Nitrate contamination in drinking water and adverse reproductive and birth outcomes: a systematic review and meta-analysis

Exposure to low levels of nitrate in drinking water may have adverse reproductive effects. We reviewed evidence about the association between nitrate in drinking water and adverse reproductive outcomes published to November 2022. Randomized trials, cohort or case–control studies published in English that reported the relationship between nitrate intake from drinking water and the risk of perinatal outcomes were included. Random-effect models were used to pool data. Three cohort studies showed nitrate in drinking water is associated with an increased risk of preterm birth (odds ratio for 1 mg/L NO3-N increased (OR1) = 1.01, 95% CI 1.00, 1.01, I2 = 23.9%, 5,014,487 participants; comparing the highest versus the lowest nitrate exposure groups pooled OR (ORp) = 1.05, 95% CI 1.01, 1.10, I2 = 0%, 4,152,348 participants). Case–control studies showed nitrate in drinking water may be associated with the increased risk of neural tube defects OR1 = 1.06, 95% CI 1.02, 1.10; 2 studies, 2196 participants; I2 = 0%; and ORp = 1.51, 95% CI 1.12, 2.05; 3 studies, 1501 participants; I2 = 0%). The evidence for an association between nitrate in drinking water and risk of small for gestational age infants, any birth defects, or any congenital heart defects was inconsistent. Increased nitrate in drinking water may be associated with an increased risk of preterm birth and some specific congenital anomalies. These findings warrant regular review as new evidence becomes available.

Selection process 8 Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

Methods: study selection
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process.

Synthesis methods
13a Describe the processes used to decide which studies were eligible for each synthesis (e.g. tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)).
Methods: study quality 13b Describe any methods required to prepare the data for presentation or synthesis, such as handling of missing summary statistics, or data conversions.
Methods: statistical analysis 13c Describe any methods used to tabulate or visually display results of individual studies and syntheses. Methods: statistical analysis 13d Describe any methods used to synthesize results and provide a rationale for the choice(s). If meta-analysis was performed, describe the model(s), method(s) to identify the presence and extent of statistical heterogeneity, and software package(s) used.
Methods: statistical analysis 13e Describe any methods used to explore possible causes of heterogeneity among study results (e.g. subgroup analysis, meta-regression).

Methods: statistical analysis
13f Describe any sensitivity analyses conducted to assess robustness of the synthesized results.

Methods: statistical analysis
Reporting bias assessment 14 Describe any methods used to assess risk of bias due to missing results in a synthesis (arising from reporting biases).

Methods: statistical analysis
Certainty assessment 15 Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome. N/A

Study selection
16a Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram.

Results:
Search results and study characteristics 16b Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded.

Results:
Search results and study characteristics; Figure 1 Study characteristics 17 Cite each included study and present its characteristics. Table 1 Risk of bias in studies 18 Present assessments of risk of bias for each included study. Table 2 Results of individual studies 19 For all outcomes, present, for each study: (a) summary statistics for each group (where appropriate) and (b) an effect estimate and its precision (e.g. confidence/credible interval), ideally using structured tables or plots.

Nitrate contamination in drinking water and adverse reproductive outcomes: protocol for a systematic review and meta-analysis Background
Nitrate is a naturally occurring ion that is part of the nitrogen cycle and used mainly in inorganic fertilisers. The amount of nitrate ingested from drinking water varies based on its concentration in drinking water and an individual's consumption habits. The increasing use of artificial fertilisers, the disposal of wastes, particularly from animal farming, and changes in land use have become significant contributors to the progressive increase in nitrate levels in groundwater supplies [12].
While there is some evidence that nitrates in drinking water are associated with colorectal cancer [13,14], the potential adverse reproductive effects of chronic exposure to low levels of nitrate have been emphasised recently. Animal studies have indicated that nitrate from the mother can cross the placenta, affect the foetus in utero, and increases adverse outcomes [15][16][17]. In addition, several epidemiological studies have indicated associations between prenatal nitrate exposure and other adverse reproductive outcomes, including congenital abnormalities, preterm birth, low birth weight and small-for-gestational-age (SGA) [18][19][20][21][22].
Therefore, the purpose of this study is to systematically review the available data and assess the association between human exposure to nitrate in drinking water and adverse reproductive outcomes.

Criteria for considering studies for this review
We will include randomised trials, cohort and case-control studies published in English. Studies that report the relationship between nitrate intake from drinking water and the risk of perinatal outcomes will be included.
The exposure of interest is nitrate intake from drinking water during the antenatal period.
The primary outcome for this review will be a composite of any of the following outcomes: preterm birth; SGA infant; low birth weight infant; stillbirth; miscarriage; and neonatal death.
The secondary outcomes will be: For infants: preterm birth, SGA, low birth weight, stillbirth, neonatal death, perinatal death, hypoglycaemia, need for respiratory support after birth, infection, congenital abnormality, necrotising enterocolitis, bronchopulmonary dysplasia, intraventricular haemorrhage, neonatal lung disease, neonatal intensive care unit (NICU) admission, jaundice, methemoglobinemia (as defined by the authors).
For women: any pregnancy complications (miscarriage, high blood pressure, preeclampsia, gestational diabetes, infection, obstetric haemorrhage as defined by the authors).
We will resolve disagreements by discussion and if necessary, a third review author will mediate for differences in interpretation.
We will record the selection process in sufficient detail to complete a PRISMA flow diagram [24].

Data extraction and management
We will develop a data extraction form to extract data from eligible studies. Two review authors will independently extract data from each eligible study. Information extracted will include, but not be limited to: source details, eligibility assessment, methodological details, characteristics of participants, details of intervention and outcomes reported. Any disagreement will be resolved by discussion and if necessary in discussion with a third review author.

Assessment of risk of bias in included studies
We will assess the quality of case-control and cohort studies according to the Newcastle-Ottawa Scale (NOS) [25]. The NOS evaluates nine methodological items and their reporting (participant selection, comparability of groups, and ascertainment of exposure/outcome), with values ≥ 7 compatible with good study quality (least bias, results are considered valid), between 2 and 7 with moderate study quality (susceptible to some bias but probably not enough to invalidate the results), and ≤ 2 with poor study quality (significant bias that may invalidate results).
We will assess the quality of randomized trials using the methods specified in the Cochrane Handbook for Systematic Reviews of Interventions[26]: (1) random sequence generation (selection bias); (2) allocation concealment (selection bias); (3) blinding of participants, personnel and outcome assessment (performance and detection bias); (4) incomplete outcome data (attrition bias); (5) selective reporting (reporting bias); (6) other bias (checking for bias due to problems not covered by (1) to (5) above).

Statistical analysis
The relationship between nitrate intake from drinking water and the risk of adverse birth outcomes will be examined based on the effect estimate. Nitrate intake from drinking water, odds ratios (ORs), risk ratios (RRs), hazard ratios (HRs), with 95% confidence intervals (CIs) will be extracted (both crude and adjusted). The median intake of nitrate will be calculated from the range given, (median intake = lowest dosage (lower limit) + highest dosage (upper limit) divided by 2 in each given quartile). When the median intake is given, the data will be used directly. When the interval of a quartile of any category of nitrate intake is not provided, the width of the class interval of quartile before this quartile will be used to calculate and estimate the interval, (when the lowest or highest category is open-ended, we assume that the open-ended interval length has the same length as the adjacent interval).
Meta-regression analysis will be conducted to assess the associations between nitrate exposure and adverse outcomes and p < 0.05 will denote statistical significance for a positive meta-regression coefficient. The pooled results across included studies will be evaluated using a random-effects model. Heterogeneity test will be performed using the I-square and Q-statistic, and significant heterogeneity is defined as p < 0.010.
Statistical analyses will be performed using STATA.

Discussion
This systematic review and meta-analysis, using the available evidence, will assess whether there is an association between nitrate in drinking water and the risk of adverse birth outcomes. The findings may help guide policy decisions regarding the safe level of nitrate in drinking water.