IQR was calculated from MRI values for each conception group. The value was considered as an outlier if it fell 1.5 IQR above the upper quartile or 1.5 IQR below the lower quartile in the same measure, time and brain area. We dropped nine outliers from 6 groups (supplementary table1).
No significant difference among conception groups by comparing MRI values of six brain regions together
Before we applied statistical methods to check the significance of differences of MRI values from ICSI, ET and NAT conception groups, we did qqnorm to check the data normality. The qqnorm figures showed the data are off normal distribution (supplementary figure 1). And so, we decided to use nonparametric test Wilcox to find out if the differences in MRI values were significant. The p values of MRI values: ADC, T2WI, T1, T2, and FA at 6 time points for the comparisons of ICSI vs NAT and ET vs. NAT are summarized in Table 1 and Table 2. All the p values of ADC and T2 at the ages of fetuses 28-32 weeks, 32-37 weeks, and newborn infants are greater than 0.05 (Table1) and the corresponding values of ADC and T2WI were shown as mean±SD in supplementary table1-3. All the p values of T1, T2, and FA at the ages of infants 5-7 months, 11-13 months and 23-25 months are greater than 0.05 as well (Table 2) and the corresponding values of T1 and T2 were shown as mean±SD in supplementary table4-6. These results suggest that the differences between ISCI and NAT and the differences between ET and NAT are not significant.
No significant difference in the comparisons of MRI values from six brain regions separately except ICSI vs. NAT of fetus FWM in PLIC
The Wilcox tests for MRI values from all brain regions reveals that ISCI and ET are not significantly different from NAT. However, whether the MRI values would be different among conception groups in a particular brain region or at a certain age are still unknown.
Therefore we did the Wilcox test for MRI values from each brain region separately and the p values were shown in Table 4. Except that the comparison in PLIC of ICSI vs. NAT has p-value 0.09, all other 81 comparisons with p values >0.05 indicating that it is not significantly different among conception groups by comparing MRI values in different brain regions separately.
Fluctuation of MRI values with brain developmental stages
Comparisons of MRI values among the conception groups show that ICSI and ET are not significantly different from NAT in all combined brain regions or each investigated brain region separately. However, whether the MRI values changes at different time points are still not clear. Therefore, we made candle plots of MRI values at the 3-time points of fetus or infant for each brain region (Figure 2).
ADC values in the ALIC, CR, and OWM regions (red candles) were dispersed at 28-32 weeks of the fetus from any conception group (Figure 2). ADC values in the PWM region fluctuate largely with lowest ADC values at 33-37 weeks of the fetus. ADC values in the PLIC region show an increasing trend with increasing gestational ages. The probable reasons causing the dispersion of AD values described below. During the process of myelinization of normal WM, the contents of cholesterol and lipid gradually increase, and its hydroxyl and ketone are based on the set of water molecules. The development of neuronal cells and colloids leads to a decrease in the activity space of water molecules, and the ADC value should be reduced. However, the individual difference of fetal myelination is relatively large, and the stability of ADC value for moving fetal imaging measurement is difficult to be guaranteed. Therefore, the observations of the variation pattern of ADC values during the period of the fetus still need to be verified by a large number of live study data.
The results of T2 showed that ALIC, PLIC, and PWM increased first and then decreased with the increase of gestational ages, while FWM and OWM showed a gradual decline with the increase of gestational ages. Although the T2 values in CR region at 28-32 weeks had the largest dispersions which masked the trend along gestational ages, the average T2 values in CR region at 28-32 weeks had a similar pattern as in ALIC, PLIC, and PWM regions.
The results of routine brain scans on T1WI and T2WI showed that there was no significant difference among the 5-7, 11-13, and 23-25 months of infants among ET, ICSI, and NAT groups. T1 values in ALIC, PLIC, and FWM regions of infants decreased gradually with the increase of months. The data of T1 in the PWM region at 5-7 months in the ICSI group is relatively dispersed, however, the overall trend in all 3 infant ages declines. The T2 values of 5-7 months and 11-13 months infants were the same except for the region of OWM, and the T2 values of 23-25 months infants were significantly lower than those of 5-7 months and 11-13 months infants. OWM decreased gradually with the increase of infant ages. These results indicated that the myelin formation in the OWM region might be earlier than other parts.
In addition to 6 brain regions: ALIC, PLIC, CR, FWM, PWM and OWM, we collected values from FA map in another 11 regions including: genu of corpus callosum (GCC), splenium of corpus callosum (SCC), external capsule (EC), longitudinal fasciculus (SLF), Inferior longitudinal fasciculus (ILF), corticospinal tract (CST), optic radiations (OR), ftemporal white matter (TWM), caudate nucleus head (CNH), thalamus (THA), lenticular nucleus (LN).
In the 5-7, 11-13, and 23-25 months of infants born through ET, ICSI and NAT, the values in the 17 brain regions on the FA map indicate are not significantly different between infants with different conception methods at the same time point. The FA values in PLIC, GCC, and TWM regions all showed an increasing trend with the increase of ages. The FA values in SCC, SLF, ILF, CSF, and OR regions were mostly the same between 5-7 and 11-13months infants but slightly increased in 23-25 months. FA values in ALIC region at 5-7 months are higher than 11-13months and 23-25months, while the values of 11-13 and 23-25 months are almost equal. It is noteworthy that FA values in FWM region decreased with the increase of infants ages. Overall, the MRI values fluctuate at different time points indicating that they may change with the development of the brain. However, they are on a similar level for different conception groups supporting our previous statistical analysis that MRI values of ICSI and ET groups are not significantly different from NAT.