Screening for Down syndrome is a part of routine obstetric care and was performed previously during the second trimester, based on two to four serum markers. Recently, a first-trimester screening test (FTST), including serum measurement of free β-human chorionic gonadotrophin (free β-hCG), pregnancy-associated plasma protein-A (PAPP-A) and a sonographic measurement of fetal nuchal translucency, has been shown to improve sensitivity and specificity . Combined with maternal age, these values are converted in multiples of the median (MoM) to calculate an estimated risk of fetal aneuploidy. If the estimated risk is greater than a certain cut-off (usually from 1/250 to 1/380), an amniocentesis or choriocentesis is proposed to confirm or exclude the diagnosis of Down syndrome. This screening strategy was shown to have a 90% detection rate (sensitivity) with 5% of invasive procedures . Neural tube defects (NTD) screening is performed during the second trimester based on the level of α-fetoprotein (AFP) in the maternal serum. When the value is greater than 2.5 MoM, a fetal ultrasonography, focused on the skull and the spine, is performed.
Several recent publications  have shown that maternal HIV infection and/or antiretroviral treatment (ART) modify the values of the biochemical markers used for second-trimester screening for Down syndrome (β-hCG, AFP, unconjugated estriol). High maternal serum β-hCG and AFP levels were found to be associated with high viral loads and low CD4 cell counts . Therefore, the risk for Down syndrome might be overestimated. Another study  found that AFP levels were lower in patients treated with protease inhibitors, thus underestimating the calculated risks for Down syndrome and NTD. However, choriocentesis and amniocentesis are associated with a risk of miscarriage of about 0.5%  and the risk of HIV mother-to-child transmission (MTCT) cannot be excluded [7,8].
An increasing number of HIV-infected women are becoming pregnant , because of the low risk of MTCT, and improved life expectancy and quality of life with ART [10,11]. It is therefore important to validate the screening tests for fetal anomalies in HIV-infected pregnant women so as to be able to offer the same standard of care as uninfected women. Underestimation of the risk will miss some cases with fetal anomaly, whereas overestimation would increase the use of antenatal invasive techniques with a possible risk of HIV MTCT and miscarriage. The aim of our study was to assess the reliability of first-trimester screening for Down syndrome and for NTD in the second trimester in HIV-infected women compared with uninfected pregnant women.
Patients and methods
We conducted a multicenter study including 312 HIV-infected women with singleton pregnancies from eight centers located in Switzerland (n = 6), Spain (n = 1) and Belgium (n = 1) and 1692 HIV-uninfected women from one Swiss center. Women were included if they had a FTST for Down syndrome and/or a second-trimester measurement of AFP. The exclusion criterion was a diagnosis of fetal chromosomal abnormality or fetal malformation (n = 3). The study was approved by the ethics committee of Geneva University Hospitals. Data were collected and analyzed anonymously.
The Swiss Mother to Child HIV Cohort Study (MoCHiV), in collaboration with the adult Swiss HIV Cohort Study (SHCS), has collected data from HIV-infected women and their children since 1998. Women having undergone prenatal screening between January 1998 and February 2007 were included in our study (n = 120). Data were extracted from the MoCHiV study database and from medical charts. In the Hospital Clinic of Barcelona, data from HIV-infected pregnant women have been prospectively collected since 1987. We included women having prenatal screening between 2000 and 2006 (n = 161). Some of these women participated in a descriptive study evaluating the feasibility of a screening programme in HIV-infected women . Data on HIV-infected pregnant women have been collected since 1992 at St Pierre Hospital in Brussels. Only nuchal translucency was assessed until the end of 2005 because of concern over a high false-positive rate with the second-trimester screening test. From 2006, serum markers have also been measured and thus these data were included (n = 31).
We randomly selected a group of HIV-negative pregnant women from the database of the University of Geneva Hospitals that contained 6666 patients having had a FTST for Down syndrome or a NTD screening between 2000 and 2006. Among these, four control women for each HIV-positive woman, matched for age and ethnicity, were randomly selected. A total of 214 HIV-infected women and 856 controls having a FTST for Down syndrome and 209 HIV-infected women and 836 controls with a risk evaluation for NTD during the second trimester were included; 114 HIV-infected women had both screening tests.
Data were collected on age, ethnicity, maternal weight, diabetes and smoking habits. Viral load, CD4 cell counts and ART grouped by type of drugs were recorded at the same gestational ages of screening. We collected also data on chronic hepatitis because of its possible influence on AFP levels. Maternal serum sampling and nuchal translucency assessment for Down syndrome screening were performed between 11 and 13 weeks. Serum sampling for NTD screening was performed between 14 and 19 weeks of gestation.
All Swiss laboratories used the same Kryptor immunoassay analyzer (Brahms, Henningsdorf, Germany). Risk calculation for Down syndrome in the first trimester was conducted with CISline Prenat'screen software (Brahms), which is certified by the Fetal Medicine Foundation. Quality control showed a variation of 4% or less in the results of dosage between the different Swiss centers. Risk calculation was performed in Barcelona by the Delfia Wallac Fluorometer (Perkin Elmer Company, Norwalk, Connecticut, USA) instruments and software, and by the Immulite 2000 analyzer (DPC Biermann, Bad Nauheim, Germany) and Prisca software (DPC Biermann) in Belgium.
We collected the values of free β-hCG, PAPP-A, nuchal translucency and AFP expressed as MoM. Because the values of serum markers change rapidly and have an asymmetric (skewed) distribution for each week of gestation, these values are standardized as MoM for a given gestational age. A MoM value equal to 1 corresponds to the median of the population. A β-hCG MoM greater than 1 and a PAPP-A MoM lower than 1 indicate a higher risk for Down syndrome. Theoretically, the median of the MoM for a given marker in a population should be equal to 1. These values were computed in each center using local software and their own medians. Levels for subgroups (CD4 level, viral load, type of treatment and chronic hepatitis) were also calculated. Statistical analyses were performed with SPSS (SPSS Inc., Chicago, Illinois, USA) and MedCalc software (MedCalc Software, Broekstraat, Mariakerke, Belgium). MoM medians and their 95% confidence intervals were reported. Medians were compared between HIV-infected women and controls and differences were tested using the Kruskal–Wallis test. To achieve a power of 80%, we calculated that 197 cases were required to detect a difference of one fifth of a standard deviation of the MoM distribution between HIV-infected women and uninfected women.
Characteristics of the HIV-positive women are presented in Table 1. CD4 cell counts and viral loads at the same gestational ages of screening were only available for a subgroup of women.
MoM medians of AFP and nuchal translucency measurement were similar between cases and controls [1.06 (0.95–1.15) vs. 1.09 (1.05–1.12); P = 0.38, and 0.88 (0.82–0.94) vs. 0.79 (0.77–0.82); P = 0.81, respectively]. Medians of the MoM of PAPP-A and β-hCG were lower in HIV-positive women then controls [0.88 (0.77–1.00) vs. 1.05 (1.00–1.11); P = 0.0004, and 0.84 (0.72–0.93) vs. 1.09 (1.02–1.16); P < 0.0001, respectively]. Nevertheless, there was a large overlap between the distributions of MoM for all markers, and the percentage of outliers was similar in HIV-positive women and in controls (Fig. 1).
In the group of HIV-positive women, we found no association between CD4 cell count, viral load, ART use at the time of screening, type of antiretroviral drug and chronic hepatitis, and the level of any biochemical screening marker (data not shown but available upon request).
These study findings provide further evidence that HIV infection has a slight effect on some of the markers used for the screening of fetal anomalies. To the best of our knowledge, this is the largest study on this topic and the only one evaluating the influence of HIV infection on the FTST for Down syndrome.
In our study, we found small differences in β-hCG and PAPP-A levels, the serum markers used for first-trimester screening, between HIV-infected women and controls. These differences, although statistically significant, are modest in absolute value and have no impact on the risk calculation. As an example, using the CISline Prenat' screen software, if β-hCG is 0.8 MoM instead of 1 MoM in a 28-year-old woman, the calculated risk will be close to 1 of 11 000 with both levels. We have also determined the estimated risks for Down syndrome in high-risk and low-risk situations by introducing the different values of β-hCG and PAPP-A found in our study in the calculations performed using other softwares (Delfia, the one used in Barcelona and Astraia, the one developed by the Fetal Medicine Foundation). There was no impact on the estimated risks. Moreover, the distribution of outliers was similar in the two populations, thus showing that very abnormal results are found in both populations.
Our findings for the FTST contrast with those of previous studies evaluating the influence of HIV infection on the biochemical markers used for the second-trimester screening tests. Yudin et al. found that β-hCG levels were significantly elevated in HIV-positive pregnant women compared with uninfected women, increasing thereby the calculated risk for Down syndrome. However, only 34 HIV-infected women were included, thus not allowing for any firm conclusions. Another study , including 53 HIV-positive women undergoing a second-trimester screening test, reported that 18% of women had positive screening results for Down syndrome and 11% for NTD. In this cohort, there were no cases of Down syndrome or NTD. The authors concluded that the false-positive rate exceeds that of the general obstetric population, thus preventing the use of these tests in HIV-positive pregnant women. Others have shown that AFP levels were lower among HIV-positive women treated with protease inhibitors .
The risk for a positive screen has been shown to be associated with the immune status in several studies [4,13]. Women with CD4+ cell counts below 200 cells/μl were more likely to have an abnormal result. When looking at individual markers, increased β-hCG levels have also been shown to be associated with a decreased CD4+ cell count. Nevertheless, other studies, as well as ours, have not shown this association . There are currently no data suggesting an effect of HIV infection on the trophoblast cell to explain a modification of the level of the serum markers used for fetal screening. The discrepancy between our results and those of previous studies might be due to the fact that we evaluated the first-trimester screening for Down syndrome and that we included a much larger number of HIV-infected women, thereby reducing the likelihood of chance findings.
We have shown that first-trimester screening for Down syndrome and second-trimester screening for NTD are reliable in HIV-positive pregnant women. The minor differences found in some markers do not have any clinical impact and concern over an overestimation of risks is no longer justified. In conclusion, we consider that HIV-positive pregnant women should be offered the same screening tests for fetal anomalies as those who are HIV-negative.
This study was financed in the framework of the Swiss HIV Cohort Study supported by the Swiss National Science Foundation (grant no. 499).
We thank Drs G. Drack, D. Wunder and C. Grawe for providing part of the Swiss data.
The members of the Swiss HIV Cohort Study and the Swiss Mother and Child HIV Study are C. Aebi, M. Battegay, E. Bernasconi, J. Böni, P. Brazzola, HC Bucher, Ph. Bürgisser, A. Calmy, S. Cattacin, M. Cavassini, J.-J. Cheseaux, G. Drack, R. Dubs, M. Egger, L. Elzi, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne), HJ. Furrer, C. Fux, A. Gayet-Ageron, S. Gerber, M. Gorgievski, C. Grawe, H. Günthard, Th. Gyr, H. Hirsch, B. Hirschel, I. Hösli, M. Hüsler, L. Kaiser, Ch. Kahlert, U. Karrer, C. Kind, Th. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez, N. Müller, D. Nadal, M. Opravil, F. Paccaud, G. Pantaleo, A. Rauch, S. Regenass, M. Rickenbach, C. Rudin (Chairman of the MoChiV Substudy, Basel UKBB, Römergasse 8, CH-4058 Basel), P. Schmid, D. Schultze, J. Schüpbach, R. Speck, P. Taffé, A. Telenti, A. Trkola, P. Vernazza, R. Weber, D. Wunder, C.-A. Wyler and S. Yerly.
Author's contributions: P.B., M.B., B.M.deT., O.C., P.B., C.A.-P. and P.B. collected the data. P.B., B.M.deT. and M.B. did the statistical analysis. All the authors participated in the writing and the reading of the article.
Data were presented at the Swiss Annual Congress of Infectiology during a symposium: challenges in HIV. Zürich, 13 June 2007.
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