PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M.Laurence & P. Gregory

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M. LAURENCE M.A. M.R.C.P. F.R.C.Path. P. GREGORY B.Sc. Department of Child Health Welsh National School of Medicine, Cardiff 1 2 3 4 5

Amniocentesis Amnion cell culture: method and difficulties Indications for amnion cell culture and cytogenetic results Ethical considerations Future developments and problems References

The incidence of chromosome abnormalities is approximately 1.5/1000 births. Amongst these is the Down syndrome, which is the most common cause of severe mental retardation in the UK. At present the factors responsible for chromosome mutations are unknown and the only means of reducing their incidence at birth is by early prenatal detection followed by selective abortion of fetuses shown to be abnormal. Since the first fetal karyotyping by Steele & Breg in 1966, the introduction of second trimester transabdominal amniocentesis and the development of reliable culture methods for the exfoliate fetal cells in the amniotic fluid, prenatal diagnosis is becoming a routine procedure. An increasing number of centres have gained experience in this field and are giving such a service, which has become an integral part of genetic counselling. Several series have now been published (Ferguson-Smith, Ferguson-Smith, Nevin & Stone, 1971; Gerbie, Nadler & Gerbie, 1971; Hsu, Dubin, Kerenyi & Hirschhom, 1973; Milunsky, 1973; WahlstrSm, Bartsch & Lundberg, 1974; Niermeijer, Sachs, Jahodova, Tichelaar-KleppeT, Kleijer & Galjaard, 1976). We here present our method for early pregnancy cytogenetics and some of the problems and pitfalls that may be encountered.

1. Amniocentesis Many of the patients seeking advice about prenatal diagnosis are already pregnant, sometimes beyond 20 weeks. Earlier counselling, with a decision about embarking on a further pregnancy and the possibility of amniocentesis for chromosome analysis should, ideally, have been carried out (Dewhurst & Lucas, 1973). If this has been done, just before amniocentesis the parents are interviewed and counselled again. It should be ascertained that both parents appreciate the slight risks of the procedure, the scope and limitations of the tests offered— especially that a normal baby cannot be guaranteed—and the possible need for a termination of pregnancy should an abnormal fetus be identified. It ought also to be explained that the results of the test may not be available for 3-4 weeks and that occasionally a second amniocentesis may be required.

VoL 32 No. 1

Occasionally amnion cell culture fails even after a second amniocentesis and then no diagnostic help can be given. However, if a culture is obtained chromosome abnormalities can be almost entirely excluded. Transabdominal amniocentesis, which is an out-patient procedure, has been shown to be the most satisfactory method of obtaining amniotic fluid. Although the uterus becomes palpable abdominally from at least 12 weeks, it is not until after 14-15 weeks that sufficient amniotic fluid is present for some to be removed with relative safety. Amniocentesis, now that the per vaginam route—with the associated risks of abortion and amniotic infection (Fuchs & Cederqvist, 1966)— has been abandoned, seems to be arelativelysafe technique in specialist centres. The number of sequential abortions is estimated at 1 % (Nadler & Gerbie, 1970; Milunsky & Atkins, 1974; Niermeijer et al. 1976); and the results of the current Medical Research Council study indicate that the number of abortions following amniocentesis seems to be little different from those occurring in "control" pregnancies. It appears that now this investigation tends not to be carried out before the 15th week. After this there is less difficulty in obtaining a successful tap, and fetal damage does not occur. However, the long-term effects have yet to be evaluated fully by a follow-up of children born after prenatal diagnosis, though preliminary results seem to be favourable (Allen, Sergovich, Stuart, Pozsonyi & Murray, 1974). The timing of the procedure is vital, not only because of the problems presented by too early an amniocentesis, but also because of the time taken for the laboratory work and the need for any termination to be carried out before the 20th week of gestation if at all possible. An ultrasonic B-scan is carried out at 15-16 weeks to measure the fetal biparietal diameter for confirmation of the gestational length. It is also essential to locate the placenta as accurately as possible and to exclude twins. Anencephaly can be identified when the normal head outline is unobtainable (Campbell, 1974). If there is any doubt as to the gestational length, an earlier B-scan is a wise precaution. There appear to be no obvious risks in the use of ultrasound. In-vitroresultssuggesting that ultrasound produces chromosome damage (Macintosh & Davey, 1970) have not only failed to be confirmed (Coakley, Hughes, Slade & Laurence, 1971; Coakley, Slade, Braeman & Moore, 1972), but have now been shown to be due largely to the techniques used (I. J. C. Macintosh, personal communication, 1974). Before carrying out the amniocentesis itself, the bladder must be emptied; the fetal heart is then monitored, using Doppler ultrasound, and the lower abdomen is cleansed. The amniocentesis is performed with a 9 cm 20-gauge spinal needle with a stilette in place, the needle being inserted into the amniotic cavity so as to avoid the placenta. It is not generally necessary to use any local anaesthetic. Once within the amniotic cavity, the stilette is removed and, with the use of a sterile plastic syringe, 10-15ml of amniotic fluid are withdrawn and placed in a sterile plastic container with a tapered bottom. Glass containers should be avoided, as viable amnion cells tend to adhere to the surface. After withdrawal of the needle, the fetal heart is once more monitored, a blood sample is taken for a Kleihauer test to exclude fetomaternal haemorrhage, and the patient is allowed to leave an hour later. The fluid should be straw coloured and free from macroscopic blood, but it may be turbid if it contains a large number of cells. The amniocentesis should preferably be carried out in the same institution as the subsequent amnion cell culture. However, if the fluid

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M. Laurence & P. Gregory has to be transported, this should be rapid, at room temperature. Refrigeration should be avoided. In this Department, 427 amniocenteses have been performed on 409 patients since April 1972. In one patient, where the placenta extended over the whole of the anterior wall of the uterus, the amniocentesis was abandoned. In two instances, amniocentesis attempted at the 14th week was unsuccessful, but it was successful one week later. There were three sequential abortions. The first was of a macerated fetus, 4 days after a tap at 17 weeks. The second occurred after amniocentesis at 16} weeks, which was repeated 10 days later as the cultures were not growing. The second amniotic fluid was discoloured and contained considerably raised a-fetoprotein concentrations. A macerated, but anatomically normal, fetus was passed on the day following. The third sequential miscarriage followed a tap at 16 weeks, which yielded somewhat discoloured amniotic fluid with elevated a-fetoprotein concentrations. An abortion of a macerated fetus occurred 7 days later, which was too distorted for neural tube malformation to be excluded with certainty. Three further miscarriages occurred in the series, which were probably unrelated to the amniocentesis. These were of fresh abortuses 3, 5 and 6 weeks after the tap. Nearly all amniocenteses were performed between the 15th and 18th weeks of gestation, though a few were carried out before the 15th week and between the 19th and 24th weeks, and there was one in which the tap was carried out just before delivery at 39 weeks. Most were carried out in this Department by one individual, but some of the fluids were obtained elsewhere and sent to the laboratory, generally within 4 hours of carrying out the tap.

The sample offluid,ideally of about 15 ml, is centrifuged for lOmin at 500-1000rev./min. The supernatant liquor is removed using sterile precautions and kept for specific biochemical tests, including the estimation of a-fetoprotein concentrations. The presence of red blood cells generally slows down the rate of growth; they are therefore best removed by the method of Lee, Gregson & Walker (1970). The centrifuged fetal cells are re-suspended in 2 ml culture medium composed of TC 199 enriched with 30% fetal bovine serum. The cell suspension is inoculated into two glass Leighton tubes which will represent the primary cultures. The tubes are gassed with a 95 % air-5 % CO2 mixture for about 15 seconds, sealed with a rubber bung and incubated undisturbed for 7 days at 37.5 °C. At the end of this time, these vessels are examined for cell growth, with the use of an inverted microscope. The way that the cultures are then handled depends on the amount of growth that has occurred. When there is a complete absence of cell growth or only an occasional small, isolated colony, the obstetrician is warned that a second sample of amniotic fluid may be required. However, after changing the culture medium as described below, the primary cultures are re-incubated for a further week, as in a number of instances adequate growth does eventually take place. If at the end of that time no colonies are visible, the cultures are then discarded and the obstetrician is informed. With our own cases, a decision to undertake a second amniocentesis when the cells from the first amniocentesis appeared not to grow was generally made only when the indication for the amniocentesis was primarily a cytogenetic one. With some repeat amniocenteses a result was eventually obtained in 90.7 % of all the pregnancies, but in those where a cytogenetic result was essential the success rate was 95.6%. If there are only isolated growing cells, even if numerous ones, the culture medium is transferred into a sterile centrifuge tube which is sealed and spun for 10 min at 500-1000 rev./min. The spent medium is decanted, the cell button re-suspended in 1 ml of fresh culture medium and the cells re-inoculated into the primary culture Leighton tubes which are gassed, sealed and re-incubated, and examined at 3- or 4-day intervals. If, at • the end of a further week's incubation, growth has not progressed, the obstetrician is informed that a result may not be obtained or may be delayed. When, on the other hand, several colonies of growing cells are present, adherent to the Leighton tubes, the floating cells in the culture medium are recovered by centrifugation and re-inoculated into fresh Leighton tubes to make back-up cultures. The original Leighton tubes are given fresh culture medium, gassed and re-incubated, and examined every 3 or 4 days. At about 10-20 days, sufficient growth should be present in the original culture vessels for subculture to be undertaken. The exact time for subculture is a matter of judgement, but in general it may be undertaken when growth covers between a third and a half of the culture vessel surface. Occasionally, subculture can be undertaken after the initial 7-day incubation. To subculture, the culture medium is first decanted from the Leighton tubes into a 15 ml conical centrifuge tube, and the adherent cells are washed with 2 ml of phosphate buffered saline which is decanted into the centrifuge tube. The remaining adherent cells are then treated with 1 ml of trypsinVersene solution, placed in an incubator for 2 min, then examined under a microscope. The colonies will be seen to be partially detached and a sharp tap is generally sufficient to remove those still adherent. The trypsin-Versene cell suspen-

2. Amnlon Cell Culture: Method and Difficulties Amniotic fluid contains numerous exfoliate cells, mostly squames from the skin, but some desquamated from the fetal membranes and from the respiratory, alimentary, gastrointestinal and renal tracts, and also macrophages. It is this latter group of potentially viable cells that one hopes to culture. Which of these cells eventually grow is not known for certain, but they are probably macrophages (Cutz & Conen, 1973). Although it is notoriously difficult to use cell-culture techniques developed in other laboratories, an attempt was made to do so between 1969 and 1972. Amniotic fluids obtained at termination of pregnancy, and also from 73 patients where a diagnostic result would have been helpful, gave uniformly poor results (Table I) (Laurence, Gregory, Stark & Turnbull, 1974). In view of this consistently disappointing experience, a different method of cell culture was developed which so far has been satisfactory. TABLE I. Results from amnion cell culture before March 1972 No growth

Experimental Diagnostic Total

Some growth

Useful cytogenetic result

Total

38 17

79 40

24 16

141 73

55

119

40

214

10

Br. Med. Bull. 1976

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M.Laurence &P. Gregory TABLE II. Quality of liquor and culture success in 428 samples of amniotic fluid

sion is then added to the centrifuge tube. The Leighton tubes are washed 3 or 4 times further with culture medium, each wash being added to the centrifuge tube. Finally, 1 ml of culture medium is added to the Leighton tubes, which is re-incubated so that any growing cells still in the Leighton tubes may resettle and provide a further back-up culture. The cell suspension in the centrifuge tube is spun at 500-1000 rev./min for lOmin, the supernatant discarded, and the cell deposit resuspended in 1 ml of culture medium. This suspension is placed very carefully on to the surface of three 22 mm square cover glasses placed in separate small Petri dishes. The suspension should not be allowed to run off the surface of the cover glass, so ensuring that the growing cells are confined to the upper surface of the glass. The Petri dishes are placed in a plastic sandwich box and gassed with a 95% air-5% COj mixture for about lOmin; the box is sealed and then incubated for 24 hours. Following the addition of 2-3 ml of fresh culture medium to each Petri dish, the cultures are examined microscopically. If the subculture has been successful, the Petri dishes are incubated for a further 24 hours in plastic boxes which are re-gassed. Occasionally, an additional 24 hours' incubation is required. Cell division is arrested at metaphase by the addition of one drop of colchicine (40mg/100ml) to each Petri dish, which isreturnedto the sandwich box, re-gassed and incubated for a further 4-5 hours. Microscopical examination should now reveal a large number of cells which have accumulated at the metaphase stage of mitosis. The cover glasses are removed from the Petri dishes and placed in 0.75M-potassium chloride diluted 1:7 with deionized water, pre-warmed to 37 °C in glass Columbia jars and left for 8-12min. This hypotonic treatment causes swelling of the cell envelope. Each cover glass is then removed andfixedin ice-cold methanol-acetic acid mixture (3:1), first being held over the fixative for 15 seconds and then immersed for a further 15min. At the end of that time the excessfixativeis drained off and the cover glasses are passed rapidly above a Bunsen flame. The cells are stained in aceto-orcein at room temperature for 15 min. After rapid dehydration, the slides are mounted in Canada balsam and examined in the usual way. Our cultures grew sufficiently successfully for a diagnostic result to be obtained in 371 out of 427 samples, an over-all success rate of 86.9%. All the taps carried out were preceded by ultrasonic placental localization and were nearly all free from blood contamination or contained only a very few red blood cells, while some of those carried out elsewhere, often without a prior scan, were blood stained (Doran, Rudd, Gardner, Lowden, Benzie & Liedgren, 1974). The clear fluids without red blood cells or cloudy fluids without red blood cells had successful culture rates of 95.1 % and 96.6%, respectively. When only a few or a moderate number of red blood cells were present after centrifugation, the culture success rates were 88.7% and 81 %, respectively. When the blood contamination was greater and required ammonium chloride haemolysis, the rate dropped to 76.2%, and to 63.2% when it contained actual blood clots (Table II). The commonest cause of culture failure was the presence of blood in the amniotic fluid (Table HI). Infection with yeast accounted for a further group of 10 failures; two failed to grow because the fetus appeared to have been dead at the time of the amniocentesis, and one sample from a late pregnancy failed, presumably because it contained only a few or no viable cells. Two cultures from second amniocenteses were discarded because the cells from the first tap ultimately grew. In 19 no reason for the culture failure could

Number of samples

Success (%)

Average time to report (days)

Clear liquor, no red blood cells (RBCs) Clear liquor, few RBCs seen after centrifugation Clear liquor, few RBCs* Cloudy liquor, no RBCs Cloudy liquor with moderate number of RBCs seen after centrifugation Cloudy liquor with moderate number of RBCs seen after centrifugation* Very bloody liquor with RBCs visible macroscopically* Very bloody liquor with RBCs visible macroscopically and clots*

122

95.1

24.2

79

88.7

22.6

15 86 21

93.4 96.6 81.0

24.8

•43

76.2

23.8

43

67.5

28

19

63.2

27

•223.

23.8

• RBCs removed by haemolysis

be found, though a few of these were from gestations of less than 15 weeks; these have been noted as less successful in culture (Wahlstrom, Brosset & Bartsch, 1970; Nelson, 1973). In 3.5 % of cases the result was available in less than 14 days and a further 31.6 % at between 14 and 20 days. The bulk of the reports were made between 21 and 27 days (47.4%). In 17.5 %, mostly the cases whichrequireda second tap, a longer time was required. The presence of red blood cells not only appeared to cause the culture success rate to fall, but also to lengthen the time that it took to obtain a result. Removal of the red cells by haemolysis seems to improve the culture success rate, but only at the expense of slowing further the rate of cell growth. The cloudy fluids without red blood cells seemed on average to be, those that yielded results most quickly. TABLE III. Causes for failure to obtain results in amnion cell culture Reason

Number of cultures

Heavy blood staining Yeast infection Intra-uterine death Failure to subculture Laboratory accident Sparse unsatisfactory spreads Failure to spread Late sample Discarded culture No reason found

17 10 2 2 1 1 1 1 2* 19

Total

56

• These were repeat amniocenteses where the Initial sample ultimately yielded a result

11 Vol. 32 No. 1

Naked eye appearance

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M.Laurence & P. Gregory TABLE IV. Indication for amniocentesis Reason for amniocentesis

Successful prenatal diagnosis

Advanced maternal age Previous child with the Down syndrome Family history of the Down syndrome Translocatlon carrier parent Previous abnormal child with confirmed chromosome abnormality Previous abnormal child with unconfirmed chromosome abnormality Carrier of X-linked disorder History of central nervous system malformation Maternal anxiety only

102 25 14 4 3

3 0 3 0 0

10 5 196

Total

3.

Prenatal diagnosis failed

Positive cytogenetic finding

Total cases

7 2 2 1 0

7 2 0 2 0

105 25 17 4 3

2

2

1

12

0 30

0 4

0 3

5 226

Repeat amniocentesis

12

12

371

38

18

409

16

suggest a risk of greater than 1 in 14 for the whole group, and a risk of almost 1 in 8 for pregnancies in women over 40 years— risks considerably higher than those generally used for genetic counselling or found in individual series or the two large collected series by Milunsky (1973) and Polani & Benson (1973). Twenty-five pregnancies were investigated because the mother, though young, had had a previous baby with the Down syndrome, of the trisomic variety. In every instance both parents were chromosomally normal. Two of the fetuses were shown to be abnormal, one with the Klinefelter syndrome and the other with trisomy G, suggesting a risk of about 1 in 12. Both pregnancies were terminated. Four pregnancies were investigated where one parent was a translocation carrier. Two of the fetuses were also shown to be carriers, the others were cytogenetically normal and all four pregnancies were allowed

Indications for Amnion Cell Culture and Cytogenetic Results

The commonest cytogenetic indication was advanced maternal age, accounting for 105 cases, with 102 successful prenatal diagnoses (Table IV). Of these patients, eight were aged 45 and over, 55 were between 40 and 44, and 36 were between 35 and 39 years. Fetal chromosome abnormality was shown in one, five and one case in these age-groups, respectively. These included three cases of trisomy G, one each of trisomy D and E, both of which have been reported separately (Turnbull, Gregory & Laurence, 1973; Laurence et al. 1974), one of the Klinefelter syndrome and one which seemed to be a male mosaic, with one cell line having an extra F chromosome (Table V). In all seven patients the pregnancy was terminated. These numbers of fetuses with abnormal chromosomes would

TABLE V. Abnormal cytogenetic findings on amnion cell culture Reason for request

Chromosome abnormality found

Percentage chromosome abnormalities Present series

c> 45 years (8 cases) 40-44 year* (55 cases) Maternal age • 35-39 years (36 cases) [Age unspecified (3 cases) History of central nervoussyrtem malformations

46,XY/47,XY,F+ 47.XY.E+ 2 47.XX.G + 47.XXY 47.XX.D + 47.XY.G + 47.XX, G +

Previous Down syndrome Abnormal child of unspecified type Maternal anxiety Translocatlon carrier parent

Milunsky & Atkins (1974)

12.5 9.1

mean = 8-2

2.9 *«1

46.XX/47.XX.20+ ** 47.XY.G + 47.XXY 47.XY.G + 46,XY.Gp+ 47.XY.G + 45,XX,t(Dp+Gp) 45,XY,t(Gp+Gp)

Polani & Benson (1973)

mean= 1.9

mean = 2.0

1.5

1.6 1.6 8.7 10.0 8.3 50§

1.0

1.0

21.2

18.2

* Pregnancy terminated t Abnormal external appearance i Chromosome abnormality not confirmed after termination or birth § Phenotyplcally normal

12 Br. Med. Bull. 1976

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M. Laurence & P. Gregory Epstein, Golbus, Abbo-Halbasch & Rogers, 1974). Whenever mosaicism occurs in culture mycoplasma infection should be excluded. All the culture material should reflect the aberration, as should cultures from a second tap, and many cells should be examined before a termination is undertaken. No case of 46,XX/46,XY mosaicism was observed which might have suggested an admixture of maternal and fetal cells, nor was a male child diagnosed as a female, even though there were a number of instances with both raised Kleihauer test values and serum a-fetoprotein concentrations; this suggests some fetomaternal transfusion, and obviously also the possibility of some maternal blood spillage into the amniotic fluid. Contamination with viable maternal cells seems to be rare, though Nadler (1972) estimated that this occurs in about 0.5 % of cultures. The possibility has always to be kept in mind, especially in cases where rapid early growth is seen, and checks of such contamination are essential (Polani & Benson, 1973). Some polyploidy is seen in most cultures and is of no clinical significance. It probably reflects the tetraploid nature of the human amnion (Burton, Gerbie & Nadler, 1974). In no case that has been allowed to go to term did the child have an unexpected chromosome abnormality. Of the six trisomy G fetuses detected, only one showed some of the stigmata associated with this syndrome. The remainder had normal external characteristics, including normal palmar creases, and they would probably not have been recognized as having the Down syndrome were it not for the knowledge of the cytogenetic results.

to go to term, ending in phenotypically normal infants. None of the 14 successful amnion cultures from 17 pregnancies of young women with a family history of the Down syndrome in the absence of a balanced translocation showed any chromosome abnormality. Three pregnancies in young women who had had a child with chromosome aberration other than the Down syndrome were investigated and all had chromosomally normal infants. Twelve women, who previously had had a child with multiple congenital malformations not known to have a chromosome basis, had an amniocentesis. The 10 successful cultures revealed one fetus with trisomy G, which was aborted. Five women who are carriers for a serious X-linked disorder, two for Duchenne muscular dystrophy and one each for the Hunter syndrome, haemophilia and Christmas disease, had an amniocentesis with a view to sexing the fetus. Allfivefetuses proved to be chromosomally normal females and were, therefore, not aborted. Had any of them been males, with a 1 in 2 risk of having the disease, the pregnancy would have been terminated. Although it is possible to make a rapid preliminary identification of the sex of the fetus by staining the fetal cells with carbol fuchsin to demonstrate the Barr body in females, and with quinacrine dihydrochloride to show a Y chromosome under fluorescence, these are unreliable methods on which to base a decision as to whether or not to abort (Nelson & Emery, 1970; Rook, Hsu, Gertner & Hirschhorn, 1971; Hsu et al. 1973). Amnion cell culture followed by positive identification of the X and Y chromosomes is essential. Amnion cell cultures were carried out for 226 women at high risk of having babies with neural tube malformations. This policy was adopted after the birth of a surviving infant with the Down syndrome, following a pregnancy investigated for neural tube malformation only by amniocentesis and a-fetoprotein estimation. However, a second amniocentesis is not normally considered when the cell culture is not growing. In the 196 in which a successful culture result was obtained, two fetuses with trisomy G and one mosaic where one cell line had an extra no. 20 chromosome were found. All three pregnancies were terminated. In 12 pregnancies of women without high risk for either chromosome or neural tube malformation, amniocenteses were carried out because of extreme maternal anxiety. In one of these trisomy G was identified and the pregnancy was terminated. Three amniotic fluids were referred to the laboratory for amnion cell culture from elsewhere, without information about the pregnancy. All proved to be of fetuses with normal chromosome constitution. No pregnancy was investigated because of habitual abortion (Lucas, Wallace & Hirschhorn, 1972). All but two of the aborted cytogenetically abnormal fetuses were examined and the result was confirmed. Of these two, in one instance the trisomy G female fetus was placed in fixative, but this had many of the external features of the Down syndrome and a ventricular septal defect of the heart. In the other instance, that of the 46,XX/47,XX:20+ mosaic, the phenotypically normal female fetus had a normal 46,XX chromosome constitution in the cord blood and skin. Other tissues should perhaps have been examined, but it would appear probable that a pregnancy of a normal fetus was terminated. This has been reported previously (Kardon, Chernay, Hsu, Martin & Hirschhorn, 1972; Niermeijertf/a/. 1976). Mosaicism occurring in an amnion cell culture should be regarded with extreme suspicion, for it may arise spontaneously in culture or possibly as a result of mycoplasma infection (Schneider, Stanbridge,

4. Ethical Considerations There can be little argument as to whether prenatal diagnosis involving an amniocentesis—which carries a 1-2% risk of precipitating an abortion—should be carried out in a pregnancy at high risk of ending in an abnormal fetus, when there is a reciprocal translocation in one of the parents or when there is advanced maternal age. Even when the risk is somewhat less, as in those cases where the mother has previously had a child with the Down syndrome or some previous chromosome abnormality, the assurance that the fetus is chromosomally normal will enable the parents to plan or continue a pregnancy with some relief of anxiety (Laurence, 1974a). For the same reasons an amniocentesis is probably justified when there is in fact not an increased risk, but a fear of malformation. Under all circumstances the parents should know of the risks and the limitations of the tests offered and they should be quite clear about the possible need for a termination of pregnancy. If, however, they are unable to accept a termination should an abnormal fetus be identified, one would have to think very carefully as to whether the investigation ought to be proceeded with, as an intolerable situation might be precipitated. No difficulties should arise regarding the decision to terminate a pregnancy when one of the autosomal trisomies is found in the fetus. Nor should doubts arise when an XXY (Klinefelter) fetus or an XXX female is present, as most of these grow up to be mentally dull, sexually abnormal or malformed. On the other hand, when there is a balanced translocation present, the pregnancy ought to be allowed to go to term. There may be some worry when this has arisen de novo in the fetus, as in the process some deletion may have occurred which is not detectable even by banding techniques, in which case the fetus may be phenotypically abnormal. 13

Vol. 32 No. 1

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M. Laurence & P. Gregory The same problem may arise with regard to marker chromosomes seen in several phenotypically normal members of a family and also in the fetus. A final decision must be arrived at by discussion with all those concerned. The difficulties which arise in mosaicism have already been referred to in section 3. On the other hand, the situation posed by the discovery of a fetus with the XYY complement is probably less difficult. It appears that some 0.1 % of Caucasian communities have this chromosome constitution (Jacobs, Melville, Ratcliffe, Keay & Syme, 1974), only a small minority of which ever seem to show any aggressive deviant behaviour (Hook, 1973). Such pregnancies should probably not be interfered with further; whether the parents are even told about such a finding is a matter for discussion. Twin pregnancies present the obstetrician and the geneticist with a dilemma. Such pregnancies ought always to be identified by a prior ultrasound scan and should then not be subjected to prenatal diagnostic procedures. Not only is it difficult to guarantee that both amniotic cavities can be tapped (Dewhurst & Lucas, 1973), but when an abnormal fetus is identified a termination of the pregnancy is almost certainly likely to lead to the death of both fetuses, although one is likely in fact to be normal. Such a problem was posed for one pregnancy of twins, for which an amniocentesis and an a-fetoprotein estimation for neural tube malformations was performed without prior ultrasound scan. A male fetus was shown to have anencephaly and the second amniotic cavity could not be entered. This pregnancy was allowed to go to term in the hope that the other fetus would be normal. The identification of a male fetus with a view to termination in a woman who is a carrier for a serious X-linked disorder seems acceptable to only a minority of couples coming for genetic counselling, as there is a 1 in 2 chance of terminating a normal fetus and a similar risk of passing the same problem on to any daughters. Fetal sexing should be undertaken only for genetic reasons, and rarely agreed to simply because the parents wish for a child of one particular sex only. Prenatal diagnostic services should be concentrated in specialist centres where this type of investigation is carried out frequently and those carrying it out are highly practised. In this way the sequential abortion rate is likely to be kept to a minimum and the success rate of amnion cell culture will remain

high. Probably no centre having a success rate of less than 90 % ought to be offering prenatal diagnostic cytogenetics as a service. As a general principle, all amniotic fluids taken from pregnancies of less than 20 weeks should have an amnion cell culture for cytogenetics to exclude chromosome abnormalities; and a-fetoprotein estimation for the detection of open neural tube malformations should be carried out whatever the indication for the amniocentesis might have been (Laurence, 1974a). Other biochemical tests should probably be carried out only when there is some special reason for suspecting a specific abnormality. All tests should be completed quickly so that any termination is carried out before 20 weeks wherever possible. 5. Future Developments and Problems By screening all pregnancies with an amniocentesis, followed by amnion cell culture, it ought to be possible to eliminate the Down syndrome, with its ever-increasing prevalence, almost entirely. Such a total policy, although desirable, is at present unlikely to be attainable, as amniocentesis carries a risk and both universal amniocentesis and amnion cell culture are unlikely to be logistically possible (Laurence, 1974b). However, this service ought to be extended to all those women who are especially at risk and should be offered to pregnant women who have had a previous infant with the Down syndrome or other chromosome abnormality, where one parent is known to be a translocation carrier and the woman is over the age of 35 years. In due course the age-limit should probably be reduced to 30 years, thereby leading to a reduction in the incidence of the Down syndrome to less than half the present figure (Stein, Susser & Guterman, 1973). Such a policy would call for a greatly increased number of safe amniocenteses and also for a better and quicker laboratory method of amnion cell culture, better direct methods of examining amnion cells without culture (Hughes, Rink, Griffiths & Paintin, 1971) or perhaps some reliable non-invasive technique involving the examination of fetal cells in the maternal circulation (Schindler, Graf & Martin-du-Pan, 1972). Any considerable extension of fetal cytogenetics would not be really feasible without some relatively inexpensive satisfactory automated technique for karyotyping.

REFERENCES

Allen, H. H., Sergovich, F., Stuart, E. M., Pozsonyi, J. & Murray, B. (1974) Am. J. Obstet. Gynecol. 118,310-313 Burton, B. K., Gerbie, A. B. & Nadler, H. L. (1974) Am. J. Obstet. Gynecol. 118,718-746 Campbell, S. (1974) In: Motulsky, A. O. & Lenz, W., ed. Birth defects: Proceedings of the Fourth International Conference, Vienna, 2-8 September 1973, pp. 240-247 (International Congress Series, no. 310). Excerpta Medica, Amsterdam Coakley, W. T., Hughes, D. E., Slade, J. S. & Laurence, K. M. (1971) Br. Med. J. 1,109-110 [Letter] Coakley, W. T., Slade, J. S., Braeman, J. M. & Moore, J. L. (1972) Br. J. Radiol. 45, 328-332 Cutz, E. & Conen, P. E. (1973) In: Arcencaux, C. J., ed. Proceedings: Thirty-First Annual Meeting, Electron Microscopy Society of America, New Orleans, August 14-17, 1973, pp. 424-425. Claitor's Publishing Division, Baton Rouge, La Dewhurst, C. J. & Lucas, M. (1973) Br. J. Hosp. Med. 10,735-743 Doran, T. A., Rudd, N. L., Gardner, H. A., Lowden, J. A., Benzie, R. J. & Liedgren, S. I. (1974) Am. J. Obstet. Gynecol. 118,314-321

Ferguson-Smith, M. E., Ferguson-Smith, M. A., Nevin, N. C. & Stone, M. (1971) Br. Med. J. 4, 69-74 Fuchs, F. & Cederqvist, L. L. (1966) din. Obstet. Gynecol. 13,178201 Gerbie, A. B., Nadler, H. L. & Gerbie, M. V. (1971) Am. J. Obstet. Gynecol. 109,765-768 Hook, E. B. (1973) Science {New York) 179,139-150 Hsu, L. Y. F., Dubin, E. C , Kerenyi, T. & Hirschhorn, K. (1973) / . Med. Genet. 10,112-119 Hughes, D. T., Rink, E., Griffiths, S. & Paintin, D. B. (1971) Lancet, 2,1319 [Letter] Jacobs, P. A., Melville, M., Ratcliffe, S., Keay, A. J. & Syme, J. (1974) Ann. Hum. Genet. 37, 359-376 Kardon, N. B., Chernay, P. R., Hsu, L. Y., Martin, J. L. & Hirschhorn, K. (1972) / . Pedlatr. 80,297-299 Laurence, K. M. (1974a) Dev. Med. Child Neurol. suppl. no. 32, pp.117-121 Laurence, K. M. (1974b) Lancet, 2,939-942 Laurence, K. M., Gregory, P., Stark, N. J. & TurnbulL A. C. (1974) In: Jacoby, F. & Rajan, K. T., ed. Tissue culture in

14 Br. Med. Bull. 1976

PRENATAL DIAGNOSIS OF CHROMOSOME DISORDERS K. M.Laurence & P. Gregory medical research, pp. 100-113 (Proceedings of the symposium held in Cardiff, 11-13 April 1973). Heinemann Medical, London Lee, C L. Y., Gregson, N. M. & Walker, S. (1970) Lancet, 2, 316-317 [Letter] Lucas, M Wallace, I. & Hirschhom, K. (1972) / . Obstet. Gynaecol. Br. Commonw. 79,1119-1127 Macintosh, I. J. C. & Davey, D. A. (1970) Br. Med. J. A, 92-93 Milunsky, A. (1973) The prenatal diagnosis of hereditary disorders. Thomas, Springfield, 111. Milunsky, A. & Atkins, L. (1974)/. Am. Med. Assoc. 230,232-235 Nadler, H. L. (1972) Adv. Hum. Genet. 3,1-37 Nadler, H. L. & Gerbie, A. B. (1970) New Engl. J. Med. 282,596599 Nelson, M. M. (1973) In: Emery, A. E. H., ed. Antenatal diagnosis ofgenetic disease, pp. 69-81. Churchill Livingstone, Edinburgh Nelson, M. M. & Emery, A. E. H. (1970) Br. Med. J. 1, 523526

15

VoL 32 No. 1

Niermeijer, M. F., Sachs, E. S., Jahodova, M., Tichelaar-Klepper, C , Kleijer, W. J. & Galjaard, H. (1976) / . Med. Genet. (In press) Polani, P. E. & Benson, P. F. (1973) Guy's Hosp. Rep. 122,65-89 Rook, A., Hsu, L. Y., Gertner, M. & Hirschhom, K. (1971) Nature (London) 230, 53 [Letter] Schindler, A. M., Graf, E. & Martin-du-Pan, R. (1972) Obstet. Gynecol. 40, 340-346 Schneider, E. L., Stanbridge, E. J., Epstein, C. J., Golbus, M., Abbo-Halbasch, G. & Rogers, G. (1974) Science (New York) 184,477-480 Stede, M. W. & Breg, W. R., jr (1966) Lancet, 1, 383-385 Stein, Z., Susser, M. & Guterman, A. V. (1973) Lancet, 1,305-310 Turnbull, A. C , Gregory, P. J. & Laurence, K. M. (1973) Proc. R. Soc.Med. 66,1115-1118 WahlstrOm, J., Bartsch, F. K. & Lundberg, J. (1974) Clin. Genet. 6,184-191 WahlstrOm, J., Brosset, A. & Bartsch, F. (1970) Lancet, 2, 1037 [Letter]