Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis.

BACKGROUND: Sulfonamides are widely used to treat infectious diseases during pregnancy. However, the safety of maternal exposure to sulfonamides is controversial. This study aims to systematically review the available studies and examine the effect of maternal sulfonamides use on adverse pregnancy outcomes.
METHODS: We searched PubMed, Science Direct, Web of Science, ClinicalTrials.gov, CNKI and Wanfang Database (in Chinese). The meta-analysis used random effects model or fixed effects model to obtain the total odds ratio (OR) for each outcome through Stata11.0 software. Study on the relationship between sulfonamide exposure during pregnancy and adverse pregnancy outcomes. The study design covered randomized controlled trials, cohort studies and case-control studies. The study protocol was registered in PROSPERO with protocol number CRD42020178687.
RESULTS: A total of 10 studies, and 1096350 participants were included for systematic review. Maternal exposure to sulfonamides was found to be possibly associated with increased risk of congenital malformations (OR = 1.21, 95% CI 1.07-1.37). The use of sulfonamides in the first trimester of pregnancy and during the entire pregnancy might be associated with congenital malformations.
CONCLUSIONS: Maternal exposure to sulfonamides may be associated with offspring' s congenital malformations. Prescription of sulfonamides for pregnant women is suggested to be carefully censored.

Introduction

Sulfonamides are a very important class of drugs, with antibacterial, diuretic, hypoglycemic, antithyroid activity and other pharmacological effects [1]. They are usually used as human medicines, agriculture, aquaculture and animal husbandry [2]. People can be exposed to sulfonamides in every aspect of daily life. Studies have shown that sulfonamides were detected in drinking water, air, dust, soil vegetables and grains, and some animalistic food like meat, egg, milk, etc [3].

Sulfonamides are also the first drugs to be systematically used to prevent and treat human bacterial infections [4]. Because infections during pregnancy often cause serious maternal and fetal complications, sulfonamides have also been used as first-line agents in the second and third trimesters to treat and prevent urinary tract infections and other infections caused by susceptible microorganisms. The most commonly used sulfonamides during pregnancy is trimethoprim-sulfamethoxazole (TMP-SMX) [5, 6]. The American College of Obstetricians and Gynecologists also advised that sulfonamides could be used in the first trimester of pregnancy when other antibiotics are not available [7, 8].

Due to the widespread use of sulfonamides, the amount of sulfonamides exposed during pregnancy is also considerable. A large German study reported that the rate of TMP-SMX use in early pregnancy was 0.54% [9]. Data from the National Ambulatory Medical Care Survey (NAMCS) and National Hospital Ambulatory Medical Care Survey (NHMACS) had shown that 22% of pregnant women with uncomplicated urinary tract infections used sulfonamides during 2002–2011 [10]. In recent years, a study investigated 536 pregnant women aged 16−42 years from two geographically different study sites in Eastern China in 2015. Urinary antibiotics were overall detected in 41.6% of pregnant women, of which, the detection rate of sulfonamides antibiotics in urine was 13.6% [11]. A human bio-monitoring data including 369 pregnant women showed that sulfonamides were detected in 0.8% of newborn meconium, and the maximum level was found to be 75.7μg/kg [12].

The Food and Drug Administration (FDA) classifies a drug into five categories: A, B, C, D, and X [13]. Sulfonamides belong to the FDA’ s Class C drugs, that is, animal studies have shown adverse effects on the fetus, and evidence from human studies is not sufficient [14, 15]. Currently, sulfonamides are not listed as prohibited drugs and are highly used during pregnancy. The potential effect of sulfonamides exposure during pregnancy on adverse pregnancy outcomes is inconclusive. Current evidence has shown that sulfonamides were more likely to cause adverse pregnancy outcomes than other antibacterial drugs [16-19]. There are also some controversies about the safety of using sulfonamides during pregnancy. However, to the best of our knowledge, there has been no integrated study to examine the impact of maternal sulfonamides exposure on pregnancy outcomes until now. Whether maternal exposure to sulfonamides will lead to adverse pregnancy outcomes, what kind of adverse pregnancy outcomes will be caused by sulfonamides exposure during pregnancy, and during which key gestational period will maternal sulfonamides exposure result in adverse pregnancy outcomes are issues worthy of research. Therefore, this study aims to provide an up-to-date systematic review and meta-analysis on whether maternal exposure to sulfonamides during pregnancy is associated with major adverse pregnancy outcomes, and to provide valuable information on the safety of sulfonamides usage as well as the rationale of sulfonamides prescription during pregnancy.

Methods

This meta-analysis was evolved according to the recommended Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline [20].

Search strategy

We searched PubMed, Science Direct, Web of Science, ClinicalTrials.gov, CNKI (China National Knowledge Infrastructure) and Wanfang Database (in Chinese). We used the following retrieval formula in Pubmed: (pregnancy OR pregnant OR conception OR fetation OR gestation) AND (sulfonamides OR sulfamethoxazole OR sulfametoxydiazine OR sulfaquinoxaline OR sulfamethazine OR sulfadiazine OR sulfasalazine OR sulfamethizole OR cotrimoxazole), there are no other special restrictions. We determined the types of adverse pregnancy outcomes based on the findings of the articles. The main adverse pregnancy outcomes for our meta-analysis were congenital malformations (including congenital malformations of the circulatory system, congenital malformations of the musculoskeletal system and Cleft lip ± palate), and the secondary adverse outcomes were spontaneous abortion, preterm birth/low birth weight.

Study selection and quality evaluation

The criteria for eligible articles were as follows: (1) There was information on maternal exposure to sulfonamides and adverse pregnancy outcomes in titles and/or abstracts. (2) The study design covered case-control studies, cohort studies and randomized controlled trial. We searched articles in both Chinese and English database but finally did not find any articles that met the criteria in the Chinese database. After the retrieval, all articles were firstly imported into Note Express and duplicate literature was removed. Then, the literature was initially screened by overviewing the titles and abstracts, articles unrelated to the subject were excluded, and full-text studies were retained. Finally, for further reading, animal experiments, duplicate patient data-set, case reports and studies with no interested outcomes were excluded. This process was carried out independently by two researchers, and different opinions were reached an agreement according to the discussion. Two researchers independently extracted literature information. The information we extracted from the included literature mainly included first author, country, publication year, research type, outcome indicators, number of cases(exposed)/controls(unexposed) and total score of NOS (Jadad), as shown in Table 1.

Table 1

The basic characteristics and effect values of the included studies.

Number	Author	Year of publication	Country	Study period	Study design	Pregnancy outcomes	Number of cases(exposed)/controls(unexposed)	Total score of NOS (Jadad)
1	Yang J	2011	Canada	1997–2000	a retrospective cohort study	Preterm birth Low birth weight	447/14537	7
2	Czeizel AE	2004	Hungary	1980–1996	a case–control study	congenital malformations	22843/38151	9
3	Prasad MH	1996	India	-	a cohort study	spontaneous abortion preterm birth congenital malformations	564/636	6
4	Ratanajamit C	2003	Denmark	1991–2001	cohort study case–control study	congenital malformations Low birth weight Preterm birth spontaneous abortion	3484/60175 3347/22599	7
5	Muanda FT	2018	Canada	1998–2009	a nested case–control study	spontaneous abortion	6612/65613	7
6	Hansen C	2016	the United States	2001–2008	a cohort study	congenital malformations	6688/6688	7
7	Crider KS	2009	the United States	1997–2003	a case-control study	congenital malformations	13155/4941	8
8	Hill L	1988	United Kingdom	1983	a case-control study	congenital malformations	676/676	6
9	Brumfitt W	1973	England	1973	a randomized controlled trial	congenital malformations	120/66	3
10	Damkier P	2019	Denmark	2000–2015	a cohort study	congenital malformations	22684/801648	8

According to the Cochrane guidelines, the quality of the included studies and the risk of bias were measured by the Newcastle-Ottawa Scale (NOS) [21] and Jadad Score [22]. The NOS scale was used to evaluate observational studies and the Jadad Score was used to evaluate randomized controlled trials, and all included literature was independently assessed by two reviewers. The NOS was based on the three sub scales, selection, comparability and outcome/exposure. The total NOS score ranged from 0 to 9, a score of 7 or higher was regarded as high quality. The total Jadad score ranged from 1 to 5, a score of 3 or higher was regarded as high quality. The quality assessment of all included studies was presented in Table 1. GRADEprofiler software was used to evaluate the GRADE level of evidence for each outcome [23]. The level of evidence is divided into four levels: high, moderate, low and very low. Observational studies were initially defined as low quality. The level of evidence will be reduced due to the following five factors: (1) risk of bias; (2) inconsistency; (3) indirectness; (4) imprecision; (5) publication bias. The following three factors may increase the quality level of evidence: (1) large effect; (2) dose response; (3) all plausible residual confounding.

Data analysis

Adverse pregnancy outcomes in our study included: (1) Congenital malformations were the main outcome we were interested in this study, which indicated structural developmental abnormalities that occurred at birth. We extracted the first author, publication year and dichotomous data of included literature, imported them into Stata11.0 software, then we got forest plot with pooled odds ratios. In addition, we performed two subgroup analysis on the association between maternal exposure to sulfonamides and the major congenital malformations respectively by different gestational ages and by different types of malformations. The different gestational ages were divided into pre-pregnancy and first trimester of pregnancy and during the entire pregnancy. The different types of malformations were classified into 3 different types, including congenital malformations of the circulatory system, congenital malformations of the musculoskeletal system and Cleft lip±palate; (2) Spontaneous abortion. It was defined as a pregnancy with a diagnosis or procedure before the 20th week of gestation (ICD-10 codes O01-O03); (3) Preterm birth. It was defined as a gestational age below 37 completed weeks at birth; (4) Low birth weight, which was defined as newborn’ s birth weight less than 2500g. Due to the limited numbers of articles on the outcome of spontaneous abortion, preterm birth/low birth weight, we had not conducted subgroup analysis by gestational week.

Dichotomized variables were extracted and imported into Stata11.0 software, and these variables were expressed by odds ratio (OR) and 95% confidence interval (CI). Forest plot was used to present the pooled OR value. In the meta-analysis, I was used as an indicator to quantify the degree of heterogeneity. We used I to evaluate the statistical heterogeneity between studies. If I ≤ 50%, and the heterogeneity was low and acceptable, then fixed effect model was used for meta-analysis; if I > 50% and heterogeneity between studies was high, random effect model was adopted [24, 25]. We would also conduct further research on the included articles through subgroup analysis or sensitivity analysis [26]. Publication bias was detected by the funnel plot and Egger’ s/Begg’ s test. Sensitivity analysis was introduced to assess the effect of each individual study on the overall meta-analysis summary estimate. We also used sensitivity analysis to investigate the stability of the outcome of meta-analysis. We observed the pooled odds ratios after removing each study in turn, if the pooled odds ratios exceeded the original confidence interval, the outcome of meta-analysis was unstable; otherwise, the outcome of meta-analysis was regarded to be stable.

Protocol registration

The protocol for this systematic review and meta-analysis has been registered in the PROSPERO (Protocol No. CRD42020178687).

Results

Characteristics of the selected studies

From the 12118 retrieved studies, 10 studies matching the inclusion criteria were selected for meta-analysis, including 4 case-control studies, 4 cohort studies, 1 randomized controlled trial and 1 including both case-control and cohort studies. A total of 1096350 participants were included in the recruited 10 studies. The studies selection process was shown in Fig 1.

Fig 1

Flow diagram of publications screening.

Maternal exposure to sulfonamides and congenital malformations (CM)

Eight studies were selected to analyze the association between maternal exposure to sulfonamides and CM [4, 18, 19, 27–31]. The combined analysis of 8 studies showed that the heterogeneity was 53.4% (P = 0.036), thus the random effects model was adopted. The results showed that maternal exposure to sulfonamides was associated with a high risk of congenital malformations (OR = 1.21, 95% CI 1.07–1.37). Publication bias was detected by the funnel plot (S1 Fig) and the Egger’ s test (t = 2.82, P = 0.030, 95% CI 0.19–2.71), and it indicated that there was publication bias. The sensitivity analysis results showed that the pooled odds ratios did not exceed the original confidence interval after removing each study in turn, and indicated that the result was stable (Fig 2).

Fig 2

Forest plot with pooled odds ratios of the effect of maternal exposure to sulfonamides on offspring’s congenital malformations.

Two subgroup analyses were further conducted. Firstly, we had performed the analysis on maternal exposure to sulfonamides by different gestational ages according to the available data. The periods of “pre-pregnancy and first trimester of pregnancy” and “during the entire pregnancy” were defined. The period of “pre-pregnancy and first trimester of pregnancy” referred to “1 month before pregnancy till the end of the first trimester”. The period of “during the entire pregnancy” referred to “at any time during pregnancy”. The results showed that maternal exposure to sulfonamides in various periods of gestation were associated with congenital malformations, overall OR (95% CI) being 1.20(1.08–1.34). Maternal exposure to sulfonamides in pre-pregnancy and first trimester of pregnancy and during the entire pregnancy was both associated with congenital malformations, and the OR (95% CI) was 1.18(1.04–1.33), and 1.26(1.01–1.57), respectively (Fig 3).

Fig 3

Forest plot with pooled odds ratios of the effect of maternal exposure to sulfonamides on offspring’s congenital malformations-subgroup analysis by periods of gestation.

Secondly, we performed the analysis on the relationship between maternal exposure to sulfonamides and different types of congenital malformations. We extracted the dichotomized data from 3 articles for pooled analysis, as shown in Fig 4. It showed that maternal exposure to sulfonamides was associated with CM of the circulatory system (OR = 1.34, 95% CI 1.07–1.68) and the musculoskeletal system (OR = 1.54, 95% CI 1.10–2.16).

Fig 4

Forest plot with pooled odds ratios of the effect of maternal exposure to sulfonamides on offspring’s congenital malformations-subgroup analysis by types of malformations.

Maternal exposure to sulfonamides and spontaneous abortion

Three studies were included for the analysis [4, 16, 29]. The heterogeneity was higher than 50% (I = 91.4%, P<0.05), so the random effects model was adopted. There was no significant association between maternal exposure to sulfonamides and spontaneous abortion (OR = 1.63, 95% CI 0.91–2.91). Funnel plot (S2 Fig) and Egger’s test (t = 3.24, P = 0.191, 95% CI -29.75–50.07) was used to detect publication bias, and it indicated that publication bias was relatively small. It had shown from the sensitivity analysis that the outcome of meta-analysis was stable (Fig 5).

Fig 5

Forest plot with pooled odds ratios of the effect of maternal exposure to sulfonamides on spontaneous abortion.

Maternal exposure to sulfonamides and preterm birth/low birth weight

Three studies were included for the analysis [4, 17, 29]. No significant heterogeneity was found (I = 83.9%, P = 0.002), the random effects model was thus adopted. It showed that maternal exposure to sulfonamides was possibly not associated with preterm birth (OR = 1.38, 95% CI 0.94–2.03). Funnel plot (S3 Fig) and Egger’ s test (t = 1.10, P = 0.470, 95% CI -35.48–42.19) was used to detect publication bias, and it indicated that publication bias was relatively small. It had shown from the sensitivity analysis that the outcome of meta-analysis was stable (Fig 6).

Fig 6

Forest plot with pooled odds ratios of the effect of maternal exposure to sulfonamides on preterm birth/low birth weight.

GRADE quality of evidence

Three outcomes, congenital malformations, spontaneous abortion, preterm birth/low birth weight were presented in the current study. The level of evidence for each outcome was relatively low. The GRADE system evidence level and the reasons for promotion and demotion for each outcome were shown in Table 2.

Table 2

A summary table based on GRADE regarding the level of evidence for each outcome.

Quality assessment							No of patients		Effect		Quality	Importance
No of studies	Design	Risk of bias	Inconsistency	Indirectness	Imprecision	Other considerations	Sulfonamides	Control	Relative (95% CI)	Absolute
congenital malformations (follow-up mean 1 years; assessed with: structural developmental abnormalities that occur at birth.)
8	observational studies1	no serious risk of bias	no serious inconsistency	no serious indirectness	no serious imprecision	reporting bias2	36674 cases 43768 controls and 1708/33540 exposed 44680/869213 unexposed		OR 1.21 (1.07 to 1.37)	-	VERY LOW	CRITICAL
								4.9%		10 more per 1000 (from 3 more to 17 more)
spontaneous abortion (follow-up mean 1 years; assessed with: a pregnancy with a diagnosis or procedure before the 20th week of gestation (ICD-10 codes O01-O03))
3	observational studies1	no serious risk of bias	serious3	no serious indirectness	serious4	none	9959 cases 88212 controls and 166/564 exposed 138/636 unexposed		OR 1.63 (0.91 to 2.91)	-	VERY LOW	CRITICAL
								10%		53 more per 1000 (from 8 fewer to 144 more)
preterm birth / low birth weight (follow-up mean 1 years; assessed with: a gestational age below 37 completed weeks at birth; newborn’s birth weight less than 2500g)
3	observational studies	no serious risk of bias	serious5	no serious indirectness	serious4	none	350/4495 (7.8%)	5204/75348 (6.9%)	OR 1.38 (0.94 to 2.03)	24 more per 1000 (from 4 fewer to 62 more)	VERY LOW	IMPORTANT

1 case-control and other study designs together

2 Egger’s test (t = 2.82, P = 0.030, 95% CI 0.19–2.71)

3 I2 = 91.4%, P<0.05

4 95% confidence interval of the pooled of effect includes both 1) no effect and 2) appreciable benefit or appreciable harm

5 I2 = 83.9%, P<0.05

Discussion

This meta-analysis revealed that maternal exposure to sulfonamides at different perinatal time (pre-pregnancy, first trimester of pregnancy and during the entire pregnancy) might increase the risk of offspring’ s congenital malformations. As far as we know, this is the first systematic review and meta-analysis that focuses on the association between maternal exposure to sulfonamides and adverse pregnancy outcomes.

In analysis on the relationship between maternal exposure to sulfonamides and fetal congenital malformations, in order to better distinguish the time of sulfonamides exposure prior to pregnancy and in first trimester of pregnancy, in the only study that separately described the use of sulfonamides before pregnancy, we found that maternal exposure to sulfonamides before pregnancy was associated with congenital malformations (OR = 2.4, 95% CI 1.1–5.3). As for the association between exposure to sulfonamides during pregnancy and offspring’ s congenital malformations, in all recruited studies, only one randomized controlled experiment was found with regards to the teratogenicity of using TMP-SMZ during pregnancy. It had not shown strong evidence that TMP-SMZ could cause serious teratogenic risks, which was inconsistent with the results of our study. Due to the small sample size and non-representative participants in the randomized controlled trial more experimental studies are needed to provide higher level evidence in the teratogenic effect of maternal exposure to sulfonamides.

Preterm birth is usually related to low birth weight. In our study, there were only three included articles concerning maternal exposure to sulfonamides and preterm birth/low birth weight. Among them, Ratanajamit C’ s study clearly indicated that low birth weight was restricted to full-term deliveries, while in Yang J’ s study, low birth weights covered preterm birth, and vice versa, and Prasad MH’ s study just included preterm birth without mentioning low birth weight. As it was difficult to clearly identify isolated preterm birth or low birth weight, we combined preterm birth and low birth weight as one outcome in analysis and did not found significant effect of maternal exposure to sulfonamides on preterm birth/low birth weight. We believe that more research data is needed to identify the relationship between maternal exposure of sulfonamides and preterm birth/low birth weight.

Some potential mechanisms might explain our findings that maternal exposure to sulfonamides was likely to increase the risk of fetal congenital malformations. Studies had shown that sulfonamides could cross the placental barrier [32]. Experimental studies revealed that sulfonamides had obvious teratogenic effects on mammalian animals [33, 34]. Folate is important for fetal development as it plays a key role in DNA synthesis during the rapid division of fetal cells [35]. Sulfonamides can inhibit cell proliferation by affecting folic acid synthesis. The process of folic acid synthesis is that dihydropteridine pyrophosphate and p-amino benzoic acid are condensed by dihydropteroate synthase to form dihydropterin, and glutamate is added to dihydropteroate by dihydrofolate synthase to form dihydrofolate, and then converts dihydrofolate to tetrahydrofolate by dihydrofolate reductase. Tetrahydrofolate is necessary for the synthesis of DNA and purines. The structure of sulfonamides is similar to p-amino benzoic acid, and compete with this compound for the biosynthesis of dihydropteroate, thereby reducing the synthesis of dihydrofolic acid [36, 37]. TMP-SMX, as the most commonly used sulfonamides during pregnancy, is a folic acid antagonist [14]. It may cross the placenta, consume folic acid and act on trophoblast cells to affect folate metabolism, thereby inhibit DNA synthesis [38, 39]. Further studies have suggested that pre-pregnancy and the first trimester of pregnancy may be a critical period for most organ and phylogenetic development, and exposure to sulfonamides during this period may lead to a higher risk of congenital malformations [40]. Since most of the studies we included were observationally designed, confounding by indication was crucial to explain the results. The use of sulfonamides during pregnancy was mostly aimed to treat infectious diseases of the pregnant woman. Adverse pregnancy outcomes in pregnant women may be caused by a variety of factors, and researchers had found that urinary tract infections during pregnancy had also been linked to an increased risk of multiple adverse pregnancy outcomes [41-44]. Therefore, whether the adverse pregnancy outcomes were caused by sulfonamides exposure or infections during pregnancy aroused our attention. Two studies presented strategies for controlling protopathic bias, they used alternative antibiotics with the same indications and without adverse pregnancy outcomes as controls [16, 28], among which Muanda FT’ s research results showed that TMP-SMX exposure was associated with an increased risk of spontaneous abortion after controlling for indication bias and protopathic bias. Control of indication bias was described in other studies, and the results show no significant association between observed adverse pregnancy outcomes and urinary tract infections and that confounding caused by urinary tract infection does not exist [45, 46]. We admit that this part of the controversy has never stopped, but we still want to emphasize that there might be a great risk of maternal exposure to sulfonamides during pregnancy.

Our study has several strengths. First of all, it is the first systematic analysis on the association between maternal exposure to sulfonamides and multiple adverse pregnancy outcomes. Our study included many high-quality articles with large study populations. We adopted the GRADE system to grade the evidence of the outcome indicators. Adverse pregnancy outcomes in our study included congenital malformations, spontaneous abortion, preterm birth/low birth weight as involved in both adversities in live births and fetal loss, and covered visible structural malformations (birth defects in organs and system) as well as “functional” manifestations (abortion, preterm birth/low birth weight), although abortion might be also due to potential malformations. Further detailed subgroup analyses were performed to clarify the impact of sulfonamides exposure during critical period of pregnancy on different types of congenital malformations.

Of course, there are some limitations in our study. First of all, few articles described the effect of sulfonamides exposure on a certain adverse pregnancy outcome, instead, multiple adverse pregnancy outcomes were often reported in one article. When we examined the effect of sulfonamides exposure during pregnancy on a specific adverse pregnancy outcome, data were limited and heterogeneity of combined outcomes was somehow difficult to control. We thus adopted the random effect model to merge the effect size, and carried out subgroup analysis and sensitivity analysis on the main results. Secondly, due to the limited information on the exact period when women were exposed to sulfonamides, we could not clearly distinguish the first, second or the third trimester of pregnancy. It also hindered us in identifying the possible key time windows that maternal sulfonamides exposure might affect adverse pregnancy outcomes. Thirdly, most of our data were extracted from maternal and infant database, prescription database, birth registrations and drug registries. Although the data were relatively complete and reliable, there still might be differences between the records and the actual medication intake of pregnant women. For instance, we were not sure whether women had taken the sulfonamides strictly as the way prescribed by doctors. Fourthly, some of the drugs we studied are sulfonamides alone, some are sulfonamides together with trimethoprim, which may cause some misclassifications of exposure group. We conducted a separate analysis to examine the effect of single sulfonamides use during pregnancy on children’ s congenital malformations, and it showed that maternal exposure to sulfonamides alone during pregnancy was associated with congenital malformations (OR = 1.20, 95% CI 1.03–1.40) (S4 Fig). At the same time, we were unable to distinguish the effects of different sulfonamides and different doses on the pregnant outcome. In our recruited studies, one article reported the teratogenic effects of five different sulfonamides [19], such as sulfamethazine, sulfathiourea, sulfamethoxypyridazine, sulfamethoxydiazine and the combination of sulfamethazine-sulfathiourea-sulfamethoxypyridazine. It argued that offspring’ s ventricular septal defects were associated with sulfamethoxazine exposure during the second and third months of pregnancy. Clubfoot was associated with sulfathiourea use during the entire period of pregnancy as well as in the second and third months of pregnancy. Further studies are needed to confirm the impact of different kinds of sulfonamides during pregnancy and adverse pregnancy outcomes. Finally, there were also limited cohort studies and only one randomized controlled trial in the included literature, this is an important reason why the evidence level of GRADE is relatively low. We need more prospective research or experimental research that can provide a higher level of evidence to prove our research conclusions. The adverse pregnancy outcomes in this study were just reported from the time of delivery till one year after birth. It was also reported that maternal exposure to antibiotics during pregnancy was associated with adverse outcomes in the postnatal and later developmental stages of the fetus, such as eczema, overweight and obesity in infant [47-49]. Long term effects of maternal exposure to sulfonamides are worth to be investigated, such as the physical and psychological development in childhood or even later life periods.

Conclusion

In conclusion, maternal exposure to sulfonamides is possibly associated with offspring’s congenital malformations. As infectious disease is harmful to both maternal and fetus health, meanwhile it’s not easy to get the most useful drugs in time based on the possible or definite side effects, prescription of sulfonamides for pregnant women is suggested to be carefully censored.

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Raw data.

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Funnel plot for the association between maternal exposure to sulfonamides and congenital malformations.

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Funnel plot for the association between maternal exposure to sulfonamides and spontaneous abortion.

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Funnel plot for the association between maternal exposure to sulfonamides and preterm birth/low birth weight.

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Forest plot with pooled odds ratios of the effect of maternal exposure to single sulfonamides on offspring’ s congenital malformations.

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1 Sep 2020

PONE-D-20-21921

Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis

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8. 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.

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Reviewers' comments:

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Comments to the Author

1. 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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. 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

**********

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: No

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: Major comments:

1.Preterm birth usually is related to low birth weight, in this manuscript only preterm birth but not low birth weight was associated to maternal exposure to sulfonamides, its unusual. Author need to analysis the data again and make sure the results are reliable.

2.Infection disease is very harmful to both maternal and fetus, for pregnant women, its not easy to get the most useful drugs in time based on the possible or definite side effects. Authors of this manuscript side “some controversies about the safety of using sulfonamides during pregnancy”, but we can only see the author focused on the bad side of using sulfonamides during pregnancy, such as congenital malformations and preterm birth. Meanwhile, the OR for both congenital malformations and preterm birth are only a little high than 1 although. So, the conclusion of this article is a tendency or possible suggestion only.

Minor comments:

1. Some grammar mistake need to be corrected by a native English expert in the revised version.

Reviewer #2: 1. Please specify what time range were used for the article selection.

2. Please clarify the final sample size of the selected studies.

3. When evaluated the Maternal exposure to sulfonamides and preterm birth, no significant heterogeneity was found (I2=35.4%, P=0.213), why did the author still choose the random effects model?

4. When evaluated the Maternal exposure to sulfonamides and congenital malformations, what’s the rationale for choosing the two subgroups while the first trimester was involved in both group (“pre-pregnancy and first trimester” and “during the entire pregnancy”)?

5. In the discussion section, folate deficiency was considered to be one of the potential mechanisms for sulfonamide related adverse outcomes. Have the reviewed studies provide any information of folate levels?

6. Molloy, Anne M et al; (Food and nutrition bulletin 2008: S101-11; discussion S112-5) have reported the association between folate deficiency and lower birth weight, however, the authors find no significant relationship between sulfonamide exposure and lower birth weight. Please provide more explanation on this.

**********

6. 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: Yes: Zehuan Ding

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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19 Oct 2020

PONE-D-20-21921

Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis

PLOS ONE

Dear Dr. Xie,

On behalf of all co-authors, we thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis”.

We have studied all the comments carefully and have made revision which marked in yellow in the paper. In the following pages are our point-by-point responses to each of the comments of the reviewers as well as your own comments.

We shall look forward to hearing from you at your earliest convenience.

Yours sincerely,

Kun Huang

Corresponding Author

E-mail Address: ahmuhuangk@163.com

Responds to Journal Requirements:

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

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

Response: We have carefully checked the entire manuscript for typography, format, and filename, and made sure that it met PLOS ONE’ s style requirements.

2. Please note that PLOS does not allow reference to data not shown (pages 11 and 16). Thus, before we proceed, we kindly ask you provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, please remove any references to these data.

Response: We have provided the relevant data instead of “data not shown” (pages 11 and 16) as the Fig 6 and the S4 Fig. In Fig 6, we have combined preterm birth and low birth weight as one outcome based on the reviewer’ s comments and re-made the forest plot.

3. Please present the full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

Response: We have presented the full electronic search strategy that had been used as the following retrieval formula in Pubmed: (pregnancy OR pregnant OR conception OR fetation OR gestation) AND (sulfonamides OR sulfamethoxazole OR sulfametoxydiazine OR sulfaquinoxaline OR sulfamethazine OR sulfadiazine OR sulfasalazine OR sulfamethizole OR cotrimoxazole). There are no other special restrictions.

4. In addition to the results from Begg’s and Egger’s tests, please assess publication bias by graphical means, e.g funnel plot.

Response: We completely agree with this valuable suggestion. We added funnel plot as a supplementary information (S1 S2 and S3 Fig) to further assess the judgment of publication bias.

5. 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 information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Response: We have added the raw data as the excel file named “raw data”.

6. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: We have provided the relevant data instead of “data not shown” (pages 11 and 16) as the Fig 6 the S4 Fig. In Fig 6, we have combined preterm birth and low birth weight as one outcome based on the reviewer’ s comments and re-made the forest plot.

7.Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

Response: We have added our tables to the main manuscript and uploaded the supplementary table (excel file) as separate “Supporting information” file.

8. 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.

Response: We have added captions for Supporting Information files at the end of the manuscript, and updated in-text citations to match accordingly.

Responds to the reviewer’s comments:

Reviewer #1: Major comments:

1.Preterm birth usually is related to low birth weight, in this manuscript only preterm birth but not low birth weight was associated to maternal exposure to sulfonamides, its unusual. Author need to analysis the data again and make sure the results are reliable.

Response: Thank you for the reviewer’ s comments. Indeed, preterm birth is usually related to low birth weight. While in some cases, some full-term infants are low birth weight, and some preterm infants have normal birth weights. That’ s why in Ratanajamit C’ s study, the authors had clearly indicated that low birth weight was restricted to full-term deliveries. In our meta-analysis, only three included articles concerning maternal exposure to sulfonamides and preterm birth/low birth weight (including Ratanajamit C’ s study). In Yang J’ s study, low birth weights covered preterm birth, and vice versa. Prasad MH’ s study just included preterm birth without mentioning low birth weight.

We fully considered the reviewer’ s comments and made a new analysis by combining preterm birth and low birth weight as one outcome. The findings revealed that there was no statistically significant effect of maternal exposure to sulfonamides on preterm birth/low birth weight (OR=1.38, 95% CI 0.94-2.03). We have also added some words in the discussion (Page 15-16). We believe that more research data is needed to identify the relationship between maternal exposure of sulfonamides and preterm birth/low birth weight.

2.Infection disease is very harmful to both maternal and fetus, for pregnant women, its not easy to get the most useful drugs in time based on the possible or definite side effects. Authors of this manuscript side “some controversies about the safety of using sulfonamides during pregnancy”, but we can only see the author focused on the bad side of using sulfonamides during pregnancy, such as congenital malformations and preterm birth. Meanwhile, the OR for both congenital malformations and preterm birth are only a little high than 1 although. So, the conclusion of this article is a tendency or possible suggestion only.

Response: Thank you for your valuable comments. We fully agree that it’ s not easy to get the most useful drugs in time based on the possible or definite side effects for pregnant women, although Muanda FT. et al. had shown that sulfonamides were associated with adverse pregnancy outcomes after controlling for indication bias and protopathic bias. We have taken the reality into account and rephrased the words in the discussion (Page 15-20) and conclusion (Page 20). We admit that this part of the controversy has never stopped and then interpret the conclusion more prudently.

3.Minor comments:

1. Some grammar mistake need to be corrected by a native English expert in the revised version.

Response: We apologize for the mistakes in the manuscript. We have carefully checked, corrected and polished the entire manuscript.

Reviewer #2:

1.Please specify what time range were used for the article selection.

Response: In the initial literature search, we had limited the time range as the recent ten and fifteen years and there were few related articles. The time rage was then not used in the final article selection.

2.Please clarify the final sample size of the selected studies.

Response: We have listed the individual sample size for each study in Table 1. According to the reviewer’ s comments, we have added up all the numbers of case and the final sample size is 1,096,350. We have also added this number in the text (Page 10).

3.When evaluated the Maternal exposure to sulfonamides and preterm birth, no significant heterogeneity was found (I2=35.4%, P=0.213), why did the author still choose the random effects model?

Response: Thank you for the reviewer’ s comments. In the revised manuscript, we have combined preterm birth and low birth weight as one outcome, and used the random effects model (Page 12-13) in the analysis.

4.When evaluated the Maternal exposure to sulfonamides and congenital malformations, what’s the rationale for choosing the two subgroups while the first trimester was involved in both group (“pre-pregnancy and first trimester” and “during the entire pregnancy”)?

Response: Thank you for the reviewer’ s comments. It’ s also what had troubled us during the analysis. We tried to divide the group of maternal sulfonamides into pre-pregnancy, the first, second and third trimester of pregnancy in initial analysis plan. After careful examining the included articles, we found that it did not meet the grouping criteria. Most articles used “pre-pregnancy and first trimester” as an independent exposure period, and no study clearly distinguished the second, third trimester of pregnancy. So we could not clearly divide the three trimesters of pregnancy, and had adopted “pre-pregnancy and first trimester” and “during the entire pregnancy” based on the existed grouping methods in the included articles. Indeed, the first trimester was involved in both groups. It’s one of the limitations in the paper, and we had added some words in the discussion section (Page 18-19).

The period of pre-pregnancy and first trimester is the key time window for folate supplementation to prevent fetal malformations, and sulfonamides may affect folic acid synthesis and metabolism.

5.In the discussion section, folate deficiency was considered to be one of the potential mechanisms for sulfonamide related adverse outcomes. Have the reviewed studies provide any information of folate levels?

Response: We have carefully checked the original literatures we included. It is a pity that they did not provide information on folate levels. Most of the included articles explained information on folate antagonism in terms of mechanisms. We detailed the mechanism of sulfonamides related to folate deficiency in the discussion.

6.Molloy, Anne M et al; (Food and nutrition bulletin 2008: S101-11; discussion S112-5) have reported the association between folate deficiency and lower birth weight, however, the authors find no significant relationship between sulfonamide exposure and lower birth weight. Please provide more explanation on this.

Response: Thank you for the reviewer’ s comments. Folate deficiency might be one of the potential metabolisms that sulfonamides may lead to adverse pregnancy outcome. As we have understood, it does not mean that maternal sulfonamides exposure will necessarily cause folate deficiency, and that folate deficiency will necessarily lead to low birth weight. As shown in Ratanajamit C’ s study, there was no significant correlation between maternal exposure to sulfonamides and low birth weight. And also, just as the reviewer had pointed out, we did not have information on folate levels in the included articles, which made it difficult to clearly identify the relationship among sulfonamides exposure. folate deficiency and adverse pregnant outcomes.

Preterm birth is usually related to low birth weight. In our meta-analysis, only three included articles concerning maternal exposure to sulfonamides and preterm birth/low birth weight. Ratanajamit C’ s study had clearly indicated that low birth weight was restricted to full-term deliveries. While in Yang J’ s study, low birth weights covered preterm birth, and vice versa. And Prasad MH’ s study just included preterm birth without mentioning low birth weight. In the revised version, we have made a new analysis by combining preterm birth and low birth weight as one outcome. The findings revealed that there was no statistically significant effect of maternal exposure to sulfonamides on preterm birth/low birth weight (OR=1.38, 95% CI 0.94-2.03). We have also added some words in the discussion (Page 15-16). We believe that more research data is needed to identify the relationship between maternal exposure of sulfonamides and preterm birth/low birth weight.

4 Nov 2020

Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis

PONE-D-20-21921R1

Dear Dr. Huang,

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,

Linglin Xie

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: 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: I Don't Know

**********

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: (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.

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

13 Nov 2020

PONE-D-20-21921R1

Maternal exposure to sulfonamides and adverse pregnancy outcomes: A systematic review and meta-analysis

Dear Dr. Huang:

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. Linglin Xie

Academic Editor

PLOS ONE