STIGMA: Single-cell tissue-specific gene prioritization using machine learning.

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues. Here, we introduce single-cell tissue-specific gene prioritization using machine learning (STIGMA), an approach that leverages single-cell RNA-seq (scRNA-seq) data to prioritize candidate genes associated with rare congenital diseases. STIGMA prioritizes genes by learning the temporal dynamics of gene expression across cell types during healthy organogenesis. To assess the efficacy of our framework, we applied STIGMA to mouse limb and human fetal heart scRNA-seq datasets. In a cohort of individuals with congenital limb malformation, STIGMA prioritized 469 variants in 345 genes, with UBA2 as a notable example. For congenital heart defects, we detected 34 genes harboring nonsynonymous de novo variants (nsDNVs) in two or more individuals from a set of 7,958 individuals, including the ortholog of Prdm1, which is associated with hypoplastic left ventricle and hypoplastic aortic arch. Overall, our findings demonstrate that STIGMA effectively prioritizes tissue-specific candidate genes by utilizing single-cell transcriptome data. The ability to capture the heterogeneity of gene expression across cell populations makes STIGMA a powerful tool for the discovery of disease-associated genes and facilitates the identification of causal variants underlying human genetic disorders.

Ferret Pediatrics.

Ferrets are bred to be pets, utilized for hunting, and as laboratory models. Despite the fact that ferrets in some areas of the world are neutered by the breeder before entering the pet trade, the importance of pediatric management should not be overlooked. Pregnant, whelping, and lactating jills should be closely monitored and kept in a quiet, stress-free environment. Hand-rearing baby kits is very challenging due to their requirement for ferret milk. Minimizing maternal stress and disease can prevent the need to hand rear kits. Infectious diseases in juvenile ferrets include canine distemper virus, rotavirus, coccidiosis, feline panleukopenia virus (experimental only), and Toxoplasma-like disease. All juvenile ferrets should be vaccinated against canine distemper and rabies. Congenital diseases are reported to affect the auditory, ocular, cardiovascular, urogenital, central nervous, and musculoskeletal systems. Early detection of these diseases is important to prevent the progression of curable diseases.

Congenital lower urinary tract obstruction with spontaneous fetal bladder rupture due to posterior urethral valves: a case report.

BACKGROUND: Congenital lower urinary tract obstruction (LUTO) is a rare but significant condition affecting fetal urinary tract development. LUTO has a range of etiologies, with posterior urethral valves (PUV) being the most common cause. The prenatal diagnosis of LUTO plays a crucial role in recognizing the condition and guiding management decisions. Prenatal ultrasound serves as the primary tool for identifying LUTO, with key findings including megacystis, bladder wall thickening, oligohydramnios, hydronephrosis, and the 'keyhole sign' indicating dilatation of the posterior urethra. We present a case of congenital LUTO with a rare complication of spontaneous fetal bladder rupture and urinary ascites, treated by peritoneo-amniotic shunt placement. CASE PRESENTATION: A 27-year-old pregnant Caucasian women was referred at 28 weeks of pregnancy due to the presence of megacystis and bilateral hydronephrosis on routine ultrasound and suspicion of LUTO. Repeat ultrasound at 29 weeks showed significant fetal ascites, oligohydramnios and resolution of megacystis and hydronephrosis, after which diagnosis of spontaneous bladder rupture was made. Despite ascites aspiration and amnio-infusion, there was persistent ascites and oligohydramnios. A peritoneo-amniotic shunt was placed with resolution of ascites and normalization of the amniotic fluid volume. At 35 weeks, relapse of the megacystis was observed with bilateral pyelectasis and oligohydramnios, possibly due to healing of the bladder rupture, after which elective cesarean section was planned. Cystography confirmed spontaneous healing of the bladder rupture and the presence of posterior urethral valves, which were resected in the neonatal period with cold knife incision. Total follow-up of 8 years continued to show positive ultrasonographic results and good renal function, but the child suffers from bladder dysfunction, manifesting as overactive bladder disease. CONCLUSIONS: LUTO might lead to important renal dysfunction and pulmonary hypoplasia in case of increasing disease severity. Spontaneous bladder rupture might improve renal prognosis, acting as a pop-off mechanism by decompression of the urinary tract. However, fetal bladder rupture is rare and only few cases have been reported. Prenatal intervention can be considered for moderate or severe LUTO, but the benefit for long-term outcome remains uncertain and further studies are needed.

[Genomic newborn screening-research approaches, challenges, and opportunities]. / Genomisches Neugeborenenscreening ­ Forschungsansätze, Herausforderungen und Chancen.

The application of high-throughput sequencing methods for population-based genomic newborn screening offers numerous opportunities for improving population health. The use of genome-based sequencing technology holds potential to enable the diagnosis of virtually any genetic disorder at an early stage and offers great flexibility when it comes to selection and expansion of target diseases. National and international efforts are therefore being made to investigate the ethical, legal, social, psychological, and technical aspects of genomic newborn screening. In addition to the many opportunities, there are numerous challenges and questions that remain to be answered: When and how should legal guardians be informed about such screening? Which diseases should be screened for? How should incidental findings or identification of a genetic predisposition be dealt with? Should data be stored long term and if so, how can this be done securely? Provided there is an appropriate regulatory framework and a transparent consent process, genomic newborn screening has the potential to fundamentally change the way in which we screen for congenital diseases. However, there is still much to be done. To achieve understanding and acceptance of genomic newborn screening amongst all stakeholders and thus to maximize its benefits for the population, a public discourse on the possibilities and limitations of genomic newborn screening is of critical importance. This article aims to provide an overview of the innovative technical developments in the field of human genetics, describe national and international approaches, and discuss challenges and opportunities of genomic newborn screening development.

Surgical resection therapy of a rare presentation of persistent Mullerian duct syndrome: a case review.

Persistent Mullerian Duct Syndrome (PMDS) is an extremely rare disease with less than 300 cases recorded in medical literature. Our patient was a 37 year old male who presented at the medical office with hematospermia as his sole complaint. He had previously undergone left orchidopexy and presented with hypotrophic left testicle and right testicle agenesis. PMDS differential was considered with the clear observation of a uterus-like structure during pelvic ultrasonography. The organs were later studied in magnetic resonance imaging and confirmed by post-surgery anatomopathological examination. Patient was discharged 24 h after surgery and developed azoospermia post-surgery.

Operative correction of an extremely rare condition called Persistent Mullerian Duct Syndrome (PMDS). What is PMDS? PMDS is a disease which has less than 300 cases in medical literature. It is a congenital condition characterized by the development of female genital organs such as the uterus and ovaries, in an otherwise normal male individual. The fetal development of these structures begins when the male fetus develops his genitalia, during the period when he must produce a hormone (anti-mullerian hormone), which suppresses female genitalia growth. Since this fetal stage is the turning point for genital development, lack of this hormone commonly results in the presence of functional female genital organs in an adult male, which characterizes the syndrome. Multiple reports also associate the syndrome with ectopic testis (cryptorchidism) or gonadal absence and dysfunctional sexual cell production. What was the aim of the report? The aim is to present a rare presentation of an already extremely rare disease in order to enrich the literature with another case of PMDS and the outcome of surgical correction. How was the patient treated? After discovering female organs in the male pelvis during an ultrasound scan, an elective surgery was performed to evaluate the removal of the uterus, fallopian tubes, ovary and vaginal canal through video laparoscopy. Why is this case important? The overall medical knowledge about PMDS is rather limited due to the reduced number of cases and the relatively wide variety of presentations. This article is useful to present a rather rare presentation, in which cryptorchidism and testicular agenesis were concomitant with hematospermia. Other than that, the diagnosis was done late in the patient's life, having lived over three decades with female genitals in his pelvis without any malignant (cancerous) mutations. The case report can also provide a record for the outcome of azoospermia, which is the absence of motile (and hence viable) sperm in the semen, following a non-complicated post-surgical recovery, which suggests unknown mechanisms may be involved in gonadal development after birth, and a different endocrine balance in patients with the syndrome.

Insights into the malfunctioning of the mitochondrial citrate carrier: Implications for cell pathology.

The mitochondrial citrate carrier (CIC) is a member of the mitochondrial carrier family and is responsible for the transit of tricarboxylates and dicarboxylates across the inner membrane. By modulating the flux of these molecules, it represents the molecular link between catabolic and anabolic reactions that take place in distinct cellular sub-compartments. Therefore, this transport protein represents an important element of investigation both in physiology and in pathology. In this review we critically analyze the involvement of the mitochondrial CIC in several human pathologies, which can be divided into two subgroups, one characterized by a decrease and the other by an increase in the flux of citrate across the inner mitochondrial membrane. In particular, a decrease in the activity of the mitochondrial CIC is responsible for several congenital diseases of different severity, which are also characterized by the increase in urinary levels of L-2- and D-2-hydroxyglutaric acids. On the other hand, an increase in the activity of the mitochondrial CIC is involved, in various ways, in the onset of inflammation, autoimmune diseases, and cancer. Then, understanding the role of CIC and the mechanisms driving the flux of metabolic intermediates between mitochondria and cytosol would potentially allow for manipulation and control of metabolism in pathological conditions.

Stochastic epigenetic mutations as possible explanation for phenotypical discordance among twins with congenital hypothyroidism.

PURPOSE: The elevated frequency of discordance for congenital hypothyroidism (CH) phenotype between monozygotic twins suggests the involvement of non-mendelian mechanisms. The aim of the study was to investigate the role of epigenetics in CH pathogenesis. METHODS: A genome-wide DNA methylation analysis was performed on the peripheral blood of 23 twin pairs (10 monozygotic and 13 dizygotic), 4 concordant and 19 discordant pairs for CH at birth. RESULTS: Differential methylation analysis did not show significant differences in methylation levels between CH cases and controls, but a different methylation status of several genes may explain the CH discordance of a monozygotic twin couple carrying a monoallelic nonsense mutation of DUOX2. In addition, the median number of hypo-methylated Stochastic Epigenetic Mutations (SEMs) resulted significantly increased in cases compared to controls. The prioritization analysis for CH performed on the genes epimutated exclusively in the cases identified SLC26A4, FOXI1, NKX2-5 and TSHB as the genes with the highest score. The analysis of significantly SEMs-enriched regions led to the identification of two genes (FAM50B and MEG8) that resulted epigenetically dysregulated in cases. CONCLUSION: Epigenetic modifications may potentially account for CH pathogenesis and explain discordance among monozygotic twins.

Zebrafish Model of Stickler Syndrome Suggests a Role for Col2a1a in the Neural Crest during Early Eye Development.

Most cases of Stickler syndrome are due to autosomal-dominant COL2A1 gene mutations leading to abnormal type II collagen. Ocular findings include axial eye lengthening with vitreal degeneration and early-onset glaucoma, which can result in vision loss. Although COL2A1 is a major player in cartilage and bone formation, its specific role in eye development remains elusive. We investigated the role of Col2a1a in neural crest migration and differentiation during early zebrafish eye development. In situ hybridization, immunofluorescence, live imaging, exogenous treatments [10 µM diethylaminobenzaldehyde (DEAB), 100 nM all-trans retinoic acid (RA) and 1-3% ethanol (ETOH)] and morpholino oligonucleotide (MO) injections were used to analyze wildtype Casper (roy-/-;nacre-/-), TgBAC(col2a1a::EGFP), Tg(sox10::EGFP) and Tg(foxd3::EGFP) embryos. Col2a1a colocalized with Foxd3- and Sox10-positive cells in the anterior segment and neural crest-derived jaw. Col2a1a expression was regulated by RA and inhibited by 3% ETOH. Furthermore, MO knockdown of Col2a1a delayed jaw formation and disrupted the ocular anterior segment neural crest migration of Sox10-positive cells. Interestingly, human COL2A1 protein rescued the MO effects. Altogether, these results suggest that Col2a1a is a downstream target of RA in the cranial neural crest and is required for both craniofacial and eye development.

Phenotypic spectrum of FGF10-related disorders: a systematic review.

FGF10, as an FGFR2b-specific ligand, plays a crucial role during cell proliferation, multi-organ development, and tissue injury repair. The developmental importance of FGF10 has been emphasized by the identification of FGF10 abnormalities in human congenital disorders affecting different organs and systems. Single-nucleotide variants in FGF10 or FGF10-involving copy-number variant deletions have been reported in families with lacrimo-auriculo-dento-digital syndrome, aplasia of the lacrimal and salivary glands, or lethal lung developmental disorders. Abnormalities involving FGF10 have also been implicated in cleft lip and palate, myopia, or congenital heart disease. However, the exact developmental role of FGF10 and large phenotypic heterogeneity associated with FGF10 disruption remain incompletely understood. Here, we review human and animal studies and summarize the data on FGF10 mechanism of action, expression, multi-organ function, as well as its variants and their usefulness for clinicians and researchers.

Hepatoblastoma in molecularly defined, congenital diseases.

Beckwith-Wiedemann spectrum, Simpson-Golabi-Behmel syndrome, familial adenomatous polyposis and trisomy 18 are the most common congenital conditions associated with an increased incidence of hepatoblastoma (HB). In patients with these genetic disorders, screening protocols for HB are proposed that include periodic abdominal ultrasound and measurement of alpha-fetoprotein levels. Surveillance in these children may contribute to the early detection of HB and possibly improve their chances of overall survival. Therefore, physicians must be aware of the high HB incidence in children with certain predisposing genetic diseases.

Hematological indices in pediatric patients with acyanotic congenital heart disease: a cross-sectional study of 248 patients.

Background: Congenital heart disease CHD is a significant cause of mortality and morbidity in children worldwide. Patients with congenital heart disease may develop hematological problems, including thrombocytopenia and neutropenia. In addition, several studies indicate the higher frailty of patients with CHDs to infections and malignancies. Nevertheless, the mechanisms of immune system changes in these patients have remained in the shadow of uncertainty. Moreover, very few studies have worked on cytopenia in CHD. This study has assessed the frequency of thrombocytopenia, neutropenia, lymphopenia, and anemia in pediatric patients with acyanotic congenital heart disease ACHD prior to open-heart surgery. Methods: This cross-sectional study was handled in the Pediatric Cardiology Clinic, Tehran University of Medical Sciences, during pre-operation visits from 2014 till 2019. Two hundred forty-eight children and adolescents with acyanotic congenital heart disease before open-heart surgery met the criteria to enter the study. Results: A total of 191 (76.7%) patients with Ventricular Septal Defects (VSD), 37 (14.85%) patients with Atrial Septal Defects (ASD), and 20 (8.11%) patients with Patent Ductus Arteriosus (PDA) were enrolled in this study. The median age was 23.87 months. Thrombocytopenia and neutropenia were found, respectively, in 3 (1.2) and 23 (9.2%) patients. Hemoglobin level and lymphocyte count were significantly lower in patients with neutropenia than patients with normal neutrophil count (P value = 0.024 and P value = 0.000). Significant positive correlations were found between neutropenia and anemia. There were no correlations between neutrophil count and Platelets. Also, anemia was found in 48 patients (19.3%). The study also found a statistically significant correlation between the co-existence of VSD and neutropenia in the patients (P value = 0.000). Conclusion: Although most were mildly neutropenic, there was a significant correlation between neutropenia and Ventricular Septal Defect compared to PDA and ASD groups. Regarding the importance of neutropenia to affect the prognosis of congenital heart defects in infections, it is important to consider further studies on the status of immune system function in these patients.

Patterns of paediatric massive blood transfusion protocol use in trauma and non-trauma patients.

BACKGROUND: Massive blood transfusion is infrequently required by children but can be a lifesaving intervention for haemorrhage or coagulopathy. Product volumes and ratios administered during the initiation of paediatric massive blood transfusion protocol (MBTP) are highly variable and the optimal component ratio is unknown. METHODS/MATERIALS: We performed a single-centre retrospective chart review of patients (<20 years) who received MBTP activation from August 2012 through January 2018. Logistic regression was used to determine the association between MBTP use characteristics (including blood product type and volume transfused, extracorporeal membrane oxygenation [ECMO] support, and cardiac arrest occurrence) and 24-h mortality. "Low" product ratio was defined as a ratio of plasma or platelets to red blood cells (RBCs) of <1:2 and "high" as ≥1:2. RESULTS: Ninety-eight MBTPs were activated for 89 patients (range 1-4 per patient). The most common underlying diagnoses were congenital heart disease (CHD, n = 28, 31.5%), followed by cardiopulmonary disease, and trauma. CHD patients required the greatest volume of RBCs (226.3 ml/kg, 95%CI [160.0, 292.7], p = 0.002) and platelets (46.7 ml/kg, 95%CI [33.2, 60.2], p < 0.001). A "low" product ratio was more common for the MBTP, with its incidence similar among the underlying diagnoses. CONCLUSION: An MBTP developed for trauma patients can be applied to non-trauma patients but standard MBTP components may not be optimal for all children. These findings show that underlying patient diagnoses may be a factor when designing an MBTP for a heterogeneous paediatric population.

Persistence of the craniopharyngeal canal.

Craniopharyngeal canal persistence is a congenital abnormality characterized by a bone canal extending from the nasopharynx to the pituitary fossa. Ascension of microorganisms through the channel can occur, triggering meningitis.

First occurrence of Arnold Chiari type II malformation and associated abnormalities in a Gir calf produced in vitro from Brazil - case report / [Primeira ocorrência de malformação de Arnold Chiari tipo II e anormalidades associadas em uma bezerra Gir de produção in vitro no Brasil - relato de caso]

This study characterized the clinical, radiological, ultrasound, and necroscopic findings of a case of Arnold-Chiari type II malformation in a Gir breed calf from Brazil. The animal was hospitalized at sixty days of age, in permanent sternal recumbency, cutaneous appendix at the 4th lumbar vertebra and kyphoscoliosis of the caudal and lumbosacral thoracic spine. Radiographic examination of the spine and skull revealed spina bifida and suspected occipital hypoplasia. Upon examination of myelography with an injection of lumbar and atlantooccipital contrast, it was possible to visualize the meningocele at the 4th lumbar vertebra region and findings at the rhombencephalon level of increased regional pressure with failure to fill the contrast in the posterior fossa, in the presence of clear demarcation of the circumvolutions of the cerebral cortex and the subarachnoid space of the cervical spinal cord. Ultrasonographic examination of the cerebellum showed an insinuation of the cerebellar worm through the foramen magnum. The animal did not show changes in complete blood count, biochemical series, and cerebrospinal fluid and was negative for Pestivirus. There was a worsening of the clinical conditions and the animal died. This malformation of unknown etiology must be studied as a differential diagnosis of the nervous system disorders.(AU)

Este estudo caracterizou os achados clínicos, radiológicos, ultrassonográficos e necroscópicos de um caso de malformação de Arnold-Chiari tipo II em uma bezerra Gir no Brasil. O animal foi hospilatizado aos 60 dias de idade, apresentando decúbito esternal permanente, apêndice cutâneo na altura da quarta vértebra lombar e cifoescoliose da coluna vertebral torácica caudal e lombossacra. Ao exame radiográfico da coluna e do crânio, foram observadas espinha bífida e suspeita de hipoplasia occipital. Ao exame de mielografia com injeção de contraste lombar e atlanto-occipital, foi possivel visualizar a meningocele na altura da quarta vértebra lombar e achados em nível rombencefálico de aumento da pressão regional com falha de preenchimento do contraste na fossa posterior, na presença de nítida demarcação das circunvoluções do córtex cerebral e do espaço subaracnoide da medula espinhal cervical. Ao exame ultrassonográfico do cerebelo, foi observada insinuação do verme cerebelar através do forame magno. O animal não apresentou alterações em hemograma completo, série bioquímica e fluido cérebro-espinhal e foi negativo para Pestivirus. Houve uma piora do quadro clínico e o animal morreu. Essa malformação de etiologia desconhecida deve ser estudada como um diagnóstico diferencial.(AU)

The Molecular Bases Study of the Inherited Diseases for the Health Maintenance of the Beef Cattle.

The article highlighted the problem of meat cattle genetic defects. The aim was the development of DNA tests for some genetic defects diagnostics, the determination of the animal carriers and their frequencies tracking in time. The 1490 DNA samples from the Aberdeen Angus (n = 701), Hereford (n = 385), Simmental (n = 286) and Belgian Blue (n = 118) cattle have been genotyped on the genetic defects by newly created and earlier developed DNA tests based on AS-PCR and PCR-RFLP methods. The Aberdeen Angus cattle genotyping has revealed 2.38 ± 0.31% AMC-cows and 1.67 ± 0.19 % AMC-bulls, 0.65 ± 0.07% DDC-cows and 0.90 ± 0.10% DDC-bulls. The single animals among the Hereford cattle were carriers of MSUD and CWH (on 0.27 ± 0.05%), ICM and HY (on 0.16 ± 0.03%). The Simmental cattle were free from OS. All Belgian Blue livestock were M1- and 0.84%-CMD1-carriers. The different ages Aberdeen Angus cattle genotyping has shown the tendency of the AMC- and DDC frequencies to increase in the later generations. The statistically significant increase of DDC of 1.17% in the cows' population born in 2019 compared to those born in 2015 allows concluding the further development of the DNA analysis-based measures preventing the manifestation of the genetic anomalies in meat cattle herds is necessary.

Recent approaches in clinical applications of 3D printing in neonates and pediatrics.

Neonates and pediatric populations are vulnerable subjects in terms of health. Proper screening and early optimal treatment would reduce infant and child mortality, improving the quality of life. Researchers and clinicians all over the world are in pursuit of innovations to improve the medical care delivery system. Infant morphometrics changes drastically due to the rapid somatic growth in infancy and childhood, demanding for patient-specific customization of treatment intervention accordingly. 3D printing is a radical technology that allows the generation of physical 3D products from digital images and addresses the patient-specific requirement. The combination of cost-effective and on-demand customization offers a boundless opportunity for the enhancement of neonates and pediatric health.Conclusion: The advanced technology of 3D printing proposes a pioneering breakthrough in bringing physiologically and anatomically appropriate treatment strategies addressing the unmet needs of child health problems. What is Known: • The potential application of 3D printing is observed across a multitude of fields within medicine and surgery. • The unprecedented effect of this technology on pediatric healthcare is still very much a work in progress. What is New: • The recent clinical applications of 3D printing provide better treatment modalities to infants and children. • The review provides an overview of the comparison between conventional treatment methods and 3DP regarding specific applications.

Radiology of Anomalies of Pulmonary Veins.

A study of 43 cases of suspected congenital diseases of heart was performed in Sahyadri Hospital, Pune, over a period of 5 to 6 years with dual source computed tomography (CT) in adolescents as well as children. Only the images of anomalies of pulmonary veins are presented. Compared with different radiological techniques, CT offers many advantages, as it can be undertaken even in neonates, yields more information than MR in a very little time, is better than 2D echo, when there is a small inter-costal window in some infants and is noninvasive. This study proved useful for further medical/surgical management.

Prenatal exposure to traffic-related air pollution and risk of congenital diseases in South Korea.

BACKGROUND: Previous studies have suggested links between exposure to ambient air pollutants and increased risk of congenital heart defects. However, few studies have investigated the association between other congenital diseases and traffic-related air pollution. In this study, we assessed the relationship between prenatal exposure to fine particulate matter (PM2.5) and nitrogen dioxide (NO2) with congenital diseases in South Korea. METHODS: Patients with one or more congenital diseases and a control group of patients with non-infective gastroenteritis and colitis with a case:control ratio of 1:3 were obtained from the National Health Insurance Service data for 2008-2013 in South Korea. We estimated the associations of PM2.5 and NO2 exposures with congenital diseases using generalized estimation equations after controlling for covariates. RESULTS: Maternal PM2.5 exposure during the first and second trimester showed positive associations with overall congenital diseases, with changes of 14.7% (95% confidence intervals (CI), 9.3%, 20.3%) and 16.2% (95% CI, 11.0%, 21.7%), respectively, per 11.1 µg/m3 and 10.2 µg/m3 increase of PM2.5 interquartile range (IQR). Similarly, NO2 exposure during the first and second trimester was associated with increased numbers of overall congenital anomalies, with 8.2% (95% CI, 4.2%, 12.3%) and 15.6% (95% CI, 9.3%, 22.2%) more cases, respectively, per 10.6 ppb increase of NO2. We found that maternal PM2.5 exposure during the first and second trimesters of pregnancy was significantly associated with increased risk of specific congenital diseases, including subtypes affecting the circulatory, genitourinary, and musculoskeletal system. However, no significant associations were observed during the third trimester. Maternal NO2 exposure across the entire pregnancy was associated with malformations of the musculoskeletal system. CONCLUSIONS: Our study identified significant links between in utero exposure to PM2.5 and NO2 and certain congenital diseases, and suggests that stricter controls on PM2.5 and NO2 concentrations are required.