The aim was to formulate practice guidelines for the diagnosis and management of congenital hypothyroidism (CH).A systematic literature search was conducted to identify key articles relating to the screening, diagnosis, and management of CH. The evidence-based guidelines were developed with the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system, describing both the strength of recommendations and the quality of evidence. In the absence of sufficient evidence, conclusions were based on expert opinion.Thirty-two participants drawn from the European Society for Paediatric Endocrinology and five other major scientific societies in the field of pediatric endocrinology were allocated to working groups with assigned topics and specific questions. Each group searched the literature, evaluated the evidence, and developed a draft document. These papers were debated and finalized by each group before presentation to the full assembly for further discussion and agreement.4 supplementation and frequent monitoring to ensure dose adjustments to keep thyroid hormone levels in the target ranges, a trial of treatment in patients suspected of transient CH, regular assessments of developmental and neurosensory functions, consulting health professionals as appropriate, and education about CH. The harmonization of diagnosis, management, and routine health surveillance would not only optimize patient outcomes, but should also facilitate epidemiological studies of the disorder. Individuals with CH require monitoring throughout their lives, particularly during early childhood and pregnancy.The recommendations include: worldwide neonatal screening, approaches to assess the cause (including genotyping) and the severity of the disorder, the immediate initiation of appropriate L-T Early detection and treatment of congenital hypothyroidism (CH) through neonatal screening prevents neurodevelopmental disability and optimizes developmental outcomes (1|⊕⊕⊕).Screening for primary CH should be introduced worldwide. The initial priority of neonatal screening for CH should be the detection of all forms of primary CH: mild, moderate, and severe. The most sensitive test for detecting primary CH is TSH determination (1|⊕⊕⊕).A strategy of second screening should be considered for the following conditions: preterm neonates; low-birth weight (LBW) and very low-birth weight (VLBW) neonates; ill and preterm newborns admitted to neonatal intensive care units (NICU); specimen collection within the first 24 hours of life; and multiple births (particularly same-sex twins); (2|⊕⊕○).If capillary TSH concentration from blood obtained on neonatal screening is ≥ 40 mU/L whole blood, we recommend starting treatment as soon as a good venous sample can be obtained, without waiting for the venous blood test result, unless venous thyroid function test (TFT) results are available on the same day (1|⊕⊕○).If capillary TSH concentration is < 40 mU/l of whole blood, the clinician may wait for the results of venous TFT, provided that these results are available on the following day (1|⊕⊕○).The detection of a high TSH concentration on screening should be communicated by an experienced person (eg screening laboratory staff or pediatric endocrine team) either by telephone or in person (2|⊕○○).When the child reaches nursery or school age, educators and teachers need not be informed about the child having CH to avoid stigmatization due to “labeling” (2|⊕○○).4 (FT4) concentration is below norms for age, treatment should be started immediately (1|⊕⊕⊕).If venous free TIf venous TSH concentration is > 20 mU/L, treatment should be started, even if FT4 concentration is normal (2|⊕⊕○).If venous TSH concentration is ≥ 6 to 20 mU/l beyond 21 days in a well baby with a FT4 concentration within the limits for age, we suggest a) investigation, which should include diagnostic imaging, to try to obtain a definitive diagnosis; b) consideration, in discussion with the family, of either initiating thyroxine supplementation immediately and retesting, off treatment, at a later stage; or withholding treatment but retesting two weeks later (2|⊕⊕○).X-ray of the knee may be carried out to assess the severity of intrauterine hypothyroidism by the presence or absence of femoral and tibial epiphyses (2|⊕⊕⊕).The thyroid gland should be imaged using either radioisotope scanning (scintigraphy) with or without the perchlorate discharge test; or ultrasonography; or both (1|⊕⊕○).Imaging should never be allowed to delay the initiation of treatment (1|⊕⊕○).All neonates with high TSH concentrations should be examined carefully for congenital malformations (particularly cardiac) and for dysmorphic features (1|⊕⊕⊕).4 alone is recommended as the medication of choice for treating CH (1|⊕⊕○).L-T4 treatment should be initiated as soon as possible and no later than 2 weeks after birth or immediately after confirmatory serum test results in infants in whom CH is detected by a second routine screening test (1|⊕⊕○).L-TAn initial L-T4 dose of 10–15 μg/kg per day should be given (1|⊕⊕○).4 or FT4 concentration, should be treated with the highest initial dose (1|⊕⊕○).Infants with severe disease, as defined by a very low pretreatment TT4 should be administered orally; if intravenous treatment is necessary the dose should be no more than 80% of the oral dose, The dose should then be adjusted according to TSH and FT4 determinations (1|⊕⊕○).L-T4 tablets should be crushed and administered via a small spoon, in a few milliliters of water or breast milk (1|⊕⊕○).L-T4 tablets should be used, particularly during infancy and in severe cases (2|⊕⊕○).Brand rather than generic L-T4 liquid should only be used if pharmaceutically produced (1|⊕⊕○).L-T4 treatment (1|⊕○○).Parents should be provided with written instructions on L-T4 administration (1|⊕⊕○).Serum or plasma FT4 (or TT4) and TSH concentrations should be determined at least 4 hours after the last L-T4 or FT4 concentration should be maintained in the upper half of the age-specific reference range (1|⊕⊕○).TSH concentration should be maintained in the age-specific reference range; TT4 dose should not be based on a single increase in FT4 concentration during treatment (1|⊕⊕○).Any reduction of L-T4 treatment (1|⊕○○).The first follow-up examination should take place 1–2 weeks after the start of L-TSubsequent evaluation should take place every 2 weeks until a complete normalization of TSH concentration is reached; then every 1 to 3 months thereafter until the age of 12 months (1|⊕○○). Between the ages of one and three years, children should undergo frequent clinical and laboratory evaluations (every 2 to 4 months) (1|⊕○○). Thereafter, evaluations should be carried out every 3 to 12 months until growth is completed (1|⊕○○).More frequent evaluations should be carried out if compliance is questioned or abnormal values are obtained (1|⊕○○).4 dose or formulation (1| ⊕○○).Additional evaluations should be carried out 4–6 weeks after any change in L-TAdequate treatment throughout childhood is essential and overtreatment should be avoided (1|⊕⊕⊕).Re-evaluation of the thyroid axis is indicated when no diagnostic assessment was carried out in infancy, and particularly when the infant was preterm/sick at the time of referral (1|⊕⊕⊕).4 treatment should be phased out over a 4- to 6-week period, and a full reevaluation should be carried out, with both biochemical testing and thyroid imaging if hypothyroidism is confirmed (2|⊕⊕○).For a precise diagnosis, L-T4 by 30% for 2–3 weeks and then rechecking thyroid function. If an increase in TSH concentrations to ≥ 10 mU/L is demonstrated, CH is confirmed. Otherwise the dose can be reduced further, with retesting after another 2–3 weeks. (2|⊕⊕○).If the presence or absence of primary CH is being assessed, rather than an exact diagnosis being sought, re-evaluation may be carried out by decreasing the dose of L-TWe recommend an immediate increase in L-T4 dose by 25–30% following a missed menstrual cycle or positive home pregnancy test (1|⊕○○).4 [TT4]) levels should be monitored every 4–6 weeks during the pregnancy, aiming at TSH concentrations < 2.5 mU/L in the first trimester and < 3 mU/L later in pregnancy (1|⊕○○).TSH and FT4  (1|⊕⊕⊕).Psychomotor development and school progression should be monitored and recorded in all children with CH, and particularly in at-risk cases .Concerns about behavior should be addressed from the time of diagnosis until school age (2|⊕⊕○).Memory deficits may be corrected by targeted training (2|⊕⊕○).Repeated  hearing tests should be carried out before school age and, as required (2|⊕⊕○).We recommend assessment for evidence of visual processing problems  (2|⊕○○).We recommend screening for speech delay and referral for speech therapy by 3 years if required (2|⊕⊕○).Compliance with treatment should be promoted throughout life (1|⊕⊕⊕).There is a risk of subtle decrease in HrQOL in young adulthood, particularly if treatment is suboptimal (2|⊕○○).Medical education about CH should be improved at all levels, with regular updates (1|⊕⊕⊕).The education of both parents and patients is essential particularly during transition to adult care and during pregnancy (1|⊕⊕⊕).Adherence to treatment influences growth and should be promoted (1|⊕⊕⊕).Normal growth, puberty, and fertility can be anticipated, if adherence is reasonably good (1|⊕⊕⊕).Patients with CH should be adequately treated with thyroxine, consume 800–1200 mg of calcium daily, and receive supplements added if intake is insufficient (2|⊕○○).We recommend lifestyle interventions, including diet and exercise, to optimize weight and health in individuals with CH (2|⊕○○).Genetic counseling should involve explaining the risk of recurrence of CH in an affected family, based on family history and thyroid morphology (1|⊕⊕○).Each family with an affected child should have access to information about the two major forms of CH (dysgenesis and dyshormonogenesis) and should if possible receive an explanation of their inheritance and recurrence rate (1|⊕⊕○).Genetic counseling should be targeted rather than general (2|⊕⊕○).Molecular genetic study should be preceded by a careful phenotypic description of CH patients (including morphology of the thyroid gland) (1|⊕⊕○).Any syndromic association should be studied genetically, to identify new CH genes and to make it possible to provide appropriate genetic counseling (1|⊕⊕○).The presence of familial cases of dysgenesis in siblings or parents should lead to a search for TSH receptor or PAX8 mutations, respectively (2|⊕⊕○).We recommend antenatal diagnosis when goiter is fortuitously discovered during fetal ultrasound, with a family history of dyshormonogenesis and with known defects of genes involved in thyroid function or development (1|⊕⊕○).The therapeutic management of affected fetuses should comply with the laws of the country concerned (1|⊕⊕○).Cordocentesis, rather than amniocentesis, should be the reference method for assessing fetal thyroid function but only performed if prenatal intervention is considered (1|⊕⊕⊕).In a euthyroid pregnant woman, a large goiter in the fetus with progressive hydramnios and a risk of premature labor and delivery and/or concerns about tracheal occlusion are criteria in favor of fetal treatment in utero (1|⊕⊕○).Interventions such as intra-amniotic L-T4 injection should be performed only by multidisciplinary specialist teams (1|⊕⊕⊕).Further research is required to improve our understanding of the pathophysiology and management of this heterogenous disorder.Thyroid hormones play a crucial role in early neurodevelopment so that untreated severe CH results in neurological and psychiatric deficits, including intellectual disability, spasticity, and disturbances of gait and co-ordination. CH is one of the most common preventable causes of mental retardation. Screening programs, which have been in operation over the last 30 years in most industrialized countries, have led to the successful early detection and treatment of infants with CH and have eliminated the severe neurodevelopmental deficits resulting from late diagnosis. Studies on cognitive function in patients with CH treated soon after birth have shown that normal development can be achieved in most patients, although some may have subtle neurocognitive deficits .Estimates of the prevalence of CH vary according to the method of ascertainment: about 1 in 2000 to 3000 live births in countries with neonatal screening vs about 1 in 6700 live births before the screening era .The results from neonatal screening programs have also helped to identify a broad spectrum of thyroid dysfunctions with different underlying etiologies. CH can be classified according to site: primary (thyroid) or secondary/central ; to severity: FT4 levels within the normal range for age (compensated) or subnormal (decompensated); and to age of onset. The most common form of CH is primary hypothyroidism, with high TSH levels reflecting various types of abnormal thyroid gland development or dyshormonogenesis. Secondary hypothyroidism is much less frequent, either with isolated TSH deficiency due to mutations inactivating the TSH β-subunit, the TRH receptor, or IGSF1 (ImmunoGlobulin SuperFamily member 1), or more commonly with TSH deficiency associated with other pituitary hormone deficiencies.Impaired thyroid hormone production may also be temporary or permanent, the latter requiring lifelong treatment, and thyroid dysfunction may change in a given individual with growth and development stages . TransieGiven the importance of optimal screening, prompt diagnosis, and adequate treatment of CH, and in recognition of the considerable variations in its management worldwide, and the stated need for a consensus-building conference , the EurThe target audience for these guidelines includes general and specialist pediatricians, other professionals providing care for patients with CH, and policy makers, particularly in countries with developing economies currently in the process of initiating neonatal screening programs for CH.Participants included individuals from Europe, North America (United States and Canada), Latin America, Asia, and Australia, with a balanced spectrum of professional seniority and expertise. In addition, an expert on the development of evidence-based guidelines was recruited to serve in an advisory capacity. Panel members declared whether they had any potential conflict of interest at the initial meeting of the group.Thirty-two participants were assigned to one of five groups to which topics 1–5 were allocated, and a chairperson was designated for each group. Each participant prepared a summary of the literature relating to a particular question distributed before the conference (which was held over 2 days in November 2011). Each group revised the summaries, which were then presented to the full conference. This report is based on the questions addressed.A detailed description of the grading scheme has been published elsewhere . RecommeFor each point, recommendations and evidence are described, with a modification in the grading evidence as follows: 1 = strong recommendation ; and 2 = weak recommendation .Qualify of evidence is indicated as follows: ⊕⊕⊕, high quality ; ⊕⊕○, moderate quality ; and ⊕○○, low quality .1.1.1 Neonatal screening programs for CH have been highly successful and economically beneficial over the last four decades. Affected children are detected very soon after birth, mostly before clinical symptoms and signs become evident. Early detection and treatment prevent morbidity, particularly neurodevelopmental disabilities (1|⊕⊕⊕).Many studies have confirmed the early success of CH screening for normalizing the cognitive outcomes of children with severe primary CH , 9, and 1.2.1 Screening for primary CH worldwide should be performed wherever possible on the basis of national resources. For new programs, there is a need to decide on the scope of screening to define the strategy for selecting neonatal screening tests. The goal of neonatal screening should be to detect all forms of primary CH—mild, moderate, and severe—but particularly those patients with severe CH in whom morbidity is high. The most sensitive test for detecting primary CH is the determination of TSH concentration (1|⊕⊕⊕).4 screening. Primary T4 screening with confirmatory TSH testing entails a risk of missing some cases of mild forms of primary CH but can detect some cases of central CH (CCH). Screening strategies for the detection of CCH are based on two approaches: 1) a combination of primary T4 and primary TSH screening; and 2) a combination of primary T4 screening with secondary TSH testing followed by T4 binding protein determination. The inclusion of T4 binding protein determinations decreases the number of false positives. The criteria defining a positive result must be adapted to the target disease definition and the resources of the screening program (1|⊕⊕⊕).1.2.2 Primary CH screening has been shown to be effective for the testing of cord blood or blood collected after the age of 24 hours, although the best “window” for testing is 48 to 72 hours of age. Blood is spotted onto filter paper, allowed to dry, and eluted into a buffer for TSH analysis. This method detects primary CH more effectively than primary T4 levels and whether these individuals have permanent or transient thyroid dysfunction  C–1.3.1 Specific biochemical criteria should be used for screening special categories of neonates at risk of transient and permanent CH and in whom initial screening tests may be inappropriate or provide normal results. A strategy of second screening may be required in the following conditions: preterm neonates with a gestational age (GA) of less than 37 weeks; LBW and VLBW neonates; ill and preterm neonates admitted to NICU; specimen collection within the first 24 hours of life; and multiple births, particularly in cases of same-sex twins. The repeat specimen should be collected at about 2 weeks of age, or 2 weeks after the first screening test was carried out. The interpretation of screening results should take into account the results of all specimens analyzed in a multiple sampling strategy. The criteria defining a positive screening test result should be adapted for the analytical parameters measured, the method used, and the age at sampling and maturity (GA/birth weight) of the infant (2|⊕⊕○).Several studies have generated data that argue for multiple sampling in preterm neonates with a GA of less than 37 weeks, LBW and very LBW neonates, ill and preterm neonates admitted to NICU, infants from whom a specimen is collected within the first 24 hours of life, and neonates from multiple births, particularly in case of monozygotic twins , 25–29. 25–34–4 alone in the initial screen, whereas TSH may also be assessed on the first specimen. However, most programs initially assessing T4 only subsequently evaluate TSH concentration for the neonates with the lowest T4 values . If TSH concentration is high, the infant is recalled for evaluation and testing. Repeat DBS specimens are collected if the T4 value is below a defined cutoff value for GA .2.1.2 We recommend starting treatment immediately if serum FT4 concentration is below the norm for age, regardless of TSH concentration (1|⊕⊕⊕).2.1.3 We suggest that treatment should be started if venous TSH concentration is persistently > 20 mU/L, even if serum FT4 concentration is normal (2|⊕○○).4 supplementation immediately and retesting, off treatment, at a later stage; or retesting 2 weeks later without treatment (2|⊕○○).2.1.4 When venous TSH concentration is between 6 and 20 mU/L in a well baby with an FT4 concentration within the normal limits for age, we suggest diagnostic imaging to try to establish a definitive diagnosis (2|⊕○○). If TSH concentration remains high for more than 3 to 4 weeks, we suggest (in discussion with the family) either starting L-T4 treatment must be started immediately if venous FT4 or TT4 levels are low, given the known adverse effect of untreated decompensated CH on somatic growth and neurodevelopment , such as a member of the screening laboratory staff or one of the pediatric endocrine team.2.2.2 The high TSH concentration should be communicated to the family either by telephone or in person as soon as possible (2|⊕○○).2.2.3 Communication may be directly with the family or via the family doctor, health visitor, or midwife (2|⊕○○).2.2.4 If possible, the baby should be seen for clinical assessment and venous thyroid function testing on the day of referral or on the next day at the latest (1|⊕○○).2.2.5 Centers should have relevant information materials about the diagnosis and management of CH in the appropriate languages for their community (2|⊕○○).4, by either the clinician or the pharmacist (2|⊕○○).2.2.6 The parents should be shown how to give the first dose of L-T2.2.7 The baby's family doctor and local pediatrician should be notified either by telephone or by letter to outline the provisional diagnosis and management (1|⊕○○).2.2.8 Options regarding shared care should be discussed with the clinician at the local level (1|⊕○○).2.2.9 When the child reaches nursery or school age, the consensus group advises against informing educators and teachers about the child having CH, to prevent “labeling” and stigmatization (2|⊕⊕○).4 treatment as soon as possible after birth to prevent irreversible neurocognitive impairment.ESPE guidelines provide little information about contacting and counseling the families of neonates with high TSH concentrations . A surve2.3.1 CH severity can be assessed clinically—on the basis of symptomatic hypothyroidism; biologically—as severe, moderate, or mild on the basis of serum FT4 levels of <5, 5 to <10, and 10 to 15 pmol/L, respectively; on the basis of delayed epiphyseal maturation on knee x-ray; and in terms of the etiology of CH (1|⊕⊕⊕).A serum thyroglobulin concentration below the detection threshold is highly suggestive of athyreosis or a complete thyroglobulin synthesis defect (2|⊕⊕⊕).4 concentration at diagnosis; and 2) IQ outcome, and is thus a reliable index of intrauterine hypothyroidism  Tyroidism , 48. A tyroidism . Data foyroidism —making i2.4.1 We recommend performing imaging studies to determine the specific etiology (1|⊕⊕○).2.4.2 Both scintigraphy and ultrasound should be considered in neonates with high TSH concentrations (2|⊕⊕○).4 treatment (1|⊕⊕○).2.4.3 Imaging should never be allowed to delay the initiation of treatment. Scintigraphy should be carried out within 7 days of starting L-T2.4.4 Infants found to have a normal-sized gland in situ in the absence of a clear diagnosis should undergo further reassessment of the thyroid axis at a later age (1|⊕○○).99mTc) or 1–2 MBq of iodine-123 (123I). 99mTc is more widely available, less expensive, and quicker to use than 123I. However, 123I is specifically taken up by the thyroid gland and gives a clearer scan than 99mTc (123I dose when perchlorate is administered at 2 hours (the perchlorate discharge test) indicates an organification defect  (Scintigraphy may be carried out with either 10–20 MBq of technetium-99m (an 99mTc . Scintign defect . Scintiger (NIS) , 53.The thyroid gland is a superficial structure that can be imaged by ultrasonography with a high-frequency linear array transducer 10–15 MHz) at a resolution of 0.7 to 1.0 mm. Ultrasound imaging, performed in the longitudinal and axial planes, can be used to investigate the absence or presence, size, echogenic texture, and structure of a thyroid gland in situ. However, it cannot always detect lingual and sublingual thyroid ectopy –56, alth15 MHz atCombining scintigraphy and thyroid ultrasound in the individual patient helps to: 1) improve diagnostic accuracy , 60; 2) 2.5.1 A thorough physical examination should be carried out in all neonates with high TSH concentrations for the detection of congenital malformations, particularly those affecting the heart, and in children for the identification of any underlying dysmorphic syndrome or neurodevelopmental disorders (1|⊕⊕⊕).The prevalence of congenital malformations, particularly cardiac malformations, including septal defects, renal abnormalities, and the risk of neurodevelopmental disorders is higher in subjects with CH than in the general population –65. Howe–Down syndrome is associated with a mild increase in TSH concentration from the neonatal period onward, although it is usually too small for detection by neonatal screening, as well as a shift to the left of the FT4 distribution so that mean values are lower when compared with the general population . Pendred4 alone is recommended as the treatment of choice for CH (1|⊕⊕○).3.1.1 L-T4 should be started as soon as possible, and no later than the first 2 weeks of life or immediately after confirmatory serum test results in infants identified in a second routine screening test. We recommend an initial L-T4 dose of 10 to 15 μg/kg per day. Infants with severe disease, as defined by a very low pretreatment TT4 or FT4 concentration, should be treated with the highest initial dose, and those with a mild to moderate hypothyroidism with a lower dose (1|⊕⊕○).3.1.2 Treatment with L-T4 should be administered orally. If oral administration is not possible, it can also be administered iv, in which case the iv dose should be no more than 80% the oral dose. The dose should then be adjusted according to TSH and FT4 determinations (1|⊕⊕○).3.1.3 L-T4 in tablet form. In neonates and infants, the tablets can be crushed and administered via a small spoon, with suspension, if necessary, in a few milliliters of water or breast milk. L-T4 can also be administered in liquid form, but only if pharmaceutically produced and licensed L-T4 solutions are available (1|⊕⊕○). A brand rather than a generic formulation is recommended in CH, at least in infancy (2|⊕⊕○).3.1.4 We recommend the administration of L-T4 can be administered or taken in the morning or evening, either before feeding or with food, but it should be administered in the same way every day. The dose should then be adjusted according to TSH and FT4 determinations to establish the appropriate dose in each setting. Caution should be taken with the administration of vitamin D during the first weeks of life, and the intake of soy, iron, and calcium at the time of L-T4 administration should be avoided (1|⊕⊕⊕).3.1.5 L-T4 treatment to avoid uncertainties that might hinder compliance (1|⊕○○).3.1.6 Parents should be provided with written instructions on L-T3 is the biologically active hormone, but there is no evidence that combined therapy with L-T4 and L-T3 is more beneficial than treatment with L-T4 alone, probably due to the high degree of efficiency of endogenous deiodinases, which break T4 down into T3 (4 is available in tablet form (the most widely used form) or as a pharmaceutically produced and licensed liquid form. Unlike suspensions prepared by pharmacists, this licensed L-T4 solution allows reliable dosing and is a convenient way of administering L-T4, particularly to infants and young children  or minerals . In infants with CH, hypersensitivity to vitamin D administration with hypercalcemia has been described during the first few weeks of L-Titamin D , 76. In ng state .4 treatment, within the first 2 weeks of life, has been shown to be crucial for neurodevelopment and for the achievement of a normal intellectual outcome in affected children  and TSH concentrations. The blood samples for laboratory evaluation should be collected at least 4 hours after the last L-T4 administration (1|⊕⊕○). We recommend maintaining TSH in the age-specific reference range (but to avoid undetectable TSH < 0.05 mU/L) and serum concentrations of TT4 or FT4 in the upper half of the age-specific reference range (1|⊕⊕○). If necessary, the treatment should be adjusted according to the hormone concentrations measured, but decreases in LT4 dose should not be based on a single high FT4 concentration during treatment (1|⊕⊕○). Clinicians should be familiar with the reference ranges for the methods used by the laboratory carrying out the tests (1|⊕○○).3.2.1 The monitoring of L-T4 treatment, with intense follow-up over the first year of life . Between the ages of 1 and 3 years, children should undergo frequent clinical and laboratory evaluations (every 2 to 4 months), with regular evaluations every 3 to 12 months thereafter until growth is completed. Measurements should be performed at more frequent intervals if compliance is questioned or abnormal values are obtained, and 4 to 6 weeks after any change in L-T4 dose or L-T4 formulation  (1|⊕○○).3.2.2 We recommend performing the first follow-up examination 1 to 2 weeks after the start of L-T4 treatment is very low. The careful monitoring of thyroid hormone parameters, during both initial and maintenance treatment, is recommended to minimize the risk (1|⊕○○).3.2.3 The incidence of adverse events during L-T4 at 50% of the target replacement dose and increasing this in accordance with FT4 levels after 2 weeks (2|⊕○○).3.2.4 In children with pre-existing cardiac insufficiency, we suggest introducing L-T4 dosage, it is appropriate to recheck thyroid function, and the recommended interval of 4–6 weeks is in keeping with the American Academy of Pediatrics guidelines (–4 treatment), patients with pre-existing health conditions or with a very late diagnosis may require special attention during treatment  and the maintenance of relatively higher FT4 concentrations during the first year of life lead to a better intellectual outcome , 92, 93.94–idelines . The adeidelines –99. Basereatment , 101.4 dose since infancy, and in children in whom no enzyme defect has been identified, either because no molecular genetic investigations have been carried out or because investigations have proved negative for all mutations tested (1|⊕⊕○).3.3.1 We recommend re-evaluation of the thyroid axis in cases in which no etiological diagnostic assessment was carried out during early infancy and/or when treatment was started in the context of the infant being ill . Re-evaluation is also mandatory when initial evaluation has shown a normally located gland, with or without goiter, in neonates with positive thyroid antibodies, in children who have required no increase in L-T4 underdosage or poor compliance with treatment (1|⊕⊕○).3.3.2 Retesting off treatment may be waived if venous TSH concentration has risen after the first year of life, due to either L-T3.3.3 Re-evaluation of the thyroid axis is not indicated when thyroid dysgenesis has been conclusively shown on imaging or (with the exception of DUOX.2 mutations or Pendred syndrome) when dyshormonogenesis has been confirmed by molecular genetic testing (1|⊕⊕⊕).Thyroid re-evaluation is important if no definitive diagnosis was made during the neonatal period; it has been shown that one-third of patients with CH and normally located glands may have transient thyroid dysfunction , 60. Re-Re-evaluation is essential in subjects who were preterm or sick during the neonatal period . Other i3.3.4 Re-evaluation of the thyroid axis, off treatment, should normally take place after the age of 3 years (1|⊕⊕○).3.3.5 Earlier re-evaluation may be indicated if the clinical context renders transient increases in TSH concentration highly probable; for example: 1) in the case of newborns in whom thyroid peroxidase or TSH receptor antibodies are detectable in the blood; and 2) when a eutopic, normally sized gland is found on ultrasound scans (2|⊕⊕○).Magnetic resonance imaging studies have shown that the myelination of the central nervous system is completed by 36 to 40 months of age , at whic4 should be phased out over a 4 to 6-week period (depending on the size of the maintenance dose) and a full re-evaluation carried out at the end of this period, with biochemical testing and thyroid imaging if hypothyroidism is confirmed (2|⊕○○).3.3.6 If a precise diagnosis is sought, L-T4 dose may be decreased by 30% for 2 to 3 weeks. If an increase in TSH concentration to ≥ 10 mU/L is observed during this period, then continuing hypothyroidism can be assumed. By contrast, if thyroid function remains normal, the dose may be reduced further, followed by retesting (2|⊕⊕○).3.3.7 If the clinician wishes to establish the presence or absence of primary hypothyroidism rather than to obtain an exact diagnosis, L-TBy the age of 2 to 3 years, the severity of thyroid impairment may be evident from a lower dose requirement , 103 or,4 dose by 25 to 30% after a missed menstrual cycle or positive home pregnancy test (1|⊕○○).3.4.1 For women with CH who are planning a pregnancy, even greater efforts should be made to ensure that maternal thyroid hormones are optimal. In newly pregnant patients, we recommend an immediate increase in L-T4) levels should be evaluated as soon as pregnancy is confirmed, every 4 to 6 weeks during the pregnancy, and 4 weeks after every change in dose (1|⊕⊕⊕).3.4.2 TSH and FT4 .There is currently no specific evidence for treatment schedules in pregnant women with CH. We therefore refer to the guidelines of the American Thyroid Association and The Endocrine Society regarding the management of thyroid disease during pregnancy –109.–4 and very high TSH concentrations at diagnosis) or poor endocrine control, particularly during the first year, and in those from economically disadvantaged families (1|⊕⊕⊕).4.1.1 Psychomotor and language development as well as school progression should be monitored and recorded in all children with CH (1|⊕⊕○). Clinicians should pay particular attention to developmental delays or learning difficulties and to attention problems in children with severe CH .4.1.3 Memory deficits may be corrected by targeted training (2|⊕⊕○).4.1.4 Concerns about behavior should be addressed from the time of diagnosis until school age (2|⊕⊕○).4.1.5 Adequate treatment throughout childhood is essential, and overtreatment should be avoided (1|⊕⊕⊕).4.1.6 The determinants of remaining deficits require further study.4 dose  has disappeared from cohorts screened for CH, and the mean global IQ is now 10 to 30 points higher in these patients than in the prescreening era , 111. Co4 dose ; school 4 dose . Cogniti4 dose .Behavior scores on initial admission to school are within the normal range,  and the Subtle and specific memory deficits and reduced hippocampal volumes may be observed . There i4.1.7 Repeated  hearing tests should be carried out before school age and as required (2|⊕⊕○).4.1.8 Assessing patients for evidence of visual processing problems  is suggested (2|⊕○○).4.1.9 We recommend screening for delays in speech acquisition by the age of 3 years and propose speech therapy as required (2|⊕⊕○).Even after excluding patients with Pendred syndrome, a higher prevalence of hearing impairment has been observed in patients with CH than in the reference population, possibly necessitating the use of hearing aids in childhood. Substantial adverse effects on speech development, school performance, and social interactions may occur if hearing impairment is undiagnosed . This im4.2.1 Compliance with treatment should be promoted throughout life (1|⊕⊕⊕).4.2.2 In future studies of HrQOL, “focusing illusion” should be considered (a form of stigmatization similar to “labeling” at school) (2|⊕○○).There is a risk of a subtle decrease in HrQOL, particularly in mental dimensions and if treatment is suboptimal , 123.4.3.1 Medical education about CH should be improved at all levels, with regular updates (1|⊕⊕⊕).4.3.2 We suggest identifying resources in the community to which patients can be referred for continuing education about self-management of their condition and to update their knowledge of CH. The education of both parents and patients is essential, with particular attention paid to the transition to adult care and management during pregnancy (1|⊕⊕⊕).The adherence of physicians to guidelines is low . Poor ad4.4.1 Adherence to treatment influences growth and should be promoted (1|⊕⊕⊕).4.4.2 Provided treatment is adequate, it is appropriate to provide patients and their parents with reassurance about growth, puberty, and fertility (1|⊕⊕⊕).Length and height increase within normal limits in patients with adequately treated CH . Patient4 and consume 800–1200 mg of calcium daily, ideally from dietary sources, with calcium supplements added if dietary calcium intake is insufficient (2|⊕○○).We recommend that patients with CH should be adequately treated with L-T4 display higher levels of bone resorption than of bone formation, leading to progressive bone loss. The goal of therapy is to render the patient euthyroid, with a normal TSH concentration. Only a few studies have evaluated the impact of long-term L-T4 treatment on bone mineral density. Two studies reported that bone mineral density in children and young adults with CH is within the normal range .4.6.2 Optimal treatment of CH is essential for cardiovascular health (1|⊕○○).Patients with CH have a higher risk of being overweight and, thus, of metabolic complications . In addi5.1.1 Genetic counseling should provide information about the risk of recurrence of CH in a family with CH, based on family history and thyroid morphology. Each family with an affected child should have access to information on the two major forms of CH (dysgenesis and dyshormonogenesis) and should receive an explanation of inheritance and recurrence rate. A certified geneticist or a genetic counselor  should be made available in some cases. In such cases, given the current state of knowledge, we propose targeted rather than general genetic counseling in the context of certain clinical situations, described in SCL5A5/NIS ; pendrin, SCL26A4/PDS ; thyroglobulin, TG (OMIM No. 274700); thyroid peroxidase, TPO (OMIM No. 274500); dual oxidase 2, DUOX2 (OMIM No. 607200); dual oxidase maturation factor 2, DUOX2A (OMIM No. 274900); or iodotyrosine deiodinase, IYD/DEHAL1 (OMIM No. 274800). These mutations are inherited in an autosomal recessive manner and are associated with no other malformations, other than deafness in Pendred syndrome ; Forkhead box E1 , Paired box gene 8 , NK2 homeobox 5 , TSH receptor ; and Gs α  genes  genes , 138 (Ta0) genes .TSHR and PAX8 gene mutations (2|⊕⊕○).5.2.1 Careful phenotypic description of CH patients  is required, and we suggest that any syndromic association should be studied genetically to identify new genes involved in CH and to ensure that healthcare staff are in a position to offer appropriate genetic counseling. The presence of familial cases of dysgenesis should lead to a search for The genetic diagnosis of CH may facilitate the targeting of specific interdisciplinary follow-up and supportive care for patients (1|⊕⊕○). The relevant features to consider are listed in NKX2–1 mutation implies that special attention should be paid to neurological development and to lung disease in the follow-up of affected children . In such circumstances, the discussion of the prenatal diagnosis should be open. The therapeutic management of affected fetuses should comply with the laws in force in the country concerned (1|⊕⊕○). The familial recurrence of CH due to dysgenesis  requires further study to determine the feasibility and clinical relevance for antenatal detection.5.3.1 We recommend antenatal diagnosis in cases of goiter fortuitously discovered during systematic ultrasound examination of the fetus, in relation to thyroid dyshormonogenesis (1|⊕⊕⊕); a familial recurrence of CH due to dyshormonogenesis (25% recurrence rate) (1|⊕⊕⊕); and known defects of genes involved in thyroid function or development with potential germline transmission (1|⊕⊕○). Special issues should be considered for syndromic cases with potential mortality and possible germline mosaicism .5.3.3 We recommend cordocentesis, rather than amniocentesis, as the reference method for assessing fetal thyroid function. Norms have been established as a function of GA. This examination should be carried out only if prenatal intervention is considered (see below) (1|⊕⊕⊕).5.3.4 In most cases, fetal thyroid function can be inferred from context and ultrasound criteria, and fetal blood sampling is, therefore, only exceptionally required (2|⊕⊕○).5.3.5 In a euthyroid pregnant woman, a large goiter in the fetus with progressive hydramnios and a risk of premature labor and delivery and/or concerns about tracheal occlusion are criteria in favor of fetal treatment in utero (1|⊕⊕○).4 (1|⊕⊕○).5.3.6 In a hypothyroid pregnant woman, the initial approach should be to treat the pregnant woman, rather than the fetus, with L-T4 have been reported to decrease the size of the fetal thyroid gland. However, experience with this procedure is limited, and the risk of provoking premature labor or infections should be evaluated with care. Thus, follow-up studies are very important to determine the efficacy and possible adverse long-term consequences of medical interventions on the fetus. Such interventions should be performed only by multidisciplinary specialist teams  (1|⊕⊕⊕).5.3.7 For goitrous nonimmune fetal hypothyroidism leading to hydramnios, intra-amniotic injections of L-T4 injection and strongly suggest that this treatment is effective for decreasing goiter size. However, none of the many L-T4 regimens used ensures euthyroidism at birth. It is therefore not possible to formulate guidelines from current data. The expert panel proposes the use of 10 μg/kg estimated fetal weight/15 days in the form of intra-amniotic injections. The risks to the fetus and the psychological burden on the parents should be factored into the risk/benefit evaluation (2|⊕○○).5.3.8 Studies have confirmed the feasibility and safety of intra-amniotic L-T5.3.9 Determination of the indications and optimal modes of prenatal treatment for nonimmune fetal goitrous hypothyroidism will require larger, well-designed studies that would be best conducted via international co-operation between multidisciplinary medical teams. Alternative ways of treating the fetus by administering drugs to the mother should also be investigated (2|⊕○○).4  and fetal hormonology have made it possible to identify thyroid function disorders in the fetus that could potentially be treated in utero by administering drugs to the mother. Several interventions have also been proposed for improving the fetal outcomes of fetal hypothyroid disorders by considering the fetus as the patient to be treated and gaining direct access to the amniotic cavity. These approaches range from public health interventions with clear benefits and negligible risks, such as increasing the iodine intake of all pregnant women, to procedures with a much less clear benefit-to-risk ratio, such as cordocentesis for determining thyroid function in a fetus with goiter and repeated intra-amniotic injections of L-T4 –152 (Tab4 –.4 treatment throughout the lifespan for most patients, with a particular emphasis on treatment in the first years of life and of treatment optimization in pregnant women with CH. Careful neurodevelopmental and neurosensory evaluations should be started early in life and repeated at important critical developmental phases, taking into account disease severity at diagnosis and providing appropriate interventions as required. Subsequent efforts should focus on educating both patients and caregivers to ensure that adequate treatment is continued into adulthood. Future research should aim to improve our understanding of the pathophysiology of this heterogeneous disorder and to determine whether knowledge of the specific defect in thyroid development or function is likely to improve patient care and outcomes.Patients with CH benefit from neonatal screening, which makes it possible to initiate essential replacement therapy immediately. This consensus highlights the need to identify clear cutoff points for CH screening, without increasing the number of false-positive results. However, the results of long-term prospective studies in subjects with false-positive results are not yet available for the formulation of evidence-based recommendations for diagnosis and management. Based on current outcome data, an immediate treatment should be initiated, and it appears necessary to maintain adequate L-TPaulina Alexander, Department of Pediatric Endocrinology, and Diabetes, Charite Children's Hospital, Berlin, Germany; Miguel Alvarez, Institute of Neurology and Neurosurgery, Havana, Cuba; Oliver Blankenstein, Department of Pediatric Endocrinology, and Diabetes, Charite Children's Hospital, Berlin, Germany; Alessandra Cassio, Department of Pediatrics, University of Bologna, Bologna, Italy; Mireille Castanet, Department of Pediatrics, University of Rouen, Rouen, France; Graziano Cesaretti, Department of Pediatric Endocrinology, University of Pisa, Pisa, Italy; Tim Cheetham, Department of Paediatric Endocrinology, Newcastle University, Newcastle-upon-Tyne, United Kingdom; Anna Chiesa, Endocrinology Division, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina; Carlo Corbetta, Neonatal Screening Laboratory, Ospedale dei Bambini “Vittore Buzzi,” Milan, Italy; Maria E. Craig, Institute of Endocrinology and Diabetes, Sydney Children's Hospital Network and University of Sydney, Sydney, Australia; Thomas P. Foley, Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania; Scott Grosse, Atlanta, Georgia; Marlies Kempers, Radboud University Nijmegen Medical Centre, Department of Genetics, Nijmegen, The Netherlands; Claire de Labriolle-Vaylet, UPMC Univ Paris IV and Nuclear Medicine, Hopital Trousseau, Paris, France; Stephen LaFranchi, Department of Pediatrics, Oregon Health and Sciences University, Portland, Oregon; Dominique Luton, Department of Obstetrics and Gynecology, University Diderot Paris 7, Clichy, France; Masanori Minagawa, Division of Endocrinology, Department of Pediatrics, Chiba Children's Hospital, Chiba, Japan; Palany Raghupathy, Department of Child Health, Christian Medical College, Vellore, India; Robert Rapaport, Department of Pediatric Endocrinology, Mount Sinai School of Medicine, New York, New York; Joanne Rovet, Departments of Psychology and Paediatrics, University of Toronto, Toronto, Canada; Diet S. Rustama, Department of Child Health, Padjadjaran University School of Medicine, Bandung, Indonesia; Mariacarolina Salerno, Department of Pediatrics, University “Federico II,” Naples, Italy; Anna Simon, Department of Pediatrics, Christian Medical College, Vellore, India; Gabor Szinnai, Pediatric Endocrinology and Diabetology, University Children's Hospital Basel, University of Basel, Basel, Switzerland; Toshihiro Tajima, Department of Pediatrics, Hokkadio University School of Medecine, Sapporo, Japan; and Paul van Trotsenburg, Department of Paediatric Endocrinology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.


Although a high photoresponsivity of 880–2000 AW−1 and photogain up to 5000 have been demonstrated in MoS2-based photodetectors, the light absorption and gain mechanisms are two fundamental issues preventing these materials from further improvement. In addition, it is still debated whether monolayer or multilayer MoS2 could deliver better performance. Here, we demonstrate a photoresponsivity of approximately 104 AW−1 and a photogain of approximately 107 electrons per photon in an n-n heterostructure photodetector that consists of a multilayer MoS2 thin film covered with a thin layer of graphene quantum dots (GQDs). The enhanced light-matter interaction results from effective charge transfer and the re-absorption of photons, leading to enhanced light absorption and the creation of electron-hole pairs. It is feasible to scale up the device and obtain a fast response, thus making it one step closer to practical applications.Molybdenum disulphide (MoS High quality and large area multilayer MoS2 films were grown on SiO2/Si substrates by the reaction of gaseous MoO3 and sulphur via atmospheric pressure chemical vapour deposition (APCVD). This growth technique affords a simple device fabrication process that does not require material transfer. Because of fewer defects and impurities, multilayer MoS2 films demonstrate surprisingly good performance for photodetection, which is comparable to the best record achieved on mechanically exfoliated MoS2In this work, we hybridised a multilayer MoS2 thin film by incorporating nitrogen-doped graphene quantum dots (GQDS), which possess strong light absorption, long carrier lifetime and a sizable bandgap upon excitation232425n-n type van der Waals heterostructure consisting of GQDs and MoS2 is formed, where the photoexcited holes can be induced successfully by MoS2 and are subsequently trapped because of a small energy barrier in the heterojunction. The long carrier lifetime of the GQDs will cause the photoexcited carriers to recirculate several times at the interface. Moreover, light emission from the GQDs will be re-absorbed by MoS2 to generate additional photocarriers. Consequently, the MoS2-GQDs hybrid system is an extremely efficient photoelectrical material that can achieve impressive photodetection performance. Indeed, this material ushers in an innovative approach for fabricating highly responsive and low cost photodetectors for practical applications.To explore the performance limit of this material, we modified the MoS2 films on SiO2/Si substrates. Details of the experimental setup and growth process are provided in the Methods and 2 substrate for the production of MoS2. Briefly, MoO3 is largely reduced by sulphur vapour to form MoO3-x species, which are further sulphurised and accompanied by the formation of MoS2 on the SiO2 substrate72 follows a typical two-dimensional growth mode consisting of nucleation, growth and coalescence, as shown in 2 crystals, and the surrounding area with bright contrast is the SiO2 substrate. It is found that shorter growth times  of bilayer and trilayer MoS2 are 21.4 and 23.1 cm−1, respectively, which are consistent with a previous reportE12g peak redshifts whereas the A1g peak blueshifts obviously. This observation is attributed to columbic interactions and possible stacking-induced changes in the intra-layer bonding2 by integrating the A1g peak to verify the thickness uniformity, as shown in A1g peak, which scales linearly with the thickness, is homogeneous over the entire scanning area (approximately 20 μm).Spectroscopic characterisations are performed to systematically study the quality and uniformity of the MoS2 film. Two absorption peaks at approximately 620 nm and 670 nm are observed, which correspond to direct excitonic transitions at the Brillouin zone K point in MoS22 film is relatively uniform. The X-ray photoelectron spectroscopy (XPS) result in i.e., Mo-3d3/2 at 232.9 eV and Mo-3d5/2 at 229.9 eV) and the S-related peaks , which are consistent with the reported values for MoS2 crystals2024Id) demonstrates enhancement under light illumination and that it increases at higher light power. At an excitation power of 30.1 μW, the device drain current reaches 10.15 μA when the source-drain voltage Vds = 1.68 V, which is 1000 times higher than the current in the dark state . Such a high optical ON-OFF ratio is comparable to the highest ratio reported for single or multilayer MoS2 phototransistors102 and the underlying SiO2 layer, which usually occurs for MoS2 grown with the CVD method10i.e., at  nA. Such2 or between MoS2 and the SiO2 substrates10112 and the Au electrodes indicates the band bending at the interface. An energy barrier hinders the carrier injection from the electrodes under dark conditions. However, electrons inside the valence band (VB) can be easily excited to the conduction band (CB) by photons when the device is biased and exposed to incident light, which will greatly increase the concentration of transported carriers.2 phototransistor has good gate tunability, which shows higher drain current at higher gating voltage, as shown in e.g., Vg = 70 V), the device can generate a photocurrent as high as 16.2 μA, even at a very low excitation light power of 50 nW. The photoresponsivity is estimated to be on the order of 102 AW−1 while the gate bias is applied, as shown in −1 (Vg = 70 V), which is comparable to the best reported performance of a phototransistor based on monolayer or multilayer MoS2101213Vg) and the threshold voltage (Vt), the Fermi level in MoS2 can be shifted up (Vg > Vt) or down (Vg < Vt) while sweeping the back gate voltage, which results in the ON or OFF switch of the phototransistor.The multilayer MoSn-doped GQDs were hybridised with MoS2 to form a heterostructure device, as shown in 2-GQDs phototransistor is noteworthy, as shown in I-V characterisations shown in 2-GQDs phototransistor has a much larger drain current under illumination, which is approximately 5 times higher than a pure MoS2-based phototransistor. Time-dependent photoresponse measurements have also been performed, as shown in Iph1 and τ2, are reduced by a factor of ten. It is generally believed that the fast decay time component τ1 is related to the direct recombination of photoexcited carriers and that the slow decay time component τ2 is caused by sub-bandgap emissions resulting from the charge impurity and trap states inside the bandgap of MoS2112 and the GQDs.With the aim to further increase the photoresponsivity and shorten the response time, 2-based phototransistor, the MoS2-GQDs phototransistor has a superior gate tunability in terms of higher photocurrent, as shown in 2-GQDs device can be effectively modulated by the back gate voltage, and the photocurrent value can reach as high as 0.55 mA under an adequate gate bias (Vg = 80 V), which is 5 times that of a MoS2 device. 2 device, the responsivity decreases exponentially as the light power increases, and the maximum responsivity is 1.6 × 104 AW−1 under an excitation power of 50 nW (Vg = 80 V), which is two orders of magnitude higher than a pure MoS2 device, and among the highest responsivity recorded for MoS2 photodetectors10Relative to a MoSτ is the carrier life time extracted from the current decay curve (2 = 1.10 s) and tL is the carrier transit time. The carrier transit time can be calculated by L is the channel length (L = 20 μm for our devices), μ is field effect carrier mobility and Vds is source-drain voltage. The carrier mobility ε is the dielectric constant of SiO2, d is the thickness of SiO2 (d = 300 nm), and W is the channel width. Accordingly, we can determine a photoconductive gain up to approximately 2.4 × 107, which is comparable to other reported atomically thin graphene-MoS2 heterostructure phototransistors34To further evaluate the performance of the device, we calculated the photoconductive gain using 2 device and the mechanism of photocurrent generation, we propose a schematic energy band diagram, as illustrated in 2 and GQDs are semiconductors with sizable bandgaps, it is important to establish the relative energy levels to determine the band bending and carrier transfer direction when they interact with each other. According to previous reports1036372 is approximately 3.9 eV, which is larger than that of GQDs (2.9 eV)2 film possesses 2–3 atomic layers, we assume its bandgap is close to that of the monolayer MoS2 (1.8 eV)n-type doped, we propose an energy diagram before the formation of the heterojunction  from the GQDs by MoS2 is the origin of the PL quenching. Furthermore, this process will induce new electron-hole pairs, which in return serve as charge carriers to increase the photocurrent. Moreover, the photoexcited electrons in the valence band of the GQDs may be injected into MoS2 through a tunnelling process (process III), which will also contribute extra electrons to increase the photocurrent. The time-resolved PL decay transients show that the decay time of MoS2-GQDs is decreased to 85 ps, which is much shorter than the decay times of MoS2 (224 ps) and GQDs (337 ps)  and is stimulated by an endogenous electrical field. Consequently, the photoresponse is faster in MoS2-GQDs photodetectors, as evidenced in The light-matter interaction in the MoS(337 ps) . This in2 and GQDs have been demonstrated, which have a photoresponsivity of 1.6 × 104 AW−1 and a photogain of 2.4 × 107. The incorporation of GQDs into MoS2 forms an n-n type heterostucture with significantly improved carrier mobility on account of the injection of electrons from the GQDs to MoS2. The improved photodetection performance is attributed to enhanced light-matter interactions resulting from the tunnelling of photoexcited carriers from the GQDs to MoS2, the re-absorption of emitted photons from the GQDs by MoS2 and the transfer of holes from the GQDs to MoS2. Our work demonstrates an effective and applicable way to optimise the photodetection performance of MoS2 and other analogous two-dimensional TMD materials.In summary, highly responsive photodetectors based on the hybrid of MoS2 films were grown directly on a SiO2/Si substrate in a 2-inch quartz tube. High purity MoO3 powder  and sulphur  were used as the precursors. The sulphur powder was placed outside the hot zone and mildly sublimated by heating belts at 140 °C. The distance between the sulphur and MoO3 was approximately 25 cm. The SiO2/Si substrates were placed downstream along the MoO3 source. Argon (99.999%) was used as the carrier gas to convey MoO3–x onto the SiO2/Si substrates. The tube was flushed with argon to maintain normal pressure. The growth conditions were 700 °C for 30 min with an argon gas flow rate of 50 standard-state cubic centimetres per minute (sccm). The GQDs were synthesised following a previously reported methodologyThe MoS2 films were wet-transferred onto a quartz substrate by etching away the SiO2 in a potassium hydroxide solution for subsequent UV-visible spectral measurements on a UV-visible-infrared spectrometer . Raman and PL spectra were measured on a micro-Raman system  with a 514 nm excitation laser. The Raman and PL images were obtained with a confocal micro-Raman system  with a 532 nm laser. The spatial resolution is approximately 250 nm with a focused laser by a 100× objective lens. The time-resolved PL decay transients were measured with a transient state fluorescence spectrometer . Chemical composition analysis was performed using XPS . The surface morphology of the samples was examined by SEM  and AFM . The microstructures of MoS2 and GQDs were investigated using HRTEM . For the preparation of the TEM samples, the MoS2 film was also wet-transferred onto TEM grids.The MoS2/Si substrates after material growth. The SiO2/Si substrate consists of an oxidisation layer (thickness: 300 nm) and highly doped n-type silicon, which serves as the back gate electrode. A typical device fabrication process involves UV lithography to define the device pattern and electron-beam evaporation to deposit the source and drain electrodes . The GQDs solution was drop-casted onto the MoS2 phototransistor device followed by a gentle heat treatment at 70 °C for 15 min.The phototransistors were fabricated directly on SiO2). The excitation light power mentioned in this work is referred to the actual light power shinning onto the device.Photoelectrical measurements were performed on a probe station (Cascade M150) equipped with a semiconductor property analyser (Keithley 2400) at room temperature in ambient conditions. A tapered optical fibre connected with laser diodes  was used to capture the illumination of the photodetector devices. The area size of the laser illumination is much larger than the detection area of the devices .


Preeclampsia (PE), a hypertensive disorder of pregnancy, is hypothesized to be associated with, if not mechanistically related to abnormal placental function. However, the exact mechanisms regulating the pathogenesis of PE remain unclear. While many studies have investigated changes in gene expression in the PE placenta, the role of epigenetics in PE associated placental dysfunction remains unclear. Using the genome-wide Illumina Infinium Methylation 450 BeadChip array, we analyzed gene-specific alterations in DNA methylation in placental biopsies collected from normal pregnant women delivering at term (n = 14), with term PE  or with preterm PE . Of the 485,582 gene loci on the array, compared to controls, 229 loci were differentially methylated in PE placentas and 3411 loci were differentially methylated in preterm PE . Functional annotation of the differentially methylated genes in preterm PE placentas revealed a 32 gene cluster in the cadherin and cell adhesion functional groups (Benjamini p<0.00001). Hypermethylation of CDH11 (p = 0.0143), COL5A1 (p = 0.0127) and TNF (p = 0.0098) and hypomethylation of NCAM1 (p = 0.0158) was associated with altered mRNA expression in preterm PE placentas. Demethylation of first trimester extravillous trophoblast cells resulted in altered CDH11 (p = 0.0087), COL5A1 (p = 0.0043), NCAM1 (p = 0.0260) and TNF (p = 0.0022) mRNA expression. These studies demonstrate aberrant methylation, correlating with disease severity, in PE placentas. Furthermore, we provide evidence that disruption of gene-specific methylation in preterm PE placentas and first trimester trophoblasts is significantly associated with altered gene expression demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE. Preeclampsia (PE), a hypertensive disorder of pregnancy, is one of the leading causes of maternal and fetal morbidity and mortality worldwide. Affecting 5–10% of pregnancies In an attempt to help clarify the molecular mechanisms regulating PE associated placental dysfunction, many studies have investigated alterations in gene function and expression within the placenta using large scale microarray-based gene expression profiling Recently, studies have focused on the contribution of placental epigenetic modifications to the development of PE. Epigenetics is defined as both heritable and transient changes in gene expression that do not entail a change in the primary DNA sequence SERPINA3MMP-9STOX1Syncytin-1CYP24A1) can affect placental function, specifically, regulating the level of active vitamin D at the feto-maternal interface The field of epigenetics is rapidly evolving as an increasing number of complex diseases have recently been shown to be associated with alterations in DNA methylation including diverse types of cancer In this study, we hypothesized that preeclamptic placentas have an altered DNA methylation status resulting in abnormal placental gene expression that may contribute to the development of PE. To test this hypothesis, we identified gene specific changes in DNA methylation in preeclamptic placentas compared to controls. We then assessed if those genes with an altered methylation status resulted in a change in placental gene expression. Further, to determine if the methylation associated gene expression changes seen in third trimester PE placentas were also present in the first trimester, we investigated the expression of these genes before and after demethylation in primary extravillous trophoblast cells.This study was performed with approval from the University of Pennsylvania Institutional Review Board. Written informed consent was obtained from all study participants prior to sample collection.A prospective case-control study (Preeclampsia: Mechanisms and Consequences) was performed between March 2005 and October 2009 at the Hospital of the University of Pennsylvania. Controls were defined as women without hypertension-related complications that presented for delivery at term  (n = 14). Cases (n = 31) were identified according to standard American College of Obstetricians and Gynecologists (ACOG) criteria for diagnosing preeclampsia. Based on these pre-specified criteria, case eligibility was determined at the time of enrollment by the study investigators and not by the treating physician. Immediately after delivery, placental biopsies were taken from cases and controls. After removal of fetal membranes and remaining decidual tissue, four biopsies (one from each “quadrant” of the placenta) were taken from the fetal side of the placenta by a trained research coordinator. The tissues were washed in sterile saline to remove maternal blood and immediately frozen in liquid nitrogen and stored at −80°C until use.Approximately 25 mg of placental tissue was incubated with proteinase K (Qiagen) and RNase A (Qiagen) at 56°C overnight. Genomic DNA was then extracted from the lysed placental tissue following the manufacture's spin column protocol included with the DNeasy extraction kit (Qiagen).Genomic DNA (250 ng) underwent sodium bisulfite conversion according to the manufacture's protocol using the EZ DNA Methylation Kit . The resulting bisulfite converted DNA was hybridized to the Illumina Infinium Human Methylation 450 BeadChip  (referred to as “methylation 450 array” for the remainder of the manuscript) which provides genome wide coverage containing greater than 450,000 methylation sites per sample. Gene probes are targeted across gene regions with sites in the promoter region, 5′UTR, first exon, gene body, and 3′UTR including CpG islands, island shores, and island shelves (sites flanking island shores), CpG sites outside of CpG islands and miRNA promoter regions among others. Amplification, hybridization, washing, labeling and scanning of the Illumina 450 BeadChip was performed by the Molecular Profiling Core Facility at the University of Pennsylvania following the manufacture's protocols. Methylation levels are quantified by beta (β) values derived from the ratio of intensities between methylated and unmethylated alleles. Average β values reported from each gene probe range from 0 (unmethylated) to 1 (completely methylated). Partek Genomics Suite version 6.5  was used for data analysis of the β values including average methylation, fold change, percent methylation and the generation of a “step-up” adjusted p-value which corrects for any false negative results. Principle component analysis (PCA) plots were created based on the methylation 450 array results. The full methylation 450 array dataset was submitted to Gene Expression Omnibus (accession # GSE57767).http://david.abcc.ncifcrf.gov) Probe specific methylation differences between placental tissues from cases and controls were analyzed. Any probe with a step-up adjusted p-value greater than 0.50 was excluded from further analysis. In order to identify those genes that were most likely to show a significant difference in biological function, methylation differences were defined as specific gene probes with a change in β values (Δβ) between cases and controls greater than 0.05 (5% methylation difference) in addition to a step-up adjusted p-value less than 0.05. In the comparisons between preterm preeclamptic placentas versus controls, the cut off criteria were further tightened to Δβ values greater than 0.05 and a step-up adjusted p-value less than 0.01 in order to decrease the number of significantly different gene probes between these groups. Gene probes meeting the cut off criteria in each comparison were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resource version 6.7 (CDH11), collagen type V, alpha 1 (COL5A1), neural cell adhesion 1 (NCAM1) and tumor necrosis factor (TNF) by designing primers (pyromark assay design software) around the gene sequence associated with the specific array probes of interest for each gene . Placental genomic DNA (600 ng) from control (n = 14), term (n = 19) and preterm (n = 12) PE placentas was bisulfite treated using the EpiTect Bisulfite Kit (Qiagen) following the manufacturer's protocol. Utilizing the PyroMark PCR kit (Qiagen), 50 ng of bisulfite treated DNA was used for PCR according to the manufacturer's protocol. Forward and reverse primer sequences for each gene are listed in Pyrosequencing was performed to confirm the methylation status of four genes of interest identified by the methylation 450 array and downstream bioinformatics analysis. We analyzed the methylation status of cadherin 11  versus PE (n = 31) placental genomic DNA was measured using pyrosequencing. The LUMA protocol has been described in detail previously Primary extravillous trophoblast cells (EVT) were isolated from first trimester villous tissue using a modified protocol originally described by Graham et al. 6 cells/ml in 10 cm plates and allowed to grow overnight. EVTs were treated with the demethylating agent 5-aza-2′-deoxycytidine  (5 umol/L) (n = 6) or dimethyl sulfoxide (DMSO) vehicle control (n = 6) for 4 days. The media was aspirated and Trizol  was added to each well, transferred to tubes and stored at −80°C for RNA extraction.EVTs were plated at 1×10CDH11, COL5A1, NCAM1 and TNF along with TaqMan Universal PCR Master Mix were purchased from Applied Biosystems . QPCR reactions were carried out using equivalent dilutions of each cDNA sample on the Applied Biosystems Model 7900 sequence detector PCR machine. Target mRNA was normalized to the amount of 18S RNA in each sample. The relative abundance of the target of interest was divided by the relative abundance of 18S in each sample to generate a standardized abundance for the target transcript of interest.Total RNA was extracted from placenta tissues (100 mg) and EVTs treated with or without AZA using Trizol (Invitrogen) and cDNA was generated using a high capacity cDNA reverse transcription kit . TaqMan Gene Expression Assays for For all experiments, excluding results from the methylation 450 array which were analyzed using methods described above, statistical analyses were performed with GraphPad Prism Software . For data that were normally distributed, t-tests or one way analysis of variance (ANOVA) were used. If the data were not normally distributed then the non-parametric Mann-Whitney test was used. Results with p <0.05 were considered statistically significant.Using the results from the methylation 450 array, we compared the β values between control, term and preterm PE placentas. Because there are no set standards for analyzing large datasets from methylation arrays, we investigated combinations of Δβ values and significant p-value cutoffs in order to identify a subset of genes for further studies . OverallAfter eliminating gene probes with Step-up values of greater than 0.05 and focusing on those gene probes with Δβ values greater than 0.05 (5% change in methylation) , we foun−5 to 1.7×10−12)  . Similar7×10−12) . 32 of t7×10−12) . From th1 or TNF .CDH11, COL5A1, NCAM1 and TNF) identified by the methylation 450 array to be significantly different between control placentas and preterm PE placentas  between control and preterm PE placentas. In agreement with the methylation 450 array, CDH11 (p = 0.037) and TNF (p = 0.030) methylation were significantly increased while NCAM1 (p = 0.001) methylation was significantly decreased. While showing a similar trend in an increase in methylation between control and preterm PE placentas, percent change in methylation of COL5A1 did not reach statistical significance (p = 0.129) as was shown in the methylation 450 array. Interestingly, although the methylation 450 array found no significant alterations in methylation in these four genes in the term PE placentas when compared to control (CDH11 (p = 0.002) and NCAM1 (p = 0.002). COL5A1 (p = 0.144) and TNF (p = 0.190) methylation remained not significant . In term PE placentas, no significant change in CDH11 methylation status was seen with the stringent statistical requirements of the methylation 450 array (p = 0.455744), however, pyrosequencing did suggest an alteration in methylation (p = 0.002) (CDH11 mRNA expression was significantly increased in preterm PE placentas (p = 0.0143) but not term PE placentas (p = 0.4393) when compared to control (= 0.002) , Table 3 control  suggestiCOL5A1: COL5A1 showed a Δβ fold change of 1.223 correlating to a 13.7% increase in methylation (p = 0.005201) in preterm PE placentas. In term PE placentas, no significant changes in COL5A1 methylation  were seen when compared to controls  but not term PE placentas (p = 0.8922) when compared to control placentas . In term PE placentas, no significant changes in NCAM1 methylation were found with the methylation 450 array (p = 0.905300), however, pyrosequencing suggested a significant change in methylation status (p = 0.002) (NCAM1 mRNA was significantly decreased in both preterm (p = 0.0158) and term (p = 0.0042) PE placentas when compared to control (= 0.002) , Table 3 control .TNF: In preterm PE placentas, TNF showed a Δβ fold change of 1.339 correlating to a 7.8% increase in methylation (p = 0.005227). In term PE placentas, TNF methylation status was not significantly altered  (TNF mRNA was significantly decreased in preterm PE (p = 0.0098) but not term PE (p = 0.5845) placentas when compared to control (uencing) , Table 3 control .CDH11, COL5A1, NCAM1 and TNF in third trimester preeclamptic placentas, we wanted to investigate if changes in methylation of these specific genes would result in an alteration of mRNA expression in first trimester extravillous trophoblast cells - the cell type whose primary function is invasion of the uterine wall and maternal spiral arteries. First trimester EVT cells treated with the demethylating agent, AZA, resulted in a significant decrease in CDH11 (p = 0.0087) (COL5A1 (p = 0.0043) (NCAM1 mRNA expression (p = 0.0260) (TNF mRNA expression (p = 0.0022) (Because we identified significant changes in methylation within the cell adhesion genes  0.0087) , COL5A1  0.0043)  and NCAM 0.0260) . On the  0.0022) .In order to determine if there were alterations in global methylation status between control and PE placentas, we performed the LUMA assay . There wGreat strides have been made in understanding the complex genetic contributions to the development of PE; however, investigations into the role of placental epigenetic modifications and their association with placental dysfunction in PE remain in their infancy. While it is generally agreed that alterations in gene methylation lead to changes in gene expression and thus functional alterations within the placenta, a direct link between altered gene-specific DNA methylation and PE-associated placental dysfunction remains to be fully elucidated. In this study, utilizing a large scale genome wide DNA methylation array, we have shown that 1) significant changes in gene methylation status are present in PE placentas when compared to those with uncomplicated term pregnancies, 2) preterm PE placentas have an increased number of differentially methylated genes when compared to control and term PE placentas, 3) those genes with an altered methylation status in preterm PE placentas cluster most significantly in the cell adhesion and cadherin functional groups, 4) altered methylation status of specific cell adhesion genes results in a change in placental mRNA expression, and 5) disruption of methylation in first trimester trophoblasts alters expression of these same genes. These studies demonstrate that differential methylation results in changes in placental mRNA expression suggesting that epigenetic regulation of these genes contributes to placental dysfunction and/or disease development. Furthermore, our results indicate that epigenetic modifications early in pregnancy could have effects on trophoblast function contributing to PE.Identifying alterations in placental DNA methylation has garnered more interest in the past several years as many believe that epigenetic alterations may play a significant role in the development of adverse obstetrical outcomes, including IUGR and PE. While several studies have sought to investigate changes in DNA methylation in PE placentas, many have focused on global DNA methylation changes We found a significantly higher number of genes with an altered methylation status in preterm PE placentas as compared to term PE placentas (vs control). Interestingly, in the term PE placentas, methylation status (as shown by array or pyrosequencing) was variable in terms of similarity to controls or preterm PE placentas depending on the specific gene investigated. Notably, women being delivered preterm for a diagnosis of PE are by definition going to have a more severe form of this disease. This is in contrast to women with a diagnosis of PE at term as there is significant variability in the clinical presentation of women with term PE. Hence, it is likely that methylation status of the placenta would vary by disease severity even at term. Understanding that there is more phenotypic variability with PE diagnosed at term than preterm, our findings of more significant methylation changes in preterm PE make biological sense. The different epigenetic profiles shown in preterm PE and term PE placentas suggests that epigenetic modification of placental genes may contribute to the varying disease phenotype of PE. Importantly, altered gene methylation may be mechanistically involved in the pathogenesis of more severe forms (and preterm) of PE. Our findings would suggest that the placental epigenetic modifications observed in our study could occur early in pregnancy contributing to the placental dysfunction that ultimately leads to the development of PE.A potential limitation of our study is that we cannot ascertain if placental methylation status is altered due to gestational age. While some previous studies have used aged matched spontaneous preterm birth placentas as controls CDH11 had the largest alteration in DNA methylation with an 18.3% increase in preterm PE placentas. The CDH11 hypermethylation identified by the methylation 450 array was further validated by pyrosequencing. It is interesting to note that while CDH11 methylation levels were not significantly altered in term PE placentas in the methylation 450 array; pyrosequencing did show a significant increase in methylation similar to the preterm PE samples. CDH11 has been shown to play a role in anchoring trophoblasts to the decidua CDH11 contributes to the regulation of trophoblast movement. Highlighting the complexity of epigenetic regulation, in our study, hypermethylation of CDH11 resulted in an increase in CDH11 mRNA expression in preterm PE placentas but not in term PE placentas suggesting that the level of methylation may play a role in the downstream regulation of biological function. Interestingly, in agreement with our results, the effects of TGF-β1, a growth factor known to decrease trophoblast invasiveness, are mediated through CDH11CDH11 is reduced in choriocarcinoma CDH11 in the regulation of trophoblast proliferation. Furthermore, the observed decrease in CDH11 expression in demethylated first trimester EVT cells suggest that altered methylation of CDH11 early in pregnancy has the ability to alter trophoblast function. Therefore, we hypothesize that hypermethylation of CDH11 resulting in increased CDH11 mRNA expression could contribute to the decreased trophoblast invasiveness associated with PE.Interestingly, in our study, the genes with the largest changes in DNA methylation in preterm PE placentas clustered into the cadherin and cell adhesion functional groups. The placental genes regulating cell adhesion could significantly contribute to alterations in the migration and invasion of the placental trophoblast cells, a process known to be abnormal in PE placentas COL5A1, an extracellular matrix (ECM) protein known to be involved in connective tissue disorders, has been identified in placental stromal cells and has been shown to be increased in PE placentas COL5A1 in preterm PE placentas resulted in increased mRNA expression. Of note, COL5A1 hypermethylation was seen in both the methylation 450 array and the pyrosequencing assay, however, the increase in methylation between control and preterm PE placentas did not reach statistical significance in the pyrosequencing assay. While there is some assumed variability between the two techniques, the overall trend for increased methylation of COL5A1 and the resulting change in mRNA expression would suggest that this is a mechanistically relevant gene and should not prevent further investigation into the role of COL5A1 in placental function. However, it is also interesting to note that COL5A1 methylation and mRNA expression was unchanged in term PE placentas. These results, taken together with our data showing decreased COL5A1 expression in demethylated first trimester EVT cells, suggests that increased expression of COL5A1 due to hypermethylation may be associated with alterations in first trimester trophoblast function and, consequently, contributing to disease severity. Although the exact function of COL5A1 in the PE placenta remains unknown, interactions between the ECM and integrins are known to regulate trophoblast migration COL5A1 expression due to alterations in gene methylation early in pregnancy may hinder trophoblast motility.NCAM1 has been shown to play a role in trophoblast-trophoblast interactions and trophoblast cell adhesion during maternal artery migration NCAM1 is decreased in the extravillous trophoblast cells of preeclamptic placentas NCAM1 was the only gene demonstrating a decrease in DNA methylation  between controls and preterm PE placentas. In term PE placentas, while NCAM1 methylation was unchanged in the methylation 450 array, pyrosequencing showed a significant decrease in methylation that was similar to preterm PE samples. Decreased methylation of NCAM1 resulted in a decrease in mRNA expression in both preterm and term PE placentas suggesting that alterations in methylation result in a change in NCAM1 gene expression possibly leading to alterations in downstream placental function. Additionally, demethylation of first trimester EVTs resulted in decreased NCAM1 expression indicating that NCAM1 may play a specific role in regulating first trimester trophoblast invasion of the maternal spiral arteries and hence may be contributing to the development of preeclampsia from an early time point in pregnancy.TNF has been well studied in pregnancy and PE and has been associated with a myriad of bioactivities including immune system activation, cell survival, proliferation, migration and differentiation TNF has been shown to inhibit trophoblast migration and invasion TNF has also been shown to stimulate matrix metalloproteinase-9 (MMP-9) TNF in regulating placental/trophoblast function remains unclear. While there are conflicting reports of altered TNF expression in PE placentas TNF resulted in a decrease in TNF mRNA expression in preterm PE placentas. Interestingly, there were no changes in TNF methylation (array or pyrosequencing) or mRNA expression in term PE placentas providing evidence that epigenetic regulation of TNF may be associated with disease severity and may be mechanistically involved in early trophoblast dysfunction leading to a more severe phenotype. Due to the many biological functions attributed to TNF, it is difficult to pinpoint the exact effect of decreased TNF expression on placental function. However, demethylation resulted in increased TNF expression in first trimester EVTs indicating that alteration of TNF methylation status early in pregnancy could alter trophoblast function.Lastly, CDH11, COL5A1, NCAM1 and TNF are an end effect of the disease or are causative of the disease, our studies do suggest that these genes are mechanistically involved the development of PE. We acknowledge that additional studies would be needed in order to investigate the effects of these genes (increased or decreased gene expression) on first trimester trophoblast function including EVT invasion and migration.While this study cannot determine if differential methylation of TNF resulted in decreased gene expression. However, in instances where the gene probe was located in promoter-distal sites (TSS1500 (1.5Kb from TSS) or gene body as designated by Illumina), hypermethylation of CDH11 and COL5A1 resulted in an increase in mRNA expression. Furthermore, hypomethylation of NCAM1 resulted in decreased gene expression when the gene probe was located in the 3′UTR region of the gene. Previous studies have found several examples in both human and mouse where methylation in the gene body is positively associated with gene expression including Igf2rAPOEMyod1IGF2BCR-ABLp16 Ex2CDH11 and COL5A1 may activate the binding of TFs to gene specific enhancers resulting in increased gene expression. On the other hand, NCAM1 hypomethylation may target TFs to gene specific repressors or block the binding of TFs to enhancers via insulators In the cancer field, it is generally accepted that DNA hypermethylation represses gene transcription through the promoter region of tumor suppressor genes CDH11, COL5A1, NCAM1 and TNF gene expression indicates that alterations in methylation could contribute to functional changes in the placenta. As gene expression in term placentas may not reflect what is occurring during early pregnancy, we investigated the effects of demethylation of first trimester EVTs on gene expression. These studies provide evidence that disruption of methylation in first trimester trophoblasts alters expression of these genes demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE. In addition to increasing our understanding of the biological mechanisms contributing to the development of PE, our studies suggest that epigenetic alterations may be targets for future therapeutic interventions in those women with PE.In summary, our results of an altered methylation status in PE placentas suggest that placental epigenetic modifications are associated with the development of PE. Furthermore, the increase in the number of genes methylated in preterm PE placentas points to a correlation between epigenetic regulation and disease severity. Methylation associated changes in Figure S1Global methylation levels in control and preeclamptic placentas.(TIF)Click here for additional data file.Table S1Pyrosequencing primers.(DOCX)Click here for additional data file.Table S2Number of differentially methylated probes at specific Δβ and p-value cutoffs in control versus preterm preeclamptic placental samples.(DOCX)Click here for additional data file.Table S3Functional annotation clusters containing differentially methylated genes in control versus preterm preeclamptic placentas.(DOCX)Click here for additional data file.Table S4Gene Ontology (GO) Terms for differentially methylated genes in control versus preterm preeclamptic placentas.(DOCX)Click here for additional data file.Table S5Full list of genes associated with the Cell Adhesion Gene Ontology functional annotation cluster which showed differential methylation between control and preterm preeclamptic placentas.(DOCX)Click here for additional data file.