patients notseen before labour or festal death, death due to an acuteepisode such as cord prolapse or abruptio placentas, deathdue to gross fcetal malformation or rhesus isoimmunisationand unrelated to possible placental failure). In a further106 of these stillbirths there was no substantial evidencethat cestriol determinations would have been helpful (e.g.,deaths associated with post-maturity and multiplepregnancy). In the remaining 54, in which oestriol assaysmight have forecast the likelihood of intrauterine death,only 30 had clinical features which could have indicateda need for assay. From our subsequent experience, 75%of these intrauterine deaths could have been predicted byoestriol estimations (Heys et al. 1968a). On this basis, 22 of

* Present address: Halifax General Hospital, Halifax, Yorkshire.

these 30 could have been forecast and possibly avoided-9% stillbirths after thirty-two weeks’.

In the period when oestriol assays were in use, 11 % ofbabies delivered prematurely after low oestriol excretiondied (Heys et al. 1968a). It must be assumed that a lossof this order would have occurred and that only 19 of thebabies would have survived if they had been born aliveafter premature delivery. Thus, we have taken as a

conservative estimate that 15 of these 30 stillborn babiesin our hospital would have survived had oestriol levelsbeen available for guidance-i.e., 5 per annum.

Number of CEstriol Assays RequiredWe estimated that 1100 assays annually would have

been required to prevent these deaths. This calculationwas based upon a review of all deliveries in the three-yearperiod, 1960-62, to find how many women had evidenceof previous unexplained stillbirths, retarded foetal growth,or severe toxacmia-the features which we now take as anindication for oestriol assay (Heys et al. 1968b). Weassumed that once-weekly oestriol estimations would havebeen carried out from thirty-one weeks’ gestation or fromthe time when the indication first arose. The totalarrived at on this basis was increased by 20% to allow forincreased frequency of assay when results were in the" borderline " range.

Technical and Laboratory Resources RequiredOur technique (Oakey et al. 1967) is within the scope

of a general hospital laboratory and does not require aspecial steroid unit. It is less complex than 17-keto-steroid and 17-hydroxycorticosteroid estimations now

widely used in general laboratories. A means of measuringoptical density is the most complex item of equipmentused. To be of value, the results must be obtained on theday of completion of the urine collection. The assaysmust therefore be carried out daily and this will requirethe services of one full-time technician.

Cost

Allowing for a technician’s salary, and the small cost ofchemicals, we calculated that 1100 assays in an existinglaboratory would cost about E800 per annum or El 60 foreach baby saved. This represents 0-3% of the cost ofrunning our hospital (E313,167 in 1966). However,since one technician can carry out 15 assays per day induplicate with internal standards, he could provide resultson approximately 4000 specimens per annum. If theresources could be fully employed in a central service, thecost per baby saved would drop to about a third, say, E50.

Discussion

In common with almost all medical services, chemicalpathology is circumscribed by limitations of finance,staff, and accommodation. Obstetricians have hithertomade relatively few demands on special biochemical

services, possibly because they have inherited a mechan-istic approach to their discipline. Physicians and surgeons,on the other hand, have in recent decades increasinglyexploited the skills of the chemist, and a doubling of thenumber of requests for chemical pathology investigationevery four to five years has become standard (Eaton andLathe 1963). Most of the demands for complex bio-chemical tests are concerned with diagnostic problems;often, the test only excludes a particular diagnosis and thespecific benefit is almost impossible to evaluate. In

obstetrics, however, with the development of rapidmethods for urinary oestriol estimation (Schindler and