Male fetus susceptibility to maternal inflammation: C-reactive protein and brain development

Abstract

Background:

Maternal inflammation in early pregnancy has been identified epidemiologically as a prenatal pathogenic factor for the offsprings’ later mental illness. The early manifestations in the newborn of the effects of maternal inflammation on fetal brain development are largely unknown.

Methods:

Maternal infection, depression, obesity, and other factors associated with inflammation were assessed at 16 weeks gestation. Maternal CRP and cytokines and maternal choline were measured then. Cerebral inhibition was assessed by inhibitory P50 sensory gating at one month of age, and infant behavior was assessed by maternal ratings at three months of age.

Results:

Maternal CRP diminished the development of cerebral inhibition in newborn males but paradoxically increased inhibition in females. Similar sex-dependent effects were seen in mothers’ assessment of their infant’s self-regulatory behaviors at three months of age. Higher maternal choline levels partly mitigated the effects of CRP in male offspring.

Conclusions:

The male fetal-placental unit appears to be more sensitive to maternal inflammation than females. Effects are particularly marked on cerebral inhibition. Deficits in cerebral inhibition one month after birth, similar to those observed in several mental illnesses, including schizophrenia, indicate fetal developmental pathways to later mental illness. Deficits in early infant behavior follow. Early intervention before birth, including prenatal vitamins, folate, and choline supplements, may help prevent fetal development of pathophysiological deficits that can have life-long consequences for mental health.

Graphical Abstract

Introduction

Retrospective epidemiological studies established maternal infection during early second trimester as a risk factor for schizophrenia (Clarke 2009, Brown, 2010). Animal models identify maternal immune activation (MIA) with cytokine induction as a pathophysiological mechanism (WL Wu 2015). Maternal inflammation, often in response to respiratory and genitourinary infections in the early second trimester, decreases the development of inhibitory sensory gating and increases impulsivity (Meylbye 2004, Ghassabian 2018, Freedman 2019). Pathogenic effects of maternal cytokines on both the placenta and the brain have been proposed (Brown 2018). In addition to infection, maternal inflammation has been associated with obesity, maternal depression and anxiety, and environmental pollution (Madan 2009, Osborne 2013, van den Hooven 2012). Elevation in plasma C-reactive protein (CRP) at 16 weeks gestation and Interleukin-6 (IL-6) later in pregnancy in women whose offspring develop schizophrenia, adds molecular evidence for the pathogenic role of inflammation (Canetta 2014, Goldstein 2014). These epidemiological findings are now well-accepted, but limited information exists for which specific aspects of human fetal brain development are altered by maternal inflammation to produce mental illness in later life (Graham 2018, Spann 2018). The first pathophysiological signs of schizophrenia are apparent as early as birth (Erlenmeyer-Kimmling 1987, Walker 1994). RDoC provides a framework that can be applied prospectively to study the earliest development origins of schizophrenia by examining pathophysiological components of the illness that appear before diagnostic symptoms are apparent (Ross 2015).

This study assessed newborn cerebral inhibitory physiology as a primary outcome of fetal brain development using an auditory sensory gating measure, a biomarker of schizophrenia in adults, that first appears in newborns (Freedman 1979, Kisley 2003). Development of inhibitory neurons was characterized by sensory gating of the P50 cerebral evoked response in a paired-auditory stimulus paradigm (S1, S2). P50 amplitude is normally decremented in response to S2, because of inhibitory mechanisms activated by the response to S1. This inhibition is impaired in persons with schizophrenia (Adler 1982; Supplement A). P50 sensory gating is a physiological measure in the RDoC Cognition Domain, Perception Construct, Auditory Sub-Construct. P50 sensory gating has been used in adults with schizophrenia to detect familial or genetic risk (Freedman 1979, Hall 2011; Quednow 2012), but P50 inhibition can be recorded as soon as one month after birth (Kisley 2003). Diminished newborn P50 inhibition predicts early childhood problems in self-regulation, attention, and social function (Hutchison 2007, Ross 2016). These early childhood behaviors are recognized as early developmental symptoms in children who late develop psychosis (Rossi 2000, Rutter 2006; Pine 2015). Newborn P50 inhibitory deficits are increased if either parent has schizophrenia, a finding replicated by another group (Hunter 2011, E. Smith 2018).

Increased maternal choline promotes the development of newborn P50 inhibitory gating, and therefore, we also assessed the possible interaction of the maternal inflammatory response with choline in this study.

Methods

1. Maternal assessment and recruitment

Women were enrolled from a public safety-net prenatal clinic at 14–16 weeks gestation from July 2013 until July 2016. Gestational age was timed from the last menstrual period and by ultrasound. Exclusions were fetal anomaly and major maternal medical morbidity. The Colorado Multiple Institution Review Board approved the study; all participants gave informed consent. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.

Women were asked to participate in a prospective study of stress in pregnancy on their child’s development. Psychiatric diagnoses were made using the Structured Clinical Interview for DSM-IV Axis I Disorders with DSM-5 criteria. Self-ratings on Center for Epidemiological Studies of Depression-R (CESD-R), State-Trait Anxiety Inventory-State Version (STAI-S), and the Perceived Stress Scale (PSS) were acquired. Maternal sociodemographics and health, including infections, substance use, BMI, and prenatal vitamin use, were assessed. Labor, delivery, and neonatal parameters were recorded from the medical record. Investigators were blinded to maternal choline levels during assessments.

2. Assessment of maternal infection

The medical record for all prenatal care was reviewed. A mother’s self-report of infection was considered significant if it was entered as a problem in the medical record. Treatment was provided for all reported genitourinary infections. Most respiratory infections were viral and were therefore not treated. In addition, mothers had an in-person review of systems for symptoms of infection at 16 weeks by research personnel. The correlation between symptoms rated by the mother as moderate to severe on a custom-made scale from 1–8 in the interview and problems in the medical record is r_s = 0.96, P < 0.001 (Freedman 2019).

3. Choline measurements

Maternal plasma was assayed for choline and its metabolite betaine at 16 weeks gestation by the Colorado Translational Research Center Metabolomics Laboratory using mass spectroscopy. Blood samples were obtained at least two hours after breakfast. Plasma was quickly separated by refrigerated centrifugation to prevent platelet phosphatidylcholine release.

Stable isotope standards for betaine (N,N,N-trimethylglycine, cat no D-3352) and choline (cat no D-2464) were purchased from CDN Isotopes. Serum samples were thawed on ice, then 20 μL was extracted with 480 μL of ice cold extraction buffer (5:3:2 MeOH:MeCN:H2O) containing 0.1 μM each of N,N,N-trimethylglycine-D9 (betaine) and [1,1,2,2-D4]choline. Extraction was performed by vigorous agitation at 4⁰C for 30 min followed by centrifugation at 12,000 rpm, 4⁰C for 10 min. A 100 μL aliquot of supernatant was transferred to a glass vial, dried under N₂ flow, and resuspended in an equal volume of water containing 0.1% (v/v) formic acid. Aqueous extracts were analyzed by ultra high pressure liquid chromatography-mass spectrometry (UHPLC-MS) on a Thermo Vanquish UHPLC (San Jose, CA) coupled to a Thermo Q Exactive mass spectrometer (Bremen, Germany) via positive electrospray ionization. Solvents were water (phase A) and acetonitrile (phase B) supplemented with formic acid (0.1%) and flow rate was 0.25 mL/min. Metabolites were separated using a Kinetex C18 (Phenomenex, Torrance, CA) column (2.1 × 150 mm, 1.7 μm) with a 6 minute gradient of 0–2 min 2% B; 2–2.5 min increase to 25% B; 2.5–4 min hold at 25% B; 4–4.01 min decrease to 2% B; 4.01–6 min hold at 2% B. The Q Exactive mass spectrometer was operated in full scan mode over the range of 65–950 m/z. Samples were randomized and a quality control sample was injected every 10 runs. The coefficient of variation was < 10%. Data analysis was performed using Maven Metabolomic Analysis and Visualization Engine (Princeton University) following file conversion by MassMatrix (Case Western Reserve University). Absolute concentrations were obtained using the following equation:

$$\left[\text{light}\right]=\left(\text{peakarealight/peakareaheavy}\right)\left[\text{heavy}\right]*\text{DF}$$

where DF = dilution factor, in this case, 25 (i.e. 20μ of serum in a total 500μ volume).

Mothers received information on diets higher in choline, but dietary intake was not estimated because of the low relationship of self-reported intake to maternal choline levels in pregnant women, r = 0.2 (BTF Wu 2012). The placental choline transporter CLT1 produces amniotic fluid levels approximately twice maternal plasma levels (Ilcol et al., 2002; Baumgartner et al., 2015). Uptake is proportional to maternal plasma concentration, which suggests that higher peak levels may be important determinants of amniotic fluid levels (Iwao et al., 2016). Maternal levels obtained in non-fasting conditions, as in the present study, can be elevated, but only after high-choline meals that exceed the recommended daily intake (Zeisel et al., 1980; Holm et al., 2003; Abratte et al., 2009). Only choline activates α7-nicotinic receptors (Alkondon et al., 1997). We found no effects of its metabolite betaine.

4. Maternal CRP and cytokines

Plasma CRP at 16 weeks gestation was assayed by the Beckman-Coulter high sensitivity assay at the Colorado Translational Research Center, Colorado Children’s Hospital. Tumor Necrosis Factor-alpha (TNFα) and Interleukin-6 (IL-6) and Interleukin-8 (IL-8) were assayed by R&D Systems high sensitivity assays at the CTRC Core Laboratory, Children’s Hospital of Colorado. Il-6 values above the detectable limit (1.9 pg/ml) were found for 9 of 91 samples from women without infection and 17 of 61 women with infection (Fisher’s exact test p = 0.007). TNF levels above detectable limits were observed in 81 of 91 women without infection and 60 of 61 women with infection (1.6 pg/ml). Levels below detectable limits were set to zero.

5. Neonatal physiological recording of cerebral inhibition

Newborns were studied one month (44 weeks) after birth adjusted for gestational age. Vertex electroencephalogram, electro-oculogram, submental electromyogram, and respiration were continuously recorded while infants napped (Kisley et al., 2003; Hunter et al., 2011). Recording of the cerebral auditory evoked potential P50, a positive EEG wave 50ms post stimulus, occurred in the second active sleep episode, the precursor of REM sleep, identified by low voltage desynchronized vertex activity with the absence of K-complexes, change in respiration, and large eye movements with submental atonia (Anders et al., 1971). The second active sleep episode was reached approximately 45 minutes after sleep onset. In adults, P50 inhibition in REM and waking are equivalent (Griffith and Freedman, 1995).

Two identical auditory stimuli are delivered 500 ms apart to elicit P50_S1 and P50_S2. P50 inhibition is often assessed as amplitude ratios P50 _S2/P50 _S1 or (P50_S1-P50_S2)/P50_S1 (Adler et al., 1982). However, the skew inherent in ratios limits their power for correlation with risk factors. P50_S2 amplitude, covaried for P50_S1, which is normally distributed, has therefore also been used (DA Smith et al., 1994). A meta-analysis of 28 independent studies found that P50_S2 amplitude, covaried for P50_S1 distinguishes adults with schizophrenia from healthy controls with high effect size (d’ = 1.45, P<0.001, Supplement A). A recent study using magnetoencephalography also reported that the sensory gating defect in schizophrenia is robust to the measurement method (Schubring 2017). Regardless of the method used, higher P50_S2 amplitudes indicate decreased inhibition. The assumption is that P50_S1 variance is small, compared to P50_S2 variance. In 151 newborns, effect sizes for P50_S1 differences between newborns whose mothers had no known risk versus women with depression or schizophrenia ranged from 0–0.16. Effect sizes for the decrease in P50_S2 amplitude were 0.21–0.50 (Hunter et al., 2011). The effect of maternal schizotypy on newborn P50 inhibition has been replicated by another group, who also found increased P50_S2 amplitudes (E. Smith et al., 2018). Intraclass correlation between two newborn recordings 1 week apart is r_ICC = 0.84. Other technical aspects of recordings have been published (Hunter et al., 2008; Hunter et al., 2015).

6. Childhood behavioral assessments

Parents completed the Infant Behavior Questionnaire-Revised Short Form (IBQ-R) when the infant was three months of age (Gartstein and Rothbart, 2003; Putnam et al., 2014). The Parental Distress Subscale of the Parenting Stress Index was also completed as a possible covariate for parental bias (Abidin, 2012). The 91-item IBQ-R Short Form, commonly used to study behavior in children at this age, rates 14 aspects of child behavior, which the IBQ-R developers clustered into 3 indices by factor analysis: Surgency summarizes the child’s level of activity and positive affect; Negativity summarizes fearfulness and anxiety; and, Regulation summarizes duration of attention, responsiveness to parents, and enjoyment of quiet play. Two components of Regulation are also in Surgency, smiling and soothability, and the two indices are highly correlated in the present sample (r = 0.51, P <0.001). Covariation with Surgency isolates elements of Regulation that are more specific to the early development of attention and less attributable to the child’s general psychomotor activation. A similar covariance between Regulation and Surgency (0.80) has been documented by another group, who have also proposed revisions to the factor structure (Bosquet-Enlow et al., 2016). Lower IBQ-R Regulation at one year of age is associated with decreased reading readiness at age four years and decreased conscientiousness, organization, and increased distractibility at age 9 years of age (Putnam 2014, Slobodskaya 2016).

7. Statistical analyses

Neonatal P50_S2 amplitude and childhood IBQ-R Regulation were the two principal outcomes, based on a previous work that found P50 inhibition was a biomarker of both infection and choline’s effects and that regulatory behaviors were the most affected outcome (Ross et al., 2016, Freedman et al., 2019). Kolmogorov-Smirnoff tests for each outcome did not find a significant deviation from normal distributions. General Linear Models analyzed CRP levels and choline levels as a continuous effect. For P50_S2 amplitude, maternal smoking was a covariate because of its previously established effect on P50 inhibition (Hunter 2011). For IBQ-R analyses, maternal education was a covariate because of its correlation with both CRP levels (r = −0.169, P = 0.038) and IBQ-R Regulation (r = 0.196, P = 0.021). Follow-up analyses investigated the possible confounding effects of marijuana and alcohol use.

Choline’s effect size on P50 inhibition in a previous study was Cohen’s d’ = 0.7 (Ross 2013). We expected 20% of the women would have adequate choline levels and 30% attrition (BTF Wu 2012, Hoffman 2019). Therefore, we enrolled 200 women to have power 1-β > 0.95, α = 0.05, 1-tail to observe an overall choline effect.

Results

Mothers’ inflammatory response during early gestation

Of 316 mothers screened, 201 were enrolled by 16 weeks gestation, as dated from the last menstrual period and verified by ultrasound measurements. Of these, 162 brought their newborns to the one-month postnatal assessment, where the auditory evoked potentials were obtained (and as previously reported; Freedman et al, 2019). CRP levels were assessed in plasma samples obtained at 16 weeks gestation from 150 of the women (Figure 1). When the infants reached three months of age, 127 women brought them in for behavioral assessment. Attrition during the study after enrollment generally reflected mothers who moved from the pre-birth residence to their mothers’ homes to care for their infants and were then lost to follow-up. The significant differences between male and female babies were only in greater male birth weights and head circumferences (Table 1).

Figure 1.

Flow of mothers and offspring over the course of gestation and post-birth development.

Table 1.

Maternal status during gestation, labor, and delivery, neonatal outcomes stratified by offspring sex.

	Males = 76 Mean (SD) or N (%)	Females = 74 Mean (SD) or N (%)	P = (t-test or Fisher’s exact test)
Maternal demographics
Maternal age yrs	27.6 (6.3)	29.8 (5.7)	0.88
Maternal education yrs	13.7 (3.0)	13.4 (3.1)	0.54
Pre-pregnancy BMI	27.0 (6.7)	27.6 (6.3)	0.58
Living with biological father N	56 (69%)	61 (76%)	0.38
Maternal mental illness and drug use
Bipolar Disorder N	3 (4%)	4 (5%)	0.22
Major Depression N	14 (17%)	10 (13%)	0.51
Anxiety Disorder N	2 (2%)	5 (6%)	0.28
Schizophrenia N	0	2 (3%)	0.24
Alcohol use N	14 (17%)	8 (10%)	0.25
Cannabis use N	16 (20%)	9 (11%)	0.19
Current smoker N	7 (9%)	4 (5%)	0.53
Cocaine N	7 (9%)	5 (6%)	0.27
Opioids N	2 (2%)	2 (2%)	0.89
Obstetrical history
Gravidity N	3.25 (1.87)	2.88 (1.80)	0.22
Pre-term delivery N	12 (15%)	18 (23%)	0.23
Miscarriage, ectopic, aborted N	56 (69%)	54 (67%)	0.87
Living children N	1.52 (1.42)	1.20 (1.25)	0.33
Pregnancy, Labor, Delivery
Prenatal vitamins with folic acid N	68 (89%)	66 (89%)	1.00
Choline μM	6.31 (1.78)	6.49 (1.87)	0.54
Betaine μM	11.1 (2.9)	11.9 (4.2)	0.18
Pre-pregnancy BMI	27.0 (6.7)	27.6 (6.3)	0.58
Obesity BMI≥30 N	19 (23%)	28 (35%)	0.12
Common infections N	35 (43%)	30 (38%)	0.52
Infection severity	2.05 (2.74)	1.91 (2.70)	0.75
Hypertension N	4 (5%)	6 (8%)	0.53
Gestational diabetes N	4 (5%)	4 (5%)	0.99
Preeclampsia N	7 (9%)	6 (8%)	0.99
Premature labor N	8 (10%)	3 (4%)	0.21
Premature delivery <37 weeks N	7 (7%)	3 (4%)	0.50
Vaginal delivery N	64 (67%)	63 (66%)	0.99
Maternal self-ratings
Center for Epidemiological Studies of Depression-R	14.5 (10.3)	13.6 (8.6)	0.53
State-Trait Anxiety Inventory	35.6 (12.2)	35.9 (9.6)	0.89
Perceived Stress Scale	23.4 (9.1)	23.8 (6.8)	0.75
Parenting Stress Index 3 mos post birth	26.8 (7.4)	25.7 (7.2)	0.81
Neonatal Status
APGAR 5 min	8.86 (0.45)	8.69 (0.80)	0.32
Small for gestational age N	3(4%)	4 (5%)	0.72
Large for Gestational age N	11 (14%)	9 (71%)	0.81
Meconium fluid N	16 (20%)	20 (25%)	0.57
Nuchal cord N	24 (30%)	14 (66%)	0.09
Days in NICU>1	5 (6%)	5 (6%)	0.99
Jaundice N	32 (40%)	36 (45%)	0.52
Gestational age at birth days	273.8 (15.7)	271.4 (18.6)	0.38
Birth weight g	3266.9 (585.0)	3049.4 (607.7)	0.022
Birth length cm	49.7 (4.2)	48.4 (5.3)	0.086
Birth head circumference cm	35.0 (2.7)	34.1 (2.7)	0.045
Maternal inflammation
CRP mg/ml	7.75 (7.01)	9.68 (8.00)	0.12
IL-6 pg/ml	0.46 (1.6)	0.63 (1.21)	0.46
IL-8 pg/ml	1.18 (1.36)	1.71 (2.38)	0.09
TNFα pg/ml	3.32 (1.59	3.52 (1.95)	0.48
Newborn cerebral physiology 1 month post birth
P50S1 μV	1.60 (0.67)	1.81 (1.00)	0.10
P50S2μV	0.83 (0.60)	0.83 (0.65)	0.96
Infant behavior 3 months post birth	N = 72	N = 65
Surgency	4.19 (1.11)	4.30 (1.12)	0.58
Negativity	3.09 (0.89)	3.13 (0.94)	0.80
Regulation	5.26(0.68)	5.21 (0.68)	0.67

CRP levels correlated with maternal CESD-R self-ratings of depression, pre-pregnancy BMI, severity of gestational infection, and maternal education (Table 2). There was no correlation with infant sex. More educated women were older (r = 0.324, P < 0.001 and had lower self-ratings of depression (r = −0.190, P = 0.015) and lower BMI (r = −0.283, P < 0.001), which has been found in other populations (Bui 2018). CESD-R ratings correlated with both infection (r = 0.264, P = 0.001) and BMI (r = 0.210, P = 0.007). Multiple regression analysis found significant effects for CRP of infection severity (β = 0.202 (95% CI 0.047, 0.356) P = 0.011) and BMI (β = 0.329 (95% CI 0.176, 0.482) P < 0.001), but not depression or maternal education. If the ratings were dichotomized to identify mothers likely to come to clinical attention as depressed (CESD-R>15), obese (BMI > 30), or as having a significant infection (severity > 5), then only infection was a significant factor for CRP elevation (β = 0.237 (95% CI 0.076, 0.398) P = 0.004; Supplement Table 1).

Table 2.

Maternal factors associated with CRP elevation at 16 weeks gestation

Parameters	Pearson r	P
Child sex	−.129	0.12
Pre-pregnancy BMI	.358	<0.001
Infection severity rating 16 wks	.245	0.002
Clinician-reported moderately severe infection 16 wks	.235	0.004
CESD-R 16 wks	.175	0.03
Maternal educations yrs	−.169	0.04
Maternal age yrs	.051	0.53
Bipolar disorder	−.049	0.55
Depressive disorder	.078	0.34
Anxiety disorder	−.103	0.21
Schizophrenia	.045	0.58
Nicotine use	.058	0.48
Alcohol use	.036	0.66
Cannabis use	.094	0.26
CESD-R > 15	.094	0.26
Antidepressant	.107	0.19
STAI 16 wks	.018	0.82
PSS 16 wks	.102	0.22
Antibiotic use	.022	0.79
Obesity BMI≥30	.030	0.71
Other maternal cytokines
IL6 16 wks	.543	<0.001
IL8 wks	.198	0.02
TNF wks	.186	0.02

CRP levels were not associated with either complications of labor and delivery, such as premature delivery, diabetes, or chorioamniotis, or any neonatal outcomes, such as lower APGAR scores or small size for gestational age (Table S1). CRP levels were correlated with other maternal cytokines, specifically IL-6, IL-8, and TNF-α (Table 2).

Newborn expression of cerebral inhibition

A paired-stimulus auditory paradigm (S1, S2) delivered during active sleep was used to assess the newborn’s development of cerebral inhibition, measured as the decrease in amplitude of P50_S2, relative to P50_S1 (Figure 2). There were no differences P50_S1 or in P50_S2 amplitude between male and female newborns (Table 1).

Figure 2.

Top: Examples of P50_S1 and P50_S2 auditory evoked responses recorded 1 month after birth in a female newborn. P50 amplitude is measured from the most positive peak relative to the preceding negativity. The mother’s 16-week gestation CRP was 15.3mg/ml and her choline was 5.87μM. P50_S1 was 1.50μV and P50_S2 was 0.27μV. Bottom: Effects of CRP on P50_S2 for male and female infants.

Maternal CRP levels had a sex-dependent effect on P50_S2 amplitude (F_df1 = 4.597, P = 0.034; Table S2); P50_S1 was unaffected. CRP levels correlated with increased P50_S2 amplitude in males (β = 0.205 (95% CI 0.023, 0.387) P = 0.03), but decreased P50_S2 amplitude in females (β = −0.214 (95% CI −0.390,−0.038) P = 0.016). Only CRP levels were associated with this effect on P50 amplitudes; the other cytokine levels had no significant association (Table S3). Cigarette, marijuana, and alcohol use during early pregnancy did not affect the significance of the effects of CRP on P50_S2 amplitude (Table S4).

Infant behavior at three months of age

Maternal ratings of their infants’ behavior showed no differences between male and female infants on the three principal indices, Surgency, Regulation, and Negativity (Table 1). Multivariate analysis of effects on CRP on the three IBQ-R indices found that maternal CRP increased 3-month IBQ-R Surgency regardless of the sex of the infant (F_df1 =5.088, P = 0.026; β = 0.207 (95% CI 0.036, 0.378). The two most affected components of this scale were Vocalizations (Wald χ²_df1 = 6.299 P = 0.012) and High Intensity Pleasure (Wald χ²_df1 = 7.478, P = 0.006). The effects of CRP on IBQ-R Regulation were sex-dependent (CRP*sex: F_df1 = 4.244, P = 0.042; Figure 3; Table S5). There were no significant effects of CRP on IBQ-R Negativity. In males, CRP decreased Regulation (β = −0.209 (95% CI −0.429, 0) P = 0.049), but there was no significant effect in females (β = 0.062, P = 0.6). The two most affected IBQ-R components were Soothability (CRP*sex: Wald χ²_df1 = 4.842, P = 0.042) and Cuddliness (CRP*sex: Wald χ²_df1 = 6.043, P = 0.014). Cigarette, marijuana, and alcohol use during early pregnancy did not affect the significance of the effects of CRP on IBQ-R Regulation (Table S6).

Figure 3.

Top left: Effects of maternal CRP on infant IBQ-R Regulation in male and female infants. Top right: Relation of newborn P50_S2 amplitude to infant IBQ-R Regulation in males and females. Bottom: Effects of maternal CRP on infant IBQ-R Surgency.

Multiple regression found that newborn P50_S2 amplitude was related to 3-month old infant Regulation, also in a sex-dependent manner (P50_S2*sex: Wald χ²_df1 = 7.052, P = 0.008; Figure 3, Table S7).

IL-6 levels correlated with infant higher Negativity across both sexes (r= 0.229, P = 0.009).

Interaction of maternal choline levels and prenatal vitamins with the maternal inflammatory response

Maternal choline levels at 16 weeks gestation did not directly affect CRP levels, but maternal choline levels decreased the effect of CRP levels on P50_S2 amplitude. Multiple regression found effects of maternal choline for all newborns ((β = −0.121 (95% CI −0.227, −0.015) Wald χ²_df1 = 2.017, P = 0.029) and sex specific effects also (Wald χ²_df1 =7.888, P = 0.019; Figure 5, Table S8). In males, choline significantly diminished P50_S2 amplitude (β = −0.216 (95% CI −0.297, −0.035) P = 0.019); the effect of CRP did not change appreciably (β = 0.195, P = 0.032, compared to P = 0.201, P = 0.020) when choline was not considered). In females, the effect of choline was small and not significant (β = 0.010, P = 0.9). To demonstrate the 3-way interaction of choline with CRP and child sex, choline levels were dichotomized based on the mean value in the sample 6.4 μM. For males whose mothers had choline levels > 6.4 μM, the effect of CRP on P50 _S2 amplitude was considerably decremented, until CRP levels rose above 15 mg/ml (Figure 4).

Figure 4.

Top: Effects of maternal choline levels and maternal CRP on male newborn P50_S2 (left) and IBQ-R Regulation (right). Bottom: Effects of maternal choline levels and maternal CRP on female newborn P50_S2 (left) and IBQ-R Regulation (right).

Prenatal vitamins with folic acid decreased P50_S2 amplitude in both sexes, but the effects did not reach significance (in males, β = −0.168, P = 0.07; in females, β = −0.148, P = 0.09).

Multivariate analysis of the 3 IBQ-R indices found a significant interaction of child sex, CRP level, and choline level for Regulation (F_df1 = 3.186, P = 0.045) and no effects on the other indices (Table S9). For males whose mothers had choline levels below 6.4 μM, the effect of CRP on Regulation was β = 0.352 (95% CI 0.044, 0.660) P = 0.028. If the mother had choline levels above 6.4 μM, the effect of CRP levels became non-significant (β = 0.093, P = 0.6; Figure 5). The effect of CRP on Regulation was non-significant for females regardless of maternal choline level.

Discussion

Increased maternal CRP levels in early gestation were associated with decreased development of P50 cerebral inhibition in males but not females. The decrease in P50 sensory inhibitory gating was further reflected in decreased infant Regulation behaviors rated on the IBQ-R at three months of age. CRP levels were increased by maternal infections, obesity, and depression, with infection being predominant (Freedman et al, 2019). Unlike inhibitory gating and Regulation, increases in both infant Surgency, also related to elevated CRP, and infant Negativity, related to elevated IL-6, were observed in both sexes. Effects on both the placenta’s integrity and neuronal development have been investigated in the maternal immune response (Brown and Meyer 2018). The sex-specific effects on inhibition in male fetuses suggest that there may be specific effects of maternal inflammation on the development of inhibitory interneurons, particularly since their development appears to be facilitated in females by the inflammatory response. Other aspects of early behavior, Surgency and Negativity, are similarly affected in both sexes, however, suggesting that there may be both sex-dependent and sex-independent mechanisms affecting fetal brain development.

In animal models, cerebral interneurons are specifically sensitive to maternal inflammation and hypoxia (Lacaille 2019). Parvalbumin neurons of the hippocampus are specifically affected (Canetta 2016). A pathophysiological mechanism involving the astroglia response has been proposed (Sobue 2018). Male mice have a greater reduction in hippocampal volume and more chronic macrophage infiltration than females after maternal immune activation (Dada 2015). Male mice are also more sensitive to other insults, such as prenatal radiation, with greater loss of hippocampal pyramidal and interneurons (Granapathi 2017). However, female mice are more sensitive to the apoptotic effects of prenatal dexamethasone (Zuloaga 2012), and effects on NMDA receptors in the hippocampus after prenatal glucocorticoids are more marked in females than males (Owen 2014). A second hit has also been proposed as a pathophysiological mechanism. Animals exposed to maternal immune activation during gestation are more likely to show deficits after pubertal stressors. This combination reduced expression of parvalbumin inhibitory interneurons, but the analyses were performed only in male mice (Giovanali 2013).

In humans the retrospective epidemiological data are not as clear. Maternal CRP levels at about 16 weeks gestation are associated with increased risk of schizophrenia and autism spectrum disorder in a Finnish cohort (Canetta 2014; Brown 2014), but decreased autism spectrum disorder in a California cohort (Zerbo 2016). In a Dutch cohort, higher maternal CRP levels at 13 weeks gestation were related to autism traits in the general population (Koks 2016). There were no sex-specific effects in any of these cohorts, despite the preponderance of schizophrenia and autism disorders in males. In a New England cohort, however, mothers of males with schizophrenia had higher Il-6 levels in early third trimester (Goldstein 2014). The double prenatal-adolescent hit is also apparently pathogenic in humans. In a Danish cohort, females had increased risk for schizophrenia after prenatal exposure to infection compared to males, but additional pubertal trauma resulted in males having a markedly increased risk (Debost 2017). Like animal models, human females are also more sensitive to the effects of corticosteroids released naturally when mothers become depressed (DJ Kim 2017). Thus, there may be a second pathway by which female fetuses are affected because the mother’s infection is often accompanied by depression (Freedman 2019).

CRP does not generally cross from the maternal circulation to directly affect the fetus (Malek 2006). A possible mechanism of the effects of maternal immune activation is cytokine-mediated macrophage attack of the placenta, which the mother’s immune system may treat as a foreign body. Placental cytokines are increased in the placenta in animal models of stress during pregnancy, specifically in males (Bronson 2014). In humans, elevated CRP in early gestation is associated with chronic placental villitis (Ernst 2013). Maternal CRP is deposited in the human placenta during pregnancy, where increased levels are associated with chorioamnionitis, pre-eclampsia, and preterm delivery (EN Kim 2015). Genes expressed in the placenta are associated with schizophrenia in the subset of patients who had prenatal complications, including significant infection. The expression of these genes in the placenta is specifically upregulated in males compared to females (Ursini 2018). Maternal immune activation of umbilical vein macrophages is also greater in males than in females (Kim-Fine 2012).

This maternal immune attack on the placenta, although not directly transmitted to the fetus, triggers a reaction in the fetus itself that directly affects fetal brain development, including interneuron migration specifically (Oskvig 2012). These interneurons are responsible for inhibition of the cerebral auditory evoked response (Miller 1995). The timing of measurement of maternal CRP levels, early in pregnancy versus closer to term, is critical for assessing pathogenic versus normal effects. Cerebral interneurons are in a critical stage of development at 16 weeks gestation, when we found effects in this study and when effects are also seen on the later risk for schizophrenia (Bayatti 2008, Zecevic 2011, Canetta 2014). Later in pregnancy, inflammation and CRP are involved in the normal parturition process. The placenta at term synthesizes CRP (Malek 2006). Increased inflammatory cytokines in the last trimester are associated with enhanced brain development (Spann 2018, Graham 2018). Maternal IL-6 levels at 16 weeks gestation, which we found associated with Infant Negativity, were not similarly associated when levels across the entire pregnancy were considered (Rudolph 2018).

The effects of choline on the development of cerebral inhibition are mediated by activation of α7-nicotinic receptors (Alkondon 1997, Ross 2013). α7-nicotinic receptors on hippocampal neurons are required for the induction of the chloride ion gradient necessary for neuronal inhibition (Liu 2006). From the graph in Figure 5, the effects of choline on inhibition appear to be competitive with the effects of maternal inflammation in males. In an animal model, deletion of CHRNA7, the gene associated with α7-nicotinic receptors, completely blocks the effects of choline on the development of inhibition (Stevens 2014). Direct comparison of CHRNA7 deletion with prenatal maternal immune activation found similar effects of both insults on the offsprings’ development (Giovanoli 2018), providing further evidence for the possible competing effects of α7-nicotinic receptor activation and maternal inflammatory effects on brain development. α7-nicotinic receptor activation mediates the anti-inflammatory action of the vagus nerve (Wang 2003). In pregnant wild-type C57BL/6N mice subjected to maternal immune activation, choline supplementation did not change IL-6 levels in the placenta, but did lower IL-6 levels in the fetal brain, suggesting no general anti-inflammatory effect but rather a specific effect in the fetus itself. Effects were seen in both males and females (WL Wu 2015). Effects of choline on maternal cytokine levels were observed in a rat gestational model with nearly 5-fold dietary choline supplementation (Zhang 2018), but there were no effects on mothers’ cytokine levels observed in our study with normal human diets. A study of 2-fold choline supplementation on the expression of placental angiogenic factors and pro-inflammatory factors in a mouse model with a hemizygous Dlx3+/− gene deletion model of placental insufficiency found effects of supplementation in early gestation were greater in male placentas and in later gestation, greater in females (King 2019). Thus, like maternal immune activation, the sites of choline’s effects appear to be complex, with different but interacting effects in mother, placenta, and fetal brain.

Effects of choline in the human infants in this study were most marked on the development of cerebral inhibition and behavioral Regulation where sex-dependent effects of inflammation were also seen. Sex-specific effects of choline have not been generally observed in other human studies of choline’s effects (Ross 2013, Ross 2016). Nonetheless, for the more vulnerable male fetus in particular higher choline levels would seem to be a useful intervention to prevent the initial steps in the pathophysiology of later mental illness from occurring in utero, when the mother experiences inflammation.

Table 3.

Effects of maternal factors on CRP levels at 16 weeks gestation.

Maternal factors	Standardized Coefficients Beta	95% Wald Confidence Interval		Wald Chi-Square df1	P
CESD-R 16 weeks	0.033	−0.125	0.191	.177	0.679
Infection severity 16 weeks	0.202	0.047	0.356	6.841	0.011
Pre-pregnancy BMI	0.329	0.176	0.482	18.537	<0.001
Maternal education yrs	−0.050	−0.208	0.108	−.635	0.527