Association between birth weight and first-trimester free b–human chorionic gonadotropin and pregnancy-associated plasma protein A Silvana Canini, B.Sc.,a Federico Prefumo, M.D., Ph.D.,b Daniela Pastorino, M.D.,b Lucia Crocetti, M.D.,b Calogero Gallo Afflitto, B.Sc.,a Pier Luigi Venturini, M.D.,b and Pierangela De Biasio, M.D.b a Laboratorio di Analisi, and b U.O. Ostetricia e Ginecologia, Istituto G. Gaslini, Università di Genova, Genova, Italy Study Objective: To assess the relationship between first-trimester maternal serum PAPP-A and free b-hCG and birth weight. Design: Observational study. Setting: Teaching hospital. Patient(s): Singleton pregnancies (n ¼ 1,630) at 10–14 weeks of gestation. Intervention(s): Fluorimetric immunoassays for maternal serum pregnancy-associated plasma protein A (PAPPA) and free b-hCG. Main Outcome Measure(s): Customized birth weight percentiles, calculated taking into account maternal height, weight, ethnic origin, parity, smoking status, and fetal gender. Result(s): There was a significant positive correlation between birth weight and PAPP-A, but not free b-hCG levels. Maternal serum levels of PAPP-A were significantly lower in small-for-gestation (SGA) newborns than in control subjects and were significantly higher in large-for-gestation (LGA) newborns than in control subjects. Maternal serum free b-hCG levels were lower in pregnancies complicated by pre-eclampsia than in normotensive ones. Multivariable analysis found PAPP-A to be an independent predictor of absolute birth weight, SGA, and LGA. Free b-hCG was found to be an independent predictor of gestational hypertension and pre-eclampsia. Neither of the two markers was associated with preterm delivery. Conclusion(s): Maternal serum PAPP-A levels in the late first trimester of pregnancy are associated with subsequent fetal growth (including both physiologic variation and abnormal growth), and decreased free b-hCG is more predictive of hypertensive disorders of pregnancy. (Fertil SterilÒ 2008;89:174–8. Ó2008 by American Society for Reproductive Medicine.) Key Words: First trimester, free b-hCG, PAPP-A, fetal growth, SGA, LGA, pre-eclampsia, gestational hypertension, preterm delivery Several recent reports suggested that maternal serum levels of PAPP-A and free b-hCG at 9–14 weeks of gestation are associated with a number of adverse pregnancy outcomes. Lower maternal serum PAPP-A and free b-hCG levels were described in association with hypertensive complications of pregnancy by some but not all studies (1–9). Low levels of PAPP-A have more consistently been reported to be associated with fetal growth restriction (1–3, 5, 6, 8–12). However, only one study has reported a difference in PAPP-A levels in human pregnancies delivering either small or large babies (7). The relationship between PAPP-A, free b-hCG, and the physiologic variation in birth weight has never been investigated. The aim of the present study was to investigate the relationship between first-trimester maternal serum markers Received November 23, 2006; revised and accepted February 12, 2007. Dr. Prefumo was supported by a Marie Curie Reintegration Fellowship of the European Community under contract number MERG-CT-2004– 006365. Reprint requests: Dr Federico Prefumo, U.O. Ostetricia e Ginecologia, Istituto G. Gaslini, Largo Gaslini, 5, 16132 Genova, Italy (FAX: þ39 010 5636382; E-mail: fprefumo@sgul.ac.uk). 174 and subsequent fetal growth in physiologic pregnancies and to clarify the association between PAPP-A and free b-hCG levels and adverse pregnancy outcome. MATERIALS AND METHODS From a computerized database, all singleton pregnancies were identified that had undergone first-trimester combined screening for Down syndrome at the Gaslini Institute in Genova, Italy, between January 2000 and February 2002. When booking for the test, all patients were asked to fill in a questionnaire with their demographic details and past medical and obstetric history. The parity, height, weight, and ethnic group of the mother were recorded. The PAPP-A and free b-hCG were assayed on maternal serum by fluorimetric immunoassays (Kryptor; Brahms, Hennigsdorf, Germany). All marker reference medians were previously determined in the local population (13). Institutional Review Board approval was obtained for this study, and all women gave their written informed consent. Pregnancy outcome was ascertained through a follow-up questionnaire given to each woman to return after delivery. Fertility and Sterilityâ Vol. 89, No. 1, January 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc. 0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.02.024 When available, hospital records and regional cytogenetic registries were also reviewed to determine the postnatal outcome for each subject. All fetuses with incomplete outcome data, abnormal karyotype, or major structural abnormalities were excluded from the study, as well as cases of pregnancy loss. Preterm delivery was defined as delivery before 37 completed weeks of gestation, small-for-gestational-age (SGA) as a birth weight lower than the 10th customized percentile, and large-for-gestational-age (LGA) as a birth weight higher than the 90th customized percentile. Customized birth weight percentiles (14) were calculated using freely downloadable software (Gardosi J, Francis A; Customized birth weight percentiles. Version 2.0.8, 2000; http://www.gestation.net). Customized birth weight percentiles take into account maternal (height, weight, ethnic origin, parity, smoking status) and fetal (gender) factors and are more predictive of neonatal outcome than population-based standards (15). We also identified normotensive pregnancies and pregnancies complicated by gestational hypertension. Gestational hypertension was defined as a blood pressure >140/90 mm Hg of new onset after 20 weeks of gestation. Within hypertensive pregnancies, a subgroup of cases where pre-eclampsia developed was identified. Pre-eclampsia was defined as a blood pressure >140/90 mm Hg and proteinuria of R300 mg in 24 hours or two readings of at least 1þ on dipstick analysis of midstream or catheter urine specimens if no 24-hour urine collection was available. During the study period, 2,203 examinations were performed. Of these, 15 (0.7%) were in twin pregnancies, and 33 (1.5%) were complicated by an abnormal karyotype, major structural abnormalities, or miscarriage. In 525 pregnancies (23.8%) delivered in other institutions, follow-up data allowed exclusion of abnormal karyotype or major structural abnormalities but were not considered satisfactory to describe other pregnancy outcomes. As a result, a total of 1,630 pregnancies (74.0%) satisfied the entry criteria. The multiples of the median (MoMs) of PAPP-A and free b-hCG were compared by using a two-tailed pooled t test after logarithmic transformation. Logarithmic transformation of the data allowed the use of parametric tests, because the marker measurements seemed to fit a gaussian distribution. The correlation between biochemical markers and birth weight customized percentiles was assessed by Spearman correlation coefficients. Birth weight percentiles in different groups were compared by Mann-Whitney test or KruskalWallis test as appropriate. Multivariable linear regression analysis was performed to assess the relationship between absolute birth weight, PAPP-A and free b-hCG levels, as well as other maternal and fetal confounders. Binary logistic regression analysis was performed to assess the relationship between the same variables and pregnancy complications. All calculations were performed using the SPSS software package (release 10.0.5; SPSS, Chicago, IL). P values less than .05 were considered to be statistically significant. RESULTS A total of 1,630 pregnancies satisfied the entry criteria. The median [range] maternal age was 31.6 [18.8–46.3] years, maternal weight 60 [40–145] kg, and maternal height 164 [142–186] cm. All women were of caucasian origin, and 75.5% of them were nulliparous. The median gestational age at sampling was 12.3 [9.9–13.9] weeks. The median gestational age at delivery was 39.9 [25.0–42.7] weeks. The gender of the newborn was male in 51.5% and female in 48.5% of the cases. An SGA infant was delivered in 9.4% (n ¼ 153) of the pregnancies and LGA in 9.4% (n ¼ 154). Preterm deliveries were 4.2% (n ¼ 69) of the total. Gestational hypertension was diagnosed in 2.5% (n ¼ 41) of pregnancies and pre-eclampsia in 1.0% (n ¼ 17). There was a significant positive correlation between customized birth weight percentiles and PAPP-A but not free b-hCG levels. The Spearman correlation coefficient between TABLE 1 ln MoM PAPP-A and ln MoM free bhCG in different groups with adverse pregnancy outcome (two-tailed pooled t test). SGA vs. nonSGA LGA vs. nonLGA Gestational hypertension vs. normal blood pressure Pre-eclampsia vs. normal blood pressure Preterm vs. term delivery ln MoM PAPP-A ln MoM free bhCG 0.164  0.541 vs. 0.014  0.495; P< .001 0.110  0.451 vs. 0.014  0.505; P¼ .02 0.094  0.529 vs. 0.005  0.501; P¼ .21 0.102  0.558 vs. 0.005  0.501; P¼ .38 0.025  0.536 vs. 0.096  0.501; P¼ .70 0.037  0.581 vs. 0.088  0.582; P¼ .30 0.1319  0.640 vs. 0.078  0.575; P¼ .33 0.224  0.650 vs. 0.079  0.579; P¼ .11 0.366  0.740 vs. 0.079  0.579; P¼ .04 0.001  0.531 vs. 0.089  0.584; P¼ .21 Note: MoM ¼ multiples of the median. Canini. Birth weight, PAPP-A, and free b-hCG. Fertil Steril 2008. Fertility and Sterilityâ 175 customized percentiles and first-trimester markers was 0.192 (P<.001) and 0.044 (P¼.11) for ln MoM PAPP-A and ln MoM free b-hCG, respectively. The marker levels in different groups with abnormal pregnancy outcome are shown in Table 1. Maternal serum levels of PAPP-A were significantly lower in SGA newborns than in controls and significantly higher in LGA newborns than in controls. Maternal serum free b-hCG levels were lower in pregnancies complicated by pre-eclampsia than in normotensive ones. FIGURE 2 Customized birth weight percentiles according to first-trimester maternal serum free b-hCG deciles. Data are shown as median and interquartile range. There is no significant correlation between birth weight and free b-hCG deciles (P¼ .27). The increase in birth weight with increasing PAPP-A levels was confirmed along the whole range of PAPP-A deciles (P<.001; Kruskal-Wallis test; Fig. 1) but not for free bhCG (Fig. 2; P¼.27). In pregnancies with the lowest decile of PAPP-A compared with pregnancies with the highest decile, the odds ratio (OR) for delivering an SGA newborn was 1.67 (95% confidence interval [CI] 1.33 to 2.09). In contrast, pregnancies with the highest decile of PAPP-A had an OR for delivering an LGA newborn of 1.65 (95% CI 1.31 to 2.07). However, the OR for pre-eclampsia in cases with the lowest decile of free b-hCG compared with those with the highest decile was not significant (OR 1.50, 95% CI 0.84 to 2.66). The results of multivariable analysis (Tables 2 and 3) found PAPP-A to be an independent predictor of absolute birth weight, SGA, and LGA. Free b-hCG was found to be an independent predictor of gestational hypertension and preeclampsia. Neither of the two markers was associated with preterm delivery. DISCUSSION The results of the present study suggest that maternal serum PAPP-A levels in the late first trimester of pregnancy are pos- FIGURE 1 Customized birth weight percentiles according to first-trimester maternal serum PAPP-A deciles. Data are shown as median and interquartile range. There is a significant increase in birth weight with increasing PAPP-A deciles (P< .001). Canini. Birth weight, PAPP-A, and free b-hCG. Fertil Steril 2008. itively associated with birth weight. This correlation is observed after correcting for maternal and fetal confounding factors. It was not possible to demonstrate a significant association between PAPP-A levels and the development of hypertensive complications of pregnancy or preterm delivery. The latter finding reflects similar negative results described in part of the literature (1, 6, 7) but might be more likely due to the low prevalence of these complications and small sample size of the present study population. Two much larger studies with adequate statistical powers were able to demonstrate a correlation between low PAPP-A levels, gestational hypertension, and preterm delivery (8, 9). Finally, free b-hCG levels TABLE 2 Results of multiple regression analysis for the prediction of birth weight. Variable Maternal age (yrs) Maternal weight (kg) Maternal height (cm) Smoker Nulliparity PAPP-A (MoM) Free b-hCG (MoM) Male fetus Gestation at delivery (wks) Standardized P coefficient value 0.024 0.131 0.123 0.062 0.100 0.134 0.008 0.158 0.571 .259 < .001* < .001* .003* < .001* < .001* .722 < .001* < .001* * P< .05. Canini. Birth weight, PAPP-A, and free b-hCG. Fertil Steril 2008. 176 Canini et al. Birth weight, PAPP-A, and free b-hCG Canini. Birth weight, PAPP-A, and free b-hCG. Fertil Steril 2008. Vol. 89, No. 1, January 2008 .008 .603 .017 .182 .469 .950 .300 .920 1.03–1.20 0.98–1.04 0.92–0.99 0.22–1.33 0.67–2.42 0.60–1.62 0.55–1.20 0.55–1.70 The association between first-trimester maternal serum PAPP-A levels and fetal growth can be explained, at least in part, by the role of placenta-derived PAPP-A as an IGF binding protein 4 (IGFBP-4) protease. The IGFBP-4 is a potent inhibitor of IGF action, and its cleavage by PAPP-A greatly increases the availability of IGF (16). The PAPP-A is produced in great amounts during pregnancy by the syncytiotrophoblast (17) and can be detected in placental tissue, decidua, maternal serum, and amniotic and coelomic fluids (18, 19). In the mouse, PAPP-A was also shown to be expressed by a number of tissues at all embryonic stages investigated. In the same animal model, individuals homozygous for targeted disruption of the PAPP-A gene were viable but their size at birth was 60% that of wild-type littermates (20). This growth-regulatory activity may explain the association between maternal serum PAPP-A levels and fetal growth observed in the present study. Although the continuum of the correlation of birth weight with PAPP-A was not examined before, two other series showed both a significantly increased adjusted odds ratio for SGA and a decreased adjusted odds ratio for LGA at low levels of PAPP-A (7, 8). 0.90–1.12 1.04–1.10 0.88–0.99 0.25–14.34 0.44–2.46 0.82–2.45 1.08–1.97 0.47–2.15 In summary, our results seem to suggest that the levels of PAPP-A are associated with subsequent fetal growth (including both physiologic variation and abnormal growth) while decreased free b-hCG is more predictive of hypertensive disorders of pregnancy, as also confirmed by two of the largest previous reports (8, 9). Although maternal serum levels of PAPP-A and free b-hCG do not appear to be a useful clinical predictor of pregnancy complications when used in isolation, their combination with other markers of placental function may improve their predictive efficiency (21). REFERENCES Canini. Birth weight, PAPP-A, and free b-hCG. Fertil Steril 2008. Note: CI ¼ confidence interval; OR ¼ odds ratio. 1.01 1.01 1.00 1.71 1.32 1.51 1.17 1.66 Maternal age (yrs) Maternal weight (kg) Maternal height (cm) Smoker Nulliparity PAPP-A (MoM) Free ßhCG (MoM) Male fetus 1.01 1.01 1.01 0.49 1.20 0.51 1.02 0.98 0.96–1.07 0.99–1.03 0.98–1.04 0.27–0.89 0.77–1.87 0.34–0.77 0.81–1.29 0.67–1.43 .691 .208 .713 .020 .411 .001 .845 .908 0.96–1.06 0.99–1.03 0.99–1.03 0.68–4.32 0.85–2.07 1.13–2.02 0.97–1.42 1.14–2.43 .766 .230 .864 .257 .219 .006 .107 .009 1.00 1.07 0.93 1.90 1.04 1.42 1.46 1.00 .993 .001 .017 .534 .928 .211 .015 .996 0.93 1.05 0.93 0.68 0.94 1.17 1.72 0.58 0.84–1.03 0.99–1.10 0.84–1.03 0.08–5.49 0.24–3.74 0.48–2.81 1.21–2.47 0.17–2.00 .426 .084 .142 .718 .932 .731 .003 .387 1.11 1.01 0.95 0.54 1.27 0.98 0.81 0.97 P 95% CI OR 95% CI OR OR 95% CI P 95% CI P OR P OR 95% CI P Preterm delivery Pre-eclampsia Gestational hypertension LGA SGA Results of a logistic regression model for the prediction of different obstetric outcomes. TABLE 3 Fertility and Sterilityâ were lower in pregnancies which subsequently developed gestational hypertension and pre-eclampsia, but the magnitude of this difference was quite small. Again, this latter finding might be explained by the small number of cases of pre-eclampsia in the present series and deserves further investigation in a larger study. 1. Morssink LP, Kornman LH, Hallahan TW, Kloosterman MD, Beekhuis JR, de Wolf BT, et al. Maternal serum levels of free beta-hCG and PAPP-A in the first trimester of pregnancy are not associated with subsequent fetal growth retardation or preterm delivery. Prenat Diagn 1998;18:147–52. 2. Ong CY, Liao AW, Spencer K, Munim S, Nicolaides KH. First trimester maternal serum free beta human chorionic gonadotrophin and pregnancy associated plasma protein A as predictors of pregnancy complications. BJOG 2000;107:1265–70. 3. Smith GC, Stenhouse EJ, Crossley JA, Aitken DA, Cameron AD, Connor JM. Early pregnancy levels of pregnancy-associated plasma protein A and the risk of intrauterine growth restriction, premature birth, preeclampsia, and stillbirth. J Clin Endocrinol Metab 2002;87:1762–7. 4. Tsai MS, Lee FK, Cheng CC, Hwa KY, Cheong ML, She BQ. Association between fetal nuchal translucency thickness in first trimester and subsequent gestational hypertension and preeclampsia. Prenat Diagn 2002;22:747–51. 5. Yaron Y, Heifetz S, Ochshorn Y, Lehavi O, Orr-Urtreger A. Decreased first trimester PAPP-A is a predictor of adverse pregnancy outcome. Prenat Diagn 2002;22:778–82. 177 6. Kwik M, Morris J. Association between first trimester maternal serum pregnancy associated plasma protein-A and adverse pregnancy outcome. Aust N Z J Obstet Gynaecol 2003;43:438–42. 7. Tul N, Pusenjak S, Osredkar J, Spencer K, Novak-Antolic Z. Predicting complications of pregnancy with first-trimester maternal serum free-betahCG, PAPP-A and inhibin-A. Prenat Diagn 2003;23:990–6. 8. Dugoff L, Hobbins JC, Malone FD, Porter TF, Luthy D, Comstock CH, et al. First-trimester maternal serum PAPP-A and free-beta subunit human chorionic gonadotropin concentrations and nuchal translucency are associated with obstetric complications: a population-based screening study (the FASTER trial). Am J Obstet Gynecol 2004;191:1446–51. 9. Krantz D, Goetzl L, Simpson JL, Thom E, Zachary J, Hallahan TW, et al. Association of extreme first-trimester free human chorionic gonadotropin-beta, pregnancy-associated plasma protein A, and nuchal translucency with intrauterine growth restriction and other adverse pregnancy outcomes. Am J Obstet Gynecol 2004;191:1452–8. 10. Smith GC, Stenhouse EJ, Crossley JA, Aitken DA, Cameron AD, Connor JM. Early-pregnancy origins of low birth weight. Nature 2002;417:916. 11. Pedersen JF, Sorensen S, Ruge S. Human placental lactogen and pregnancy-associated plasma protein A in first trimester and subsequent fetal growth. Acta Obstet Gynecol Scand 1995;74:505–8. 12. Smith GC, Crossley JA, Aitken DA, Pell JP, Cameron AD, Connor JM, et al. First-trimester placentation and the risk of antepartum stillbirth. Jama 2004;292:2249–54. 13. De Biasio P, Siccardi M, Volpe G, Famularo L, Santi F, Canini S. Firsttrimester screening for Down syndrome using nuchal translucency measurement with free beta-hCG and PAPP-A between 10 and 13 weeks of pregnancy—the combined test. Prenat Diagn 1999;19:360–3. 14. Gardosi J, Mongelli M, Wilcox M, Chang A. An adjustable fetal weight standard. Ultrasound Obstet Gynecol 1995;6:168–74. 178 Canini et al. Birth weight, PAPP-A, and free b-hCG 15. Clausson B, Gardosi J, Francis A, Cnattingius S. Perinatal outcome in SGA births defined by customised versus population-based birthweight standards. BJOG 2001;108:830–4. 16. Lawrence JB, Oxvig C, Overgaard MT, Sottrup-Jensen L, Gleich GJ, Hays LG, et al. The insulin-like growth factor (IGF)–dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancyassociated plasma protein-A. Proc Natl Acad Sci U S A 1999;96: 3149–53. 17. Guibourdenche J, Frendo JL, Pidoux G, Bertin G, Luton D, Muller F, et al. Expression of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro. Placenta 2003;24:532–9. 18. Grudzinskas JG, Obiekwe BC, Perry LA, Houghton DJ, Sinosich MJ, Bolton AE, et al. The relation of pregnancy associated plasma protein A (PAPP-A) in the umbilical circulation of the human fetus to oestriol production by the placenta. Asia Oceania J Obstet Gynaecol 1985;11: 425–8. 19. Iles RK, Wathen NC, Sharma KB, Campbell J, Grudzinskas JG, Chard T. Pregnancy-associated plasma protein A levels in maternal serum, extraembryonic coelomic and amniotic fluids in the first trimester. Placenta 1994;15:693–9. 20. Conover CA, Bale LK, Overgaard MT, Johnstone EW, Laursen UH, Fuchtbauer EM, et al. Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development. Development 2004;131:1187–94. 21. Prefumo F, Canini S, Casagrande V, Pastorino D, Venturini PL, De Biasio P. Correlation between first-trimester uterine artery Doppler indices and maternal serum free b–human chorionic gonadotropin and pregnancy-associated plasma protein A. Fertil Steril 2006;86: 977–80. Vol. 89, No. 1, January 2008