screening for trisomy 21 in twins using first trimester ultrasound and maternal serum biochemistry...

Screening for trisomy 21 in twins using first trimester ultrasoundand maternal serum biochemistry in a one-stop clinic: a review

of three years experience

Kevin Spencera,*, Kypros H. Nicolaidesb

Objective To evaluate the performance of screening for fetal trisomy 21 in the first trimester of twinpregnancies by a combination of maternal serum biochemistry and ultrasonography.

Design Three year retrospective review of screening performance.

Setting District General Hospital maternity unit.

Population All women booked to receive routine antenatal care at Harold Wood Hospital between 1 June1998 and 30th September 2001. The population included 13,940 women of all ages presenting withpregnancies between 10 weeks 3 days and 13 weeks 6 days gestation. Of these, 230 had a twin pregnancy.

Methods Women booked into the clinic were offered screening using a combination of maternal serum freeh-hCG and pregnancy-associated plasma protein-A (PAPP-A) and fetal nuchal translucency thickness.Women at increased risk of carrying a fetus with trisomy 21 or trisomy 13/18 (�1 in 300 at sampling) wereoffered counselling and an invasive diagnostic procedure. Follow up of the outcome of all pregnancies wascarried out. For women who on examination were at 14 weeks of gestation or greater, or for womenpresenting as late bookers beyond 14 weeks, screening was performed in the same time frame using onlymaternal serum free h-hCG and a-fetoprotein.

Main outcome measures The first trimester detection rate for trisomy 21 and all aneuploides, false positiverate, uptake of screening, uptake of invasive testing in women identified at increased risk and fetal loss ratesafter invasive testing.

Results Overall, 97.4% of the women with twins (224/230) accepted first trimester screening. The rate ofdetection of trisomy 21 was 75% (3/4). Fetal death at presentation was found in 3.4% of fetuses (16/460). Ofwomen who accepted screening, 4.3% (10/230) presented too late for fetal nuchal translucencymeasurement and 10.0% of women (23/230) presented too early. A risk for trisomy 21 was calculatedfor each fetus based on the individual fetal nuchal translucency thickness and the maternal biochemistry.The false positive rate among those eligible for first trimester screening was 9.0% (19/206) of pregnanciesand 6.9% of fetuses (28/412). Uptake of invasive testing was 59% (10/17) with chorionic villus sampling ineight cases and amniocentesis in two. No fetal loss occurred within 28 days of chorionic villus sampling andno loss occurred after amniocentesis. One case of trisomy 21 was identified for every three invasiveprocedures.

Conclusion First trimester screening for trisomy 21 in twin pregnancies is both theoretically possible andpractically achievable using a combination of nuchal translucency thickness and maternal serumbiochemistry. However, dilemmas for the mother and health professionals when both nuchal translucencythickness measurements are normal might suggest that greater reliance be placed on the nuchal translucencythickness risk alone when counselling women about invasive testing.

INTRODUCTION

In the first trimester of pregnancy, screening by a

combination of fetal nuchal translucency thickness and

maternal serum free h-human chorionic gonadotrophin

(h-hCG) and pregnancy-associated plasma protein-A

(PAPP-A) has been shown in retrospective1 and prospec-

tive studies2,3 to identify approximately 90% of cases of

trisomy 21 in singleton pregnancies for a 5% false positive

rate.

The incidence of twin pregnancy is increasing in the UK,

partly as a result of the increased use of assisted repro-

ductive techniques. Furthermore, the pregnancy population

is getting older and the rate of twinning increases with

maternal age. In 1979, the incidence of multiple pregnan-

cies was 0.97%, this has increased to 1.43% by 19994.

In the second trimester, biochemical screening of twin

BJOG: an International Journal of Obstetrics and GynaecologyMarch 2003, Vol. 110, pp. 276–280

D RCOG 2003 BJOG: an International Journal of Obstetrics and Gynaecology

doi:10.1016/S1470-0328(02)02922-1 www.bjog-elsevier.com

aDepartment of Clinical Biochemistry, Harold Wood

Hospital, Romford, Essex, UKbHarris Birthright Research Centre for Fetal Medicine,

King’s College Hospital, London, UK

* Correspondence: Dr K. Spencer, Department of Clinical Biochemistry,

Harold Wood Hospital, Gubbins Lane, Romford, Essex RM3 0BE, UK.

pregnancies was shown to be possible using a pseudo risk

approach5,6. The detection rate in twins discordant for

trisomy 21 was expected to be lower than in singleton

pregnancies. Nevertheless, this approach identified twins

concordant for trisomy 21 in prospective screenings7.

In the first trimester, both fetal nuchal translucency

thickness8 and maternal serum biochemistry can be com-

bined together9 to provide detection rates approaching

those achieved in singleton pregnancies. Such algorithms

do not seem to be dependent upon chorionicity10 and an

example case of twins discordant for trisomy 21 that

had been identified by prospective screening has been

described11. Screening of twin pregnancies is, however,

still considered by many to be problematical because of

the significant clinical, technical and ethical challenges

posed for the diagnosis and clinical management of such

pregnancies6,12,13. Here we examine the outcome of screening

or trisomy 21 in the first trimester of twin pregnancies over a

three-year period in our OSCAR clinic2,3.

METHODS

The maternity unit under study is located in the borough

of Havering at the eastern boundary of Greater London.

The screening population is predominantly white Cauca-

sian from the London Borough of Havering (84.2% with an

RM postcode) and the Local Authority of Brentwood

(13.2% with a CM postcode), with a small proportion of

women from the London Borough of Barking and Dagen-

ham (2.3% with an IG postcode) and 0.3% from other

adjacent areas. The two major areas comprise a relatively

affluent population with approximately half the population

in social class I or II. The 2000 Index of Multiple Depriva-

tion provided by the Department of Environment, Transport

and the Regions14 showed Havering with a rank of 233/354

districts in England and Wales, while that of Brentwood

was 311/354. Barking and Dagenham on the other hand has

a higher proportion of manual skilled, partly skilled and

unskilled workforce. The Index of Multiple Deprivation in

this Borough was ranked 24/354, showing considerably

higher deprivation.

All women booked for maternity care at Harold Wood

Hospital, Essex are given an appointment to attend the

antenatal clinic for the first time at around 12 weeks.

Screening using the first trimester one-stop approach began

in June 199815. In the first year of screening, the qualifying

gestational age was between 10 weeks 3 days and 13 weeks

6 days by ultrasound dating. During the second and third

year, the minimum gestation was increased to 11 weeks

0 days to allow for better identification of structural

anomalies at the slightly later gestations. Women are

initially seen in the community by a member of the

community midwifery team (65% of cases) and they

receive an information leaflet about the one-stop clinic

service and the screening tests that are available with their

initial booking appointment letter. On attending the clinic

at a specified appointment time, women have a consultation

with a midwife concerning the available tests and, if they

elect to have prenatal screening for chromosomal anomalies,

all relevant clinical information is recorded on the request

forms. After pre-test counselling, women opting into the

screening programme go to the phlebotomy room where

blood samples are taken for serum biochemical screening, in

addition to samples for other routine antenatal investiga-

tions. Prenatal screening blood samples are passed through

to the adjacent clinic laboratory and the women then move

on to the ultrasound suite where fetal nuchal translucency

thickness and crown–rump length are measured using

standard procedures16 (www.fetalmedicine.com) by sono-

graphers certified by the Fetal Medicine Foundation. At

the same time, a mini anomaly scan is also performed.

The whole ultrasound process can be completed within a

20-minute period in 99% of the cases. All relevant clinical

information is recorded on a networked fetal database

(ViewPoint, Webling, Germany). If the ultrasound dating

reveals the fetus has a gestation prior to 11 weeks (45 mm

crown–rump length—38 mm in year one), then the mother

is sent back to the reception to be rebooked at the

appropriate period. If the gestation is beyond 13 weeks

6 days (84 mm by crown–rump length), then the labo-

ratory is informed and a-fetoprotein is measured instead of

PAPP-A.

During the time the mother is in the ultrasound suite, the

serum from the blood sample is separated and analysed for

free h-hCG and PAPP-A using the Kryptor analyser

(Brahms Diagnostica, Berlin—formerly CIS) in the clinic

laboratory. The quality performance of this system has

previously been described1,17. When the results are avail-

able (within 20 minutes) these are logged onto the fetal

database and a composite risk report is produced by the

time the woman has returned from the ultrasound suite.

This risk report is then available for the midwife/counsellor

to discuss with the woman. Appropriate, further follow up

and management is arranged. Patient-specific risks were

calculated by a multivariate approach using populations

parameters established in our retrospective study1, after

correction of the maternal serum biochemistry for maternal

weight18 and for twin pregnancies10, and the age-related

risk of the trisomy in singleton pregnancies at the time of

sampling 19,20. Although we have previously shown that in

the first trimester free h-hCG levels are significantly

increased (21%) in singleton pregnancies achieved by

assisted reproduction21, no correction was made for this

as no data have been published in twin pregnancies.

Similarly, although the incidence of raised nuchal trans-

lucency thickness is higher in monochorionic twins than in

dichorionic twins8, an appropriate correction algorithm has

yet to be developed.

Women with an increased risk in either fetus (�1 in

300) for trisomy 21 are referred to the Harris Birthright

Centre for Fetal Medicine for chorionic villus sampling or

SCREENING FOR TRISOMY 21 IN TWINS 277

D RCOG 2003 Br J Obstet Gynaecol 110, pp. 276–280

amniocentesis and fetal karyotyping. Provisional results

from quantitative PCR were available within 48 hours

and a confirmed diagnosis by conventional karyotyping

within seven days.

Outcome of all pregnancies was ascertained from deliv-

ery room records, hospital PAS system and Child Health

records and was cross checked with the fetal database.

Cytogenetics records were obtained from the laboratory,

the Child Health computer records and the National

Down’s Syndrome register.

RESULTS

Within a three-year and four-month period (1st June

1998 to 30th September 2001), a total of 13,940 pregnant

women were offered first trimester screening in the

OSCAR clinic. Of these 230 cases were twin pregnancies

(1.65%) of which 21% were conceived by assisted concep-

tion. The uptake of first trimester screening among women

with a twin pregnancy was 97.4% (224/230). The popu-

lation consisted of 84% Caucasian, 2% Afro-Caribbean, 3%

Asian and 11% of either unknown or other ethnic origin.

The median gestational age was 12 weeks 1 day (range 10

weeks 4 days to 13 weeks 6 days) and median crown–rump

length was 59 mm (range 38–84). The median maternal

age was 31.5 years (range 19.1–42.7). The median mater-

nal weight of the population was 66 kg (range 43–185).

Cigarette smoking was self-reported in 18.3%, with 80.2%

reporting themselves as non-smokers and the status was

unknown in 1.5% of cases.

Fetal death was noted on ultrasound examination in

3.4% of fetuses (16/460). According to the measurement

of fetal crown–rump length, the gestation was prior to

the minimum acceptable period in 10% of cases (23/230);

these women were rebooked for repeat examination at

the appropriate gestation. In addition, 4.3% of women

(10/230) had a gestation too late for nuchal translucency

thickness measurement and these women had conventional

second trimester screening22 with a-fetoprotein and free

h-hCG. In total, after exclusion of those women with fetal

demise at presentation, those declining screening and

those with crown–rump length beyond 84 mm, some

206 with a twin pregnancy had first trimester screening

performed.

Of the twin fetuses screened in the OSCAR clinic, 6.8%

(28/412) had risks greater than the 1 in 300 cutoff and 9.2%

of pregnancies (19/206) had at least one fetus with an

increased risk. After counselling, 37% of women (7/19)

declined the offer of an invasive diagnostic test, while 63%

(12/19) accepted invasive testing. Chorionic villus sam-

pling was the procedure chosen by 83% of women (10/12)

requesting invasive testing while 17% (2/12) decided to

wait until 14 weeks for amniocentesis. In all cases of

invasive testing when the fetus was shown to be of normal

karyotype, the pregnancy continued with a viable fetus at

least beyond 28 days after the procedure.

The individual combined risks and the fetal nuchal

translucency thickness in those women declining invasive

testing are shown in Table 1, indicating a predominance of

women with risks very close to the cutoff and with normal

nuchal translucency thickness in both fetuses.

Among the twin study population, four cases with

trisomy 21 were ascertained (Table 2). A combination of

maternal age, fetal nuchal translucency thickness, maternal

serum free h-hCG and PAPP-A identified 75% (3/4) cases

with trisomy 21. There was one case of trisomy 21 detected

per three invasive procedures. All women with an iden-

tified fetus with trisomy 21 elected to undergo embryo

reduction, which was carried out by ultrasound-guided

injection of potassium chloride in the chest of the affected

fetus. In each of the three cases, the surviving twin

progressed to a normal healthy delivery.

Table 3 provides a brief summary of the obstetric history

to each case.

Among the 19 women identified ‘at increased risk’, in

addition to the three cases with trisomy 21, fetal death

occurred in two sets of twins at 24 weeks. In one of these,

Table 1. Risk and NT MoM in twins when the mother declined invasive

testing.

Case Risk fetus 1 Risk fetus 2 NT fetus 1 NT fetus 2

1 124 124 0.76 0.76

2 219 301 1.04 0.94

3 236 236 0.64 0.64

4 273 273 1.24 1.24

5 17 29 1.06 0.72

6 269 319 1.44 1.49

7 289 362 1.23 1.17

Table 2. Cases of twins discordant for trisomy 21.

Case Maternal

age

(years)

Gestation

(week þ day)

Chorionicity NT

MoM

1

NT

MoM

2

NT

risk

1

NT

risk

2

Free

h-hCG

MoM

PAPP-A

MoM

Biochemical

risk

Combined

risk 1

Combined

risk 2

Outcome

1

Outcome

2

1 35 11 þ 5 Dichorionic – diamniotic 1.43 1.86 212 46 1.86 0.64 120 122 27 N T21




278 K. SPENCER & K.H. NICOLAIDES


twin-to-twin transfusion had occurred. In one further case,

preterm delivery occurred at 26 weeks with neither twin

surviving, and in yet a further case, one of the twins had a

missing hand.

The median MoM free h-hCG after weight correction

and before correction for twin pregnancy was 2.15 with a

mean log10 of 0.3306 and a log10 standard deviation of

0.2544. The median MoM PAPP-A after weight correction

and before correction for twin pregnancy was 1.93 with a

mean log10 of 0.2628 and a log10 standard deviation of

0.2178. These values are very similar to those published in

a previous series9.

DISCUSSION

The prevalence of twin pregnancies increases with

maternal age. In addition to being twice at risk of structural

defects, one would expect the risk for chromosomal abnor-

malities to be higher than in singletons23. However, when

congenital malformation rates in twins have been studied, it

has been difficult to demonstrate this for trisomy 21 and

some studies have reported a lower rate of trisomy 21 in

twins compared with singletons24. The difficulty with such

studies is related to population size, as the incidence of

twins is only 15 per 1000 births, even the largest study only

included 42 cases with trisomy 21.

In the second trimester, maternal serum biochemical

markers on average appear twice as high in twins compared

with that of a singleton pregnancy at the same gestational

age5,6,25. Using the ‘pseudo risk’ approach correcting for

twins5, the provision of risks in twin pregnancies leads to

detection rates in twins some 15% lower than in singleton

pregnancies6,25. Although such approaches have been suc-

cessful in routine practice7, many centres still consider the

ethical and technical difficulties too problematical in rou-

tine obstetric units13. As a consequence, many do not offer

screening in twin pregnancies.

In the first trimester, the use of individual nuchal

translucency thickness has allowed the calculation of spe-

cific risks for each fetus. This physical marker can specif-

ically identify the fetus at increased risk8 and thus be used

as a guide when undertaking chorionic villus sampling and

selective fetocide in twins discordant for trisomy 21. In

addition, it has been suggested that the risk based on nuchal

translucency thickness and maternal age can be used as

the basis for making decisions regarding the appropriate

diagnostic procedure to be followed in such circumstances26.

The projected detection rate using fetal nuchal trans-

lucency thickness and maternal age alone was 75.2%,

although this did not take into account chorionicity, in

which the incidence of increased nuchal translucency

thickness is 1.5 times greater in monochorionic than in

dichorionic twins8.

Levels of first trimester maternal serum biochemical

makers are similarly on average twice as high in twins

than in singleton pregnancies of the same gestation9.

Using a ‘pseudo risk’ approach, combining fetal nuchal

translucency thickness and maternal serum biochemistry

was predicted to identify 80% of cases of twins discor-

dant for trisomy 21 (at a 5% false positive rate) and

81.5% in concordant twins. Thus, it was argued that while

maternal serum biochemistry alone could not specifically

identify the fetus at risk in the presence of twins discor-

dant for trisomy 21, it would be possible to enhance the

detection rate in twins by some 5–6% yet still retain the

benefits of nuchal translucency thickness in identifying

the specific affected twin9. Furthermore, it has been

shown that chorionicity has no impact on the maternal

serum biochemical marker levels in twin pregnancy10.

The first practical example of this new first trimester twin

algorithm was presented in a report11 of the first case in

Table 2.

In this paper, we have outlined our first 3.3 years

experience in screening twin pregnancies. Our incidence

of twin pregnancies is a little above the national average

but not greatly so. However, our incidence of twins

discordant for trisomy 21 is far greater than one might

have expected (1 in 58); based on the maternal age

distribution of our twin pregnancies and the risk of

trisomy 21 in singleton pregnancies at 12 weeks of

gestation, we would have expected to see 1.5 fetuses with

trisomy 21 rather than 4. Our detection of 75% of cases

(3/4) is in line with our theoretical projection. The rate of

acceptance of first trimester screening among twin preg-

nancies was no different from that observed in singleton

pregnancies in which a very high uptake was achieved3.

Fetal death was twice as common among twin pregnancies

than in our singleton series, reflecting the increased

perinatal mortality in multiple pregnancy. Uptake of

invasive testing was lower than in the case with singleton

pregnancies3 when 80% of women accept invasive testing.

This reflects both the added risk and complexity of the

invasive procedure in twins and the increased potential

fetal loss of the normal twin as a result of selective

fetocide. Clearly, instances when nuchal translucency

thickness is normal in both twins, yet the biochemistry

is abnormal, present a further dilemma in that even if

chorionic villus sampling was performed on both twins —

fetal reduction would be inadvisable as it would be

difficult to identify the affected twin. Although adding

maternal serum biochemistry does improve detection,

clearly, nuchal translucency thickness risk alone is the

Table 3. Obstetric history of twin pregnancies discordant for trisomy 21.

ICSI ¼ intracytoplasmic sperm injection.

Case Gravidity Parity IUD <15 weeks Conception Smoker

1 4 2 1 Normal No

2 1 0 0 Normal No

3 1 0 0 Normal No

4 3 1 1 ICSI No

SCREENING FOR TRISOMY 21 IN TWINS 279


predominant factor by which women should be counselled

regarding invasive testing.

Acknowledgements

The OSCAR clinic is a multidisciplinary clinic which

functions because of the dedication of all contributors to the

team. The authors would like to thank the support and

contributions from all those in midwifery, obstetrics,

clerical, sonography, pathology and support workers.

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Accepted 11 November 2002

280 K. SPENCER & K.H. NICOLAIDES


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