Maternal cytomegalovirus infection and delayed language development in children at 3 years of age–a nested case-control study in a large population-based pregnancy cohort

Regine Barlinn; Susanne G Dudman; Halvor Rollag; Lill Trogstad; Jonas C Lindstrøm; Per Magnus

doi:10.1371/journal.pone.0278623

. 2022 Dec 1;17(12):e0278623. doi: 10.1371/journal.pone.0278623

Maternal cytomegalovirus infection and delayed language development in children at 3 years of age–a nested case-control study in a large population-based pregnancy cohort

Regine Barlinn ^1,^*,^#, Susanne G Dudman ^2,^#, Halvor Rollag ², Lill Trogstad ³, Jonas C Lindstrøm ⁴, Per Magnus ^5,⁶

Editor: Zemenu Yohannes Kassa⁷

PMCID: PMC9714838 PMID: 36455052

Abstract

Introduction

Maternal cytomegalovirus (CMV) infection in pregnancy may result in vertical transmission of CMV to the child. Long-term effects of congenital CMV infection include visual, cognitive as well as neurological impairment. The aim of this study was to estimate the odds ratios for CMV seropositivity and seroconversion in mothers, with and without delayed language development in 3 year old children, nested within a large cohort.

Material and methods

The Norwegian Mother, Father and Child Cohort Study (MoBa) is a prospective population-based pregnancy cohort that includes 95 200 mothers and 114 500 children. Blood samples were obtained from mothers during pregnancy weeks 17 or 18 in pregnancy and after birth. We included 300 women from MoBa with children suffering from delayed language development at three years of age, based on validated questionnaires. Within the cohort, 1350 randomly selected women were included as controls to perform a nested case-control study. The cases and controls were tested for CMV IgG antibodies by an enzyme-linked immunosorbent assay.

Results

Among mothers of cases, 63.2% were CMV-IgG positive in the sample at birth, as compared to 55.9% among controls; OR 1.36, (95% CI; 1.05 to 1.76). Also, among case mothers, 8/118 (6.8%) initially seronegative cases, seroconverted. Among initially seronegative controls, seroconversion occurred in 23/618 (3.7%) mothers. The OR for seroconversion in cases as compared to control mothers was 1.88 (CI; 0.82 to 4.31), thus not statistically significant different.

Conclusion

This study shows a higher risk of delayed language development at three years of age in children born by mothers seropositive for CMV, compared to children born from seronegative mothers.

Introduction

Maternal cytomegalovirus (CMV) infection in pregnancy may result in vertical transmission of CMV to the fetus [1]. CMV is the most common congenital infection and affects 0.7% of all newborns [2]. The virus may be vertically transmitted both in primary and non-primary (reinfection and reactivation) CMV-infections. The transmission rate is between 30–40% during primary infection, which is high compared to around 1% in a non-primary infection [2, 3]. Despite the lower transmission rate, non-primary infections contribute to the majority of congenital infections, given that the seroprevalence is above 30% [1]. The CMV seroprevalence varies in different parts of the world and is influenced by age and socioeconomic factors [4]. In Norway, the CMV-IgG seroprevalence in pregnant women lies around 60% with a seroconversion rate of 3.7% [5, 6]. Of all infected newborns, 15–20% suffer from long-term sequelae. CMV is a leading cause of birth defects and developmental disabilities [7]. Congenital CMV infection (cCMV) is the most common cause of nonhereditary sensorineural hearing loss in childhood. Around 12% of cCMV cases will experience hearing loss [8]. Long-term impairment after cCMV may also include visual, cognitive as well as other neurological disabilities [7]. A Dutch study showed that speech and language problems were common in both children with cCMV and children without cCMV. Nevertheless, children with cCMV were most frequently affected [9]. Studies with long follow up are needed to gain knowledge on outcomes developing over several years in newborns with cCMV.

The aim of this nested case-control study was to estimate the association between CMV seropositivity and seroconversion in pregnant women with and without children who had delayed language development at the age of 3 years, within a large, population-based pregnancy cohort.

Materials and methods

Study population

The Norwegian Mother, Father and Child Cohort Study (MoBa) is a prospective population-based pregnancy cohort conducted by the Norwegian Institute of Public Health (NIPH). The main aim of MoBa is to provide a comprehensive database for understanding the causes of serious diseases among Norwegian women and children. Participants were recruited from all over Norway from 1999–2008 after giving informed consent [10, 11]. The cohort includes 95 200 mothers, 75 000 fathers and 114 500 children. Blood (plasma) samples were obtained from mothers in week 17 or 18 during pregnancy (M1) when inclusion was done and after birth (M2) [12]. The data used in this nested case-control study is based on Medical Birth Registry of Norway (MBRN) records and three questionnaires (Q) that the women responded to during pregnancy, and one when the child was 3 years old.

Case definition

We included 300 women who had children suffering from delayed language development at three years of age. The case definition was defined as severe (tick on point 1–3) or moderate (tick on point 4) problems in question 18, and a simultaneous score of ≤40 in question 21 in the 3 year questionnaire [13]. One case mother later withdrew from the study and was excluded, leaving 299 cases eligible for analysis (Tables 1 and 2).

Table 1. In question 18 of the questionnaire at 3 years of age, mothers are asked which of the six response categories best described the speech performance of their child.

Response categories in question 18
1- Not yet talking
2- He/she is talking, but you can’t understand him/her
3- Talking in one-word utterances, such as “milk” or “down”
4- Talking in 2- to 3-word phrases, such as “me got ball” or “give doll”
5- Talking in complete sentences, such as “I got a doll” or “can I go outside?”
6- Talking in long and complicated sentences, such as “when I went to the park, I went on the swings” or “I saw a man standing on the corner”.

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Table 2. In question 21, in the questionnaire at 3 years of age, mothers are asked to answer the six questions listed under in either “yes, often”, “sometimes”, or “not yet”, according to whether the child can do the activity (“yes” scored 10 points, “sometimes” = 5 points and “not yet” scored 0 points).

Question 21
1.Without showing him/her first, does your child point to the correct picture when you say, “Where is the cat” or “Where is the dog”?
2.When you ask your child to point to his/her eyes, nose, hair, feet, ears, and so forth, does he/she correctly point to at least seven body parts?
3.Does your child make sentences that are three or four words long?
4.Without giving him/her help by pointing or using gestures, ask your child to “Put the shoe on the table” and “Put the book under the chair”. Does your child carry out both of these directions correctly?
5.When looking at a picture book, does your child tell you what is happening or what action is taking place in the picture? (For example, “Barking”, “Running”, “Eating” and “Crying”?) You may ask, “What is the dog (or boy) doing?”
6.Can your child tell you at least two things about an object he/she is familiar with?

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Controls

Within the MoBa cohort, 1350 randomly selected women, with the birth registered in the MBRN were included as controls. One control mother later withdrew from the study and was excluded, leaving 1349 controls eligible for analysis. After starting to analyse, it became evident that one control had only the M2 sample.

Further inclusion and exclusion criteria for cases and controls

All included women had given birth to a live born child, had available plasma samples and had completed questionnaire 1 (completed at week 17) and questionnaire 6 (completed at 3 years of age). We excluded children from multiple pregnancies, children with chromosomal abnormalities as registered in the MBRN, and children selected to participate as cases or controls in another ongoing study within MoBa, the autism birth cohort (ABC) study [14].

Outcome measures

The outcome measure based on the maternal response to the questionnaire at 3 years of age. The questions were derived from the Ages and Stages Questionnaire (ASQ) communication scale, which is found to show good test-retest agreement and concurrent validity [15, 16].

In addition we used a language grammar rating scale, in which the mother was asked to choose 1 of 6 categories, ranging from no word production to full sentences with complete grammatical markings [17]. A validation was conducted to assess the questionnaire’s ability to detect language development by comparing the parent`s response to concomitant psychological examination of the child at 3 years showing high correlation [18]. The six categories of question 21 were derived from the Ages and Stages Questionnaires and were designed to assess the child’s ability to comprehend and convey.

Other variables

The impaired hearing variable was based on the questionnaire at 3 years of age with the question; has your child ever suffered or is currently suffering from impaired hearing.

Maternal educational attainment was scored as a dichotomous variable as completed / ongoing education level ≤12 years or completed / ongoing education level ≥13 years.

Serological tests

Samples collected from the pregnant women at birth (M2 sample) were analysed for CMV IgM and IgG antibodies by an enzyme-linked immunosorbent assay (Institut Virion/Serion Gmbh, Wuerzburg, Germany). No further testing was done if the M2 sample had an IgG and IgM negative result. The M1 sample was analysed for IgG and IgM in the remaining mothers. Further laboratory details have been described previously [6]. CMV seropositivity was defined as the proportion of women who had detectable IgG antibodies in the sample at birth. Serology results with an equivocal IgG result in M2 samples were interpreted as seronegative (applicable for 1 case and 2 controls). Seroconversion was defined as CMV-IgG antibody negative result in the sample taken in week 17–18 of pregnancy combined with a CMV-IgG antibody positive result in the sample taken at birth.

Statistical analyses

Logistic regression analysis was used to estimate the effect of CMV IgG seropositivity and seroconversion on delayed language development. Odds ratios (ORs) with accompanying 95% confidence intervals were calculated with univariate logistic regression models, and adjusted ORs were calculated by multivariate logistic regression adjusting for hearing impairment, age, parity, maternal education level, and hearing impairment in the child. Student`s t-test was used to calculate p-values for continuous variables. Separate analyses without adjustment for hearing impairment was done because it is not clear if it is a mediator between CMV and language development. In addition, sensitivity analyses where children with hearing impairment were excluded was also done. The sample size needed to perform this study was calculated based on the CMV seroprevalence. A sample of 300 cases and 1350 controls was needed to detect an odds ratio (OR) for CMV-infection of 1.5 with a significance level of 0.05 and power of 80% (OpenEpi.com). Statistical analyses were conducted using SPSS for windows, 27, SPSS Inc. Chicago, IL.

Details of ethical approval

Informed consent was obtained from each MoBa participant upon recruitment. The establishment and data collection in MoBa obtained a license from the Norwegian Data Inspectorate (01/4325) and approval from The Regional Committee for Medical Research Ethics (S-97045, S-95113). The current study was approved by the Regional Committee for Medical Research Ethics in South-Eastern Norway (2012/374B).

Results

Maternal age was similar for case mothers and control mothers, and for seropositive and seronegative women. The mean gestational length was comparable in the two groups. The maternal education level was lower in case mothers (49%) ≤12 years than in controls (27%). The seropositivity rate was higher in the mothers with lower education level. Fewer case mothers (36.5%) were primiparous compared to controls (48%) and primiparous women were more often seronegative. Mean BMI were higher in cases versus controls, 24.7 and 23.7 respectively. Among cases 43.5% had BMI > 25, compared to 30.9% in controls. In all analyses, parity was positively associated with delayed language development while mother’s education level was negatively associated. Hearing impairment in children was strongly associated (OR = 4,32, CI 2.79–6.75, p<0.001) with delayed language development. In children of the case group 13.5% had impaired hearing compared to only 3.5% in children of control mothers. Further characteristics are reported in Table 3.

Table 3. Maternal and pregnancy characteristics in 299 delayed language delay cases and 1349 population based controls from the Norwegian Mother, Father and Child Cohort Study (MoBa).

Maternal and pregnancy characteristics		Cases	Controls	P-values³	CMV-IgG	CMV-IgG
		n = 299	n = 1349		seropositive	seronegative
		(%) [SD]	(%) [SD]		n = 943 (%)	n = 705 (%)
Mean age(years)¹		30.9[4.6]	30.4[4.4]	0.14	30.6	30.4
Primiparous (%)¹	Yes	109 (36.5)	647 (48.0)	<0.001	400 (42.4)	356 (50.5)
	No	190 (63.5)	702 (52.0)		543 (57.6)	349 (49.5)
Folic acid intake during pregnancy²	Yes	165 (55.2)	806 (59.7)	0.17	545 (57.8)	426 (60.4)
	No	134 (44.8)	543 (40.3)		398 (42.2)	279 (39.6)
*Maternal education level^¹**	≤12 years	147 (49.2)	372 (27.6)	<0.001	322 (34.1)	197 (27.9)
	≥13 years	146 (48.8)	957 (70.9)		603 (63.9)	500 (70.9)
Missing		6	20		8	18
BMI ^**
Mean (range) ¹		24.7[6.5]	23.7[5.4]	0.002	23.8	24.0
BMI >25	Yes	127 (43.5)	409 (30.9)	<0.001	307 (32.6)	229 (32.5)
	No	165 (56.5)	913 (69.1)		612 (65)	466 (66.1)
Missing		7	27		24	10
Gestational length, (mean) weeks, (min-max) ²		39.4(30–43) [1.9]	39.7 (31–44) [1.6]	0.014	39.6 (31–44)	39.7 (30–43)
Birth weight (mean)kg²		3.567[612.0]	3.628[506.1]	0.07	3.613	3.635
Missing		15	53		39	29
Placenta weight (mean)kg²		0.668[150.6]	0.683[194.4]	0.212	0.681	0.680
Missing		15	53		39	29
Impaired hearing^¹*	Yes	39 (13.5)	47 (3.5)	<0.001	50 (5.4)	36 (5.2)
	No	249	1292		878	663
Missing		11	10		15	6

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n = number, SD = standard deviation.

¹ Data from questionnaires.

² Data from MBRN.

³ p-values comparing cases and controls.

*Completed or ongoing education level.

** Pre-pregnancy body mass index, (BMI).

***Data collection is based on the questionnaire when the child was 3 years of age; has your child ever suffered, or is currently suffering from hearing problems.

Among the case mothers, 63.2% were CMV-IgG positive in the sample at birth, as compared to 55.9% among controls, OR 1.36, (CI; 1.05 to 1.76) (Tables 4 and 5, model 1). A similar, but not statistical significant estimate was found when adjusted for maternal age, parity, and education level (OR = 1.29, 95% CI 0.99–1.69, p = 0.06) (Table 5, model 2). Additional adjustment for hearing impairment also gave similar results (Table 5, model 3).

Table 4. Maternal seroprevalence of CMV-IgG antibodies at birth and seroconversion comparing children with or without delayed language development at 3 years of age.

Maternal characteristics	Cases n = 299	Controls n = 1349
CMV seroprevalence
CMV antibodies	189	754
Yes (IgG+)	63.2%	55.9%
CMV antibodies	110	595
No (IgG-)	36.8%	44.1%
CMV seroconversion ^a	Cases	Controls
CMV seroconversion ^a	n = 118	n = 618
CMV seroconversion	8	23
Yes	6.8%	3.7%
CMV seroconversion	110	595
No	93.2%	96.3%

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^a Only seroconversion samples and seronegative (IgG-negative) in the M1 samples are taken into calculation (118 cases and 618 controls).

Table 5. Results from logistic regression modelling, with crude and adjusted odds ratios for the association between CMV IgG+ and delayed language development.

	OR (95% CI)	P-value
Model 1
CMV IgG+	1.36 (1.05–1.76)	0.021
Model 2
CMV IgG+	1.29 (0.99–1.69)	0.061
Age	1.02 (0.99–1.05)	0.134
Parity	1.42 (1.08–1.88)	0.013
12 + years of education	0.39 (0.29–0.50)	<0.001
Model 3
CMV IgG+	1.29 (0.99–1.71)	0.064
Hearing impairment	4.26 (2.67–6.81)	<0.001
Age	1.03 (0.99–1.06)	0.087
Parity	1.39 (1.05–0.86)	0.022
12 + years of education	0.39 (0.29–0.51)	<0.001

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Since hearing impairment can be either a confounder or a mediator for delayed language development, we performed the analysis both with and without adjusting for child’s hearing impairment giving very similar results (Table 5). A separate analysis of hearing impairment as the outcome, showed a non-significant association between CMV-IgG seropositivity, both in adjusted (OR = 1.08, CI: 0.69–1.70, p = 0.73) and unadjusted (OR = 1.05, CI: 0.68–1.61, p = 0.83) analyses.

In addition, we performed a sensitivity analysis, and excluded mothers of cases and controls with hearing impairment. Among mothers of cases without hearing impairment, 63.7% were CMV- IgG positive, as compared to 55.7% among controls without hearing impairment, still showing a significant positive association between CMV-IgG positivity and delayed language development (OR = 1.40, CI: 1.06–1.85, p = 0.02).

Among case mothers, 8/118 (6.8%) initially seronegative mothers seroconverted. Among initially seronegative control mothers, seroconversion occurred in 23/618 (3.7%), (OR = 1.88, 95% CI 0.82 to 4.31, p = 0.135) (Tables 4 and 6, model 1).

Table 6. Results from logistic regression modelling, with crude and adjusted odds ratios for the association between CMV IgG seroconversion and delayed language development.

	OR (95% CI)	P-value
Model 1
CMV seroconversion	1.88 (0.82–4.31)	0.135
Model 2
CMV seroconversion	2.18 (0.92–5.17)	0.076
Age	1.01 (0.96–1.06)	0.61
Parity	1.28 (0.83–1.97)	0.26
12 + years of education	0.32 (0.21–0.49)	<0.001
Model 3
CMV seroconversion	1.91 (0.77–4.74)	0.166
Hearing impairment	4.69 (2.30–9.59)	<0.001
Age	1.02 (0.97–1.07)	0.51
Parity	1.26 (0.81–1.97)	0.31
12 + years of education	0.31 (0.20–0.48)	<0.001

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When adjusted for maternal age, parity, and education level variables, the association between seroconversion during pregnancy and delayed language development was quite similar to the unadjusted analyses (OR = 2.18, 95% CI: 0.92–5.17, p = 0.08) (Table 6, model 2). Additional adjustment for hearing impairment also gave similar results (Table 6, model 3).

Discussion

In this nested case-control study from a large, population-based pregnancy cohort, we observed an association between development of language impairment in children at three years of age and CMV IgG positive status of the mother during pregnancy, also present when adjusted for maternal age, parity, and education level. We found a higher seroconversion rate of 6.8% in initially seronegative case mothers compared to controls (3.7%), although the difference was not statistically significant.

Speech and language problems are common in children and have multiple causes, the most severe are caused by chromosomal anomalies, inherited disorders and injuries at birth. Hearing loss, which is a common outcome in children with cCMV, can lead to speech and language impairment. However, studies performed in both asymptomatic and symptomatic subjects have shown that impairment in language development may also be present in cCMV children without hearing loss [19, 20]. This support that different parts of the brain has been affected in children with delayed language development compared to those with hearing loss [21]. That delayed language development in cCMV can occur in children without hearing loss is also described in the DECIBEL study by Korver and coworkers [22]. They found a greater delay in language comprehension in children with both permanent childhood hearing impairment and cCMV, than in children with permanent childhood hearing loss without cCMV. Another recent study also showed that both children with an asymptomatic or a symptomatic cCMV infection developed long-term sequelae, particularly in the behavioral and communicative areas [23]. Both the asymptomatic and the symptomatic children received valganciclovir treatment, and the long-term sequelae was not affected by which of the three trimesters that the maternal infection occurred (21). Our results show a trend similar with these previous studies, although not statistical significant. The estimate of the association when hearing impairment was not adjusted for in the regression model was similar to the estimate when it was included. A similar result was also seen when we removed the hearing impaired children from the analysis. These results agree with the hypothesis that CMV could cause delayed language development independent of hearing impairment.

Seropositivity and seroconversion rates in our study are comparable to other studies among pregnant women. We found that variables, like parity and education level, influenced the CMV seropositivity and seroconversion rates [24, 25]. In contrast to other countries comparable to Norway, age did not influence the risk of being IgG positive in this and our previous study within MoBa [6]. This is consistent with observations in previous Norwegian studies and has been explained partly by the decades-long high percentage of breastfeeding women in Norway, and high attendance to day care centers for children where there is a high risk of CMV transmission [5, 26].

The strength of this study is the population-based cohort design. The cases and controls were drawn among more than 90 000 pregnant women participating in MoBa, a large prospective population based cohort over a 10- year time span. Another strength is that the participants had blood sampled at two occasions during their pregnancy, at week 18 and after birth, making it possible to test for seropositivity at two time points and checking for seroconversions. A limitation is the timing of inclusion of participants in the MoBa study. The participants were invited to MoBa during routine hospital examination with ultrasound in pregnancy weeks 17 or 18, when the first blood samples were taken. Thus, seroconversion occurring early in pregnancy or in the peri-conceptional period may not be detected, and incorrectly assumed to be a past CMV-infection. Another weakness is the exclusion of mothers of children selected as cases or controls in the ABC-study within MoBa, this may have influenced the eligible number of cases with delayed language development for this study [14]. This study was designed to only look at CMV in pregnancy as a cause of language development delay. Potential causes like chromosomal abnormalities was not investigated as this was an exclusion criteria in our study. Other disorders like inherited diseases or infections other than CMV were not investigated, and the study was not designed to have enough power to fully investigate the potential mediating effects of hearing impairment.

Investigation of CMV in newborns was not part of this study. Previously we have shown that cCMV is under-diagnosed and awareness on this matter should be strengthened [6]. There is lack of data on long-term outcome of newborns with cCMV infection particularly for those asymptomatic at birth. The influence of maternal CMV-infection, both primary and non-primary, upon language impairment should be further investigated, both in asymptomatic and symptomatic cCMV-children, especially without concomitant hearing impairment.

Conclusion

This population-based study supported the previous study finding of a higher risk of delayed language development in children at three years of age in mothers seropositive for CMV than in mothers seronegative for CMV although no statistical significant association was found connected with CMV seroconversion.

Acknowledgments

We are grateful to all the participating families in Norway who take part in The Norwegian Mother, Father and Child Cohort Study. We would like to thank Synnve Schjolberg for excellent advice regarding the language delay cases and, Hege Fremstad, and Moustafa Gibory, at the Norwegian Institute of Public Health for excellent technical assistance.

Data Availability

Data from this study are available only upon request as there are legal and ethical restrictions on sharing data publicly. Data cannot be made publicly available because it contains sensitive participant information. Additionally, participants did not give consent for data to be made publicly available in a repository. Interested researchers can request access to the relevant datasets via request to: datatilgang@fhi.no. The request will require approval from an ethics committee and formal contract with Norwegian Mother and Child Cohort study (MoBa).

Funding Statement

Funding The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NINDS (grant no.1 UO1 NS 047537-01 and grant no.2 UO1 NS 047537-06A1). This study was funded by a grant (213916/H10) from the Norwegian Research Council.

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PLoS One. doi: 10.1371/journal.pone.0278623.r001

Decision Letter 0

Zemenu Yohannes Kassa

9 Aug 2022

PONE-D-22-17172Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohort

PLOS ONE

Dear Dr. Regine Barlinn, MD PhD,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR COMMENTS

The topic of the manuscript is interesting. Nevertheless, the reviewers raised several concerns: considering this point, I invite authors to perform the required major revisions.

A# Abstract

1. Introduction line 7-8, “Maternal CMV infection in pregnancy may lead to vertical transmission of CMV”. This sentence is incomplete and revised it.

The abbreviation is not recommended in abstract

2. Line 10-11…. with and without delayed language development in 3-year-old children. Why do you specifically take three years and below? It needs strong justification.

B. Methods

1. What are the abbreviations of MoBa?

The abbreviation is not recommended in abstract

3. Line 14-15 “Blood samples were obtained from mothers during pregnancy weeks 17 or 18 in pregnancy and after birth”. It needs clarification why you took blood at 17 or 18 weeks of gestations.

4. Elaborate on how to select control for your cohort study.

C. Results

1. Line 23-24 “the OR for seroconversion in cases as compared to control mothers was 1.88 (CI; 0.82 to 4.31). Seroconversion in cases is not associated with control mothers”. Please see again your analysis part.

D. Revised your conclusion based on your pertinent findings.

# Introduction

General comments

You put your introduction in two paragraphs

30-47 one paragraph and 48-51

Based on the above comments, you should revise your paragraph and split the ideas into different paragraphs.

* Introduction line 7-8, “Maternal CMV infection in pregnancy may lead to vertical transmission of CMV”. This sentence is incomplete and revised it.

* line 48 nested case-control study

# Methods

* Elaborate on how to calculate the sample size of cases and why you took 300 why not other numbers.

*Elaborate on how to calculate the sample size of controls and why you took 1350 why not other numbers.

*Line 82 you mean questionnaire 21 or where is the questionnaire?

Statistical analyses

1. Please elaborate “A sample of 300 cases and 1350 123 controls were needed to detect an odds ratio (OR) for CMV-infection of 1.5 with a significance level of 0.05 and power of 80%”. It is a sample size calculation?

2. How do control confounding and mediating variables? Elaborate it.

3. Which model do you use to cut off your points? Did you check your model’s fitness?

# Result

1. The maternal education level was lower in case mothers (49%) ≤12 years than in controls (27%).

2. It is not clear to me that≤12 years, you mean grade two?

3. Put confidence interval (OR > 4, p<0.001)

4. you should recategorize “maternal education level” again

#conclusion

Revised your conclusion based on your pertinent findings

you should write a declaration based on PLOS ONE guidelines,

Put all abbreviations

==============================

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Zemenu Yohannes Kassa, Msc

Academic Editor

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: I found this article very interesting aiming to describe the association between cCMV and delayed language development. However, there is main concerns in the statistical analysis.

The authors mentioned that CMV seropositive does not associate with hearing impairment. Then, hearing impairment is less likely a mediator between cCMV and delayed hearing development. if the authors think hearing impairment is the mediator variable, there are some other analysis methods, such as path analysis or mediator analysis.

Did authors try to conduct multi-variate logistic regression between delayed language development and seropositivity/seroconversion adjusting for hearing impairment? Is there any collinearity? Please clarify if those attempts were made.

I think hearing impairment will be a confounder when analyzing the association between cCMV infection and delayed language development if hearing impairment is not caused by cCMV only. Hearing impairment should be included in the model to adjust the effect rather than conducting sensitivity analysis.

The authors may misinterpret 95%CI of OR. There are several sentences that they have judged there is an association between outcome variable and explanatory variable, but 95%CI is crossing 1, which means there is no statistical significance in associations, e.g., lines 156 and 205.

I understand that maternal age, parity and education level were associated with CMV seropositivity and seroconversion (explanatory variables) but not all were associated with delayed language development (outcome variable), then they are not necessarily to be included in the model. On the other hand, BMI seems to have an association with seropositive, then why not including BMI? What is the variable selection strategy?

Table 3 should include crude ORs (and p-values) as a result of univariate logistic regression. CMV seropositive and negative should be in the row as one explanatory variable rather than separate column. Statistical analysis and Line 133-144 should be also rewritten according to the results in table 3.

Isn't it possible to include before 30 weeks of gestational age? I wonder why there is no children before 30 weeks. Or is there any cases with other known virus infection, such as rubella?

There are several minor comments:

line 36, is this seroprevalence maternal or women in reproductive age? please specify.

line 48-49, the association between CMV seropositivity and seroconversion in pregnant women with their children who had delayed language development. (delayed language development should be used consistently in the article as a outcome variable)

line 84 , please clarify the abbreviation of MBRN

Line 86 outcome measured. Is this different from case? this section should be combined with Case definition. (or deleted if duplicated.)

line 100, other variables

Is hearing impairment defined only by questionnaire? Any test result of any referral history to the otolaryngologist?

Can you briefly describe when BMI was measured and when GA, GW and other factors included in univariate analysis were collected?

line 121, hearing impairment was included in covariates to adjust the association, but the results Table 3,4,5 did not include this variable, rather the authors did separate analysis. It needs to be clarified.

In my understanding, only when M2 sample's IgG or IgM were positive, therefore M1 samples were tested, these samples were included in the analysis, correct? I think it is a bit unclear, so please clarify.

In discussion, it should be considered other explanations for delayed language development except cCMV. And if cCMV causes language development delay, what the potential mechanisms are. Even without any chromosomal abnormalities, are there any other development delay or growth retardation or other factors such as asphyxia at birth or inherited disorder and other reasons for admissions to the NICU? Whether children have siblings?

Reviewer #2: - The topic is so relevance and timely to improve maternal and child health

- The paper is well written , but need to address points raised in detail below

- Tables should be revised according to the comments below

- The discussion should address the limitation

- The result should be revised in brief description of the cohort outcome specifically to CMV incidence

My comments for authors in details are attached. Please forward the comments to authors to revise it.

**********

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Reviewer #1: No

Reviewer #2: Yes: Abel Gedefaw( MD, MPH), Associate Professor of Obstetrics and gynecology, Hawassa University , Ethiopia

**********

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Attachment

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PLoS One. 2022 Dec 1;17(12):e0278623. doi: 10.1371/journal.pone.0278623.r002

Author response to Decision Letter 0

6 Sep 2022

Response to academic editor and reviewers

Ref. No.: PONE-D-22-17172

Title: Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohort.

First, I would like to thank the reviewers and the editor for the time and effort that has been put into assessing the previous version of the manuscript. Please find our point-by-point responses to the comments below.

ACADEMIC EDITOR COMMENTS

The topic of the manuscript is interesting. Nevertheless, the reviewers raised several concerns: considering this point, I invite authors to perform the required major revisions.

A# Abstract

1. Introduction line 7-8, “Maternal CMV infection in pregnancy may lead to vertical transmission of CMV”. This sentence is incomplete and revised it.

The abbreviation is not recommended in abstract. We have revised the introduction line 7-8 and included an explanation of the abbreviation.

2. Line 10-11…. with and without delayed language development in 3-year-old children. Why do you specifically take three years and below? It needs strong justification.

The language development status was investigated at the age of three. Below this age the natural variation of language development is great making comparisons more difficult. A validation was conducted initially to assess the questionnaire’s ability to detect language development by comparing the parent`s response to concomitant psychological examination of the child at 3 years showing high correlation. This validation justified the use of questionnaire data collected at the age of 3 years.

B. Methods

1. What are the abbreviations of MoBa? The abbreviation is not recommended in abstract.

The abbreviation is explained in the article text and we have omitted the abbreviation from the abstract. The short name of the cohort study is MoBa as it is made up of the Norwegian words for mother (Mor) and child (Barn).

3. Line 14-15 “Blood samples were obtained from mothers during pregnancy weeks 17 or 18 in pregnancy and after birth”. It needs clarification why you took blood at 17 or 18 weeks of gestations.

As part of the pregnancy follow-up, all pregnant women in Norway are invited to ultrasound examination in week 17 to 18, when inclusion was done. It was convenient to draw the blood samples at this visit.

4. Elaborate on how to select control for your cohort study.

Using a statistical soft-ware (SPSS), the controls were randomly selected from the total cohort.

C. Results

We have revised the sentence.

D. Revised your conclusion based on your pertinent findings.

We have revised the conclusion in the manuscript and result section in the abstract.

# Introduction

General comments

You put your introduction in two paragraphs

30-47 one paragraph and 48-51

Based on the above comments, you should revise your paragraph and split the ideas into different paragraphs.

We kept the introduction in two paragraphs, since we believe it is easier for the reader.

* Introduction line 7-8, “Maternal CMV infection in pregnancy may lead to vertical transmission of CMV”. This sentence is incomplete and revised it.

We have revised the introduction line in abstract and introduction.

* line 48 nested case-control study

This has been revised accordingly.

# Methods

* Elaborate on how to calculate the sample size of cases and why you took 300 why not other numbers.

*Elaborate on how to calculate the sample size of controls and why you took 1350 why not other numbers.

A power calculation was performed initially in the planning of this study. The numbers of cases and controls needed to achieve a power of 80% and 95% confidence level were calculated based on 4% exposure to CMV infection among cases. The result of the power calculation showed that 300 cases and 1350 controls were needed using OpenEpi version 2013.

*Line 82 you mean questionnaire 21 or where is the questionnaire?

We are referring to MoBa questionnaire number 1 from pregnancy week 17-18 at inclusion of the study. Please see reference number 13. Question 21 was part of the 3 years of age questionnaire. We have revised the manuscript to clarify this matter.

Statistical analyses

2. How do control confounding and mediating variables? Elaborate it. Please see explanation below.

3. Which model do you use to cut off your points? Did you check your model’s fitness?

Cut off were based on questionnaire variables, and no model was used. The questionnaires have been previously validated for the purpose, please see reference 15.

# Result

1. The maternal education level was lower in case mothers (49%) ≤12 years than in controls (27%).

2. It is not clear to me that≤12 years, you mean grade two?

4. you should recategorize “maternal education level” again

We have used dichotomous variable for education. The junior and high school last 12 years in Norway. We categorized the education level into ≤12 years and above 12 years of education.

3. Put confidence interval (OR > 4, p<0.001)

We have added the confidence interval as suggested.

#conclusion

Revised your conclusion based on your pertinent findings

you should write a declaration based on PLOS ONE guidelines,

Put all abbreviations

Thanks for these points, we have updated accordingly.

Reviewer #1: I found this article very interesting aiming to describe the association between cCMV and delayed language development. However, there is main concerns in the statistical analysis.

We have included results from the statistical analysis adjusted for hearing impairment. It is also important to note that the study was not designed nor powered to investigate any hypothetical mediation effects of hearing impairment, so we have not investigated this in any more depth. We have now mentioned this in the discussion as well.

We have added results of regression analyses both with and without hearing impairment as a predictor. Thus, we accommodate for hearing impairment being either mediator or confounder. If hearing impairment is a mediator, we also get what might be interpreted as a direct effect of CMV infection when hearing is adjusted for.

We agree with the reviewer on the interpretation of 95% CI of OR. We have clarified in the discussion and results that the results are not statistically significant.

We believe that it is important to study potential association of age, parity and education level, therefore these variables were included in the statistical analyses. When looking at risk factors for maternal CMV infection, BMI is not likely to be of impact for acquiring CMV in contrast to age, parity and education level, which are factors previously found to be linked to higher rates of CMV seroprevalence. We judge that the observed association between BMI and CMV is confounded by the other variables.

Table 3 has been changed accordingly. Also, a paragraph has been added in statistical analysis section and in the text.

Isn't it possible to include before 30 weeks of gestational age? I wonder why there is no children before 30 weeks. Or is there any cases with other known virus infection, such as rubella?

Women in the MoBa were invited to participate at week 17-18, and their pregnancy outcome were recorded for all. In our study, there were no exclusion criteria in case of premature birth. It is a coincidence that there were no premature birth prior week 30 among cases and controls. In Norway approximately 0.4 % of pregnancies result in birth between week 22-27, and 5% in birth between week 28-36 according to Medical Birth Registry of Norway.

As the objective of our study is cmv-infection, we did not investigate other virus infections such as rubella. However, it is unlikely that any rubella infection occurred among our study participants since rubella was eliminated many years ago from Norway (www.fhi.no). We have included a paragraph about this limitation in the discussion.

There are several minor comments:

line 36, is this seroprevalence maternal or women in reproductive age? please specify.

Seroprevalence reference is in women of reproductive age. We have added a new reference for the CMV seroprevalence among women of reproductive age.

We agree with the reviewer about referring to delayed language development as an outcome variable and have reviewed the text to check that it is used consistently.

line 84 , please clarify the abbreviation of MBRN

Medical Birth Registry of Norway. This has been explained in line 63 in the revised article.

Line 86 outcome measured. Is this different from case? this section should be combined with Case definition. (or deleted if duplicated.)

We have deleted duplicated text from this section.

line 100, other variables

Is hearing impairment defined only by questionnaire? Any test result of any referral history to the otolaryngologist?

Yes, it is correct that data on hearing impairment came from the questionnaire. Medical test results from otolaryngologist were not available.

Can you briefly describe when BMI was measured and when GA, GW and other factors included in univariate analysis were collected?

BMI was measured prior to the pregnancy through questionnaire 1. GA, GW and placenta weight data was collected from birth records in the MBRN.

We have now added this information in footnote of table 3.

We have included hearing impairment in the statistical analysis as shown in the new tables 5 and 6. Also the text in the results and discussion section is revised accordingly.

The laboratory analysis started with IgG and IgM in M2 sample. If there were any positive IgG or IgM results, the M1 sample was also analyzed. There were no need to analyze the M1 samples in case of negative IgG and IgM in M2 as explained in line 111-114.

Other explanations for delayed language development than cCMV can be other congenital diseases e.g. inherited disorders and injuries at birth. We have added a paragraph in the discussion explaining about this and that a limitation to our study is that we did not examine for all these explanations. Chromosomal abnormalities were part of the exclusion criteria for this study as stated in material and methods section. The factor whether the children have siblings is covered by the parity status of the cases and controls.

Reviewer #2: - The topic is so relevance and timely to improve maternal and child health

- The paper is well written , but need to address points raised in detail below

- Tables should be revised according to the comments below

- The discussion should address the limitation

- The result should be revised in brief description of the cohort outcome specifically to CMV incidence

My comments for authors in details are attached. Please forward the comments to authors to revise it.

General comment

- The topic is so relevance and timely to improve maternal and child health

- The paper is well written , but need to address points raised in detail below

Abstract:

Conclusion: Only the odds of CMV seroprevalence positivity, which had a significant association, are included in the conclusion part. The non-significant association of seroconversion odds should also be addressed in the conclusion.

We have included a sentence concerning this lack of association in the conclusion of the article.

Introduction:

Only one study, a Dutch study, mentioned the effects of CMV infection and language development. It is good to include more studies on CMV infection and language development to justify the importance of the study.

There are high numbers of articles investigating the connection between CMV and hearing impairment in contrast to the very few studies looking at delayed language development. We have cited five articles previously published with this subject as a main focus.

Other risk factors for delay language development like other infections could be mentioned briefly and the importance of CMV infection study can be elaborate.

Other explanations have been added to the discussion.

Materials and methods

i. Study ( Cohort ) population description

1. Briefly describe the objective of the cohort for a better understanding of the paper

The objective of the MoBa study has been added to this part of the article.

2. It is good to describe briefly what was done or measured for the cohort population related to CMV infection and outcome despite it was mentioned in other papers. The only measurement stated in the manuscript was blood sample was taken for seroconversion and 2nd trimester CMV positivity analysis. Despite we assumed nearly half of the primary infection end up with congenital infection based on previous data, it is good to include the following if it was measured.

- Diagnosis of congenital CMV infection

- Diagnosis of symptomatic CMV infection at birth

- Whether there were follow-ups of infants born with CMV converted mothers for short and long-term sequel

Examination of cCMV at birth and follow-ups of infants was not part of this study. Standard operating procedures on the diagnosis of cCMV and CMV with symptoms at birth has been briefly explained below. We have added some lines in the discussion about cCMV.

ii. Selection of cases need clarification.

It is stated only as 300 cases of delayed language development. Among the 95,200 pregnant women cohort

- How many children did have delayed language development

- How did you select the 300 cases? How many cases were excluded from analysis due to other exclusion criteria ( Multiple pregnancies, chromosomal disorders, other study participation )

Please see explanation above.

Other variables:

Only educational status mentioned. But in the result part, age, parity, gestational age, BMI, mentioned.

We have added explanations of the following variables: age, parity, gestational age, BMI in table 3.

Statistical analysis

It is good to add the reference for sample size calculation

Please see explanation above.

Result:

Despite it is case –control study, it is good to include incidence data related to CMV infection among the cohort population since it is a national and large sample cohort

- What is the incidence of CMV positivity at the 2nd trimester (could be primary infection in the first trimester or reactivation of latent infection) and seroconversion rate at birth?

We have previously studied the incidence of CMV positivity at 2nd trimester and found 54.1% seropositivity (Barlinn et al. APMIS 2018). We further investigated seroconversion rate in the cohort between week 17/18 and birth which was 3.7%. We have added this information in the text and included this study as a reference.

- Among the total seroconverted women

How many of them had been diagnosed with congenital CMV infection?

How many of them were symptomatic at birth for CMV infections?

If there was no congenital infection diagnosis done, it should be stated in the discussion part regard to the clinical benefit of only maternal seroconversion at birth without a diagnosis of congenital infection. If we decrease the cases by half considering only half of the primary infection will have a congenital infection, do the risk of delayed language development association still persist

Investigation of cCMV at birth was not part of this study and this is now stated in the discussion. All new-borns in Norway are examined for the symptoms of cCMV and CMV PCR will be performed in saliva, urine and blood samples for diagnosis of CMV infection if this is suspected clinically. Additionally, all new-borns are subjected to hearing test and those failing this test will be without delay investigated for CMV by PCR in the same types of specimens as stated above.

- Table 3: add the P-Value for each variable to know the presence of association

- Table 4: it is good to show the confounding variables crud and adjusted odd ratio too( parity, age, education, hearing impairment, etc for delayed language development

This is now included in the new tables 5 and 6.

Discussion

It states “ Seropositivity and seroconversion rates in our study are comparable to other studies among pregnant women”. This is difficult to conclude in case-control study. As mentioned above, we need to know the cohort result.

It is good to discuss the limitation of

1. Do we exclude other congenital infections such as syphilis, toxoplasmosis, and rubella based on maternal and infants’ serological testing? How do we control the confounding effect of other infections? Despite it is a case-control study and we need to mention the importance of excluding other infections in the limitation part.

Other causes of congenital infections have not been investigated in our study, this has now been added to the limitations.

2. Ascertainment of congenital infection ( Seroconversion Vs congenital infection and language development delay )

Please see explanation above.

Conclusion: see above in the abstract

Attachment

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PLoS One. doi: 10.1371/journal.pone.0278623.r003

Decision Letter 1

Zemenu Yohannes Kassa

26 Sep 2022

PONE-D-22-17172R1Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohortPLOS ONE

Dear Dr. Regine Barlinn, MD PhD,,

Please submit your revised manuscript by Nov 10 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Zemenu Yohannes Kassa, Msc

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Dr. Regine Barlinn, MD PhD,

Academic editors’ comments

The topic of the manuscript is interesting and your manuscript is improved. However, the reviewers have raised concerns your statistical analysis how to control a confounder, you should check Multicollinearity and multivariable analysis. if you have a mediator variables, you should use other methods of analysis: considering this point, I invite authors to perform the required major revisions.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

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: (No Response)

Reviewer #2: (No Response)

**********

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

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The manuscript became clearer and improved.

I recognized that the authors main interest is the association between maternal CMV infection defined as serological positive or seroconversion and delayed language development regardless of the co-existence of hearing impairment.

The outcome is defined as moderate or severe problem of in ASQ question 18 (speech delay) AND simultaneous score <=40 in ASQ question 21 (cognitive developmental delay).

Authors answered all the questions, but I would like to clarify the major issue on the statistical analysis again.

1. If the authors think that hearing impairment is a mediator, they should not include the hearing impairment in the multivariate logistic regression in (Table 5 and 6). Just use model 1 and model 2.

2. If the authors think that hearing impairment is a confounder, they can keep the hearing impairment in the multivariate logistic regression and probably model 2 is just CMV IgG+ or seroconversion and hearing impairment, which is the authors' main interest. It is up to them to include other variables in model 3.

So please change the wording from mediator to confounder if authors would like to keep the current analysis. (line 198)

Alsos no need to mention mediator analysis in lines 269-271 if authors are going to take hearing impairment as a confounder.

I have one question in the definition of the case. ASQ question 21 is detecting the mental delay. I think the authors would like to remove this question 21 from the case definition as the language development delay without cognitive disorder was excluded in the current definition.

I also suggest that column of case and controls in table3 and table 4 should be combined as a result of univariate logistic regression analysis. CMV 1gG seropositive and seronegative should be in an independent table before Table 3.

Table 3 should include the age as a potential confounder.

In statistical analysis lines 147-149, it should be "Odds ratios with 95% confidence intervals were calculated with univariate logistic regression models, and adjusted ORs were calculated multivariate logistic regression adjusting for hearing impairment (if hearing impairment is recognized as a confounder), age, parity and maternal educational status." IF sensitivity analysis is going to be conducted, please mention that in the statistic section.

Line 250 authors said that age did not influence the risk of being IgG positive in our study. This result was not mentioned in the result section. I suggest including the age in table 3 or new table.

Conclusion said that "this population based study showed a higher risk of delayed language development ..." But I suggest "showed" is a bit strong conclusion while this study did not show the association between them. The expression may be,, "this study supported the previous study finding of ......."

Reviewer #2: (No Response)

**********

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.

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Reviewer #1: No

Reviewer #2: Yes: Abel Gedefaw ( MD,MPH), Associate Professor of Obstetrics and Genecology, Hawassa University Collage of Medicine and Health Sciences , Hawassa , Ethiopia.

**********

PLoS One. 2022 Dec 1;17(12):e0278623. doi: 10.1371/journal.pone.0278623.r004

Author response to Decision Letter 1

19 Oct 2022

Reviewer response letter

Ref. No.: PONE-D-22-17172R1

Title: Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohort.

We would again like to thank the reviewers and the editor for the time and effort that has been put into assessing the previous version of the manuscript. Please find our point-by-point responses to the comments below.

Review Comments to the Author

Reviewer #1: The manuscript became clearer and improved.

The outcome is defined as moderate or severe problem of in ASQ question 18 (speech delay) AND simultaneous score <=40 in ASQ question 21 (cognitive developmental delay).

Please see explanation below about question 18 and 21.

Authors answered all the questions, but I would like to clarify the major issue on the statistical analysis again.

1. If the authors think that hearing impairment is a mediator, they should not include the hearing impairment in the multivariate logistic regression in (Table 5 and 6). Just use model 1 and model 2.

So please change the wording from mediator to confounder if authors would like to keep the current analysis. (line 198)

Alsos no need to mention mediator analysis in lines 269-271 if authors are going to take hearing impairment as a confounder.

Response to poins 1 and 2:

The reviewer is correct that our main interest of the study is to ascertain the effect of CMV on speech development, but because the role of hearing impairment is unclear we have tried to account for this in our analyses and how we present our results. We do not think it is wise to commit to one of the two possibilities and pretend the other is irrelevant. We would also like to point out that if hearing impairment is indeed a mediator, it still makes sense to include an analysis where hearing impairment is a predictor. In the mediator case this would make the coefficients for CMV interpretable as direct effects. Hence, the model with hearing impairment included is relevant for both the mediator and confounder scenarios.

We revised Results and Discussion to clarify this issue. Please see lines 153-155, 185, 205 and 251 – 254.

The two questions are both exploring the language development in children. They were not developed to detect mental delay. Question 18 describes the speech performance and question 21 the child’s ability regarding language comprehension. The two questions are used in combination to select cases in the most appropriate way to ensure a consolidated group of children with language problems. A validation was conducted initially to assess the questionnaire’s ability to detect language development by comparing the parent`s response to these questions to a concomitant psychological examination of the child at 3 years showing high correlation. We have included a paragraph in the re-revised manuscript about this comparison and a new reference.

The selection of cases in this study is based on both Question 18 and 21 to obtain a consolidated group of children with language problems. This means that both questions are required for the analyses and Question 21 cannot be omitted from this study.

Table 3 should include the age as a potential confounder.

In the revised Table 3 we have included a column with p-values for comparing all the variables between cases and controls. We have included a new footnote with an explanation to this variable. This table also compares mean age between cases and controls as well as between CMV seropositives and seronegatives. No difference was found comparing age between the groups.

Results section also includes Table 4 with a comparison of CMV IgG antibody status between cases and controls.

We have revised accordingly in the Statistical analyses.

Line 250 authors said that age did not influence the risk of being IgG positive in our study. This result was not mentioned in the result section. I suggest including the age in table 3 or new table.

We thank the reviewer for the constructed comment and we understand that the sentence could be misunderstood. We have revised this sentence and included the reference to the previous study.

It is also referred to age in the beginning of the result section, stating that age was similar for CMV seropositive status compared to seronegative. Table 3, already includes age as a variable.

We thank the reviewer for suggesting a revised Conclusion, which we have implemented.

Attachment

Submitted filename: PONE-D-22-17172R2_Response_letter.docx

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PLoS One. doi: 10.1371/journal.pone.0278623.r005

Decision Letter 2

Zemenu Yohannes Kassa

21 Nov 2022

Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohort

PONE-D-22-17172R2

Dear Dr. Regine Barlinn,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Zemenu Yohannes Kassa, Msc

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: (No Response)

**********

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

Reviewer #1: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

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

Reviewer #1: No

Reviewer #3: No

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: I am very sorry that my intention was not clearly understood by the authors.

I do understand that hearing impairment could be a mediator in this study design, but I cannot accept that a mediator is included as a covariate in a multivariate logistic regression model. I do not have adequate statistical knowledge how to handle this case if authors do not take hearing impairment as a confounder. At this point, I need to reject this manuscript, but if it is a too strict decision, I would like to ask editors or another reviewer who is more familiar with the cCMV and statistical analysis to make an appropriate decision.

Reviewer #3: A research study was conducted which aimed to estimate the odds ratios for CMV seropositivity and seroconversion in mothers, with and without delayed language development in 3 year old children. The observed odds ratio was 1.36 and the 95% CI did not contain zero, indicating a higher risk of delayed language development at three years of age in children whose mothers were seropositive for CMV, compared to children of mothers who were seronegative.

Minor revisions

Table 3:

A. In the statistical analysis section, state and describe the statistical methods used to estimate the p-values.

B. For continuous factors, provide standard deviations that correspond to means.

**********

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 #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0278623.r006

Acceptance letter

Zemenu Yohannes Kassa

23 Nov 2022

PONE-D-22-17172R2

Maternal cytomegalovirus infection and delayed language development in children at 3 years of age – a nested case-control study in a large population-based pregnancy cohort

Dear Dr. Barlinn:

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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Zemenu Yohannes Kassa

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

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PERMALINK

Maternal cytomegalovirus infection and delayed language development in children at 3 years of age–a nested case-control study in a large population-based pregnancy cohort

Regine Barlinn

Susanne G Dudman

Halvor Rollag

Lill Trogstad

Jonas C Lindstrøm

Per Magnus

Roles

Abstract

Introduction

Material and methods

Results

Conclusion

Introduction

Materials and methods

Study population

Case definition

Table 1. In question 18 of the questionnaire at 3 years of age, mothers are asked which of the six response categories best described the speech performance of their child.

Controls

Further inclusion and exclusion criteria for cases and controls

Outcome measures

Other variables

Serological tests

Statistical analyses

Details of ethical approval

Results

Table 3. Maternal and pregnancy characteristics in 299 delayed language delay cases and 1349 population based controls from the Norwegian Mother, Father and Child Cohort Study (MoBa).

Table 4. Maternal seroprevalence of CMV-IgG antibodies at birth and seroconversion comparing children with or without delayed language development at 3 years of age.

Table 5. Results from logistic regression modelling, with crude and adjusted odds ratios for the association between CMV IgG+ and delayed language development.

Table 6. Results from logistic regression modelling, with crude and adjusted odds ratios for the association between CMV IgG seroconversion and delayed language development.

Discussion

Conclusion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Zemenu Yohannes Kassa

Roles

Author response to Decision Letter 0

Decision Letter 1

Zemenu Yohannes Kassa

Roles

Author response to Decision Letter 1

Decision Letter 2

Zemenu Yohannes Kassa

Roles

Acceptance letter

Zemenu Yohannes Kassa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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