- Email: [email protected]
In combined first-trimester Down syndrome screening, the false-positive rate is not higher in pregnancies conceived after assisted reproduction compared with spontaneous pregnancies
In combined first-trimester Down syndrome screening, the false-positive rate is not higher in pregnancies conceived after assisted reproduction compared with spontaneous pregnancies The maternal serum levels of pregnancy-associated plasma protein-A (PAPP-A) were reduced in hormonally stimulated pregnancies in the in vitro fertilization and intracytoplasmic sperm injection groups (N ¼ 176; PAPP-A: 0.82) and in the entire assisted reproduction group (N ¼ 282; PAPP-A: 0.83) as compared with controls (N ¼ 24,783; PAPP-A: 0.94). However, the false-positive rate of first-trimester combined screenings was not statistically significantly increased in assisted reproduction pregnancies after adjustment for maternal age. (Fertil Steril 2011;95:378–81. 2011 by American Society for Reproductive Medicine.) Key Words: Assisted reproduction, Down syndrome, fb-hCG, first-trimester screening, PAPP-A
Prenatal screening for Down syndrome (DS) has become routine during antenatal care. Pregnancies conceived by assisted reproduction techniques (ART) have been reported to be associated with changes in the biochemical parameters of screening for DS, leading to an increased false-positive rate (FPR) in the second trimester (1–6). The effect of ART on first-trimester Down screening, which combines maternal age, nuchal translucency thickness (NT), maternal serum free b subunit human chorionic gonadotropin (fb-hCG), and pregnancy-associated plasma protein-A (PAPP-A), has been examined, but the results have been inconclusive. In this study, we examined whether the levels of first-trimester maternal biochemical markers and the FPR were affected by conception after ART in various forms. This topic is important because an increasing number of women wish to have a risk estimation for DS after ART, and results from the previous studies have been controversial. The Research-Ethics Committee of Oulu University approved the study (accordance with the Helsinki Declaration of 1975). Maarit Matilainen, M.B. Sini Peuhkurinen, M.Sc. Paivi Laitinen, Ph.D. Ilkka Jarvela, M.D. Laure Morin-Papunen, M.D. Marku Ryynanen, M.D., Ph.D. Department of Obstetrics and Gynaecology, Oulu University Hospital Laboratory, Oulu University Hospital, Oulu, Finland Received March 23, 2010; revised July 4, 2010; accepted July 9, 2010; published online August 21, 2010. M.M. has nothing to disclose. S.P. has nothing to disclose. P.L. has nothing to disclose. I.J. has nothing to disclose. L.M-P. has nothing to disclose. M.R. has nothing to disclose. €nen, M.D., Ph.D., Department of ObstetReprint requests: Marrku Ryyna rics and Gynecology, Oulu University Hospital, PL 24, 90029 OYS, Finland (FAX: þ358-8-315-4310; E-mail: [email protected]).
378
This retrospective, cohort study with a control group was performed between January 1, 2002, and January 21, 2009. We collected data from 333 women who had conceived after ART and had participated in first-trimester combined DS screening at a tertiary in vitro fertilization (IVF) referral center (the University Hospital of Oulu, Finland) and the Family Federation Institution, Oulu Department, two institutions that work in close collaboration. Gestational ages ranged from 9 to 13 weeks and were calculated from crown–rump length (CRL) measured at NT scan, by last menstrual period, or by embryo transfer day. After exclusion criteria (multiple pregnancies, vanishing twin, ovum donation, spontaneous abortions, or incomplete data), 282 ART pregnancies were studied. The control group comprised 24,783 spontaneous singleton pregnancies over the same period of time. No signs of chromosomal abnormality were detected in the infants from the ART groups. The control group had 61 DS cases. Patients were divided into four groups according to the type of conception, as follows: controls, hormonally stimulated IVF/intracytoplasmic sperm injection (IVF/ICSI), spontaneous frozen embryo transfer (FET), and hormonally stimulated (hormone replacement therapy) FET (HRT-FET). Ovarian stimulation was performed by use of a long protocol of either gonadotropinreleasing hormone (GnRH) agonist or GnRH antagonist, as previously described elsewhere (7–9). The serum samples were collected in the laboratories of the health care centers, and they were transported, centrifuged, and refrigerated/frozen by the laboratory of Oulu University Hospital. The samples were analyzed by use of the PerkinElmer AutoDELFIA time-resolved fluoroimmunoassay kit (PerkinElmer, Wallac Oy, Turku, Finland) for the measurement of PAPP-A and fb-hCG. The analytical sensitivities of fb-hCG and PAPP-A were 0.2 ng/mL and 5 mIU/L, respectively. Three-level quality control samples were used as internal quality controls in each run, and an additional quality control sample
Fertility and Sterility Vol. 95, No. 1, January 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.07.1048
was analyzed as the patient sample for the assessment of the risk calculation. The within-assay and between-assay variations for fb-hCG were both <3.4%, and for PAPP-A they were <1.4% and <4.8%, respectively. For external quality assessment, samples were acquired from an international quality assurance company (UK NEQAS Chemistry, Birmingham, UK) for the evaluation of the laboratory’s performance against other laboratories using the same method. The measured marker levels were presented as multiples of medians (MoM) after adjusting for maternal weight, smoking, and diabetes status. The marker levels were not corrected for ethnicity because more than 99% of the population is Caucasian in Northern Finland. The NT was measured in maternity clinics and health centers by doctors, midwives, and nurses with formal training, but it was variable and not standardized. For NT, regression analysis was performed to derive the relationship with CRL; the expected values of NT were calculated, and the measurements converted into MoM. LifeCycle software (PerkinElmer LifeSciences, Wallac, Finland) was used for calculating the risk figure. The program compares a patient result with a population model described by a set of multivariate Gaussian distributions, which takes into account maternal age, NT, and CRL. In all cases in which combined test was R1:250, an invasive procedure was offered.
The statistical analysis was performed using the SPSS program (version 15.0; SPSS Inc., Chicago, IL). Continuous data are expressed as the median. The distribution of markers was tested using the Kolmogorov-Smirnov test of fit. Even after logarithmic transformation, the data did not achieve normal distribution; therefore, a nonparametric test was used. The odds ratio (OR) for the risk of DS R1:250 was calculated using logistic regression in which the study groups were the variables of interest and age was the confounding variable. P<.05 was considered statistically significant. The baseline characteristics of the study population and main results are presented in Table 1. The women in each ART group were statistically significantly older compared with controls. The median MoM PAPP-A levels were statistically significantly lower in the entire ART group and the hormonally stimulated IVF/ICSI groups when compared with controls. In women pregnant after spontaneous FET and HRT-FET, the PAPP-A levels were lower compared with controls but did not reach statistical significance. There was no difference between the different ART groups. The median MoM fb-hCG, NT, NT MoM, gestational age, or CRL in the ART and spontaneous pregnancies showed no statistically significantly differences. The FPRs are presented in Table 1. There was no statistically significant difference in OR for elevated risk for DS in ART pregnancies compared with controls after adjustment for maternal age.
TABLE 1 Baseline parameters of the study population and the main results. Parameter Maternal characteristics N Age (y) Age R35 y (%) Weight (kg) Smoking (%) Caucasian (%) Diabetes (%) Gestational age (d) CRL (mm) PAPP-A Median MoM fb-hCG Median MoM NT (mm) Median NT MoM Median MoM FPR (%) OR 95% CI
Controls
All ART
IVF/ICSI
FET
HRT-FET
24,783 29.5 (15.6–48.0) 17.7 67 (40–150) 12.4 99 0.2 82 (63–79) 54.0 (22.0–78.0)
282 32.1 (21.1–43.3)a 25.9 66 (43–123) 5.3 100 0.0 80 (63–97) 54.0 (22.0–78.0)
176 31.7 (21.1–42.9)b 23.9 66 (48–123) 5.1 100 0.0 80 (63–97) 54.0 (22.0–78.0)
87 33.3 (23.2–41.9)c 32.2 65 (43–110) 6.9 100 0.0 80 (63–97) 54.0 (22.0–78.0)
19 32.8 (25.8–43.3)d 15.8 66 (52–82) 0.0 100 0.0 79 (63–97) 54.0 (22.0–78.0)
0.94 (0.01–8.39)
0.83 (0.13–5.89)e
0.82 (0.13–5.89)f
0.78 (0.13–4.23)
0.66 (0.24–3.95)
1.02 (0.01–11.89)
0.98 (0.24–4.01)
1.00 (0.24–4.01)
0.94 (0.28–3.75)
0.83 (0.47–3.43)
1.2 (0.1–8.4)
1.2 (0.6–3.1)
1.3 (0.6–2.5)
1.2 (0.6–3.1)
1.2 (0.8–1.8)
0.97 (0.06–7.58) 4.3 — —
1.00 (0.50–2.38) 6.7 1.3 0.8–2.0
1.03 (0.54–1.83) 6.3 1.2 0.7–2.3
1.00 (0.50–2.38) 6.9 1.2 0.5–2.7
0.96 (0.64–1.27) 10.5 1.9 0.4–8.5
Notes: Values are given as medians, ranges, and percentages. The odds ratios (OR) and 95% confidence intervals (CI) for the elevated risk for trisomy 21 were calculated using logistic regression analysis. ART ¼ assisted reproduction technology; CRL ¼ crown–rump length; fb-hCG ¼ maternal serum free b subunit human chorionic gonadotropin; FET ¼ frozen embryo transfer; FPR ¼ false-positive rate (proportion of women with a 21-trisomy risk R1:250); HRT ¼ hormone replacement therapy; ICSI ¼ intracytoplasmic sperm injection; IVF ¼ in vitro fertilization; MoM ¼ multiples of medians; NT ¼ nuchal translucency thickness; PAPP-A ¼ pregnancy-associated plasma protein-A. a P%.001, all ART versus controls, Mann-Whitney U test. b P%.001, IVF/ICSI versus controls, Mann-Whitney U test. c P%.001, FET versus controls, Mann-Whitney U test. d P%.001, HRT-FET versus controls, Mann-Whitney U test. e P%.003, all ART versus controls, Mann-Whitney U test. f P%.01, IVF/ICSI versus controls, Mann-Whitney U test. Matilainen. Correspondence. Fertil Steril 2011.
Fertility and Sterility
379
In our study, we found that the PAPP-A level was reduced in the overall ART group versus the control group. The PAPP-A level in the control group was 6% lower (0.94) than expected; at the same time, the fb-hCG level was approximately 1. Despite of the lower value of PAPP-A, the detection rate was 80.1%, and the FPR was 4.3%, both of which are acceptable. There was a 2-day difference in the gestation ages between the ART and the control group, but this difference was not statistically significant. Changes in the maternal serum PAPP-A levels happened weekly not daily. The PAPP-A level was also reduced in the previous studies by Gjerris et al. (10), Amor et al. (11), and Kagan et al. (12), which was the largest study to date. The study reported that the PAPP-A levels were reduced 10% in IVF pregnancies. Our study also found a statistically significant reduction in the PAPP-A concentration in hormonally stimulated IVF/ ICSI pregnancies compared with controls. This is in agreement with previous studies (10, 11, 13–17). In spontaneous FET and HRTFET pregnancies, the PAPP-A levels were reduced but did not reach statistical significance compared with controls. Our study group, however, did not contain a large number of cases. Other studies have reported that statistically, the PAPP-A concentration was not significantly reduced in FET pregnancies (10, 18). There is also a study in which statistically, the PAPP-A levels were significantly reduced in ICSI pregnancies in the fresh and the frozen-thawed embryo subgroups and in the IVF fresh embryo subgroups as compared with controls (15). In the study by Amor et al. (11), the PAPP-A levels were reduced both in fresh and frozen-thawed embryos. We found no difference in the fb-hCG concentrations between the ART and control groups. This is in agreement with most previous studies (6, 10, 11, 14, 17–20). Some studies have reported the fb-hCG to be increased (13, 15, 21–23). In the largest study to date, Kagan et al. (12) found the median fb-hCG was 9% higher in the IVF pregnancies.
There was no difference in the size of NT in ART pregnancies compared with controls; this is in agreement with the majority of previous studies (6, 13, 14, 20, 22, 23). Two studies have reported a larger NT (15, 17), and one study found the NT MoM was statistically significantly smaller in ART pregnancies (10). In our study, the ORs for a FPR in the combined first-trimester Down screening were not increased in ART pregnancies, after adjustment for maternal age. This is in agreement with many other studies (13, 17, 20, 24). Several studies have reported a higher FPR in the ART group even after adjustment for maternal age (10, 11, 14). Tul et al. (16) found a higher FPR in pregnancies conceived after ICSI. Amor et al. (11) found a higher FPR in both fresh and frozenthawed embryos, but only in hormonally stimulated pregnancies. There are many possible confounding factors that could lead to contradictory results on screening markers in ART pregnancies. Multiple corpora lutea and implantation sites have been suggested to be a reason for altered marker levels (25). Also, a functional delay in fetal and placental development and the higher risk of obstetric complications associated with ART can lead to changes in marker concentrations (15, 19). Several studies have suggested that exogenous hormone treatment is the main reason for reduced PAPP-A levels (11, 15, 16, 21). In our study, we found that PAPP-A levels were reduced in hormonally stimulated IVF/ICSI pregnancies, but we also found that PAPP-A levels were decreased in nonstimulated FET cycles. We found that the PAPP-A MoM was statistically significantly decreased in stimulated IVF/ICSI pregnancies. Nevertheless, the FPR was not higher in ART pregnancies compared with controls. However, the contradictory results from previous published works will require larger studies. As more information accumulates on serum marker variations, procedure-specific medians for serum markers need to correct changes in pregnancies conceived after ART.
REFERENCES 1. Barkai G, Goldman B, Ries L, Chaki R, Dor J, Cuckle H. Down’s syndrome screening marker levels following assisted reproduction. Prenat Diagn 1996;16:1111–4. 2. Ribbert LS, Kornman LH, De Wolf BT, Simons AH, Jansen CA, Beekhuis JR, et al. Maternal serum screening for fetal Down syndrome in IVF pregnancies. Prenat Diagn 1996;16:35–8. 3. Frishman GN, Canick JA, Hogan JW, Hackett RJ, Kellner LH, Saller DN Jr. Serum triple-marker screening in in vitro fertilization and naturally conceived pregnancies. Obstet Gynecol 1997;90: 98–101. 4. Wald NJ, White N, Morris JK, Huttly WJ, Canick JA. Serum markers for Down’s syndrome in women who have had in vitro fertilisation: implications for antenatal screening. BJOG 1999;106:1304–6. 5. Raty R, Virtanen A, Koskinen P, Anttila L, Forsstrom J, Laitinen P, et al. Serum free betahCG and alphafetoprotein levels in IVF, ICSI and frozen embryo transfer pregnancies in maternal mid-trimester serum screening for Down’s syndrome. Hum Reprod 2002;17:481–4. 6. Lambert-Messerlian G, Dugoff L, Vidaver J, Canick JA, Malone FD, Ball RH, et al. First- and second-trimester Down syndrome screening
380
Matilainen et al.
Correspondence
7.
8.
9.
10.
11.
markers in pregnancies achieved through assisted reproductive technologies (ART): a FASTER trial study. Prenat Diagn 2006;26:672–8. Vilska S, Tiitinen A, Hyden-Granskog C, Hovatta O. Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum Reprod 1999;14:2392–5. Tiitinen A, Halttunen M, Harkki P, Vuoristo P, HydenGranskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001;16:1140–4. Veleva Z, Jarvela IY, Nuojua-Huttunen S, Martikainen H, Tapanainen JS. An initial low response predicts poor outcome in in vitro fertilization/intracytoplasmic sperm injection despite improved ovarian response in consecutive cycles. Fertil Steril 2005;83:1384–90. Gjerris AC, Loft A, Pinborg A, Christiansen M, Tabor A. First trimester screening markers are altered in pregnancies conceived after IVF/ICSI. Ultrasound Obstet Gynecol 2009;33:8–17. Amor DJ, Xu JX, Halliday JL, Francis I, Healy DL, Breheny S, et al. Pregnancies conceived using assisted reproductive technologies (ART) have low levels of pregnancy-associated plasma protein-A (PAPP-A) leading to a high rate of false-positive results in first trimester screening for Down syndrome. Hum Reprod 2009;24:1330–8.
12. Kagan KO, Wright D, Spencer K, Molina FS, Nicolaides KH. First-trimester screening for trisomy 21 by free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A: impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol 2008;31:493–502. 13. Liao AW, Heath V, Kametas N, Spencer K, Nicolaides KH. First trimester screening for trisomy 21 in singleton pregnancies achieved by assisted reproduction. Hum Reprod 2001;16: 1501–4. 14. Orlandi F, Rossi C, Allegra A, et al. First trimester screening with free beta-hCG, PAPP-A and nuchal translucency in pregnancies conceived with assisted reproduction. Prenat Diagn 2002;22:718–21. 15. Hui PW, Lam YH, Tang MH, Ng EH, Yeung WS, Ho PC. Maternal serum pregnancy-associated plasma protein-A and free beta-human chorionic gonadotrophin in pregnancies conceived with fresh and frozen-thawed embryos from in vitro fertilization and intracytoplasmic sperm injection. Prenat Diagn 2005;25:390–3. 16. Tul N, Novak-Antolic Z. Serum PAPP-A levels at 10–14 weeks of gestation are altered in women after assisted conception. Prenat Diagn 2006;26: 1206–11.
Vol. 95, No. 1, January 2011
17. Maymon R, Shulman A. Serial first- and second trimester Down’s syndrome screening tests among IVF-versus naturally-conceived singletons. Hum Reprod 2001b;17:1081–5. 18. Anckaert E, Schiettecatte J, Sleurs E, Devroey P, Smitz J. First trimester screening for Down’s syndrome after assisted reproductive technology: non-male factor infertility is associated with elevated free beta-human chorionic gonadotropin levels at 10– 14 weeks of gestation. Fertil Steril 2008;90:1206–10. 19. Maymon R, Shulman A. Integrated first- and second-trimester Down syndrome screening test among unaffected IVF pregnancies. Prenat Diagn 2004;24:125–9.
Fertility and Sterility
20. Wojdemann KR, Larsen SO, Shalmi A, Sundberg K, Christiansen M, Tabor A. First trimester screening for Down syndrome and assisted reproduction: no basis for concern. Prenat Diagn 2001;21:563–5. 21. Bersinger NA, Wunder D, Vanderlick F, Chanson A, Pescia G, Janecek P, et al. Maternal serum levels of placental proteins after in vitro fertilisation and their implications for prenatal screening. Prenat Diagn 2004;24:471–7. 22. Niemimaa M, Heinonen S, Seppala M, Hippelainen M, Martikainen H, Ryynanen M. First-trimester screening for Down’s syndrome in in vitro fertilization pregnancies. Fertil Steril 2001;76:1282–3.
23. Ghisoni L, Ferrazzi E, Castagna C, Levi Setti PE, Masini AC, Pigni A. Prenatal diagnosis after ART success: the role of early combined screening tests in counselling pregnant patients. Placenta 2003;24(Suppl B):S99–103. 24. Bellver J, Lara C, Soares SR, Ramirez A, Pellicer A, Remohi J, et al. First trimester biochemical screening for Down’s syndrome in singleton pregnancies conceived by assisted reproduction. Hum Reprod 2005;20:2623–7. 25. Weisz B, Rodeck CH. An update on antenatal screening for Down’s syndrome and specific implications for assisted reproduction pregnancies. Hum Reprod Update 2006;12:513–8.
381
Prenatal screening for Down syndrome (DS) has become routine during antenatal care. Pregnancies conceived by assisted reproduction techniques (ART) have been reported to be associated with changes in the biochemical parameters of screening for DS, leading to an increased false-positive rate (FPR) in the second trimester (1–6). The effect of ART on first-trimester Down screening, which combines maternal age, nuchal translucency thickness (NT), maternal serum free b subunit human chorionic gonadotropin (fb-hCG), and pregnancy-associated plasma protein-A (PAPP-A), has been examined, but the results have been inconclusive. In this study, we examined whether the levels of first-trimester maternal biochemical markers and the FPR were affected by conception after ART in various forms. This topic is important because an increasing number of women wish to have a risk estimation for DS after ART, and results from the previous studies have been controversial. The Research-Ethics Committee of Oulu University approved the study (accordance with the Helsinki Declaration of 1975). Maarit Matilainen, M.B. Sini Peuhkurinen, M.Sc. Paivi Laitinen, Ph.D. Ilkka Jarvela, M.D. Laure Morin-Papunen, M.D. Marku Ryynanen, M.D., Ph.D. Department of Obstetrics and Gynaecology, Oulu University Hospital Laboratory, Oulu University Hospital, Oulu, Finland Received March 23, 2010; revised July 4, 2010; accepted July 9, 2010; published online August 21, 2010. M.M. has nothing to disclose. S.P. has nothing to disclose. P.L. has nothing to disclose. I.J. has nothing to disclose. L.M-P. has nothing to disclose. M.R. has nothing to disclose. €nen, M.D., Ph.D., Department of ObstetReprint requests: Marrku Ryyna rics and Gynecology, Oulu University Hospital, PL 24, 90029 OYS, Finland (FAX: þ358-8-315-4310; E-mail: [email protected]).
378
This retrospective, cohort study with a control group was performed between January 1, 2002, and January 21, 2009. We collected data from 333 women who had conceived after ART and had participated in first-trimester combined DS screening at a tertiary in vitro fertilization (IVF) referral center (the University Hospital of Oulu, Finland) and the Family Federation Institution, Oulu Department, two institutions that work in close collaboration. Gestational ages ranged from 9 to 13 weeks and were calculated from crown–rump length (CRL) measured at NT scan, by last menstrual period, or by embryo transfer day. After exclusion criteria (multiple pregnancies, vanishing twin, ovum donation, spontaneous abortions, or incomplete data), 282 ART pregnancies were studied. The control group comprised 24,783 spontaneous singleton pregnancies over the same period of time. No signs of chromosomal abnormality were detected in the infants from the ART groups. The control group had 61 DS cases. Patients were divided into four groups according to the type of conception, as follows: controls, hormonally stimulated IVF/intracytoplasmic sperm injection (IVF/ICSI), spontaneous frozen embryo transfer (FET), and hormonally stimulated (hormone replacement therapy) FET (HRT-FET). Ovarian stimulation was performed by use of a long protocol of either gonadotropinreleasing hormone (GnRH) agonist or GnRH antagonist, as previously described elsewhere (7–9). The serum samples were collected in the laboratories of the health care centers, and they were transported, centrifuged, and refrigerated/frozen by the laboratory of Oulu University Hospital. The samples were analyzed by use of the PerkinElmer AutoDELFIA time-resolved fluoroimmunoassay kit (PerkinElmer, Wallac Oy, Turku, Finland) for the measurement of PAPP-A and fb-hCG. The analytical sensitivities of fb-hCG and PAPP-A were 0.2 ng/mL and 5 mIU/L, respectively. Three-level quality control samples were used as internal quality controls in each run, and an additional quality control sample
Fertility and Sterility Vol. 95, No. 1, January 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.07.1048
was analyzed as the patient sample for the assessment of the risk calculation. The within-assay and between-assay variations for fb-hCG were both <3.4%, and for PAPP-A they were <1.4% and <4.8%, respectively. For external quality assessment, samples were acquired from an international quality assurance company (UK NEQAS Chemistry, Birmingham, UK) for the evaluation of the laboratory’s performance against other laboratories using the same method. The measured marker levels were presented as multiples of medians (MoM) after adjusting for maternal weight, smoking, and diabetes status. The marker levels were not corrected for ethnicity because more than 99% of the population is Caucasian in Northern Finland. The NT was measured in maternity clinics and health centers by doctors, midwives, and nurses with formal training, but it was variable and not standardized. For NT, regression analysis was performed to derive the relationship with CRL; the expected values of NT were calculated, and the measurements converted into MoM. LifeCycle software (PerkinElmer LifeSciences, Wallac, Finland) was used for calculating the risk figure. The program compares a patient result with a population model described by a set of multivariate Gaussian distributions, which takes into account maternal age, NT, and CRL. In all cases in which combined test was R1:250, an invasive procedure was offered.
The statistical analysis was performed using the SPSS program (version 15.0; SPSS Inc., Chicago, IL). Continuous data are expressed as the median. The distribution of markers was tested using the Kolmogorov-Smirnov test of fit. Even after logarithmic transformation, the data did not achieve normal distribution; therefore, a nonparametric test was used. The odds ratio (OR) for the risk of DS R1:250 was calculated using logistic regression in which the study groups were the variables of interest and age was the confounding variable. P<.05 was considered statistically significant. The baseline characteristics of the study population and main results are presented in Table 1. The women in each ART group were statistically significantly older compared with controls. The median MoM PAPP-A levels were statistically significantly lower in the entire ART group and the hormonally stimulated IVF/ICSI groups when compared with controls. In women pregnant after spontaneous FET and HRT-FET, the PAPP-A levels were lower compared with controls but did not reach statistical significance. There was no difference between the different ART groups. The median MoM fb-hCG, NT, NT MoM, gestational age, or CRL in the ART and spontaneous pregnancies showed no statistically significantly differences. The FPRs are presented in Table 1. There was no statistically significant difference in OR for elevated risk for DS in ART pregnancies compared with controls after adjustment for maternal age.
TABLE 1 Baseline parameters of the study population and the main results. Parameter Maternal characteristics N Age (y) Age R35 y (%) Weight (kg) Smoking (%) Caucasian (%) Diabetes (%) Gestational age (d) CRL (mm) PAPP-A Median MoM fb-hCG Median MoM NT (mm) Median NT MoM Median MoM FPR (%) OR 95% CI
Controls
All ART
IVF/ICSI
FET
HRT-FET
24,783 29.5 (15.6–48.0) 17.7 67 (40–150) 12.4 99 0.2 82 (63–79) 54.0 (22.0–78.0)
282 32.1 (21.1–43.3)a 25.9 66 (43–123) 5.3 100 0.0 80 (63–97) 54.0 (22.0–78.0)
176 31.7 (21.1–42.9)b 23.9 66 (48–123) 5.1 100 0.0 80 (63–97) 54.0 (22.0–78.0)
87 33.3 (23.2–41.9)c 32.2 65 (43–110) 6.9 100 0.0 80 (63–97) 54.0 (22.0–78.0)
19 32.8 (25.8–43.3)d 15.8 66 (52–82) 0.0 100 0.0 79 (63–97) 54.0 (22.0–78.0)
0.94 (0.01–8.39)
0.83 (0.13–5.89)e
0.82 (0.13–5.89)f
0.78 (0.13–4.23)
0.66 (0.24–3.95)
1.02 (0.01–11.89)
0.98 (0.24–4.01)
1.00 (0.24–4.01)
0.94 (0.28–3.75)
0.83 (0.47–3.43)
1.2 (0.1–8.4)
1.2 (0.6–3.1)
1.3 (0.6–2.5)
1.2 (0.6–3.1)
1.2 (0.8–1.8)
0.97 (0.06–7.58) 4.3 — —
1.00 (0.50–2.38) 6.7 1.3 0.8–2.0
1.03 (0.54–1.83) 6.3 1.2 0.7–2.3
1.00 (0.50–2.38) 6.9 1.2 0.5–2.7
0.96 (0.64–1.27) 10.5 1.9 0.4–8.5
Notes: Values are given as medians, ranges, and percentages. The odds ratios (OR) and 95% confidence intervals (CI) for the elevated risk for trisomy 21 were calculated using logistic regression analysis. ART ¼ assisted reproduction technology; CRL ¼ crown–rump length; fb-hCG ¼ maternal serum free b subunit human chorionic gonadotropin; FET ¼ frozen embryo transfer; FPR ¼ false-positive rate (proportion of women with a 21-trisomy risk R1:250); HRT ¼ hormone replacement therapy; ICSI ¼ intracytoplasmic sperm injection; IVF ¼ in vitro fertilization; MoM ¼ multiples of medians; NT ¼ nuchal translucency thickness; PAPP-A ¼ pregnancy-associated plasma protein-A. a P%.001, all ART versus controls, Mann-Whitney U test. b P%.001, IVF/ICSI versus controls, Mann-Whitney U test. c P%.001, FET versus controls, Mann-Whitney U test. d P%.001, HRT-FET versus controls, Mann-Whitney U test. e P%.003, all ART versus controls, Mann-Whitney U test. f P%.01, IVF/ICSI versus controls, Mann-Whitney U test. Matilainen. Correspondence. Fertil Steril 2011.
Fertility and Sterility
379
In our study, we found that the PAPP-A level was reduced in the overall ART group versus the control group. The PAPP-A level in the control group was 6% lower (0.94) than expected; at the same time, the fb-hCG level was approximately 1. Despite of the lower value of PAPP-A, the detection rate was 80.1%, and the FPR was 4.3%, both of which are acceptable. There was a 2-day difference in the gestation ages between the ART and the control group, but this difference was not statistically significant. Changes in the maternal serum PAPP-A levels happened weekly not daily. The PAPP-A level was also reduced in the previous studies by Gjerris et al. (10), Amor et al. (11), and Kagan et al. (12), which was the largest study to date. The study reported that the PAPP-A levels were reduced 10% in IVF pregnancies. Our study also found a statistically significant reduction in the PAPP-A concentration in hormonally stimulated IVF/ ICSI pregnancies compared with controls. This is in agreement with previous studies (10, 11, 13–17). In spontaneous FET and HRTFET pregnancies, the PAPP-A levels were reduced but did not reach statistical significance compared with controls. Our study group, however, did not contain a large number of cases. Other studies have reported that statistically, the PAPP-A concentration was not significantly reduced in FET pregnancies (10, 18). There is also a study in which statistically, the PAPP-A levels were significantly reduced in ICSI pregnancies in the fresh and the frozen-thawed embryo subgroups and in the IVF fresh embryo subgroups as compared with controls (15). In the study by Amor et al. (11), the PAPP-A levels were reduced both in fresh and frozen-thawed embryos. We found no difference in the fb-hCG concentrations between the ART and control groups. This is in agreement with most previous studies (6, 10, 11, 14, 17–20). Some studies have reported the fb-hCG to be increased (13, 15, 21–23). In the largest study to date, Kagan et al. (12) found the median fb-hCG was 9% higher in the IVF pregnancies.
There was no difference in the size of NT in ART pregnancies compared with controls; this is in agreement with the majority of previous studies (6, 13, 14, 20, 22, 23). Two studies have reported a larger NT (15, 17), and one study found the NT MoM was statistically significantly smaller in ART pregnancies (10). In our study, the ORs for a FPR in the combined first-trimester Down screening were not increased in ART pregnancies, after adjustment for maternal age. This is in agreement with many other studies (13, 17, 20, 24). Several studies have reported a higher FPR in the ART group even after adjustment for maternal age (10, 11, 14). Tul et al. (16) found a higher FPR in pregnancies conceived after ICSI. Amor et al. (11) found a higher FPR in both fresh and frozenthawed embryos, but only in hormonally stimulated pregnancies. There are many possible confounding factors that could lead to contradictory results on screening markers in ART pregnancies. Multiple corpora lutea and implantation sites have been suggested to be a reason for altered marker levels (25). Also, a functional delay in fetal and placental development and the higher risk of obstetric complications associated with ART can lead to changes in marker concentrations (15, 19). Several studies have suggested that exogenous hormone treatment is the main reason for reduced PAPP-A levels (11, 15, 16, 21). In our study, we found that PAPP-A levels were reduced in hormonally stimulated IVF/ICSI pregnancies, but we also found that PAPP-A levels were decreased in nonstimulated FET cycles. We found that the PAPP-A MoM was statistically significantly decreased in stimulated IVF/ICSI pregnancies. Nevertheless, the FPR was not higher in ART pregnancies compared with controls. However, the contradictory results from previous published works will require larger studies. As more information accumulates on serum marker variations, procedure-specific medians for serum markers need to correct changes in pregnancies conceived after ART.
REFERENCES 1. Barkai G, Goldman B, Ries L, Chaki R, Dor J, Cuckle H. Down’s syndrome screening marker levels following assisted reproduction. Prenat Diagn 1996;16:1111–4. 2. Ribbert LS, Kornman LH, De Wolf BT, Simons AH, Jansen CA, Beekhuis JR, et al. Maternal serum screening for fetal Down syndrome in IVF pregnancies. Prenat Diagn 1996;16:35–8. 3. Frishman GN, Canick JA, Hogan JW, Hackett RJ, Kellner LH, Saller DN Jr. Serum triple-marker screening in in vitro fertilization and naturally conceived pregnancies. Obstet Gynecol 1997;90: 98–101. 4. Wald NJ, White N, Morris JK, Huttly WJ, Canick JA. Serum markers for Down’s syndrome in women who have had in vitro fertilisation: implications for antenatal screening. BJOG 1999;106:1304–6. 5. Raty R, Virtanen A, Koskinen P, Anttila L, Forsstrom J, Laitinen P, et al. Serum free betahCG and alphafetoprotein levels in IVF, ICSI and frozen embryo transfer pregnancies in maternal mid-trimester serum screening for Down’s syndrome. Hum Reprod 2002;17:481–4. 6. Lambert-Messerlian G, Dugoff L, Vidaver J, Canick JA, Malone FD, Ball RH, et al. First- and second-trimester Down syndrome screening
380
Matilainen et al.
Correspondence
7.
8.
9.
10.
11.
markers in pregnancies achieved through assisted reproductive technologies (ART): a FASTER trial study. Prenat Diagn 2006;26:672–8. Vilska S, Tiitinen A, Hyden-Granskog C, Hovatta O. Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum Reprod 1999;14:2392–5. Tiitinen A, Halttunen M, Harkki P, Vuoristo P, HydenGranskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001;16:1140–4. Veleva Z, Jarvela IY, Nuojua-Huttunen S, Martikainen H, Tapanainen JS. An initial low response predicts poor outcome in in vitro fertilization/intracytoplasmic sperm injection despite improved ovarian response in consecutive cycles. Fertil Steril 2005;83:1384–90. Gjerris AC, Loft A, Pinborg A, Christiansen M, Tabor A. First trimester screening markers are altered in pregnancies conceived after IVF/ICSI. Ultrasound Obstet Gynecol 2009;33:8–17. Amor DJ, Xu JX, Halliday JL, Francis I, Healy DL, Breheny S, et al. Pregnancies conceived using assisted reproductive technologies (ART) have low levels of pregnancy-associated plasma protein-A (PAPP-A) leading to a high rate of false-positive results in first trimester screening for Down syndrome. Hum Reprod 2009;24:1330–8.
12. Kagan KO, Wright D, Spencer K, Molina FS, Nicolaides KH. First-trimester screening for trisomy 21 by free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A: impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol 2008;31:493–502. 13. Liao AW, Heath V, Kametas N, Spencer K, Nicolaides KH. First trimester screening for trisomy 21 in singleton pregnancies achieved by assisted reproduction. Hum Reprod 2001;16: 1501–4. 14. Orlandi F, Rossi C, Allegra A, et al. First trimester screening with free beta-hCG, PAPP-A and nuchal translucency in pregnancies conceived with assisted reproduction. Prenat Diagn 2002;22:718–21. 15. Hui PW, Lam YH, Tang MH, Ng EH, Yeung WS, Ho PC. Maternal serum pregnancy-associated plasma protein-A and free beta-human chorionic gonadotrophin in pregnancies conceived with fresh and frozen-thawed embryos from in vitro fertilization and intracytoplasmic sperm injection. Prenat Diagn 2005;25:390–3. 16. Tul N, Novak-Antolic Z. Serum PAPP-A levels at 10–14 weeks of gestation are altered in women after assisted conception. Prenat Diagn 2006;26: 1206–11.
Vol. 95, No. 1, January 2011
17. Maymon R, Shulman A. Serial first- and second trimester Down’s syndrome screening tests among IVF-versus naturally-conceived singletons. Hum Reprod 2001b;17:1081–5. 18. Anckaert E, Schiettecatte J, Sleurs E, Devroey P, Smitz J. First trimester screening for Down’s syndrome after assisted reproductive technology: non-male factor infertility is associated with elevated free beta-human chorionic gonadotropin levels at 10– 14 weeks of gestation. Fertil Steril 2008;90:1206–10. 19. Maymon R, Shulman A. Integrated first- and second-trimester Down syndrome screening test among unaffected IVF pregnancies. Prenat Diagn 2004;24:125–9.
Fertility and Sterility
20. Wojdemann KR, Larsen SO, Shalmi A, Sundberg K, Christiansen M, Tabor A. First trimester screening for Down syndrome and assisted reproduction: no basis for concern. Prenat Diagn 2001;21:563–5. 21. Bersinger NA, Wunder D, Vanderlick F, Chanson A, Pescia G, Janecek P, et al. Maternal serum levels of placental proteins after in vitro fertilisation and their implications for prenatal screening. Prenat Diagn 2004;24:471–7. 22. Niemimaa M, Heinonen S, Seppala M, Hippelainen M, Martikainen H, Ryynanen M. First-trimester screening for Down’s syndrome in in vitro fertilization pregnancies. Fertil Steril 2001;76:1282–3.
23. Ghisoni L, Ferrazzi E, Castagna C, Levi Setti PE, Masini AC, Pigni A. Prenatal diagnosis after ART success: the role of early combined screening tests in counselling pregnant patients. Placenta 2003;24(Suppl B):S99–103. 24. Bellver J, Lara C, Soares SR, Ramirez A, Pellicer A, Remohi J, et al. First trimester biochemical screening for Down’s syndrome in singleton pregnancies conceived by assisted reproduction. Hum Reprod 2005;20:2623–7. 25. Weisz B, Rodeck CH. An update on antenatal screening for Down’s syndrome and specific implications for assisted reproduction pregnancies. Hum Reprod Update 2006;12:513–8.
381