論文

Background & aims Little is known about short-term vitamin D dynamics during the immediate neonatal period. Most neonatal studies have relied on single-point measurements of 25-hydroxyvitamin D (25(OH)D), limiting understanding of early postnatal changes. In Japan, healthy full-term infants typically remain hospitalized for 4–5 days, receiving minimal vitamin D intake from small amounts of formula and experiencing virtually no sunlight exposure. This clinical setting provides a unique opportunity to investigate early postnatal vitamin D dynamics under conditions of minimal exogenous input. This study aimed to examine short-term changes in serum 25(OH)D from birth to day 4 of life (Δ25(OH)D) and determine how baseline vitamin D status influences these changes. Methods We analyzed 40 infants with paired cord blood and day 4 serum 25(OH)D measurements obtained by chemiluminescence immunoassay. Linear regression evaluated the association between cord blood 25(OH)D concentrations and Δ25(OH)D. Results Estimated vitamin D intake during hospitalization was 64 ± 18 IU/day, substantially below the 400 IU/day recommended by the American Academy of Pediatrics. Baseline-dependent bidirectional changes in serum 25(OH)D were observed: infants with lower cord blood concentrations increased, whereas those with higher baseline concentrations declined. Cord blood 25(OH)D concentrations were strongly inversely correlated with Δ25(OH)D (r = −0.745, p < 0.001). Regression analysis indicated an equilibrium point of approximately 21.4 ng/mL, at which no net change in serum 25(OH)D was expected. Conclusion This study provides the first quantitative description of short-term changes in neonatal 25(OH)D after birth and suggest dynamic physiological regulation of vitamin D during early life.

Background: For preventing Vitamin D (VD) insufficiency, several VD supplementation guidelines were established worldwide. In Japan, no nationwide guidelines for preventing VD insufficiency have been implemented, whereas guidelines for preventing vitamin K (VK) deficiency-related bleeding recommend weekly supplementation of VK. The aim of this study is to clarify whether weekly VD plus VK supplementation during the early neonatal period prevents VD insufficiency at one month of age.MethodsWe retrospectively analyzed serum 25-hydroxyvitamin D (25(OH)D) levels of 555 one-month-old infants born between 2017 and 2023. Infants were classified into the control group (not supplemented), weekly group (1000 IU/week), and daily group (240 IU/day). We compared serum 25(OH)D levels among the three groups. Multivariable logistic regression analyses adjusted for formula intake and BMI were performed to better estimate the effect of VD supplementation on the prevention of VD insufficiency.ResultsWe included 414, 55, and 86 infants in the control, weekly, and daily groups, respectively. All infants received weekly supplementation of VK. Serum 25(OH)D levels in the weekly and daily groups were higher than those in the control group (median (ng/mL): control 9.7 vs weekly 22.2, P < 0.001; control vs daily 23.0, P < 0.001). The frequencies of VD insufficiency were 370/414 (89.4 %), 11/55 (20.0 %), and 22/86 (25.6 %) in the control, weekly, and daily groups, respectively. Adjusted odds ratios of VD insufficiency compared to the control were 0.038 (95 % confidence intervals (95 %CI): 0.017, 0.085) and 0.036 (95 %CI: 0.019, 0.067) in the weekly and daily groups, respectively. No infant with VD excess was observed.ConclusionOur results demonstrated that combined weekly supplementation of VD and VK during early infancy can prevent VD insufficiency at one month of age without causing VD excess. This finding may provide evidence for the development of nationwide prophylaxis for VD insufficiency in regions lacking specific guidelines.

Vitamin D deficiency during infancy has been associated with increased bone turnover rate and bone mineral loss. However, few studies have examined bone turnover markers (BTMs) for both bone formation and resorption in infants with vitamin D deficiency. Here, we analyzed serum concentrations of 25OHD, intact parathormone (iPTH), and BTMs including total alkaline phosphatase (ALP), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), and serum type I collagen N-telopeptide (NTx) as well as basic clinical characteristics of 456 infants (626 samples) aged less than 12 mo born at Saitama City Hospital, Japan (latitude 35.9? North) between January 2021 and December 2022. One hundred sixteen infants (147 samples) were classified as having vitamin D deficiency (25OHD < 12.0 ng/mL), and 340 infants (479 samples) had sufficient vitamin D levels (25OHD = 12.0 ng/mL). In addition to 25OHD and ALP, both TRACP-5b and sNTx were measured in 331 infants (418 samples), while 90 infants (105 samples) had only TRACP-5b measured and 101 infants (103 samples) had only sNTx measured. Statistical comparison of 104 subjects each in the vitamin D deficiency and sufficiency groups after matching for the background characteristics revealed that the vitamin D deficiency group had significantly higher levels of ALP and iPTH compared with the sufficiency group (P = <.0001, .0012, respectively). However, no significant differences were found in TRACP-5b and NTx levels between the 2 groups (P = .19, .08, respectively). Our findings suggest discordant responses between bone formation and resorption markers in subclinical vitamin D deficiency during infancy.

Background: Imprinted genes are expressed in a parental-origin–specific manner. The imprinted regions including imprinted genes have differentially methylated regions (DMRs) with different 5-methylcytosine (5mC) patterns for CpGs on each parental allele, and DMRs function as imprinting control centers. Aberrant expression of the imprinted genes caused by structural variants involving DMRs, single-nucleotide variants in imprinted genes, uniparental disomy, and epimutation lead to imprinting disorders (IDs). Nanopore-based targeted long-read sequencing (T-LRS) can obtain sequence reads 10–100 kb long together with information on DNA methylation in each CpG and is cost-effective compared to whole-genome LRS. T-LRS is a valuable tool for efficient genetic testing for IDs and has great potential to elucidate the regulatory mechanisms in the imprinted regions. However, there is no T-LRS system targeting all ID-related regions. Methods: We conducted T-LRS targeting 78 DMRs and 22 genes in peripheral blood leukocytes from six healthy controls and set the normal range of methylation index (MI) for each CpG within the DMRs. To clarify the properties of DMRs, we compared MIs in CpGs within DMRs between haplotypes in 78 DMRs. To evaluate the usefulness of T-LRS, we conducted T-LRS on two previously reported patients with multi-locus imprinting disturbance (MLID) having pathogenic variants in MLID-causative genes and compared the MIs in CpGs within DMRs with those measured by array-based methylation analysis. Results: The median number of reads with 5mC and unmethylated cytosine in all DMRs in the six controls was over 40. We defined the normal range of MI for all CpGs in each allele and the total, and classified 78 DMRs into three categories, namely, 33 Complete-DMRs, 25 Partial-DMRs, and 20 Non-DMRs, based on the average of six controls for the median of differences of MIs in CpGs between haplotypes. We confirmed by T-LRS pathogenic variants in MLID-causative genes in patients with MLID. The patients’ methylation defect patterns in T-LRS were similar to those in array-based methylation analysis, although T-LRS showed additional aberrantly methylated DMRs. Conclusions: We established a T-LRS system targeting all ID-related regions, defined standard MI ranges in CpGs on each parental allele, and demonstrated the usefulness of T-LRS.

Vitamin D (VD) status is assessed by measuring serum 25-hydroxyvitamin D (25(OH)D) levels using immunoassays. In December 2024, a Japanese diagnostic laboratory transitioned from chemiluminescent immunoassay (CLIA: LIAISON® 25 OH Vitamin D Total Assay) to an electrochemiluminescent immunoassay (ECLIA: Elecsys® Vitamin D Total III assay). Although these methods yield comparable results in adults, it remains unclear whether this applies to children, particularly newborns and infants. This study aims to clarify the impact of transition from CLIA to ECLIA on 25(OH)D levels during early infancy. Serum 25(OH)D levels were measured in 84 infants under three months old (67 under four days old) using both CLIA and ECLIA. Correlation and agreement were assessed using Passing–Bablok regression and Bland–Altman analyses, respectively. The obtained regression equation was applied to 252 age- and sex-matched cases from 2,253 historical controls in the registry to evaluate changes in VD classification. The regression equation was YECLIA = 0.846XCLIA – 2.281 (τ = 0.53, p < 0.001). Bland–Altman analysis revealed a constant negative bias of –4.89 ± 3.53 ng/mL [–5.66, –4.12] (mean ± standard deviation [95% confidence interval]) for ECLIA versus CLIA. Reclassification revealed an increase in VD deficiency (25(OH)D < 12 ng/mL) from 27.8% to 61.9%, and VD insufficiency (25(OH)D < 20 ng/mL) from 75.4% to 91.7%. The transition from CLIA to ECLIA led to lower measured 25(OH)D levels in newborns and infants and increased VD deficiency and insufficiency diagnoses. Method-specific differences should be considered when evaluating VD status during early infancy, particularly in infants under four days.

Aberrant expression of imprinted genes results in imprinting disorders (IDs). Differentially methylated regions (DMRs) reveal parental–origin–specific DNA methylation on CpGs and regulate the expression of the imprinted genes. One etiology of IDs is epimutation (epi-IDs) induced by some error in the establishment or maintenance of methylation imprint during the processes of gametogenesis, fertilization, or early embryonic development. Therefore, it has been a concern that assisted reproductive technologies (ART) increase the risk for the development of IDs, particularly epi-IDs. We review the effects of ART on DNA methylation of the genome, including DMRs in gametes, embryos, and offspring, and the risk of advanced parental age (a confounding factor of ART) and infertility itself for the development of IDs, particularly epi-IDs.

Background: Multi-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes. Results: Of 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART). Conclusions: The frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients.

Vitamin D (VD) insufficiency in pregnant women is a serious health problem worldwide. To prevent VD insufficiency during pregnancy, several guidelines recommend 600 IU/day VD for all pregnant women. In Japan, no national guidelines for preventing VD insufficiency have been implemented, and no study has evaluated adequate VD intake in pregnant women; however, the number of pregnant women taking commercial dietary supplements containing VD has increased in recent years. This study aimed to examine the effects of maternal commercial supplementation of VD on 25-hydroxyvitamin D (25(OH)D) levels in newborns. We retrospectively analyzed the serum 25(OH)D levels in 279 four-days-old newborns born at the Saitama City Hospital from 2022 to 2023. Newborns were classified into a supplement group (mothers who took VD-containing commercial supplements regularly throughout pregnancy; n = 103) and a non-supplement group (mothers who did not take any supplements during pregnancy; n = 176). The study findings revealed that serum 25(OH)D levels in newborns in the supplement group were higher than those in the non-supplement group (median [interquartile range]: supplement group 17.2 [14.6, 22.9] vs. non-supplement group 14.3 [11.6, 16.7], p < 0.001). In the supplement group, approximately 70% of newborns still showed VD insufficiency. Although the maternal use of VD-containing commercial supplements during pregnancy increased the serum 25(OH)D levels in newborns at four days of age, additional measures, such as VD supplementation for newborns, are needed to improve neonatal VD status.

Background: Episignatures are disease-specific, genome-wide DNA methylation patterns identified in more than 100 genetic syndromes caused by mutation of genes related to epigenetic modifiers. The utility of episignatures as a disease diagnostic tool has been demonstrated in recent years. However, studies evaluating episignatures in imprinting disorders (IDs) are very limited. We hypothesized that identifying episignatures in Silver–Russell syndrome (SRS), one of the representative IDs, could lead to the development of a new diagnostic tool for SRS. We investigated methylation profiles in patients with different molecular etiologies to clarify the presence or absence of DNA methylation episignatures in SRS. Results: We conducted genome-wide methylation analysis (GWMA) using the HumanMethylationEPIC array in 27 patients consisting of five groups based on molecular etiologies satisfying clinical diagnostic criteria: hypomethylation of H19-differentially methylated region (DMR) (n = 9), maternal uniparental disomy chromosome 7 (n = 11), IGF2 variant (n = 3), PLAG1 variant (n = 2), and deletions including KCNQ1OT1:TSS-DMR (n = 2). We compared the methylation levels of CpGs (β values) between each group and control group (94 pediatric samples) and extracted aberrantly methylated CpGs satisfying the following criteria: (1) Bonferroni-corrected p value < 0.05, and (2) the absolute value of ∆β (|∆β|), the difference between the average β value of each ID group and controls at each probe, was above 0.1. Based on the extracted aberrantly methylated CpGs, we examined (1) aberrantly methylated patterns, (2) aberrantly methylated regions, and 3) CpG-enriched genes and their functions, shared by multiple groups. Although several CpGs were commonly hypermethylated or hypomethylated in two of the five groups, the number of CpGs was too small to form characteristic methylation patterns. Several aberrantly methylated regions, including disease-responsible DMRs of SRS, were identified in each group, but no regions common in multiple groups were identified. Previously reported aberrantly methylated regions other than disease-responsible DMRs were not identified. In addition, we found no CpG-enriched genes or their functions common in multiple groups. Conclusions: GWMA in SRS patients with different molecular etiologies identified no characteristic episignatures. Only methylation changes of CpGs within disease-responsible DMRs, not genome-wide methylation changes, occur in some SRS etiologies.

To prevent the worsening of bronchopulmonary disease (BPD), early extubation is desirable. However, in extremely preterm infants, BPD tends to become severe, making early extubation difficult and leading to prolonged intubation. Even if the intubation period is prolonged, feasible respiratory strategies for extubation in extremely preterm infants during the chronic phase of severe BPD are necessary. In preterm infants, nasal high-frequency oscillatory ventilation (NHFOV) can support breathing after extubation immediately after birth, but whether NHFOV is effective as respiratory support after extubation in the chronic phase of severe BPD in extremely preterm infants is unclear. Especially for extremely preterm births or infants with extremely low birth weights, early extubation is difficult. Although such infants' postmenstrual age and weight increase during long-term ventilator support, their respiratory function is very poor compared with that of preterm infants born at a gestational age equivalent to such infants' postmenstrual age owing to substantial lung damage caused by the ventilator. For this reason, extubation in the chronic phase of BPD may also be challenging. In this report, we describe a case of a marginally viable infant who was born at 23 weeks’ gestation weighing 374 g, required 2 months of intubation after birth owing to severe BPD, and was successfully extubated using NHFOV. This case report suggests that NHFOV may be an effective respiratory strategy for very low birth weight infants with severe BPD.

Silver-Russell syndrome (SRS) is a congenital disorder characterized by prenatal and postnatal growth failure and craniofacial features. Hypomethylation of the H19/IGF2:IG-differential methylated region (H19LOM) is observed in 50% of SRS patients, and 15% of SRS patients with H19LOM had multilocus imprinting disturbance (MLID). Schimke immuno-osseous dysplasia (SIOD), characterized by spondyloepiphyseal dysplasia and nephropathy, is an autosomal recessive disorder caused by mutations in SMARCAL1 on chromosome 2. We report a patient with typical SRS-related features, spondyloepiphyseal dysplasia, and severe nephropathy. Molecular analyses showed H19LOM, paternal uniparental isodisomy of chromosome 2 (iUPD(2)pat), and a paternally inherited homozygous frameshift variant in SMARCAL1. Genome-wide methylation analysis showed MLID in this patient, although it showed no MLID in another patient with SIOD without SRS phenotype. These results suggest that iUPD(2)pat unmasked the recessive mutation in SMARCAL1 and that the SMARCAL1 gene mutation may have no direct effect on the patient’s methylation defects.

Background Two imprinting control centres, H19/IGF2:IG-differentialy methylated region (DMR) and KCNQ1OT1:TSS-DMR, reside on chromosome 11p15.5. Paternal deletions involving the KCNQ1OT1:TSS-DMR result in variable phenotypes, namely, normal phenotype, Silver-Russel syndrome (SRS) and fetal demise. However, expression analyses for CDKN1C in these patients are very limited. Cases Patient 1 (adult woman) and patient 2 (boy in early childhood) showed prenatal and postnatal growth failure and clinical suspicion of SRS. Molecular analyses Both patients showed hypermethylation of the KCNQ1OT1:TSS-DMR caused by the paternal heterozygous de novo deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers. The deletion sizes were 5 kb and 12 kb for patients 1 and 2, respectively. CDKN1C gene expressions in immortalised leucocytes of both patients were increased compared with those of controls. Conclusion Paternal deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers, disrupt KCNQ1OT1 expression, strongly activate CDKN1C expression and consequently cause severe growth failure.

Context: Children born small-for-gestational-age with short stature (SGA-SS) is associated with (epi)genetic defects, including imprinting disorders (IDs), pathogenic copy number variants (PCNVs), and pathogenic variants of genes involved in growth. However, comprehensive studies evaluating these 3 factors are very limited. Objective: To clarify the contribution of PCNVs and candidate pathogenic variants to SGA-SS. Design: Comprehensive molecular analyses consisting of methylation analysis, copy number analysis, and multigene sequencing. Methods: We enrolled 140 patients referred to us for genetic testing for SGA-SS. Among them, we excluded 42 patients meeting Netchine- Harbison clinical scoring system criteria for Silver-Russell syndrome and 4 patients with abnormal methylation levels of the IDs-related differentially methylated regions. Consequently, we conducted copy number analysis and multigene sequencing for 86 SGA-SS patients with sufficient sample volume. We also evaluated clinical phenotypes of patients with PCNVs or candidate pathogenic variants. Results: We identified 8 (9.3%) and 11 (12.8%) patients with PCNVs and candidate pathogenic variants, respectively. According to the American College of Medical Genetics standards and guidelines, 5 variants were classified as pathogenic and the remaining 6 variants were classified as variants of unknown significance. Genetic diagnosis was made in 12 patients. All patients with PCNVs or candidate pathogenic variants did not correspond perfectly to characteristic clinical features of each specific genetic cause. Conclusion: We clarified the contribution of PCNVs and pathogenic variants to SGA-SS without IDs. Comprehensive molecular analyses, including copy number analysis and multigene sequencing, should be considered for patients with unknown SGA-SS etiology.

Background: Our previous study suggested that assisted reproductive technology (ART) may be a possible risk factor for the development of epimutation-mediated imprinting disorders (epi-IDs) for mothers aged ≥ 30 years. However, whether ART or advanced parental age facilitates the development of uniparental disomy-mediated IDs (UPD-IDs) has not yet been investigated. Results: We enrolled 130 patients with aneuploid UPD-IDs including various IDs confirmed by molecular studies and obtained ART data of the general population and patients with epi-IDs from a robust nationwide database and our previous report, respectively. We compared the proportion of ART-conceived livebirths and maternal childbearing age between patients with UPD-IDs and the general population or patients with epi-IDs. The proportion of ART-conceived livebirths in patients with aneuploid UPD-IDs was consistent with that in the general population of maternal age ≥ 30 years and was lower than that in the patients with epi-IDs, although there was no significant difference. The maternal childbearing age of patients with aneuploid UPD-IDs was skewed to the increased ages with several cases exceeding the 97.5th percentile of maternal childbearing age of the general population and significantly higher than that of patients with epi-IDs (P < 0.001). In addition, we compared the proportion of ART-conceived livebirths and parental age at childbirth between patients with UPD-IDs caused by aneuploid oocytes (oUPD-IDs) and that by aneuploid sperm (sUPD-IDs). Almost all ART-conceived livebirths were identified in patients with oUPD-IDs, and both maternal age and paternal age at childbirth were significantly higher in patients with oUPD-IDs than in patients with sUPD-IDs. Because maternal age and paternal age were strongly correlated (rs = 0.637, P < 0.001), higher paternal age in oUPD-IDs was explained by the higher maternal age in this group. Conclusions: Different from the case of epi-IDs, ART itself is not likely to facilitate the development of aneuploid UPD-IDs. We demonstrated that advanced maternal age can be a risk factor for the development of aneuploid UPD-IDs, particularly oUPD-IDs.

Vitamin D seasonality has been reported in adults and children, suggesting that sunlight exposure has effects on 25(OH)D production. While vitamin D deficiency among infants has received significant attention, little is known about the extent to which vitamin D status during early infancy is affected by sunlight exposure. Here, we retrospectively analysed serum 25(OH)D levels of 692 samples obtained from healthy infants aged 1-2 months born at Saitama City Hospital, Japan (latitude 35·9° North) between August 2017 and September 2021. Data regarding the frequency of outdoor activities, formula intake and BMI were also collected and analysed. Month-to-month comparisons of vitamin D levels revealed significant variation in 25(OH)D levels in breastfed infants starting at 2 months, with maximal and minimal levels in September and January, respectively. An outdoor activity score of 0 was most common at 1 month (83·9 %) and a score of 3 was most common at 2 months (81·2 %), suggesting an increased amount of sunlight exposure at 2 months. Multiple linear regression analysis revealed the amount of formula intake to be significantly associated with vitamin D status at both 1 (t = 17·96) and 2 months (t = 16·30). Our results comprise the first evidence that seasonal variation of vitamin D begins at 2 months among breastfed infants from East Asia, though dietary intake appears to be the major determinant of vitamin D status. These findings provide new insights into the influence of dietary and non-dietary factors on vitamin D status during early infancy.

Background: Several cut-off points for 25-hydroxyvitamin D (25(OH)D) levels have been proposed to determine vitamin D deficiency or insufficiency. However, the level for 25(OH)D deficiency in early infancy remains unclear. The serum 25(OH)D value at which parathyroid hormone level plateaus, called the “inflection point,” is considered the most appropriate criterion for defining an adequate vitamin D status. Methods: This was a single-center retrospective study involving 305 1-month-old and 252 2-month-old Japanese infants. Nonlinear segmented regression analysis was performed based on the correlation between 25(OH)D and parathyroid hormone levels to determine vitamin D deficiency cut-off points. Results: Inflection points were 7.90 ng/mL for 1-month-old (95% confidence interval, 6.31–9.49) and 6.74 ng/mL for 2-month-old (95% confidence interval, 5.80–7.68) Japanese infants, which were lower than previously reported. Cut-off values were also lower in the high-body mass index (BMI) group than in the low-BMI group for both 1-month and 2-month-old infants. Conclusion: These results imply the need for nutritional rickets prevention via policy recommendations in most full-term newborns in Japan. Although validation studies are required, these results can still be used to guide vitamin D insufficiency treatment options in early infancy.

Intestinal volvulus without malrotation is a rare disease that causes volvulus of the small intestine despite normal intestinal rotation and fixation. We encountered a neonate with this disease who developed early jaundice and was suspected to have a fetal onset. This patient was characterized by early jaundice complicating intestinal volvulus without malrotation and is considered to have exhibited reduced fetal movement and early jaundice as a result of volvulus, necrosis, and hemorrhage of the small intestine in the fetal period. If abdominal distention accompanied by early jaundice is noted in a neonate, intestinal volvulus without malrotation and associated intraabdominal hemorrhage should be suspected and promptly treated.

Backgrounds: The proportion of assisted reproductive technology (ART)-conceived livebirths of patients with imprinting disorders (IDs) is higher than that of the general population. Whether this is due to ART or confounding effects of advanced parental age was not investigated. We examined the association of ART and parental ages at childbirth for the development of eight epimutation-mediated imprinting disorders (epi-IDs). Results: We enrolled 136 patients with epi-IDs and obtained general population ART data from the Japanese robust nationwide registry. We compared the proportion of ART-conceived livebirths and maternal childbearing ages between patients with epi-IDs and the general population. The proportion of ART-conceived livebirths in patients with epi-IDs was higher than that in mothers aged ≥ 30 years, the age group in which more than 90% of ART procedures performed. The maternal childbearing ages of patients with epi-IDs were widely distributed from 19 to 45 (median: 32) within the approximate 2.5th to 97.5th percentiles of maternal childbearing ages of the general population. In addition, we compared the proportion of ART-conceived livebirths and parental ages at childbirth across patients with eight epi-IDs. We demonstrated that more than 90% of ART-conceived patients with epi-IDs were found in Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) patients, and parental ages were almost consistent in patients with eight epi-IDs, except Prader-Willi syndrome. Conclusions: According to the prerequisite that most of the ART procedures in Japan are performed on mothers aged ≥ 30 years, ART can be a risk factor for the development of epi-IDs, particularly SRS and BWS, for mothers aged ≥ 30 years.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold standard for the measurement of serum 25-hydroxyvitamin D (25(OH)D) levels instead of the conventional method, radioimmunoassay (RIA). However, there was no study that compared RIA and LC-MS/MS for measuring serum 25(OH)D levels in infants and their mothers. The aim of this study was to assess the agreement of RIA and LC-MS/MS for measuring the serum levels in infants and postpartum women. This study enrolled 70 preterm infants, 113 term infants (134 samples), and 120 postpartum women. Serum concentration of 25(OH)D was measured by RIA and LC-MS/MS. We evaluated the correlation between RIA and LC-MS/MS. Also, we evaluated the bias between RIA and LC-MS/MS using Bland-Altman analysis. Sixty percent of preterm infants had serum 25(OH)D levels below the lower limit of quantification (LOQ) (4 ng/mL) and 90% of them were classified as vitamin D deficient. The serum 25(OH)D levels measured by RIA were significantly correlated with those measured by LC-MS/MS in all groups. According to the Bland-Altman plot, the serum 25(OH)D levels of infants measured by RIA had constant positive bias (mean±standard deviation [SD] [95% confidence interval, CI], preterm: +4.8± 2.4 ng/mL [4.2-5.4], term: +5.8±4.0 [5.1-6.5]) and proportional bias (preterm: r=0.44, p<0.01, term: r=0.50, p<0.01) compared with LC-MS/MS. The serum 25(OH)D levels of postpartum women measured by RIA had constant positive bias compared with LC-MS/MS, but no proportional bias was found. RIA demonstrated falsely high 25(OH)D levels when used for infants and postpartum women.