Concentrations of remdesivir and GS-441524 in human milk from lactating individuals diagnosed with COVID-19.

IMPACT: Findings from this study provide further reassuring evidence that infant exposure through human milk received from lactating individuals who require treatment with remdesivir is negligible.

Characterization of SARS-CoV-2 antibodies in human milk from 21 women with confirmed COVID-19 infection.

BACKGROUND: One potential mechanism for protection from SARS-CoV-2 in children is through passive immunity via breast milk from a mother infected with the novel coronavirus. The primary objectives of this study were to establish the presence of SARS-CoV-2-specific IgA and IgG and to characterize the antigenic regions of SARS-CoV-2 proteins that were reactive with antibodies in breast milk. METHODS: Between March 2020 and September 2020, 21 women with confirmed SARS-CoV-2 infection were enrolled in Mommy's Milk. Participants donated serial breast milk samples around their time of illness. Breast milk samples were used to probe a multi-coronavirus protein microarray containing full-length and variable-length overlapping fragments of SARS-CoV-2 proteins. Samples were also tested against S and N proteins by electrochemiluminescence assay. RESULTS: The breast milk samples contained IgA reactive with a variety of SARS-CoV-2 antigens. The most IgA-reactive SARS-CoV-2 proteins were N (42.9% of women responded to ≥1 N fragment) and S proteins (23.9% responded to ≥1 fragment of S1 or S2). IgG responses were similar. A striking observation was the dissimilarity between mothers in antibody recognition, giving distinct antibody reactivity and kinetic profiles. CONCLUSIONS: Individual COVID-19 cases had diverse and unique milk IgA profiles following the onset of symptoms. IMPACT: In this observational longitudinal case series of 21 women with confirmed SARS-CoV-2 infection, IgA binding to SARS-CoV-2 proteins detected by orthologous proteome microarray and electrochemiluminescence assays was observed in >75% of women, but there was heterogeneity in which antigens and how many were reactive between women. Immunological profiles of protein regions recognized by each woman were distinct. Diverse repertoires of mucosal breast milk antibody to SARS-CoV-2 reflect heterogeneous passive transfer of maternal antibody to exposed breastfeeding infants.

Metabolomic and exposomic biomarkers of risk of future neurodevelopmental delay in human milk.

BACKGROUND: The chemical composition of human milk has long-lasting effects on brain development. We examined the prognostic value of the human milk metabolome and exposome in children with the risk of neurodevelopmental delay (NDD). METHODS: This retrospective cohort study included 82 mother-infant pairs (40 male and 42 female infants). A total of 59 milk samples were from mothers with typically developing children and 23 samples were from mothers of children at risk. Milk samples were collected before 9 months of age (4.6 ± 2.5 months, mean ± SD). Neurocognitive development was assessed by maternal report at 14.2 ± 3.1 months using the Ages and Stages Questionnaires-2. RESULTS: Metabolome and exposome profiling identified 453 metabolites and 61 environmental chemicals in milk. Machine learning tools identified changes in deoxysphingolipids, phospholipids, glycosphingolipids, plasmalogens, and acylcarnitines in the milk of mothers with children at risk for future delay. A predictive classifier had a diagnostic accuracy of 0.81 (95% CI: 0.66-0.96) for females and 0.79 (95% CI: 0.62-0.94) for males. CONCLUSIONS: Once validated in larger studies, the chemical analysis of human milk might be added as an option in well-baby checks to help identify children at risk of NDD before the first symptoms appear. IMPACT: Maternal milk for infants sampled before 9 months of age contained sex-specific differences in deoxysphingolipids, sphingomyelins, plasmalogens, phospholipids, and acylcarnitines that predicted the risk of neurodevelopmental delay at 14.2 months of age. Once validated, this early biosignature in human milk might be incorporated into well-baby checks and help to identify infants at risk so early interventions might be instituted before the first symptoms appear.

No infectious SARS-CoV-2 in breast milk from a cohort of 110 lactating women.

BACKGROUND: Genomic RNA of severe acute respiratory syndrome-associated coronavirus type 2 (SARS-CoV-2) has been detected in the breast milk of lactating women, but its pathological significance has remained uncertain due to the small size of prior studies. METHODS: Breast milk from 110 lactating women was analyzed by reverse transcription-polymerase chain reaction (285 samples) and viral culture (160 samples). Those containing SARS-CoV-2 viral RNA (vRNA) were examined for the presence of subgenomic RNA (sgRNA), a putative marker of infectivity. RESULTS: Sixty-five women had a positive SARS-CoV-2 diagnostic test, 9 had symptoms but negative diagnostic tests, and 36 symptomatic women were not tested. SARS-CoV-2 vRNA was detected in the milk of 7 (6%) women with either a confirmed infection or symptomatic illness, including 6 of 65 (9%) women with a positive SARS-CoV-2 diagnostic test. Infectious virus was not detected in any culture and none had detectable sgRNA. In control experiments, infectious SARS-CoV-2 could be cultured after addition to breastmilk despite several freeze-thaw cycles, as it occurs in the storage and usage of human milk. CONCLUSIONS: SARS-CoV-2 RNA can be found infrequently in the breastmilk after recent infection, but we found no evidence that breastmilk contains an infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. IMPACT: This article goes beyond prior small studies to provide evidence that infectious SARS-CoV-2 is not present in the milk of lactating women with recent infection, even when SARS-CoV-2 RNA is detected. Recent SARS-CoV-2 infection or detection of its RNA in human milk is not a contraindication to breastfeeding.

The Association Between Maternal Psychological Health and Human Milk Oligosaccharide Composition.

Background and Objective: Human milk oligosaccharides (HMOs) are carbohydrates abundant in human breast milk. Their composition varies widely among women, and prior research has identified numerous factors contributing to this variation. However, the relationship between maternal psychological health and HMO levels is currently unknown. Thus, our objective was to identify whether maternal stress, anxiety, or depressive symptoms are associated with HMOs. Methods: Data originated from 926 lactating individuals from the UC San Diego Human Milk Biorepository. Nineteen prevalent HMOs were assayed using high-performance liquid chromatography. Participants self-reported measures of the Edinburgh Postnatal Depression Scale (n = 495), State-Trait Anxiety Inventory S-Scale (n = 486), and/or Perceived Stress Scale (n = 493) within 60 days of their milk collection; their results were categorized using standard screening cutoffs. HMOs were assessed individually and grouped by principal component analysis (PCA), and associations with maternal psychological symptoms were analyzed using multivariable linear regression adjusted for covariates. Results: After Bonferroni correction (p < 0.002), the following HMOs significantly varied with maternal psychological distress in multivariate analysis: lacto-N-fucopentaose III (LNFP III) and lacto-N-hexaose (LNH) among Secretors with depressive symptoms and difucosyllactose (DFLac), LNFP III, and disialyl-LNH (DSLNH) among Secretors with stress. In PCA, depressive symptoms and stress were associated with one principal component among Secretors. No HMOs varied with anxiety symptoms. Conclusions: Several HMOs varied with maternal depressive symptoms and stress, suggesting a relationship between maternal psychological health and breast milk composition. Additional studies are needed to determine the impact of this variation on infant health.

Consistency and Variability of the Human Milk Oligosaccharide Profile in Repeat Pregnancies.

Human milk oligosaccharides (HMOs) are a set of complex carbohydrates and the third largest solid component of human milk, after lactose and lipids. To date, over 150 HMOs have been identified and the diversity of structures produced by lactating women is influenced by maternal genetics as well as other maternal, infant, and environmental factors. While the concentrations of individual HMOs have been shown to vary between individuals and throughout the course of lactation, the variability of HMO concentration profiles following different pregnancies occurring in the same woman is presently unknown. As such, the objective of this study was to compare HMO concentrations in human milk samples provided by the same women (n = 34) following repeat pregnancies. We leveraged existing human milk samples and metadata from the UC San Diego Human Milk Research Biorepository (HMB) and measured the concentrations of the 19 most abundant HMOs using high-performance liquid chromatography with fluorescence detection (HPLC-FL). By assessing dissimilarities in HMO concentration profiles, as well as concentration trends in individual structures between pregnancies of each participant, we discovered that HMO profiles largely follow a highly personalized and predictable trajectory following different pregnancies irrespective of non-genetic influences. In conclusion, this is the first study to assess the interactions between parity and time following delivery on variations in HMO compositions.

Expansion of mammary intraepithelial lymphocytes and intestinal inputs shape T cell dynamics in lactogenesis.

Pregnancy brings about profound changes to the mammary gland in preparation for lactation. Changes in immunocyte populations that accompany this rapid remodeling are incompletely understood. We comprehensively analyzed mammary T cells through all parous stages, revealing a marked increase in CD4+ and CD8+ T effector cells in late pregnancy and lactation. T cell expansion was partly dependent on microbial signals and included an increase in TCRαß+CD8αα+ cells with strong cytotoxic markers, located in the epithelium, that resemble intraepithelial lymphocytes of mucosal tissues. This relationship was substantiated by demonstrating T cell migration from gut to mammary gland in late pregnancy, by TCR clonotypes shared by intestine and mammary tissue in the same mouse, including intriguing gut TCR families. Putative counterparts of CD8αα+ IELs were found in human milk. Mammary T cells are thus poised to manage the transition from a non-mucosal tissue to a mucosal barrier during lactogenesis.

The Association Between Adverse and Positive Childhood Experiences and Marijuana Use During Lactation.

Background: Adverse childhood experiences (ACEs) are associated with substance use later in life, including marijuana use. It is unknown whether these behaviors extend to lactating women. Our objective was to examine the association between childhood ACE and marijuana use in lactating individuals and determine whether positive childhood experiences (PCEs) modified this association. Methods: This study included 617 lactating individuals from the UC San Diego Human Milk Research Biorepository enrolled from 2015 to 2020. ACE and PCE histories were assessed by the Positive and Adverse Childhood Experiences questionnaire. Past 2-week marijuana use was self-reported at enrollment. Multivariable log-linear regressions were used to calculate adjusted risk ratios (aRRs) and 95% confidence intervals (CIs) for ACE history and marijuana use, and to assess modification by PCE. Results: Marijuana use during lactation was higher among individuals who reported three or more ACEs (aRR = 2.58, 95% CI = 1.23-5.44), household dysfunction (aRR = 3.08, 95% CI = 1.17-8.10), sexual abuse (aRR = 2.25, 95% CI = 1.08-4.68), or physical abuse (aRR = 2.10, 95% CI = 1.02-4.13). There was no association between emotional abuse and marijuana use during lactation. There was no effect modification by PCEs. Conclusion: Higher ACE frequency, and specifically history of household dysfunction, physical abuse, or sexual abuse increased risk for marijuana use during lactation. Because of marijuana's potential adverse effects on the infant through human milk, postpartum ACE screening is warranted.

The Prevalence of Nonserious Events in a Cohort of Breastfed Infants.

Introduction: Data on baseline rates of nonserious events in breastfed infants in the general population are sparse. This results in difficulty determining if there is an increase in infant nonserious events potentially due to prescription medication exposure through human milk. In this study, we determined the prevalence of nonserious events in infants consuming human milk whose mothers reported no exposure to any prescription medications, tobacco, or recreational drugs in the previous 14 days. Materials and Methods: Between August 2014 and December 2019, 487 breastfeeding mothers without any recent exposure to prescription medications, tobacco, or recreational drugs enrolled in the Human Milk Research Biorepository at the University of California, San Diego. Participants completed a semistructured telephone interview with trained research staff and provided information on maternal and child health, breastfeeding habits, recent medication, and lifestyle exposures, and completed a standard checklist of infant adverse reactions. Results: We found 131 (44.1%) participants reported one or more infant nonserious adverse events in the past 14 days at the time of their study interview. The most commonly reported nonserious events were rash (12.1%), irritability (9.4%), constipation (7.8%), poor sleep (7.1%), and fever (6.3%). Conclusions: These baseline frequencies provide a benchmark for rates of recent nonserious events in breastfed infants in the general population. These data can be used as a reference point for studies that examine adverse events in breastfed infants following maternal use of prescription medications or exposures due to other lifestyle habits such as tobacco or other substances. Clinical Trial Registration Number: NCT05553743.

Cannabidiol Exposure Through Maternal Marijuana Use: Predictions in Breastfed Infants.

BACKGROUND AND OBJECTIVE: Knowledge about exposure to cannabidiol (CBD) in breastfed infants can provide an improved understanding of potential risk. The aim was to predict CBD exposure in breastfed infants from mothers taking CBD and CBD-containing products. METHODS: Cannabidiol concentrations in milk previously attained from data collected through an existing human milk research biorepository were used to simulate infant doses and identify subgroups. A developed pediatric physiologically based pharmacokinetic model produced virtual breastfed infants administered the simulated CBD doses. Predicted breastfed infant exposures and upper area under the curve ratios were compared to the lowest therapeutic dose for approved indications in children. RESULTS: The existing human milk research biorepository contained 200 samples from 181 unique breastfeeding mothers for whom self-reported administration data and CBD concentrations had previously been measured. Samples that were above the lower limit of quantification with only one maternal administration type revealed that administration type, i.e., joint/blunt or edible versus oil or pipe, resulted in significantly different subgroups in terms of milk concentrations. Resulting simulated infant doses (ng/kg) were described by lognormal distributions with geometric means and geometric standard deviations: 0.61 ± 2.41 all concentrations, 0.10 ± 0.37 joint/blunt or edible, and 2.23 ± 8.15 oil or pipe. Doses administered to breastfed infants had exposures magnitudes lower than exposures in children aged 4-11 years administered the lowest therapeutic dose for approved indications, and low upper area under the curve ratios. CONCLUSIONS: Based on real-world use, breastfeeding infants are predicted to receive very small exposures of CBD through milk. Studies examining adverse reactions will provide further insight into potential risk.

An untargeted metabolomics analysis of exogenous chemicals in human milk and transfer to the infant.

Human milk is the optimal infant nutrition. However, although human-derived metabolites (such as lipids and oligosaccharides) in human milk are regularly reported, the presence of exogenous chemicals (such as drugs, food, and synthetic compounds) are often not addressed. To understand the types of exogenous compounds that might be present, human milk (n = 996) was analyzed by untargeted metabolomics. This analysis revealed that lifestyle molecules, such as medications and their metabolites, and industrial sources, such as plasticizers, cosmetics, and other personal care products, are found in human milk. We provide further evidence that some of these lifestyle molecules are also detectable in the newborn's stool. Thus, this study gives important insight into the types of exposures infants receiving human milk might ingest due to the lifestyle choices, exposure, or medical status of the lactating parent.

Allergen Content and Protease Activity in Milk Feeds from Mothers of Preterm Infants.

Rationale: There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have not performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Methods: To evaluate feasibility, we initially performed mass spectrometry on 4 human milk (HM) samples (2 term and 2 preterm) from the Mommy's Milk Human Milk Biorepository (HMB) and analyzed the results against the University of Nebraska FASTA database and UniProt for a total of 2,211 protein sequences. We then further analyzed five samples from the Microbiome, Atopy, and Prematurity (MAP) study including peptidomic and protease activity analysis. Results: Each HMB sample had between 806 and 1,007 proteins, with 37-44 nonhuman proteins/sample encompassing 26 plant and animal species. In the preterm MAP samples, 784 digested nonhuman proteins were identified, 30 were nonbovine in origin. Proteins from 23 different species including aeroallergens, food, and contact allergens were identified. Protease activity was highest in HM samples without human milk fortifier and lowest in preterm formula. Conclusions: These findings represent the first preterm milk feed mass spectrometry and protease analysis with identification of known allergenic proteins to food, contact, and aeroallergens. These results raise questions of whether the composition of milk feeds in the neonatal intensive care unit impact the development of atopic disease in the preterm population and whether the complex interaction between allergens, proteases, and other HM components can serve to induce sensitization or tolerance to allergens in infants. Clinical Trial Registration Number: NCT04835935.

Maternal and Child Outcomes Reported by Breastfeeding Women Following Messenger RNA COVID-19 Vaccination.

Background: In December 2020, two novel messenger RNA (mRNA) vaccines for severe acute respiratory syndrome coronavirus-2 received emergency use authorization from the U.S. Food and Drug Administration; however, the early trials excluded lactating women. Methods: Breastfeeding women residing in the United States who received either of the two mRNA vaccines were enrolled into the Mommy's Milk Human Milk Research Biorepository at the University of California, San Diego. From December 14, 2020 to February 1, 2021, 180 women who received two doses of either mRNA vaccine were recruited into the study. Results: Similar proportions of women reported any one or more symptoms following vaccination with either mRNA vaccine. In addition, the frequency by specific type of symptom did not differ by brand. However, following the second dose of vaccine, women who received the Moderna brand were significantly more likely to report symptoms. A small proportion of women following the first dose of either vaccine brand reported a reduction in milk supply, and significantly, more women reported a reduction in milk supply following the second dose of Moderna. Few infant events were reported for either vaccine brand following either dose, and no serious adverse events were reported. Conclusions: These data are reassuring regarding the safety of vaccination in breastfeeding women and their breastfed children with either of the mRNA COVID-19 vaccines.

No Evidence of Infectious SARS-CoV-2 in Human Milk: Analysis of a Cohort of 110 Lactating Women.

BACKGROUND: SARS-CoV-2 infections of infants and toddlers are usually mild but can result in life-threatening disease. SARS-CoV-2 RNA been detected in the breast milk of lactating women, but the potential role of breastfeeding in transmission to infants has remained uncertain. METHODS: Breast milk specimens were examined for the presence of the virus by RT-PCR and/or culture. Specimens that contained viral RNA (vRNA) were examined for the presence of subgenomic coronavirus RNA (sgRNA), a putative marker of infectivity. Culture methods were used to determine the thermal stability of SARS-CoV-2 in human milk. RESULTS: Breast milk samples from 110 women (65 confirmed with a SARS-CoV-2 diagnostic test, 36 with symptoms but without tests, and 9 with symptoms but a negative SARS-CoV-2 diagnostic test) were tested by RT-PCR (285 samples) and/or viral culture (160 samples). Although vRNA of SARS-CoV-2 was detected in the milk of 7 of 110 (6%) women with either a confirmed infection or symptomatic illness, and in 6 of 65 (9%) of women with a positive SARS-CoV-2 diagnostic test, virus was not detected in any culture. None of the 7 milk specimens with detectable vRNA contained sgRNA. Notably, when artificially added to human milk in control experiments, infectious SARS-CoV-2 could be cultured despite several freeze-thaw cycles, as occurs in the storage and usage of human milk. CONCLUSIONS: SARS-CoV-2 RNA can be found infrequently in the breastmilk of women with recent infection, but we found no evidence that breastmilk contains infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. KEY POINTS: Question: SARS-CoV-2 RNA has been detected in a small number of human milk samples collected from recently infected women. The role of breastfeeding in transmission of the virus to infants has remained uncertain due to the small number of specimens analyzed in any study published thus far.Findings: In a total study group of 110 women, SARS-CoV-2 RNA was detected in milk from 6 of 65 women (9.2%) with recent confirmed infection. Neither infectious virus nor subgenomic RNA (a marker of virus infectivity) were detected in any of the samples.Meaning: We found no evidence that infectious SARS-CoV-2 is present milk from recently infected women, even if SARS-CoV-2 PCR tests are positive, providing reassurance of the safety of breastfeeding.

The Design and Mechanics of an Accessible Human Milk Research Biorepository.

Introduction: Human milk is the normative standard for infant/toddler nutrition. To better understand human milk's imprinting on health, and inform complex decisions about maternal medication, substance use, and other exposures during lactation, researchers at University of California San Diego (UC San Diego) established Mommy's Milk, a Human Milk Research Biorepository (HMB). Materials and Methods: The HMB was founded in 2014 with the goal of building a constant but rotating inventory of 3,000 human milk samples available for future research. Following informed consent, women in the United States or Canada provide 50 mL up to a full pump of expressed breast milk. Participants are also interviewed about their sociodemographic characteristics, pregnancy history, dietary intake, maternal stress, anxiety and depression, breastfeeding behaviors, and signs and symptoms of potential adverse reactions in the offspring. Data on growth of the infant/toddler are captured from medical records, and neurodevelopmental assessments are conducted longitudinally. Sample collections occur at UC San Diego, community sites, or the woman's home, and are aliquoted and stored at -80°C. Results: To date, 1,362 unique women have contributed to the HMB. The majority of mothers were between the ages of 31-35, and identified as White. The range of ages of breastfed offspring was well-represented through 23 months. Conclusions: The HMB is a well-characterized, accessible research resource that can contribute to better understanding of the characteristics of human milk, and potential effects of maternal medications, substances, and other environmental agents on the health and development of the breastfed infant/toddler.

Evaluation of SARS-CoV-2 in Breastmilk from 18 Infected Women.

To The Editor, Currently, the U.S. Centers for Disease Control and Prevention, American Academy of Pediatrics and the World Health Organization advise that women who are infected with SARS-CoV-2 may choose to breastfeed with appropriate protections to prevent transmission of the virus through respiratory droplets. However, the potential for exposure to SARS-CoV-2 through breastfeeding is currently unknown. To date, case reports on breastmilk samples from a total of 24 SARS-CoV-2-infected women have been published. Of those, viral RNA was detected in ten breastmilk samples from four women. In some but not all cases, environmental contamination as the source of the virus or retrograde flow from an infected infant could not be ruled out. We established a quantitative RT-PCR assay for SARS-CoV-2 in breastmilk with a limit of detection of 250 copies per mL and validated it by spiking breastmilk from uninfected women with known amounts of viral RNA. In addition, we established tissue culture methods to detect replication-competent SARS-CoV-2 in breastmilk. No viral RNA nor culturable virus was detected after Holder pasteurization of breastmilk samples that had been spiked with replication-competent SARS-CoV-2 (see Supplement). Between March 27 and May 6, 2020, we collected and analyzed 64 serial breastmilk samples from 18 SARS-CoV-2-infected women residing in the U.S. (see Supplement for clinical characteristics). Breastmilk samples were collected before and after women had a positive SARS-CoV-2 RT-PCR test and all but one woman had symptomatic disease (see Figure). One of the 64 breastmilk samples had detectable SARS-CoV-2 RNA by RT-PCR. The positive sample was collected on the day of symptom onset but one sample 2 days prior to symptom onset and two subsequent samples, collected 12 and 41 days later, tested negative for viral RNA. In addition, a subset of 26 breastmilk samples from nine women were tested for the presence of replication-competent virus using our established culture methods, and all were negative including the one sample that tested positive for viral RNA by RT-PCR. Although SARS-CoV-2 RNA was detected in one milk sample from one of eighteen infected women, the viral culture for that sample was negative. This suggests that SARS-CoV-2 RNA does not represent replication-competent virus and that breastmilk itself is likely not a source of infection for the infant. Furthermore, when control breastmilk samples spiked with replication-competent SARS-CoV-2 virus were treated by Holder pasteurization, a process commonly performed by donor milk banks, no replication-competent virus nor viral RNA was detectable. Further research to confirm these findings is needed, as well as an examination of convalescent milk for the presence of antibodies against SARS-CoV-2.

Vedolizumab Does Not Affect Antibody Secreting Cell Recruitment to the Lactating Mammary Gland of Mothers With Inflammatory Bowel Disease.

Despite a known role for α4ß7 and MAdCAM-1 for the recruitment of antibody secreting cells to the lactating mammary gland, vedolizumab which targets integrin α4ß7 did not lower breastmilk IgA in lactating mothers with IBD receiving the drug. It is likely that antibody secreting cells alternatively employ α4ß1 to arrest on VCAM-1 also expressed by the lactating mammary gland.

Marijuana Use by Breastfeeding Mothers and Cannabinoid Concentrations in Breast Milk.

: media-1vid110.1542/5799877373001PEDS-VA_2018-1076Video Abstract BACKGROUND AND OBJECTIVE: Marijuana is the most commonly used recreational drug among breastfeeding women. With legalization of marijuana in several US states and a 1990 study in which authors documented psychomotor deficits in infants breastfed by mothers using marijuana, there is a need for information on potential exposure to the breastfed infant. Our objective with this study was to quantify cannabinoids in human milk after maternal marijuana use. METHODS: Between 2014 and 2017, 50 breastfeeding women who reported marijuana use provided 54 breast milk samples to a research repository, Mommy's Milk. Concentrations of Δ-9-tetrahydrocannabinol (∆9-THC), 11-hydroxy-Δ-9-tetrahydrocannabinol, cannabidiol, and cannabinol were measured by using liquid chromatography mass spectrometry electrospray ionization. RESULTS: ∆9-THC was detectable in 34 (63%) of the 54 samples up to ∼6 days after last reported use; the median concentration of ∆9-THC was 9.47 ng/mL (range: 1.01-323.00). Five samples had detectable levels of 11-hydroxy-Δ-9-tetrahydrocannabinol (range: 1.33-12.80 ng/mL) or cannabidiol (range: 1.32-8.56 ng/mL). The sample with the highest concentration of cannabidiol (8.56 ng/mL) did not have measurable ∆9-THC. Cannabinol was not detected in any samples. The number of hours since last use was a significant predictor of log ∆9-THC concentrations (-0.03; 95% confidence interval [CI] -0.04 to -0.01; P = .005). Adjusted for time since last use, the number of daily uses and time from sample collection to analysis were also significant predictors of log ∆9-THC concentrations (0.51; 95% CI 0.03 to 0.99; P = .039; 0.08; 95% CI 0.00 to 0.15; P = .038, respectively). CONCLUSIONS: ∆9-THC was measurable in a majority of breast milk samples up to ∼6 days after maternal marijuana use.