BackgroundMultisite MRI studies have become prevalent given their advantage in revealing reliable biological or clinical findings. Adult and adolescent multisite studies have demonstrated inevitable site-related non-biological effects that introduce bias. However, the site effect on fetal brain MRI remains unknown.PurposeTo identify crucial acquisition factors affecting fetal brain structural measurements and developmental patterns, while assessing the effectiveness of existing harmonization methods in mitigating site effects.Materials and MethodsBetween May 2017 and March 2022, T2-weighted fast spin-echo sequences in-utero MRI were performed in healthy fetuses from prospectively recruited pregnant volunteers on four different scanners at four sites. A generalized additive model (GAM) was used to quantitatively assess site effects, including field strength (FS), manufacture (M), in-plane resolution (R), and slice thickness (ST), on subcortical volume and cortical morphological measurements, including cortical thickness, curvature, and sulcal depth. Growth models were selected to elucidate developmental trajectories of these morphological measurements. Welch’s test was performed to evaluate the influence of site effects on developmental trajectories. ComBat-GAM harmonization method was applied to mitigate site-related biases.ResultsThe final analytic sample consisted of 340 MRI scans from 218 fetuses (mean gestational age, 30.1 weeks ± 4.4 [range, 21.7–40 weeks]). GAM results showed that low FS and low spatial resolution led to overestimations in selected brain regions of subcortical volumes and cortical morphological measurements, and cortical measurements were more susceptible to site effects than subcortical volumes. Only the peak cortical thickness in developmental trajectories was significantly influenced by the effects of FS and R. Notably, ComBat-GAM harmonization effectively removed site effects while preserving developmental patterns.ConclusionOur findings pinpointed the key acquisition factors in in-utero fetal brain MRI and underscored the necessity of data harmonization when pooling multisite data for fetal brain morphology investigations.