Diagnostic value of maternal alpha-fetoprotein variants in second-trimester biochemical screening for trisomy 21 and 18

To evaluate the clinical predictive value of serum alpha-fetoprotein variants (AFP-L2, AFP-L3) in combination with maternal serum prenatal screening biomarkers in predicting fetal trisomy 21 and trisomy 18. We analyze the data of singleton pregnant women at 15–20+6 weeks of 731,922 gravidas from October 2007 to September 2019. The research objects were separated into the following groups: control (n = 569), trisomy 21 (n = 116), and trisomy 18 (n = 52). The cases were diagnosed by chromosomal karyotypic analysis of amniotic fluid cells. Level of AFP-L2 and AFP-L3 were detected in maternal serum among control women and patients. Receiver operator characteristic analysis, detection rate, false positive rate, false negative rate, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio, comprehensive discriminant improvement, net weight classification improvement, decision curve analysis and Hosmer–lemeshow (H-L) test were used to investigate the predictive value of free β-hCG, AFP, AFP-L2 and AFP-L3 on the risk models of trisomy 21, 18. There was a statistically significant difference in maternal serum AFP-L2 and AFP-L3 multiple of the median (MoM) among the trisomy 21, trisomy 18, and control groups. The AUCs of AFP-L2 and AFP-L3 for the screening trisomy 21 and trisomy 18 fetus were 0.785, 0.758 and 0.775, 0.754. According to ROC, the optimal cut-off values of AFP-L2 and AFP-L3 for predicting trisomy 21 and trisomy 18 fetuses all were 1.09 MoM and 1.30 MoM, respectively. The risk-calculation model constructed by AFP-L2 + AFP-L3 MoM manifested better efficiency than the original single-value truncation method using AFP MoM alone. Compared with different modeling methods, the AUC of trisomy 21 fetuses predicted by AFP-L2 + AFP-L3 + free β-hCG achieved an optimal value (0.938), while the AUC of trisomy 18 fetus predicted by AFP-L2 + free β-hCG was the best (0.991). Compared with AFP, the IDI of AFP-L2 or AFP-L3 alone increased 9.56% and 12.34%; the NRI increased 26.50% and 26.70 in predicting trisomy 21. For trisomy 18, the IDI of AFP-L2 or AFP-L3 alone declined with 8.12% and 1.52%; the NRI declined with 13.84% and 8.54%. In the combined model, the model with best detection rate, false positive rate and positive likelihood ratio was AFP-L2 + AFP-L3 + free β-hCG, followed by AFP-L2 + free β-hCG and AFP-L3 + free β-hCG, and finally AFP + free β-hCG. Maternal serum AFP-L2 and AFP-L3 in the second trimester is a good marker for screening trisomy 21 and trisomy18 with high sensitivity and specificity. The combined screening results are better than the single marker, and the efficiency of AFP-L2 + AFP-L3 + free β-hCG is the best.

www.nature.com/scientificreports/ informed consent was waived by Hangzhou Women's Hospital (Hangzhou Maternity and Child Health Care Hospital) ethics committee due a retrospective nature of the study. These cases were diagnosed according to the criteria established by Chinese Birth Defect Monitoring Network and International Classification of Diseases (ICD-10) 21,22 . The exclusion criteria were: (1) women undergoing multiple pregnancy, (2) patients with severe medical conditions and other pregnancy complications, (3) habitual smokers, (4) infants conceived from assisted reproductive technology, (5) patients in whom follow-up results showed NTDs or other serious birth defects, (6) patients with incomplete individual information, and (7) patients whose pregnancy information did not match its cognate serum sample.
Second-trimester screening. 2-3 mL fasting venous blood of pregnant women was collected for the detection of prenatal screening markers. The blood samples were centrifuged at 2500 r/min for 5 min approximately 30 min after collection, and the remaining specimens were stored in a -80 °C refrigerator within 1 week of the initial examination. Before measurement, serum samples and corresponding data were matched with the case group and the control group. The DELFIA method was used to measure the levels of pregnancy-associated plasma protein A (PAPP-A) and free β-hCG levels in maternal serum. We then measured the levels of AFP-L2 and AFP-L3 by ELISA 20,23 . Calculation of multiples of the median (MoMs) and establishment of risk models. Maternal aneuploidy screening markers varied with maternal weight and gestational age (GA) in unaffected pregnancies. AFP, free β-hCG, AFP-L2, and AFP-L3 concentrations were represented as MoMs to reduce the deviation caused by GA and maternal weight 20,24-26 .
The formula for maternal-age risk values was as follows. Age represented the maternal age and risk age was risk value for maternal age 24,26 : Likelihood ratio calculation 25,27 : The models below were constructed using the above steps. The single-index models comprised: Model A: free β-hCG; Model B: AFP; Model C: AFP-L2; Model D: AFP-L3; The dual-index models comprised: Model E: AFP + free β-hCG; Model F: AFP-L2 + free β-hCG; Model G: AFP-L3 + free β-hCG; Model H: The triple-index model comprised:

Results
Comparison of basic data. Although we observed no significant difference in pregnancy weight between the case and control groups, maternal age in the control group was significantly lower than that in the trisomy 21 group (P = 0.004). Maternal age in the trisomy 18 group showed a tendency to be lower than that in the control group, but there is no statistically significant difference (P = 0.810, Table 1).
Comparison of serum AFP-L2, AFP-L3, free β-hCG, and AFP levels among the case and control groups. The AFP-L2 levels of gravidas with fetal trisomy 21 and trisomy 18 were 1.48 (0.56-3.27) and 1.46 (0.55-5.75) MoMs, which were respectively higher than the 0.83 (0.24-2.62) 20 in the control subjects (all P < 0.001). The levels of AFP-L3 in gravidas carrying trisomy 21 and trisomy 18 fetuses were 1.73 (0.05-6.84) and 1.66 (0.60-5.96) MoMs, respectively, which were also higher than the 0.85 (0.24-2.48) MoM 20 in the control subjects (all P < 0.001). However, the AFP levels of pregnant women with trisomy 21 and trisomy 18 fetuses were lower than in the control subjects (all P < 0.001). The free β-hCG of the trisomy 21 group was elevated relative to the control subjects, and that of the trisomy 18 group was inferior to that of the control subjects (all P < 0.001, Table 2 and Fig. 1).
The screening value of individual-and combined-index for trisomy 21, 18. The AUCs for AFP-L2 in the screening of trisomy 21, 18 fetuses were higher than those for AFP in trisomy 21, and lower than those for trisomy 18 (0.785 and 0.758 vs, 0.613; 0.775 and 0.754 vs, 0.869, respectively). In addition, the optimal threshold values for AFP-L2 and AFP-L3 in predicting T21 and T18 in fetuses all were 1.09 and 1.30 MoMs, respectively. The AUCs for free β-hCG in trisomy 21 and 18 fetuses were 0.793 and 0.986, respectively, higher than for AFP-L2, AFP-L3, or AFP (as shown in Table 3). The risk-calculation model constructed with AFP-L2 + AFP-L3 MoM exhibited better screening efficiency than the original single-value truncation method using AFP MoM. Compared with different modeling methods, the AUC for trisomy 21 fetuses as predicted by Model I was the highest (AUC = 0.938, Fig. 2a), while for trisomy 18, Model F achieved the optimal AUC (AUC = 0.991, Fig. 2b).
The evaluation of risk models. The AUCs of the single-index model for predicting trisomy 21 in decreasing order were AFP-L2 > AFP-L3 > AFP, and the AUCs of the single-index model for predicting trisomy 18 were AFP > AFP-L2 > AFP-L3. In the multi-index combination model, the ranking of DR, false-positive rate (FPR), and +LR evaluation indices were Model I > Model F > Model G > Model E, Table 3. www.nature.com/scientificreports/ The replacement of AFP with AFP-L2 or AFP-L3 increased the IDI by 9.56% and 12.34%, respectively, and with NRI by 26.50% and 26.70, respectively, in predicting trisomy 21.
The replacement of free β-hCG by AFP-L2 or AFP-L3 combined with free β-hCG increased the IDI by 7.86% and 12.15%, respectively; and NRI by 15.61% and 17.21%, respectively. The IDI and NRI for T21 as predicted by Model I instead of Model H were 5.22% and 13.53%, respectively ( Table 4).
The replacement of AFP by AFP-L2 or AFP-L3 decreased the IDI by -8.12% and -1.52%, respectively, and NRI by − 13.84% and − 8.54%, respectively, in predicting trisomy 18. The replacement of free β-hCG by AFP-L2 or AFP-L3 combined with free β-hCG increased the IDI by 17.05% and 2.10%, respectively; and NRI by 9.81% and − 1.57%, respectively. The IDI and NRI for T21 as predicted by Model I instead of Model H were 10.44% and 33.84%, respectively.
When the risk threshold was < 0.40, the decision curve analyses for predicting T21 using different models were ranked Model H > Model F > Model E > Model D > Model C > Model B > Model A > Model G > Model I (Fig. 3a). There were different models that comprised DCA predictive ability for trisomy 18, and the top models were Model B, Model E, Model I, and Model D (Fig. 3b).

Discussion
Trisomy 21 and trisomy 18 are the most common chromosomal abnormalities observed in neonatal birth defects. After birth, children with these anomalies cannot provide for themselves, and this engenders serious economic and social demands from families and society. Therefore, early prenatal diagnosis and intervention with certain measures are particularly important. The use of highly sensitive and specific markers for prenatal screening   30 . In the present study, we evaluated the effect of combining free β-hCG after replacing AFP with AFP-L2 or AFP-L3 on predicting trisomies 21 and 18. The research showed that the AFP-L2 levels of gravidas screening for trisomy 21 and 18 fetuses in second trimester were higher than in the control subjects (1.48 and 1.46 vs. 0.83 MoM, respectively) (P < 0.001). Yu et al. 17 also showed that the average level of serum AFP-L2 with trisomy 21 fetuses was higher than that of normal subjects. The consequences of our research also revealed that the AFP-L3 levels of gravidas carrying trisomy 21, 18 fetuses were augmented relative to those in the control subjects (1.73 MoM, 1.66 MoM vs. 0.85 MoM, respectively) (all P < 0.001). Similarly, Feng et al. 31 demonstrated that the ratio of serum AFP-L3 to AFP in gravidas with a DS fetus was higher than that in pregnant women carrying a healthy fetus. However, Huai et al. 32 showed that the level of serum AFP-L3 MoM in the normal control subjects was significantly higher than that in gravidas with DS fetuses (P < 0.05); this diverged from the results of our study, and it may be related to the different detection methods used to determine AFP-L3. However, the preliminary results of the present study revealed that maternal serum AFP-L2 and AFP-L3 levels of the control subjects were significantly lower than those of DS fetuses (all P < 0.01) 19 . Similarly, Wu et al. 33 demonstrated that the concentrations of AFP-L2 Table 4. NRI and IDI for assessing improvement in model performance after adding AFP-L2, AFP-L3 to predicting Trisomy 21,18. AFP, alpha-fetoprotein; free β-hCG, free beta subunit of human chorionic gonadotropin; AFP-L2, α-fetoprotein heterogeneity L2; AFP-L3, α-fetoprotein heterogeneity L3; IDI, Integrated Discrimination Improvement; NRI, Net Reclassification Improvement. *P < 0.001, **P < 0.05.

New method
Old method IDI (%) P value for IDI NRI (%) P value for NRI www.nature.com/scientificreports/ and AFP-L3 in women with healthy fetuses were lower than that of DS fetuses, which was resemble to the data from the current study.
In the current research, we determined the levels of AFP-L2 and AFP-L3 by ELISA, and we showed that the AUC screening for trisomy 21, 18 fetuses using AFP-L2 was higher than that with AFP for trisomy 21, and lower than that for trisomy 18 (0.785 and 0.775 vs. 0.613 and 0.869, respectively). The AUCs screening for trisomy 21, 18 fetuses with AFP-L3 were 0.758 and 0.754, respectively, which were higher than AUCs using AFP (0.613 and 0.869). These results showed that the diagnostic values of AFP-L2 and AFP-L3 for trisomy 21, 18 were better than that of AFP. After denaturation of AFP, it can be easily refolded into both forms of recombinantly produced AFP under the conditions of dilution and redox reaction, and it is detectable by ELISA 34 .
Compared with the study by Long, our AUC for AFP-L3 screening of trisomy 21 in our study was higher than the ratio of AFP-L3 to AFP (0.758 vs. 0.710) 32 , but lower than the concentration of AFP in gravidas as measured by liquid-phase combination assay. Yamamoto found that the AUCs of AFP MoM (0.750), AFP-L3% (0.868), L3 MoM (0.949), and L3 MoM/AFP MoM (0.946), respectively, as determined by liquid-phase binding assay 35 . This revealed that different detection methods may produce different diagnostic values. However, the AUC for trisomy 21 as predicted by AFP-L2 and AFP-L3 in the present study showed a tendency to be lower than that predicted in our preliminary study (0.891, 0.824) 19 . The apparent discrepancy between the former and latter may have been due to our subsequent expansion of the number of control cases, or it may have related to our modeling after calibrating for gestational age and weight in the current study.
The AFP levels in the control group were higher than gravidas with trisomy 21, 18 fetuses (all P < 0.001), while the levels of AFP-L2 and AFP-L3 were higher than those in the control subjects (all P < 0.001). The maternal serum AFP-L2 and AFP-L3 levels with trisomy 21, 18 fetuses were also higher than in the control subjects, which was opposite to the diminution in AFP levels. At present, the mechanism (s) underlying this discrepant phenomenon is unclear.
Yamamoto et al. posited that the placental transfer of the AFP-L3 component in women carrying a fetus with trisomy 21 may be relatively high, which might be one of the causes for the elevated serum AFP-L3 levels in these pregnant women 36 . In addition, Yamamoto 37 did not detect a correlation between maternal serum AFP-L3 and AFP MoMs (r = 0.006) and did not observe a significant correlation between serum AFP level and AFP-L3 percentage. No significant correlation was observed between serum AFP level and AFP-l3 percentage (r = 0.160) by Khien et al. 38 .
In our last study, the MoM values of AFP-L2 and AFP-L3 were calculated by indirect simulation based on the AFP MoM values of 21,656 middle-pregnancy maternal serum samples in our laboratory 24 . So we used the 569 maternal serum samples in this study to build the risk model construction of AFP-L2 and AFP-L3. These results all disproved any correlation between the values of AFP-L2/AFP-L3 and AFP. In support of this, our data also suggested that the combination of AFP-L2 and AFP-L3 was better than that for AFP, AFP-L3, and AFP-L2 as single indicators in predicting fetal trisomies 21, 18. www.nature.com/scientificreports/ For trisomy 21, the results revealed that the substitution of AFP-L2 for AFP improved the IDI and NRI by 9.56% and 26.50%, respectively, that AFP-L3 substitution for AFP augmented the IDI and NRI by 12.34% and 26.70%, respectively. For trisomy 18, AFP-L2 replacement of AFP decreased the IDI and NRI by 8.12% and 13.84%, and that AFP-L3 replacement of AFP lowered the IDI and NRI by 1.52% and 8.54% in Table 4, respectively.
NRI is an approach that involves classifying patients into risk categories and determining how does the new model reclassify patients into risk categories compared to previous models 39 . The IDI calculation is another way of assessing reclassification that does not rely on a pre-specified risk category but represents a continuous measure 40,41 . The addition of AFP-L2 and AFP-L3 to the traditional model improved the ability to predict T21 and T18 fetuses assessed by ROC and to reclassify subjects into different risk categories by NRI and IDI. It shows that AFP-L2 and AFP-L3 can effectively increase the accuracy of previous studies.
The data in Table 4 also suggested that the utility of AFP-L3 and AFP-L2 alone in predicting trisomy 21 was better than that with AFP, while for trisomy 18, the ranked sequence was AFP-L3 > AFP > AFP-L2. If combined with DR, FPR, and +LR evaluation indicators, Model I was optimal, followed by Model F and Model E.
When both false-positive and false-negative results are inevitable, it is incumbent upon us to find a way to maximize net benefit, which is a clinical utility problem. Therefore, we introduced DCA to evaluate the predictive effects of different models for trisomy 21 and 18. It is generally postulated that the threshold probability range of the abscissa in risk threshold is 0-1. However, if a specific situation is consistent in clinical practice and the threshold probability reaches a certain value (e. g., 40%), intervention measures must be undertaken 42 and, therefore, a risk threshold > 0.4 is of little significance. Figure 3 shows depicts the ranked order as Model A > Model G > Model I. The top models are Model B, Model E, Model I, and Model D using the DCA prediction for trisomy 18. We note that these results were different from previous AUC, IDI, and NRI evaluation indices, which might be due to the small number of cases of trisomy 18 in the current study 18,19 .

Conclusion
In summary, the prediction using AFP-L2 and AFP-L3 for trisomy 21 and trisomy 18 fetuses in maternal serum exhibited high sensitivity and specificity, thus providing a favorable marker for screening these trisomies. We demonstrated that the combined screening results were better than using single markers, and that the combined predictive efficiency by Model I achieved optimal results.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).