Postpartum, patients with multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) have increased risk for disease activity. Anti‐CD20 IgG1 monoclonal antibodies (mAb) are increasingly used as disease‐modifying therapies (DMTs). Patients may wish to both breastfeed and resume DMT postpartum. This study aimed to determine the transfer of anti‐CD20 IgG1 mAbs, ocrelizumab, and rituximab (OCR/RTX), into mature breastmilk and describe maternal and infant outcomes.cis‐women receiving OCR/RTX after 59 pregnancies and their infants were enrolled and followed up to 12M postpartum or 90 days post‐infusion. Breastmilk was collected pre‐infusion and serially up to 90 days and assayed for mAb concentration. Medical records and patients' questionnaire responses were obtained to assess neurologic, breastfeeding, and infant development outcomes.Fifty‐seven p > 0.05) infants; neither did the proportion with normal development . After postpartum infusion, two mothers experienced a clinical relapse.The median average concentration of mAb in breastmilk was low . Concentration peaked 1–7 days post‐infusion in most (77%) and was nearly undetectable after 90 days. Median average relative infant dose was <1% . Forty‐three participants continued to breastfeed post‐infusion. At 8–12 months, the proportion of infants' growth between the 3rd and 97th World Health Organization percentiles did not differ for breastfed (36/40) and non‐breastfed (14/16, These confirm minimal transfer of mAb into breastmilk. Anti‐CD20 mAb therapy stabilizes MS activity before conception to the postpartum period, and postpartum treatments appears to be safe and well‐tolerated for both mother and infant. The safe and effective management of neurological disease during gestation and lactation represents major unmet treatment goals across many neurological conditions. Both multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are more prevalent in females than in males, with onset often occurring during childbearing years.This prospective study enrolled international patients with a clinical diagnosis of MS or NMOSD receiving OCR or RTX clinically prescribed by their treating neurologist while breastfeeding or weaning and who were willing to provide breastmilk samples. All participants were enrolled in the University of California, San Francisco (UCSF) Pregnancy Registry, Infants, and Milk/Serum Analysis (PRISMA) study, which includes patients followed at the UCSF and other MS centers if they sought enrollment in the study. The samples included in this analysis were collected between November 2017 and December 2021, from participants referred from 10 MS centers in the United States and Germany. A combination of maternal records (59 pregnancies), infant records , and breastmilk samples  was collected. A subset of the 29 women (30 pregnancies) receiving ocrelizumab were enrolled in an investigator‐initiated sub‐study sponsored by Genentech. Among the 59 pregnancies, only nine in which women received rituximab were previously reported.clinicaltrials.gov, NCT04387110. Written informed consent was obtained from all the enrolled participants in their preferred language by the UCSF study team.The UCSF Institutional Review Board (IRB) approved this study (17‐22422). Under the same IRB, a sub‐study of ocrelizumab in breastmilk was sponsored by Genentech as an investigator‐initiated study (IIS) and listed on Maternal medical records were reviewed to capture demographic data, medical history, mAb infusion date(s)/time and dose(s), and neurological data during the 12 months (M) before conception and up to 12M after delivery . Questionnaires were completed prospectively by participating mothers to provide pertinent pregnancy information, including medications used before and during pregnancy, pregnancy complications, MS or NMOSD relapses, infections, infusion reactions, and breastfeeding status. Telephone interviews were used to supplement missing information as needed. Clinical relapses, defined as new or worsening neurologic symptoms for ≥24 h in the absence of infection, were prospectively collected using the patient‐reported assessing relapses in MS Questionnaire at 1, 4, 8, and 12M postpartum and corroborated by the treating neurologist's note. When Expanded Disability Status Scale (EDSS) score was not explicitly included in the treating neurologist's note, this was extrapolated from the neurologic examination, documented symptoms, and reported ambulatory abilities by a neurologist (R.B.), blinded to the timing of the neurological exam relative to pregnancy.Infant medical records were reviewed to record delivery outcomes, immunizations, growth, laboratory results, infections, and developmental concerns. Weight, length, and head circumference recorded in medical records from delivery to 12M of age were plotted on the World Health Organization (WHO) clinical growth charts and reported as percentiles for age. When available, clinically assessed CD19 count and immunoglobulin levels from cord blood or peripheral blood were collected for infants with potential exposure to maternal anti‐CD20 therapy either in utero or during lactation. The Ages and Stages Questionnaire Version 3 (ASQ3) was completed prospectively by parents at 2, 4, 6, 8, 10, and 12M of age . Due to the low number, survey timing was not adjusted for prematurity. The ASQ3 evaluates five domains of child development . Each domain is scored and categorized as “below cutoff”, “monitoring range”, and “above cutoff”. Both the “monitoring range” and “above cutoff” are considered on‐schedule development. “Below cutoff” suggests delayed development and indicates need for professional assessment.For most participants, serial breastmilk samples were systematically collected before infusion (drug‐naïve) and at 8 and 24 h post‐infusion, in addition to 7, 18–21, 30, 60, and 90 days after one or two OCR (300 or 600 mg) or RTX (500 or 1000 mg) infusion(s) at the first and/or second postpartum treatment cycle. During an initial period of study feasibility (November 2017 to April 2019), four participants provided single‐ or two‐timepoint samples, and another five participants provided serial samples; these were previously reportedTo determine the breastmilk concentration of anti‐CD20 mAb therapies, fluorescent enzyme‐linked immunosorbent assay (ELISA) was used. Samples provided by participants treated with OCR were analyzed with ELISA by Syneos Health Laboratories , using affinity‐purified goat anti‐ocrelizumab polyclonal antibody as coat and a goat anti‐IgG1 antibody conjugated to horseradish peroxidase as detection. Samples provided by participants treated with RTX were analyzed by Marin Biologic Laboratories  with the Eagle Biosciences rituximab ELISA Kit, using methods previously reported.Anti‐CD20 breastmilk concentrations were measured in duplicate for each sample and averaged, and when replicates of a sample were analyzed across several assays, these replicates were averaged.AVE) was calculated by dividing the AUC by the number of days from infusion at the time of last collection for each individual set. The maximum concentration (CMAX) and the time of CMAX were also reported.Descriptive statistics were used to characterize the cohort and infant outcomes. Then, to evaluate breastmilk parameters, for the participants who provided serial (at least 3) samples, the breastmilk concentrations of mAb were analyzed using R software (4.2.2) with pharmacokinetic methods  was used to assess the difference in proportion of infants breastfed post‐maternal infusion versus not with normal growth and development  at 8–12M, reflecting methods outlined by Matro et al. 2018.cis‐women with relapsing–remitting MS and two cis‐women with NMOSD enrolled in the PRISMA study. Altogether, these 57 participants provided data corresponding to 59 pregnancies, including 33 pregnancies with postpartum treatment with OCR and 26 pregnancies with postpartum treatment with RTX. The mean age was 33.4 (SD 3.9) years, with low disability (median EDSS of 1.5) and median disease duration of 5.1 (range 0.1–14.0) years (Table n = 27 ≤ 6 M prior to conception). The median time between delivery and postpartum infusion in the samples provided was 2.6 M (range 0.1–36.0). Among the 59 total pregnancies , 51 were term pregnancies and 8 pregnancies were preterm (<37 weeks gestation).Fifty‐five n = 3: no DMT, one of whom subsequently started glatiramer acetate during pregnancy, n = 1: fingolimod discontinued before conception, n = 1: fingolimod discontinued before conception and switched to glatiramer acetate during pregnancy, with four relapses). Postpartum, a relapse occurred in 13/57 pregnancies between delivery and first anti‐CD20 infusion. Between anti‐CD20 infusion and the 12M postpartum, one relapse occurred among 57 pregnancies, 2 days after infusion.Clinical relapses and their association with DMT use from 12M before conception through 12M postpartum are visualized in Figure Focusing on patients treated using optimized windows for anti‐CD20, with treatment within 6M of conception, 1/25 experienced a relapse between infusion and delivery ; 2/25 experienced a relapse between delivery and infusion (infusion was >5 weeks postpartum in both cases); and 1/25 experienced a relapse 2 days after infusion . Of all 14 participants who were treated with anti‐CD20 within 5 weeks of delivery, 0/14 experienced a clinical relapse between delivery and 12M postpartum.Two participants with NMOSD received anti‐CD20 therapy within 6M prior to conception. Neither experienced a relapse in the year before conception or during pregnancy. Postpartum, one relapse occurred in one pregnancy 1.5M after delivery, 14 days after anti‐CD20 therapy had been resumed.Maternal infections were also prospectively collected . No maternal infections requiring hospitalization were reported. Four women reported mastitis during their participation in the study; however, none of these infections coincided with breastmilk collection.In 17 pregnancies, serum IgG, IgM, and/or IgA levels were assessed clinically postpartum and prior to maternal infusion . Among women with anti‐CD20 within 6M of conception, IgG (13/16 pregnancies), IgM (7/8 pregnancies), and IgA (14/15 pregnancies) were within normal range. The three abnormal postpartum IgG levels were: 275, 589, and 652 mg/dL . Among the women with low postpartum IgG postpartum, two women had been treated with OCR for ≥12M prior to conception and one participant was DMT naïve.n = 16 with detectable concentrations]) and 0.1 μg/mL for RTX (range 0.01–0.9 [n = 15 with detectable concentrations]). Based on CAVE, RID was 0.1% for OCR (range 0.07–0.7) and 0.04% for RTX (range 0.005–0.3)  Table .Among the participants who provided a single or two samples, the observed concentrations were similar to those of patients with serial samples sets apart from one outlier with a concentration of 1.15 μg/mL at 14 days post‐infusion (RID 3.4%). This outlying concentration and similar sample sets ending on a peak in concentration were collected at the time of weaning and subsequent samples were not available; thus the rate of decline from peak concentration could not be calculated.Eight infants with maternal anti‐CD20 treatment within 6M of conception and one infant with maternal infusion at 7 weeks and 1 day underwent clinical evaluation of cord and/or peripheral blood for IgG and CD19 levels; all were within normal range Fig. .15, 1n = 27 exclusively, n = 16 partially); median duration of breastfeeding post‐infusion and up to 12M postpartum was 6.4 M (range 0.3–11.7). When comparing the breastfed versus non‐breastfed infants, there were no differences in the proportion of infants whose 8‐12 M measurements fell within the WHO standards for normal growth . Additionally, there were no differences in the proportion of breastfed  versus non‐breastfed infants  in ASQ3 development scores up to 8–12M ,p > 0.05 in all domains . From the clinical records available for 56 infants (including both breastfed and non‐breastfed), there were minor but nonserious infections which are common in infancy , minimal concentration of mAb was found in mature breastmilk, corresponding to a very low RID, well below the theoretically acceptable threshold. Infants who were subsequently breastfed did not differ in growth or development from infants who were weaned, and immunological parameters were normal when measured, suggesting negligible uptake of any residual undigested anti‐CD20 IgG drug molecules into the infant circulation.Several aspects of the findings, reassuringly, support the safety of breastfeeding during anti‐CD20 treatment. First, similar trends were observed for both OCR and RTX, including RID (<1%) and time to peak (1–10 days). Second, the findings from the preliminary cohort of RTX patients were reaffirmed in the current larger dataset. Third, the highest concentrations were noted in the mothers who were actively weaning, which is consistent with the physiology of the mammary gland when discontinuing breastfeeding.Historically, about one third of all women with MS relapse postpartum,Clinically, these results confirm that anti‐CD20 therapy effectively reduces MS disease activity before conception through to the postpartum period, minimal transfer into breastmilk, and reassuring infant development, growth, and CD19 levels when exposed to anti‐CD20 therapies via breastfeeding. The findings may have relevance to the use of several other IgG1 mAbs in the management of pregnant and lactating women with neurological diseases, including MS, NMOSD, myasthenia gravis, and other neuroimmune conditions, as well as more common conditions such as migraines.Annika Anderson was responsible for the acquisition and analysis of data and drafted and revised the manuscript. William Rowles, Shane Poole, Ayushi Balan, Dr. Carolyn Bevan, Dr. Rachel Brandstadter, Dr. Andrea I. Ciplea, Dr. Joanna Cooper, Dr. Michelle Fabian, Dr. Thomas W. Hale, Dr. Dina Jacobs, Dr. Mihir Kakara, Dr. Kristen M. Krysko, Dr. Erin E. Longbrake, Dr. Jacqueline Marcus, Dr. Pavle Repovic, Dr. Claire S. Riley, Dr. Andrew R. Romeo, Dr. Alice Rutatangwa, Dr. Timothy West, Dr. Kristen Hellwig, and Dr. Sara C. LaHue were responsible for the aquisition and analysis of data and revised the manuscript. Dr. Riley Bove conceptualized and supervised the study and drafted and revised the manuscript.The PI (RB) is funded through a Harry Weaver Award from the National Multiple Sclerosis Society (NMSS). Support for enrollment and analysis of patients infused with ocrelizumab (only) was provided through an investigator‐initiated study sponsored by Genentech, Inc, the manufacturer of ocrelizumab.All authors’ financial disclosures are included in the attached ICMJE forms. The drugs, ocrelizumab and rituximab, are tested in this study. Genentech (a subsidiary of the Roche Group) is the manufacturer of ocrelizumab and co‐markets rituximab, a drug developed and co‐marketed by Biogen. Potential conflict of interest disclosures related to the study are outlined below: CB has received scientific advisory board fees and travel support from Genentech which manufactures and co‐markets the AIC reports speaker honoraria from Biogen which co‐markets a drug tested in this study. DJ has received research support and consulting/scientific advisory board fees from Biogen and Genentech which manufacture and co‐market drugs tested in this study. KMK is an advisory board member for Roche and has received grants from Roche which manufactures and co‐markets a drug tested in this study. KMK also reports support from an MS fellowship grant during her fellowship from Biogen, which co‐markets a drug tested in this study. EEL reports research support and consulting/advisory board fees from Genentech which manufactures and co‐markets the drugs tested in this study. PR reports consulting and/or speaking honoraria from Genentech which manufactures and co‐markets the drugs tested in this study. CSR reports consulting fees or honoraria from Genentech which manufactures and co‐markets the drugs tested in this study. TW has received honoraria for speaking and education activities through Biogen Idec which co‐markets a drug tested in this study. KH has received research support, consulting, speaking, and/or advisory board fees from Roche and Biogen which manufacture and co‐market the drugs tested in this study. RB reports research support from Biogen and Roche‐Genentech which manufactures and co‐markets the drugs tested in this study.Table S1.Click here for additional data file.


Hyperglycaemia in Type 1 diabetes (T1D) results from an absolute insulin deficiency. However, insulin resistance (IR) may exacerbate glycaemic instability in T1D and contribute to long‐term cardiovascular complications. We previously showed that IR in teenagers with obesity is associated with sex‐dependent derangements in the catabolism of branched‐chain amino acids (BCAA) and fatty acids. Here we hypothesized that byproducts of BCAA and fatty acid metabolism may serve as biomarkers or determinants of glycaemic control and IR in prepubertal or early pubertal children with T1D.Metabolites, hormones and cytokines from fasting blood samples were analysed in 28 children  with T1D. Principal components analysis (PCA) and multiple linear regression models were used to correlate metabolites of interest with glycaemic control, total daily insulin dose , adiponectin and the triglyceride (TG) to high‐density lipoprotein (HDL) ratio.Males and females were comparable in age, BMI‐z, insulin sensitivity, glycaemic control, inflammatory markers, BCAAs and C2/C3/C5‐acylcarnitines. The majority of components retained in PCA were related to fatty acid oxidation (FAO) and BCAA catabolism. HbA1c correlated positively with Factor 2  and Factor 9 (fasting glucose). TDD correlated negatively with C3 and C5 and Factor 10 (BCAA catabolism) and positively with the ratio of C2 to C3 + C5 and Factor 9 (fasting glucose).These findings suggest that glucose intolerance in prepubertal or early pubertal children with T1D is accompanied by incomplete FAO while TDD is associated with preferential catabolism of fats relative to amino acids. We hypothesized that byproducts of BCAA and fatty acid metabolism may serve as biomarkers or determinants of glycaemic control and insulin resistance (IR) in prepubertal or early pubertal children with Type 1 diabetes (T1D). Principal components analysis and multiple linear regression models were used to correlate metabolites of interest with glycaemic control and IR. Our findings suggest that glucose intolerance in prepubertal or early pubertal children with T1D is accompanied by incomplete fatty acid oxidation while total daily insulin dose is associated with preferential catabolism of fats relative to amino acids. However, resistance to insulin action may exacerbate glycaemic instability in T1D and contribute to long‐term cardiovascular complications.Mechanisms explaining the pathogenesis of IR in T1D remain poorly understood but likely involve dysregulation of insulin action in liver, skeletal muscle and adipose tissues. This in turn promotes lipolysis and proteolysis and decreases glucose uptake and utilisation.We previously showed that IR in adolescents with obesity is associated with sex‐dependent derangements in the catabolism of branched‐chain amino acids (BCAA) and fatty acids.We focused on young prepubertal and early pubertal children with T1D to minimize the effects of sex steroids and pubertal increases in GH on glycaemic control and insulin sensitivity. Our aims were to, (1) identify potential biomarkers that associate with glucose intolerance and IR in children with T1D; and (2) determine if there are any sex differences in these biomarkers in prepubertal and early pubertal children with T1D.22.1Peripheral sensitivity to insulin declines during puberty, reaching a nadir at Tanner Stage III.Twenty‐nine participants were enrolled; one patient withdrew from the study; thus, the study cohort consisted of 28 prepubertal or early pubertal youth with T1D .2.2https://www.cdc.gov/nccdphp/dnpao/growthcharts/resources/sas.htm). Body fat percentage (BF%) was estimated by electrical impedance using a Tanita BC‐418 segmental body composition analyzer. A physical examination was performed by a paediatric endocrinologist or a nurse practitioner specialized in paediatric endocrinology and diabetes.Body weight, measured to the nearest 0.1 kg, and height, measured to the nearest 0.1 cm, were measured by standard methods. BMI, BMI percentiles and BMI z‐scores were calculated using the SAS program available at Centers for Disease Control and Prevention  was collected to assess surrogate measures of insulin sensitivity, conventional metabolites, plasma acylcarnitines, amino acids, cortisol and inflammatory markers. All samples were stored until analysis was run together in a batch.2.3.1Total and high molecular weight (HMW) adiponectin were measured using ELISA kits from Alpco  for which plasma samples were diluted 1000‐fold. Leptin was measured using immunoassay kits from Meso Scale Discovery (MSD) on an SI‐2400 electrochemiluminescent imager. Reproducibility, as assessed by coefficient of variation for duplicate measurements, averaged 5.6%.2.3.2Measurements of conventional metabolites were performed using a Beckman‐Coulter DxC 600 clinical analyzer . Plasma glucose, total cholesterol, HDL, LDL and triglycerides (TGs) were measured using reagents from Beckman. Total nonesterified fatty acids (NEFA), total ketones and 3‐hydroxybutyrate were measured with reagents from Wako .2.3.3Acylcarnitines  and amino acids  were analysed by tandem mass spectrometry using a Waters TQD instrument.2.3.4Cytokines, including interferon gamma (IFN‐γ), interleukin 6 (IL‐6), interleukin 10 (IL‐10) and tumour necrosis factor alpha (TNF‐α), were measured by multiplex assay using the MSD imager. High‐sensitivity CRP (hs‐CRP) was measured using the analyser and reagents from Beckman. Cortisol was assayed using a kit from Alpco .2.3.5Fasting plasma glucose, HbA1c and insulin regimens were extracted from medical charts. Of our 28 patients, 11 were on multiple daily injections (MDI) and 17 were using insulin pumps . When available, continuous glucose monitoring (CGM) data were also extracted from medical charts, which provided time in range (TIR). All 28 patients were using CGM systems. We included 2 weeks of CGM data from the weeks preceding the study visit to reflect current metabolic status and accepted readouts reflecting >70% CGM use per established protocol.2.4n = 28) provides adequate statistical power for our primary analysis of multivariable models of IR. Between group comparisons of the participant characteristics, participant body composition measures and measures of glycaemic control/insulin sensitivity were performed by t‐test if the variable was normally distributed or by Wilcoxon rank sum test when not normally distributed. We calculated Spearman correlation coefficients to analyse bivariate relationships between surrogate measures of IR, and BCAA and FAO metabolites. All analyses were performed in R version 4.1.2 (2021‐11‐01) and SAS version 9.4 . Descriptive results in tables are reported as mean ± SD. p‐value ≤ .05 was considered statistically significant.Sample size was calculated using SAS  Proc Power to detect correlations of 0.5 or greater between metabolites of interest and TDD. In linear regression model with five explanatory variables, a sample size of 27 provides power of 80% to detect a correlation of 0.5 using a two‐sided 0.05 significance level test; thus, our sample size  with varimax rotation was used to reduce the large number of correlated metabolites into uncorrelated clusters of fewer components.All metabolites were assessed for normality and those that were not normally distributed were log‐transformed. Eleven principal components with the largest eigenvalues that explained 82% of the variance of all metabolites used for the analysis were retained for downstream analysis. Metabolites with an absolute value of factor loading of 0.4 or greater were considered to constitute that component.p = .05. All models were adjusted for age, sex, Tanner stage and diabetes duration.For modelling, the components retained in the PCA were used in multivariable models as explanatory variables. None of the dependent variables required transformation to improve the normality assumption. Model selection was performed using stepwise selection with both entry and stay criteria set to 33.1Twenty‐eight subjects were studied; 15 females and 13 males. This included 82.1% (23 out of 28) Caucasian/White, 10.7% (3 out of 28) Black/African American, 3.6% (1 out of 28) Native Hawaiian or Other Pacific Islander and 3.6% (1 out of 28) two or more races. Ethnicity was reported as 89.3% (25 out of 28) non‐Hispanic/Latino, 7.1% (2 out of 28) Hispanic/Latino and 3.6% (1 out of 28) not reported. Mean diabetes duration was 49.4 ± 30.7 months.Male participants had mean weight 33.75 ± 6.17 kg, mean height 137.55 ± 7.96 cm, mean BMI z‐score 0.367 ± 0.98 and mean BF% 19.93 ± 5.41 , but not with TIR .HbA1c  and TIR  were comparable in males and females , HMW adiponectin , TG/HDL ratio  or TDD . BF% did not correlate with HbA1c , TIR , total adiponectin , HMW adiponectin , TG/HDL ratio  or TDD .Total adiponectin , HMW adiponectin , TG/HDL ratio  and TDD  were comparable in males and females  celiac screening obtained per standard of care clinic visits.3.4r = .62, p = .0038 and r = .72, p = .0004, respectively).There were no significant differences between males and females with regard to morning cortisol levels, cytokines including IFN‐γ, IL‐6, IL‐10, TNF‐α or hs‐CRP  was calculated as a surrogate measure of the catabolism of fats relative to the catabolism of BCAAs. Levels of BCAAs, C2, C3 and C5, and the ratio C2/(C3 + C5) were similar among males and females Table .3.7r = −.36, p = .05 and r = −.43, p = .02, respectively). In contrast, TDD correlated positively with a metric of fat catabolism . There were no significant correlations between HbA1c , TIR , leptin  or BF%  and metrics of BCAA catabolism or fat catabolism.TDD  correlated negatively with metrics of BCAA catabolism including C3 and C5 acylcarnitines , validating our data. TDD (units/kg/d) correlated positively with Factor 9 (fasting glucose), and negatively with Factor 10 (BCAA catabolism), consistent with our bivariate results  correlated negatively with metrics of BCAA catabolism and positively with the metrics of fat catabolism, implicating preferential catabolism of fats relative to amino acids. Finally, there were no significant differences in metabolites between males and females in our cohort of prepubertal or very early pubertal children with T1D. This observation contrasts strongly with studies of older, more sexually mature teenagers and adults, which find that fasting BCAA levels and C3 and C5 acylcarnitines are significantly higher in males than females.Surrogate metrics of IR in this study included total and high molecular weight adiponectin and the TG to HDL ratio. Adiponectin is a marker of insulin sensitivity, as it decreases hepatic glucose output and increases glucose uptake and FAO in muscles.We found that daily insulin requirement  correlated negatively with metrics of BCAA catabolism and positively with the ratio of acetylcarnitine (C2) to the sum of propionylcarnitine (C3) and (C5), implicating preferential catabolism of fats relative to amino acids. This is in agreement with prior studies completed in pubertal children.r = −.768; p < .0001), validating our data.In contrast to daily insulin requirement, HbA1c did not associate with markers of BCAA catabolism or complete FAO. Rather, HbA1c correlated most strongly with Factor 2  and Factor 9 (glucose). The association of glycaemic control in T1D with fatty acid metabolism has been noted previously: hyperinsulinaemic euglycaemic clamp studies demonstrated that adolescents with T1D have significantly higher rates of lipolysis and endogenous glucose production and lower peripheral glucose uptake during hyperinsulinaemia compared to controls.As noted previously, there were no significant differences in metabolites between the young males and females in our cohort with T1D. This finding contrasts sharply with our previous studies of IR in adolescents with obesity: fasting levels of BCAA and products of BCAA catabolism were significantly higher in obese teenage boys than obese girls of similar age and BMI z‐score.Elevated cortisol and inflammatory cytokines have previously been demonstrated in individuals with T1D in ketoacidosis and can induce proteolysis and muscle catabolism and reduce muscle protein synthesis.Our study has some limitations. The sample size was small but provided adequate statistical power for our primary analysis. We used surrogate measures of insulin sensitivity; additional methods, including insulin and glucose clamps and iv and oral glucose tolerance tests, might have provided useful information regarding insulin secretion and tissue‐specific IR.Nevertheless, our study is one of the first to examine the relationship of BCAAs and FAO metabolites to glycaemic control and metrics of insulin sensitivity in young prepubertal or early pubertal children with T1D. Our findings suggest that TDD is associated with the preferential catabolism of fats relative to amino acids, while HbA1c is associated with incomplete FAO. Future studies are needed to examine the rates of muscle protein and fat synthesis and breakdown in healthy children and children with T1D along with their relationship to insulin administration and glycaemic control, metabolic control, growth and pubertal development.Grace Hendrix: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; writing – original draft (lead); writing – review and editing . Yuliya Lokhnygina: Data curation ; formal analysis ; investigation ; methodology ; resources ; software ; writing – review and editing . Megan Ramaker: Data curation ; formal analysis ; investigation ; methodology ; resources ; software ; writing – review and editing . Olga Ilkayeva: Data curation ; formal analysis ; methodology ; resources ; software ; supervision ; writing – review and editing . Michael Muehlbauer: Data curation ; formal analysis ; methodology ; resources ; software ; supervision ; writing – review and editing . William Evans: Conceptualization ; investigation ; methodology ; resources ; validation ; writing – review and editing . Lisa Rasbach: Investigation (supporting); methodology (supporting); project administration (supporting); resources (supporting); validation (supporting); writing – review and editing (supporting). Robert Benjamin: Investigation (supporting); methodology (supporting); project administration (supporting); resources (supporting); validation (supporting); writing – review and editing (supporting). Michael Freemark: Conceptualization ; data curation ; investigation ; methodology ; resources ; supervision ; validation ; writing – review and editing . Pinar Gumus Balikcioglu: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration ; resources (lead); software (lead); supervision (lead); validation (lead); visualization (lead); writing – review and editing (lead).P.G.B. was supported by Diabetes Research Connection (DRC), National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under the award number DK117067, Gall Family Support, Duke University Pediatric Departmental Support and Duke Strong Start Award Program. OI and metabolomics assays performed at the DMPI Metabolomics Core Laboratory were supported by National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award number P30DK124723. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.The authors have no conflict of interest to disclose.The Institutional Review Board at Duke University approved the research protocol.


Metasequoia glyptostroboides. Using both nuclear microsatellites (nSSRs) and chloroplast DNA (cpDNA) fragments, we sampled across the narrow distribution range of this species and determined its conservation units by exploring its genetic structure and historical demography. cpDNA haplotypes were classified into two groups, but mixed in space, suggesting that the existent wild trees of M. glyptostroboides cannot be divided into different evolutionarily significant units. However, using nSSRs, we detected strong spatial genetic structure, with significant genetic differentiation and weak gene flow between the samples in the east of the species' distribution range and other samples. The divergence between the two nSSR groups was dated to the Last Glacial Maximum (c. 19.6 kya), suggesting that such spatial genetic structure has been maintained for a long term. Therefore, these two nSSR groups should be considered as different conservation units, that is, management units, to protect intergroup genetic variations, which is likely to be the outputs of local adaptation. Our findings highlight the necessity to reveal small‐scale genetic structure and population demography to improve the conservation strategies of evolutionary potential of endangered plants.Identifying conservation units is crucial for the effective conservation of threatened species. Previous cases are almost exclusively based on large‐scale but coarse sampling for genetic structure analyses. Significant genetic structure can occur within a small range, and thus multiple conservation units may exist in narrowly distributed plants. However, small‐scale genetic structure is often overlooked in conservation planning especially for wind‐pollinated and wind‐dispersed trees, largely due to the absence of dense and elaborate sampling. In this study, we focused on a representative endangered relict plant,  nSSRs‐based genetic structure and population demography analysis show two spatially segregated genetic groups with limited intergroup gene flow. Therefore, two management units should be set up to protect intergroup genetic variations. It was thought to have been extinct in the early Pleistocene but was rediscovered in the 1940s in Three Gorges Mountain Region (TGMR), China. Fossil records indicate that genus Metasequoia originated in the early Late Cretaceous (c. 100 million years ago) and once distributed over most areas of the Northern Hemisphere , China, with a census size of about 5500 . Our previous studies using random amplified polymorphic DNA markers detected a low genetic diversity in both its wild and planted populations  separated by Hanpo mountain . After testing for homogeneity among sequences using a partition‐homogeneity option implemented in PAUP* v.4.0b10  for 45 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. PCR products were scanned on an ABI 3130 genetic analyzer  using GeneScan 500(‐250) LIZ as the internal lane standard (Applied Biosystems). Amplified fragments were called and binned using GeneMapper 4.0 software (Applied Biosystems).All of the 467 samples were genotyped using eight reciprocally independent microsatellite loci  developed for i et al. . PCR amp2.3H), haplotype diversity (h), and nucleotide diversity (π) using DNASP v. 6.12.01 , Taiwania cryptomerioides , and Cunninghamia lanceolata  were used as outgroups. The best model for nucleotide substitutions (the HKY + I + G model) was selected by the Akaike information criterion using jModeltest 2.1.4 ; and (3) the MRCA of C. lanceolata and other species of Taxodiaceae  ; number of effective alleles (Ne); observed heterozygosity (HO); expected heterozygosity . We allocated all sampled individuals into 18 populations according to the locations of sampling sites to perform the linkage disequilibrium test and null allele frequency analysis . Admixture model and correlated allelic frequencies were used to analyze the data set. We used the ΔK, which measures the rate of change in the log probability of the data between successive K values ) to obtain standardized measures of genetic differentiation using AMOVA  using 10,000 permutations across allele size using the program SPAGeDi  was converted to the migration rate (m) divided by mutation rate  procedure  over time; (2) the population growth model (PGM) where the ancestral effective population size Na is assumed to increase exponentially (experiencing t generations ago) with a growth rate of G, that is, Na = N × exp (G × t), to reach its current effective size (N); and (3) the size reduction model (SRM) which describes a population with an ancestral effective population size Nb experienced population decline from instantaneously t1 generations ago to reach its current effective size . Details for prior distribution of parameters for each model are shown in Table In the analysis of population size change, we calculated the average and standard deviation of the number of alleles, heterozygosity, and allele size range using ARLSUMSTAT v.3.6  as 5 × 10−4 with the prior distribution for each locus drawn randomly from gamma distribution with shape and rate parameters  ranging from 0 to 1 for eight independent loci. All priors were generated by R v.4.0.3  between the two clusters were calculated using ARLSUMSTAT, setting the other parameters using the same statistics of the best population dynamics model. The divergence model assumed the divergence between different genetic groups from their ancestral population with effective population size NANC at time T. Both unidirectional and bidirectional migration rates were set between any two genetic groups, forming three population divergence models. Each model has basic structural parameters: effective population size of a genetic group (such as NCUR1 and NCUR2), ancestral effective population size , divergence time (T), and migration rate (m). NCUR1 and NCUR2 were fixed according to the results in single population size model (Table T to the present. Prior distribution of divergence time (T), ancestral effective population size (NANC), and migration rate (m) were drawn from uniform distribution from 1 to 104, from 102 to 104, and from 10−5 to 0.3, respectively. Simulations and model choice were conducted using the same approaches in the analysis of population dynamics. For the optimal population divergence model, we conducted 105 simulations, with posterior distribution of parameters estimated using neural network regression by the R package ABC v.2.1  were identified according to the definition from Moritz  and nucleotide diversity (π) were 0.926 and 0.90 × 10−3, respectively  and Fu and Li's test  did not detect significant deviation from neutrality.Based on the cpDNA data in 172 sampled trees, we identified a total of 47 distinct haplotypes (GenBank accession: OQ415011‐OQ415039), containing 17 variants including 10 SNPs and seven indels Table . The oveM. glyptostroboides haplotypes and three taxa  for constructing phylogenetic trees. Using the Bayesian optimization approach, we found that all haplotypes of M. glyptostroboides were divided into two clades with a high posterior probability (1.00)  in Neogene  Figure , and the) Figure . In addie Figure .φST′ = 0.241), and AMOVA did not revealed a high intergroup differentiation , showing a low level of inbreeding  and FST′ (0.213)), and AMOVA also showed a significant genetic differentiation between the two groups explaining 18.34% of total genetic variance . Moreover, we detected weak but significant recent gene flow between the two groups, as of the lower boundaries of 95% confidence interval of migrate rates per generation (m) revealed by BayesAss were larger than 0, with the m from the west group to the east one being 0.0077 (95% CI: 0.0010–0.0208) and the m in the reverse direction being 0.0085 (95% CI: 0.0012–0.0192). A similar pattern of historical gene flow was also found using Migrate‐n, showing very low values of m (from the west to the east group: 0.0002 (95% CI: 0.0001–0.0003) and the reverse direction: 0.0004 (95% CI: 0.0002–0.0005)).We found high genetic differentiation between the east and the west groups  was the optimal model, which had the highest posterior probabilities for both groups. Average classification error rates of compared models were 0.295 and 0.315 of the west and east groups, respectively Table . This reNCUR1), the east group (NCUR2), and their ancestral population (NANC), respectively  from the east group to the west one was very low (1.738), with a migration rate of 0.0013  pattern ; data curation ; funding acquisition (lead); investigation ; resources ; visualization (lead); writing – original draft (lead). Min‐Yan Cui: Formal analysis ; investigation . Xiao‐Wei Le: Formal analysis ; investigation . Jun Gong: Formal analysis ; investigation . Kai Jiang: Formal analysis ; writing – original draft . Xin Tong: Formal analysis ; writing – original draft . Qian Zhang: Formal analysis ; writing – original draft . Jia‐Hui Li: Formal analysis ; investigation . Hong‐Yue Li: Formal analysis ; investigation . Ling Lu: Formal analysis ; investigation . Jie Zou: Formal analysis ; writing – original draft . Rong Wang: Conceptualization ; writing – review and editing . Xiao‐Yong Chen: Conceptualization ; methodology ; resources ; writing – review and editing .The authors declare that they have no conflict of interest.Data S1Click here for additional data file.


To assess the differences in circulating DNA methylation levels of CXCR5 between rheumatoid arthritis (RA) and osteoarthritis (OA) and healthy controls (HC), and the correlation of methylation changes with clinical characteristics of RA patients.Peripheral blood samples were collected from 239 RA patients, 30 patients with OA, and 29 HC. Target region methylation sequencing to the promoter region of CXCR5 was achieved using MethylTarget. The methylation level of cg04537602 and methylation haplotype were compared among the three groups, and the correlation between methylation levels and clinical characteristics of RA patients was performed by Spearman's rank correlation analysis.p = 1.3 × 10−3) and in the HC group (p = 5.5 × 10−4). The sensitivity was enhanced when CXCR5 methylation level combined with rheumatoid factor and anti–cyclic citrullinated peptide with area under curve (AUC) of 0.982 . The methylation level of cg04537602 in RA was positively correlated with C‐reactive protein (CRP) , and in RA patients aged 60 years and above, cg04537602 methylation levels were positively correlated with CRP , tender joint count , visual analog scales score , Disease Activity Score in 28 joints (DAS28) using the CRP level DAS28‐CRP , and DAS28‐ESR . We also observed significant differences of DNA methylation haplotypes in RA patients compared with OA patients and HC, which was consistent with single‐loci‐based CpG methylation measurement.The methylation level of cg04537602 was significantly higher in the peripheral blood of RA patients compared with OA patients  patients than in osteoarthritisand HC, and correlated with the level of inflammation in RA patients, Our study establishes a link between CXCR5 DNA methylation and clinical features that may help in the diagnosis and disease management of RA patients. However, the role of methylation levels of the CXCR5 in RA has not been well investigated. In this study, we analyzed circulating DNA methylation levels of CXCR5 in patients with RA, OA, and HC. We depicted the level of CXCR5 DNA methylation and the proportion of haplotypes in the peripheral blood of RA patients, combined the methylation changes with clinical characteristics of RA patients for correlation analysis, explored the role of CXCR5 in RA from an epigenetic perspective, and helped to deepen the understanding of the pathogenesis of RA.22.1The study included 239 RA patients, 30 patients with OA, and 29 healthy controls (HC), all from Precision Medicine Research Cohort in Shanghai Guanghua Hospital.2.2Peripheral venous blood was collected from all participants and genomic DNA was extracted using the Blood Genomic DNA Extraction Kit , a 1.5 mL sample tube was pretreated with 40 μL of Proteinase K solution at a concentration of 10 mg/mL, followed by 400 μL of the whole blood sample. The pretreated samples were subjected to automated DNA extraction in a nucleic acid purifier  (Supporting Information 2). Sample quality control required concentrations ≥20 ng/μL and sample purity ≥400 ng in total . DNA was treated with bisulfite. Targeted methylation profiling is applied for methylation measurement for a specific 200–500 bp region.2.3p < .05 were regarded as statistically significant.IBM SPSS Statistics  and Sangerbox33.1p > .05).239 participants with RA, 30 with OA, and 29 with HC were included in the analysis. Basic characteristics are listed in Table 3.2p = 1.3 × 10−3) and HC (p = 5.5 × 10−4)  Figure . The AUC) Figure .3.3r = .16, p = .01) , tender joint count (TJC) , visual analog scales score , DAS28‐CRP , and DAS28‐ESR . In RA patients aged less than 60 years, cg04537602 methylation levels were negatively correlated with DAS28‐ESR , TJC    Figure .3.4p < .01)  CTC (p < .05) and TCC (p < .01) increased significantly. Compared with OA group or HC group, the proportion of haplotypes TTT decreased significantly (p < .001). Compared with OA group, the proportion of haplotypes TCT decreased significantly (p < .05)  and a weak positive correlation between CCT and CRP   Figure . Correla4In this study, we measured altered DNA methylation of CXCR5 in the peripheral blood of RA patients, OA patients, and HC. In our results, overall methylation levels were significantly higher in RA patients than in OA and HC. This result is in some agreement with that reported in another paper, which reports that CXCR5 cg04537602 locus methylation levels differ significantly between healthy individuals and patients with undifferentiated arthritis, and that a large proportion of patients with undifferentiated arthritis progress to RA, suggesting that CXCR5 methylation may be involved in the pathogenesis of RA.Circulating levels of CXCR5 methylation in RA patients were further analyzed to determine the presence of methylation variants associated with the clinical features of RA. Age is an important risk factor for many chronic inflammatory diseases, including RA and has an impact on patient disease status, dosing regimens, and so on. Moreover, in RA, a growing trend is to stratify patients according to age.In the 300 bp region near cg04537602, we detected a total of three CpG loci, including 11893150, 11893154, and 11893192, and five haplotypes, including CCC, CCT, TTT, TCT, TCC, and CTC. We found that the methylation levels at all three cpg loci were significantly higher in RA patients than in OA patients and HC, and the proportion of TTT representing unmethylated status was significantly lower in RA and the proportion of CCC representing all methylated status was significantly higher, which was basically consistent with the analysis of individual CpGs. Correlation analysis of haplotypes with clinical characteristics of RA patients revealed a significant negative correlation between TTT and CPR, and a significant positive correlation between CCT and CRP. DNA methylation haplotype analysis provides a new vision for observing the direction of methylation change.There are limitations to our study, as confounding factors such as drug selection and different stages of the disease may have an impact on methylation levels, and our results need to be validated in a larger cohort. In conclusion, our study established a link between circulating DNA methylation levels of CXCR5 and inflammation in patient with RA and may contribute to the clinical integration of DNA methylation profiles for better classification and management of RA patients.Dongyi He: Conceptualization (lead); data curation ; funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); writing—review and editing . Rongsheng Wang: Conceptualization ; data curation (supporting); supervision ; writing—review and editing . Shicheng Guo: Conceptualization ; data curation ; supervision ; writing—review and editing (lead). Yiming shi: data curation (lead); formal analysis ; investigation ; methodology ; visualization (lead); writing—original draft (lead). Cen Chang: Data curation ; formal analysis ; investigation (supporting); methodology (supporting); visualization (supporting); writing—original draft . Lingxia Xu and Ping Jiang: Formal analysis ; investigation ; methodology (supporting). Kai Wei, Jianan Zhao, Linshuai Xu, Yehua Jin, Runrun Zhang, and Huijuan Wang: Investigation (supporting). Yi Qian, Yingying Qin, Qin Ding, and Ting Jiang: Writing—review and editing (supporting). All authors agreed to the published version of the manuscript.The authors declare no conflict of interest.This study was approved by the Ethics Committee of Guanghua Hospital of Integrated Traditional Chinese and Western Medicine (2020‐K‐06‐01) and written consent was collected before the surgery from the patients.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


PABPC1L, a predominant poly(A)‐binding protein in Xenopus, mouse, and human oocytes and early embryos prior to zygotic genome activation, plays a key role in translational activation of maternal mRNAs. Here, we identified compound heterozygous and homozygous variants in PABPC1L that are responsible for female infertility mainly characterized by oocyte maturation arrest in five individuals. In vitro studies demonstrated that these variants resulted in truncated proteins, reduced protein abundance, altered cytoplasmic localization, and reduced mRNA translational activation by affecting the binding of PABPC1L to mRNA. In vivo, three strains of Pabpc1l knock‐in (KI) female mice were infertile. RNA‐sequencing analysis showed abnormal activation of the Mos‐MAPK pathway in the zygotes of KI mice. Finally, we activated this pathway in mouse zygotes by injecting human MOS mRNA, and this mimicked the phenotype of KI mice. Our findings reveal the important roles of PABPC1L in human oocyte maturation and add a genetic potential candidate gene to be screened for causes of infertility.Oocyte maturation arrest is one of the important causes of female infertility, but the genetic factors remain largely unknown.  PABPC1L were identified in five infertile females mainly characterized by oocyte maturation arrest followed a recessive inheritance pattern. In vitro and in vivo functional studies confirmed the destructive effects and pathogenicity of PABPC1L variants.Seven rare variants in  TUBB8, PATL2, and TRIP13, but the underlying genetic factors for most affected individuals are still unknown.Oocyte maturation arrest is one of the common factors leading to repeated failures of assisted reproduction and female infertility. Studies have confirmed that genetic factors play a crucial role in oocyte maturation arrest, including PABPC1L that are responsible for female infertility mainly characterized by oocyte maturation arrest in five individuals from four independent families. PABPC1L encodes a poly(A)‐binding protein that plays a key role in translational activation of maternal mRNAs. In vitro studies demonstrated that pathogenic variants resulted in the functional impairment on PABPC1L, including truncated proteins, reduced protein abundance, altered cytoplasmic localization, and reduced mRNA translational activation by affecting the binding of PABPC1L to mRNA. In vivo, we constructed three strains of Pabpc1l KI mice corresponding to patient‐derived variants and found that female mice were infertile due to abnormal activation of the Mos‐MAPK pathway in zygotes. These results demonstrate the destructive effects and pathogenicity of the PABPC1L variants identified in affected individuals both in vitro and in vivo.We identified compound heterozygous and homozygous pathogenic variants in PABPC1L that cause oocyte maturation arrest and female infertility. Our findings provide direct evidence for the important role of PABPC1L in human oocyte maturation and thus add a new marker for the genetic diagnosis of infertility patients in the clinic.We identified and verified bi‐allelic pathogenic variants in in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI), and such failure is characterized by the repeated production of immature oocytes with resulting infertility  cause human oocyte MI arrest by impairing oocyte spindle assembly  and TRIP13 (MIM: 604507) cause oocyte GV arrest and MI arrest, respectively , ZP2 (MIM: 182888), and ZP3 (MIM: 182889) affect the formation of zona pellucida and result in female infertility  variant responsible for female infertility. Pathogenic variants in TUBB8, PATL2, and TRIP13 account for around 10% of infertility patients with oocyte maturation arrest , spindle assembly, extrusion of the first polar body (PB1), and finally arrest at meiotic metaphase II (MII) ‐binding protein cytoplasmic 1‐like) plays a key role in these processes. PABPC1L, also known as EPAB (embryonic poly(A)‐binding protein), was originally identified in Xenopus  tail to stabilize polyadenylated mRNAs  after zygotic genome activation (ZGA)  tail‐modulated translational activation of dormant maternal mRNAs stored in the cytoplasm  that are responsible for female infertility mainly characterized by oocyte maturation arrest. All five affected individuals carried biallelic variants, including missense, stop gain, or frameshift insertions, and all the five followed a Mendelian recessive inheritance pattern. We investigated the functional mechanism of the corresponding pathogenic variants in HeLa cells and Pabpc1l knock‐in (KI) mice. Our findings suggest a causal relationship between PABPC1L and female infertility and reveal important roles for PABPC1L in human oocyte maturation.In this study, we identified compound heterozygous and homozygous pathogenic variants in To investigate the genetic factors responsible for oocyte maturation arrest, we recruited 1,394 infertile female individuals diagnosed with oocyte maturation arrest. In this study, all five affected individuals had been diagnosed with primary infertility of unknown cause for several years despite their having normal menstrual cycles. Their male partners had normal sperm counts and normal sperm morphologic features and motility. The pedigrees of the individuals are shown in Fig PABPC1L (GenBank: NM_001124756.2)  and c.526C>T (p.Arg176*). The affected individual in family 2 carried the compound heterozygous variants c.410C>T (p.Ser137Phe) and c.956_957insA (p.Ile320Asnfs*122), whereas the affected individual in family 3 carried the variants c.1501C>T (p.Arg501Trp) and 3′UTR variant c.*45G>C. These results implied the genetic contribution of PABPC1L to oocyte maturation arrest and female infertility. We then performed mutational screening of PABPC1L in a cohort of 504 infertile individuals with abnormalities in fertilization and early embryonic development. We identified two affected sisters from family 4 who carried the same homozygous missense variant c.1121G>A (p.Arg374Gln). All identified variants were confirmed using Sanger sequencing  Fig . No homocing Fig . The inhcing Fig . Specifisues Fig , suggestin vitro, we first performed immunoblot analysis in HeLa cells transfected with HA‐labeled WT or mutant PABPC1L expression plasmids. Compared with WT, the c.526C>T (p.Arg176*) and c.956_957insA (p.Ile320Asnfs*122) variants resulted in truncated proteins and showed significantly reduced PABPC1L protein levels. The missense variants c.290G>A (p.Gly97Asp), c.410C>T (p.Ser137Phe), c.1501C>T (p.Arg501Trp), and c.1121G>A (p.Arg374Gln) did not lead to changes in protein levels  resulted in significantly lower luciferase activity than the WT group  tail of mRNA , c.410C>T (p.Ser137Phe), and c.1121G>A (p.Arg374Gln) identified in families 1, 2, and 4 respectively. We performed PCR and Sanger sequencing to confirm the mutated allele in the three KI mice  cRNA (a previously reported loss‐of‐function variant)  and c.956_957insA (p.Ile320Asnfs*122) affected the expression pattern and localization of PABPC1L, while the missense variants c.290G>A (p.Gly97Asp), c.410C>T (p.Ser137Phe), and c.1501C>T (p.Arg501Trp) reduced its translation activation and RNA‐binding ability. Moreover, the 3′UTR variant c.*45G > C affected the protein expression of the luciferase reporter gene. Finally, we constructed three strains of Pabpc1l KI mice corresponding to patient‐derived variants and found that female mice were infertile. The Mos‐MAPK pathway was abnormally activated in the zygotes of KI mice, which may provide an explanation the infertile phenotype. These results demonstrate the destructive effects and pathogenicity of the PABPC1L variants identified in affected individuals both in vitro and in vivo.In this study, we identified different compound heterozygous and homozygous pathogenic variants in Xenopus oocyte extracts that regulates mRNA deadenylation, and it is the predominant PABP in Xenopus oocytes and early embryos ‐binding proteins) are a family of multifunctional RNA‐binding proteins, including PABPC1, PABPC3, PABPC4, PABPC4L, PABPC5, and PABPC1L, that mediate mRNA metabolism at different stages of gametogenesis and early embryogenesis  and had normal fertility. This result suggests that the c.1121G>A (p.Arg374Gln) variant may cause the phenotype of fertilization failure following a recessive inheritance pattern. The unique fertilization failure phenotype might be related to the observation that the c.1121G>A (p.Arg374Gln) variant had a slight effect on the RNA‐binding ability of PABPC1L  female mice were previously shown to be infertile due to impaired oocyte maturation . In addition, we also found other candidate pathogenic genes through bioinformatics analysis, but the pathogenicity of these mutant genes needs to be further verified by in vitro experiments and the generation of knockout or patient‐derived mutated animal models. Nevertheless, the genetic basis involved in the majority of affected individuals remain unknown, which requires extensive exploration using different research strategies. Previous studies have shown that gene‐based burden test is an effective strategy for finding novel pathogenic genes in many other disorders , and whole‐exome sequencing was performed using the SeqCap EZ Exome Kit (Roche) on an Illumina HiSeq 3000 platform (Illumina). Sequencing analysis was compared with the human reference sequence (NCBI Genome build GRCh37). Variants were annotated with GRCh37 and the dbSNP (version 138) database. All candidate variants were filtered using the following criteria: (1) variants that conformed to the autosomal recessive inheritance pattern; (2) variants with a minor allele frequency less than 0.1% (for homozygous variants) or 1% (for compound heterozygous variants) in the public databases, including gnomAD, 1000 Genomes Project, or ExAC databases, and not existing in our control database; (3) variants predicted to be loss of function or to be damaging by SIFT, PolyPhen‐2, and MutationTaster; and (4) variants within genes that were highly expressed or specifically expressed in oocytes. The candidate pathogenic variants were subsequently verified by Sanger sequencing of the affected probands as well as all the available family members. The primers used are shown in Appendix Table PABPC1L was normalized by comparison to the expression of an internal human GAPDH control. The qRT‐PCR primers for PABPC1L and GAPDH are shown in Appendix Table Total RNA from control samples , blastocysts, and other somatic tissues) were extracted with the RNeasy Mini Kit (Qiagen). Reverse transcription was performed with the PrimeScript RT Reagent Kit (Takara) according to the manufacturer's instructions. qRT‐PCR was performed with TB Green Premix Ex Taq (Takara) in triplicate on a LightCycler 480 II System (Roche). The expression of PABPC1L was amplified from control human oocyte cDNA. The coding sequence product was then cloned into the pcDNA3 vector containing the N‐HA tag or the pCMV6‐Entry vector containing the C‐FLAG tag, and the 3′UTR product was cloned into the psiCHECK‐2 vector. Site‐directed mutagenesis was performed to introduce the identified variants  into the wild‐type (WT) vector according to the instructions of the KOD‐Plus‐Mutagenesis Kit (Toyobo Life Science). HeLa cells  were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) in a 5% CO2 atmosphere at 37°C. The WT and mutant expression plasmids were transfected into HeLa cells using the PolyJet In Vitro DNA Transfection Reagent (Signagen) according to a standard protocol.The full‐length coding sequence and 3′‐untranslated region (3′UTR) of 25 magnetic beads (NEB). After incubation at 4°C for 2 h, the beads were washed with lysis buffer three times. The bead‐bound proteins were eluted using sodium dodecyl sulfate (SDS) sample buffer.HeLa cells were harvested 36 h post‐transfection and washed three times with cold PBS. Cells were lysed in NP‐40 lysis buffer with 1% protease inhibitor cocktail (Bimake) and RNase inhibitor (NEB). After quantification with the Bicinchoninic Acid Protein Assay Kit , equal amounts of protein extracts were incubated with Oligo d(T)All immunoblot samples were heated at 100°C for 10 min. Equal amounts of protein were separated by SDS‐polyacrylamide gel electrophoresis (EZBiolab) and transferred to nitrocellulose membranes . The membranes were blocked with 5% non‐fat milk diluted in PBS with 0.1% Tween 20 (PBST) for 1 h and then incubated at 4°C overnight with primary antibodies. The membranes were washed with PBST three times and incubated with secondary antibodies for 1 h at room temperature followed by washing again with PBST three times. Finally, enhanced chemiluminescence imaging was performed on a chemiluminescent imaging system . Quantitation of immunoblot results was performed with the ImageJ software . The following primary antibodies were used for immunoblotting: antibodies against HA Tag , HuR , and pERK1/2  were used at 1:1,000 dilution, and antibodies against Vinculin  and Tubulin  were used at 1:3,000 dilution as the internal controls. The secondary antibodies were goat anti‐rabbit IgG  or goat anti‐mouse IgG  conjugated to horseradish peroxidase.PABPCL expression plasmids, and cells were fixed for immunofluorescence after culturing for 36 h. Transfected cells were first treated with 4% paraformaldehyde  for 30 min at room temperature, washed three times in PBS, and then blocked in a solution containing 1% bovine serum albumin (BSA)  and 0.1% Triton X‐100  in PBS for 1 h at room temperature. The cells were washed again and then incubated with anti‐FLAG M2‐FITC antibody  for 1 h at 37°C. After being washed three times, the cells were incubated with Hoechst  for 15 min at room temperature. Images were captured with the EVOS M7000 Microscope Imaging System (Thermo Fisher Scientific).HeLa cells were transfected with WT or mutant Mouse zygotes were fixed with 2% paraformaldehyde diluted in PBS for 30 min at room temperature and then permeabilized in PBS containing 0.5% Triton X‐100 for 20 min at room temperature. After incubation for 2 h in blocking buffer (3% BSA diluted in PBS with 0.1% Tween‐20 and 0.01% TritonX‐100) at room temperature, zygotes were stained with anti‐pERK1/2  primary antibodies diluted in blocking buffer for 1 h at 37°C. After three washes, samples were incubated with Alexa Fluor 488‐conjugated donkey anti‐rabbit  secondary antibodies for 1 h at 37°C. Pronucleus was briefly stained with Hoechst  for 15 min at room temperature. The samples were in PBS and imaged using a laser scanning confocal microscopy .PABPC1L 3′UTR were cloned into the C‐terminus of the Renilla luciferase gene in the psiCHECK™‐2 vector. Empty vector was used as the control, and Firefly luciferase expression served as the internal standard for transfection efficiency. HeLa cell extracts were prepared 36 h after transfection and assayed for the Firefly and Renilla luciferase activities. Renilla luciferase activity values were normalized to Firefly activity. Results are presented as the means obtained from three independent experiments.The luciferase assay was performed with the Dual‐Luciferase Reporter Assay System (Promega). WT and mutant Pabpc1l KI mice were generated using the CRISPR‐Cas9 gene‐targeting approach. All animal operations were approved by the Shanghai Medical College of Fudan University. The guide RNA (gRNA) target site for mouse Pabpc1l close to the disease variant was selected using the CRISPR design tool . Additionally, the donor template of G97D and R374Q contained the synonymous AAG to AAA p.K95 mutation and GAG to GAA p.E373 mutation to avoid gRNA recognition and excision. To produce the Cas9 mRNA, gRNA, and donor oligo, a T7 promoter was first fused to the Cas9 coding region of the px330 plasmid by PCR amplification. The T7‐Cas9 PCR product was purified and used as a template for in vitro transcription using the mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies). A T7 promoter was fused to the gRNA template by PCR amplification, and the T7‐gRNA PCR product was purified and used as a template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). Both the Cas9 mRNA and the gRNAs were purified using a MEGAclear kit (Life Technologies) and eluted in RNase‐free water. Oligo donors were obtained as ultramer DNA oligos from GenScript Corporation. Female C57BL/6 mice  were superovulated by injecting 10 IU of pregnant mare serum gonadotropin , followed 48 h later by injection of 10 IU of human chorionic gonadotropin . They were then mated to C57BL/6 males, and fertilized embryos were collected from the oviducts. Cas9 mRNA (100 ng/ml), gRNAs (50 ng/ml), and donor oligos (100 ng/ml) were mixed and injected into the cytoplasm of fertilized oocytes using an IM300 microinjector (Narishige). About 30 injected single‐cell embryos were transferred into the oviducts of pseudopregnant ICR females at 0.5 days after coitus. For the identification of Pabpc1l point mutation founders, DNA was extracted and the target fragment was amplified followed by sequence analysis. The primers are shown in Appendix Table 2, and embryo development was monitored at regular intervals. All mouse experiments were reviewed and approved by the Shanghai Medical College of Fudan University.To obtain GV oocytes, ovaries were isolated from female mice (10–12 weeks old). GV oocytes were isolated from ovaries by puncturing the antral follicles with a fine needle on the stage of a dissecting microscope. For IVF, female mice (10–12 weeks old) were intraperitoneally injected with 10 IU of PMSG. After 46 h, mice were injected with 10 IU of hCG. After an additional 13 h, oocyte/cumulus masses were surgically isolated from the oviducts and were mixed with sperm in human tubal fluid medium (Nanjing Aibei Biotechnology Co.) for IVF. Zygotes were cultured in drops of K‐modified simplex optimized medium (Nanjing Aibei Biotechnology Co.) under oil at 37°C in an atmosphere of 5% COPabpc1l were determined using qRT‐PCR with mouse Actb expression as the internal control. The primers are shown in Appendix Table Each sample included five zygotes. RNA‐seq libraries were prepared using the cDNA Synthesis, Amplification and Library Generation Kit . RNA quality was examined by gel electrophoresis and with Qubit (Thermo). RNA samples were sequenced on the Illumina Novaseq 6000 instrument by Genergy Biotechnology Co. Ltd. The raw data were handled by trim galore software, and data quality was checked by FastQC v0.11.2. The read length was 2 × 150 bp. Clean reads were aligned to the mouse genome mm10 using STAR and StringTie. The expression levels of the genes were quantified using normalized fragments per kilobase million (FPKM). Differential gene expression analysis was performed by DESeq2 with read counts data. The thresholds for determining differentially expressed transcripts were abs[Fold Change] > 1.5 and FDR < 0.05. Venn software was used to identify the co‐upregulated and co‐downregulated transcripts in the two databases (G97D vs. WT and S137F vs. WT). Gene Ontology (GO) term analysis was performed using the DAVID online tool. The expression of Mos‐MAPK pathway‐related genes and t‐tests when comparing experimental groups. Statistically significant values of P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 are indicated by (*), (**), (***), and (****), respectively.Experiments were performed with biological replicates as indicated in the Figure legends, and GraphPad Prism (GraphPad Software) was used to perform the statistical analyses. Differences were analyzed by Student's Lei Wang: Conceptualization; supervision; funding acquisition; writing – review and editing. Weijie Wang: Conceptualization; data curation; formal analysis; funding acquisition; investigation; visualization; methodology; writing – original draft; writing – review and editing. Jing Guo: Resources; investigation. Juanzi Shi: Resources; investigation. Qun Li: Data curation; formal analysis. Biaobang Chen: Data curation; formal analysis. Zhiqi Pan: Formal analysis; investigation. Ronggui Qu: Investigation. Jing Fu: Resources; investigation. Rong Shi: Resources; investigation. Xia Xue: Resources; investigation. Jian Mu: Methodology. Zhihua Zhang: Formal analysis. Tianyu Wu: Visualization. Wenjing Wang: Methodology. Lin Zhao: Validation. Qiaoli Li: Project administration. Lin He: Resources. Xiaoxi Sun: Resources. Qing Sang: Conceptualization; supervision; funding acquisition. Ge Lin: Resources; supervision.The authors declare no competing interests.https://www.ncbi.nlm.nih.gov/genbank/GenBank, https://gnomad.broadinstitute.org/gnomAD, http://mutationtaster.org/MutationTasterMutationTaster, https://www.omim.orgOMIM, http://genetics.bwh.harvard.edu/pph2/PolyPhen‐2, https://sift.bii.a‐star.edu.sgSIFT, https://david.ncifcrf.govDAVID online tool, AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Source Data for Expanded ViewClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 6Click here for additional data file.


Burkitt's lymphoma is a highly aggressive non‐Hodgkin's lymphoma that can affect various parts of the body, which include jaw, facial bones, retroperitoneum, and abdominal viscera, it is worth noting that breast involvement is extremely rare in Burkitt's lymphoma and has not been documented.Burkitt's lymphoma is a highly aggressive non‐Hodgkin's lymphoma that can affect various parts of the body including breast. This sarcoma is identified as a rapidly fatal malignant lymphoma syndrome and 50% of all malignant tumors in children. However, breast involvement has not been documented. A 19‐year‐old male presented to our emergency department with a left breast swelling for 2 months associated with drenching night sweats, weight loss and evening fevers. Physical examination revealed a mass measuring approximately 15 × 16 × 15 cm in the widest dimension, skin hyperpigmentation, no nipple discolorations, discharges, and non‐tender on palpation. Biopsy was done, and the histology report revealed sheets of monomorphic medium lymphocytes with a high mitotic rate and frequent apoptotic bodies showing a starry‐sky appearance. The immunohistochemistry report revealed positive staining for Ki‐67, CD‐20, and CD‐10 tumor markers and CD45 on flow cytometry. The patient was started on aggressive hydration, rasubricase administration, CODOX‐M/IVAC regimen 6 cycles, and G‐CSF and registered significant reduction in the size of the mass. Burkitt's lymphoma is a highly aggressive non‐Hodgkin's lymphoma that can affect various parts of the body. It commonly involves the jaw, facial bones, retroperitoneum, and abdominal viscera. The disease typically affects young patients in areas of high incidence, such as the jaw, whereas visceral involvement is more common in older patients in low‐incidence areas. It is worth noting that breast involvement is extremely rare in Burkitt's lymphoma. H&E stained section the cells show cytoplasmic borders with molded appearance and proliferation with increased mitotic activity and increased apoptosis sheets of monomorphic medium lymphocytes showing a starry‐sky appearance. This sarcoma is identified as a rapidly fatal malignant lymphoma syndrome and 50% of all malignant tumors in children. However, breast involvement has not been reported up to date.Involvement of the jaw and facial bones, retroperitoneum, abdominal viscera, ovaries, spinal epidural, and bone marrow has been described; however, jaw involvement is usually seen in younger patients in endemic areas of high incidence, while visceral involvement is noted more commonly in older patients in areas of low incidence.2A 19‐year‐old male presented to our emergency department with left breast swelling for 2 months with drenching night sweats, weight loss, and evening fevers. A physical examination revealed a mass measuring approximately 15 × 16 × 15 cm in the widest dimension, skin hyperpigmentation, no discharge from the nipples, no nipple skin discoloration, and non‐tender on palpation. There were also palpable, non‐tender axillary lymph nodes. The patient was admitted in our emergency department clerked and done the necessary investigations which included the biopsy of the breast mass. Figure .2O(6–25cmH20), glucose: 2.7 mmol/L(2.5–4.4 mmol/L), and total protein: 30 mg/dL.(15–60 mg/dL.) WBC: 4.0/ul(<5.0/ul). Serum LDH: 25 U/L(<40.0 U/L), renal function tests: urea 6.0 mmol/L(2.5–8 mmol/L), creatinine: 70 mmol/L, (63–115 mmol/L), potassium: 4.0 meq/L(3.5–5.5 meq/L), sodium:136 meq/L(135–155 meq/L), calcium: 6.0 mg/dL (8.5–10.5 mg/dL), complete white blood cell count 5.0 (4.0–10.0 × 109/L), chest CT‐scan was done and showed no pulmonary metastases, PET scan and bone marrow biopsy and aspirate were not done due to financial constraints.The patient underwent a pre‐chemotherapy workup that included a lumber puncture for cerebral spinal fluid analysis to rule out central nervous system involvement, For CSF, opening pressure was 18cmHBiopsy of the breast mass was done, and on hematoxylin–eosin stained section, the cells had cytoplasmic borders giving them a molded appearance and highly proliferative, which was reflected in the increased number of mitotic figures and Increased cell turnover with increased apoptosis sheets of monomorphic medium lymphocytes showing a starry‐sky appearance. Figure .The immunohistochemistry report revealed positive staining for Ki‐67, CD‐20, and CD‐10 tumor markers and CD45 on flow cytometry Figure . The CODThis form of combination has been found to be more effective than using any drugs alone. In CODOX‐M/IVAC the drugs are cyclophosphamide, vincristine (Oncovin), doxorubicin, and Methotrexate. In IVAC regimen, the drugs are isocyanide, etoposide (Viperid), and cytarabine (known as Ara‐C), Table .The drugs were delivered through a central venous access device (CVAD), and there were six cycles based on this regimen. In between the cycles, filgrastim (G‐CSF) was given to allow the blood count levels to recover. Once the blood count levels recovered, the next cycle was planned.3Burkitt lymphoma can be divided into sporadic, endemic, and immunodeficiency‐related variants. In developed countries where sporadic cases are most commonly encountered, Burkitt lymphoma can occur at any age, although the peak incidence is in the first decade of life.Burkitt lymphoma accounts for 30% of pediatric lymphomas and less than 1% of lymphoma cases in adults. The disease incidence is 2–3 times higher in equatorial Africa. Termed endemic Burkitt lymphomas, the majority of cases are Epstein–Barr virus (EBV)‐related. The immunodeficiency‐related variant is most commonly associated with the human immunodeficiency virus (HIV).Involvement of the jaw and facial bones has been described and seen usually in younger patients in endemic areas of high incidence, while visceral involvement is noted more commonly in older patients in areas of low incidence.The diagnosis is most readily made by an excisional lymph node biopsy; in cases where an excisional lymph node biopsy is difficult, multiple core needle biopsies may be done, and a hematopathology review is essential.On hematoxylin–eosin‐stained sections, the cells have cytoplasmic borders, giving them a molded appearance. The neoplastic cells are highly proliferative, which is reflected in the increased number of mitotic figures. Increased cell turnover is countered by increased apoptosis resulting in a distinct starry‐sky pattern of classic Burkitt lymphoma with histologic proliferative fraction approaching 100%. While more unusual variants may be harder to distinguish, the most commonly used measure of proliferation is the Ki‐67, where nearly 100% of the cells stain positive for Ki‐67, and most Burkitt lymphoma cells can be initially identified using a SSC versus CD45 flow cytometry plot. These cells have moderate to high SSC/FSC and bright CD45.In developing countries, the stage of the disease and abdominal ultrasound are used to determine the prognosis. Fluorodeoxyglucose positron emission tomography (PET) scans and CT scans are used to evaluate for residual disease.The rapid institution of chemotherapy, ideally within 48 h of presentation, is of utmost importance in Burkitt lymphoma. Aggressive hydration and recombinant urate oxidase (rasubricase) for hyperuricemia are currently recommended as prophylaxis against tumor lysis syndrome and to improve the patient's performance status.3.1Based on experiences with other malignancies, patients with Burkitt lymphoma may experience a rapid improvement with treatment such as fractionated cyclophosphamide or a single cycle of CHOP chemotherapy, which can improve a patient's performance status. While most patients will initially respond to treatment using CODOX‐M/IVAC regimen, investigators have reported two‐year overall survival ranging from 67%–92%.Three‐year overall survival for hyper‐CVAD has ranged from 49% to 89%. And six‐year overall survival for DA‐EPOCH‐R has been reported to be 100%. And for patients who relapse after initial therapy, salvage options should be considered palliative given few patients achieve long‐term survival.Few options exist for patients who relapse after initial therapy. Typical salvage regimens used in diffuse large B‐cell lymphoma such as ICE  or DHAP  can sometimes be used.3.2The differential diagnosis for adolescent breast masses is similar to those in adults and includes phyllodes tumors, primary breast cancer, sarcoma, lymphangioma or hemangioma, metastatic cancer, intraductal papilloma, fibroadenoma (and giant fibroadenoma), abscesses, and benign cysts.3.3For good prognosis includes isolated jaw mass and completely resectable mass, while bad prognosis includes male sex, intra‐abdominal mass, relapse, CNS involvement, and bone marrow involvement. Treatment of limited‐stage (stages I and II) Burkitt lymphomas is usually very successful, with a long‐term survival rate of over 90%. The long‐term survival rate for children and teens with more advanced (stage III or IV) Burkitt lymphoma ranges from about 80% to 90%.4Burkitt's lymphoma is a highly aggressive non‐Hodgkin's lymphoma that can affect various parts of the body. It commonly involves the jaw, facial bones, retroperitoneum, abdominal viscera, ovaries, spindle epidural, and bone marrow. The disease typically affects young patients in areas of high incidence, such as the jaw, whereas visceral involvement is more common in older patients in low‐incidence areas. It is worth noting that up to date breast involvement is extremely rare in Burkitt's lymphoma.Ronald Kato: Conceptualization; formal analysis; investigation; methodology; writing – original draft; writing – review and editing. Kibudde Solomon: Methodology; project administration; software; supervision; validation.There was no funding or any financial assistance that was contributed towards this manuscript.There is no conflict of interest between authors and all our institutions in regarding to publication of this report.I confirm that, the case report has been fully explained to me and all my questions have been answered to my satisfaction, I therefore have no objection to the publication of this report.”A written informed consent was obtained from the patient to publish this report in accordance with the Journal's patient consent policy. From the patient, “The patient's perception is that the swelling was something that might be related to witch craft from his village and also related it to cancer of the breast.


Inflammation plays a vital role in the occurrence and progression of atrial fibrillation (AF). The association between pericoronary adipose tissue attenuation (PCATA) and AF recurrence following ablation has not been fully clarified.We aimed to evaluate the association between PCATA and AF recurrence after radiofrequency catheter ablation (RFCA).Patients who underwent the first RFCA for AF and performed coronary computed tomography angiography before ablation between 2018 and 2021 were enrolled. The predictive values of PCATA for AF recurrence after ablation were investigated. The area under curve (AUC), relative integrated discrimination improvement (IDI), and categorical free net reclassification improvement (NRI) were used to assess the discrimination ability of different models for AF recurrence.p = .024), with a relative IDI of 0.043 (p = .006) and continuous NRI of 0.521 (p < .001).During 1‐year follow‐up, 34.1% patients experienced AF recurrence. The multivariable analysis model revealed that PCATA of the right coronary artery (RCA) was an independent risk factor for AF recurrence. Patients with a high level of RCA‐PCATA had a high risk of recurrence, after adjusting for other risk factors by restricted cubic splines. The performance in predicting AF recurrence was significantly improved by adding the marker of RCA‐PCATA to the clinical model  is an independent predictor for atrial fibrillation (AF) recurrence after radiofrequency ablation. PCATA surrounding the proximal RCA may identify high‐risk recurrence in AF ablation patients.The PCATA index in coronary computed tomography angiography reporting might be more meaningful for assessing AF risk stratification following ablation. The electrophysiology and structural properties of the atria are critically affected by inflammatory processes.22.1We conducted a retrospective study of AF patients who received the first catheter ablation from January 2018 to April 2021 in the First Affiliated Hospital of Zhengzhou University. The inclusion criteria were: (1) patients underwent coronary computed tomography angiography (CCTA) before ablation. (2) Patients without history of coronary heart disease. The exclusion criteria were patients with pacemaker implantation, thyroid dysfunction, renal dialysis, advanced valvular heart disease, hypertrophic cardiomyopathy, and patients with poor/insufficient CCTA images. Patients who were dead or lost to follow‐up were also excluded. The study complied with the Declaration of Helsinki. The study protocol was authorized by the local institution's ethics committee, and waived the need for written informed consent.2.2The following data were collected from hospital records: demographic parameters, comorbidities, echocardiography parameters, CCTA parameters, and medications on admission. The duration of AF was calculated by the time from the date of initial symptom onset or first diagnosis of AF to the RFCA index date.2.3All CCTA scanning was accomplished using dual‐source computed tomography (CT) of a third‐generation scanner . CCTA images were acquired with the following scan protocol: Tube voltage was 100 or 120 kV. If 100 kV voltage was used instead of 120 kV voltage, the mean Hounsfield units (HU) value was corrected dividing by 1.11485, as previously reported.2.4EAT is a low‐density margin encasing the myocardium in the pericardial space on CCTA. EAT was defined as CT density ranging from −190 to –30 HU.2.5For each patient, we measured the value of PCATA by the dedicated workstation . The weighted mean CT attenuation of PCAT (voxels with attenuation between −190 and −30 HU) within the range of the distance from the outer vascular wall, which is equal to the average diameter of the targeted vessel, was defined as PCATA.2.6Non‐vitamin K antagonist oral anticoagulants (NOACs) or warfarin with a target international normalized ratio between 2.0 and 3.0 were administered in all patients. Transoesophageal echocardiography was performed to rule out thrombus before ablation.The RFCA procedure has been described before.2.7The primary outcome was the recurrence of AF after ablation during 1 year follow‐up. The definition of AF recurrence was any atrial tachyarrhythmia lasting for more than 30 seconds in electrocardiogram (ECG) or 24‐hour Holter monitoring after the 3‐month blanking period. All patients were prescribed antiarrhythmic drugs (AADs) for 3 months after ablation to prevent early recurrence. The use of AADs was subsequently determined by the physicians and patients. Patients were scheduled for follow‐up at 3‐month intervals within the first year after ablation in the outpatient setting. Each follow‐up visit included 12‐lead ECG, 24‐hour Holter monitoring, and clinical assessment. Patients who had any symptoms related to AF were required to complete an additional outpatient visit.2.8χ2 test. The values of the continuous variables were described as the mean ± standard deviation, and compared among groups using Student's t‐test and Mann−Whitney U test, depending on whether data were normally distributed. The relation between RCA‐PCATA and LAD‐PCATA was investigated using Pearson's correlation. Univariable and multivariable Cox proportional hazards analysis were constructed to investigate risk factors for AF recurrence. The variables with p < .01 in the univariable analysis and traditional risk factors for AF recurrence were used in the multivariate Cox regression analysis model, which adjusting risk factors including heart failure, prior stroke, body mass index (BMI), type of AF, duration of AF (>2 years), LA volume, and EAT volume. Receiver operating characteristic (ROC) curve was performed to determine the cut‐off value of RCA‐PCATA. The cut‐off value (−77.45 HU) was used to make the RCA‐PCATA as a categorical value. Survival analysis was visualized by Kaplan−Meier curves and significance tests between groups were conducted by the log‐rank test. Furthermore, we used restricted cubic splines (RCS) with four knots at the 5th, 25th, 75th, and 95th centiles to model the relationship between RCA‐PCATA levels and recurrence of AF, with the cut‐off value of RCA‐PCATA serving as the reference.Categorical variables are presented as number (percentage) or median  and compared between two groups by the p‐value of <.05 was considered statistically significant.The performance of the prediction models for AF recurrence were compared used to determine the incremental discriminatory and reclassification performance of clinical characteristics for identify predictors of AF recurrence. The parameters included in the clinical model 1  were heart failure, prior stroke, BMI, type of AF, duration of AF (>2 years), LA volume, and EAT volume. The parameters included in the model 2 were a combination of clinical model 1 and the RCA‐PCATA as a continuous variable. Clinical model 3 was the combination of clinical model 1 and the RCA‐PCATA as a categorical variable. To further evaluate the discriminatory ability of the models, The discriminatory abilities of clinical models 2 and 3 were assessed by the reclassification performance of each model using the area under curve (AUC), relative integrated discrimination improvement (IDI), and the net reclassification improvement (NRI) values. Moreover, a subset of 50 patients were randomly selected to assess the intra‐ and interobserver reliability for the measurement of PCATA, and intra‐class correlation coefficient (ICC) was calculated for evaluation of intra‐ and interobserver agreement. Statistical analysis was performed using R language version 4.0.3 . A two‐sided 33.1Five hunderd and twenty‐four patients who underwent successful RFCA for AF and pre‐ablation CCTA were screened for eligibility. Two hundred and ninety‐nine patients were included in the final analysis , linear ablation, SVC ablation, and BMI between the patients with and without recurrence. Compared to patients without AF recurrence, patients with recurrence were more likely to have heart failure, persistent AF, duration of AF >2 years. The patients with recurrence also had larger LA volume, EAT volume, RCA‐PCATA, and LAD‐PCATA. However, there was not significantly different on the value of LCX‐PCATA between two groups, as shown in Table 3.2r = .687, p < .001), as described in Supporting Information: Figure r = .055, p = .435) and LAD‐PCATA . Furthermore, RCA‐PCATA  and LAD‐PCATA  were not significantly different between the patients with RCA dominance (n = 212) and with left coronary artery dominance (n = 87).The distribution of PCATA is shown in Supporting Information: Figure 3.3p < .001). The ICC of inter‐reader reliability for LAD‐PCATA, LCX‐PCATA, and RCA‐PCATA were 0.991, 0.977, and 0.983, respectively , as depicted in Supporting Information: Table Our analysis showed that the good intra‐ and inter‐reader reliability of PCATA measurements were performed in 50 randomized AF patients. The ICC of the intra‐reader reliability for LAD‐PCATA, LCX‐PCATA, and RCA‐PCATA were 0.987, 0.965, 0.984, respectively , LA volume , and duration of AF >2 years  were independently associated with AF recurrence. However, neither the value of LAD‐PCATA nor EAT volume were not independently associated with AF recurrence (Table p for nonlinearity = .489), as depicted in Figure Multivariate Cox regression analysis revealed that the value of RCA‐PCATA . The incremental reclassification efficacy for predicting AF recurrence was significantly improved by adding RCA‐PCATA . Moreover, the efficacy for predicting AF recurrence was also significantly improved by adding RCA‐PCATA as a categorical variable, as described in Supporting Information: Table The addition of RCA‐PCATA as a continuous variable significantly improved the predictive ability for AF recurrence compared with the clinical model 1 (AUC: 0.724 vs. 0.686, 4This is a pilot study to investigate the association between PCATA and AF recurrence after RFCA. Our main findings revealed that RCA‐PCATA was an independent predictor for AF recurrence. Our study may help physicians identify high risk of AF recurrence patients following catheter ablation.The high level of inflammation could regulate calcium homeostasis and connexins of cardiac myocyte, all of which could promote AF development.Pericoronary adipose tissue could secrete bioactive substances and plays an important role in vasoconstriction and dilatation, and smooth muscle cell proliferation.The previous study revealed that RCA‐PCATA is independently associated with adverse cardiac events rather than LAD‐PCATA in diabetes patients.EAT is a unique type of visceral adipose tissue located between the myocardium and visceral pericardium, without an intervening fascial plane.Some limitations of the study need to be acknowledged. First, this was a retrospective, single‐center study with a limited sample size, and selection bias may exist. Although the follow‐up was prospective and detailed, and improving the quality of data. Second, some patients with AF recurrence might have been missed for asymptomatic arrhythmia, although we encouraged patients to contact the physician in the case of suspected recurrence symptoms. Third, we did not have the local or systemic total markers of inflammation data to support the association with PCATA, as hs‐CRP was only available in 69.2% AF patients. Finally, PCATA data measurement through semiautomated software may still be biased, although we use well‐established software to measure PCATA and the reliability of the measured data has been confirmed in several previous studies.5The high level of RCA‐PCATA was an independent predictor for AF recurrence following RFCA. The PCATA may help to recognize high recurrence risk following ablation in AF patients. Further larger cohort studies to validate these findings are certainly needed to provide us with more consistent data.Zhe Wang, Ying‐Wei Chen, and Yi‐Hong Sun conceived and designed the study. Zhe Wang, Jia‐Wei Chen, Li‐Chen Ren, and He‐He Guo participated in the acquisition of data. Zhe Wang, Jia‐Wei Chen, and He‐He Guo analyzed the data. He‐He Guo and Xiao‐Jie Chen gave advice on methodology. Zhe Wang and Yi‐Jia Wang drafted the manuscript. Yi‐Hong Sun revised the manuscript. All authors read and approved the final manuscript.The authors declare no conflict of interest.Supporting information.Click here for additional data file.