Sigmaplot 8

Mouse grimace scale (MGS) and BF were performed as previously described (Wang et al., 2017a (link), 2018 (link); Guo et al., 2019b (link)). All behavioral assays and video analysis were performed in a blinded manner. For MGS assay, the mice were videotaped for 30 min in each experimental time point and 10 images per 30 min session were manually captured. The scores of the five action units in each photograph were averaged, and a mean MGS score was obtained from the 10 images, which was presumed to reflect the level of spontaneous pain. For BF assay, mice were placed in a modified 60-ml plastic syringe with a wide opening at one end to accommodate the head of the mouse. To minimize stress, the mouse was released immediately from the syringe if it vigorously moved or tried to hide inside the syringe. The syringe containing the mouse was held manually and moved slowly at 0.5–1 cm/s toward bite plates so that the mouse could bite the plates. Spike 2 software was used to measure the voltage changes from transducer displacement. SigmaPlot 8.0 was used to convert the voltage change into force based on calibration using standard weights. Bite force was recorded for 120 s per session and the top five force measurements were averaged.

All data were processed and analyzed by Sigma-Plot 8.0 software. The normality test was done before t-test. The statistical significance was evaluated by t-test and p<0.05 was considered significant.

Fetal DNA fractions were compared between the three fetal aneuploidy risk groups (HR, IR, LR) and assessed for correlation with the FTS test parameters; gestational age, crown-rump length (CRL), free β-subunit of human chorionic gonadotropin (free β-hCG), nuchal translucency (NT), pregnancy-associated plasma protein A (PAPP-A) as well as maternal weight using SigmaStat (version 3.5) software and visualized by SigmaPlot 8.0 and MedCalc (version 11.4.4.0). The measured free β-hCG, NT and PAPP-A were converted into multiples of the median (MoM) and log₁₀ transformed to obtain a Gaussian distribution. Normality was assessed by the Kolmogorov-Smirnov Z test. We examined differences among groups using analysis of variance test (ANOVA) or Mann-Whitney Rank Sum Test as appropriate, with P value <0.05 to indicate statistical significance. Regression analysis (Pearson's Product-Moment Correlation) was used to assess the relationship between fetal DNA fraction and maternal weight as well as FTS parameters (gestational age, CRL, log₁₀ free β-hCG MoM, log₁₀ NT MoM, log₁₀ PAPP-A MoM).

For the statistical analysis, Student's t tests (comparing two groups) or one‐way analyses of variance (ANOVAs) with Tukey post hoc tests were performed using GraphPad Prism software 8 and Sigma Plot 8.0. Differences were considered significant at *p < .05, **p < .01, and ***p < .001. All experiments were replicated six times and are shown as the mean ± SEM.

In the different kinetics studies, the parameters were obtained by using the Sigma Plot 8.0 software that applies an iterative procedure, based on the Marquardt-Levenberg algorithm, which seeks the values of the parameters that minimize the sum of the squared differences between the observed and predicted values of the dependent variable.

Data were analyzed using the unpaired Student t-test tool of SigmaPlot 8.0, with P < 0.05 considered as a significant difference. The number of independent experiments (n) is indicated in each table or figure. Otherwise, figures show representative data of at least three experiments. Each independent experiment was the mean of at least three technical replicates. Several protein purifications were used during this work with low variations between purification batches (<10% variation in specific activity).