Mapping the effects of pregnancy on resting state brain activity, white matter microstructure, neural metabolite concentrations and grey matter architecture

While animal studies have demonstrated a unique reproduction-related neuroplasticity, little is known on the effects of pregnancy on the human brain. Here we investigated whether pregnancy is associated with changes to resting state brain activity, white matter microstructure, neural metabolite concentrations and grey matter architecture using a comprehensive pre-conception cohort study. We show that pregnancy leads to selective and robust changes in neural architecture and neural network organization, which are most pronounced in the Default Mode Network. These neural changes correlated with pregnancy hormones, primarily third-trimester estradiol, while no associations were found with other factors such as osmotic effects, stress and sleep. Furthermore, the changes related to measures of maternal-fetal bonding, nesting behavior and the physiological responsiveness to infant cues, and predicted measures of mother-infant bonding and bonding impairments. These findings suggest there are selective pregnancy-related modifications in brain structure and function that may facilitate peripartum maternal processes of key relevance to the mother-infant dyad.


Supplementary Material
Supplementary Note. Results of the main model comparing grey matter volume changes between the Pre and Post sessions in the women who were pregnant between sessions in comparison to the nulliparous control group. Statistics are extracted from two-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. PRG = nulliparous women who were pregnant between sessions, CTR = nulliparous women who were not pregnant between sessions, L = left, R = right. Note. Following up on the significant group differences obtained in the PRG>CTR contrast reported in Supplementary Table 1, this table reports the results for contrasts representing the increases in the CTR group and decreases in the PRG group between the pre-conception and post-pregnancy sessions, allowing us to examine whether the results observed in the CTR>PRG contrast reflect grey matter volume increases in the CTR group or grey matter volume decreases in the PRG group. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. PRG = nulliparous women who were pregnant between sessions, CTR = nulliparous women who were not pregnant between sessions, L = left, R = right.
Supplementary Figure 1. Effect sizes (Cohen's d) for the PRE to POST changes in GM volume. Effect sizes are presented for the changes between sessions in the women who were pregnant between sessions in comparison to the control women. All depicted effect sizes correspond to large effect sizes (Cohen's d>0.8). Effect sizes were extracted using the VBM8 toolbox (http://www.neuro.uni-jena.de/vbm/) and plotted in mricron (https://www.nitrc.org/projects/mricron). N pregnant group = 40, N control group = 40.
Supplementary Note. Main model comparing grey matter volume changes between the Pre and Post sessions in the PRG and CTR groups, excluding the 3 women who initially did not participate in the Post session and therefore had a delayed time interval (>800 days) between the sessions (leaving a PRG sample size of N = 37 Statistics are extracted from two-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. PRG = nulliparous women who were pregnant between sessions, CTR = nulliparous women who were not pregnant between sessions, L = left, R = right.
Supplementary Note. Main model comparing grey matter volume changes between the Pre and Post sessions in the PRG and CTR groups, excluding the women who underwent fertility treatment or delivered twins (leaving a PRG sample size of N = 36). Statistics are extracted from two-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. PRG = nulliparous women who were pregnant between sessions, CTR = nulliparous women who were not pregnant between sessions, L = left, R = right.
Supplementary Note. Total grey matter, white matter and total brain volume of the two groups at the Pre and Post sessions. Between-group differences were analyzed using two-sided two-sample t-tests. Repeated Measures General Linear Models comparing the change in total tissue volumes across sessions between the groups were also performed, which revealed significant group*session interaction effects for the changes in grey matter (F=24.39, p<0.001) and total brain volume (F=22.67, p<0.001) but not for white matter (F=0.221, p=0.640). Pre = pre-pregnancy session; Post = post-pregnancy session; L = liter; GM = grey matter; WM = white matter; TBV = total brain volume. Note. Values of total grey matter, white matter and total brain volume for the late postpartum sessions in the women who became pregnant during this study are provided in this table. L = liter; GM = grey matter; WM = white matter; TBV = total brain volume. Note. Main model comparing grey matter volume changes between the Pre and Post sessions in the PRG and CTR groups corrected for changes in total brain volume. Statistics are extracted from two-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. PRG = nulliparous women who were pregnant between sessions, CTR = nulliparous women who were not pregnant between sessions, L = left, R = right. and the brain's cognitive networks ). The overlap of our results with these functional networks was extracted by computing the intersection between each of these maps and the GM volume changes of pregnancy (column 'Observed overlap'). The percentages of each of the maps represented by the overlap were subsequently determined and reported in percentage of the functional map (column 'Observed overlap (%Map1))' and in percentage of the map of GM volume changes of pregnancy (column 'Observed overlap (%ẟPRG))'. The expected overlap based on a random distribution across the brain was determined by multiplying the percentages of the brain's total GM represented by each of the 2 maps. The percentage of expected overlap was then multiplied by total GM (column 'Expected overlap'), and the expected overlap was divided by the observed overlap (column 'Observed>Expected overlap'). Comp= component, ẟPRG = changes in GM volume across pregnancy. Other colors represent the visual network (purple), the somatosensory network (blue), the dorsal attention network (green), the ventral attention network (violet), the limbic network (cream) and the frontoparietal network (orange). Note. The overlap of the changes in GM volume across pregnancy were quantified with the networks of intrinsic functional connectivity defined by Yeo et al. 2 . The overlap of our results with these functional networks was extracted by computing the intersection between each of these maps and the GM volume changes of pregnancy (column 'Observed overlap'). The percentages of each of the maps represented by the overlap were subsequently determined and reported in percentage of the functional map (column 'Observed overlap (%Map1))' and in percentage of the map of GM volume changes of pregnancy (column 'Observed overlap (%δPRG))'. The expected overlap based on a random distribution across the brain was determined by multiplying the percentages of the brain's total GM represented by each of the 2 maps. The percentage of expected overlap was then multiplied by total GM (column 'Expected overlap'), and the expected overlap was divided by the observed overlap (column 'Observed>Expected overlap' Note. The overlap of the changes in GM volume across pregnancy were quantified with the networks of intrinsic functional connectivity defined by Smith et al. 3 . The overlap of our results with these functional networks was extracted by computing the intersection between each of these maps and the GM volume changes of pregnancy (column 'Observed overlap'). The percentages of each of the maps represented by the overlap were subsequently determined and reported in percentage of the functional map (column 'Observed overlap (%Map1))' and in percentage of the map of GM volume changes of pregnancy (column 'Observed overlap (%δPRG))'. The expected overlap based on a random distribution across the brain was determined by multiplying the percentages of the brain's total GM represented by each of the 2 maps. The percentage of expected overlap was then multiplied by total GM (column 'Expected overlap'), and the expected overlap was divided by the observed overlap (column 'Observed>Expected overlap' Note. Changes across pregnancy in within-network connectivity in each of the networks. The other neural networks rendered no significant results. Results are extracted from one-sided t-tests performed in SPM12 and reported at a threshold of P<0.05 FWE-corrected (P value peak voxel). * also present in group*session interaction effect.

Supplementary
Supplementary Note. Results of one-sided two-sample t-tests performed within the framework of the SPM12 General Linear Model testing for baseline differences in DMN connectivity. A region of interest (ROI) analysis involving the region of change observed in the main analysis indicated that there is no overlap between the observed cluster and the region undergoing changes across pregnancy. Note. Comparisons between the metabolite concentrations in the Pre session between women who were pregnant between scans (PRG) in comparison to women who were not (CTR) in the Posterior Cingulate Cortex. In case of any deviations from normality, non-parametric Mann-Whitney U tests were performed. The U values and p-values of these tests are reported between brackets in the table. None of these effects survive a correction for multiple comparisons. tNAA = N-acetylaspartate (including contributions from N-acetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol.

Supplementary
Supplementary Note. Changes in metabolite concentrations between Pre and Post sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR) in the Posterior Cingulate Cortex. In case of any deviations from normality in one of the groups or sessions, non-parametric Mann-Whitney U tests were performed on the Post-Pre difference values. The U values ("U") and p-values ("p U") of these tests are reported between brackets in the table. None of these effects survive a correction for multiple comparisons. tNAA = Nacetylaspartate (including contributions from N-acetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol. Note. Partial eta squared values for the changes in metabolite concentrations between Pre and Post sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR) in the Posterior Cingulate Cortex Volume of Interest. tNAA = N-acetylaspartate (including contributions from Nacetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol.

Supplementary
Supplementary Note. Changes in metabolite concentrations between Pre-and Post-pregnancy sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR), excluding the women who initially did not participate in the Post session and therefore had a delayed time interval (>800 days) between the sessions (leaving a PRG sample size of N = 36). Session (pre-post pregnancy) * group (PRG, CTR) interaction effects are reported. In case of any deviations from normality in one of the groups or sessions, non-parametric Mann-Whitney U tests were performed on the Post-Pre difference values. The U values ("U") and p-values ("p U") of these tests are reported between brackets in the table. None of these effects survive a correction for multiple comparisons. tNAA = N-acetylaspartate (including contributions from N-acetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol.
Supplementary Note. Changes in metabolite concentrations between Pre and Post sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR), excluding the woman who had participated in this study both as a nulliparous control and subsequently also as a pregnant women (leaving a PRG sample size of N = 38). Session (pre-post pregnancy) * group (PRG, CTR) interaction effects are reported. In case of any deviations from normality in one of the groups or sessions, non-parametric Mann-Whitney U tests were performed on the Post-Pre difference values. The U values ("U") and p-values ("p U") of these tests are reported between brackets in the table. None of these effects survive a correction for multiple comparisons. tNAA = N-acetylaspartate (including contributions from N-acetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol. Note. Changes in metabolite concentrations between Pre-and Post-pregnancy sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR), excluding the women who underwent fertility treatment or who had twins (leaving a PRG sample size of N = 35). Session (pre-post pregnancy) * group (PRG, CTR) interaction effects are reported. In case of any deviations from normality in one of the groups or sessions, non-parametric Mann-Whitney U tests were performed on the Post-Pre difference values. The U values ("U") and p-values ("p U") of these tests are reported between brackets in the table. None of these effects survive a correction for multiple comparisons. tNAA = N-acetylaspartate (including contributions from Nacetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate , Ins = myo-Inositol.

Supplementary
Supplementary Note. Changes in metabolite concentrations between Pre-and Post-pregnancy sessions in women who were pregnant between scans (PRG) in comparison to women who were not (CTR), while correcting for a previous history of medical or psychiatric disorders. Session (pre-post pregnancy) * group (PRG, CTR) interaction effects are reported. None of these effects survive a correction for multiple comparisons. tNAA = N-acetylaspartate (including contributions from N-acetylaspartylglutamate), Cho = Choline (phosphorylcholine and glycerophosphorylcholine), tCr = Creatine (creatine and phosphocreatine), Glu = Glutamate, Ins = myo-Inositol.
Supplementary Note. Correlation results between the observed changes across pregnancy. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table. The correlations with grey matter were performed using multivariate regression analyses using Kernel Ridge Regression. GM=observed changes in grey matter volume, rsfMRI=observed changes in temporal coherence of the Default Mode Network, tCr=observed changes in creatine concentrations, Cho=observed changes in choline concentrations, Ins=observed changes in myo-inositol. Note. Grey matter volume changes between the late postpartum session and the pre-conception baseline in the PRG participants. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). P-value at peak voxel (whole-brain FWE corrected) is reported extracted from one-sample t-tests perform. L = left, R = right.

Supplementary
Supplementary Figure 3. Plots of volume changes from pre-conception to late postpartum of the PRG and CTR sample. Mean (± SEM) grey matter volume changes at each Post session (Post and Post+1y) relative to the Prepregnancy baseline of the most significant clusters (i.e., T > 8), extracted from the smoothed normalized Jacobian difference images for each cluster. Red line represents control group (no Post+1y data is available of the control group). Blue bar represents pregnancy. GM = grey matter, L = left, R = right, PP = Postpartum.
Supplementary Note. Changes in grey matter volume across the postpartum period in primiparous mothers. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right.
Supplementary  Note. Correlation results between the Nesting Questionnaire and the observed changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all prenatal tests (correlation-adjusted Bonferroni correction for the number of performed tests with prenatal measures for this modality). Note. Correlation results between the Prenatal Attachment Inventory (PAI) and Maternal Antenatal Attachment Scale (MAAS) and the observed changes in default mode network coherence. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all prenatal tests (correlation-adjusted Bonferroni correction for the number of performed tests with prenatal measures for this modality). Note. Correlation results between the Prenatal Attachment Inventory (PAI) and Maternal Antenatal Attachment Scale (MAAS) and the changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all prenatal tests (correlation-adjusted Bonferroni correction for the number of performed tests with prenatal measures for this modality). Note. Correlation results between the physiological responses (interval between heart rates and skin conductance response (SCR)) to movies of laughing and crying babies and the observed changes in default mode network coherence. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the Note. Correlation results between the physiological responses (interval between heart rates and skin conductance response (SCR)) to movies of laughing and crying babies and the changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all prenatal tests (correlation-adjusted Bonferroni correction for the number of performed tests with prenatal measures for this modality).

Postpartum Mother-Infant Bonding and Bonding Impairments
In addition to the association with gestational changes in a mother that prepare for motherhood, we wanted to investigate whether the observed brain changes across pregnancy could predict a mother's bonding to her infant after birth and problems in the mother-infant relationship. Mother-infant bonding was measured using the Maternal Postnatal Attachment Scale (MPAS) in the early (Post) and late (Post+1y) postpartum session. In addition, impairments in the mother-infant relationship were assessed with the Postpartum Bonding Questionnaire (PBQ) in the early (Post) and late (Post+1y) postpartum session. First, correlation analyses were performed with the total MPAS and PBQ measures acquired in the early and late postpartum period to examine the time point when potential associations could be observed. These analyses revealed no associations with the early postpartum measures, while changes in default mode activity across pregnancy significantly predicted both mother-infant bonding and bonding impairments in the late postpartum period (Supplementary Figure 7, Supplementary Tables 40 -43), with stronger brain changes being associated with more mother-infant bonding and less impairments in the mother-infant relationship. Subsequent analyses of the subscores of these scales in the late postpartum period did not render significant results, although a trend was observed with impaired bonding and the risk of infant rejection (Supplementary Tables 44 -45).

Development of Mother-Infant Bonding and Bonding Problems across the Postpartum Period
Based on the observed associations with these measures in the late but not the early postpartum period, we hypothesized that neurally-regulated effects on bonding may actually only become evident in the postpartum period. Therefore, we performed supplementary analyses to test whether pregnancy-related changes in DMN coherence relate to subsequent developments in the mother-infant relationship that take place in the postpartum period ( Supplementary Figure 8 and 9). These analyses showed that stronger pregnancy-related increases in DMN coherence predicted a stronger increase in bonding and a decrease in bonding impairments across the postpartum period (Supplementary Table 46). Associations were additionally observed with changes in the degree of pleasure experienced by the mother in the interaction with her baby and the absence of hostility (Supplementary Tables  47). Furthermore, pregnancy-related changes in DMN coherence were associated with postpartum changes in the risk of infant rejection and pathological anger across the postpartum period (Supplementary Tables 48). It should be noted that these changes were not associated with the neural changes across the postpartum period, only with the preceding changes across pregnancy. When applying a Bonferroni correction across all postnatal measures, the correlation between the changes in DMN coherence and the postpartum changes in mother-infant bonding and infant rejection and pathological anger remained significant. These findings thus reveal associations between pregnancy-related neural changes and mother-infant bonding and bonding impairments across the postpartum period, suggesting that neurogestational effects on aspects of maternal caregiving may affect the subsequent bonding of a mother to her infant across the postpartum period.  Note. Correlation results between the Maternal Postnatal Attachment Scale (MPAS) scores in the early and late postpartum period with the observed changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all postpartum tests (correlation-adjusted Bonferroni correction for the number of performed tests with postpartum measures for this modality). Note. Correlation results between the Postpartum Bonding Questionnaire (PBQ) scores in the early and late postpartum period with the observed changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of tests within the research question). **effect also survives correction for multiple comparisons for all postpartum tests (correlation-adjusted Bonferroni correction for the number of performed tests with postpartum measures for this modality).   Note. Correlation results between hormone levels across pregnancy and the changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of hormones). Note. Correlation results between hormone levels across pregnancy and the observed changes in DMN coherence. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table. *P<0.05 corrected for multiple comparisons (correlation-adjusted Bonferroni correction for the number of hormones).

Supplementary
Supplementary Figure 10. Correlation between mean pregnancy Estradiol levels and the changes in brain structure across pregnancy (PRE to POST). a) Scatter plot depicting results from the multivariate regression analyses examining the relation between mean Estradiol levels (pg/ml) divided by creatinine (mg/dl) across pregnancy and changes in Grey Matter volume across pregnancy. It should be noted that for these analyses the direction of the depicted correlation cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. b) Weight map depicting the relative contribution of each voxel to the multivariate regression. Note that blue colors depict a negative contribution to the regression, reflecting that higher Estradiol levels in pregnancy are associated with stronger volume reductions within the blue regions. Post-PreΔGM-based predictions = Predicted covariate values based on changes in grey matter between Pre and Post sessions defined by multivariate regression analyses. .27 .080 Note. Correlation results between Estradiol levels across pregnancy and the changes in grey matter volume based on multivariate regression analyses. Note that correlations were not performed with any hormone levels extracted from week 40 due to the low number of available samples. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. *P<0.05 corrected for multiple comparisons. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in default mode network coherence.*P<0.05 corrected for multiple comparisons. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in Choline across pregnancy. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in Creatine across pregnancy. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in glutamate across pregnancy. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in myo-inositol across pregnancy. Note. Correlation results between mean osmolality levels averaged across whole pregnancy and for each of the trimesters of pregnancy with the changes in tNAA across pregnancy.

Supplementary
Supplementary Note 2

Stress
Becoming a mother represents a life-changing transition involving many drastic changes in a woman's biology and environment and this can be a stressful period for many mothers. To measure psychological distress, we applied the K10 questionnaire during pregnancy and in the postpartum period. In addition, a questionnaire measuring subjective stress during these periods was applied to obtain an indication of the overall distress experienced by women during their pregnancy and in the postpartum period between delivery and the early postpartum session. Correlation analyses involving each of these measures during pregnancy and the postpartum period rendered no significant results (see Supplementary Tables 59-62), suggesting that stress did not represent a major factor in the induction of the observed neural changes. In addition, for completeness, the main models rendering the changes in brain structure and function were repeated while correcting for each of these stress variables, which indicated that the observed brain changes were also evident when correcting for the degree of stress experienced during pregnancy and the postpartum period (Supplementary Tables 63-70).

Sleep
Furthermore, when becoming a mother, many women experience drastic changes in sleep, especially in the early postpartum period. Therefore, women were asked to keep track of their sleep duration and the number of sleep disruptions, which were used as an indication of the women's sleep quality. An average number of hours of sleep and an average number of sleep disruptions per night in the week preceding the pregnancy and postpartum sessions were included in correlation analyses. In addition, indications of the women's sleep across pregnancy (in terms of the number of hours and sleep disruptions, until week 36 of pregnancy) and the early postpartum period (until the first postpartum session) were included in correlation analyses. All measures of sleep were included in correlation analyses with the observed changes in brain structure and function, which rendered no significant results (Supplementary Tables 71 and 72). In addition, the main models were repeated while correcting for each of these sleep variables, which rendered highly similar results (Supplementary Tables 73-84), suggesting that the women's sleep does not represent a major factor contributing to the observed brain changes.

Duration of Exposure to Postpartum Factors
To further examine the contribution of postpartum factors to the observed changes, correlations were additionally performed with the time between delivery and the Post session, which represents the duration of exposure to postpartum factors until the women's participation in the early postpartum session. These analyses indicated no significant correlations between this variable and the observed changes in brain structure and function (Supplementary Tables 85and 86). Accordingly, models including this variable as a confounding factor also rendered similar results to the main models (Supplementary Tables 87 and 88).

Breastfeeding
Breastfeeding, which involves intense contact with the infant and its own repertoire of hormonal fluctuations, could also be hypothesized to potentially contribute to the observed neural changes. To examine the potential role of breastfeeding, we compared the changes in brain structure and function between women who breastfed their infant and women who did not. These analyses did not render any significant results. In addition, within the group of women who breastfed their children, correlations were performed with the number of feedings per 24 hours in the period of the postpartum session, which also did not render significant results (see Supplementary Tables 89  and 90). Accordingly, including this variable as a confounding factor in the main analyses did not significantly alter the results (see Supplementary Tables 91 and 92).
Interestingly, a supplementary analysis examining the association between the total months of breastfeeding until the Post+1y session with changes in grey matter volume and DMN coherence across the postpartum period revealed a positive correlation between reversal of DMN coherence across the postpartum period and the duration of breastfeeding (Supplementary Tables 93 and 94), suggesting that prolonged breastfeeding may play a role/may stimulate the maintenance of these changes in DMN coherence.
Type of delivery Finally, to investigate whether the type of delivery plays an important role in the observed brain changes, we compared the changes in brain structure and function between the women who gave birth by means of vaginal delivery to the women who delivered by means of a caesarean section. These comparisons not show significant differences in brain changes based on the type of childbirth.
Supplementary Note. Correlation results between K10 scores acquired during pregnancy and the postpartum period and the observed changes in default mode network coherence. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table.
Supplementary Note. Correlation results between K10 scores acquired during pregnancy and the postpartum period and the observed changes in grey matter volume. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. Note. Correlation results between the women's subjective stress experienced during pregnancy and the postpartum period and the observed changes in default mode network coherence. Note. Correlation results between the women's subjective stress experienced during pregnancy and the postpartum period and the observed changes in grey matter volume. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain. Note. Increases or decreases in within-network connectivity in the DMN in primiparous women between the preconception and the early postpartum session corrected for the K10 scale in the pregnancy period. Results are reported at a statistical threshold of p<0.05 FWE-corrected. L = left, R = right.

Supplementary
Supplementary Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for K10 in the pregnancy session. Statistics are extracted from onesample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for K10 of the early postpartum session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for subjective degree of stress during pregnancy. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the subjective degree of stress since birth. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Correlation results between variables representing the women's sleep quality during pregnancy and the postpartum period and the observed changes in grey matter volume. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain.

Supplementary
Supplementary Note. Correlation results between variables representing the women's sleep quality during pregnancy and the postpartum period and the observed changes in default mode network coherence. In case of deviations from normality, a non-parametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table.
Supplementary Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the average number of hours of sleep per night in the week before the pregnancy session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the average number of sleep disruptions per night in the week before the pregnancy session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the average number of sleep disruptions per night in the week before the early postpartum session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right.
Supplementary Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the average number of hours of sleep per night between birth and the early postpartum session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Increases or decreases in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the average number of sleep disruptions per night between birth and the early postpartum session. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Correlation results between variables representing the duration of exposure to postpartum factors and the observed changes in grey matter volume. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain.

Supplementary
Supplementary Note. One sample t-test to explore the direction of change in grey matter volume in primiparous (PRG) participants across Pre and Post1 sessions, while adjusting for the days between birth and Post session. . Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Increases or decreases in within-network connectivity in the DMN in primiparous women between the preconception and the early postpartum session corrected for the duration of exposure to postpartum factors (the number of days between birth and the post session). Results are reported at a statistical threshold of p<0.05 FWEcorrected. L = left, R = right. Note. Correlation results between the number of feedings per 24 hours in the women who breastfed their children and the observed changes in grey matter volume. It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain.

Supplementary
Supplementary Note. Correlation results between the number of feedings per 24 hours in the women who breastfed their children and the observed changes default mode network coherence. In case of deviations from normality, a nonparametric Spearman's correlation test was performed rather than a Pearson's test. Spearman's rho ("rho") and the p-values for this test ("p rho") are then also reported in the table.
Supplementary Note. Changes in regional grey matter volumes in primiparous women between the pre-conception and the early postpartum session corrected for the number of feedings per 24 hours in breastfeeding women. Statistics are extracted from one-sample t-tests performed within the framework of an SPM12 General Linear Model and are one-sided (as is standard in SPM). Results are reported at a statistical threshold of p<0.05 FWE-corrected. P-value at peak voxel (whole-brain FWE corrected) is reported. L = left, R = right. Note. Correlation results between the total number of months of breastfeeding until the Post+1yr session and the observed changes in grey matter volume across the postpartum period (between Post and Post+1yr sessions). It should be noted that for these analyses the R cannot be interpreted as a reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain.

Supplementary
Supplementary Note. Correlation results between the total number of months of breastfeeding until the Post+1yr session and the observed changes in default mode network coherence across the postpartum period (between Post and Post+1yr sessions). Note. Correlation results between age (at Pre session), the time interval between the pre-conception and postpregnancy scans and the educational level according to the Verhagen scale and the observed changes in default mode network coherence. Note. Correlation results between age (at Pre session), the time interval between the pre-conception and postpregnancy scans and the educational level according to the Verhagen scale and the observed changes in grey matter volume based on multivariate regression analyses. It should be noted that for these analyses the R cannot be interpreted as a direct reflection of the direction of the biological effect, since this statistic is based on patterns of brain changes across the whole brain.

Supplementary
Supplementary Figure 11. Positions of the VOIs used during the Magnetic Resonance Spectroscopy acquisitions. These depict the PCC VOI (a, in yellow) and the STG VOI (b, in blue) of a representable subject at the baseline session, shown in subject T1 space. VOI = Volume of Interest, PCC = Posterior Cingulate Cortex, STG = Superior Temporal Gyrus.
Supplementary Figure 12. Position of the PCC VOI used during the Magnetic Resonance Spectroscopy acquisitions. These images depict the VOI of a representable subject at the baseline session, shown in MNI space. The position of this VOI is at the mean center-of-gravity of all VOIs (x=0 mm, y=-57 mm, z=38 mm). I= Inferior, S=Superior, L=Left, R=Right, A=Anterior, P=Posterior, VOI = Volume of Interest, PCC= Posterior Cingulate Cortex.