Holoprosencephaly in Human Embryos: Epidemiologic Studies of 150 Cases EI MATSUNAGA AND KOHEI SHIOTA Department of Human Genetics, National Institute of Genetics, Mishima. Shizuoka-Ken411, Japan

ABSTRACT One hundred fifty embryos with holoprosencephaly were found among the total of 36,380 conceptuses obtained through induced abortion in the period from 1962 to 1974, giving an overall incidence of 0.4%.The occurrence was largely a t random through time, and no "epidemic" was noted in particular years or months, but there appeared more cases derived from conceptions in winter than in summer months. The mean maternal age did not differ significantly from that of the general embryonic population, indicating that, although none of our cases were karyotyped, chromosome aberrations such as trisomies 13 and 18 that are closely associated with maternal age may not constitute a major part as causes of holoprosencephaly in human embryos. Maternal age did not differ by the presence or absence of associated external anomalies. No association was found with paternal age, parental consanguinity nor with maternal medical history, including irregularity of menstrual cycles, and smoking and drinking habits. There was a n indication that the mothers were prone to have repeated miscarriages, supporting the view that some kind of maternal predisposition is responsible for the causation of holoprosencephaly. Argument was made that, apart from various chromosome aberrations well documented as causes of this malformation, polygenic mechanism probably accounts for the majority of the cases with normal karyotype. Holoprosencephaly (cyclopia-arhinencepha- '69) and 69, XXX (Emberger et al., '76) have ly series) is a group of malformations charac- been found, while others showed apparently terized by deformities of median structures of normal karyotype (DeMyer et al., '63; Landau the head due to developmental defects of the et al., '63; DeMyer, '64; Hintz et al., '68; Mcbrain and face. It includes various degrees of Donald, '68; James and van Leeuwen, '70; facio-cerebral malformations ranging from Valkeakari and Anttila, '70; Warkany, '71; cyclopia to arhinencephaly with almost nor- Holmes et al., '74; Walter et al., '74; Lazjuk mal appearance of the face. As pointed out by et al., '76). Holoprosencephaly with normal Warkany ('71) and &hen et al. ('711, this karyotype may be due either to a single museries of malformations is quite heterogene- tant gene or to polygenes or to some specious in origin. In some newborn and stillborn fic environmental factors. Familial cases have cases, abnormal karyotypes such as trisomy been reported repeatedly. In some families the 13 (McKusick, '61; von Buhler et al., '62; Mil- parents were consanguineous (Klopstock, '21; ler et al., '63; Smith et al., '63; Conen et al., Grebe, '54), indicating that the deformity can '66; Snodgrass et al., '66; Toews and Jones, be caused by homozygosity of a rare autosomal '68; Arakaki and Waxman, '69; Halbrecht et recessive gene. In one reported pedigree, the al., '71; Taysi and Tinaztepe, '72; Fujimoto et mothers of cyclops were found to be carriers of al., '73; Holmes e t al., '74; Lazjuk et al., '76; a balanced translocation (Pfitzer and MunMing et al., '76), trisomy 18 (Holmes et al., '74; tefering, '68). However, little is known about Lang et al., '76), 18p- (Faint and Lewis, '64; the relative significance of each of the preUchida et al., '65; Nitowski et al., '66; Gorlin sumed causes responsible for holoprosencephet al., '68; McDermott et al., '68; Schinzel et aly in man. al., '74), 18r (Yanoff et al., '70; Neu et al., '71; Received Feb. 4, '77. Accepted July 13, '77. Cohen et al., '72), trisomy C (Lejeune et al., TERATQLOGY, 16: 261-272.

261

262

EI MATSUNAGA AND KOHEI SHIOTA

Although holoprosencephaly is relatively rare a t birth (Mitani, ’54;Emanuel et al., ’72), it is one of the most common malformations in the embryo population. Nishimura (‘70) found 17 cases of cyclopia and cebocephaly among 3,381 embryos obtained through induced abortion; the incidence was 5.0 per 1,000 against 0.06 per 1,000 newborn, which implies that almost all of the affected embryos are destined to be eliminated prenatally. Thus the embryo population could provide us with an opportunity for epidemiologic investigations of holoprosencephaly in man. On the basis of 81 affected embryos collected in the Nishimura’s laboratory, Tanimura and Uwabe (‘71) reported that the incidence was 7.3 per 1,000 embryos a t Carnegie stages XIV-XXIII, and that the mothers of the affected embryos had significantly lower parity and more often genital bleeding than controls. However, these authors gave no account concerning whether or not the lowered parity is of etiological significance. Since that time, the collection of embryos has been expanded and data are now available for 150 cases of holoprosencephaly in all. The purpose of this paper is to present some epidemiologic characteristics of these cases. MATERIALS AND METHODS

Materials used in the present study are subsets of a large collection of human concep tuses, derived from artificial termination of pregnancy, kept a t the Human Embryo Center for Teratological Studies, Kyoto University, Kyoto. This collection has been initiated since 1961 by Professor H. Nishimura and his associates with the help of several hundred obstetricians, and details of the sources and the procedures of acquisition of the specimens were given elsewhere (Nishimura et al., ’68). For each specimen, information concerning medical, reproductive and family history of the mother was obtained directly by the cooperating obstetricians, using a standard interview form consisting of ca. 20 items, before or soon after operation. The conceptuses were fixed in Bouin’s fluid immediately after operation, and these with the recorded form were periodically brought to the laboratory. In a great majority of cases pregnancy was terminated by cervical dilatation and curettage during the first trimester for “socio-economic” reasons under the Eugenic Protection Law. They contained, however, occasionally those cases with maternal genital bleeding or threatened abortion as judged by the obstetri-

cians. The term “threatened abortion” does not necessarily mean a beginning abortion; it is used if the obstetricians consider the probability of spontaneous abortion be more or less high, depending upon the degree and duration of genital bleeding. Furthermore, the collection included a small number of the cases from strictly therapeutic abortions conducted by means of hysterectomy or salpingectomy with such indications as ectopic pregnancy or uterine myoma. At any rate, it should be noted that the sampling was not biased with respect to the outcome of the embryos, because the obstetricians supplied the specimens without examining them for external defects or signs of intrauterine death. For this and other reasons, Nishimura (‘70) regarded the material to be an almost non-selected sample of the total embryonic population, in the sense that it consists of an approximately random series of viable as well as inviable embryos that may otherwise be aborted spontaneously. The number of conceptuses collected between January 1962 and December 1974 amounted to 36,380, including empty sacs. However, specimens containing intact embryos represented only about lo%,i.e., 3,411 in number, the rest being more or less damaged by the procedure of curettage. Holoprosencephaly was diagnosed in 150 instances in all, which involved cyclopia, ethmocephaly, cebocephaly and some lesser degree of orbital hypotelorism with a flat nose. Photographs of some of the cases are shown elsewhere (Nishimura and Okamoto, ’76).They were diagnosed by careful examination with the aid of a dissecting microscope. Doubtful cases were subjected to more detailed studies including histological examination. No case in the present series was karyotyped. As controls, three groups of embryos were drawn from the embryo collection. (1) The 3,411 intact embryos were used for analysis of the effects of parity and maternal age. Because the procurement of a n intact embryo by operation usually depends upon chance only, this group may be regarded as representative of the whole set of embryo collection; hence this control will be referred to as “the general embryonic population.” Table 1 shows joint distribution of maternal age and parity for these embryos. The term “parity” here used refers to the number of pregnancies, including the last one which resulted in the embryo under study. The mean parity was 3.78 and the mean maternal age was 30.2 years; the latter is close to the mean of 30.9 years for all

263

EPIDEMIOLOGY OF HOLOPROSENCEPHALIC EMBRYOS

induced abortuses (757,389 in number) officially notified in Japan in 1968 (Annual Report of the Eugenic Protection Statistics, The Ministry of Health and Welfare). In this connection, i t is interesting to note that in Japan mothers asking for induced abortion are generally older and of higher parity than those giving birth to children. Taking the vital statistics data for all live-births (1,870,000 in number) in 1968, for example, the mean maternal age was 27.6 years and the mean parity 1.78. (2) One case of normal embryo, for each affected one, matched for maternal age and month of the onset of the last menstruation, was chosen as controls for analysis of paternal age effect. (3) For each affected case, two cases of normal embryos were taken t h a t were matched for parity and month of the onset of the last menstruation. This group was used for analysis of several factors other than parental age and parity. RESULTS

Source of the specimens The source of the 150 holoprosencephalics and of the 300 cases of parity-matched con-

trols is shown in table 2. About half of the malformed embryos were obtained by induced abortion performed in mothers having a sign of threatened abortion, while this was the case in about 15% of the control group. Further, more than half of the malformed group were found to be dead in utero, as judged by maceration. These findings clearly indicate that most of the conceptions with a holoprosencephalic embryo tend to terminate spontaneously.

Developmental stages and associated anomalies As shown in table 3, the developmental stages of the 150 holoprosencephalics a t the time of pregnancy interruption ranged from XI11 and XXIII according to the Carnegie system (O'Rahilly, '72). The proportion of intrauterine dead cases increased with advancing developmental stage, in either group of the embryos with or without a mat.erna1 sign of threatened abortion. This again substantiates that holoprosencephalic embryos are usually eliminated from an early stage of intrauterine life. Among well preserved specimens, nearly

TABLE 1

Joint distribution of maternal age and parity for 341 1 intact embryos ~

Maternal age Parity

-19

20.24

25.29

30-34

35-39

40-44

83 22 7 3

329 197 110 43 26 9 5

88 207 225 156 88 53 29 10 11 867

26 68 173 186 161 98 67 30 38 847

19 39 92 113 113 102 61 47 46 632

3 8 30 42 41 29 23 11 26 213

Total

45-

~

1 2

3 4 5 6 7 8 9Total

115

1 720

548 541 639 544 433 294 186 100 126 3411

TABLE 2

Source of the 150 holoprosencephalic embryos and of the parity-matched controls Holoprosencephaly Method of acquisition

Induced abortion, without a sign of threatened abortion Induced abortion, with a sign of threatened abortion Hysterectomy Ectopic pregnancy Unknown Total I

Significantly different from controls (P < 0.001).

Total

No.

Control

-~ No.

Live

Dead

47

22

69

46.0'

228

76.0

20 2 0

51 0 0 8 81

71 2 0

47.3' 1.4 0.0 5.3 100.0

46

15.3 1.0 0.7 7.0 100.0

0

69

8

150

%

3

2! 21 300

%

264

EI MATSUNAGA AND KOHEI SHIOTA TABLE 3

Distribution of the holoprosencephalbc embryos by deuelopmental stages, method of acquisition, and intrauterine status Induced abortion in cases Carnegie stage

13 14 15 16 17

With a sign of threatened abortion

Without a sign of threatened abortion Live

Dead

6 5 3

?

:a

(%)

Live

0

0 3 2

3

(25.8)

3 8

9

(35.7)

3 0 1

(100.0)

(12.5)

18 19 20

5 3 1

21 22 23

0 0 0

0

0 0 0

Unknown I Total

1 47

3 22

0 20

I

1

'I

Hysterectomy

Source not known

Live

Dead

(58.3)

0 1 0

0 0 0

9 12 8

6.0 8.0 5.3

(63.3)

0 0

1

1

29 34

19.3 22.7

0

0 1 1

13 12 7

8.7 8.0 4.7

0 0 0

2 0 1

11 1

7.3 4.7 0.7

0 2

1 8

7 150

4.7 100.0

-

(%>I

Dead

1 3

;]

(73.3)

3

(100.0)

2 51

0 1

Total No.

7

X

Severely damaged or macerated

TABLE 4

Associated external anomalies in holoprosencephalic embryos, excluding damaged specimens In utero status Live (Y,)

No. of cases examined No. of cases with associated anomalies CNS defects Cleft lip Branchial arch anomalies Polydactyly Limb anomalies Others

half of the holoprosencephalics were associated with various external malformations (table 4). The most commonly associated anomalies were polydactyly (hand and/or foot) and additional defects of t h e central nervous system such a s spina bifida a n d nuchal blebs. I t should be pointed out t h a t t h e frequency of combined anomalies in dead cases (34.9%) must be taken as a n underestimate because detailed examination was often difficult in macerated embryos. Malformations of internal organs were not summarized here because some of the cases were too young to detect internal anomalies. Zncidence and distribution in time As described earlier, the 150 holoprosencephalics were found among the total of 36,380 conceptuses collected during 13 years.

Dead (%I

50

63

30 (60.0) 8 (16.0) 1 (2.0) 8 (16.0) 16 (32.0) 4 (8.0) 0 (0.0)

22 (34.9) 8 (12.7) 6 (9.5) 2 (3.2) 3 (4.8) 2 (3.2) 4 (6.3)

Total I,%)

113 52 (46.0) 16 (14.2) 7 (6.2) 10 (8.8) 19 (16.8) 6 (5.3) 4 (3.5)

If we divide t h e former by the latter, the overall incidence would be 4.1 per 1,000 conceptions. Admittedly this figure is a n underestimate, since holoprosencephalic embryos that had been too much damaged to be detected did not come to our attention. On t h e other hand, t h e figure of 7.3 per 1,000 embryos reported by Tanimura and Uwabe ('71) seems to be a n overestimate because they excluded intact sacs without embryonic proper tissues. Thus t h e average incidence of holoprosencephaly in early conceptuses may be estimated to be somewhere between 0.4-0.7%. Assuming t h a t the proportion of severely damaged embryos in our samples was uniformly distributed throughout the time under study, we examined t h e variation in the incidence of holoprosencephaly by year and month. As shown in table 5, no "epidemic" was

EPIDEMIOLOGY OF HOLOPROSENCEPHALIC EMBRYOS TABLE 5

Annual incidence of holoprosencephalic embryos, 1962-1974 Holoprosencephaly Year

Approximate number of specimens collected

1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 Total

No. of cases

330 100 970 4,960 7,190 4,650 3,980 3,270 2,600 1,800 2,450 1,850 2,230 36,380

Incidence (Y,)

1

0.30

0 2 12 21 19 16 16 17 9 17 11 9 150

0.00

0.21 0.24 0.29 0.41 0.40 0.49 0.65 0.50 0.69 0.59 0.40 0.41

TABLE 6

Distribution of months by number of holoprosencephalics found in a month No. of months, 1965.19'74 No of cases in a month

0 1 2 3 4 5 and more Total Mean Variance

Observed

34 49 23 13 1 0

120 1.15 0.97

Expected

I

38.0 43.7 25.1

;::! 0.8) 120.0 1.15 1.15

xz

0.421 0.643 0.176 0.049 1.289 d.f. = 3

P > 0.50

' Based on the Poisson model

noticed for a particular year or years. However, apart from the first two years in which the sample size was too small, the incidence increased slowly from 0.21% in 1964 up to 0.65% in 1970 and thereafter fluctuated and finally declined to 0.40% in 1974. Although the cause of the upward trend in the first several years is not clear, it seems to be due to improvement in detectability on the side of the investigators rather than to some other environmental factors. Table 6 shows the distribution of months by number of holoprosencephalics from 1965 to 1974, during which the number of specimens collected per year was fairly large. The mean number of cases occurred in one month was 1.15, which was rather close to the variance of 0.969. The distribution of months by number of cases agrees well with the expectation from the Poisson model. This finding implies that

265

holoprosencephaly occurs randomly through time and that the number in one month does not influence the number in other months. Seasonal variation Of the 150 holoprosencephalics, exact date of the last menstruation of the mother was known for 138 cases. Table 7 represents the distribution of months of conception for these cases, together with the expected ones calculated on the basis of vital statistics data for all live-births in Japan for the year of 1968; it is assumed that distribution of months of conception does not differ according to whether the conception ends in induced abortion or live-birth. There was a slight excess in March and April and a slight deficit in June, August and October. Pooling the monthly data by seasons of the year (table 81, there seems to be more cases in winter and less in summer months than expected, though the difference was statistically not significant. On the other hand, assuming homogeneous distribution through months and applying Edwards' method ('61)of testing for a cyclic trend, 0 was found to be 73O, implying that the maximum incidence was in the middle of March and the minimum in the middle of September, and the ratio of the highest to the lowest incidence was 1.55. However, the discrepancy from a homogeneous distribution did not reach the significance level 0:' = 3.23, d.f. 2, P = 0.20). Parity effect, after exclusion of maternal age effect Data concerning both parity and maternal age were available for 128 cases. Table 9 represents the distribution of parit,y of the case mothers, as compared with the expected one in the general embryonic population. The latter was computed from the data shown in table 1, corrected for maternal age; that is, partitioning the observed number in each maternal age group in proportion to the parity distribution a t the corresponding maternal #agedistribution in table 1, the respective figures were summed up over all maternal age groups. The mean parity of the mothers of holoprosencephalics (3.59) was lower than expected (3.84), though the difference was not significant (t = 1.298, 0.3 > P > 0.1). If we divide the mothers into two groups, one with parities 1and 2 and the other with parities 3 and more, then in the case mothers there were significantly more with low parity and less with high

2 66

EI MATSUNAGA AND KOHEI SHIOTA TABLE 7

Distribution of holoprosencephalic embryos by month of conception Expected No of cases

Difference Obs Exp

0.9 0.1

11.1 10.9 11.9 12.1 11.1 11.7 11.9 11.4 10.7 12.0 11.8 11.3 137.9

8.06 7.91 8.63 8.80 8.03 8.46 8.65 8.25 7.77 8.71 8.56 8.17 100.00

12 11 15 15 13 9 13 7 11 7 12 13 138

January February March April May June July August September October November December Total I

% distribution of total conceptions in Japan I

Observed No of cases

Month of conception

3.1

2.9 1.9 - 2.7 1.1 -4.4 0.3 -5.0 0.2 1.7

Based on the vital statistics data for all live-births (1,870,000 in number) in 1968, the Ministry of Health and Welfare.

TABLE 8

Seasonal trend Seeson of conception

holoprosencephalic embryos, 1962.1974

of

Observed No. of cases

Expected No. of cases

Difference 0bs.-Exp.

December-March June-September April, May October, and November

51 40

45.2 45.7

- 5.7

47

47.0

0.0

December-May June-November

79 59

68.4 69.6

10.6 - 10.6

Significance

xZ = 1.46

5.8

d.f. = 2 0.5 > P > 0.3

xZ = 3.26 d.f. = 1 0.10> P > 0.05

TABLE 9

TABLE 10

Distribution ofparity in the study group and in thegeneral embryonic population corrected for maternal age

Distribution of maternal ages in the study group and in the general embryonic population corrected for parity

No. of Parity

cases

1 2 3 4 5 6 7 and more Total Mean Variance

23 31 19 12 16 12 15 128 3.59 4.86

1

Expected No.

16.3 20.9 26.0 21.9 16.8 11.2 14.9 128.0 3.84 4.27

X'

2.75 4.88 1.88 4.48 0.04 0.06 0.0 14.09

Relative incidence

Maternal age

No. of cases

1.42 1.48 0.73 0.55 0.95 1.07 1.00 1.oo

-19 20-24 25-29 30-34 35-39 40-44 45Total Mean Variance

0 22 44 37 20 5

P < 0.05

I

parity than expected (x2 = 10.7, d.f. = 1, P < 0.01).However, the relative incidence showed no consistent pattern with increasing parity. Because the risk does not show a decreasing tendency for the mothers with parity 5 and more, there seems to be no effect of parity upon the production of holoprosencephaly.

P

Expected No.

5.0 30.8 32.9 29.0 22.5

1

0

128 30.23 28.68

127.9 29.72 42.58

x2

Relative incidence

5.32

0.61

3.74 2.21 0.28

1.34 1.27 0.89

0.95

0.64

12.5l

1.00

< 0.05.

Maternal age effect, after exlusion of parity effect Table 10 shows the distribution of maternal ages for the 128 cases, as compared with the expected one which was constructed again from the data in table 1 after correction of the variation in parity. The mean age of the case

267

EPIDEMIOLOGY OF HOLOPROSENCEPHALIC EMBRYOS TABLE 11

Distribution of maternal ages by parity in the study group and in thegeneral embryonic population Parity 1 and 2 Maternal age

Obs.

Exp.

Parity 3 and over Relative incidence

Obs.

Relative incidence

Exp.

-19 20-24

11

4.7 25.1)

0.57

0 5

25-29 30-34

29 5

15.6 5.0

1.86 1.00

15 32

17.3 24.0

35-39 40-44 45-

2

3.0

I

0.6 0.0

0.84

18 4

19.5

1 0

0.92 0.56

54 27.04 18.18

54.0 25.49 27.16

1.00

73.9 32.82 31.99

1.00

Total

Mean Variance

0

0.84

:::1

0

mothers was slightly raised but did not differ significantly from expected (t = 1.078, P > 0.10). Incidentally, among the case mothers, the mean age of 61 mothers with a sign of threatened abortion was 29.7 years and that of 61 without such a sign was 30.4 years, the difference being not significant (t = 0.774, P > 0.20). Referring again t o table 10, the proportion of young mothers (under 25 years of age; 17.2%)was significantly lower than expected (28.0%)(xz = 7.39, d.f. = 1, P < 0.01). However, the relative incidence showed no consistent pattern with advancing maternal age; in particular, the risk apparently decreases for mothers after 30 years of age. In order to explore the complicated pattern as revealed with regard to parity and maternal age, the distribution of maternal age was divided into two parts by parity (table 11). Whereas the mean age of the mothers with high parity in the study group was almost the same as expected, the mean of the mothers with low parity (27.0 years) was higher than expected (25.5years), the difference being significant (t = 2.67, P < 0.01). However, the relative incidence for the mothers with low parity showed no consistent pattern with advancing maternal age. Since the mothers of holoprosencephalics contained, when maternal age was adjusted, significantly more with low parity than expected, and since information is more reliable for maternal age than for parity, the above finding may best be accounted for by assuming that some of the mothers with “low” parity underestimated the number of previous pregnancies they had experienced. DeMyer e t al. (’64) and Holmes e t al. (’74) suggested that arhinencephaly or cebocephaly with normal karyotype is an isolated malfor-

14 32.57 23.63

0.87 1.33

mation or is combined with only a few extracranial anomalies, while a complex of extracranial malformations is typical of cases with an abnormal karyotype. Since many of the reported chromosome aberrations found in the holoprosencephaly series consist of trisomy 13, trisomy 18 and 18p- that are known to increase with advancing maternal age (Lenz and Lenz, ’68; Lenz, ’70), it seemed of some interest to see if there is a difference in the maternal age distribution of our cases between those with and without associated anomalies. A s shown in table 12, however, there was no difference in the mean nor in the distribution of maternal age between the two subgroups.

Paternal age effect, after exclusion of maternal age effect Data on paternal age were available for only 71 cases. Table 13 shows the distribution of TABLE 12

Maternal age distribution for holoprosencephalics, with or without associated anomalies Holoprosencephaly Maternal age

With other anomalies

No.

-19 20-24 25-29 30-34 35-39 40-44 45Total Mean Variance

%

Without other anomalies

No.

%

0

0.0

0

0.0

8 17 14 5 3

17.0 36.2 29.8 10.6 6.4 0.0 100.0

14 27 23 15 2

17.3 33.3 28.4 18.5 2.5

0

47 30.16 30.27

0

81 30.28 28.13

0.0

100.0

268

EI MATSUNAGA AND KOHEI SHIOTA TABLE 13

Paternal age distribution for holoprosencephalics and controls. matched for maternal age Cases Paternal age

20-24 25-29 30-34 35-39 40-44 45Total Mean Variance

Controls Relative incidence

~

No.

4 20

No

'',,

5.6 28.2

X

3 18

20 28.2 13 18.3 12 16.9 2 2.8 100.0 71 33.6 39.94

4.2 25.4)

"14

24 33.8 14 19.7 8 11.2 4 5.6 71 100.0 33.8 38.37

0.83 0.93 1.17 1.00

TABLE 14

History of previous miscarriages of the multiparous mothers in the study group and in the controls (parity matched) No. of previous miscarriages

Mothers of holoprosencephalics

No.

0 1

2 3 Total Mean Variance

i,

67 16 8 4 95 0.463 0.679

70.5 16.8 8.4 4.2 100.0

Controls

No.

X

150 26 10 4 190 0.305 0.446

78.9 13.7 5.3 2.1 100.0

TABLE 15

Regularity ofmenstrual cycles in the mothers o f t h e study group and of the controlgroup (parity-matched) Mothers of holoprosencephalics

Controls

Menstrual cycles

Regular Irregular Total

No

A,

No

%

114 22 136

83.8 16.2 100.0

232 40 272

85.3 14.7 100.0

primiparae. For the remaining 95 multiparous mothers and 190 control mothers matched for parity, the distribution of number of previous miscarriages is shown in table 14.The proportion of the mothers having had a t least one pregnancy wastage prior to the index pregnancy was 29.5% in the study group, whereas it was 21.0% in the control group, though the difference was not significant (xz = 2.47, d.f. = 1, 0.20 > P > 0.10). The mean number of miscarriages in the study group (0.463) was greater than in the controls (0.3051, the difference being a t the borderline of significance (t = 1.623, d.f. = 283, P = 0.05 by 1-tailed test). This finding suggests that the mothers having conceived a holoprosencephalic embryo are prone to have miscarriages in other pregnancies as well. Menstrual cycles

As shown in table 15, no difference was noted in the regularity of menstrual cycles between the mothers of holoprosencephalics and the parity-matched controls. Thus, i t appears that possible causes and consequences of irregular menstruation, e.g., delayed ovulation and delayed fertilization, do not increase the risk of holoprosencephaly. Maternal ill health during pregnancy The frequency of maternal diseases during the index pregnancy did not differ significantly from that of the parity-matched controls (table 16). Association was not proved between holoprosencephaly and chronic diseases or acute infections. The history of epilepsy, goiter, and hyperemesis was reported only by the case mothers, but it is premature to make any hypothesis only from these data. Approximately 70% of the case mothers TABLE 16

paternal age of these cases, together with that of normal controls matched for maternal age. There was virtually no difference in the mean nor in the distribution of paternal ages between the cases and the controls, indicating that paternal age has no effect upon the production of.holoprosencephaly. Maternal history of miscarriages Of the 128 mothers of holoprosencephalics whose parity was known, information concerning previous miscarriages (spontaneous abortions and stillbirths) was available for 118 cases. Of these, 23 mothers were allegedly

Maternal medical history duringpregnancy in the study group and in the control group (parity-matched) Mothers of holoprosencephalic8

No.

No. of cases examined No. of cases with positive history Chronic diseases Acute infections Epilepay Hyperemesis Myoma uteri Unknown

P,

128 20 6 7 1 1 2 3

Controls

No.

%

256 15.6 4.7 5.5 0.8

0.8 1.6 2.3

30

11.7

9

3.5 3.9 0.0 0.0

10 0 0 1 10

0.4 3.9

269

EPIDEMIOLOGY OF HOLOPROSENCEPHALIC EMBRYOS TABLE 17

Maternal genital bleeding during the terminated pregnancy in the study group and in the control group (parity-matched) Holoprosencephaly Genital hleeding

(-1 (+) ( + +)

Total

Total

Csmtrol

-~

Live

Dead

No.

x

26 4 17 41

7 11 44 62

33 15 61 109

30.3 13.8 56.0 100.1

' Accompanied by symptoms of threatened abortion.

'Significantly different from controls ixp

=

62.7, d.f. = 1. P

No.

x.

162 25 31 218

74.3 11.5 14.2 100.0

< 0.001).

TABLE 18

Maternal drug consumption and irradiation during pregnancy in the study group and in the control group (parity-matched) Mothers of holoproeencephalics

No.

No. of cases examined No. of cases with positive history of dlwgs Progestagens Antipyretics Antibiotics Vitamines Hemostatistics Antiemetics Vaccines Other drugs Unknown No. of cases with positive history of irradiation

had genital bleeding, including a sign of threatened abortion, during the index pregnancy and this figure was far greater than that (25.7%)for the control group (table 17). Obviously genital bleeding is a symptom of having a malformed embryo, because i t is closely associated with those holoprosencephalics that have been found dead in utero.

Maternal exposure to drugs and ionizing radiation As shown in table 18, maternal drug intake during the index pregnancy was found to be more frequent in the study group as compared to the parity-matched controls, though the difference was statistically not significant (x' = 3.21, d.f. = 1, 0.10 > P > 0.05). This involved a large grouping of progestagens which, in all the instances, were given to the mothers as a treatment of threatened abortion. Since threatened abortion has been found more frequently in the case mothers,

9L

Controls

-~ No. x 246

123

75 38 5 3 5 2 2

1 4 9

39.8 22.8 3.3 0.8 0.8 1.6 1.6 0.8 6.5 7.3

10 14

4.1

0

0.0

5

2.0

49 28 4 1 1 2 2

1

30.5 15.4 2.0 1.2 2.0 0.8 0.8 0.4 5.7

this association must not be a causal one. No difference was noted in the consumption of other kinds of drugs between the mothers of holoprosencephalics and the control mothers. None of the mothers in the study group had a history of X-ray exposure during the index pregnancy.

Maternal smoking and alcohol consumption There was no difference in the frequency of reported smoking and alcohol consumption during t h e pregnancy between t h e case mothers and the control mothers (tables 19, 20).

Family history and parental consanguinity Information on the family was available for only 57 mothers in the study group. The father of a holoprosencephalic embryo had some congenital malformation (details were not given),

270

El MATSUNAGA AND KOHEI SHIOTA TABLE 19

Smoking habit of the mothers in the studygroup and in the controlgroup (parity-matched) Mothers of holoprosencephalics

x

No.

With history of smoking Without history of smoking Total

Controls No.

7,

12

11.7

33

16.0

91 103

88.3 100.0

173 206

84.0 100.0

TABLE 20

Drinking habit of the mothers in the study group and in the control group (parity-matched) Mothers of holoprosencephalics No.

With history of alcohol consumption Without history of alcohol consumption Total ’

V,

Controls

x

No.

17

33.3

30

31.3

34 51

66.7 100.0

66

68.7 100.0

’ Include all the cases for which information

96

was available.

while the remaining 56 mothers did not recall any abnormalities in their close relatives. Among 115 mothers in the study group, there was only one (0.9%;second cousin marriage) who admitted consanguinity between spouses. In the control group of 231 mothers, all but one (0.4%;details unknown) gave negative answer with respect to consanguinity. Thus, there was no indication of elevated parental consanguinity in our series of holoprosencephaly. DISCUSSION

From the data presented above, it has become clear that holoprosencephaly, which was found in the human embryo collection with a frequency of a t least once per every 250 conceptuses, occurred largely at random through time. During the ten years from 1965 to 1974, no “epidemic” was noted in particular years or months. However, the incidence rose slowly from 0.21%in 1965 up to 0.65%in 1970 and thereafter fluctuated and finally declined to 0.40% in 1974. The upward trend in the first several years seemed to be due to improvement in ascertainment rather than to some other environmental factors. On the other hand, there was a suggestive indication that holoprosencephaly arises more frequently

from conception in winter and less frequently in summer months than expected. However, the number of cases was too small to allow to examine the consistency of the pattern for each year under study. Clearly more data are needed to interpret the observed seasonal variation. In our data, the mean parity of the mothers of holoprosencephalic embryos was found, as noted by Tanimura and Uwabe (‘711, to be lower than expected. However, the relative incidence showed no consistently decreasing pattern with increasing parity, suggesting that parity has no effect upon the production of holoprosencephaly. The lowered mean parity of the mothers was ascribed to the involvement of significantly more mothers with low (1 and 2) parity, whose mean age was significantly higher than the controls. In view of the finding that the mothers of holoprosencephalics had more frequently miscarriages in the past pregnancies, it was concluded that some of the mothers with “low” parity had underestimated the number of miscarriages they had experienced. Our data disclosed no evidence of parental age effect. In addition, apart from the history of genital bleeding and intake of progestagens as a treatment of threatened abortions, no association was found between holoprosencephaly and maternal medical history, including irregularity of menstrual cycles, and smoking and drinking habits. Presumably the commonly used drugs listed in table 18 do not cause holoprosencephaly in man. Among the negative findings perhaps the most striking is that we failed to demonstrate the effect of maternal age upon our series of holoprosencephaly. As described at the outset, there have been a number of reported cases of arhinencephaly or cebocephaly in which trisomies 13 and 18 or 18p- were detected. On the other hand, all of these chromosome aberrations are known to occur in close association with advancing maternal age (Lenz and Lenz, ‘68; Lenz, ’70). Consequently, if our series had contained an appreciable fraction of embryos with these abnormal karyotypes, a proportionate rise in maternal age should be expected. For example, if all of our holoprosencephalic embryos were of trisomy 13, which is the most common abnormal karyotype reported in association with holoprosencephaly among newborn or stillborn cases (Cohen et al., ’71), and if the relative incidence of trisomy 13 rises with advancing maternal age in a fashion sim-

EPIDEMIOLOGY OF HOLOPROSENCEPHALIC EMBRYOS

ilar to trisomy 21, i t can be shown that the mean age of the mothers should be raised to about 35 years. Referring to table 10, the observed mean age of our mothers was 30.2 years which was higher than expected only by 0.5 year. Therefore, our data suggest that the chromosome aberrations that are dependent upon maternal age may not constitute a major part as causes of holoprosencephaly in early human embryos. However, this should be confirmed by cytogenetic survey of holoprosencephalic embryos in future. Incidentally, Boue et al. (’75) found trisomy 16 to be the most frequent autosomal trisomy in spontaneous abortuses, and Pawlowitzki (‘69) demonstrated no increase in maternal age for this specific type of autosomal trisomies (cited from Vogel, ’701, Whether embryos of trisomy 16 show holoprosencephaly a s part of the phenotype remains to be answered. The only positive clue disclosed by the present study to the etiology of human holoprosencephaly was that the mothers of holoprosencephalics had more frequently miscarriages in previous pregnancies than the control mothers. In view of a number of reports on familial occurrence of arhinencephaly and cebocephaly, this finding supports the view that there is some kind of maternal predisposition, genetic andlor environmental, to this malformation. Formally, such predisposition may involve chromosomal mosaicism or structural aberrations in a balanced form, as has been described by Pfitzer and Munterfering (‘681, recessive mutant genes, polygenes, or chronic maternal illness or intoxication. In our data, however, there was no indication of increased rate of parental consanguinity, which suggests that most of our cases were to be attributed to causes other than rare autosomal recessive genes. With respect to the possibility of chronic maternal illness or intoxication, there was virtually no suggestion a t all. Dekaban (‘59) reported two cases of arhinencephaly born to diabetic mothers (cited from Warkany, ’711, but none of our mothers were known to be diabetic. Review of the literature indicates that most of the reported cases of holoprosencephaly occurred apparently sporadically. Holmes e t al. (’74) and Ming e t al. (’76) showed that the reported cases for which karyotyping was performed were nearly equally divided into two groups, i.e., one with normal karyotype and the other with various kinds of chromosomes aberrations. However, since cases with abnor-

271

mal karyotypes are likely to be reported more frequently, they represent probably less than half of all newborn cases, though the situation may be different in holoprosencephalic embryos. Sporadic cases with normal karyotype can of course be caused by the same genic or environmental factors as proposed for familial cases. In addition, fresh dominant mutations are conceivable as a cause, but this must be the case only in rare instances because the mutation rate per locus per generation is genTaking erally as low as of the order of into account the high frequency of this lethal malformation in the embryonic population, and its random occurrence through time in the rapidly changing environment of our society, i t may well be assumed that a majority of the cases with normal karyotype are, as many other common congenital malformations, caused by polygenic mechanism with multiple thresholds corresponding to various degrees of the defects. ACKNOWLEDGMENTS

We are deeply indebted to Professor H. Nishimura for helpful comments. We acknowledge the collaboration of the members of Department of Anatomy and Human Embryo Center for Teratological Studies, Kyoto University. The help of a large number of obstetricians who provided embryonic specimens is also acknowledged. This study, contribution 1132 from the National Insititute of Genetics, was supported by a grant from the Ministry of Education, Japan. LITERATURE CITED Arakaki, D. T., and S. H. Waxman 1969 Trisomy D in a cyclops. J. Pediat., 74: 620-622. h u e , J., A. Boue and P. Lazar 1975 Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous human abortions. Teratology, 12: 11-26. von Buhler, E., I. Bodis, R. Rossier and G. Stalder 1962 Trisomie 13-15 mit Cebocephalie. Ann. Paediat., 199; 198205. Cohen, M. M., Jr., J. E. Jirasek, R. T. Guzman, R. J. Gorlin and M. Q. Peterson 1971 Holoprosencephaly and facial dysmorphia: Nosology, etiology and pathogenesis. Birth Defects: Orig. Art. Ser., 7(7): 125-135. Cohen. M. M., D. F. Storm and V. J. Capraro 1972 A ring chromosome (no. 18)in a cyclops. Clin. Genet., 3: 249-252. Conen, P. E., B. Erkman and C. Metaxotou 1966 The “D” syndrome. Am. J. Dia. Child., 111: 236.247. DeMyer, W. 1964 A 46-chromosome cebocephaly with remarks on the relation of 13-15 trisomy to holoprosencephaly (arhinencephaly). Ann. Paediat., 203: 169-177. DeMyer, W., W. Zeman and C. D. Palmer 1963 Familial alobar holoprosencephaly (arhinencephaly) with median cleft lip and palate. Neurology, 13: 913-918. Edwards, J. H. 1961 The recognition and estimation of cyclic trends. Ann. Hum. Genet., 25: 89-86.

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Emanuel. I.. S. W. Huang, L. T. Gutman, F. C. Yu and C. C. Lin 1972 The incidence of congenital malformations in a Chinese population. Teratology, 5: 159-169. Emberger, J.-M., C. Marty-Double, D. Pincemin and J. Caderas de Kerleau 1976 Holoprosencephalie par triploidie 69, XXX chez un foetus de 5 mois. Ann. Genet., 19: 191-193. Faint, S., and F. J. W. Lewis 1964 Presumptive deletion of the short arms of chromosome 18 in a cyclops. Human Chromosome Newsletter, 14: 5. Fujimoto, A,, A. J. Ebbin, J. W. Towner and M. G. Wilson 1973 Trisomy 13 in two infants with cyclops. J. Med. Genet., 10: 294-296. Gorlin, R. J., J. Yunis and V. E. Anderson 1968 Short arm deletion of chromosome 18 in cebocephaly. Am. J. Dis. Child., 115: 473-476. Grebe, H. 1954 Familienbefunde bei letalen Anomalien der Korperform. Acta Genet. Med. Gemellol., 3: 93-111. Halbrecht, I., 0. Kletzky, L. Komlos. M. Lotker and N. Gersht 1971 Trisomy D in cyclops. Obst. Gyn., 37: 391393. Hintz, R. L., M. Menking and J. F. Sotos 1968 Familial holoprosencephaly with endocrine dysgenesis. J. Pediat.. 72: 81-87. Holmes, L. B., S. Driscoll and L. Atkins 1974 Genetic heterogeneity of cebocephaly. J. Med. Genet., 1 I: 35-40. James, E.. and G. van Leeuwen 1970 Familial cebocephaly. Case description and survey of the anomaly. Clin. Pediat., 9: 491-493. Klopstock, A. 1921 Familiares Vorkommen von Cyklopie und Arrhinencephalie. Mschr. Geburtsh. Gyn., 56: 59-71. Landau, J. W., J. M. Barry and R. Koch 1963 Arhinencephaly. J . Pediat., 62: 895-900. Lang, A. P., F. M. Schlager and H. A. Gardner 1976 Trisomy 18 and cyclopia. Teratology, 14: 195-204. Lazjuk, G. I., 1. W. Lurie and M. K. Nedzved 1976 Further studies on the genetic heterogeneity of cebocephaly. J . Med. Genet., 13: 314-318. Lejeune, J., B. Dutrillaux, M. 0. Rethore, R. Berger. H. Dehrey, P. Veron, F. Gorce and A. Grosslord 1969 Sur trois cas de trisomie C. Ann. Genet., 12: 28-35. Lenz, W. 1970 Birth defects - genetic aspects. In: Congenital Malformations. Excerpta Medica International Congress Series. Vol. 204. F. C. Fraser and V. A. McKusick. eds. Excerpta Medica, Amsterdam and New York. pp. 402-406. Lenz. W., and F. Lenz 1968 Grundlinien der Humangenetik. Zu Definition, Terminologie und Methoden. In: Humangenetik. P. E. Becker, ed. Georg Thieme Verlag, Stuttgart. Bd. 111, pp. 1-76. McDermott, A,. J . Insley, M. E. Barton, P. Rowe. J . H. Edwards and A. H. Cameron 1968 Arrhinencephaly associated with a deficiency involving chromosome 18. J. Med. Genet., 5: 60-67. McDonald, R. 1968 Median facial cleft with hypotelorism. Am. J. Dis. Child., 115: 728-731. McKusick, V. A. 1961 Medical genetics 1960. J. Chr. Dis., 14; 1-198. Miller, J. Q., E. K. Picard, M. K. Alkan, S. Warner and P. S. Gerald 1963 A specific congenital brain defect (arhinencephaly) in 13-15 trisomy. New Eng. J. Med., 286: 120124. Ming, P.-M. L.. D. M. Goodner and T. S. Park 1976 Cytogenetic variants in holoprosencephaly. Am. J. Dis. Child., 130- 864-867.

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