Identification of potential NAD-related biomarkers of recurrent miscarriage risk

Do women with a history of recurrent miscarriage have altered nicotinamide adenine dinucleotide (NAD)-related metabolites that can be detected in blood, plasma, or urine samples?

Women with a history of recurrent miscarriage have higher blood, plasma, and urine concentrations of NAD Salvage Pathway excretion products, and urinary excretion of nicotinamide (NAM) is also elevated, compared to control women.

Recurrent miscarriage risk is associated with advancing age, high and low BMI, dietary factors, various medical conditions including inflammation, as well as environmental exposures, e.g. to chemicals and pollution. Perturbation of NAD synthesis due to genetic and/or environmental factors causes NAD deficiency, which is implicated in Congenital NAD Deficiency Disorder (CNDD) characterized by recurrent pregnancy loss and congenital anomalies. In CNDD mouse models, foetal anomalies and embryo loss are prevented if NAD levels are raised by supplementing the mother's diet with an NAD precursor, such as vitamin B3, during pregnancy.

This prospective pilot cohort study included 88 non-pregnant women between 20 and 40 years of age, 37 with and 51 without a history of recurrent miscarriage. Recurrent miscarriage was defined as a history of two or more consecutive spontaneous miscarriages <20 weeks' gestation, with the last miscarriage between 6 weeks and 2 years prior to recruitment. The study was conducted at the Royal Hospital for Women, Sydney, Australia, between March 2022 and December 2023.

Participants completed a questionnaire about their socio-demographic characteristics, health, lifestyle, diet, medication, and vitamin use; and provided morning fasting blood and urine samples. Levels of NAD and 25 related metabolites were measured in whole blood, plasma, and urine using a validated ultra-high performance liquid chromatography-tandem mass spectrometry method. Differences in NAD metabolism between the groups were assessed by volcano plots and partial least-squares discriminant analysis. Characteristics of women between the two groups were compared using chi-squared statistics. Multivariable generalized additive models were used to assess the association between NAD metabolites and miscarriage. Predictive accuracy of metabolites alone and with age was examined using three machine learning models, including Logistic Regression, Random Forest, and Gradient Boosting Classifier and assessed using the area under the receiver operating characteristic curve (AUROC).

Elevated levels of the metabolites 1-methylnicotinamide (1MNA), N-methyl-2-pyridone-5-carboxamide (2PY), and N-methyl-4-pyridone-3-carboxamide (4PY), representing excretion metabolites of the NAD Salvage Pathway, were associated with a higher risk of recurrent miscarriage. These metabolites showed a strong positive correlation among the three tested biological matrices, confirming the suitability of all three matrices to quantify these markers. Whole blood anthranilic acid and urine NAM levels were also elevated in women with recurrent miscarriage. 1MNA in plasma was associated with recurrent miscarriage on univariate analysis, with the effect sustained after taking into account maternal age (adjusted odds ratio 1.02; 95% CI 1.01, 1.03) with every one-unit increase in 1MNA, the odds of miscarriage increasing by 2%. The predictive accuracy using machine learning approaches was highest when all three metabolites 1MNA, 2PY, and 4PY, and age were included in the model (AUROC 0.89; 95% CI 0.83, 0.95).

This study was conducted in a single hospital, which assures high internal validity but may limit external validity. Sample size was small, and findings should be replicated in larger studies. The measured levels of NAD-related metabolites represent a snapshot at the time of sample collection but might not be reflective of when the women had been pregnant.

Our novel finding of elevated excretion of NAM and its derived products in women experiencing recurrent miscarriage suggests differences in the NAD synthesis pathway are linked to adverse pregnancy outcomes. The significant differences in 2PY and 4PY levels in the circulation and urine between the study groups indicate that these metabolites are potential biomarkers associated with recurrent miscarriage. Several conditions which are associated with adverse pregnancy outcomes also affect NAD metabolism and cause elevated levels of these metabolites. Thus, these metabolites may not simply be biomarkers but also be an indicator of the underlying mechanisms in many cases of recurrent miscarriage. Further evaluation of NAD metabolism in women with recurrent miscarriage in other populations and of other associations including diet is required.

This research was supported by funds to S.L.D. from: the National Health and Medical Research Council (NHMRC), Principal Research Fellowship (ID1135886), Leadership Level 3 Fellowship (ID2007896), and Project Grant (ID1162878); a New South Wales (NSW) Health Cardiovascular Research Capacity Program Senior Researcher Grant; philanthropic support from The Key Foundation, The Ross Trust, and Steven and Linda Harker. N.N. was supported by NHMRC Leadership Level 1 Fellowship (ID1197940) and Financial Markets Foundation for Children. We gratefully acknowledge the Victor Chang Cardiac Research Institute Innovation Centre, funded by the NSW Government, as well as funding from the Freedman Foundation for the Metabolomics Facility. The authors declare no conflicts of interest.

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