Serial first- and second-trimester Down's syndrome screening tests among IVF-versus naturally-conceived singletons

R. Maymon1,3 and A. Shulman2

1 Departments of Obstetrics and Gynecology, Assaf Harofe Medical Center, Zerifin and 2 Sheba Medical Center, Tel Hashomer, both affiliated with the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It has been reported that second-trimester serum markers may be affected by assisted reproduction, leading to a higher false-positive rate. METHODS: A total of 285 naturally and 71 IVF-conceived singletons which underwent a serial disclosure Down's syndrome screening programme were compared. The study protocol included first-trimester combined [nuchal translucency (NT), free ß-HCG and pregnancy-associated plasma protein-A (PAPP-A)] testing. The second-trimester triple serum screening included {alpha}-fetoprotein (AFP), intact HCG and unconjugated estriol (uE3). After excluding aneuploidies, miscarriages, anatomical anomalies and cases with incomplete follow-up, the serum samples of normal cases were assessed and correlated. RESULTS: NT measurement was not significantly changed in either group. However, the IVF group had lower PAPP-A [0.96 versus 1.05 multiples of normal median (MoM)] and higher AFP (1.13 versus 1.07 median MoM). Both groups had similar rates of first-trimester false-positive results (FPR; 7 and 9% respectively), but the IVF group had a significantly higher mid-gestation FPR rate (10 versus 5%; Pearson {chi}2, P = 0.029). This has contributed to amniocentesis uptake rates of 15 and 13% for the IVF and natural conception pregnancies respectively. CONCLUSIONS: The IVF group tended to have a significantly higher second-trimester FPR rate. To counterbalance this phenomenon, integrated first- and second-trimester screening tests or the use of NT alone might be a reasonable option that deserves further investigation.

Key words: assisted reproduction/combined test/Down's syndrome/triple test


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Routine multi-marker screening for Down's syndrome (DS) is now an established practice in many countries. In the second trimester, the most common markers are maternal serum HCG or its free ß-subunit (FßHCG), {alpha}-fetoprotein (AFP) and unconjugated estriol (uE3). Large studies using combinations of HCG or FßHCG and either or both of the other markers have confirmed model predictions that about two-thirds of DS-affected pregnancies can be detected with a false-positive result (FPR) rate of ~5% (Wald et al., 1997Go; Cuckle, 1998Go). In the first trimester, the combination of maternal serum pregnancy-associated placental protein-A (PAPP-A) and FßHCG can achieve similar performance (Haddow et al., 1998Go).

Prospective studies have assessed the combination of first-trimester maternal serum analytes with the ultrasonographic finding of nuchal translucency (NT), which is an echo-free area at the back of the fetal neck. This sonographic marker was reported to be associated with fetal chromosomal abnormalities (Pandya et al., 1995Go; Snijders et al., 1998Go). It has been proposed (Wald and Hackshaw, 1997Go) that combining NT with first-trimester biochemical serum markers should result in a combined screening test. It has also been reported that such a combination may allow an ~85% DS detection rate with a 5% FPR rate (De Biasio et al., 1999Go; de Graff et al., 1999; Spencer et al., 1999Go), which is a significant improvement on other DS screening tests.

Over the past decade, both maternal age and the wide use of various assisted reproduction technologies (ART) and medications have risen dramatically (Maymon et al., 1999aGo). This has been reflected upon all mid-gestation serum markers, but mainly affected serum HCG levels, which were found to be increased in ART pregnancies, leading to a higher FPR rate in singletons (Barkai et al., 1996Go; Heinonen et al., 1996Go; Ribbert et al., 1996Go; Frishman et al., 1997Go; Hsu et al., 1999Go; Wald et al., 1999aGo; Bar-Hava et al., 2001Go; Maymon and Shulman, 2001Go) as well as in twins (Maymon et al., 1999bGo; Raty et al., 2000Go).

Recently, two studies (Liao et al., 2001Go; Wøjdeman et al., 2001Go) have evaluated the first-trimester markers profile among ART singletons and reported conflicting results. Although both of these groups agreed that NT was not influenced, one group (Liao et al., 2001Go) found decreased PAPP-A and increased FßHCG among their IVF cases. In contrast, the other group (Wøjdeman et al., 2001Go) found those serum markers not to be significantly changed.

The aim of the current study was to assess among a preselected group of unaffected IVF pregnancies the marker profile which constitutes the combined test (Wald and Hackshaw, 1997Go), followed by the second-trimester triple test (TT) and the FPR rate of each test. These results were compared with data from a group of women who conceived naturally, and underwent the same investigation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This longitudinal study was carried out between January 1999 and September 2000 in a private-practice facility. The pregnant women attending were invited to participate in a sequential disclosure DS screening approach. Those who fulfilled the entry criteria and gave their consent comprised the study population. This assessment was carried out among women of a similar age (±3 years). Exclusion criteria included more than one gestational sac seen at the first-trimester scan, cases with chromosomal aneuploidy, early to mid-gestation pregnancy loss (<=24 completed weeks), anatomical abnormalities and those cases for which essential information was not available. After they were given a leaflet which explained the nature and implications of this test, the parturient women underwent NT screening. Following the scan, blood for monitoring of the first-trimester serum markers (FßHCG and PAPP-A) was withdrawn, and the women were offered mid-gestation TT for assessing AFP, total HCG and uE3. The women were informed of the results for each test upon completion of each screening test (i.e. combined first trimester and TT). Those women who had one positive screening test result were advised to have mid-gestation amniocentesis for fetal karyotyping.

The results of the screening tests are presented in Israel as risk for DS live birth, and the threshold for recommending fetal karyotyping in both tests is >=1:380. This is the equivalent risk of a 35-year-old gravida at term pregnancy, and is the common practice in the Israeli national healthcare system (Legum et al., 1994Go).

IVF protocol
The procedure for the assisted conception in this programme was carried out in accordance with a previously reported protocol (Shulman et al., 1999Go; Maymon and Shulman, 2001Go).

Screening tests
Only singleton fetuses with a crown–rump length (CRL) of 38–84 mm were included. This corresponds with a gestational age of between 10 weeks and 3 days, and 13 weeks and 7 days.

Measurement of CRL to estimate gestational age was performed concomitantly with NT assessment, and was carried out according to a previously described method (Pandya et al., 1995Go). The scans were performed by experienced examiners (R.M. and A.S.). First-trimester PAPP-A and FßHCG assays were measured as previously reported (Maymon et al., 2001Go). Calculation of first-trimester DS risk was based on the multivariate Gaussian distribution (Wald and Hackshaw, 1997Go), using the `alpha' software (Logical Medical Systems Ltd, London, UK). Second-trimester TT were assessed as reported previously (Maymon and Shulman, 2001Go). Second-trimester DS risk was derived from the combination of triple serum markers and maternal age, and was calculated using commercial software. The serum samples in both sessions were tested in a routine analytical run together with regular maternal serum samples, all at the same antenatal DS screening programme in Zer Medical Laboratories (ISO 9002 UK, certified and authorized by the Ministry of Health, Israel). Testing was carried out in a fashion blinded to group classification; that is, samples from the study cases and those of other pregnant women were assessed simultaneously in the same laboratory. The measured marker levels were expressed as multiples of the gestation-specific normal medians (MoM). Median values for each serum analyte were calculated against completed menstrual weeks and adjusted for maternal weight. Reference MoM values were calculated from the local population as established in Zer Medical Laboratories during the same time period. Gestational age in the IVF cases was calculated from the date of oocyte retrieval minus 2 weeks (or embryo transfer minus 16 days) to convert the menstrual dating. The gestational age in all cases was confirmed by first-trimester scan, as this was performed simultaneously with the NT measurement. Data on the sequential DS screening tests results and pregnancy outcomes were recorded prospectively on a database.

Statistical analysis
Standardized kurtosis showed that the data were derived from a normal distribution, and were expressed as mean (± SD). Frequencies were expressed as percentages. The DS screening test was estimated based both on NT values and five serum markers (data were transformed using natural logarithms) and maternal age. The data all fitted a Gaussian distribution after log10 transformation. After excluding all women who did not fulfil the study criteria, the remaining parturient women with normal pregnancy outcome were further subdivided according to first-versus second-trimester screening results. Differences between various variables of pregnancy follow-up were compared using the Pearson {chi}2-test. Student's t-test was applied in order to compare the first- and second-trimester markers in MoM values between the IVF- and naturally conceived pregnancies. A P-value < 0.05 was considered significant in both tests. Statistical analysis was performed by the Tel Aviv University statistical department using Statistics Package for Social Sciences software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study was limited to normal pregnancies from a general obstetric population. This included 285 naturally and 71 IVF-conceived singletons for whom the results of both screenings tests and complete data on pregnancy outcome were available.

The mean log10 MoMs between each marker within each screening tests were compared, and median MoM values are listed in Table IGo. The mean age of women in the IVF group was greater than that of the natural conception group, and their fetuses had a smaller mean CRL value. The IVF group also had a significantly lower PAPP-A in the first trimester, and increased AFP levels in the second trimester (Table IGo). Based on maternal age alone (>=35 years), 24 and 14% of the IVF and natural conception parturient women respectively were defined as screen positive (Table IIGo). The FPR rate among the IVF parturient women increased from 7% during the first trimester to 10% in the second (Table IIGo). Similar screen-positive results were found throughout both tests (1.4%) in both groups (Table IIGo). The amniocentesis uptake due to screen-positive test results (avoiding repetition) was 15 and 13% for the IVF- and natural conceptions respectively (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table I. Comparison of maternal age, crown–rump length (CRL) and first- and second-trimester screening testsa between natural versus IVF-conceived singleton pregnancies
 

View this table:
[in this window]
[in a new window]
 
Table II. Comparison of first and second trimester screening tests results and pregnancy follow-up between natural versus IVF-conceived singleton pregnancies. Values in parentheses are percentages
 
There were no fetal or neonatal deaths in either group. No prenatal anomalies or traits that would have justified karyotyping were detected post-natally. Long-term follow-up (mean 8 months; range 3–18) was available for all infants included in this study and for whom there was an uneventful pregnancy and delivery outcome. No other genetic abnormalities were detected, and the motor and mental development of the infants appeared normal. The condition of all infants was checked by an experienced paediatrician.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The current study addresses serial first- and second-trimester serum screening as applied to preselected IVF-achieved pregnancies. With regard to first-trimester assessment, the IVF fetuses had a smaller CRL, though this may have reflected an earlier uptake of testing. The NT measurements among IVF cases were slightly increased, but not in any statistically significant manner. These findings were compatible with cross-sectional data which demonstrated an increase in NT measurement between 9 and 12 weeks gestation, followed by a decrease at 13–14 weeks (Braithwaite et al., 1996Go). NT measurements were all within the normal range for gestation (Herman et al., 2000aGo) in both groups of the study population; NT was consistently not influenced by ART and this is in agreement with previous reports (Maymon et al., 1999bGo; Lara et al., 2000Go; Liao et al., 2001Go; Wøjdeman et al., 2001Go). The most marked difference between the naturally conceived pregnancies and those obtained after IVF was hormonal treatment and multiple corpora lutea, all of which persisted throughout the first trimester of gestation. Thus, it might be expected that marker changes induced by this treatment would be more marked during the first trimester, and be reflected mainly by high HCG levels (Wøjdeman et al., 2001Go).

Surprisingly, no significant changes were found in this marker in both trimesters; likewise, with regard to the first trimester this was in agreement with previously published data (Wøjdeman et al., 2001Go). In addition, low levels of PAPP-A were found, in agreement with other data (Liao et al., 2001Go). The lower PAPP-A might be attributed to early testing in the IVF group, as it has been reported that PAPP-A levels increase significantly with gestation (Ong et al., 2000Go). Another theory, however—and one which deserves further investigation—is that the lower PAPP-A level in IVF cases is secondary to metabolic impairment, which also reflects infertility cases. Lower maternal serum PAPP-A levels at 10–14 weeks gestation were recently reported as being associated with the subsequent development of various complications of pregnancy (Ong et al., 2000Go). The differential diagnosis between such complications, or the constitutional characteristic of the IVF cases, should be further investigated. A FPR rate of 7–9% was found at a threshold of 1:380 for first-trimester screening in both groups. This was lower than the 16% FPR rate found for the same test previously reported elsewhere among IVF cases (Liao et al., 2001Go), but was higher when compared with data obtained at other centres (Wøjdeman et al., 2001Go) or when the NT screening test was used alone in ART pregnant women (Maymon et al., 1999cGo).

Interestingly, a significantly higher rate of second-trimester FPR (10%) was found among IVF cases in comparison with the natural conception group (5%). Again, this observation was in agreement with a previous report which identified a similar mid-gestation FPR rate among ART pregnancies (Maymon et al., 1999bGo,cGo; Maymon and Shulman, 2001Go) whereas Bar Hava et al., have reported 18% mid-gestations FPR from a different centre in Israel (Bar-Hava et al., 2001Go).

After having corrected for gestational age by dating scan, and having checked for maternal age (which did not change from the first- to the second-trimester test), it was contended that the high second-trimester FPR rate in IVF patients could be attributed mainly to other factors that are directly associated with the pregnancy itself. The reasoning behind this was that such pregnancies in infertile couples might have another underlying pathology that may lead to various (as yet unknown) metabolic disturbances, and these might also affect the serum screening results. It has been suggested (Wald et al., 1999aGo) that a high HCG level might be explained by progesterone levels remaining high in IVF pregnancies. In contrast, the findings of a previous report (Maymon and Shulman, 2001Go) and data from the present study are in agreement with the outcome of a recent in-vivo study which demonstrated an elevated maternal progesterone serum level, but without a concomitant increase in HCG concentration in early first-trimester IVF pregnancies (Costea et al., 2000Go). The present data are in agreement with two other studies on IVF cases (Muller et al., 1993Go; Lam et al., 1999Go) in which the mid-gestation serum HCG marker levels were similar to those of the natural conception group. Three studies which assessed mid-gestation serum markers among IVF cases have reported, in addition to a higher HCG level, a higher rate of complicated obstetric outcome (Maymon and Shulman, 2001Go), including proteinuric pre-eclampsia (Heinonen et al., 1996Go), premature deliveries, and smaller infants (Ribbert et al., 1996Go). In this respect, it has been found that pregnant women who later in gestation developed pre-eclampsia or have growth-impaired infants tend also to have both higher HCG and AFP levels (Ogle et al., 2000Go). Thus, it is possible that the high mid-gestation HCG level found among IVF pregnancies provides a warning for such complications rather than for a DS risk.

A significantly increased AFP level was also found among the current IVF pregnancy cases, and this was even higher than shown previously in this population (Maymon and Shulman, 2001Go). There was no clear explanation for this finding. Several problems emerged during the present study. One problem involved counselling the patient in whom the results were conflicting, and another problem involved the high number of invasive procedures, partly because the screen-positive results could not be adjusted by other screen-negative reports. This summation of the two screening test results led to a suboptimal interpretation of the sequential disclosure screening results. Currently, 15 and 13% of the IVF and natural conception pregnancies respectively were found to be screen-positive, and the women subsequently underwent amniocentesis. Based on previously published data, it has been reported recently (Hackshaw and Wald, 2001aGo) that ~0.07% of all women found screen-positive on the combined test remain screen-positive after the integrated test. In the present study, higher values of 1.4% screen-positive tests were found in both groups which underwent the disclosure screening tests.

The advantage of a method which combines the results of different screening tests in order to reach a single value is clear. For example, it may significantly reduce the FPR rate and even increase the detection rate. Previously, a risk of >=1:120 was used as the threshold to define a positive result on the integrated first- and second-trimester screening test (Wald et al., 1999bGo). The model used by these authors consisted of NT and PAPP-A from the first-trimester test, and uE3, HCG, AFP and inhibin A from the second-trimester test. Accordingly, the detection rate was expected to be 85% with a FPR rate of 0.9% (Wald et al., 1999bGo). This approach seems very promising, mainly for IVF-conceived pregnancies which bear a high second-trimester FPR rate. As yet, it is not possible to comment on the detection rate as the present study was focused only on normal cases, and it is not possible to provide integrated results as both tests included either FßHCG or HCG. It has been shown previously that the two markers correlate well, both in unaffected and DS cases (Maymon et al., 2001Go). A recent report (Hackshaw and Wald, 2001bGo), using published data, suggested that if the correct age-specific risk and screening marker distribution were used, then risk estimation would be accurate. However, the use of two tests would be less efficient than integrating all the information into a single test. Therefore, if an integrating screening programme is planned, based on our previous experience (Herman et al., 2000bGo) it is contended that a combination of NT (which is unaffected by IVF pregnancies) and mid-gestation TT offers a reasonable and simple alternative.

Otherwise, first-trimester screening using either NT alone or combined with serum screening seems to be an efficient option (Liao et al., 2001Go; Wøjdeman et al., 2001Go), and this approach—at least in our experience—yields the lower FPR rate. Larger prospective studies are required to verify the best screening policy for IVF pregnancies.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank the Zer Medical Laboratories (Israel) for their co-operation and support, and Ms Esther Eshkol for editorial assistance.


    Notes
 
3 To whom correspondence should be addressed. E-mail: intposgr{at}post.tau.ac.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bar-Hava, I., Yitzhak, M., Krissi, H. et al. (2001) Test screening in in vitro fertilization pregnancies. J. Assist. Reprod. Genet., 18, 226–229.[ISI][Medline]

Barkai, G., Goldman, B., Ries, L. et al. (1996) Down's syndrome screeni ng marker levels following assisted reproduction. Prenat. Diagn., 16, 1111–1114.[ISI][Medline]

Braithwaite, J.M., Morris, R.W. and Economides, D.L. (1996) Nuchal translucency measurements: frequency distribution and changes with gestation in a general population. Br. J. Obstet. Gynaecol., 103, 1201–1204.[ISI][Medline]

Costea, D.M., Gunn, L.K., Hargreaves, C. et al. (2000) Delayed luteo-placental shift of progesterone production in IVF pregnancy. Int. J. Gynaecol. Obstet., 68, 123–129.[ISI][Medline]

Cuckle, H. (1998) Antenatal screening. Semin. Neonatol., 3, 1–8.

De Biasio, P., Siccardi, M., Volpe, G. et al. (1999) First-trimester screening for Down syndrome using nuchal translucency measurement with free ß-hCG and PAPP-A between 10 and 13 weeks of pregnancy—the combined test. Prenat. Diagn., 19, 360–363.[ISI][Medline]

de Graaf, I.M., Pajkart, E., Bilardo, C.M. et al. (1999) Early pregnancy screening for fetal aneuploidy with markers and nuchal translucency. Prenat. Diagn., 19, 458–462.[ISI][Medline]

Frishman, G.N., Canick, J.A., Hogan, J.W. et al. (1997) Serum triple-marker screening in in vitro fertilization and naturally conceived pregnancies. Obstet. Gynecol., 90, 98–111.[Abstract/Free Full Text]

Hackshaw, A.K. and Wald, N.J. (2001a) Inaccurate estimation of risk in second trimester serum screening for Down's syndrome among women who have already had first trimester screening. Prenat. Diagn., 21, 741–746.[ISI][Medline]

Hackshaw, A.K. and Wald, N.J. (2001b) Assessment of the value of reporting partial screening results in prenatal screening for Down's syndrome. Prenat. Diagn., 21, 731–740.

Haddow, J.E., Palomaki, G.E., Knight, G.J. et al. (1998) Screening of maternal serum for fetal Down's syndrome in the first trimester. N. Engl. J. Med., 338, 955–961.[Abstract/Free Full Text]

Heinonen, S., Ryynanen, M., Kirkinen, P. et al. (1996) Effect of in vitro fertilization on human chorionic gonadotropin serum concentrations and Down's syndrome screening. Fertil. Steril., 66, 398–403.[ISI][Medline]

Herman, A., Dreazen, E., Samandarov, A. et al. (2000a) On-to-on versus on-to-out nuchal translucency measurements. Ultrasound Obstet. Gynecol., 15, 126–130.[ISI][Medline]

Herman, A., Weinraub, Z., Dreazen, E. et al. (2000b) Combined first-trimester nuchal translucency and second-trimester biochemical screening tests among normal pregnancies. Prenat. Diagn., 20, 781–784.[ISI][Medline]

Hsu, T.Y., Hsu, C.Y., Ou, J.J. et al. (1999) Maternal serum screening for Down syndrome in pregnancies conceived by intra-uterine insemination. Prenat. Diagn., 19, 1012–1014.[ISI][Medline]

Lam, Y.H., Yeung, W.S.B., Tang, M.H.Y. et al. (1999) Maternal serum alpha-fetoprotein and human chorionic gonadotrophin in pregnancies conceived after intracytoplasmic sperm injection and conventional in vitro fertilization. Hum. Reprod., 14, 2120–2123.[Abstract/Free Full Text]

Lara, C., Beever, J., Iberico, G. et al. (2000) Application to early sonographic marker of Down syndrome to pregnancies achieved by assisted reproduction techniques. Ultrasound Obstet. Gynecol., 16 (Suppl.), 43.

Legum, C., Glassmer, M., Peretz, H. et al. (1994) Screening for Down syndrome in the second trimester of pregnancy. Eur. J. Obstet. Gynecol. Reprod. Biol., 55, 15–16.[ISI]

Liao, A.W., Heath, V., Kametas, N. et al. (2001) First-trimester screening for trisomy 21 in singleton pregnancies achieved by assisted reproduction. Hum. Reprod., 16, 1501–1504.[Abstract/Free Full Text]

Maymon, R. and Shulman, A. (2001) Comparison of triple serum screening and pregnancy outcome in oocyte donation versus IVF pregnancies. Hum. Reprod., 16, 691.[Abstract/Free Full Text]

Maymon, R., Dreazen, E., Weinraub, Z. et al. (1999a) Antenatal screening for Down's syndrome in assisted reproductive pregnancies. Hum. Reprod. Update, 5, 530–534.[Abstract/Free Full Text]

Maymon, R., Dreazen, E., Rozinsky, S. et al. (1999b) Comparison of nuchal translucency measurement and second-trimester triple serum screening in twin versus singleton pregnancies. Prenat. Diagn., 19, 727–731.[ISI][Medline]

Maymon, R., Dreazen, E., Rozinsky, S. et al. (1999c) Comparison of nuchal translucency measurement and midgestation serum screening in assisted reproduction versus naturally conceived singleton pregnancies. Prenat. Diagn., 19, 1007–1011.[ISI][Medline]

Maymon, R., Bergman, M., Segal, S. et al. (2001) Sequential first and second trimester screening tests: correlation of the markers' levels in normal versus Down's syndrome affected pregnancies. Prenat. Diagn., 21, 1175–1177.[ISI][Medline]

Muller, F., Aegerter, P. and Boue, A. (1993) Prospective maternal serum human chorionic gonadotropin screening for the risk of fetal chromosome anomalies and the subsequent fetal and neonatal death. Prenat. Diagn., 13, 29–43.[ISI][Medline]

Ogle, R., Jauniaux, E., Pahol, G.S. et al. (2000) Serum screening for Down syndrome and adverse pregnancy outcome: a case-controlled study. Prenat. Diagn., 20, 96–99.[ISI][Medline]

Ong, C.Y.T., Liao, A.W., Spencer, K. et al. (2000) First trimester maternal serum free ß human chorionic gonadotropin and pregnancy associated plasma protein A as predictors of pregnancy complications. Br. J. Obstet. Gynaecol., 107, 1265–1270.[ISI]

Pandya, P.P., Snijders, R.J.M., Johnson, S.J. et al. (1995) Screening for fetal trisomies by maternal age and fetal nuchal translucency thickness at 10 to 14 weeks of gestation. Br. J. Obstet. Gynaecol., 102, 957–962.[ISI][Medline]

Raty, R., Virtanen, A., Kaskinen, P. et al. (2000) Maternal midtrimester serum APF and free ß-hCG levels in in vitro fertilization twin pregnancies. Prenat. Diagn., 20, 221–223.[ISI][Medline]

Ribbert, L.S.M., Kornmann, L.H., de Wolf, B.T.H.M. et al. (1996) Maternal serum screening for fetal Down syndrome in IVF pregnancies. Prenat. Diagn., 16, 35–38.[ISI][Medline]

Shulman, A., Frenkel, Y., Dor, J. et al. (1999) The best donor. Hum. Reprod., 14, 2493–2496.[Abstract/Free Full Text]

Snijders, R.J.M., Noble, P.L., Sebire, N.J. et al. (1998) UK multicenter project on assessment of risk of trisomy 21 by maternal age and nuchal translucency thickness at 10–14 weeks of gestation. Lancet, 352, 343–346.[ISI][Medline]

Spencer, K., Souter, V., Tul, N. et al. (1999) A screening program for trisomy 21 at 10–14 weeks using fetal nuchal translucency, maternal serum free ß-human chorionic gonadotropin and pregnancy-associated plasma protein-A. Ultrasound Obstet. Gynecol., 13, 233–237.

Wald, N.J. and Hackshaw, A.K. (1997) Combining ultrasound and biochemistry in first-trimester screening for Down syndrome. Prenat. Diagn., 17, 821–829.[ISI][Medline]

Wald, N., Kennard, A., Hackshaw, A. et al. (1997) Antenatal screening for Down's syndrome. J. Med. Screening, 4, 181–246.[Medline]

Wald, N., White, N., Morris, J.K. et al. (1999a) Serum markers for Down's syndrome in women who have had in vitro fertilisation: implications for antenatal screening. Br. J. Obstet. Gynaecol., 106, 1304–1306.[ISI][Medline]

Wald, N., Watt, H.C. and Hackshaw, A.K. (1999b) Integrated screening for Down syndrome based on tests performed during the first and {sigma}econd trimesters. N. Engl. J. Med., 341, 461–467.[Abstract/Free Full Text]

Wøjdeman, K.R., Larsen, S.O., Shalmi, A. et al. (2001) First trimester screening for Down syndrome and assisted reproduction: no basis for concern. Prenat. Diagn., 21, 563–565.[ISI][Medline]

Submitted on November 27, 2000; resubmitted on August 24, 2001; accepted on November 21, 2001.