The advent of motion robust fetal DTI permitted in vivo characterization of brain microstructural development11. The construction of spatiotemporal atlases of fetal brain DTI have further enabled analysis of many developmental changes at early GAs despite the small size of the structures and the relatively low SNR of the individual acquisitions. In this study we utilized an atlas-based approach to evaluate tract-specific microstructural changes and their relationship to other important cytoarchitectonic processes. Our atlas-based diffusion tractography shows a series of tract-specific, age-related changes in FA and MD. While the changes in the later part of gestation (> 30 weeks) conform to expected post-natal trajectories depicting an increase in FA and a decrease in MD, these trends are unapparent or appear reversed in several major white matter tracts earlier in gestation (22–29 weeks). This divergence indicates that at earlier developmental stages different biologic processes drive diffusivity and anisotropy in the parenchyma and consequently along the traversing white matter tracts. In addition, our segment-based analysis of selected commissural, projection, and association tracts demonstrates considerable within-tract heterogeneity in maturation, with distinct local developmental trajectories.
Two anatomically overlapping yet biologically distinct and asynchronous processes take place in the brain in the second half of gestation: (a) the evolution of the transient zones of the fetal telencephalon and (b) the onset and progression of myelination. Early in the second trimester, the telencephalon is organized in concentric layers, most of which are visible on MRI. The outermost layer or cortical plate (CP) contains post-migratory neurons and shows high FA and low MD early in gestation; as the CP matures, dendritic arborization and synapse formation mediate an increase in intercellular space that leads to loss of FA and higher diffusivity22. Beneath the CP, there is a water rich and relatively acellular layer characterized by high MD and very low FA, known as the subplate (SP)8,23. The SP increases in thickness and volume during the second and early third trimester, with a reported peak at approximately 30 weeks of GA when it occupies up to 45% of the volume of the telencephalon 6. The intermediate zone (IZ) is the deep layer subjacent to the SP, which contains migrating neurons and organized axonal bundles that result in moderately low MD and high FA. Overtime, the progressive loss of the radial glial scaffolding and the dispersion of the migrating neurons results in a loss of FA and increase in MD8,23. After 30 weeks of GA, the subplate and intermediate zone undergo reorganization and become difficult to differentiate on MRI; this process has been described by Kostovic et al as the “resolution of the subplate”24, which ultimately results in consolidation of these two layers to form the “mature” fetal cerebral white matter 25. Independent of these changes, myelination begins at approximately 25 weeks of GA, with the earliest detectable histologic findings in the posterior limb of the internal capsules, posterior globus pallidus, and ventral lateral thalamus26,27. From there on, myelination progresses along white matter tracts in caudal-to-rostral, central-to-peripheral, and anterior-to-posterior direction28,29. It is well known that myelination results in increase in FA and decreased MD30.
The non-linear GA- and tract-specific trends observed in our tractography analysis reflect the varying influence of these processes on the diffusivity characteristics of the fetal brain. The distinct GA-related increase in FA and decrease in MD that is seen in all tracts after 30 weeks of GA is consistent with the anatomic progression of myelination11. Even though some pre-myelination and myelination changes are known to occur prior to this age, the consolidation of this trend appears to coincide chronologically with the “resolution stage” of the fetal subplate and intermediate zone as well as more widespread and robust myelination on histology3,27. At GA less than 30 weeks, the microstructural changes of the transient fetal zones play a dominant role. For most tracts (forceps minor, ILF, IFOF, UF), there was a significant decrease in FA and increase in MD with GA in this time period. These changes correspond with the layer-specific diffusion and volumetric changes described above (see paragraph above). Specifically, the age-related decrease in FA in the cortex and intermediate zone and the expansion of the subplate (with its low FA and high MD), contribute to these observations. Given that the long white matter bundles traverse these layers, the large effects of the layer-specific microstructural properties on whole-tract estimates is expected (Supplementary Fig. 1).
Tractography of the CST deserves special mention. In fetuses < 27 weeks, the CST tracking terminates within the intermediate zone, without appreciable fiber tracking along the subplate or extension to the primary motor cortex. Review of the raw data, with lax deterministic tractography parameters (no exclusion ROIs), shows crossing fibers headed toward the lateral parietal and frontal regions. This is probably secondary to fiber tracking along the radial glial scaffolding that predominates in the second trimester; the former, in conjunction with the inherent limitation of the diffusion tensor model to resolve crossing fibers, results in the incomplete delineation of the tract 8 (Supplementary Fig. 2). The missing segment of the tract corresponds to the SP and CP voxels; given the large relative volume of the SP in this developmental stage and the dominant diffusion features of this layer, we believe that this contributes to the lack of significant change in FA or MD before 30 weeks of GA.
We also observed substantial within tract heterogeneity in microstructural development of the CST, forceps major, and IFOF. The presence of distinct developmental trajectories for segments within a single white matter tract suggests that tract averaged MD and FA are insufficient to capture the complex developmental changes of fetal brain development. The advantage of more precise spatial analyses has been documented postnatally. Colby et al., showed that within-tracts analysis outlined microstructural differences in children with fetal alcohol spectrum disorders that were not apparent on the tract-averaged analyses31. Our data, albeit limited to selected white matter tracts, are similar. For instance, most of the peripheral /telencephalic components of tracts (CST: superior segment; IFOF: rostral and caudal segments; forceps major: peripheral segments), showed an inflection point or nadir in FA at 29–32 weeks of GA. These findings were not apparent on tract-averaged analysis of the CST or forceps major as they were probably averaged with other components of the tracts. The central components of the tracts (CST: mid segment; IFOF: mid segment; forceps major: central segment) showed different trends. In all of these, the FA showed a continued linear increase with age, an effect that is likely dominated by density of axonal packing and myelination, as these segments do not traverse the telencephalic layers. The forceps major does show a peak around 30 weeks, that is likely driven by the other effects of axonal organization. Finally, the caudal segment of the CST shows the fastest rate of increase in FA of all CST segments at < 30 weeks of GA, which is consistent with the early myelination of the brainstem which precedes supratentorial myelination; subsequently, the rate of change decreases as myelination of these segment of the CST is largely complete by the mid third trimester. The set of ROIs chosen for this analysis are based solely on anatomy; however, future work will be focused on developing robust tools for along tract analysis that incorporate growth and change in geometry of individual white matter tracts. Improvement in these tools will improve our ability to correlate these findings with patterns of gene expression, early functional specialization, and selective vulnerability to injury.
This study has several limitations. Our analysis is based on a single-tensor model of the diffusion signal. This approach is sufficient to characterize tissue coherence along major white matter tracts; however, it imposes limitations related to crossing fibers which, as our data show, can impact the ability to delineate the anatomy of fetal white matter tracts and impact the reliability of the microstructural analysis. Future work utilizing other models of diffusion signal processing, including diffusion compartment imaging, are expected to improve our ability to resolve these structures32. Additionally, our analysis of within tract changes are based on anatomic parcellations of the white matter; although these convincingly demonstrate the heterogeneity of the white matter maturation, they do not perform direct spatial correspondence or enable group wise comparisons. Development of novel methods that allow for such analyses, analogous to those existing for post-natal pipelines31, will significantly improve our ability to study brain maturation in utero.