45
Mutation Research, 33 (1975) 45--54
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SOME EPIDEMIOLOGICAL ASPECTS OF T E R A T O G E N DETECTION
JAMES R. MILLER Department of Medical Genetics, The University of British Columbia, Vancouver, B.C. (Canada)
(Received July 10th, 1975) (Accepted August 25th, 1975)
Introduction
Numerous reports have been published on the epidemiology of congenital defects and I see no virtue in re-examining the strengths and weaknesses of such studies. I think it is a fair summary to say that while they have yielded much information relating to the association of defects with such variables as geographic location, season, birth order, social class, occupation and ethnic origin, these studies have n o t identified a single teratogen. Since the implicit theme of this symposium is the rapid detection of environmental agents which are mutagenic, oncogenic or teratogenic, I propose to focus on problems associated with the monitoring of newborns and the potential value of including embryos and fetuses in such monitoring schemes. It would be pleasant if there was no need to worry about such monitoring, because adequate in vitro or in vivo tests using experimental animals existed. Shepard [31] has pictured the problem as a series of defence walls with gaps through which undetected teratogens can pass. At present, no defence system is perfect and, since potentially deleterious agents may be introduced into the environment without passing through any screen, it must be recognized that the pregnant human female is the ultimate test organism. Once this is recognized, it is essential that methods be devised for the constant evaluation of agents to which pregnant w o m e n are exposed and the development of screening methods for the monitoring of as many conceptuses as possible. Although one of the principal themes of this symposium is the similarity of oncogenic, mutagenic and teratogenic processes, it is equally important to recognize critical differences between them. Some of these have been c o m m e n t e d upon already but, in my opinion, the critical issue revolves around a much neglected organ, the placenta, "the organ of metabolic interchange between the fetus and mother". It is primarily because of the placenta that in vitro testing for teratogens is still only a theoretical possibility and that in vivo tests remain a matter of considerable debate. As Wilson [35] has pointed out, aside from
46 the pregnant woman, the ideal test organism does not exist. If this fact were acknowledged, the approach to monitoring programs, clinical trials etc. would be more realistic. General epidemiological considerations Oakley [25] has summarized succinctly a general epidemiological approach to the detection of teratogens (Fig. 1). Of course, the transition across time from the recognition of an epidemic (point B) to the final withdrawal of the offending agent (point G) is not smooth. Invariably, difficulties and delays develop at points C and D; i.e., with respect to the development of multiple hypotheses about the cause of the epidemic and the testing of these hypotheses. The message is clear: even though a system may be devised to detect the presence of a new teratogen, there is no guarantee that the offending agent will be uncovered and removed rapidly. Using thalidomide as a model, Oakley has demonstrated how two types of testing of hypothesis can be employed at point D (Fig. 2). McBride's cohort approach was possible because a group of w o m e n in his hospital began to receive thalidomide as a therapy for morning sickness. On the other hand, because the epidemic already existed in Germany in 1959, Lenz had to carry out a retrospective case control study. In a way, McBride's rapid implication of thalidomide was almost fortuitous -- the outcome of a clinical trial for another purpose. The usual course of events is that observed in Germany; i.e., a long time lag between the concern about the presence of a teratogen and its removal from the environment. Unfortunately, there is no escaping this fact, even in the most ideal situation.
Monitoring In reference to congenital defects, the term "monitoring" denotes an early warning system designed to detect evidence of a new teratogen in the environment either b y the appearance of a new malformation or malformation syndrome, or by an increase in the incidence rate of known conditions. N e w c o m b e [22] has proposed the following three key elements in a monitoring system: (1) a high degree of specificity in diagnosis and classification; (2) early detection,
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47 Recognition
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and (3) improved methods for the recording, rapid accumulation and communication of data. Although the principal feature of such a system is urgency b o t h in accumulating data and reporting rates and trends, this must not be accomplished at the expense of accurate diagnosis. Perhaps the specificity stress in point 1 should be extended to include accuracy, completeness, and uniformity of diagnosis. The question o f the types of malformations to be monitored is controversial. Some monitoring systems use all observed anomalies [3,7,9], while others [ 5,6] record only a few cardinal surface defects which are easily and reliably diagnosed in the newborn, not only b y physicians b u t by qualified paramedical personnel. It is n o t easy to be dogmatic a b o u t this matter because, as will be discussed below, there are no uniform standards b y which different monitoring systems can be compared at present. Although the use of only cardinal defects has the virtue that almost any health care personnel can be trained to obsei-ve and record them, it must be recognized that almost all of the known environmental teratogens in man, rather than increasing the frequency of c o m m o n surface malformations, produce instead a specific array of defects which are sufficiently unique to be designated as a syndrome. Syndrome identification has become a complex field requiring skilled diagnostic training. The statistical methods used to measure rates and trends are of critical importance in a monitoring system. The whole success of the operation depends on sounding a warning when a genuine increase occurs in the rate of some or all of the defects being monitored, and on not doing so when a non-relevant change occurs. Some false alarms will be tolerated b u t t o o many will result in a loss of confidence in the system. In any population, long-term analyses of incidence rates exhibit considerable variation (Fig. 3). A surveillance system must be able to detect and differentiate changes in rates produced b y a new teratogen from this normal random variation or from an artificial change caused by overzealous reporting or b y changes in the methods of registration. Since a
48
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teratogenic agent may be introduced gradually and cause a steady, rather than a sharp, increase in rates most existing schemes have multiple tests to detect both types of change [3,7,9]. Although none of the surveillance systems in existence at present have discovered a new teratogen, most, if not all, have demonstrated that they are sensitive enough to detect a change in the rate of a specific defect. Since there is a singular lack of uniformity in these systems, it is reassuring that there appears to be available a multiplicity of methods which share some degree of sensitivity. I should n o w like to discuss some existing monitoring systems and highlight some of their unique features, some of the statistical methods used, and the results they have achieved. This is neither an overview of these systems nor a formal comparison of their strength and weaknesses. A monitoring system has been in existence in Finland since 1963. This scheme comprises two levels: a basic obligatory notification, which provides rough incidence rates, and a more closely studied group, selected to be a register of cases designed as the monitoring system. The unique feature of this scheme is that each case has a paired control. The excellent vital and health records in Finland make it possible to obtain relevant prenatal histories on both affected and control cases. Saxdn and his colleagues have demonstrated the value of this scheme in a variety of epidemiological investigations [28--30]. Kiillen and Winberg [13] have claimed that the Swedish monitoring system, if it had been in operation in 1963, would have detected the thalidomide episode. In a later report [14] the same authorsdemonstrated that the system was able to pick up a "new" syndrome in 1965, although no specific agent could be identified. A well-run surveillance system operates from the Center for Disease Control in Atlanta under the direction of W. Flynt [7]. Incidence rates for 1968 to 1970 serve as base line statistics to calculate the expected numbers for 1, 2, 4,
49 6 and 12-month cumulative periods. When the observed number of cases exceeds that expected b y t w o standard deviations, using the Poisson distribution, the defects are listed on a c o m p u t e r printout for further studies. Since this system has been in operation, 23 defects have shown increases at the 0.1% level of significance. In no instance has an environmental agent been identified to explain these increases. In Canada, a monitoring system has been functioning in Ottawa since 1966, under the direction of Philip Banister, using data from four Canadian provinces (a fifth was added in 1974). Early in its development, this system detected a significant increase in the incidence rate of limb reduction anomalies in t w o of the four provinces. This event was followed up promptly and, while no specific teratogen was identified, the exercise did demonstrate that the system was sensitive and that once an alarm bell was rung, appropriate follow-up procedures could be instituted rapidly [2,32]. A surveillance scheme for England and Wales has operated since 1964 [9]. The frequency of anomalies is examined in two ways. In the first, current figures in each of 173 counties and boroughs are compared with the current totals for the whole of England and Wales. In the second, the trend in the notification of each of approximately 80 abnormalities in each of the 173 areas is examined, using the " c u s u m " (cumulative sum) chart employed in quality control work. This system was sensitive enough to detect changes initiated b y more thorough registration of specific defects. A monitoring scheme has been in operation in Norway since January, 1971. Bjerkedal and Bakketeig [5] have described this system, which has successfully detected two frequency increases (congenital dislocation of the hip and anomalies of the limbs), attributable to alterations in ~nethods of registration. However, in the autumn of 1972, an increase occurred in the registration of urogenital system anomalies. This "epidemic" still persists and is being investigated by means of a case control study. In this monitoring system, 16 easily detected anomalies are analyzed separately and the Shewhart chart method is used for statistical evaluation. This method is based .on quality control analysis and has b o t h upper and lower control limits which signify that something is awry. The control limits are based on discrete Poisson distributions (for items with expected numbers equal to or less than 10) or the standardized normal distribution (for items with expected numbers greater than 10). As it is designed, this system will yield about 1--2 "false alarms" per year. In their report, Bjerkedal and Bakketeig state,-"However, this population is t o o small to provide a quick answer to urgent questions. To overcome this obstacle, international collaboration is essential". At present, there is no formal mechanism established for collecting, distributing, and developing unified international tests. However, WHO is beginning to collect data from a few monitoring centers, and the National Foundation has established an International Clearing House f o r Birth Defects Monitoring Systems. In view of the multiplicity of problems associated with diagnosis and coding of congenital malformations, and the lack of uniformity of monitoring systems, it is going to be a major task to develop international standards in order that information from various monitoring systems throughout the world can be interchanged and compared. However, it is reassuring that a beginning has been made.
50 Monitoring of embryos and fetuses The malformations observed in term infants are akin to the tip of a m a m m o t h iceberg. The submerged mass of the iceberg is represented b y the large numbers of malformations present in spontaneously aborted embryos and fetuses. Estimation of the magnitude of this loss is not easy, b u t all evidence from various sources suggests that it is considerable. The most recent estimate is that 78% of conceptions in w o m e n 20--29 years are lost and that the vast majority of these conceptuses have anomalies [27]. This estimate is in general agreement with those of others [10,36] and with observations in non-human mammals [1]. Of course many of these spontaneous losses occur very early in pregnancy and in such a way that there is no opportunity for recovery and examination of the specimen. Nevertheless, many conceptuses are simply discarded without receiving even a causal examination. In addition, liberalization of abortion laws in many industrialized countries has meant that large numbers of embryos and fetuses obtained b y surgical induction are potentially available for study. In recent years, several investigators have pointed out that this considerable volume of material could be used as a valuable adjunct to a monitoring program based on newborns [17--19,25,34]. The principal justification for the use of abortuses in a surveillance system is the possibility of detecting the activity of a new teratogen several months before such activity would be suspected in a newborn monitoring program. In addition, it is hoped that the detection of such activity, six or seven months earlier, would aid in locating the teratogenic agent, because of the reduced interval between the initiation of the teratogenic action and its detection, i.e., between point A and B. in Fig. 1. Problems of maternal and physician memory biases about such things as drug exposure, while still formidable, would be reduced. Admittedly, it would be difficult at present, to meet any of Newcombe's criteria outlined earlier except the second, i.e., early detection. It is going to be some time before ahigh degree of specificity in the diagnosis and the classification of developmental defects in human conceptuses is a realistic possibility. Some pathologists might argue that it will never be possible, except in a very small percentage of cases, because of the damage that is done to the conceptus during the course of surgical induction and the natural autolytic processes present in almost all specimens derived spontaneously. However, Nishimura and his colleagues have demonstrated the feasibility of accurately describing developmental defects in a large number of embryos derived by induction [23,24,34]. While the difficulties are considerable, careful dissection and documentation of true developmental defects in spontaneously aborted embryos is also possible [18,37]. Reasonably reliable incidence rates of malformation frequencies in both induced and spontaneous abortuses are beginning to accumulate (Tables I and II). Newcombe's third criterion (improved methods for the recording, rapid accumulation, and communication of data) presents a major hurdle, because so few centers are equipped to carry o u t studies on human abortions. Since abortions, unlike live and stillbirths, are not reportable by law in most countries, there is no formal basis for the collection of reliable baseline statistics, let alone for the careful examination o f the aborted material. It has been suggested that
51 TABLE I FREQUENCY
OF ABNORMAL ABORTUSES BY SOURCE
Source of specimen Spontaneous Induced Ectopic Self-induced Total
Total source
Number abnormal
Percent abnormal
902 388 45 150
478 38 19 21
53.0 9.8 42.2 14.0
1485
556
37.4
all abortions should be notifiable and that the notification should include information on the specimen. Given the difficulties associated with the routine reporting of data related to stillbirths, it would seem to be a major undertaking to institute a reliable system of abortion reporting. Despite these difficulties, Miller and Poland have outlined such surveillance methods using embryos from spontaneous abortion [19] and Tanimura has presented compelling evidence that a system could be developed using specimens derived by surgical intervention [34]. In neither instance has there been any attempt to work out the economics of the inclusion of abortions in a monitoring system. Both proposals are based on research projects on prenatal development, and the only way cost effectiveness could be determined would be by the establishment of a pilot project. Both studies, although not designed as monitoring schemes, have results which suggest that, with a little modification, they could be adapted to be effective systems for detecting fluctuation in anomalies. Miller and Poland were able to demonstrate an increase in the occurrence of limb reduction anomalies amongst their spontaneously derived abortions at a time when the Canadian monitoring scheme had detected apparent increase in similar defects in newborns [18]. Nishimura and his colleagues in Kyoto have demonstrated apparently real, although unexplained, fluctuations in anomalies amongst their collection of induction derived abortions [24]. Despite differences in philosophy, both these proposals have a common T A B L E II COMPARISON OF THE FREQUENCY OF CASES WITH EXTERNAL MALFORMATION AMONGST JAPANESE EMBRYOS, FETUSES AND NEWBORNS (MODIFIED FROM NISHIMURA, 1974)
Embryos a
Early f e t u s e s a Newborns c
A g e group
Total
Number malformed
Percent malformed
Stages 8 - - 1 5 b Stages 16--17 Stages 1 8 - - 2 3 (Crown-rump length 40 ram--160 ram)
1794 1870 2169 595 155222 64569
23 44 46 18 1295 659
1.28 2.35 2.12 3.02 0.83 1.02
aDerived f r o m i n d u c e d a b o r t i o n s . bStreeter's d e v e l o p m e n t a l h o r i z o n s . O r w o n e w b o r n s t u d i e s in Japan - - see N i s h i m u r a for r e f e r e n c e s .
52 theme, namely, that neglect of the study of abortions represents an appalling waste of biological material which could yield valuable information on early human development and the factors and mechanisms by which this development is altered adversely. The study of such abortions could also yield very valuable information for an ongoing monitoring system. Conclusion In a way, this general consideration of monitoring makes for pessimism. Known environmental teratogens play a small role in the total picture of developmental defects. Proven genetic factors and complex polygenic or multifactorial mechanisms apparently account for the overwhelming number of congenital malformations. The most important of the non-genetic factors seem to be several viruses, and, given their ubiquity and the complexity of their relation with other living forms, it would not be surprising if future investigation were to yield more important information on the teratogenicity of viruses. However, since thalidomide, not one new dramatic teratogen has been observed. Considerable debate surrounds the potential teratogenic agents receiving most attention over the past several years, such as diphenylhydantoin [ 2 1 ] , meprobamate [16], hormones [8,11] and alcohol [12]. The controversy involves the action of the drug as opposed to the reasons for taking it, the biological and social attributes of the w o m e n concerned, etc. Smithells, in a recent editorial [33], expresses skepticism of the value of monitoring. "The concept behind monitoring is that by watching hawk-eyed for unexplained increases (or decreases) in the incidence of specific defects, we shall be alert to pick up new environmental teratogens. It is a worthwhile ambition, though m y guess is that the expense involved may not justify itself in terms of temtogens identified. We have an unfortunate (perhaps inevitable) tendency to be one jump behind. After Gregg's discovery of rubella e m b r y o p a t h y in 1941, we turned our investigative guns on viruses, only to be taken in the rear b y a teratogenic drug twenty years later. We remain acutely thalidomide-conscious, b u t whilst we monitor birth defects we are again taken in the rear by estrogen producing, not a malformation, b u t a cancer." Is it more by good luck than by good management that no agent with the teratogenic potential of thalidomide has been detected? Not necessarily because we d o n ' t know how many agents have been detected b y animal tests long before they have achieved the level of clinical trial or are put into the environment. The point is that monitoring schemes by themselves may be costly and those who finance them impatient because there are no obvious positive results. For this reason, it has been argued that monitoring should be just one phase of a general program of population studies on congenital defects [7]. Others have questioned the merit of monitoring systems. The most explicit of these skeptics is R.W. Miller, who points out that all known teratogens have been detected by astute clinical observers. "Who discovered the major human teratogens? Radiation-induced microcephaly with mental retardation (Philadelphia obstetricians), rubella e m b r y o p a t h y (an Australian ophthalmologist), thalidomide-induced phocomelia (an Australian obstetrician; a West German human geneticist), methyl mercury-induced cerebral palsy (Japanese pediatri-
53 cians)" [ 2 0 ] . Miller argues, therefore, that a better education of the physician is the key to the detection of teratogenic activities and the institution of rapid follow-up. He believes that surveillance studies, registries, etc., should be used to test hypotheses generated by clinical and experimental observation. There is already one excellent example of such use o f monitoring data. In 1972, McBride in Australia suggested that imiprimine, a tricyclic anti-depressant might produce limb reduction defects [ 1 5 ] . The Atlanta and Canadian monitoring systems were able to make a rapid analysis of their data and produce strong evidence that the drug was not a teratogen [4,26]. To some extent, I share the pessimism of Smithells, Miller and others who write on this subject. Nevertheless, until better animal tests are available and no harmful agents pass through the defence walls, I believe we are obligated to devote our time to the monitoring of newborns (and ideally embryos and fetuses) for the detection of the presence of agents which might damage many hundreds or thousands of conceptuses. To neglect this screen and rely solely on the astute clinical observer is unrealistic. In my opinion, there is no way out of this dilemma. Acknowledgements The original work on embryos and fetuses by Poland and Miller was supported by grant MA-2740 f r o m the MRC (Canada). The help of Mrs. Ann Rosevear and Miss Sheila Manning in preparing the manuscript is gratefully acknowledged. References 1 Austen, C.R., Pregnancy losses and birth defects, in C.R. Austen and R.V. Short (Eds.), R e p r o d u c t i o n in Mammals, Cambridge Univ. Press~ Cambridge, vol. 2 ( E m b r y o n i c and Fetal Development), 1972, pp. 134--153. 2 Banister, P., Congenital malformations; preliminary report of an investigation of reduction d e f o r m i t i e s of the limbs, triggered by a pilot surveillance system, Can. Med. Ass. J., 103 (1970) 466--472. 3 Banister, P., Evaluation of vital record usuage for congenital anomaly surveillance, in E.B. Hook, D.T. Janerich and I.H. Porter (Eds.), Monitoring, Birth Defects and Envi ronme nt : The Problem of Surveillance, Academic Press, N e w York, 1971, pp. 119--135. 4 Banister, P., C. Dafoe, E.S.O. Smith and J. Miller, Possible teratogenicity of tricyclic antidepressant, Lancet, 1 (1972) 838--839. 5 Bjerkedal, T., and L.S. Bakketeig, Surveillance of congenital ma l forma t i ons and other conditions of the newborn, Int. J. Epid., 4 (1975) (in press). 6 Cziezl, A,, The Hungarian congenital anomaly monitor, Teratology 10 (1974) 307. 7 F l y n t Jr., J.W., Trends in surveillance of congenital malformations, in D.T. Janerich, R.G. Skalko and I.H. Porter (Eds.), Congenital Defects: N e w Directions in Research, Academic Press~ New York, 1974, pp. 119--128. 8 Harlap, S., R. Prywes and A.M. Davies, Birth defects and oestrogens and progesterones in pregnancy, Lancet 1, (1975) 682--683. 9 Hill, G.B., C.C. Spicer and J,A.C. Weatheral], The c o m p u t e r surveillance of congenital malformations, Brit. Med. Bull., 24 (1968) 215--218. 10 James, W.H., The incidence of spontaneous abortion, Pop. Studies, 24 (1970) 241--245. 11 Janerich, D.T., J.M. Piper a n d D.M. Glebatis, Oral contraceptives and congenital limb reduction defects, N e w EngL J. Med., 291 (1974) 697--700. 12 Jones, K.L., D.W. Smith, A. Streissguth and N.C. Myrianthopoulos, Outcome of offspring of chronic alcoholic women, Lancet, 1 (1974) 1076--1078. 13 K~illen, B., and J. Winberg, A Swedish register of e o n g e n i t a l m a l f o r m a t i o n s : experience with continuous registration during t w o years w i t h special reference to multiple malformations, Pediatrics, 41 (19681 765---776.
54
1 4 K[illen, B., a n d J. W i n b e r g , M u l t i p l e m a l f o r m a t i o n s s t u d i e d w i t h a n a t i o n a l r e g i s t e r o f m a l f o r m a t i o n s , Pediatrics, 44 (1969) 410--417. 1 5 M c B r i d e , W.G., L i m b d e f o r m i t i e s a s s o c i a t e d i m i n o d i b e n z y l h y d r o c h l o r i d e , Med. J. A u s t . , 1 ( 1 9 7 2 ) 4 9 2 . 1 6 M i l k o v i c h , L., a n d B.J. v a n d e n Berg, E f f e c t s o f p r e n a t a l m e p r o b a m a t e a n d c h l o r d i a z e p o x i d e h y d r o c h l o r i d e o n h u m a n e m b r y o n i c a n d f e t a l d e v e l o p m e n t , N e w Engl. J. M e d . , 2 9 1 ( 1 9 7 4 ) 1 2 6 8 - - 1 2 7 1 . 1 7 Miller, J . R . , a n d B.J. P o l a n d , T h e v a l u e o f h u m a n a b o r t u s e s in t h e s u r v e i l l a n c e o f d e v e l o p m e n t a l a n o m a l i e s , I. G e n e r a l o v e r v i e w , C a n . Med. Ass. J., 1 0 3 ( 1 9 7 0 ) 5 0 1 - - 5 0 2 . 1 8 Miller, J . R . , a n d B.J. P o l a n d , T h e value o f h u m a n a b o r t u s e s in t h e s u r v e i l l a n c e o f d e v e l o p m e n t a l a n o m a l i e s , II. R e d u c t i o n d e f o r m i t i e s o f t h e l i m b s , C a n . Med. Ass. J., 1 0 3 ( 1 9 7 0 ) 5 0 3 - - 5 0 5 . 19 Miller, J . R . , a n d B.J. P o l a n d , M o n i t o r i n g o f h u m a n e m b r y o n i c a n d f e t a l w a s t a g e , in E.B. H o o k , D . T . J a n e r i c h a n d I.H. P o r t e r (Eds.), M o n i t o r i n g B i r t h D e f e c t s a n d E n v i r o n m e n t : T h e P r o b l e m o f Surveill a n c e . A c a d e m i c Press, N e w Y o r k , 1 9 7 1 , p p . 6 5 - - 8 1 . 2 0 Miller, R . W . , D i s c u s s i o n , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s ( E d s . ) , M e t h o d s f o r D e t e c t i o n o f E n v i r o n m e n t a l A g e n t s w h i c h p r o d u c e C o n g e n i t a l D e f e c t s . N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 21 M o n s o n , R . R . , L. R o s e n b e r g , S.C. H a r t z , S. S h a p i r o , O.P. H e i n o n e n a n d D. S l o n e , D i p h e n y l h y d a n t o i n a n d s e l e c t e d m a l f o r m a t i o n s , N e w Engl. J. M e d . , 2 8 9 ( 1 9 7 3 ) 1 0 4 9 - - 1 0 5 2 . 2 2 N e w e o m b e , H . B . , V a l u e o f C a n a d i a n h o s p i t a l i n s u r a n c e r e c o r d s in d e t e c t i n g i n c r e a s e s in c o n g e n i t a l ' a n o m a l i e s , C a n . Med. Ass. J., 1 0 1 ( 1 9 6 9 ) 1 2 1 - - 1 2 8 . 23 N i s h i m u r a , H., I n c i d e n c e o f m a l f o r m a t i o n s in a b o r t i o n s , in F.C. F r a s e r a n d V . A . M c K u s i c k (Eds.), Congenital Malformations: Proceedings of the Third International Conference, Excerpta Medica, Amsterdam, 1970, pp. 275--283. 2 4 N i s h i m u r a , H., D e t e c t i o n of e a r l y d e v e l o p m e n t a l a n o m a l i e s in h u m a n a b o r t u s e s , in P r o c . X I V C o n g . Ped. B u e n o s Aires, 1 0 ( 1 9 7 4 ) 1 5 9 - - 1 7 0 . 2 5 O a k l e y J r . , G.P., T h e use o f h u m a n a b o r t u s e s in t h e s e a r c h f o r t e r a t o g e n s , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s (Eds.), M e t h o d s f o r D e t e c t i o n o f E n v i r o n m e n t a l A g e n t s w h i c h p r o d u c e C o n g e n i t a l D e f e c t s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 26 R a c h e l e f s k y , G . S . , J.W. F l y n t , A . J . E b b i n a n d M.G. Wilson, Possible t e r a t o g e n e c i t y o f t r i c y c l i e a n t i depressants, Lancet, 1 (1972) 838. 2 7 R o b e r t s , C.J., a n d C . R . L o w e , W h e r e h a v e all t h e c o n c e p t i o n s g o n e ? , L a n c e t , 1 ( 1 9 7 5 ) 4 9 8 - - 4 9 9 . 2 8 SaxOn, L., P o p u l a t i o n s u r v e i l l a n c e f o r b i r t h d e f e c t s , in A . G . M o t u l s k y a n d W. L e u z ( E d s . ) , B i r t h Defects: Fourth International Conference, Excerpta Medica, Amsterdam, 1974, pp. 177--186. 29 S a x d n , L., A. K l e m e t t i a n d A.S. Hiir6, A. m a t c h e d p a i r r e g i s t e r f o r s t u d i e s of s e l e c t e d c o n g e n i t a l d e f e c t s , A m . J. E p i d e m i o l . , 1 0 0 ( 1 9 7 4 ) 2 9 7 - - 3 0 6 . 3 0 SaxOn, L., N e w b o r n m o n i t o r i n g , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s (Eds.), M e t h o d s f o r Detection of Environmental Agents which Produce Congenital Defects, North-Holland, Amsterdam, 1 9 7 5 (in press). 31 S h e p a r d , T . H . , I n t r o d u c t i o n , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s (Eds.), M e t h o d s f o r D e t e c t i o n of E n v i r o n m e n t a l A g e n t s w h i c h P r o d u c e C o n g e n i t a l D e f e c t s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 3 2 S m i t h , E . S . O . , C.S. D a f o e , J . R . Miller a n d P. B a n i s t e r , A n e p i d e m i o l o g i c a l s t u d y o f c o n g e n i t a l r e d u c t i o n d e f o r m i t i e s of t h e l i m b s , (in press). 3 3 S m i t h e l l s , R.W., E p i d e m i o l o g y o f m a l f o r m a t i o n s : i n s p i r a t i o n a n d p e r s p i r a t i o n , T e r a t o l o g y , 1 0 ( 1 9 7 4 ) 217--220. 3 4 T a n i m u r a , T., T h e u s e o f i n d u c e d a b o r t u s e s f o r m o n i t o r i n g , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s (Eds.), M e t h o d s f o r D e t e c t i o n o f E n v i r o n m e n t a l A g e n t s w h i c h p r o d u c e C o n g e n i t a l D e f e c t s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 3 5 W i l s o n , J . G . , C r i t i q u e o f c u r r e n t m e t h o d s f o r t e r a t o g e n i c i t y t e s t i n g in a n i m a l s a n d s u g g e s t i o n s f o r t h e i r i m p r o v e m e n t , in T . H . S h e p a r d , J . R . Miller a n d M. M a r o i s (Eds.), M e t h o d s f o r D e t e c t i o n o f E n v i r o n m e n t a l A g e n t s w h i c h p r o d u c e C o n g e n i t a l D e f e c t s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 36 Witschi, E. T e r a t o g e n i c e f f e c t s f r o m o v e r r i p e n e s s o f t h e egg, in F.C. F r a s e r a n d V . A . M c K u s i c k (Eds.), Congenital Malformations: Proceedings of the Third International Conference, Exeerpta Medica, Amsterdam, 1970, pp. 157--169. 3 7 Y a s u d a , M., a n d B.J. P o l a n d , A 6 3 - r a m h u m a n f e t u s w i t h r a d i a l aplasia, tibial p o l y d a c t y l y , rib a n d vertebral fusions, and right aortic arch, Teratology, 10 (1974) 119--124.
Mutation Research, 33 (1975) 45--54
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SOME EPIDEMIOLOGICAL ASPECTS OF T E R A T O G E N DETECTION
JAMES R. MILLER Department of Medical Genetics, The University of British Columbia, Vancouver, B.C. (Canada)
(Received July 10th, 1975) (Accepted August 25th, 1975)
Introduction
Numerous reports have been published on the epidemiology of congenital defects and I see no virtue in re-examining the strengths and weaknesses of such studies. I think it is a fair summary to say that while they have yielded much information relating to the association of defects with such variables as geographic location, season, birth order, social class, occupation and ethnic origin, these studies have n o t identified a single teratogen. Since the implicit theme of this symposium is the rapid detection of environmental agents which are mutagenic, oncogenic or teratogenic, I propose to focus on problems associated with the monitoring of newborns and the potential value of including embryos and fetuses in such monitoring schemes. It would be pleasant if there was no need to worry about such monitoring, because adequate in vitro or in vivo tests using experimental animals existed. Shepard [31] has pictured the problem as a series of defence walls with gaps through which undetected teratogens can pass. At present, no defence system is perfect and, since potentially deleterious agents may be introduced into the environment without passing through any screen, it must be recognized that the pregnant human female is the ultimate test organism. Once this is recognized, it is essential that methods be devised for the constant evaluation of agents to which pregnant w o m e n are exposed and the development of screening methods for the monitoring of as many conceptuses as possible. Although one of the principal themes of this symposium is the similarity of oncogenic, mutagenic and teratogenic processes, it is equally important to recognize critical differences between them. Some of these have been c o m m e n t e d upon already but, in my opinion, the critical issue revolves around a much neglected organ, the placenta, "the organ of metabolic interchange between the fetus and mother". It is primarily because of the placenta that in vitro testing for teratogens is still only a theoretical possibility and that in vivo tests remain a matter of considerable debate. As Wilson [35] has pointed out, aside from
46 the pregnant woman, the ideal test organism does not exist. If this fact were acknowledged, the approach to monitoring programs, clinical trials etc. would be more realistic. General epidemiological considerations Oakley [25] has summarized succinctly a general epidemiological approach to the detection of teratogens (Fig. 1). Of course, the transition across time from the recognition of an epidemic (point B) to the final withdrawal of the offending agent (point G) is not smooth. Invariably, difficulties and delays develop at points C and D; i.e., with respect to the development of multiple hypotheses about the cause of the epidemic and the testing of these hypotheses. The message is clear: even though a system may be devised to detect the presence of a new teratogen, there is no guarantee that the offending agent will be uncovered and removed rapidly. Using thalidomide as a model, Oakley has demonstrated how two types of testing of hypothesis can be employed at point D (Fig. 2). McBride's cohort approach was possible because a group of w o m e n in his hospital began to receive thalidomide as a therapy for morning sickness. On the other hand, because the epidemic already existed in Germany in 1959, Lenz had to carry out a retrospective case control study. In a way, McBride's rapid implication of thalidomide was almost fortuitous -- the outcome of a clinical trial for another purpose. The usual course of events is that observed in Germany; i.e., a long time lag between the concern about the presence of a teratogen and its removal from the environment. Unfortunately, there is no escaping this fact, even in the most ideal situation.
Monitoring In reference to congenital defects, the term "monitoring" denotes an early warning system designed to detect evidence of a new teratogen in the environment either b y the appearance of a new malformation or malformation syndrome, or by an increase in the incidence rate of known conditions. N e w c o m b e [22] has proposed the following three key elements in a monitoring system: (1) a high degree of specificity in diagnosis and classification; (2) early detection,
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47 Recognition
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and (3) improved methods for the recording, rapid accumulation and communication of data. Although the principal feature of such a system is urgency b o t h in accumulating data and reporting rates and trends, this must not be accomplished at the expense of accurate diagnosis. Perhaps the specificity stress in point 1 should be extended to include accuracy, completeness, and uniformity of diagnosis. The question o f the types of malformations to be monitored is controversial. Some monitoring systems use all observed anomalies [3,7,9], while others [ 5,6] record only a few cardinal surface defects which are easily and reliably diagnosed in the newborn, not only b y physicians b u t by qualified paramedical personnel. It is n o t easy to be dogmatic a b o u t this matter because, as will be discussed below, there are no uniform standards b y which different monitoring systems can be compared at present. Although the use of only cardinal defects has the virtue that almost any health care personnel can be trained to obsei-ve and record them, it must be recognized that almost all of the known environmental teratogens in man, rather than increasing the frequency of c o m m o n surface malformations, produce instead a specific array of defects which are sufficiently unique to be designated as a syndrome. Syndrome identification has become a complex field requiring skilled diagnostic training. The statistical methods used to measure rates and trends are of critical importance in a monitoring system. The whole success of the operation depends on sounding a warning when a genuine increase occurs in the rate of some or all of the defects being monitored, and on not doing so when a non-relevant change occurs. Some false alarms will be tolerated b u t t o o many will result in a loss of confidence in the system. In any population, long-term analyses of incidence rates exhibit considerable variation (Fig. 3). A surveillance system must be able to detect and differentiate changes in rates produced b y a new teratogen from this normal random variation or from an artificial change caused by overzealous reporting or b y changes in the methods of registration. Since a
48
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teratogenic agent may be introduced gradually and cause a steady, rather than a sharp, increase in rates most existing schemes have multiple tests to detect both types of change [3,7,9]. Although none of the surveillance systems in existence at present have discovered a new teratogen, most, if not all, have demonstrated that they are sensitive enough to detect a change in the rate of a specific defect. Since there is a singular lack of uniformity in these systems, it is reassuring that there appears to be available a multiplicity of methods which share some degree of sensitivity. I should n o w like to discuss some existing monitoring systems and highlight some of their unique features, some of the statistical methods used, and the results they have achieved. This is neither an overview of these systems nor a formal comparison of their strength and weaknesses. A monitoring system has been in existence in Finland since 1963. This scheme comprises two levels: a basic obligatory notification, which provides rough incidence rates, and a more closely studied group, selected to be a register of cases designed as the monitoring system. The unique feature of this scheme is that each case has a paired control. The excellent vital and health records in Finland make it possible to obtain relevant prenatal histories on both affected and control cases. Saxdn and his colleagues have demonstrated the value of this scheme in a variety of epidemiological investigations [28--30]. Kiillen and Winberg [13] have claimed that the Swedish monitoring system, if it had been in operation in 1963, would have detected the thalidomide episode. In a later report [14] the same authorsdemonstrated that the system was able to pick up a "new" syndrome in 1965, although no specific agent could be identified. A well-run surveillance system operates from the Center for Disease Control in Atlanta under the direction of W. Flynt [7]. Incidence rates for 1968 to 1970 serve as base line statistics to calculate the expected numbers for 1, 2, 4,
49 6 and 12-month cumulative periods. When the observed number of cases exceeds that expected b y t w o standard deviations, using the Poisson distribution, the defects are listed on a c o m p u t e r printout for further studies. Since this system has been in operation, 23 defects have shown increases at the 0.1% level of significance. In no instance has an environmental agent been identified to explain these increases. In Canada, a monitoring system has been functioning in Ottawa since 1966, under the direction of Philip Banister, using data from four Canadian provinces (a fifth was added in 1974). Early in its development, this system detected a significant increase in the incidence rate of limb reduction anomalies in t w o of the four provinces. This event was followed up promptly and, while no specific teratogen was identified, the exercise did demonstrate that the system was sensitive and that once an alarm bell was rung, appropriate follow-up procedures could be instituted rapidly [2,32]. A surveillance scheme for England and Wales has operated since 1964 [9]. The frequency of anomalies is examined in two ways. In the first, current figures in each of 173 counties and boroughs are compared with the current totals for the whole of England and Wales. In the second, the trend in the notification of each of approximately 80 abnormalities in each of the 173 areas is examined, using the " c u s u m " (cumulative sum) chart employed in quality control work. This system was sensitive enough to detect changes initiated b y more thorough registration of specific defects. A monitoring scheme has been in operation in Norway since January, 1971. Bjerkedal and Bakketeig [5] have described this system, which has successfully detected two frequency increases (congenital dislocation of the hip and anomalies of the limbs), attributable to alterations in ~nethods of registration. However, in the autumn of 1972, an increase occurred in the registration of urogenital system anomalies. This "epidemic" still persists and is being investigated by means of a case control study. In this monitoring system, 16 easily detected anomalies are analyzed separately and the Shewhart chart method is used for statistical evaluation. This method is based .on quality control analysis and has b o t h upper and lower control limits which signify that something is awry. The control limits are based on discrete Poisson distributions (for items with expected numbers equal to or less than 10) or the standardized normal distribution (for items with expected numbers greater than 10). As it is designed, this system will yield about 1--2 "false alarms" per year. In their report, Bjerkedal and Bakketeig state,-"However, this population is t o o small to provide a quick answer to urgent questions. To overcome this obstacle, international collaboration is essential". At present, there is no formal mechanism established for collecting, distributing, and developing unified international tests. However, WHO is beginning to collect data from a few monitoring centers, and the National Foundation has established an International Clearing House f o r Birth Defects Monitoring Systems. In view of the multiplicity of problems associated with diagnosis and coding of congenital malformations, and the lack of uniformity of monitoring systems, it is going to be a major task to develop international standards in order that information from various monitoring systems throughout the world can be interchanged and compared. However, it is reassuring that a beginning has been made.
50 Monitoring of embryos and fetuses The malformations observed in term infants are akin to the tip of a m a m m o t h iceberg. The submerged mass of the iceberg is represented b y the large numbers of malformations present in spontaneously aborted embryos and fetuses. Estimation of the magnitude of this loss is not easy, b u t all evidence from various sources suggests that it is considerable. The most recent estimate is that 78% of conceptions in w o m e n 20--29 years are lost and that the vast majority of these conceptuses have anomalies [27]. This estimate is in general agreement with those of others [10,36] and with observations in non-human mammals [1]. Of course many of these spontaneous losses occur very early in pregnancy and in such a way that there is no opportunity for recovery and examination of the specimen. Nevertheless, many conceptuses are simply discarded without receiving even a causal examination. In addition, liberalization of abortion laws in many industrialized countries has meant that large numbers of embryos and fetuses obtained b y surgical induction are potentially available for study. In recent years, several investigators have pointed out that this considerable volume of material could be used as a valuable adjunct to a monitoring program based on newborns [17--19,25,34]. The principal justification for the use of abortuses in a surveillance system is the possibility of detecting the activity of a new teratogen several months before such activity would be suspected in a newborn monitoring program. In addition, it is hoped that the detection of such activity, six or seven months earlier, would aid in locating the teratogenic agent, because of the reduced interval between the initiation of the teratogenic action and its detection, i.e., between point A and B. in Fig. 1. Problems of maternal and physician memory biases about such things as drug exposure, while still formidable, would be reduced. Admittedly, it would be difficult at present, to meet any of Newcombe's criteria outlined earlier except the second, i.e., early detection. It is going to be some time before ahigh degree of specificity in the diagnosis and the classification of developmental defects in human conceptuses is a realistic possibility. Some pathologists might argue that it will never be possible, except in a very small percentage of cases, because of the damage that is done to the conceptus during the course of surgical induction and the natural autolytic processes present in almost all specimens derived spontaneously. However, Nishimura and his colleagues have demonstrated the feasibility of accurately describing developmental defects in a large number of embryos derived by induction [23,24,34]. While the difficulties are considerable, careful dissection and documentation of true developmental defects in spontaneously aborted embryos is also possible [18,37]. Reasonably reliable incidence rates of malformation frequencies in both induced and spontaneous abortuses are beginning to accumulate (Tables I and II). Newcombe's third criterion (improved methods for the recording, rapid accumulation, and communication of data) presents a major hurdle, because so few centers are equipped to carry o u t studies on human abortions. Since abortions, unlike live and stillbirths, are not reportable by law in most countries, there is no formal basis for the collection of reliable baseline statistics, let alone for the careful examination o f the aborted material. It has been suggested that
51 TABLE I FREQUENCY
OF ABNORMAL ABORTUSES BY SOURCE
Source of specimen Spontaneous Induced Ectopic Self-induced Total
Total source
Number abnormal
Percent abnormal
902 388 45 150
478 38 19 21
53.0 9.8 42.2 14.0
1485
556
37.4
all abortions should be notifiable and that the notification should include information on the specimen. Given the difficulties associated with the routine reporting of data related to stillbirths, it would seem to be a major undertaking to institute a reliable system of abortion reporting. Despite these difficulties, Miller and Poland have outlined such surveillance methods using embryos from spontaneous abortion [19] and Tanimura has presented compelling evidence that a system could be developed using specimens derived by surgical intervention [34]. In neither instance has there been any attempt to work out the economics of the inclusion of abortions in a monitoring system. Both proposals are based on research projects on prenatal development, and the only way cost effectiveness could be determined would be by the establishment of a pilot project. Both studies, although not designed as monitoring schemes, have results which suggest that, with a little modification, they could be adapted to be effective systems for detecting fluctuation in anomalies. Miller and Poland were able to demonstrate an increase in the occurrence of limb reduction anomalies amongst their spontaneously derived abortions at a time when the Canadian monitoring scheme had detected apparent increase in similar defects in newborns [18]. Nishimura and his colleagues in Kyoto have demonstrated apparently real, although unexplained, fluctuations in anomalies amongst their collection of induction derived abortions [24]. Despite differences in philosophy, both these proposals have a common T A B L E II COMPARISON OF THE FREQUENCY OF CASES WITH EXTERNAL MALFORMATION AMONGST JAPANESE EMBRYOS, FETUSES AND NEWBORNS (MODIFIED FROM NISHIMURA, 1974)
Embryos a
Early f e t u s e s a Newborns c
A g e group
Total
Number malformed
Percent malformed
Stages 8 - - 1 5 b Stages 16--17 Stages 1 8 - - 2 3 (Crown-rump length 40 ram--160 ram)
1794 1870 2169 595 155222 64569
23 44 46 18 1295 659
1.28 2.35 2.12 3.02 0.83 1.02
aDerived f r o m i n d u c e d a b o r t i o n s . bStreeter's d e v e l o p m e n t a l h o r i z o n s . O r w o n e w b o r n s t u d i e s in Japan - - see N i s h i m u r a for r e f e r e n c e s .
52 theme, namely, that neglect of the study of abortions represents an appalling waste of biological material which could yield valuable information on early human development and the factors and mechanisms by which this development is altered adversely. The study of such abortions could also yield very valuable information for an ongoing monitoring system. Conclusion In a way, this general consideration of monitoring makes for pessimism. Known environmental teratogens play a small role in the total picture of developmental defects. Proven genetic factors and complex polygenic or multifactorial mechanisms apparently account for the overwhelming number of congenital malformations. The most important of the non-genetic factors seem to be several viruses, and, given their ubiquity and the complexity of their relation with other living forms, it would not be surprising if future investigation were to yield more important information on the teratogenicity of viruses. However, since thalidomide, not one new dramatic teratogen has been observed. Considerable debate surrounds the potential teratogenic agents receiving most attention over the past several years, such as diphenylhydantoin [ 2 1 ] , meprobamate [16], hormones [8,11] and alcohol [12]. The controversy involves the action of the drug as opposed to the reasons for taking it, the biological and social attributes of the w o m e n concerned, etc. Smithells, in a recent editorial [33], expresses skepticism of the value of monitoring. "The concept behind monitoring is that by watching hawk-eyed for unexplained increases (or decreases) in the incidence of specific defects, we shall be alert to pick up new environmental teratogens. It is a worthwhile ambition, though m y guess is that the expense involved may not justify itself in terms of temtogens identified. We have an unfortunate (perhaps inevitable) tendency to be one jump behind. After Gregg's discovery of rubella e m b r y o p a t h y in 1941, we turned our investigative guns on viruses, only to be taken in the rear b y a teratogenic drug twenty years later. We remain acutely thalidomide-conscious, b u t whilst we monitor birth defects we are again taken in the rear by estrogen producing, not a malformation, b u t a cancer." Is it more by good luck than by good management that no agent with the teratogenic potential of thalidomide has been detected? Not necessarily because we d o n ' t know how many agents have been detected b y animal tests long before they have achieved the level of clinical trial or are put into the environment. The point is that monitoring schemes by themselves may be costly and those who finance them impatient because there are no obvious positive results. For this reason, it has been argued that monitoring should be just one phase of a general program of population studies on congenital defects [7]. Others have questioned the merit of monitoring systems. The most explicit of these skeptics is R.W. Miller, who points out that all known teratogens have been detected by astute clinical observers. "Who discovered the major human teratogens? Radiation-induced microcephaly with mental retardation (Philadelphia obstetricians), rubella e m b r y o p a t h y (an Australian ophthalmologist), thalidomide-induced phocomelia (an Australian obstetrician; a West German human geneticist), methyl mercury-induced cerebral palsy (Japanese pediatri-
53 cians)" [ 2 0 ] . Miller argues, therefore, that a better education of the physician is the key to the detection of teratogenic activities and the institution of rapid follow-up. He believes that surveillance studies, registries, etc., should be used to test hypotheses generated by clinical and experimental observation. There is already one excellent example of such use o f monitoring data. In 1972, McBride in Australia suggested that imiprimine, a tricyclic anti-depressant might produce limb reduction defects [ 1 5 ] . The Atlanta and Canadian monitoring systems were able to make a rapid analysis of their data and produce strong evidence that the drug was not a teratogen [4,26]. To some extent, I share the pessimism of Smithells, Miller and others who write on this subject. Nevertheless, until better animal tests are available and no harmful agents pass through the defence walls, I believe we are obligated to devote our time to the monitoring of newborns (and ideally embryos and fetuses) for the detection of the presence of agents which might damage many hundreds or thousands of conceptuses. To neglect this screen and rely solely on the astute clinical observer is unrealistic. In my opinion, there is no way out of this dilemma. Acknowledgements The original work on embryos and fetuses by Poland and Miller was supported by grant MA-2740 f r o m the MRC (Canada). The help of Mrs. Ann Rosevear and Miss Sheila Manning in preparing the manuscript is gratefully acknowledged. References 1 Austen, C.R., Pregnancy losses and birth defects, in C.R. Austen and R.V. Short (Eds.), R e p r o d u c t i o n in Mammals, Cambridge Univ. Press~ Cambridge, vol. 2 ( E m b r y o n i c and Fetal Development), 1972, pp. 134--153. 2 Banister, P., Congenital malformations; preliminary report of an investigation of reduction d e f o r m i t i e s of the limbs, triggered by a pilot surveillance system, Can. Med. Ass. J., 103 (1970) 466--472. 3 Banister, P., Evaluation of vital record usuage for congenital anomaly surveillance, in E.B. Hook, D.T. Janerich and I.H. Porter (Eds.), Monitoring, Birth Defects and Envi ronme nt : The Problem of Surveillance, Academic Press, N e w York, 1971, pp. 119--135. 4 Banister, P., C. Dafoe, E.S.O. Smith and J. Miller, Possible teratogenicity of tricyclic antidepressant, Lancet, 1 (1972) 838--839. 5 Bjerkedal, T., and L.S. Bakketeig, Surveillance of congenital ma l forma t i ons and other conditions of the newborn, Int. J. Epid., 4 (1975) (in press). 6 Cziezl, A,, The Hungarian congenital anomaly monitor, Teratology 10 (1974) 307. 7 F l y n t Jr., J.W., Trends in surveillance of congenital malformations, in D.T. Janerich, R.G. Skalko and I.H. Porter (Eds.), Congenital Defects: N e w Directions in Research, Academic Press~ New York, 1974, pp. 119--128. 8 Harlap, S., R. Prywes and A.M. Davies, Birth defects and oestrogens and progesterones in pregnancy, Lancet 1, (1975) 682--683. 9 Hill, G.B., C.C. Spicer and J,A.C. Weatheral], The c o m p u t e r surveillance of congenital malformations, Brit. Med. Bull., 24 (1968) 215--218. 10 James, W.H., The incidence of spontaneous abortion, Pop. Studies, 24 (1970) 241--245. 11 Janerich, D.T., J.M. Piper a n d D.M. Glebatis, Oral contraceptives and congenital limb reduction defects, N e w EngL J. Med., 291 (1974) 697--700. 12 Jones, K.L., D.W. Smith, A. Streissguth and N.C. Myrianthopoulos, Outcome of offspring of chronic alcoholic women, Lancet, 1 (1974) 1076--1078. 13 K~illen, B., and J. Winberg, A Swedish register of e o n g e n i t a l m a l f o r m a t i o n s : experience with continuous registration during t w o years w i t h special reference to multiple malformations, Pediatrics, 41 (19681 765---776.
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1 4 K[illen, B., a n d J. W i n b e r g , M u l t i p l e m a l f o r m a t i o n s s t u d i e d w i t h a n a t i o n a l r e g i s t e r o f m a l f o r m a t i o n s , Pediatrics, 44 (1969) 410--417. 1 5 M c B r i d e , W.G., L i m b d e f o r m i t i e s a s s o c i a t e d i m i n o d i b e n z y l h y d r o c h l o r i d e , Med. J. A u s t . , 1 ( 1 9 7 2 ) 4 9 2 . 1 6 M i l k o v i c h , L., a n d B.J. v a n d e n Berg, E f f e c t s o f p r e n a t a l m e p r o b a m a t e a n d c h l o r d i a z e p o x i d e h y d r o c h l o r i d e o n h u m a n e m b r y o n i c a n d f e t a l d e v e l o p m e n t , N e w Engl. J. M e d . , 2 9 1 ( 1 9 7 4 ) 1 2 6 8 - - 1 2 7 1 . 1 7 Miller, J . R . , a n d B.J. P o l a n d , T h e v a l u e o f h u m a n a b o r t u s e s in t h e s u r v e i l l a n c e o f d e v e l o p m e n t a l a n o m a l i e s , I. G e n e r a l o v e r v i e w , C a n . Med. Ass. J., 1 0 3 ( 1 9 7 0 ) 5 0 1 - - 5 0 2 . 1 8 Miller, J . R . , a n d B.J. 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M a r o i s (Eds.), M e t h o d s f o r D e t e c t i o n o f E n v i r o n m e n t a l A g e n t s w h i c h p r o d u c e C o n g e n i t a l D e f e c t s , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 5 (in press). 36 Witschi, E. T e r a t o g e n i c e f f e c t s f r o m o v e r r i p e n e s s o f t h e egg, in F.C. F r a s e r a n d V . A . M c K u s i c k (Eds.), Congenital Malformations: Proceedings of the Third International Conference, Exeerpta Medica, Amsterdam, 1970, pp. 157--169. 3 7 Y a s u d a , M., a n d B.J. P o l a n d , A 6 3 - r a m h u m a n f e t u s w i t h r a d i a l aplasia, tibial p o l y d a c t y l y , rib a n d vertebral fusions, and right aortic arch, Teratology, 10 (1974) 119--124.
