A prospective study to explore the relationship between MTHFR C677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women.

BACKGROUND: The etiology of postpartum psychopathologies are not well understood, but folate metabolism pathways are of potential interest. Demands for folate increase dramatically during pregnancy, low folate level has been associated with psychiatric disorders, and supplementation may improve symptomatology. The MTHFR C677T variant influences folate metabolism and has been implicated in depression during pregnancy.
OBJECTIVE: To conduct a prospective longitudinal study to explore the relationship between MTHFR C677T genotype, folate levels, and postpartum psychopathology in at-risk women. HYPOTHESIS: In the first three months postpartum, folate will moderate a relationship between MTHFR genotype and depression, with TT homozygous women having more symptoms than CC homozygous women.
METHODS: We recruited 365 pregnant women with a history of mood or psychotic disorder, and at 3 postpartum timepoints, administered the Edinburgh Postnatal Depression Scale (EPDS); Clinician-Administered Rating Scale for Mania (CARS-M) and the Positive and Negative Symptom Scale (PANSS) and drew blood for genotype/folate level analysis. We used robust linear regression to investigate interactions between genotype and folate level on the highest EPDS and CARS-M scores, and logistic regression to explore interactions with PANSS psychosis scores above/below cut-off.
RESULTS: There was no significant interaction effect between MTHFR genotype and folate level on highest EPDS (p = 0.36), but there was a significant interaction between genotype, folate level and log(CARS-M) (p = 0.02); post-hoc analyses revealed differences in the effect of folate level between CC/CT, and TT genotypes, with folate level in CC and CT having an inverse relationship with symptoms of mania, while there was no relationship in participants with TT genotype. There was no significant interaction between MTHFR genotype and folate level on the likelihood of meeting positive symptom criteria for psychosis on the PANSS (p = 0.86). DISCUSSION: These data suggest that perhaps there is a relationship between MTHFR C677T, folate level and some symptoms of postpartum psychopathology.

Introduction

Postpartum psychiatric disorders are urgent health concerns that have important implications for mothers, infants, and their families. Although all women are at risk for an episode of mental illness in the postpartum [1, 2], those who have a history of a mood or psychotic disorder are at greater risk compared to the general population [2-10].

Similar to non-perinatal psychiatric disorders, postpartum psychiatric disorders are thought to arise due to the combined effects of genetic and environmental factors. There is an abundance of literature investigating the role of genetic variations in psychiatric disorders such as schizophrenia and bipolar disorder [11], with accumulating research on gene-environment interactions [12]. However, in the context of postpartum psychiatric disorders, the majority of investigations focus on either environmental contributors or the role of genetics [13-18] with few studies investigating gene and environment interactions (e.g. 5HTTLPR/monoaminergic variations and stress [19, 20]).The need for studies of postpartum depression that integrate these elements has been recognized [21], and there are potential gene-environment interactions worthy of investigation in relation to postpartum psychopathology. One such potential example involves variations in the gene encoding the enzyme methylenetetrahydrofolate reductase, and folate.

Methyltetrahydrofolate reductase (MTHFR) is a folate dependent enzyme that has a common, functional, thermolabile variant—MTHFR C677T. It has been studied in the context of non pregnancy related psychiatric disorders, depression, bipolar disorder, and schizophrenia [20-22]–including a study of the relationship between psychopathology, folate, and the MTHFR C677T variant [22].

MTHFR C677T variants and low folate levels have also been separately studied, and associated with depressed mood during pregnancy [23, 24]. Folate deficiency has also been postulated as a contributor to postpartum psychosis and depression [24, 25], but no studies, to our knowledge, have explored whether the MTHFR C677T variant increases risk for postpartum psychiatric disorders and/or explored how postpartum physiological folate levels interact with these genotypes in relation to risk. Further, no studies to our knowledge have investigated the role of MTHFR C677T or folate levels in postpartum mania. Given the increased demands for folate during pregnancy, and the impact of folate deficiency on TT homozygous women [26], there is need to further understand the role of MTHFR C677T and folate levels on postpartum psychopathology, especially since there has been some suggestion of using folic acid or L-methylfolate supplements to treat low mood in the postpartum and the general population in those with MTHFR C677T variants [27, 28]. A more thorough understanding of genetic risk factors that may suggest remediatory biological interventions (e.g. perinatal folic acid supplementation tailored to MTHFR genotype) is critical to improving outcomes for women at risk for postpartum psychopathology.

The purpose of this study was to conduct a prospective, longitudinal, observational study to better understand the relationships between the MTHFR C677T variant, physiological folate levels and postpartum depression in at-risk women. We aimed to test the hypothesis that in the first three months postpartum, compared to MTHFR CC homozygous women, TT homozygous women would have increased symptoms of postpartum depression (PPD) and that this relationship would be moderated by physiological levels of red blood cell (RBC) folate. We also conducted exploratory analyses regarding the impact of MTHFR C677T genotypes and RBC folate levels on: a) postpartum mania (PPM) and b) postpartum psychosis (PPP).

Materials and methods

The study was approved by the University of British Columbia Research Ethics Board (H06-70145). All participants provided written informed consent.

Participants were recruited from the metropolitan Vancouver, Canada area between 2007 and 2016 (N = 365). Women were eligible to participate if they: a) had a history of a mood or psychotic disorder (depression, bipolar disorder, schizophrenia) as confirmed by the Structured Clinical Interview for Diagnosis (SCID) [29]; b) were pregnant; and c) were fluent in English. Details regarding recruitment methods are described elsewhere [30].

The study was observational; no experimental interventions were provided to participants. Data collection occurred at 4 timepoints: T1 (during pregnancy (>15 weeks gestation)); T2 (1–2 week(s) postpartum); T3 (1–2 month(s) postpartum); and T4 (3–4 months postpartum).

At T1, we collected demographic information, and at each timepoint, blood was drawn (for measuring RBC folate, and MTHFR genotyping). Also at each timepoint participants completed the Edinburgh Postnatal Depression Scale (EPDS) to assess depression symptomatology (see below), and a trained researcher administered the Clinician Administered Rating Scale (CARS-M) to assess mania symptomatology (see below), and the Positive and Negative Symptom Scale (PANSS) to assess psychosis symptomatology (see below). Participants also provided information regarding use of folic acid supplements and psychotropic medication at each timepoint.

Data were managed using REDCap (Research Electronic Data Capture) tools hosted at BC Children’s Hospital Research Institute. REDCap is a secure, web-based application designed to support data capture for research studies [31].

Instruments

Edinburgh Postnatal Depression Scale (EPDS)

The EPDS is a 10-item, self-administered, Likert scale-based questionnaire (each item is rated by selecting from 4 options, scored from 0 to 3) that has been validated for measuring both prenatal and postpartum depression [32]. Higher scores indicate greater depression symptomatology.

Clinician Administered Rating Scale for Mania (CARS-M)

The 10-item CARS-M is a clinician rated, reliable and valid measure of the severity of mania symptomatology with or without psychotic features. On the basis of an interview and observation, severity of symptomatology is assessed by rating each item from 0 (absent) to 5 (extreme) [33].

Positive and Negative Syndrome Scale (PANSS)

The PANSS is a well-validated instrument (completed by a clinically trained rater, on the basis of a 30–45 minute semi-structured interview) that measures the presence and severity of 30 psychiatric symptoms [34]. Each symptom is rated on a 7-point scale; a score of 1 means the symptom is not present, and a score of 7 means that it is present to an extreme degree. Five of the PANSS items can be used to assess the presence and severity of psychosis, using specific cut off scores [35, 36]. Specifically we categorized psychosis as present if a participant met at least one of the following threshold scores (delusions ≥3, conceptual disorganization ≥5 [37], hallucinations ≥3, suspiciousness ≥5, unusual thought content ≥4).

To ensure agreement between multiple raters (inter-rater concordance) for the PANSS, we conducted periodic PANSS inter-rater concordance sessions and calculated coefficient of agreement (determined by % agreement within 1 rating point amongst all raters) for each of the five PANSS items for psychosis [38].

Biological measures

Folate

RBC folate was measured using an Abbott Architect i1000 immunoanalyzer (Abbott Diagnostics,Canada, Mississauga Ont), folate reagent kit (#B1P740) and Abbott folate calibrators and quality controls. RBC folate levels were corrected for hematocrit and plasma folate.

MTHFR C677T genotyping

DNA was extracted from buffy coats from whole blood (or if blood samples were unavailable, buccal swabs) using Qiagen QIAmp DNA cultured cells protocol. Real-time PCR using Taqman primers/probes determined genotypes of MTHFR C677T (ie CC, CT, or TT).

Analysis

To allow for differences in timing for the emergence of symptoms of depression and mania we selected the highest postpartum EPDS and CARS-M scores (from all available postpartum timepoints) for each participant and the corresponding RBC folate levels (i.e. RBC folate measured at the time of the highest EPDS or CARS-M) for analysis. When the highest score persisted for more than one timepoint, we used the earliest timepoint with available corresponding RBC folate level. Similarly, to allow for differences in timing for the emergence of key positive symptoms of psychosis, we used the earliest postpartum timepoint for which the participant met criteria for psychosis (determined by the PANSS scores defined above) with available corresponding RBC folate data. If the participant never met criteria for psychosis we used the earliest timepoint with available RBC folate data.

To investigate relationships between: depression (EPDS) or mania (CARS-M) symptoms in the postpartum, MTHFR genotype, and RBC folate levels, we used robust linear regression with MM estimation to mitigate the effects of outliers and highly influential points [39, 40] as implemented in the ‘robustbase’ package [41]. The presence of outliers and influential data was assessed using diagnostic regression plots of non-robust models. Additionally, the results of the robust regressions were different enough from the non-robust regressions to justify their use. Moderation of the relationship between MTHFR and EPDS or CARS-M by RBC folate levels, was tested by including an interaction term in the models [42]. If a significant interaction term was detected, we estimated the difference in slope among the CC, CT, and TT genotypes using asymptotic Chi-square tests [43] with Benjamini-Hochberg false-discovery rate p-value correction [44]. Otherwise, the interaction term was removed and the main effects of MTHFR genotype and RBC folate were estimated. CARS-M scores were log transformed to better meet the assumptions of normality and homogeneity of variance. We calculated Cohen’s d values to estimate the effect size of the difference in mean EPDS and CARS-M scores between CC, CT, and TT genotypes, controlled for RBC folate.

We investigated relationships between: likelihood of meeting criteria for psychosis on the PANSS, MTHFR genotype, and RBC folate levels with logistic regression.

Statistical significance was assumed at p < 0.05.

We used descriptive statistics to report demographic data. To test differences in demographic and clinical variables between genotypes we used Kruskal-Wallis tests for continuous variables and Fisher Exact tests for categorical variables.

Results

The study enrolled 365 women. Sufficient postpartum data was collected from 327 participants to be included in analyses (i.e. completed the PANSS, EPDS, or CARS-M, had corresponding RBC folate data for at least one postpartum timepoint, and were genotyped for MTHFR C677T). There were no significant differences in demographic characteristics between MTHFR genotype groups, and distribution of genotype groups did not deviate from Hardy-Weinberg equilibrium (χ2 = 3.67, df = 1, p = 0.06) (see Table 1).

Table 1

Demographics for each MTHFR C677T genotype.

		MTHFR genotype
	Total	TT	CT	CC	P-value
	N = 327	n = 37	n = 125	n = 165
Age (years) (n = 326)
Mean (SD)	31.1 (±5.7)	31.0 (±6.4)	30.8 (±5.9)	31.3 (±5.4)	0.83
Annual Household Income ($ CAD) (n = 316)
<$20,000	32 (10.1%)	4 (11.1%)	12 (9.8%)	16 (10.1%)	0.16
$20,000 - $40,000	54 (17.1%)	11 (30.6%)	24 (19.7%)	19 (12.0%)
$41,000 - $60,000	55 (17.4%)	6 (16.7%)	21 (17.2%)	28 (17.7%)
$61,000 - $80,000	62 (19.6%)	6 (16.7%)	24 (19.7%)	32 (20.3%)
$81,000 - $100,000	44 (13.9%)	6 (16.7%)	19 (15.6%)	19 (12.0%)
>$100,000	69 (21.8%)	3 (8.3%)	22 (18.0%)	44 (27.8%)
Ethnicity (n = 321)
European	243 (75.7%)	24 (66.7%)	91 (72.8%)	128 (80.0%)	0.38
Asian	24 (7.5%)	3 (8.3%)	8 (6.4%)	13 (8.1%)
Mixed	42 (13.1)	7 (19.4%)	20 (16.0%)	15 (9.4%)
Other* (includes African and Aboriginal)	10 (3.1%)	2 (5.6%)	5 (4.0%)	3 (1.9%)
Highest level of education (n = 315)
High school	24 (7.6%)	4 (11.4%)	10 (8.3%)	10 (6.3%)	0.79
Less than 4 years of college/university	121 (38.4%)	10 (28.6%)	47 (38.8%)	64 (40.3%)
4 or more years of college/university	157 (49.8%)	20 (57.1%)	58 (47.9%)	79 (49.7%)
Did not complete high school	13 (4.1%)	1 (2.9%)	6 (5.0%)	6 (3.8%)
Employment (n = 326)
Not employed	48 (14.7%)	4 (10.8%)	23 (18.4%)	21 (12.8%)	0.34
Employed	278 (85.3%)	33 (89.2%)	102 (81.6%)	143 (87.2%)
Body Mass Index (BMI) (n = 296)
Mean (SD)	29.1 (±5.6)	28.7 (±5.4)	28.7 (±5.5)	29.4 (±5.7)	0.74
Gravida (n = 324)
Median (IQR)	2.0 (1.0–3.0)	2.0 (1.0–3.0)	2.0 (1.0–3.0)	2.0 (1.0–3.0)	0.79
Number of children at enrollment (n = 324)
Median (IQR)	0.0 (0.0–1.0)	0.0 (0.0–1.0)	0.0 (0.0–1.0)	0.0 (0.0–1.0)	0.83
IVF pregnancy (n = 320)
no	307 (95.9%)	33 (94.3%)	115 (94.3%)	159 (97.5%)	0.26
yes	13 (4.1%)	2 (5.7%)	7 (5.7%)	4 (2.5%)
Marital status (n = 324)
Married/Common-Law/Partnered	302 (93.2%)	33 (97.3%)	109 (88.6%)	157 (95.7%)	0.04
Single	22 (6.8%)	1 (2.7%)	14 (11.4%)	7 (4.3%)
Psychiatric Diagnosis (n = 326)
Bipolar Disorder	60 (18.4%)	7 (18.9%)	21 (16.8%)	32 (19.5%)	0.95
Depression	264 (81.3%)	30 (81.1%)	103 (82.4%)	131 (79.9%)
Schizophrenia	2 (0.6%)	0 (0.0%)	1 (0.8%)	1 (0.6%)
Previous History of psychotic symptoms (n = 326)
no	251 (77.0%)	28 (75.7%)	102 (81.6%)	121 (73.8%)	0.28
yes	75 (23.0%)	9 (24.3%)	23 (18.4%)	43 (26.2%)
Psychotropic medication in the postpartum (n = 325)
no	216 (66.5%)	24 (64.9%)	84 (67.7%)	108 (65.9%)	0.93
yes	109 (33.5%)	13 (35.1%)	40 (32.3%)	56 (34.1%)
RBC folate (ng/ml)
Mean (SD)	663.5 (261.7)	777.0 (332.3)	650.0 (198.4)	648.2 (189.8)	0.097
Taking folic acid supplement throughout postpartum (n = 326)
no	127 (39.0%)	12 (33.3%)	48 (38.4%)	67 (40.6%)	0.71
yes	199 (61.0%)	24 (66.7%)	77 (61.6%)	98 (59.4%)

Depression

There were 305 participants whose highest postpartum EPDS had a corresponding RBC folate available (see Table 2).

Table 2

Highest EPDS (depression) scores and the corresponding RBC folate levels and medication/supplement data for each MTHFR C677T genotype.

		MTHFR genotype
	Total	TT	CT	CC	p-value
	N = 305	n = 33	n = 116	n = 156
Highest postpartum EPDS score
Mean (SD)	9.4 (5.2)	10.4 (5.9)	9.1 (5.1)	9.4 (5.2)	0.48
RBC folate (ng/ml)
Mean (SD)	645.8 (218.5)	722.0 (257.2)	648.2 (217.1)	627.8 (208.4)	0.08
Taking a Folic Acid Supplement (n = 303)
no	72 (23.8%)	5 (15.6%)	23 (19.8%)	44 (28.3%)	0.14
yes*	231 (76.2%)	27 (84.4%)	93 (80.2%)	111 (71.6%)
Taking a daily Psychotropic Medication (n = 304)
no	230 (75.7%)	23 (69.7%)	90 (78.3%)	117 (75.0%)	0.57
yes	74 (24.3%)	10 (30.3%)	25 (21.7%)	39 (25.0%)

*n = 1 participant took 5-MTHF supplements (not folic acid).

RBC folate and highest postpartum EPDS scores (EPDS vs RBC folate) are shown for each MTHFR genotype in Fig 1. There was no significant interaction between MTHFR genotype and RBC folate level on highest EPDS (p = 0.36). There was also no relationship between RBC folate and EPDS on its own (coefficient = -0.002 (95%CI = -0.004 to 0.0008), p = 0.19, adjusted R-squared = 0.002), and no difference between genotypes for mean EPDS controlling for RBC folate (p = 0.59, Cohen’s d values: CC compared to CT: d = 0.02, CC compared to TT: d = 0.16, and CT compared to TT: d = 0.18).

Fig 1

(n = 305) EPDS (depression) score vs. RBC folate (ng/ml) score for each MTHFR C677T (TT, CT, CC) genotype (dashed line indicates confidence intervals).

Mania

There were 233 women whose highest CARS-M had a corresponding RBC folate level (Table 3).

Table 3

Highest CARS-M (mania) score and the corresponding RBC folate levels and medication/supplement data for each MTHFR C677T genotype.

		MTHFR genotype
	Total	TT	CT	CC	p-value
	N = 233	n = 24	n = 90	n = 119
Highest postpartum CARS-M score
Mean (SD)	6.8 (3.8)	7.46 (3.3)	7.1 (3.7)	6.5 (3.9)	0.31
RBC folate (ng/ml)
Mean (SD)	672.4 (227.9)	818.9 (342.4)	659.4 (199.1)	652.7 (210.9)	0.004
Taking a Folic Acid Supplement (n = 232)
no	52(22.4%)	4 (17.4%)	13 (14.4%)	35 (29.4%)	0.03
yes*	180 (77.6%)	19 (82.6%)	77 (85.6%)	84 (70.6%)
Taking a daily psychotropic medication
no	176 (75.5%)	16 (43.2%)	73 (81.1%)	87 (73.1%)	0.22
yes	57 (24.5%)	8 (33.3%)	17 (18.9%)	32 (26.9%)

* n = 1 participant took 5-MTHF supplements (not folic acid).

RBC folate level and log transformed CARS-M (CARS-M vs RBC folate) scores are shown for each MTHFR genotype in Fig 2. There was a significant interaction between MTHFR genotype and RBC folate level on highest (log)CARS-M scores (p = 0.02). Post-hoc analyses suggested no significant differences between the slopes (CARS-M vs RBC Folate) for MTHFR CC and CT genotypes (p = 0.65), but suggested that there was a significant difference between the slopes of CARS-M vs RBC folate for MTHFR CC and TT (p = 0.03) genotypes, and between the slopes of CARS-M vs RBC folate for MTHFR CT and TT (p = 0.03) genotypes (Fig 2). The slopes for the CC and CT genotype groups showed an inverse relationship between RBC folate and CARS-M, with higher folate levels associated with lower CARS-M scores. However, the slope for the TT genotype suggests that there was not a strong relationship between CARS-M and RBC folate within the TT genotype group.

Fig 2

(n = 233) CARS-M score vs. RBC folate (ng/ml) score for each MTHFR C677T (TT, CT, CC) genotype (dashed line indicates confidence intervals).

Psychosis

Coefficient of agreement for each of the five PANSS items (delusions, conceptual disorganization, hallucinations, suspiciousness, and unusual thought content) from PANSS interrater concordance sessions held over the course of the study were 0.85, 0.70, 0.90, 0.77, and 0.85 with an overall mean coefficient of agreement of 0.82.

There were 326 women with sufficient postpartum PANSS data and corresponding RBC folate data, for at least one postpartum timepoint (Table 4).

Table 4

Earliest presence of psychotic symptoms and the corresponding RBC folate levels and medication/supplement data for each MTHFR C677T genotype.

		MTHFR genotype
	Total	TT	CT	CC	p-value
	N = 326	n = 37	n = 124	n = 165
PANSS Psychosis Criteria Met
no	261 (80.0%)	33 (89.2%)	96 (77.4%)	132 (80.0%)	0.31
yes	65 (20.1%)	4 (10.8%)	28 (22.6%)	33 (20.0%)
RBC folate (ng/ml)
Mean (SD)	705.0 (238.7)	830.4 (369.8)	698.9 (206.9)	681.4(216.1)	0.002
Taking a Folic Acid Supplement (n = 324)
no	71 (21.9%)	6 (16.7%)	24 (19.4%)	42 (25.6%)	0.42
yes*	253 (78.1%)	30 (83.3%)	100 (80.6%)	122 (73.9%)
Taking a daily psychotropic medication (n = 324)
no	244 (75.3%)	28 (75.7%)	95 (77.2%)	121 (73.8%)	0.82
yes	80 (24.7%)	9 (24.3%)	28 (22.6%)	43 (26.2%)

* n = 1 participant took 5-MTHF supplements (not folic acid).

There was no significant interaction between MTHFR genotype and RBC folate level on the probability of meeting psychotic symptom criteria on the PANSS (p = 0.86). RBC folate levels and whether participants met criteria for psychosis for each MTHFR genotype is shown in Fig 3. There was also no relationship between RBC folate and psychotic symptoms on its own (OR = 1.00, 95%CI = 0.99 to 1.01, p = 0.09), and there was also no difference between genotypes for psychotic symptoms controlling for RBC folate (p = 0.86; Odds Ratio (OR) for CT compared to CC = 1.16 (95%CI = 0.66 to 1.03); OR for TT compared to CC = 1.12 (95%CI = 0.44 to 1.60)).

Fig 3

(n = 326) Predicted probably of meeting threshold criteria for psychosis vs. RBC folate (ng/ml) for each MTHFR C677T (TT, CT, CC) genotype (dashed line indicates confidence intervals).

Regression coefficients and data from both linear (depression and mania) and logistic (psychosis) regression analyses are displayed in S1 Table.

Discussion

This is the first study to our knowledge to explore relationships between MTHFR C677T variants, physiological levels of folate, and postpartum psychiatric symptoms, and specifically it is the first study to explore postpartum mania and any associations with MTHFR or folate levels. Our data suggest that, for women with a MTHFR C677T C allele (i.e. CC or CT genotypes), folate levels and mania symptoms may be inversely related, but that there is no relationship between folate levels and mania symptoms in women with a TT genotype. Interestingly, our data also showed that mean mania scores for women with a TT genotype did not significantly differ from those of women with CC or CT genotypes. While visually (see Fig 1) there is an appearance of an inverse relationship between depression scores and folate levels for women with CC and CT genotypes, that reflects the relationships found with mania, in this case, the relationship was not statistically significant. Given the small effect size of the difference in EPDS scores (when controlling for RBC folate levels) between TT genotypes and those with a C allele (CC and CT), it is possible that a relationship between MTHFR genotype and RBC folate levels (like we observed with mania symptoms) also exists for depressive symptoms, but the sample size was underpowered to detect it. Our data do not suggest a relationship between psychosis and folate levels and MTHFR C677T genotype.

MTHFR C677T was historically considered a strong candidate gene for a variety of psychiatric disorders (outside the perinatal setting) and was initially implicated in schizophrenia [45-50], bipolar and unipolar depression [50-53], though this association was not supported by genome wide association study (GWAS) findings [54, 55]. Our data suggest that MTHFR C677T genotype alone does not increase risk for postpartum psychopathology. Our findings are also broadly in line with previous studies that have shown that low folate (and thus subsequent high levels of homocysteine) is associated with various forms of psychopathology [56-59]. However, our findings suggest that this inverse relationship may only exist in the postpartum for women with a MTHFR C677T C allele, at least in terms of mania, and, perhaps, depressive symptoms.

One study that investigated the impact of folate levels on perinatal depression, without accounting for MTHFR genotype, did not find any relationship between plasma folate levels and depression in the postpartum period [24]. While our study also did not find a significant relationship of RBC folate with postpartum depression symptomatology, our results suggest that there may be an inverse relationship with other mood symptoms (mania), and possibly depression, when the effect of MTHFR genotype is considered.

While retrospective observational studies have suggested that self-reported perinatal folic acid supplementation can improve maternal depression symptomology several months postpartum, especially in women with a MTHFR TT genotype [28] our findings suggest that women with a TT genotype do not demonstrate an inverse relationship between physiological folate and mood symptoms (i.e. mania and perhaps, depression). Our results also suggest that overall there is no difference in physiological folate levels based on MTHFR C677T genotype (Table 1), but at times there may be higher physiological folate levels in those with TT genotypes (Tables 3 and 4), despite previous research suggesting that individuals who are TT homozygous would be more prone to folate deficiency [60]. These differences in our study may be due to the fact that a perinatal population is highly supplemented (e.g. in our study population the majority of women were taking 1 mg of folic acid daily, in addition to folate and folic acid consumed through diet) compared to studies in the general population. It is also possible that further studies of MTHFR C677T variants in pregnancy and postpartum cohorts are needed to fully understand the impact of pregnancy on enzyme function.

Limitations

Since most of the women in our study were taking folic acid supplements, and all were living in Canada (a country with folic acid fortified foods), it is possible that an inverse relationship between physiological folate levels and psychiatric symptoms does exist for women who are MTHFR TT homozygotes; however, this relationship is not observable in populations with adequate folic acid supplementation (i.e. at a certain RBC folate level there may be a ceiling effect (for TT homozygotes) for the impact of folate on psychiatric symptoms). While there was no statistical difference in RBC folate levels (overall–see Table 1) between CC, CT, and TT genotypes, those with a TT genotype consistently had the highest RBC folate levels, which reached statistical significance when there was the highest levels of manic symptoms or presence/absence of psychotic symptoms, suggesting that the TT group was more than adequately supplemented, perhaps mitigating any negative impact of the TT genotype on psychopathology that would be observed when folic acid supplementation (and RBC folate) is less abundant. It is also possible that, while overall there was no difference in the proportions of women taking a folic acid supplements across the genotype groups (Table 1), participants with a TT genotype may, by chance, have been taking a greater amount of folic acid.

Our data may be enriched for missing data at timepoints when women were the most depressed or experiencing psychotic symptoms (resulting in data only being available for a single timepoint, where symptoms may be less severe), as women may have cancelled study visits during periods of poorer mental health. This possibility increases the chance of a Type II error, whereby results indicate the absence of relationship that truly exists. Because our study population consisted of women who were already at a high risk for postpartum psychiatric disorders, due to their past history of a mood or psychotic disorder [2-10], our results may not be generalizable to other populations of postpartum women. As our study did not investigate the role of elevated homocysteine, future studies could explore relationships with MTHFR C677T genotype, psychopathology, and homocysteine levels.

Conclusions

There may be a relationship between MTHFR C677T genotype, RBC folate levels, and risk for some forms of postpartum psychopathology, at least in the context of high-risk Canadian women with adequate folic acid supplementation. Further studies with larger samples of women may be needed to characterize the nuances in relationships between postpartum depression and mania symptoms in relation to MTHFR C677T genotype and folate levels.

Regression coefficients and data from linear and logistic regression analyses.

(DOCX)

Click here for additional data file.

1 Sep 2020

PONE-D-20-16875

A prospective study to explore the relationship between MTHFRC677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women

PLOS ONE

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PLOS ONE

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors analyse the relation between MTHFR C677T genotype, folate levels, and postpartum psychopathology in at-risk women. The Authors hypothesise that, in the first three months postpartum, compared to MTHFR CC homozygous women, TT homozygous women would have increased symptoms of postpartum depression and that this relationship would be moderated by physiological levels of red blood cell (RBC) folate. In addition, they conduct an exploratory analysis to assess the impact of MTHFR C677T genotypes and RBC folate levels on postpartum mania and postpartum psychosis.

The paper could benefit from clarification about data as well as the methodologies used. The Authors should provide more details on the modelling choices to help readers in having a clear understanding on the proposed study. Some important issues are listed below.

1. The Authors conducted a longitudinal observational study on a sample of 365 pregnant women with a history of a mood or psychotic disorder and that data collection occurred at 4 timepoints. However, the longitudinal aspect of this study is not exploited in the analysis where only the highest postpartum EPDS and CARS-M scores (from all the available postpartum timepoints) and the corresponding RBC folate levels were selected. Along the same line, RBC folate levels corresponding to the first postpartum timepoint for which the participant met criteria for psychosis, or to the earliest time point for participants which never met criteria for psychosis were chosen. All these choices might be due to a high presence of missing values at different time points but this is not clear from the text, where the only reason provided is linked to differences in timing for the emergence of symptoms of depression, mania and psychosis. The Authors should discuss in more detail why they do not perform a longitudinal statistical analysis.

2. To assess the hypothesised relationships, the Authors run a moderation analysis but they do not include any references to the literature on the moderation model. They should provide a clear and rigorous description of the moderation model implemented.

3. The Authors affirm that there are outliers in the data but they do not specify if the outliers affect only the dependent variables or also the folate levels, and neither do they provide statistical results to support their claim

4. To evaluate the relationships included in the moderation model, the Authors use robust linear regression. At line 192, they refer to “minimum maximum likelihood estimation”, which to the best of my knowledge does not exist. They are probably referring to the MM-estimator introduced by Yohai (1987). The Author should provide references for this method and specify the software used to perform the analysis.

Yohai, J. V. (1987). High Breakdown-Point and High Efficiency Robust Estimates for Regression, Annals of Statistics, 17, 4, 1662-1683.

5. How were post-hoc analyses conducted? Methodological aspect should be detailed in the manuscript.

6. I would suggest to move Table 1: Demographic information for each MTHFR C677T genotype, into the Supplementary data, and to include the results provided in Supplementary Table 1 in the manuscript, along with p-values, which are not reported in this Table.

7. In the Discussion, the Authors affirm that an inverse relationship between folate and depressive symptoms may exist in the postpartum for women with a MTHFR C677T 341 C. However, this conclusion is not supported by the analysis.

8. At lines 372-375, it reads “While there was no statistical difference in RBC folate levels […] between CC, CT, and TT genotypes,”. p-values reported in Tables 2 (p=0.08), 3 (p=0.004) and 4 (p=0.002) do not support this conclusion. This has to be clarified.

9. In the Limitation section, the Authors state that “participants with a TT genotype may, by chance, have been taking a greater amount of folic acid” (line 379). Could it be worth of investigation the inclusion of covariates, related to whether participants were taking a Folic Acid Supplement or a daily psychotropic medication, into the model?

Reviewer #2: The manuscript entitled “A prospective study to explore the relationship between MTHFRC677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women” is an interesting and well structured study investigating the influence of MTHFR C677T polymorphism and folate blood levels on the onset of psychopathological disorders in the postpartum. The study design is methodologically flawless and statistical data analysis is appropriate and properly conducted. The graphs are immediately explanatory. It is also written in fluent and clear English. The topic is interesting for a wide audience. An increase in homocysteine levels resulting from folate deficiency and / or MTHFR mutations, has also been described as a possible risk factor for neuropsychiatric disorders, as well as for various other pathological situations, such as cardiovascular disease and osteoporosis, probably facilitating an increase in inflammatory factors, as suggested in a recent paper, to which the Authors could possibly refer (De Martinis M, Sirufo MM, Nocelli C, Fontanella L, Ginaldi L. Hyperhomocysteinemia is Associated with Inflammation, Bone Resorption, Vitamin B12 and Folate Deficiency and MTHFR C677T Polymorphism in Postmenopausal Women with Decreased Bone Mineral Density. Int J Environ Res Public Health 2020;17(12):4260.doi:10.3390/ijerph17124260). Are there data available on homocysteine levels in the women studied? The lack of this information should be considered among the limitations of the study. It might be interesting to evaluate them, maybe even in a future study. Since hyperomocysteinemia could be a pathogenetic mechanism that may help explain the effects of folate deficiency and MTHFR C677T polymorphism, I suggest that the Authors briefly address this topic in the discussion.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Report to the Authors.pdf

Click here for additional data file.

7 Oct 2020

We thank the reviewers for the comments and feedback on our manuscript. We have documented our responses below (in bold italics) and have made the necessary changes to the manuscript.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Thank you. Changes have been made to reflect the formatting style.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was made possible through funding from the Canadian Institutes of

Health Research (CIHR), JA was supported by BC Mental Health and Substance Use

Services, the Canada Research Chairs Program, the Michael Smith Foundation for Health

Research, and CIHR."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"JA received funding from the Canadian Institutes of Health Research (https://cihr-irsc.gc.ca/e/193.html) to conduct this work (CIHR MOP 82750). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Thank you for bringing this to our attention. We have deleted this text from our Acknowledgements. Please update our funding statement to say:

This work was made possible through funding from the Canadian Institutes of

Health Research (CIHR), JA was supported by BC Mental Health and Substance Use

Services, the Canada Research Chairs Program, the Michael Smith Foundation for Health

Research, and CIHR. JA received funding from the Canadian Institutes of Health Research (https://cihr-irsc.gc.ca/e/193.html) to conduct this work (CIHR MOP 82750). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

Our data contains potentially sensitive information. Further, in order to share de-identified data, our Research Ethics Board requires that participants explicitly consent. Unfortunately this was not included in our consent form for this study and we are unable to provide a de-identified data set to be made accessible. Data access requests can be sent to the University of British Columbia Clinical Research Ethics board (ethics.research.ubc.ca).

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Thank you for bringing this to our attention. We have included these captions in the manuscript and updated our in-text citations.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors analyse the relation between MTHFR C677T genotype, folate levels, and postpartum psychopathology in at-risk women. The Authors hypothesise that, in the first three months postpartum, compared to MTHFR CC homozygous women, TT homozygous women would have increased symptoms of postpartum depression and that this relationship would be moderated by physiological levels of red blood cell (RBC) folate. In addition, they conduct an exploratory analysis to assess the impact of MTHFR C677T genotypes and RBC folate levels on postpartum mania and postpartum psychosis.

The paper could benefit from clarification about data as well as the methodologies used. The Authors should provide more details on the modelling choices to help readers in having a clear understanding on the proposed study. Some important issues are listed below.

1. The Authors conducted a longitudinal observational study on a sample of 365 pregnant women with a history of a mood or psychotic disorder and that data collection occurred at 4 timepoints. However, the longitudinal aspect of this study is not exploited in the analysis where only the highest postpartum EPDS and CARS-M scores (from all the available postpartum timepoints) and the corresponding RBC folate levels were selected. Along the same line, RBC folate levels corresponding to the first postpartum timepoint for which the participant met criteria for psychosis, or to the earliest time point for participants which never met criteria for psychosis were chosen. All these choices might be due to a high presence of missing values at different time points but this is not clear from the text, where the only reason provided is linked to differences in timing for the emergence of symptoms of depression, mania and psychosis. The Authors should discuss in more detail why they do not perform a longitudinal statistical analysis.

We appreciate this comment and the opportunity to provide further clarity in the manuscript. Indeed, as the reviewer mentions, there are some missing values, resulting in not all participants having data at each timepoint, limiting the number of participants who have data across all timepoints. The focus of our study; however, was not on the longitudinal evolution of psychiatric symptoms but, rather to observe the most severe symptoms (depression and mania) or if they every met criteria (psychosis), in our study time frame to test differences between genotypes. We have added the limitation of missing data to our limitation section to reflect how this may have influenced our findings.

2. To assess the hypothesised relationships, the Authors run a moderation analysis but they do not include any references to the literature on the moderation model. They should provide a clear and rigorous description of the moderation model implemented.

We have modified the analysis section of our manuscript to provide more clarity on our model and have included references where necessary.

3. The Authors affirm that there are outliers in the data but they do not specify if the outliers affect only the dependent variables or also the folate levels, and neither do they provide statistical results to support their claim

We thank the reviewer for highlighting this – we have added further details to our analysis section to better explain our analyses of outliers; specifically that their presence was assessed using diagnostic regression plots of non-linear models.

4. To evaluate the relationships included in the moderation model, the Authors use robust linear regression. At line 192, they refer to “minimum maximum likelihood estimation”, which to the best of my knowledge does not exist. They are probably referring to the MM-estimator introduced by Yohai (1987). The Author should provide references for this method and specify the software used to perform the analysis.

Yohai, J. V. (1987). High Breakdown-Point and High Efficiency Robust Estimates for Regression, Annals of Statistics, 17, 4, 1662-1683.

The reviewer is correct and we have revised the manuscript and have have added appropriate references regarding our use of MM-estimation to mitigate the effects of outliers and highly influential points.

5. How were post-hoc analyses conducted? Methodological aspect should be detailed in the manuscript.

We have provided more specific details about our “post-hoc” analysis in the manuscript.

6. I would suggest to move Table 1: Demographic information for each MTHFR C677T genotype, into the Supplementary data, and to include the results provided in Supplementary Table 1 in the manuscript, along with p-values, which are not reported in this Table.

We thank the reviewers for this suggestion. The p-values associated with the Supplementary data appear in the main body of the manuscript. As table 1 contains information fundamental to interpretation of the data, we have left this in the main body of the manuscript.

7. In the Discussion, the Authors affirm that an inverse relationship between folate and depressive symptoms may exist in the postpartum for women with a MTHFR C677T 341 C. However, this conclusion is not supported by the analysis.

We appreciate the opportunity to clarify this for the reviewer. The reviewer is correct, there is no relationship. We have edited our previously ambiguous wording to make this clearer in the manuscript.

8. At lines 372-375, it reads “While there was no statistical difference in RBC folate levels […] between CC, CT, and TT genotypes,”. p-values reported in Tables 2 (p=0.08), 3 (p=0.004) and 4 (p=0.002) do not support this conclusion. This has to be clarified.

We thank the reviewer for highlighting this discrepancy. This statement in the limitations section was an oversight on our part. We have edited it to more accurately reflect our data.

9. In the Limitation section, the Authors state that “participants with a TT genotype may, by chance, have been taking a greater amount of folic acid” (line 379). Could it be worth of investigation the inclusion of covariates, related to whether participants were taking a Folic Acid Supplement or a daily psychotropic medication, into the model?

We thank the reviewer for this comment. We have included whether participants were taking a folic acid supplement, and psychotropic medication in Table 1 and compared against genotype groups, for which there were no significant differences - with the majority of individuals taking a folic acid supplement, and a minority taking medications for all genotype groups.

Reviewer #2: The manuscript entitled “A prospective study to explore the relationship between MTHFRC677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women” is an interesting and well structured study investigating the influence of MTHFR C677T polymorphism and folate blood levels on the onset of psychopathological disorders in the postpartum. The study design is methodologically flawless and statistical data analysis is appropriate and properly conducted. The graphs are immediately explanatory. It is also written in fluent and clear English. The topic is interesting for a wide audience.

An increase in homocysteine levels resulting from folate deficiency and / or MTHFR mutations, has also been described as a possible risk factor for neuropsychiatric disorders, as well as for various other pathological situations, such as cardiovascular disease and osteoporosis, probably facilitating an increase in inflammatory factors, as suggested in a recent paper, to which the Authors could possibly refer (De Martinis M, Sirufo MM, Nocelli C, Fontanella L, Ginaldi L. Hyperhomocysteinemia is Associated with Inflammation, Bone Resorption, Vitamin B12 and Folate Deficiency and MTHFR C677T Polymorphism in Postmenopausal Women with Decreased Bone Mineral Density. Int J Environ Res Public Health 2020;17(12):4260.doi:10.3390/ijerph17124260). Are there data available on homocysteine levels in the women studied? The lack of this information should be considered among the limitations of the study. It might be interesting to evaluate them, maybe even in a future study. Since hyperomocysteinemia could be a pathogenetic mechanism that may help explain the effects of folate deficiency and MTHFR C677T polymorphism, I suggest that the Authors briefly address this topic in the discussion.

We thank the reviewer for this suggestion and have edited the limitations section accordingly and will explore the possibilities to examine this in our own future work.

Submitted filename: Response to Reviewers_oct7.docx

Click here for additional data file.

1 Dec 2020

A prospective study to explore the relationship between MTHFRC677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women

PONE-D-20-16875R1

Dear Dr. Austin,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kyoung-Sae Na, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The paper is acceptable for publication, although some interesting topics that we suggested to address in this first revision have been missed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

3 Dec 2020

PONE-D-20-16875R1

A prospective study to explore the relationship between MTHFR C677T genotype, physiological folate levels, and postpartum psychopathology in at-risk women

Dear Dr. Austin:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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