Acanthamoeba sp. The goal of this study is to know the potential of cellulase in the degradation of cyst wall Acanthamoeba sp. obtained from isolate collection of Department of Parasitology FKUI of which two samples come from patient and one sample is from environment. All three samples were cultured using non-nutrient agar (NNA) media and identified by PCR and sequencing. The concentration of cellulase concentration used was 50 U, 100 U, 150 U, 200 U, 250 U, and 300 U with the incubation time used being 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours. Furthermore, treatment results with the most optimum concentration and incubation time were observed by using SEM to see changes in the surface of the walls of the cyst. A cysticidal test was performed to determine the effectiveness cysticidal action of disinfectant solution, cellulase, and the combination of disinfectant solution and cellulase in killing Acanthamoeba sp. cyst assessed by their viability value. Sample of Acanthamoeba sp. cysts was 300 U with an incubation time of 24 hours. Percentage of viability of Acanthamoeba sp. which was exposed to a disinfectant solution for 24 hours was 95%, cellulase alone for 24 hours 75%, and the combination of cellulase and disinfectant solution for 24 hours 25%. The most optimal cellulase concentration in killing Acanthamoeba sp. cyst wall. Optimal cellulase concentration in degrading Acanthamoeba sp. cyst wall is 300 U with an optimal incubation time being 24 hours. The addition of cellulase to the disinfectant solution has the potential to increase the effectiveness of the disinfectant solution because cellulase is capable of degrading the cyst wall allowing the disinfectant solution to enter and kill Acanthamoeba sp. cysts. Cellulase is capable of degrading Acanthamoeba sp. is one of free-living amoeba (FLA), which is an opportunistic pathogen widely spread in the environment [ Acanthamoeba sp. genus can be divided into 20 genotypes, T1-T20 [ Acanthamoeba T4 genotypes are the main cause of Acanthamoeba keratitis (AK), a severe progressive corneal inflammation which can lead to blindness [ironment . Accordilindness .Acanthamoeba keratitis is often associated with contact lenses misuse [s misuse . Accordis misuse . Each yes misuse . Contacts misuse . Acanthamoeba sp. cysts [ Acanthamoeba sp. [ Acanthamoeba sp. cysts [Studies were performed to evaluate the effectiveness of contact lens disinfectant solution on eliminating contaminant from bacteria or parasites. Siddiqui R et al. (2014) have shown that none out of 9 disinfectant solutions tested was effective in killingp. cysts . Similarp. cysts .Acanthamoeba sp. cyst wall consists of ectocyst  and endocyst (fibrillar layer) [β1⟶4 glycosidic bonds. Cellulase is an enzyme that can damage this glycosidic bond, which can be the target in eliminating Acanthamoeba sp. cyst [r layer) . Ectocyssp. cyst . Acanthamoeba cyst wall [Cellulose degradation is a potential therapy in targeting trophozoite wrapped by cyst wall effectively. Thus cellulase can damage theyst wall . The mor Acanthamoeba keratitis (AK) and 1 sample from the environmental water in Department of Parasitology, Faculty of Medicine, University of Indonesia.Three samples used in this study were obtained from 2 patientsAcanthamoeba sp. from Department of Parasitology collection were regrown in pages-salt agar media and stored at 30°C for 14 days to form a cyst. Total cysts formed were counted with hemocytometer [ Acanthamoeba sp. DNA extraction was performed with Qiagen kit, followed by Polymerase Chain Reaction (PCR) targeted to 18s rRNA with primers JDP1 (5'GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'TCTCACAAGCTGCTAGGGGAGTCA-3') to yield a 423-551 bp specific amplicon [ytometer . Acantha Aspergillus niger. One unit (U) will liberate 1 μm of glucose from cellulose in one hour at pH 5. Cellulase enzyme preparation was carried out by dissolving the cellulase enzyme in acetate buffer with a pH of 4.5-5.5.The cellulase used is commercial cellulase obtained from MP Biomedicals  and originating fromμL of the sample with 5x103 of total cysts in each tube. To each tube, cellulase was added with serial concentration as follows: 50U, 100U, 150U, 200U, 250U, and 300U, and then incubated in room temperature for 24h. Trypan blue 0.1% was added to the cysts for quantification with a hemocytometer.Six tubes were filled with 100μL of a sample containing 5x103 of total cysts in each tube. Cellulase was added to each tube with the optimum concentration, followed by incubation for 2h, 4h, 6h, and 24h, respectively, to each tube. Remaining cysts were counted with hemocytometer Trypan blue in 0.1%.Five tubes were prepared with 100 Acanthamoeba sp. into 2 tubes; one contains 300 U of cellulase. Samples were incubated for 24h, followed by morphological examination by SEM. To observe the sample with SEM, the sample was fixed with glutaraldehyde 2% in 0.1 M cacodylate buffer with pH between 6.8 and 7.4. The sample was centrifuged at 500 g for 2 minutes. Then the pellets were fixed again with 2% osmium tetroxide, dehydrated with ethanol and propylene oxide, filtered with a millipore filter (diameter 22 mm), dried for 24 hours, then stained with contrast uranyl acetate and citrate, and then observed under SEM.In this study, Scanning Electron Microscope used was JSM-6510LA. Morphological examination is done by dividing Acanthamoeba sp. cyst. Three tubes were prepared with 100μL of a sample containing 5x103 of total cysts. 100μL of cellulase 300U was added to the first and third tube, while 100μL of disinfectant solution was added to the second and third tube. Each tube was then incubated in room temperature for 24h. Surviving cysts were counted with hemocytometer in 0.1% Trypan blue. Acridine orange staining was also performed to evaluate the different cyst wall morphology among treatments.Effectivity assay was assessed to compare the cysticidal activity of cellulase, disinfectant solution, and a mixture of both in discarding Acanthamoeba castellani T4 genotype, and sample from biosphere was identified as Acanthamoeba lenticulata T5 genotype. According to cellulase optimization step, Acanthamoeba castellani T4 was found to be the most pathogenic genotype. Optimal cellulase concentration to discard Acanthamoeba sp. was 300U with 24h of incubation time  (μm) with imperfect round shape (μm) and irregular cyst shape  . Celluland shape . Meanwhist shape .Acanthamoeba keratitis (AK) is a corneal infection caused by a microscopical organism, a Free-Living Amoeba (FLA) known as Acanthamoeba, and normally found in the waterbody  as well as tap water, swimming pool, tub, soil, and air. Acanthamoeba has 20 genotypes identified so far which are T1-T20 [e T1-T20 . T4 genoe T1-T20 . Acanthamoeba sp. sequence published in GeneBank to calculate the percentage of similarity through NCBI BLAST.In this study, we identified 2 samples from AK patients and 1 sample from the environment. All DNA samples were amplified with PCR technique, yielded a ~450 bp amplicon length, followed by gene sequencing to reveal its genotype. Sequencing result from B, W, and S samples showed 432bp, 433bp, and 424bp amplicon length, respectively. These sequences were compared to Acanthamoeba castellani strain T4, and S sample obtained from the environment has 100% sequence similarity to Acanthamoeba lenticulata strain T5. This result is similar to the previous study which stated that Acanthamoeba T4 was the main cause of AK [ Acanthamoeba lenticulate T5 was incapable of causing AK. Spanakos G et al. in 2006 discovered the AK cases with Acanthamoeba lenticulata T5 as the main cause of this disease [B and W samples which came from AK patients have 100% sequence similarity tose of AK . Convers disease . Acanthamoeba, especially in cyst stadium. Acanthamoeba cyst has 2 layers of cyst walls which is difficult for the disinfectant solution to penetrate [ Acanthamoeba cyst wall. Cellulase addition has a high chance in degrading Acanthamoeba cyst wall and hence will allow the disinfectant solution to penetrate to the cyst [AK disease is often related to the contact lens utilization. Incorrect usage and upkeep become the main risk factor of AK . Previouenetrate . Cellulothe cyst . Acanthamoeba cyst wall was gained by optimization. The sample used in this step was B sample, which was the Acanthamoeba T4 genotype, due to its pathogenicity, and the most common cause of AK. B sample was chosen over W sample because B sample contains more cysts than W sample. Total cyst used for cellulase concentration and incubation time optimization was 5 x 103.In this research, optimum cellulase concentration to degrade3 of total cyst in 100 μL. Results of concentration optimization are as follows: 0,75 x 103, 1 x 103, 1 x 103, 1,5 x 103, 1,5 x 103, and 1,75 x 103 cysts were eliminated in 50U, 100U, 150U, 200U, 250U, and 300U, respectively. Other than cellulase concentration, the incubation time was also determined in this research. Series of incubation time as follows: 2h, 4h, 6h, 8h, and 24h, was performed to discover the optimum incubation time. 24h was found to be the optimum incubation time as it eliminated 1,75 x 103 of the cysts. These combined results showed that cellulase alone (300U) for 24h was still ineffective in banishing Acanthamoeba cyst. Only 1,75 x 103 cysts were eliminated, out of 5 x 103 of the total cysts.Series of concentration tested for optimization was 50U to 300U, incubated for 24h, with 5 x 10 Acanthamoeba morphological change after cellulase treatment. Untreated and 24h cellulase 300U-treated Acanthamoeba were noted and compared. SEM result revealed that untreated Acanthamoeba has granulated surface while cellulase-treated Acanthamoeba has smooth, degranulated, and impaired surface.Next step was to observe the Acanthamoeba cyst until 25% of viability.A further step was to determine the cysticidal capability of cellulase and disinfectant solution. Samples were incubated with cellulase, disinfectant solution, and a mixture of both for 24h. Results showed that 95%, 75%, and 25% of cyst were viable after incubation with disinfectant solution, cellulase, and a mixture of both, respectively. It was clear that a mixture of cellulase and disinfectant solution is more effective in eliminatingμm size and a round shape. Cellulase 300U for 24h treatment did reduce cyst size to 12μm and yield to irregular cyst shape, while a mixture of cellulase 300U and disinfectant solution treatment for 24h caused the cyst size reduction to 8-11μm and irregular cyst shape.Cyst morphology after cellulase; disinfectant solution; and a mixture of both cellulase and disinfectant solution-treatment was observed. Disinfectant solution treatment for 24h showed that the cyst morphology was similar to normal cyst: 10-14Acanthamoeba cyst was more susceptible to antiamoeba compared to trophozoite, due to a double layer of cyst wall in Acanthamoeba, mainly consisting of cellulose [ Acanthamoeba encystation. In other words, cellulase may rise as a useful additional treatment in eliminating Acanthamoeba [ellulose . Hence, thamoeba . Acanthamoeba encystation [ Acanthamoeba stored glycogen as the building block of cellulose synthesis. Previous studies have shown that glycogen was drained along the encystation process, resulting in interference of glycogen phosphorylase mRNA expression and eventually blocking encystation process [Cellulose was synthesized in certain organisms, including vascular plant, major algae, fungi, and certain bacterial species. Mammal, on the other hand, does not synthesize cellulose, thus bringing cellulase as a suitable antimicrobial candidate. Cellulose 2,6-bichlorobenzonitrile synthesis inhibitor was mentioned to be able to preventystation . Targeti process . Acanthamoeba, as disinfectant solution alone could not penetrate the double cyst wall, and cellulase alone is not capable of inhibiting encystation but still depends on amoebicidal activity to banish Acanthamoeba. Cellulase and disinfectant solution together allowed the cellulase to degrade the cyst wall and disinfectant solution to target the trophozoite cell membrane. Overall, Acanthamoeba trophozoite is extremely sensitive to the disinfectant solution, as well as a mixture of cellulose and disinfectant, which has proven to eliminate both trophozoite and cysts. Although we still have to discover the toxicity effect of this new formulation on the host; however, this finding showed that the addition of cyst wall degrading molecule in disinfectant solution would increase the Acanthamoeba elimination effectivity [Neither disinfectant solution nor cellulase alone was effective in eliminatingectivity . Acanthamoeba sp. cell component after treatment, due to inadequate equipment, where SEM was only able to observe the cell wall surface. Thus, this research was unable to further analyze the potency of cellulase and the effectivity of cellulase addition to the Acanthamoeba sp. disinfectant solution.This research is limited but unable to observe the morphological change on


Prediction of spontaneous preterm birth (sPTB) in asymptomatic women remains a great challenge; accurate and reproducible screening tools are still not available in clinical practice. We aimed to investigate whether the maternal serum metabolome together with clinical factors could be used to identify asymptomatic women at risk of sPTB. We conducted two case-control studies using gas chromatography-mass spectrometry to analyse maternal serum samples collected at 15- and 20-weeks’ gestation from 164 nulliparous women from Cork, and 157 from Auckland. Smoking and vaginal bleeding before 15 weeks were the only significant clinical predictors of sPTB for Auckland and Cork subsets, respectively. Decane, undecane, and dodecane were significantly associated with sPTB (FDR < 0.05) in the Cork subset. An odds ratio of 1.9 was associated with a one standard deviation increase in log (undecane) in a multiple logistic regression which also included vaginal bleeding as a predictor. In summary, elevated serum levels of the alkanes decane, undecane, and dodecane were associated with sPTB in asymptomatic nulliparous women from Cork, but not in the Auckland cohort. The association is not strong enough to be a useful clinical predictor, but suggests that further investigation of the association between oxidative stress processes and sPTB risk is warranted. Although sPTB is prevalent in both high and low/middle-income countries, the major burden of sPTB is concentrated in Asian and African countries where about 85% of preterm births occur5. Short- and long-term consequences of preterm birth include bronchopulmonary dysplasia, neurodevelopment and cognitive impairment, retinopathy, as well as substantial impact on the functional, mental and social health of the infant and its family8. Despite an increase in the research conducted into sPTB and advancements in the implementation of management and prevention strategies, significant reductions of sPTB have not been achieved10.Spontaneous preterm birth (sPTB) due to spontaneous onset of labour or premature rupture of membranes (PROM) is a major cause of neonatal mortality and morbidity14, but their benefits are disputed. According to systematic reviews and meta-analyses, the use of such interventions in selected women can reduce the risk for sPTB, but they are less likely to reduce the risk for perinatal morbidity19. The identification of a more accurate screening tool to predict which women are most at risk of sPTB could improve the selection of women that would benefit from interventions, increasing the likelihood of success and reducing the costs to the healthcare system. However, current screening tools used to identify women at risk of developing sPTB fail to accurately predict preterm birth in asymptomatic women.Natural progesterone , 17α-hydroxyprogesterone (intra-muscular), pessary and/or cervical cerclage have been variously recommended to prevent sPTB in some guidelines12, approximately 2/3 of women with short cervical length will have a term birth21. Chemical biomarkers for sPTB have also been sought, with fetal fibronectin (fFN), insulin-like growth factor-binding protein 1 (IGFPB-1), and interleukin-6 (IL-6)27 among the most studied. A systematic review of sPTB biomarkers found more than 200 studies published between 1965 and 2008, reporting more than 100 potential biomarkers27. However, no single biomarker has proven to be a reliable predictor of sPTB. The authors of the systematic review concluded that there are many heterogeneities between the studies in terms of their experimental study design, timepoint of sample collection, and sample processing methods. One of the largest prospective cohort studies to date evaluated the performance of serial transvaginal cervical length measurements and quantitative vaginal fFN levels for predicting sPTB in a sample of approximately 10,000 nulliparous women with singleton pregnancies28. Despite determination of cervical length and fibronectin levels at three different gestations, the model still showed low predictive accuracy for sPTB.Cervical length is possibly the most employed indicator for sPTB risk in clinical practice. While there is evidence for an increased risk of sPTB in women with a shortened cervix29. For instance, the cervical remodelling process, which involves softening and ripening of the cervix, invariably occurs in the spontaneous onset of labour and is considered its endpoint30. However, it does not occur in the same period of pregnancy for all women, which limits its potential as a marker for the prediction of sPTB31. The underlying complexity of the drivers of sPTB demands a robust analytical technique capable of taking into consideration the multiple pathways potentially affected in the development of sPTB.sPTB is a condition with a multifactorial aetiology, a long pre-clinical phase, and adaptive mechanisms during pregnancy35. Metabolomic measurements provide a biochemical snapshot of the physiological state of the organism36; their close relationship with the biological phenotype means they have the potential to reveal underlying mechanisms of disease. Due to the biochemical complexity of the metabolome, hyphenated analytical techniques are used. This involves subjecting sample extracts to a high-resolution separation technique such as gas chromatography (GC), high-pressure liquid chromatography (HPLC), or capillary electrophoresis (CE). Identification is then carried out using mass spectrometry (MS), or nuclear magnetic resonance (NMR).Metabolomics, the study of low molecular weight compounds in a biological system, has previously been used to successfully investigate pregnancy complications such as fetal growth restriction, preeclampsia, and gestational diabetes mellitus37, in collaboration with the SCOPE (Screening for Pregnancy Endpoints) study. SCOPE was an international multicentre prospective cohort study that collected standardized data and samples from more than 5,690 low-risk pregnant women40. The main objective of SCOPE was to develop a robust database and biobank to identify predictors and develop screening tests for preeclampsia, sPTB and small-for-gestational-age newborns. The current study describes the results of Preterm SAMBA phase 1, which used the SCOPE biobank to investigate the metabolomic profile, at 15- and 20-weeks’ gestation, amongst SCOPE participants who went on to have sPTB, when compared to matched controls. Parallel analyses were performed using participants from the Irish and New Zealand’s centres of the SCOPE study.The Preterm SAMBA study (Preterm Screening and Metabolomics in Brazil and Auckland) has been established to identify and validate metabolomic biomarkers for sPTB41.Two case-control studies were conducted, using a sub-set of women from the Auckland, New Zealand and Cork, Ireland centres of the Screening for Pregnancy Endpoints (SCOPE) study40. Collection of data and biological samples complied with standardized procedures in all participating centres and was conducted in accordance with the principles of the Declaration of Helsinki. Cases of sPTB were defined as those women who delivered before 37 weeks’ gestation, and controls as those who delivered at or after 37 weeks’ gestation.Cork, Ireland.2)*.The subset of SCOPE participants included in this study from the Cork, Ireland centre consisted of 55 cases of sPTB and 102 controls, matched to cases according to maternal age (±3 years) and maternal body mass index  05/02/08, Clinical Research Ethics Committee of the Cork Teaching Hospitals; New Zealand: study number AKX/02/00/364, Northern X Reginal Ethics Committee) and all participants gave their written informed consent. Details on enrolment and inclusion criteria for the SCOPE study have been published elsewhere2)*.The subset of SCOPE participants included in this study from the Auckland, New Zealand centre consisted of 55 cases of sPTB and 109 controls, comprising 56 controls matched to cases according to maternal age (±3 years), and 53 controls matched to cases according to both maternal age (±3 years) and BMI .Demographic information including maternal age and BMI was collected at the time of recruitment (15 ± 1 weeks’ gestation). History of vaginal bleeding and any infections during the pregnancy were also recorded via interview at the initial visit. The infant characteristics were recorded at birth and included birth weight, customised birth weight centile, and whether the infant was small, normal, or large for gestational age.Spontaneous preterm birth was defined as any birth that occurred before 37 weeks due to spontaneous onset of labour or premature rupture of membranes. Gestational age (GA) was estimated by last menstrual period (LMP) and confirmed by an ultrasound dating before 16 weeks of gestation. A discordance of seven or more days between LMP and ultrasound dating or an unsure LMP led to the estimation of GA exclusively by early ultrasound parameters. Term birth for the control group was determined as delivery after 37 weeks of gestation. Early sPTB before 34 weeks was considered for subgroup analysis.42.Maternal blood samples were collected in two 6 mL vacutainers by venepuncture at 15 and 20 weeks (±1 week). Following clot formation, the vacutainers were centrifuged at 3,000 rpm for 10 min at 4 °C, followed by a second centrifugation at 4,000 rpm for 10 min at 4 °C. After centrifugation, the resulting serum (supernatant) was pipetted into a sterile tube and aliquots of 250 µL were dispensed into cryotubes. All samples were stored at −80 °C. The study followed standard best practice procedures for repositories in all steps of sample collection and storage, registering all SPREC  data accordingly in an online databaseFor this study, two 250 µL maternal serum samples were obtained from the biobank for each participant, one from each time point; 15- and 20-weeks’ gestation (±1 week).et al.43. Briefly, samples were thawed on ice at 4 °C and transferred from cryotubes to 1.5 mL microcentrifuge tubes. An internal standard  was added to all samples and the sample-IS mix was vortexed for 1 min. Samples were dried for 4 h at 0.8 HPa in a centrifugal vacuum concentrator with a −104 °C refrigerated vapour trap (Thermo Fisher Scientific Savant SC250EXP SpeedVac Concentrator with Savant SP5121P Refrigerated Vapour trap). Metabolites were extracted using 50% and 80% cold methanol-water solution . Specifically, 500 µL of 50% cold methanol-water solution was added to all samples, followed by vortexing for 1 min and centrifugation at 3,000 rpm for 5 min at −4 °C. After centrifugation, the supernatant was transferred to a fresh chilled microcentrifuge tube (kept on dry ice). Then, 500 µL of 80% methanol-water solution was added to the pellet and this was centrifuged at 3,000 rpm for 5 min at −4 °C. The supernatants obtained from both extraction steps were combined and dried in the centrifugal vacuum concentrator for 4 hours at 0.8 HPa, with a −104 °C refrigerated vapour trap. Dried extracted samples were stored at −80 °C prior to derivatisation.The serum samples underwent extraction and derivatization procedures based on the 2010 protocol of Smart Negative controls were produced by subjecting an empty microcentrifuge tube to the same processing as the samples. Pooled quality control samples (QC) were produced by pooling a small amount from every sample, mixing, and then making aliquots of the same volume as the samples.Derivatization was carried out using methyl chloroformate (MCF). Samples were derivatised in batches of 18–24, on the same day that they were analysed on the GC-MS. Samples were re-suspended with 400 µL of 1 M sodium hydroxide and were transferred to silanised glass tubes, followed by addition of 334 µL of methanol and 68 µL of pyridine. The sample was placed on a vortexer for the remainder of the derivatization process at ~1,500 rpm. The rate limiting step began from the addition of 40 µL of MCF. A second addition of 40 µL of MCF was made, 30 sec later. After another 30 sec, 400 µL of chloroform was added to extract the alkylated derivatives from the reaction mixture. After 10 sec, 400 µL of sodium bicarbonate (50 mM) was added. Centrifugation was used to separate the aqueous layer from the chloroform layer. After centrifugation, the aqueous layer was removed and the remaining chloroform extract was dehydrated by the addition of sodium sulphate (~0.3 g). The remaining liquid was then transferred to an amber glass GC-MS vial with a glass 33 µL insert.et al.43, with modifications. One microliter of sample was injected for analysis. The injector was set to 290 °C in splitless mode. The column flow was maintained at 1.0 mL min-1 in constant flow mode. The column was a fused silica ZB-1701 30 m long, 0.25 mm inside diameters, with a 0.15 µm stationary phase constituting of 86% dimethylpolysiloxane and 14% cyanopropylphenyl (Phenomenex). Instrument grade helium  was used as the carrier gas for the analysis. The detector was run in positive-ion, electron-impact ionisation mode, at 70 eV electron energy. Identification of compounds was carried out using mass spectra acquired in scan mode from 38 to 550 atomic mass units. The Cork samples were analysed on an Agilent 7890 A gas chromatograph coupled to an 5975 C inert mass spectrometer. The Auckland samples were analysed on a Thermo Scientific Trace GC Ultra gas chromatograph coupled to an ISQ mass spectrometer.The GC-MS instrument parameters were based on Smart http://www.amdis.net/)44 from an in-house mass spectral library for MCF-derivatised metabolites (~210 compounds) developed by Silas Villas-Boas. The library contained mass spectra predominantly from certified reference standards. In addition to the in-house library, the National Institute of Standards and Technology (NIST) mass spectral library  was also used to identify peaks in the raw chromatograms. Since AMDIS is not able to batch deconvolute with the entire NIST library, a NIST subset library was constructed employing a method developed by Elizabeth McKenzie using pooled quality controls.Data processing was semi-automated. The raw files obtained from the GC-MS were converted into common data form (cdf) format for analysis and were deconvoluted and identified using the Automated Mass Spectral Deconvolution and Identification System  developed by Ting-Li Han. MassOmics used XCMS and the AMDIS report to integrate the peak areas for each of the identified metabolites. The summary report obtained from running the MassOmics script was then checked, and peaks with low ID hits, or with large retention time shift, and laboratory contaminants were removed.The NIST subset was produced using the top five results for each feature from the Agilent Chemstation PBM (Probability Based Matching) deconvolution program. MassOmics in house software (version 2.3) was used to create the subset library. MassOmics is an R script based on the XCMS R packageData was checked against negative controls to identify and remove background contaminants. Peaks that were not extracted correctly with XCMS were integrated separately using the Ion Extractor feature in MassOmics. Co-eluting peaks underwent manual integration. The relative abundance obtained for each metabolite was normalised to the internal standard (Alanine-d4). After internal standard normalisation, the remaining technical variation was corrected for by analytical batch median centering using the samples. An analytical batch was defined as ~25 injections, comprising ~18 samples, 4 QC’s, one Alkane series, one negative control, and one standard mixture.https://www.R-project.org)46. Data was analysed separately for each site. Clinical predictors were analysed for univariate associations with preterm birth, and a multivariate logistic regression model predicting preterm birth with these predictors was selected using stepwise logistic regression with AIC, starting from an intercept only model, followed by backward elimination of variables with multivariate p > 0.05. Mann-Whitney tests were used to analyse the difference in metabolite levels between cases and controls. Ratios between the 20 and 15-week levels of each metabolite were also assessed in this way, to identify situations where changes from baseline rather than absolute metabolite levels were associated with case status. To adjust for multiple comparison testing, false discovery rates (FDR) were calculated for each comparison using the Benjamini-Hochberg procedure47. Metabolites with an estimated FDR < 0.05 are reported.Statistical analysis of the normalised mass spectral data was performed using R 3.4.3 . The random forest was assessed via a permutation test based on the out-of-bag error rate.Where potential metabolite predictors were identified, we then built logistic regression and random forest models using the selected clinical factors and metabolites. For logistic regression, we assessed whether one or more metabolites improved the area under the receiver operator characteristic curve compared to the model based on clinical predictors alone50. The clinical variables, log transformed 15 and 20-week metabolite intensities, and 20 to 15-week ratios were used as candidate predictors. Up to three components with five predictors each were used. The number of components was selected based on the Mahalanobis distance balanced error rate using 10-fold cross validation, averaged over 10 repeats. This error rate was also used to assess the significance of this model via a permutation test.To assess whether expanding the set of predictors might improve accuracy, the sparse partial least squares discriminant analysis (PLS-DA) method from the mixOmics package was employedThe above procedures were repeated for comparing preterm birth <34 weeks to the entire control group.Participant demographic and outcome characteristics are shown in Table In the Cork samples, 176 compounds were detected. Of these, 77 were identified using an in-house library of reference standards and the remaining compounds were identified using mass spectrum alone . Of the in-house library matches, 61 were putatively identified (80–100% match to a reference standard) and 15 were tentatively identified . Of the NIST14 library identifications, 25 were putatively identified , 58 were tentatively identified , and three were unknown .In the Auckland samples, 142 compounds were detected. Of these, 50 were identified using an in-house library of reference standards and the remaining compounds were identified using mass spectrum alone (NIST 2014). Of the in-house library matches, 41 were identified, eight were tentatively identified, and one was unknown. Of the NIST14 library identifications, 51 were putatively identified, 34 were tentatively identified, and seven were unknown.Table Similarly, the random forest model using log(undecane) and vaginal bleeding was significantly better than random (p = 0.002) although the classification success was modest (70%). However, models also including log(decane) or log(dodecane), or both, had error rates similar to those based on permuted data .Sparse PLS-DA also produced a 1-component model for the Cork preterm birth data with an error rate that was significantly better than error rates produced for random permutations (p = 0.01). This model again included undecane, dodecane, decane, and vaginal bleeding. A fifth predictor, stearic acid measured at 15 weeks’ gestation, was present; however, its loading was small, and it did not have an odds ratio significantly different from 1 (p = 0.07) when incorporated into the logistic regression with vaginal bleeding and undecane.No metabolites or 20–15 week ratios met the false discovery rate threshold for the Cork <34 weeks data, for either preterm <37 weeks or <34 weeks in the Auckland data, nor were the sparse PLS-DA models significant .Log transformed Undecane did not meet the FDR threshold for association with preterm birth <34 weeks in the Cork data (compared to birth ≥ 37 weeks), but was associated with preterm birth <37 weeks. We therefore examined the sensitivity of our model to the threshold for preterm birth. The odds ratio associated with a 1 sd change in log(Undecane), in a model also including vaginal bleeding, when the divider between preterm birth and term birth was reduced to 36, 35 and 34 weeks were 2.1 (p = 0.0009), 2.2 (p = 0.0014), and 1.7 (p = 0.0571) respectively.We have analysed potential predictors of sPTB using serum samples and clinical data from Cork and Auckland participants of the SCOPE study, an international cohort of low-risk nulliparous women. An untargeted metabolomics approach was applied to serum samples collected at 15 and 20 weeks of gestation. More than one hundred metabolites were identified in each subset (Cork and Auckland). As expected for the metabolomics method employed, the most common classes of metabolites were fatty acids, followed by amino acids. Only three metabolites from the 20-week serum of Cork participants were found to be significantly associated with sPTB. Vaginal bleeding before 15 weeks, and smoking during pregnancy were the only clinical factors associated with sPTB, in Cork and Auckland subsets, respectively. In the Cork cohort, adding undecane to a multivariate logistic regression model for predicting sPTB improved its performance over a model with vaginal bleeding alone. This improvement is robust to the definition of preterm birth, persisting when the preterm threshold was decreased to 36 or 35 weeks; at 34 weeks the improvement is no longer significant. We note that, as depicted in Fig. 53, and oxidative stress has been associated with elevated alkane levels in gastroenteric disease, lung disease, and other chronic diseases of metabolism56. Glutathione, an important intracellular antioxidant, has been found to be decreased in maternal and umbilical cord blood of very low preterm neonates and their mothers57. Preterm birth seems to be associated with depletion of glutathione, reinforcing a possible increased oxidative status and lower antioxidant capacity. However, failure to demonstrate elevated alkanes among sPTB cases in the Auckland cohort limits our confidence, and there was no evidence these alkanes were associated with early sPTB (<34 weeks) at either study site. In addition, hypotheses describing the role of reactive oxygen species generation, metabolic and inflammatory imbalance and many other downstream mechanisms  caused by oxidative stress activation51 do not clarify whether those mechanisms are trigger factors or consequences of underlying conditions/alterations resulting in preterm PROM and/or spontaneous onset of preterm labour.There is a biologically plausible explanation for observing elevated alkanes  in the serum of mothers who had sPTB. Oxidative stress may lead to degradation of cell membranes by lipid peroxidation, followed by conversion of polyunsaturated fatty acids to volatile alkanes. The association of several oxidative stress-associated processes and sPTB have been previously reported59. Further investigation of individual pollutant exposure would be necessary to confirm whether elevated alkanes are associated with environmental exposure. It is also possible that technical rather than biological variability accounts for some of the differences observed across sites. Samples from the different sites, while analysed using the same protocols, were run on different platforms by different technical personnel. Thirty-four fewer metabolite species were detected in the Auckland samples, suggesting reduced sensitivity. In particular, two of the three metabolites elevated in cases in Cork (decane and dodecane) were not identified in the Auckland samples.The differences across study sites in the associations between sPTB and clinical factors such as smoking and vaginal bleeding in this case-control analysis also suggest that there are differences in the Cork and Auckland populations. The alkanes elevated in the Cork preterm birth group are present in outdoor and indoor air contaminants, which could potentially differ across study sites, providing an explanation for this observation. Association of environmental exposures with maternal and perinatal health have been reported for many years but are not yet well establishedWe also examined prediction of very preterm birth (<34 weeks). No clinical predictors were significant in the Auckland cohort, but vaginal bleeding before 15 weeks was associated with very preterm birth, as well as preterm birth, in the Cork cohort.62. Previous studies have used between 20 and 70 samples of a variety of biofluids , equally divided between cases and controls. There is also a diversity of pregnancy stages and analytical techniques, so it is not surprising that there is little overlap in the specific compounds identified. However, Virgiliou et al. also suggested that the changes in amino acids and lipids they observed could be related to oxidative stress62.To the best of our knowledge, there are few studies in the literature applying metabolomics techniques to understand sPTB in asymptomatic pregnant womenOur study has strengths and limitations. Cases and controls were selected from a large cohort comprised of low-risk nulliparous women enrolled in early pregnancy and containing a high standard biobank. Several procedures were employed to assure data, sample, and analysis compliance and reliability according to Standard Operating Protocols. Shortcomings of our study include lack of data regarding cervical length, a previously reported risk factor, and assaying of the metabolome of the two cohorts at different times on different equipment. Genetic factors may also be contributors to PTB risk however these were not assessed in this study. In addition, we have not investigated predictive metabolites for the different preterm birth subtypes (spontaneous onset of preterm labour or preterm premature rupture of membranes), focusing instead on an early sPTB (<34 weeks) subgroup, due to the increased morbidity in this specific group. Using data and samples from women of different populations , enabled us to compare reproducibility of our technique and also to discuss possible local drivers for sPTB . Cork and Auckland samples were analysed by different laboratory experts and instruments and as such, the reproducibility and sensitivity differed across the sites.While our finding of an association between elevated alkanes and sPTB at the Cork site is preliminary, it raises several interesting questions to pursue in the future. What differences are typical or expected across geographically distant study sites? What role might exposure to exogenous pollution sources, including alkanes, play in preterm birth? And finally, how might oxidative stress trigger, or be triggered by, processes leading to spontaneous preterm birth?Supplementary Figures


Similarly, maternal grandmothers (MGM) with higher HEI scores were less likely to have grandchildren with LBW  and more likely to have macrosomia . Higher paternal and paternal grandmothers (PGM) HEI scores were associated with lower likelihood of childhood obesity  and overweight , respectively. Mediation analysis showed significant direct relationship of MGM and PGM HEI scores on grandchildren’s birthweight and obesity, respectively. In conclusion, maternal line dietary quality appears to influence fetal growth whereas paternal line dietary quality appears to influence postnatal growth.Maternal dietary quality during pregnancy is associated with offspring outcomes. These associations have not been examined in three-generation families. We investigated associations between parental and grandparental dietary quality, determined by healthy eating index (HEI)-2015, and offspring birth outcomes and weight status at age 5. The Lifeways cohort study in the Republic of Ireland comprises 1082 index-child’s mothers, 333 index-child’s fathers, and 707 grandparents. HEI-2015 scores were generated for all adults from prenatal dietary information collected using a validated food frequency questionnaire. In an adjusted model, greater adherence to the maternal HEI was associated with lower likelihood of low birth weight (LBW) (OR: 0.72, 95% CI: 0.50–0.99,  Maternal diet plays an important role in determining birth outcomes and offspring health ,2. HowevStudies investigating maternal diet have mainly focused on adequate intake of selected macronutrients and micronutrients during pregnancy ,10,11,12Several studies have examined the associations between overall maternal diet quality and offspring outcomes ,17,18,19The HEI-2015 is the latest update of a diet quality index designed by the United States Department of Agriculture ,22. SeveThe Lifeways cross generation cohort study is a prospective family study that has been described in detail elsewhere ,29. The Ethical approval was granted by ethical committees of the Coombe University Hospital, Dublin, University College Dublin, Irish College of General Practitioners and University College Hospital, Galway, Ireland. Written informed consent was collected from all women upon recruitment and at all subsequent sweeps of the study.All adults completed the same questionnaire. Habitual dietary intakes of the women during the first trimester of pregnancy and 5 years postnatally were assessed by a validated 149-item semi quantitative food frequency questionnaire (FFQ), which has been validated for use in the Irish population . ParticiThe HEI-2015 is a measure of overall diet quality that measures alignment with the 2015–2020 dietary guidelines for Americans . The HEI2 (kg/m2). Overweight and obesity were defined according to the most recent International Obesity Task Force sex-for-age-BMI cut-offs [Information on birth outcomes and infant gender were abstracted from linked hospital records. Adverse birth outcomes: (1) Low birth weight (LBW), BW < 2500 g; (2) macrosomia, BW > 4000 g; (3) pre-term birth, delivery before 37 completed weeks of gestation; and (4) post-term birth, delivery at and after 42 completed weeks of gestation) were defined based on standard clinical cut-offs ,34,35. Acut-offs . Childrecut-offs .At recruitment, mothers provided information on age, self-reported height and pre-pregnancy weight, socioeconomic status  , and higχ2 test for categorical variables. Relationships between parental HEI scores and nutrient intakes were assessed using Spearman’s correlation. Linear and logistic regression analysis examined associations between parental and grandparental HEI scores with continuous and binary offspring outcomes, respectively. Spearman’s correlation examined associations between grandparental HEI scores and parental HEI scores. The overall trend of odds ratios (ORs) across tertiles of HEI was calculated by considering the median of HEI in each tertile for tertile analysis. Mediation analysis was conducted using the PROCESS macro v2.16 for SPSS , developed by Hayes, and bias-corrected bootstrap confidence intervals are presented [p value < 0.05.Maternal and paternal characteristics and nutrient intakes were first summarized according to tertiles of HEI-2015 scores and examined using Kruskal–Wallis test for continuous variables or resented . The regresented . In thisp = 0.04) and had lower likelihood of post-term birth  in the continuous adjusted model. Among mothers with the highest HEI tertile, risk of both LBW and post-term birth were lower  compared to mothers with the lowest HEI tertile. However, neither of these findings were statistically significant upon adjustment of covariates. No associations between maternal HEI scores and childhood overweight or obesity at age five were noted. With respect to fathers, in the multivariable adjusted analyses, higher paternal HEI scores were associated with lower odds ratios of childhood obesity at five years old . This association was even more evident when comparing fathers with highest and lowest HEI tertiles . It should be noted that the numbers of cases for these outcomes was low and the p-trends were not statistically significant for any of the birth outcomes (p = 0.03), longer birth length , and larger head circumference . However, the birth length and head circumference associations were attenuated upon adjustment of covariates  and more likely to have grandchildren with macrosomia  in the adjusted models, comparing highest to lowest HEI tertiles. The association between MGM HEI scores with lower post-term birth risk  was attenuated upon adjustment of covariates. PGMs with higher HEI scores were associated with lower risk of overweight grandchildren at five years old  in the adjusted model. No significant associations were observed between maternal and paternal grandfathers (MGF and PGF) HEI scores with any offspring outcomes at birth or at age five  (p = 0.01) and birth BMI  after adjusting for potential confounders  . Resultsfounders .r = 0.21, p < 0.01). No significant correlations were observed between maternal and MGF HEI scores or between paternal grandparents HEI and paternal HEI scores.Spearman correlation analysis revealed a significant but weak correlation between MGM HEI scores and maternal HEI scores (p = 0.035). In p = 0.034). No significant associations were observed for parental grandfathers nor for any other offspring outcome.While maternal nutrition during pregnancy has been previously studied in relation to birth outcomes ,2, to thPoor diet quality during pregnancy is known to increase neonatal adiposity ,23. RodrThese scores indicate a high quality diet with high intake of vegetables, fruits, fibre, protein, and unsaturated fats and low in saturated fats ,22,23,24The influence of paternal diet quality on offspring outcomes is less studied. We investigated possible relationships between paternal HEI scores and birth outcomes and weight at five years. We identified significant associations between higher paternal HEI scores and lower risk of childhood obesity. Poor paternal diet has been linked with increased risk of metabolic syndrome, obesity, and diabetes in offspring, possibly through epigenetic effects ,47. PosiOur study is highly novel in that it also examined the associations between grandparental HEI scores and grandchild’s outcomes and evaluated the relationships between different generations of HEI scores with birth and childhood outcomes. Mothers and MGMs with higher HEI scores were less likely to have delivered infants with LBW and to have higher birth weight, also consistent with the mediation analysis, and in keeping with an intrauterine or shared environment hypothesis. Notably, there were also associations in the paternal line, with no pattern related to birth outcomes but showing associations between paternal and PGM HEI scores and overweight and obese children at five years of age. These findings persisted in the mediation analysis also. There are no significant associations for grandfathers in either lineage. In a previous analysis of the Lifeways cohort, significant positive correlations were observed for nutrient intakes of maternal grandmother–mother–child triads, but not found in paternal lines . The matn = 333), potentially leading to lower statistical power and some self-selected bias. Also, there are few families where all four grandparents had information collected. Regarding generalizability of our findings, the Lifeways study was not designed to be representative of the general obstetric population in Ireland [n > 215,000) investigating the predictive validity of HEI-2015 scores confirmed the inverse associations with BMI, risk of mortality, and cardiovascular disease [Regarding the strengths of the study, this research provides new information about how diet quality is transmitted in different ways across three generations, influencing the health of offspring at birth and childhood. The sample size is reasonably large and we considered many potential confounding factors. The prospective study design is valuable in determining the temporal relationship between exposure (HEI) and offspring outcomes. Further, the HEI-2015 has undergone extensive validation and has been shown to capture the multidimensionality of diet as well as to have predictive validity ,32. None Ireland , though  Ireland , consist disease .In conclusion, we observed that higher HEI-2015 scores were associated with lower risk of LBW in the maternal and MGM lines, whereas higher paternal and PGM dietary quality predicted lower risk of childhood overweight and obesity. These data suggest that differential intergenerational transmission of risk exists between maternal and paternal lineages, meriting further investigation.


The Child-Mother binomial is potentially susceptible to the toxic effects of pollutants because some chemicals interfere with placental transfer of nutrients, thus affecting fetal development, and create an increased the risk of low birth weight, prematurity and intrauterine growth restriction.x) on birth weight in a cohort of Mexican newborns.To evaluate the impact of prenatal exposure to nitrogen oxides . After adjusting for maternal age and other confounders, a significant birthweight reduction was observed for each IQR of NOx increase .The average birth weight was 3217 ± 439 g and the mean of NOx exposure has a negative effect on birth weight, which may influence the growth and future development of the newborn.Our results provides evidence that prenatal NO An accelerated increase in health effect related to air pollution has been observed over recent years in developed as well as developing countries 234. Vari2368910111214Birth weight is an indicator of the duration of gestation and fetal growth rate , and is 18x emissions is vehicle exhaust, which contributes to 64% of total emissions [21x exposure on reproductive effects considering a birth cohort study.The primary source of air pollutants and NOmissions . In Meximissions 2122, how2) and nitric oxides (NOx) on birth weight in a cohort of newborns from Morelos, Mexico.Therefore, we realized the present study to evaluate the impact of prenatal exposure to nitric oxide (NO), nitrogen dioxide  cohort study; a large double blind randomized controlled trial (RCT) of prenatal DHA supplementation. Pregnant women (n = 1094) were randomized to receive a daily supplement of 400 mg of DHA or placebo from 18–22 weeks of pregnancy until delivery. A detailed description of the design and methods has been published elsewhere [This analysis is based on the POSGRAD , and outdoor exposure to pollutants .At birth: Using an ad hoc form, personnel staff obtained information about the newborn characteristics, including: condition at birth (live or stillbirth), gender, type of birth, complications at birth, presence of congenital abnormalities, and gestational age. Birth weight was obtained according to the technique proposed by Lohman [y Lohman , using ax exposure during pregnancy at home, using a standardized area specific land-use regression (LUR) models. The LUR models were based on measurements of NO2, NO and NOx for a continuous period of 15 days in 60 different sites throughout the State of Morelos. Ambient levels of air pollutants were measured with Ogawa and 3M passive samplers. The samplers were positioned outdoors, near the participants’ homes  making sure they were not being blocked by any object that could obstruct the flow of air . The samplers were cleaned before use, and transported in sealed amber-color containers before and after the measurement. After 2 weeks of continuous monitoring, the samples were collected and again placed in resealable bags in amber-colored containers and transported at 5°C at laboratory of the Mexico National Institute of Public Health where, inside a glove box, the pads were extracted and stored in refrigeration until their analysis. As part of quality control, 10% were blanks and duplicates. NOx concentrations were determined at the Harvard School of Public Health, using spectrophotometry [For each participant, we estimated the individual NOotometry .LUR models were developed for each pollutant in each study area to predict air pollution levels at the residences of the cohort participants using information about: traffic variables (type of roads and highways avenues), weather data , geography (elevation and coordinates), population density and land-use variables   obtained2 and NOx) on the weight of newborns. As potential confounders, we evaluated: parity, maternal passive or active smoking during pregnancy, pre-gestational maternal anthropometric characteristics, intervention group among others. All of them remained in the final model except those who did not change the crude association between NOx with birthweight. All of the statistical analyses were performed using Stata 13 statistical analysis software for Windows [To evaluate the quality and consistency of the data, an exploratory and univariate analysis was performed, and measures of central tendency and frequency were estimated for each of the study variables. A bivariate and multiple linear regression models were run to evaluate the impact of prenatal exposure of air pollutants  for NOx and 0.9211 (p = 0.0789) for NO2. The values of the blanks used during the monitoring campaign ranged from 0 to 0.2. The average wind speed was 2.51 m/sec and the temperature was 20.3°C during the study period. The correlation between predicted and observed values for each of the pollutants was statistically significant (p < 0.01), and were 0.91 for NO, 0.79 for NO2, and 0.94 for NOx (data not shown).Table x and birth weight. After adjusting for mother’s age, height and passive smoking, gestational age, and gender of child, a significant birth weight decrease was observed by each increment in the interquartile range of NOx  and NO . No association between prenatal NO2 and birth weight was observed.Table 2 and NOx, as estimated by land-use regression model, significantly decreases the birth weight of newborns residing in Morelos, Mexico. To our knowledge, this is the first prospective study performed in Mexico that analyzes the effects of prenatal exposure to nitrogen oxides  on birth weight using this methodology to evaluate the exposure to air pollutants. This approach provides stronger results given that most of previous studies evaluated NOx exposure according to data from fixed monitoring stations.The results from the present study suggest that prenatal exposure to NOx exposure per trimester or throughout the pregnancy and adverse effects on birth, including birth weight and/or low fetal weight and some of those studies estimated exposure using land-use regression and particulate matter variants, while others have used other types of dispersion models and local monitoring systems [293031333435Previous studies have reported an association between prenatal NOms 29303133343536.et al. found a decrease in fetal weight of 74.7 g per increase in interquartile range of NO2 (IQR = 12 μg/m3) adjusting by NOx exposure of each trimester. That association was observed when the models had been adjusted for the three trimesters of pregnancy and for women who spent over 2 hours/day in non-residential outdoor areas. Nonetheless, when analyzing this data individually, no association was found between exposure during the entire pregnancy nor per trimester [2 exposure on fetal weight during different weeks using ultrasound measurements from a sample of 562 women. When limiting the analysis to women who spent over 2 hours/day in non-residential outdoor areas (n = 255), a statistically significant weight decrease of –5.5 g was found in week 32 of gestation, and a similarly significant decrease (4.78 g) was found between weeks 20 and 32, for each increase in the IQR (13.23 μg/m3) [2, and NOx on the risk of low birth weight at term births, using a LUR exposure model and with and without seasonal adjustment [x, respectively. Nevertheless, the data was obtained from an electronic database of birth certificates.As part of INMA, a multicentric cohort study, Aguilera rimester . In addi3 μg/m3) ; howeverjustment . Researcx affects birth weight is unclear, the association between exposure to these pollutants and decreased birth weight can be explained by some of the proposed mechanisms that involve placental circulation. NOx may affect birth weight because NOx can promote blood coagulation and viscosity [Although the mechanism through which prenatal exposure to NOiscosity , pulmonaiscosity . As oxidiscosity . In addiiscosity .x limits the possible variations that could occur in the concentrations of the pollutants over the study period, assuming that the air concentrations had the same behavior throughout during the entire time of pregnancy. However, the use of a LUR model to evaluate exposure strengthened the findings and made it possible to detect small-scale variations, thereby limiting classification errors in the assignment of exposure (Figure This study, however, has some limitations that should be considered when interpreting the results. First, the number of sites monitored and the duration of the measurements NOe Figure . It is aSecond, we did not obtain detailed information about the indoor and outdoor activities of each participants. Instead, we presumed that exposure was primarily associated with the amount of time spent in outdoors. However, for both cases, the measurement error was non-differential and the observed association was underestimated.x exposure.Finally, the possibility that the results were a consequence of poor control of confounders is unlikely because the design of the study excluded women with high-risk pregnancies and/or pre-existing illness and the models were adjusted for variables that were considered to have both a possible effect on birth weight and a relationship to NOx, especially on birth weight. These results could have significant public health implications given the role of birth weight as a determinant of childhood mortality, and more recently as a determinant of chronic illnesses during adulthood. However, it is important to continue with the investigations that allow for the identification of critical windows of exposure and that strengthen the evidence related to these associations.In summary, our results provide valuable information about the adverse health effect of prenatal exposure to NO


Introduction: Clinical guidelines recommend using neuroimaging, Prechtls’ General Movements Assessment (GMA), and Hammersmith Infant Neurological Examination (HINE) to diagnose cerebral palsy (CP) in infancy. Previous studies provided excellent sensitivity and specificity for each test in isolation, but no study has examined the pooled predictive power for early diagnosis. Methods: We performed a retrospective case-control study of 441 high-risk infants born between 2003 and 2014, from three Italian hospitals. Infants with either a normal outcome, mild disability, or CP at two years, were matched for birth year, gender, and gestational age. Three-month HINE, GMA, and neuroimaging were retrieved from medical records. Logistic regression was conducted with log-likelihood and used to determine the model fit and Area Under the Curve (AUC) for accuracy. Results: Sensitivity and specificity for detecting CP were 88% and 62% for three-month HINE, 95% and 97% for absent fidgety GMs, and 79% and 99% for neuroimaging. The combined predictive power of all three assessments gave sensitivity and specificity values of 97.86% and 99.22% . Conclusion: CP can be accurately detected in high-risk infants when these test findings triangulate. Clinical implementation of these tools is likely to reduce the average age when CP is diagnosed, and intervention is started. Cerebral palsy (CP) identifies a group of developmental disorders of movement and posture, causing activity limitation, attributed to non-progressive disturbances related to brain injury early in development ,2. It isEstablishing an early diagnosis of CP is important as this can expedite early intervention, thereby maximizing the opportunities for appropriate physical and learning support, optimizing neuroplasticity, prevention of complications, and provision of adequate psychological support to parents. A recent International Clinical Guideline for the early identification of CP recommends the use of neuroimaging, Prechtls’ General Movements Assessment (GMA), and the Hammersmith Infant Neurological Examination (HINE) for early detection of CP ,6,7,8. EThe Hammersmith Infant Neurological Examination (HINE), is a simple and scorable method designed for evaluating infants between 2 months and 24 months of age . It inclGeneral movements (GMs) are part of the spontaneous movement repertoire, are present from early fetal life onwards until the end of the first half a year of life, and were first categorized by Heinz Prechtl ,11. GMs During the writhing period abnormal GMs are described as either: (a) poor repertoire (PR) GMs, where the sequence of successive movement components is monotonous, and arm, leg, trunk, and head movements do not occur in the normal rich and complex sequence; (b) cramped-synchronized (CS) GMs, where the GMs appear rigid and stiff and lack the normal, smooth, and fluent character, and all the limb and trunk muscles contract and relax almost simultaneously; (c) chaotic (Ch) GMs where all limb movements are of large amplitude, occur chaotically, and lack fluency or smoothness, or both.During the fidgety period, normal FMs are defined as circular movements of small amplitude, moderate speed, and variable acceleration of neck, trunk, and limbs in all directions. Abnormal GMs at the fidgety age are classified as follows: (a) absent FMs, when normal FMs are never observed from age 6 to 20 weeks’ post term age (PTA); (b) abnormal FMs, when FMs can be detected but their amplitude, speed, and jerkiness are moderately or greatly exaggerated. Cramped synchronized GMs in the writhing period and an absence of fidgety GMs in the fidgety period are predictive of CP with high sensitivity and have specificity values repeatedly demonstrated across multiple studies ,13. OtheHINE, GMs, and neuroimaging address different, although overlapping, aspects of the condition, such as (i) pathogenesis, mainly targeted by the assessment of neuroimaging findings, (ii) impairment, especially addressed by neurological examinations like the HINE exploring posture, tone, and reflexes, and (iii) functional limitations, as expressed by abnormalities of neurodevelopment, as measured by the quality of movement . Only a We performed a retrospective case-control study of infants born in three hospitals in Italy  between 2002 and 2016. Clinical data from infants with typical development, mild disability, or CP diagnosis at two years were included . The milIn all, gestation age at birth, birth weight, sex, neurological examination (HINE) score, GMA spontaneous movements assessments, neuroimaging data (MRI or Ultrasound), Gross Motor Function Classification System (GMFCS) in children with CP, and 24-month developmental quotient (DQ) were retrieved from medical records. Minimum required data for inclusion were gestation age at birth, date of birth, sex, HINE at 3 months, both writhing and fidgety GMA results, and 2-year outcome, including GMFCS level in children with a diagnosis of CP. We did not include records from infants with a history of congenital anomalies. Age correction was used, as per standard clinical protocol, for all evaluation and outcome data. For each group, infants’ data were matched for birth year (±1 year), gender, and gestational age at birth . A total of 720 records were evaluated, and 441 (147 in each group) were included.General Movements: We retrieved the results of GMA, which were completed as a standard of care at two time points. Writhing and fidgety periods classifications were retrieved, as previously completed according to standard Prechtls’ spontaneous movements GMA method . In all Neurologic Examination: Hammersmith Infant Neurological Examination scores were retrieved from infants’ clinical records. All infants included in the study were assessed with the HINE at 3, 6, 9, and 12 months PTA. As the aim of the present work was to compare HINE and the other techniques, only the assessment performed at 3 months was considered for the study. In all centres, HINE was scored by trained assessors who have contributed to the development of the scale  and have shown high inter-scorer reliability in previous prospective studies .Ethics: Since this is a retrospective study, no specific consent was obtained from the parents of the infants to retrieve the clinical information. Consent for video recording GMs was obtained from parents at the time of clinical assessments as per standard procedures at each of the centres. N = 182) or Griffiths (N = 118) assessments during the 24-month (±1 month) assessment visit. These tools were chosen as they are the ones routinely used for the clinical follow up at the participating centres. They both showed a high correlation with the Bayley Scales of Infant Development [Gross Motor Function Classification Scale (GMFCS) scores were retrieved from the medical record. Developmental quotient was derived from CAT-CLAMs , blinded to any other clinical information and group allocation. When reports from both imaging modalities were available, they were both considered for the analysis. Scorers assigned each patient to one of the following groups, based on current knowledge on the predictive value of early neuroimaging in infants at-risk to develop CP ,23:(i) No CP, i.e., extremely unlikely to develop CP. Children included in this group were typically those with normal imaging or with anomalies usually not associated with the development of CP, such as IVH grade 1 or transient flares(ii) Unclear, i.e., when CP was possible but not very likely. Children included in this group were those with evidence of brain damage that, however, appeared to spare the motor structures. Typical examples are children with IVH, stroke, or hypoxic-ischaemic encephalopathy showing ischemic or haemorrhagic lesions not involving the perirolandic area, the Posterior Limb of the Internal Capsule (PLIC), the pyramids, or the basal ganglia.(iii) CP, i.e., very likely to develop CP. Children included in this group were those with evidence of brain damage involving the motor structures. Typical examples were IVH grade IV, cystic PVL, HIE with BG involvement, or brain malformation with involvement of the motor cortex. Children were assigned to a different group by the two scorers, data were re-analysed, and a consensus was reached.Frequency and percentage (%) were reported for categorical variables, and the mean and standard deviation (SD) were reported for continuous variables overall and for the three groups . The median and interquartile range (IQR) were reported for the HINE for clinical interpretation. Chi-squared were investigated for associations between type of CP (Control + Mild versus CP) and General Movements (GMs), HINE, and MRI as categorical variables. n = 42) were then removed and an analysis of fidgety (n = 258) versus absent fidgety (n = 141) was conducted. In the final multiple regression, we analysed the combined predictive value of HINE at 3 months + fidgety (absent versus fidgety) + imaging.Logistic regression was undertaken with outcome variables as no CP (control + mild disability) versus CP and the HINE at 3 months to determine the cut off value using sensitivity and specificity. Logistic regression was undertaken with outcome variable as CP versus no CP and the following predictor variables: HINE 3 months continuous, HINE 3 months dichotomised (less than 57 versus 57 or more); fidgety dichotomised , imaging ordinal . Multiple logistic regression was then undertaken with outcome variable as no CP versus CP and variables: (a) HINE 3 months continuous + fidgety dichotomised, (b) HINE 3 months continuous + imaging ordinal, (c) fidgety dichotomised + imaging ordinal, (f) HINE 3 months continuous + fidgety dichotomised + imaging ordinal. Cases with abnormal fidgety movements .Akaike information criterion (AIC) was used to perform model comparisons. Area under the curve (AUC) for the Receiver Operating Characteristic (ROC) Curve was used to determine the accuracy of the test to correctly classify those with and without the outcome. The significance level was set at In this study, we purposively included and matched: 147 children with a CP diagnosis; 147 with a diagnosis of mild disability (not CP); and 147 children assessed to have a normal outcome. Of the 441 children, 221 were girls with a mean gestational age of 33.8 weeks and mean birth weight of 2.14 kg . Three pDQs were only available for 68% of the sample overall. While 76% of data was retrievable for the children with a mild disability, only 56% of the children with a CP diagnosis had a DQ calculated. In 71% of the children with a normal outcome DQ was reported, although in the majority of the cases simply as “higher than 85”, without specification on the actual score. A significant proportion of the children with CP were unable to be assessed on these norm-referenced measures due to the motor requirements of the cognitive tasks.In children with a normal outcome (DQ > 85 at two years), the GMA at writhing age were normal in 82% of the sample, while the remaining infants were classified as poor repertoire. At fidgety age, all infants without disability had normal GMs. The median HINE score at 3 months was 63 (IQR 56–66) and at 12 months was 72 (IQR 70–74). In 73% of cases, neuroimaging accurately predicted a normal motor outcome; however, the prediction of CP was unclear in 26% of cases. Only 2 of the 147 children in this group were predicted by neuroimaging to have CP.Children with CP for whom data was available had a mean DQ of 77 (SD 17.85). GMFCS data for this group are summarised in Of the children with a non-CP disability, the mean DQ was 61 (SD 11.09), indicating most of these children had a mild to moderate intellectual disability. The distribution of GMs scores was more variable in this group with just over half of the group recording poor repertoire GMs around the equivalent term age. At fidgety age, just over 70% had normal fidgety GMs while 24% scored abnormal fidgety. Seven infants scored absent fidgety. Most infants (82%) were accurately predicted from neuroimaging not to have CP in this group with an unclear outcome prediction in only 16%.Each assessment  independently demonstrated excellent predictive validity for CP . GMA at When GMA and HINE scores at 3 months, plus early neuroimaging, were combined in the model, 98.74% of children were correctly classified D. SensitWe sought to calculate the pooled predictive power of early GMA, neuroimaging, plus HINE in diagnosing CP in a population of high-risk infants via a retrospective case-control study. We found that the pooled predictive power of early GMA, neuroimaging, plus HINE was highly accurate, with a sensitivity for detecting CP of > 97% and a specificity of > 97%. The pooled sensitivity and specificity of the three instruments together was higher than the three tools in isolation, where literature reports the GMA has a sensitivity of 98%, neuroimaging  has a sensitivity of 86–89%, and the HINE has a sensitivity of 90% . The advOur findings are, therefore, clinically important because the clinical diagnosis of CP has traditionally been made late at 12–24 months of age , rather Our study has a number of limitations. First, due to the retrospective nature of our case-control study, there are likely changes in clinical care over time , which may affect the accuracy of the results and the generalisability to contemporary care. Second, our cases and controls were recruited exclusively from hospitals, which will have higher rates of CP and mild disability than the general population, meaning selection bias is likely to have occurred. Third, the individuals selected as controls may under-represent the population that produced the cases because of the data collection method, again, meaning that selection bias may have occurred. Fourth, we used highly skilled masked assessors  whose accuracy in scoring these tests may be higher than the general clinician population, affecting the generalisability of the results. Fifth, the GMA, imaging and HINE do not have perfect interrater reliability, and accordingly, our masked evaluators did not always agree on the scoring of each of the three tests. In spite of these methodological limitations and the lack of a biomarker, these retrospective observational study data can still be considered the best-available evidence for a diagnostic work-up that is likely to yield an early and accurate diagnosis of CP.Future research into early diagnosis would be beneficial in understanding the long-term outcomes of children with only two of the three tests that are in the abnormal range, and conversely one test in the normal range. Preliminary indications from clinical trials suggest that these children almost never have a normal outcome, but often have a neurological diagnosis other than CP ,32.In conclusion, the clinical diagnosis of CP can be made early at three-months corrected age, with an accuracy of greater than 97%, when the child has absent fidgety general movements, a Hammersmith Infant Neurological Score below 57, and abnormal neuroimaging in the motor areas.