Magnetic resonance imaging (MRI) based diffusion tensor imaging (DTI) can assess white matter (WM) integrity through several metrics, such as fractional anisotropy (FA), axial/radial diffusivities (AxD/RD), and mode of anisotropy (MA). Standard DTI is susceptible to the effects of extracellular free water (FW), which can be removed using an advanced free-water DTI (FW-DTI) model. The purpose of this study was to compare standard and FW-DTI metrics in the context of Alzheimer’s disease (AD). Data were obtained from the Open Access Series of Imaging Studies (OASIS-3) database and included both healthy controls (HC) and mild-to-moderate AD. With both standard and FW-DTI, decreased FA was found in AD, mainly in the corpus callosum and fornix, consistent with neurodegenerative mechanisms. Widespread higher AxD and RD were observed with standard DTI; however, the FW index, indicative of AD-associated neurodegeneration, was significantly elevated in these regions in AD, highlighting the potential impact of free water contributions on standard DTI in neurodegenerative pathologies. Using FW-DTI, improved consistency was observed in FA, AxD, and RD, and the complementary FW index was higher in the AD group as expected. With both standard and FW-DTI, higher values of MA coupled with higher values of FA in AD were found in the anterior thalamic radiation and cortico-spinal tract, most likely arising from a loss of crossing fibers. In conclusion, FW-DTI better reflects the underlying pathology of AD and improves the accuracy of DTI metrics related to WM integrity in Alzheimer’s disease. Current biomarkers for AD target the dominant pathological paradigm, characterized by beta-amyloid and tau pathologies, as well as neurodegenerative changes. While much of the pathologic evidence of AD is found in gray matter, it is well recognized that concomitant white matter (WM) alterations occur in the brains of AD populations, suggesting an expanded role for WM-based biomarkers in AD2.Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia in older adults3; most commonly, diffusion-related parameters are obtained by analysis using the diffusion tensor imaging (DTI) model. In WM, faster diffusivity along the fibers, with lower perpendicular diffusivity, yields a higher fractional anisotropy (FA). While decreased FA has been associated with demyelination, edema, gliosis, and inflammation, FA is unfortunately an indirect marker of WM integrity because the individual microscopic contributions of various non-WM components cannot be disentangled5. Other DTI-derived metrics include axial diffusivity (AxD), which may be related to axonal damage, and radial diffusivity (RD), which is correlated with myelin integrity, axonal diameter and density, and fiber coherence6. Prior DTI studies have shown that AD is associated with lower FA and higher AxD and RD values across multiple regions in the WM, including the corpus callosum (CC), fornix, cingulum, inferior longitudinal fasciculus (ILF), and superior longitudinal fasciculus (SLF)9. Moreover, changes in DTI metrics may precede both neuronal loss and symptoms onset, suggesting their potential role as early biomarkers. While several studies have shown a correlation between DTI-derived metrics and cognitive scores12, other similar investigations have failed to show a significant correlation16 suggesting methodologic and/or analytic impacts on dMRI-derived data.Diffusion-based magnetic resonance imaging (dMRI) enables the in vivo evaluation of brain microstructure and, more specifically, WM integrity by probing water diffusivity associated with axons17. The impact of PVEs may be particularly profound in AD, where neuronal loss leads to secondary ex vacuo increases in cerebrospinal fluid (CSF) and free water (FW) across the brain. To overcome the effects of extracellular FW on DTI-derived metrics, a FW correction algorithm has been developed to quantify and remove the contribution of extracellular FW18. FW-DTI has been shown to improve both tissue specificity19 and DTI-based tract reconstruction18. This model has previously been used to study several neurological diseases, including AD24, Parkinson’s disease25, and depression27.Although dMRI is a well-recognized method for assessing WM, it has several limitations, including known inaccuracies related to partial volume effects (PVEs), wherein the resulting DTI metrics reflect a weighted average of multiple diffusion components within a voxel and are thus no longer specific to a single tissue type20. FW correction may be particularly important in the fornix, due to both its proximity to the lateral ventricles and its association with the hippocampus22, and may also improve sensitivity to conversion to mild cognitive impairment (MCI) relative to uncorrected DTI metrics23. The FW index has separately been proposed as a standalone biomarker of AD-related pathology. This index has been associated with cognitive decline in multiple domains and may enable separation of vascular and neurodegenerative contributions to cognitive decline in the presence of mixed dementia21. More recently, an elevated FW index was observed in MCI and AD patients compared with healthy controls (HC), even after correction for white matter hyperintensities28. However, none of these studies have directly compared standard and FW-DTI metrics in the context of distinguishing between AD and HC. As AD-related WM degeneration leads to a significant intravoxel FW component, FW-DTI may improve sensitivity to early brain changes and provide a clearer and more specific interpretation of results compared to standard DTI.Using FW-DTI in a healthy aging population enriched for AD risk, Hoy et al. found that DTI-based markers of altered microstructure were significantly associated with CSF biomarkers of preclinical AD pathology29. This dataset was previously used to investigate AD-related changes in the size and shape of the CC31. We also investigated the complementary FW index for both groups and analyzed voxel-based correlations between all DTI-derived metrics (from DTI and FW-DTI) and the mini-mental state examination (MMSE) score32. We hypothesize that FW-DTI will improve the sensitivity and specificity for detection of WM tract abnormalities in AD. More specifically, increased FW due to neurodegeneration reduces the accuracy of diffusion-derived metrics, and thus, the removal of FW contributions improves the estimation of DTI metrics and may increase confidence in the patterns observed across DTI studies. In addition, the ability to quantify FW may yield complementary insight into sub-voxel WM neurodegenerative processes. The application of FW-DTI for AD better reflects the underlying pathology and should thus further improve interpretation of AD WM pathology.In this study, we compared FA, AxD, RD, and MA from both standard DTI and FW-DTI between AD and HC subjects obtained from the Open Access Series of Imaging Studies (OASIS-3) brain projecthttp://oasis-brains.org/), which is a neuroimaging dataset for normal aging and Alzheimer’s disease and is freely available to the scientific community. Inclusion criteria were participants between the ages of 60 and 90 years who had 3 T dMRI data available with more than 30 directions. Exclusion criteria were cognitively impaired participants with dementia not attributed to AD, healthy control participants with significant comorbidities, and participants for whom the MMSE was not available. Only one time-point was used per subject. Subsequently, this study included 30 HCs  = 73 (6) years) and 28 AD subjects  years). Four AD subjects had remote mood disorders and one had a remote hypothyroidism as secondary medical diagnoses. The Clinical Dementia Rating (CDR)33, which is a 5-point scale used to characterize multiple domains of cognitive and functional abilities, was zero for all HCs and between 0.5 and 3.0 for the AD group. The MMSE is a 30-point assessment commonly used to measure cognitive impairment. The mean value of MMSE was 29.10 (S.D. 1.24) for HC and 24.18 (S.D. 5.02) for AD. All subject characteristics are summarized in Table All data were downloaded from the OASIS-3 brain project : 24.0 × 24.0 cm; slice thickness: 2.5 mm; 64 axial slices) and one non-diffusion-weighted image (B0 image).All dMRI data collected from the OASIS-3 database were acquired using a 3 T scanner (Siemens). For all subjects, the acquisition was performed using 64 diffusion-encoding directions  software library tool 34. Following translation and rotation estimation, the raw dMRI images were corrected for eddy currents and motion35. After motion correction, the gradient orientations were compensated  to account for the rotational component of registration. A brain mask was defined for each subject using the Brain Extraction Toolbox (BET)36 for the single B0 images. DTI standard (uncorrected for FW) and FW-corrected maps were calculated using an in-house MATLAB script26, which is available upon request. The FW maps were computed by fitting the following model at each voxel18:g, and b is the b-value (1000 s/mm2). The first term reflects the tissue compartment, where D is the diffusion tensor of this compartment, f is the fractional volume of the compartment, and gT is the transpose of the vector g. The second term reflects an isotropic free-water compartment with a fractional volume of (1 − f), where the diffusion coefficient −3 mm2/s). AxD, RD, and FA were obtained from D. Standard DTI metrics are reported as AxD, RD, and FA, while FW-corrected DTI metrics are reported as AxDt, RDt, and FAt, where the t is indicative of greater tissue specificity from FW-DTI.DMRI data were downloaded in NIFTI format and processed as previously described37:While FA indicates deviation from a spherical-shaped tensor , the mode of anisotropy (MA) is a complementary measure that discriminates between linear and planar anisotropy. MA is mathematically orthogonal to FA and relates to the skewness of the DTI eigenvalues  algorithm39. The warp fields obtained from this normalization and from the B0 group-wise template creation were used to transfer all DTI maps (standard and FW-corrected) to the Illinois Institute of Technology (IIT) Human Brain Atlas (v.4.1), which contains both anatomical and DTI brain templates in International Consortium for Brain Mapping (ICBM)-152 space40. All DTI maps in standard space were smoothed using FSL with an isotropic Gaussian kernel .After quantifying all DTI-derived metrics, the B0 images from each subject were used to create a B0 group-wise template image using 41 with threshold‐free cluster enhancement (TFCE)42 and a correction for multiple comparisons via Family‐Wise Error (FWE) rate38 at a 0.01 level. A WM mask was defined using a threshold of 0.2 on the average values of FA (from standard DTI) and was applied to the other DTI maps. For each voxel, the DTI-derived metrics were fitted by an Analysis of Covariance model (ANCOVA), which included age and sex as covariates. The number of randomized permutations was set at 5,000. The clusters identified to be statistically different between groups were labeled according to the JHU DTI-based white-matter atlases44. All results are reported with a significance threshold of p value < 0.01, corrected for FWE, except where otherwise noted.Age, MMSE, and CDR scores are presented as mean and standard deviation (SD) for each group. Differences in age, sex, and cognitive test scores between groups were assessed using the Student's t-test. Comparisons of all DTI-, FW-DTI- metrics, and FW maps between groups were performed using the FSL Randomise toolp-value < 0.01 (FWE-corrected).For the cognitively impaired group (AD only), the voxel-based relationship between MMSE scores and DTI metrics was assessed through Randomise with significance at p = 0.137) or sex  between HC and AD. Significant differences were found for MMSE  and for CDR , as shown in Table Student’s t-test did not show any difference in age  are reported in Table t, AxDt, and RDt are displayed in panels (a), (b), and (c) . The clusters where AD had lower FAt values than HC correspond to the same WM locations as those observed using standard FA ; however, these clusters  are smaller than standard FA . Higher FAt values in AD compared with HC were found bilaterally for ATR, CST, limb of internal capsule (LIC), SCR, and superior fronto-occipital fasciculus (SFOF). With regard to AxDt and RDt, both higher and lower values were observed in multiple regions in the AD group . More specifically, AxDt was lower in the AD group in CC and fornix and predominantly higher in the ATR, CST, and retrolenticular part of internal capsule. Lower RDt was observed with AD mainly in the ATR, CST, PLIC, and SFOF, corresponding to similar areas with higher FAt. A small cluster inside the body of the CC showed higher RDt in the AD group, albeit at p < 0.05 FWE corrected.Figure t values overlapped with higher AxDt and RDt; however, lower FAt values overlapped with lower AxDt and RDt values in the fornix (4.8%), while lower FAt corresponded with lower AxDt in several regions including the CC and forceps minor. Higher FAt values were found to overlap with higher AxDt and lower RDt in the SFOF, right PLIC, SCR, and anterior LIC. Several clusters were observed with higher AxDt and lower RDt, including the left CST, right PLIC, and right SCR. Unlike standard DTI, no clusters demonstrated simultaneously higher AxDt and higher RDt. Finally, Supplementary Table For each brain region, the significant cluster sizes (as percent volume) are reported in Table p < 0.01 FWE corrected. The corresponding violin plots are also shown for FW values for each group . Only clusters with higher FW values in AD compared with HC were found, with clusters covering 34% of the total WM mask. These differences were observed in several WM regions, including most prominently the fornix, cingulum , and CC. The complete list of group-wise significant clusters for the FW index between HC and AD is reported in Table Figure The results for MA are reported in Fig. p < 0.05 FWE corrected level.No significant voxel-based correlation was found between MMSE and either standard or FW-DTI metrics at Supplementary Fig. t, AxD and FW-AxDt, RD and FW-RDt, respectively. By removing the isotropic diffusion contribution, the impact of FW-correction is to increase FAt, decrease AxDt, and decrease RDt, relative to the corresponding standard DTI metrics. These panels also show that FA is less affected by the FW component than AxD and RD . As AxD and RD metrics are more influenced by free-water components , this is consistent with the widespread AxD and RD changes without FW-correction. Here again, there is not a strong group difference in the correlations. Additionally, the striking differences for AxD and RD metrics between standard and FW-correction can be attributed to their biophysical origin; that is, raw diffusivity parameters AxD and RD are likely to be more impacted by intravoxel free-water diffusion than the composite metric FA.Panels (e)–(g) show the correlations between FA and FW-FA18, producing more accurate FW-corrected DTI metrics and the complementary FW index. Using both standard and FW-DTI, significant WM differences were observed between HC and AD subjects in several regions, including both higher and lower FA. Both AxD and RD were highly impacted by the effects of FW, and FW-corrected AxDt and RDt were more consistent with pathology-based expected spatial patterns of neurodegeneration. The FW index was higher in AD than HC across many regions, as expected. The FW correction method has shown promise in many neurological diseases of the WM27, particularly those with neurodegenerative aspects; similarly, we now demonstrate that the application of FW correction in AD improves the accuracy and sensitivity of the resulting DTI metrics.In this study, WM integrity was assessed in patients with Alzheimer’s disease using both standard and FW-corrected DTI metrics. Although DTI is a commonly used method for assessing WM, PVEs can reduce the accuracy of DTI metrics, ultimately limiting their interpretation. To mitigate PVE, free water‐contaminated voxels were removed by incorporating a FW component in the conventional diffusion modeling pipeline45. In the context of AD, pathological changes in the fornix may occur at an early disease stage before clinical manifestations. Lower FA and higher MD in the fornix has been a robust and consistent finding in AD47 and may correlate with cognitive decline48. Similarly, FA reductions in the fornix have been identified using skeletonized tract-based spatial statistics (TBSS) in early-symptomatic individuals and those at high risk for developing AD52. However, the fornix is particularly prone to PVEs due to its proximity to the lateral ventricles2, which may be further exacerbated by atrophy-related ventricular expansion in AD. While decreases in standard FA may reflect a complex combination of atrophy and microstructural changes, these decreases remained after correction for FW, indicating that they reflect true pathological changes in the fornix.Lower FA values, indicative of more isotropic motion, are consistent with neurodegenerative changes and were observed in AD in multiple regions, including most prominently the fornix and CC. Of particular interest, the fornix is the major output tract of the hippocampus and plays a major role in episodic memory53 and others reporting predominant changes in the genu9 or splenium54. Early involvement of the genu would support the anterior-to-posterior progression of the retrogenesis hypothesis, wherein degeneration occurs in reverse of myelination. In contrast, early changes in the splenium would be consistent the posterior-to-anterior progression of Wallerian degeneration following neuronal loss, wherein WM damage is subsequent to grey matter damage. To date, conclusive evidence of a gradient pattern has remained elusive56. Given the substantial FW component observed in the CC, the inclusion of FW correction algorithms could improve accuracy in the resulting DTI metrics in the presence of neurodegenerative changes, and future studies should consider whether the implementation of FW correction could increase confidence in the spatiotemporal patterns observed across DTI studies.Other significant changes were observed in AD in the CC, which is a major fiber bundle responsible for interhemispheric connection. Using both standard and FW-DTI, large clusters of lower FA were observed in the genu and body with minimal splenial changes, particularly using FW-DTI. Several other studies have similarly reported decreased FA in the CC; however, these studies have shown inconsistent regional changes with AD, with some studies finding changes throughout the CCt in AD compared with HC was observed seemingly paradoxically using FW-DTI in bilateral ATR and CST, as well as internal capsule and corona radiata; with standard DTI, much smaller clusters of higher FA were observed in the right hemisphere  in the ATR, CST, PLIC and SCR  in the AD group; these changes were driven by both higher AxD and RD with standard DTI, while FW correction showed that these changes in FAt may be driven by underlying lower AxDt. Using FW-DTI, lower FW-AxDt (AD < HC) were found in WM locations corresponding to lower FAt, while the fornix showed simultaneous lower FW-RDt (HC > AD) in a small cluster. With standard DTI, AxD was higher across multiple regions, consistent with prior literature62. However, the mechanism of higher AxD is unknown and contrary to prevailing theories of known AD pathology61. Using FW-DTI, regions of lower AxDt corresponded to lower FAt values, suggesting that prior reports of higher AxD may have been sensitive to the effects of FW. A similar trend of AxD decreasing with FW correction was previously reported using TBSS in the context of AD and cerebrovascular disease21. After FW correction, higher AxDt in AD, compared with HCs, was observed in regions with known crossing fibers, consistent with degeneration of select fiber tracts in these regions. Additionally, clusters with higher FW-FAt values in AD  were associated with higher FW-AxDt and lower FW-RDt. Using standard DTI analysis, no lower AxD or RD values were detected in AD subjects.Using standard DTI, non-specific higher AxD and RD were observed across WM, while FW-AxD21. Overall, FW-DTI provides not only more accurate and consistent DTI-based biomarkers, but also provides quantitative FW volume fractions, which may be a potential biomarker of sub-voxel neurodegenerative processes.The FW volume fraction was quantified using the FW-correction algorithm and, consistent with AD-related neurodegeneration, was higher in AD in many WM areas and tracts. More specifically, large clusters of higher FW were located in the cingulum , ILF and IFOF , forceps major and minor (approximately 40% each), and ATR (31.39%). Similar clusters were also found in the fornix (95.6%) and CC . Additionally, approximately 42% of the FW clusters were located in WM outside the regions specified by the atlases; in particular, FW increases were found in several regions bordering various sulcal fundi. Consistent with DTI-based metrics, the FW index further demonstrates an important connection between the fornix, CC, and AD. The high volume of FW in these regions may be related to loss of WM integrity; moreover, the contaminating effects of FW further validates the significant differences observed between standard and FW-DTI metrics. This metric may itself be a valuable biomarker, consistent with our hypothesis, as previous studies have shown that higher FW values were associated with poorer executive functioning, visual construction, and visuomotor performanceCorrelations between DTI metrics and various neuropsychology assessments could lend insight into the role of WM changes on cognitive function in AD pathology. In this study, we did not observe any significant voxel-wise correlations between MMSE and DTI metrics before or after FW correction in the AD cohort. This may be attributable to the similar group characteristics (predominantly very mild to mild AD) and the relatively narrow range of MMSE scores, while future studies could consider including more sensitive measures of cognitive functioning, including various sub-domains such as memory and executive function. Despite the lack of correlation in this study, the overall finding that FW-DTI metrics are more consistent with the pathology may lend insight into the inconsistent correlative findings across the literature. While the relationship between WM integrity as assessed by DTI and cognitive functions remains unsettled, the implementation of FW correction to yield more accurate DTI metrics should be considered as a methodological improvement for future correlation studies, particularly for the diffusivity metrics AxD and RD.63. Even so, all of these approaches necessitate the use of atlases, which may be limited in the WM regions that are represented ; this is particularly relevant for the FW index that showed widespread changes across WM, even outside of the atlas regions. In the present study, atrophy likely minimal due to the selected patient population, further enabling the use of a voxel-based approach. Second, no significant voxel-based correlations (p < 0.01 FWE corrected) were observed between standard DTI and FW-DTI metrics and the MMSE score, which is consistent with several previous studies16. This suggests that while WM is altered in AD, the relationship between global cognitive scores and WM pathology may not be linear. Future work could consider more sensitive measures of cognitive subdomains, and the inclusion of a longitudinal aspect may further pinpoint the temporal ordering of WM alterations relative to GM pathology and cognitive decline. Finally, as early biomarkers are of increasing interest, future studies should also include cohorts of MCI or subjects at high risk of developing AD.There are several limitations to this study. First, we used a voxel-based approach for WM analysis rather than a skeletonized approach, such as TBSS. As TBSS minimizes tract variance across subjects using a common skeletonized tract, it uses fewer voxels and is inherently less sensitive to partial volume effects. However, the higher number of voxels available with voxel-based approaches may increase sensitivity to subtle differences in microstructureIn conclusion, this study demonstrates that the implementation of a FW correction algorithm for DTI improves both the sensitivity and specificity of derived DTI metrics by removing PVEs, and better captures underlying AD-related pathologic changes than standard DTI approaches. We found significantly altered DTI metrics in AD compared to HC for both standard and FW-DTI, but importantly FW-DTI identified changes were more consistent with known AD pathology as hypothesized (both in terms of magnitude and direction of DTI changes). In addition, MA may improve specificity related to crossing fibers, while the FW index may improve sensitivity to sub-voxel neurodegeneration. Overall, FW-DTI improves the reliability and inter-parameter consistency of DTI metrics in the presence of atrophy, and the resulting metrics provide more sensitive and specific insight into AD-related pathological changes in white matter.Supplementary Information.


Preterm birth  is one of the global public health concerns. Epidemiologic evidence on the potential impact of perfluoroalkyl substances (PFAS) on PTB is still limited and inconsistent. We aimed to investigate the associations between prenatal PFAS exposure and PTB among singleton live births.We studied 2849 mother-infant pairs in the Shanghai Birth Cohort (SBC) from 2013 to 2016. Ten PFAS in maternal plasma in early pregnancy : 15 (13–16) weeks) were measured. Primary outcomes were duration of gestation, PTB, spontaneous PTB and clinically indicated PTB. A linear regression model was used to assess the associations between ln-transformed PFAS and duration of gestation (in weeks). Logistic regression models were applied to estimate the relative risks of these outcomes.n = 136) in this study population. In the linear regression analyses, PFAS were not associated with the duration of gestation after controlling for potential confounders. In the multiple logistic models, no significant associations were observed between PFAS and overall PTB, spontaneous or indicated PTB.The incidence of overall PTB was 4.8% (95% confidence limit: 4.0–5.6%, In this prospective cohort study, we did not observe significant associations between maternal plasma PFAS concentrations in early pregnancy and gestational length, overall PTB, spontaneous or indicated PTB. Preterm birth (PTB), defined as a birth < 37 completed weeks’ gestation, is one of the global public health problems with an average rate of 10.6% around the world . PTB is Environmental pollution, endocrine disrupting chemicals in particular, has been suspected to be one of the culprits . For exaPrevious studies demonstrated that PFAS could change lipid metabolism , disruptThe current study aimed to examine the associations between PFAS in early pregnancy and duration of gestation and risk of PTB in a large prospective cohort in Shanghai, China.The study subjects in the current analysis were from the Shanghai Birth Cohort, a large prospective study in Shanghai, China, from 2013 to 2016 . The incn = 289). Mothers or infants with missing information on maternal age at enrollment, pre-pregnancy body mass index (BMI), parental educational level, parity, chronic diseases during pregnancy or infant sex were excluded (n = 104). Finally, 2849 mother-infant pairs were included for analysis.For the current analysis, a total of 3242 women who provided blood samples in early pregnancy : 15 (13–16) weeks) and delivered a singleton live birth were eligible. Births with missing information on gestational age at birth, gestational age < 20 weeks or > 42 weeks were excluded  irrespective of mode of delivery , while clinically indicated PTB was defined as PTB for preeclampsia, fetal stress, placenta previa and other maternal, fetal or placenta indications [2), parity , pregnancy complicated by chronic diseases , and infant sex . Chronic diseases included heart, kidney or liver disease, diabetes mellitus, chronic hypertension disorders, epilepsy, malnutrition and anemia.Demographic characteristics included maternal age , parental education levels , smoking during pregnancy , gestational age at blood collection. Obstetric and medical covariates included pre-pregnancy BMI , parental education levels , parity , chronic diseases , infant sex  and gestational week at blood collection. Because only few mothers  smoked during early pregnancy, maternal smoking status was not included in the adjusted models.Distributions of demographic and pregnancy related characteristics were showed as numbers and percentages. PFAS with concentrations below the LOD were assigned LOD/ √2. The distribution of PFAS in early pregnancy was presented as medians and interquartile ranges. Multivariable restricted cubic spline model was used to examine potential non-linear relationships between PFAS and gestational age (in weeks) at birth, according to the ln-transformed PFAS at three knots of 10th, 50th, 90th percentile. Univariable and multivariable linear regression analyses were used to evaluate the associations of ln-transformed PFAS with gestational age (in weeks) at birth. Simple and multiple logistic regression analyses were applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the overall PTB, spontaneous PTB and clinically indicated PTB. In the logistic regression models, PFAS were entered as continuous variables (ln-transformed) and in tertile (untransformed) with the lowest tertile served as the reference group. Based on the directed acyclic graphs and existing literature, covariates were included in all adjusted models, including maternal age (years), pre-pregnancy BMI .Spearman’s rank correlation coefficient was used to estimate the correlations between each pair of PFAS. As multicollinearity between PFAS may bias the estimated effects, we conducted linear regression or logistic regression models with mutual adjustment of the correlated PFAS  and R software (R version 3.6.1). Results at 2) before pregnancy (74.4%). The vast majority (95.0%) of couples had more than 12 years of education and only 196 (6.9%) women complicated with chronic diseases during pregnancy. In addition, the rate of boys and girls was 51.1 and 48.9%, respectively  gave a singleton live birth before 37 weeks’ gestation, including 97 (71.3%) spontaneous PTB and 39 (28.7%) indicated PTB. More than 60% of the women aged less than 30 years and most of them were nulliparous (85.5%), nonsmokers (99.4%) and had normal weight . Median concentrations were 11.85, 9.33, 1.69, 1.69. 1.39, 0.54, 0.16, 0.06 and 0.04 ng/mL for PFOA, PFOS, PFNA, PFDA, PFUA, PFHxS, PFDoA, PFHpA and PFBS, respectively Table .Table 2Non-linearity was not detected for the association between any ln-transformed PFAS and gestational age at birth (in weeks) in a spline model  in Eastern Massachusetts reported that PFOS and PFNA exposures increased the risk of preterm birth [A previous cohort study of 3535 mother-infant pairs in Denmark found that the 3 upper quartiles of PFOA and PFOS in early pregnancy relative to the lowest quartile were significantly associated with PTB. This study also suggested an elevated risk of PTB for per doubling increase of PFHpS and PFDA concentrations . Howeverrm birth . Howevern = 1202) did not observe significant associations of maternal plasma PFOA, PFOS, PFHxS and PFNA in the first trimester with PTB [n = 252) reported that maternal serum PFOA, PFOS and PFHxS in the second trimester did not influence the risk of PTB [In contrast to the above studies, we failed to find any association, which was supported by several other studies –22. A Spwith PTB ; a cohork of PTB ; the C8 k of PTB . Likewisk of PTB , 19, 21.It should be mentioned that comparing results with previous studies was challenging because these studies were often incomparable in exposure level, study population and exposure window, all of which may affect the effect estimations. First, PFAS exposure levels among these studies varied widely. For instance, the PFOA level ranged from 1.3 ng/mL (geometric mean)  to 21.2 It is worth noting that none of the previous studies –22 diffeA recurrence risk of PTB could increase by 15 to 50% among women with a previous PTB . On the Several limitations of this study are worth noting. First, PTB was reported in one of our previous large-scale studies which conducted in 20 hospitals in Shanghai between 2015 and 2017 with an incidence rate of 5.6% . The preSecond, we did not adjust for hemodynamics in pregnancy in the analyses. Only two previous studies , 19 haveOur study found no significant associations between maternal PFAS concentrations in early pregnancy and the length of gestation or the risk of PTB.Additional file 1: Figure S1. Associations between PFAS (ng/ml) concentrations and gestational age at birth . PFAS concentrations were measured in maternal plasma in early pregnancy and have been ln-transformed before entering into the restricted cubic spline regression model. Model adjusted for maternal age, pre-pregnancy BMI, parity, parental educational levels, pregnancy complicating with chronic diseases, infant sex and gestational age at blood drawn.Additional file 2: Figure S2. Associations between non-transformed PFAS (ng/ml) concentrations and gestational age at birth . PFAS concentrations were measured in maternal plasma in early pregnancy. Model adjusted for maternal age, pre-pregnancy BMI, parity, parental educational levels, pregnancy complicating with chronic diseases, infant sex and gestational age at blood drawn.Additional file 3: Figure S3. Directed acyclic graph (DGA) illustrating confounders and modifier. (A) Path A indicated a direct effect; (B) The associations between PFAS exposure and outcomes of interest were confounded by maternal age, parity, pre-pregnancy BMI, parental educational levels and pregnancy complicating with chronic diseases and gestational age at blood drawn; (C) Fetal gender can be an effect modifier in the associations between PFAS exposure and outcomes of interest.Additional file 4: Table S1. Correlations between plasma concentrations of PFAS. Table S2. Associations between ln-transformed plasma concentrations of PFAS in early pregnancy and preterm birth. Table S3. Associations between PFAS concentrations in early pregnancy and preterm birth stratified by infant sex. Table S4. Associations between PFAS concentrations in early pregnancy and preterm birth in nulliparous women. Table S5. Associations between of PFAS concentrations in early pregnancy and preterm birth in women without chronic diseases. Table S6. PFAS median levels (ng/mL) in different areas. Table S7. Associations between ln-transformed plasma concentrations of PFAS in early pregnancy and length of gestation (weeks). Table S8. Linear regression was used to analyzed the associations between plasma concentrations of PFAS in early pregnancy and length of gestation (weeks) in three tertile groups. Table S9. Linear regression was used to analyzed the associations between plasma concentrations of PFAS in early pregnancy and preterm birth in three tertile groups. Table S10. Associations between PFAS concentrations in early pregnancy and preterm birth with adjustment of education in finer categories . Table S11. Associations between PFAS concentrations in early pregnancy and late preterm birth (34–36 weeks).


In 2007 the German government passed smoke-free legislation, leaving the details of implementation to the individual federal states. In January 2008 Bavaria implemented one of the strictest laws in Germany. We investigated its impact on pregnancy outcomes and applied an interrupted time series (ITS) study design to assess any changes in preterm birth, small for gestational age (primary outcomes), and low birth weight, stillbirth and very preterm birth. We included 1,236,992 singleton births, comprising 83,691 preterm births and 112,143 small for gestational age newborns. For most outcomes we observed unclear effects. For very preterm births, we found an immediate drop of 10.4%  and a gradual decrease of 0.5%  after implementation of the legislation. The majority of subgroup and sensitivity analyses confirm these results. Although we found no statistically significant effect of the Bavarian smoke-free legislation on most pregnancy outcomes, a substantial decrease in very preterm births was observed. We cannot rule out that despite our rigorous methods and robustness checks, design-inherent limitations of the ITS study as well as country-specific factors, such as the ambivalent German policy context have influenced our estimation of the effects of the legislation. There is strong evidence that smoke-free legislation improves adult health outcomes, such as cardiovascular health and mortality from smoking-related illnesses3. There is also evidence that smoke-free legislation improves pregnancy outcomes and child health4. For example, a recent systematic review found reductions in preterm birth rates, perinatal mortality and hospital attendance rates for asthma following smoke-free legislation4. However, findings are not fully consistent and based on less rigorous study methods4, and more rigorous studies are needed to strengthen the evidence base5.Over the past two decades, a range of policies and programmes at global, national and regional levels have been designed to reduce the detrimental harms associated with tobacco use6. Germany prohibited smoking in the workplace in 20047. In 2007 a national law was passed to protect non-smokers from the harmful consequences of SHS and required the implementation of federal state level legislation to prohibit smoking in public places8. Sargent et al.9 investigated the short-term effects of the smoke-free legislation on the national level and found a significant decrease in hospital admissions due to acute coronary events after implementation of the smoke-free legislation. No study, however, has assessed the effects of the smoke-free legislation on pregnancy outcomes in the German context.Since ratification of the World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) in 2004, Germany is obliged by international law to implement appropriate measures to reduce and prevent tobacco consumption and second-hand smoke (SHS) exposure10.In this study, we assess the impact of the smoke-free legislation on pregnancy outcomes implemented on 1 January 2008 in Bavaria, Germany’s largest and second most populous state with more than 12.5 million inhabitants and approximately one-sixth of all births in GermanyThere were 1,290,487 deliveries between 1 January 2005 and 31 December 2016. Due to the standardized data collection process related to Bavarian hospital births, there are no missing data of hospital births. We excluded 53,495 births (4.15%) because inclusion criteria were not met see Fig.  online.FOverall, there were 83,691 preterm births  during the study period. During the same time span there were 112,143 babies born small for gestational age (SGA) (< 10th percentile) and 60,763 born with low birth weight (< 2500 g); 11,001 were very preterm births  and 3,109 deliveries were stillbirths (intrauterine death > 500 g). Maternal, newborn and subgroup characteristics are specified in Table For the two primary and two out of three secondary outcomes, i.e. preterm birth, SGA, low birth weight and stillbirth, any effect on level changes or slope changes following implementation of the legislation in January 2008 was not statistically significant. We observed small effects with 95% confidence intervals (CIs) that include a decrease as well as an increase of the outcome rates. Results are presented in Table For the secondary outcome very preterm births we did observe a rate ratio of 0.8960  for level change. This represents an immediate relative decrease of very preterm births by 10.40% , corresponding to a level change from a very preterm birth rate of 0.98% in December 2007 to 0.89% in January 2008. We also observed a rate ratio of 0.9954  for a slope change in very preterm births. This represents a decrease of 0.46%  in the very preterm birth rate, corresponding to an additional relative decrease from e.g. 0.983% in July 2009 to 0.979% in August 2009, representing four very preterm births less each month.Consistent with the main analysis, most of the subgroup and sensitivity analyses showed small effects with confidence intervals suggesting that the effect could be in either direction  or gradual (slope change) effects of the smoke-free legislation on preterm birth, SGA, low birth weight and stillbirth; 95%-confidence intervals surrounding effect estimates suggest that the effect could be in either direction. Our findings were consistent across the majority of sensitivity and subgroup analyses.11, we observed no clear differential effect of the legislation according to SES.We observed statistically significant immediate and gradual reductions for very preterm births. We could replicate these findings in the sensitivity and subgroup analyses especially for smokers; however, here the effects were not statistically significant, probably due to a lack of power considering the much smaller population. Although the preterm birth and SGA rates were higher for the most deprived quintile (BIMD 5) as compared to the least deprived (BIMD 1), consistent with the literature4 observed an immediate drop (level change) in preterm birth, low birth weight, SGA and very preterm birth, but a gradual decline (slope change) only in SGA and very preterm birth. The effect estimates (risk differences) reported by the included studies were rather small for the outcomes preterm birth, SGA and low birth weight and greater for very preterm birth. In our study we observed, consistent with the meta-analysis in terms of relative effect size, unclear changes in all pregnancy outcomes following the smoke-free legislation except for very preterm birth.The literature shows mixed effects for the impact of smoke-free legislation on pregnancy outcomes, with a tendency towards a protective effect. Specifically, a recent systematic review and meta-analysis of mostly interrupted time series (ITS) studies performed by Faber et al.9. A difference-in-difference study examined short-term effects of the legislation on smoking behaviour using German socio-economic panel (SOEP) data. It found no impact of the legislation in the general population but only for those frequenting bars and restaurants more often12.In the absence of studies assessing pregnancy outcomes in Germany, up to now, only one published study employed a time series design to investigate the smoke-free legislation and found a short-term immediate decrease in hospital admissions for acute coronary events13 identified several factors, apart from methodological differences, that may explain the heterogeneous findings across studies assessing the effect of smoke-free legislation on pregnancy outcomes, namely: (i) different policy environments in terms of smoking prevalence and smoking norms, (ii) the presence of existing legislation prior to the smoke-free legislation under investigation, and (iii) differences in policy implementation and enforcement. These factors may contribute to understanding why we did not observe a clearer effect related to the Bavarian smoke-free legislation.A previous study conducted in Canada20. However, Germany, with a prevalence of 27% in 2008–2011 lies at the upper end of this range21 and Germany’s efforts in tobacco control have been poor compared to other countries24. According to the Tobacco Control Ranking Scale 201925, Germany occupies the last rank for successful implementation of tobacco control among 36 mostly European countries. For example, Germany currently is the only EU country, which still allows tobacco advertising on billboards1. Tobacco smoking is a well-established risk factor for fetal growth restriction and preterm birth. Indeed, we found large differences between smoking and non-smoking mothers regarding rates of preterm birth and especially SGA. However, we did not observe any drop or gradual decrease of preterm birth or SGA rates in either subgroup following the implementation of the smoke-free legislation. Furthermore, we did not observe changes in smoking rates related to the implementation of the legislation as assumed in our logic model and shown in other studies18. However, caution is merited, as a 5.9% smoking prevalence in our data is very likely an underestimation. On the other hand, the lack of a clear effect could, for example, also be explained by more health-conscious behaviour among pregnant women, who, prior to implementation of the smoke-free legislation, already avoided exposure to second-hand smoke.With regards to different policy environments, the smoking prevalence in other countries was similar to the prevalence in Germany, ranging from 18 to 27% among the female populationConsidering pre-existing legislation, as laid out in our logic model, the smoke-free legislation is not the only factor influencing pregnancy outcomes, and we did identify co-interventions and existing legislation prior to implementation of the legislation under investigation that may have subdued a more prevalent effect.26. While Bavaria has one of the strictest smoke-free legislations within Germany, the legislation lacked supporting interventions, such as an accompanying media campaign or tax increase. Studies in Spain, England and Scotland, where improvements in pregnancy outcomes associated with legislation were observed, also found high compliance with the legislation27. While we do not have data in Bavaria regarding actual enforcement or compliance, it is possible that the unclear effects could be explained by a lack of enforcement or compliance considering Germany’s lacking efforts in tobacco control24.Finally, regarding the role of policy implementation and enforcement, recent studies have shown that the health impact is larger when the smoke-free legislation is more comprehensive28. Beyond this design-inherent limitation, the use of rigorous a priori methods is important.The ITS study design is prone to certain methodological limitations. The lack of randomization and thus potential confounding make it difficult to definitely attribute causality to the intervention-outcome relationship29 to account for common methodological and conceptual flaws in assessing population level interventions with time series designs30. In particular, we took a complex systems approach and used a logic model to conceptualize our study and identified co-interventions and other risk factors prior to conducting our study31. We registered a detailed study protocol, in which we defined an impact model, the main statistical analysis, as well as the sensitivity and subgroup analyses. Had we used, for example, our originally planned impact model including the full range of data from 2000 to 2016, we would have come to different conclusions. However, in such a model the pre-intervention slope (and therefore level and slope change) was defined primarily by a major breakpoint in 2004 caused by several co-interventions in this year. This emphasizes the importance of choosing an appropriate impact model, as described by Lopez Bernal et al.32.We used a high quality, large dataset and followed the steps outlined in the tutorial developed by Lopez Bernal et al.34. Also, the interrupted implementation of the legislation in Bavaria from 2008 to 2010 makes it difficult to identify the exact time at which we can expect to see an effect. Some studies have investigated an immediate onset19, or even an anticipatory intervention time point17 whereas others have used an intervention time point nine months after the actual implementation of the intervention13. A study which investigated the smoke-free legislation in the different cantons of Switzerland, found that the more time a mother spent under the smoke-free legislation the fewer were the risks for preterm birth and early-term births20.Our choice of the correct impact model, however, was also associated with several uncertainties. Many aspects of biological processes of pregnancy and especially the exact window of susceptibility of pregnancy to smoking are insufficiently understood35. Indeed, a recent study has shown the limitations of single group ITS studies assessing the impact of smoke-free legislation on mortality in Spain where initial protective intervention effects from a single group ITS study were not confirmed after the addition of a comparable geographical control site36. Despite careful preparation and consideration, we may have failed to identify important confounders or co-interventions, considering the complexity of the intervention as well as of the system in which it was implemented. Additionally, little concrete guidance on choice of statistical model exists, and determining the ‘best-fit’ model among a range of alternatives remains at least partially arbitrary. Gasparrini et al.37 already concluded that the model specifications, among other factors, have a strong impact on the effect estimate when assessing smoke-free legislation on acute myocardial infarction. We aimed, however, to define statistical parameters a priori, where possible, and to comprehensively report modelling choices by publishing our code alongside the manuscript.A further design-inherent weakness of the single-arm ITS study design is the lack of a concomitant, geographical control group38. It is increasingly used in the field of healthcare and public health40. This study design usually draws on routine data collected over time to identify any underlying time trends, and can thereby observe changes after the implementation of an intervention compared to a counterfactual scenario . We pre-specified the study methods including the main impact model, main analysis, and subgroup and sensitivity analyses in a study protocol .We applied an ITS study design to assess the association between implementation of the smoke-free legislation and pregnancy outcomes using monthly data from all births in Bavaria between 2005 and 2016. The ITS study design is considered to be one of the best alternatives to assess intervention effectiveness of population-level interventions where randomization is considered infeasible42. It provides extensive information retrieved from all hospitals in Bavaria regarding maternal and neonatal demographic and health-related characteristics, clinical management, and pregnancy complications. The data are subjected to a series of formal and contextual plausibility checks43.We included aggregated data from a high-quality routine dataset of maternal and neonatal health indicators, collected and managed by the Bavarian Institute for Quality Assurance in hospital care (BAQ). This dataset contains all Bavarian in-hospital births, which constitute about 99% of all births in Bavaria44), both measured as the percentage of these outcomes among all births that occurred during a given month. Secondary outcomes included monthly percentages of low birth weight (< 2500 g), very preterm birth , and stillbirth (intrauterine death > 500 g).Our primary outcomes included preterm birth , and SGA  and other risk factors influencing pregnancy outcomes over the study period, which we describe in detail in our study protocol.47. Due to political arguments, however, the Bavarian legislation was loosened on 1 August 2009, and smoking was permitted again in a subset of restaurants, e.g. in restaurants larger than 75 m2 mainly serving drinks. Following a referendum, which allowed Bavarian citizens to vote directly for or against more restrictive smoke-free legislation, the legislation was tightened again on 1 August 2010. This iteration additionally banned smoking in beer and event tents49, making the Bavarian smoke-free legislation one of the strictest in Germany48. Violations of smoke-free legislation for smokers as well as event organisers include fines between 5 and 1,000 Euros, but information on enforcement and compliance is lacking.The 16 German federal states are responsible for the implementation of the national law for the protection of non-smokers, and individual state legislation varies in strength , as well as in timing of implementation (ranging from August 2007 to July 2008). Bavaria implemented the smoke-free legislation on 1 January 2008. Thereafter, smoking was prohibited in all public buildings and institutions, such as universities, hospitals, retirement and nursing homes, and restaurants and bars52.We hypothesized that the effects of the smoke-free legislation may be detected as an immediate drop (level change) and gradual decline (slope change) in pregnancy outcomes at the first introduction of the smoke-free legislation on 1 January 2008. The changes could be the result of an immediate reduction in maternal exposure to SHS, and/or an immediate reduction in active maternal smoking . They could also be impacted by longer-term influences on sociocultural norms, affecting smoking behaviours in different settings7, a major cigarette price increase in September 200453, as well as a documentation change initiated in January 2004. Therefore, we shortened the pre-intervention time period, using data from 2005 to 2016. The main impact model is therefore based on a pre-intervention period from January 2005 to 31 December 2007 and a post-intervention period from 1 January 2008 until 31 December 2016. We report, however, additional sensitivity analyses using data from 2000 to 2016.Originally, we planned to use data from 2000 to 2016 with equal time periods pre- and post-intervention. Upon visual inspection of the data we identified, however, a series of pronounced changes in outcome rates in the year 2004, during the pre-intervention period, that we were not able to sufficiently account for through adjustments in the analysis. These changes were potentially triggered by the smoking ban at work in August 200436. As the analysed monthly data were not independent from one another, we adjusted for seasonality through the inclusion of monthly dummy variables and/or for autocorrelation through the inclusion of auto-regressive structures in the model. We performed goodness-of-fit tests to decide whether to use a Poisson or a more flexible Negative Binomial model. We scrutinized auto-correlation function (ACF) and partial auto-correlation function (PACF) plots visually and compared Akaike’s information criteria (AIC) to see whether the model performed better after adjustment for seasonality and/or remaining autocorrelation.We performed a segmented regression analysis using a generalized linear model with log-link for all analysesThe final models were generalized linear models  including seasonal dummy variables and/or a random effect term comprising the appropriate autoregressive terms. As we were dealing with count data, we were using the population as an offset variable in order to transform back to rates. The main statistical formula is depicted below:54, and the analyses using R55. The complete R code for the main statistical analysis is available in the Supplementary Information online.We performed data management with SAS software version 9.4We specified a series of sensitivity and subgroup analyses a priori for the primary outcomes preterm birth and SGA  approved the use of the data.Supplementary Information.


Escherichia coli, or the TLR4‐dependent pro‐inflammatory lipid, platelet‐activating factor (PAF). Here, we summarise the rationale for targeting TLR4 as a master regulator of inflammation in fetal and gestational tissues, and the potential utility of TLR4 antagonists as candidates for preventative and therapeutic application in preterm delivery and fetal inflammatory injury.Every year, 15 million pregnancies end prematurely, resulting in more than 1 million infant deaths and long‐term health consequences for many children. The physiological processes of labour and birth involve essential roles for immune cells and pro‐inflammatory cytokines in gestational tissues. There is compelling evidence that the mechanisms underlying spontaneous preterm birth are initiated when a premature and excessive inflammatory response is triggered by infection or other causes. Exposure to pro‐inflammatory mediators is emerging as a major factor in the ‘fetal inflammatory response syndrome’ that often accompanies preterm birth, where unscheduled effects in fetal tissues interfere with normal development and predispose to neonatal morbidity. Toll‐like receptors (TLRs) are critical upstream gatekeepers of inflammatory activation. TLR4 is prominently involved through its ability to sense and integrate signals from a range of microbial and endogenous triggers to provoke and perpetuate inflammation. Preclinical studies have identified TLR4 as an attractive pharmacological target to promote uterine quiescence and protect the fetus from inflammatory injury. Novel small‐molecule inhibitors of TLR4 signalling, specifically the non‐opioid receptor antagonists (+)‐naloxone and (+)‐naltrexone, are proving highly effective in animal models for preventing preterm birth induced by bacterial mimetic LPS, heat‐killed  Inflammation is the central mechanism of spontaneous preterm birth, and TLR4 is an essential mediator of the microbial and endogenous pro‐inflammatory triggers. Preclinical studies identify TLR4 as an attractive pharmacological target to protect against preterm birth and fetal inflammatory injury. In this review, we discuss the biological rationale and clinical potential of novel small‐molecule inhibitors of TLR4 signalling that offer promise for preventative and therapeutic applications in preterm birth. New therapeutic options to tackle spontaneous preterm birth and mitigate its adverse impact in infants born prematurely are urgently needed.Infection is a common cause of preterm birth, but sterile factors and insults, such as exposure to oxidative stress and toxins, immune or endocrine imbalance, multiple births, and placental hypoxia and haemorrhage, are also risk factors.The inflammatory processes of term and preterm birth are broadly comparable, but in preterm birth there can be different initiating triggers, kinetics and scale of response, and a lack of coordinating regulation.in utero conditions are unfavorable for continued fetal development and viability. Substantial clinical data point to a critical role for TLR4 and innate immune activation in humans – a large genome‐wide association study (> 40 000 women) identifies rare variants in genes encoding negative regulators of innate immunity and anti‐microbial defence, as predisposing to preterm birth.Toll‐like receptors (TLRs) are pivotal upstream gatekeepers of innate immune activation and are abundantly expressed in the placenta, fetal membranes and uterus.In this review, we assemble evidence from animal models and clinical studies implicating TLR4 as a key rate‐limiting mediator in preterm birth and discuss novel compounds that inhibit TLR4 signalling and their potential utility in suppressing inflammation to extend gestation, and protect the fetus from inflammatory injury.TLR4 is one of 13 receptors, each with specific sets of cognate ligands, which make up the mammalian TLR system. Each TLR engages ligands released by different microbes, known as pathogen‐associated molecular patterns (PAMPs). The key microbial ligand for TLR4 is lipopolysaccharide  from cell walls of Gram‐negative bacteria. TLR2 recognises peptidoglycan (PGN) and lipoteichoic acid (LTA) common to several bacterial taxa, while other TLRs bind double‐stranded RNA viral motifs or bacterial flagellin.Typically, LPS activation of TLR4 signalling involves formation of a receptor complex consisting of TLR4, MD‐2 and adaptor molecules including myeloid differentiation factor 88 (MyD88) and MyD88 adaptor‐like , as well as TIR domain‐containing adaptor‐inducing interferon‐beta (TRIF)As well as microbial elements, TLRs recognise endogenously produced agents released from intracellular and extracellular compartments after cell stress or necrotic cell death. Endogenous TLR ligands are known as damage‐associated molecular patterns . DAMPs provide a physiological signal of tissue stress and damage, to initiate tissue defence and repair mechanisms.TLR4 occurs.An array of TLRs is expressed in a spatially and temporally controlled manner in the fetal and maternal compartments of the gestational tissues. TLR4 is strongly expressed in the fetal membranes, placental trophoblasts and endocervixTlr4−/−) mice have an extended gestation length and increased perinatal mortality compared to wild‐type controls.Il1b, I16, I112b and Tnf normally induced in wild‐type fetal membranes, placenta and uterus prior to term labour. Additionally, Tlr4−/− mice have delayed expression of UAGs involved in transitioning the uterus from a quiescent to contractile state, notably prostaglandin F receptor, oxytocin receptor and connexin‐43. Leucocyte accumulation is impaired in TLR4‐deficient females, with fewer neutrophils and macrophages in the placenta, and fewer dendritic cells and more regulatory T cells in the uterus, compared to wild‐type mice. Unlike TLR4 deficiency, genetic disruption of MyD88 does not delay parturition,Mouse models have been instrumental in defining TLR4 as a rate‐limiting effector at the apex of the inflammatory response driving uterine activation and controlling timing of labour. In mice, TLR4 is readily detectable in maternal tissues including uterine decidua and cervix, fetal membranes and placenta, and becomes elevated in the cervix and uterus towards the end of gestation.Tlr2 (Tlr2−/− mice) have delayed labour compared to wild‐type mice,Tlr2−/− mice in late gestation exhibit altered expression of activation markers Il1b and Arg1 mRNA.TLR2 interacts with TLR4 to regulate labour, and there is redundancy between the two receptors. Mice with a null mutation in Since infection is not usually present in healthy term labour, endogenous DAMPs including HMGB1, cell‐free DNA and oxysterols released from senescent cells in fetal membranes and placenta are implicated in TLR4 signalling in term labour. There is compelling evidence that pro‐inflammatory signals released from the maturing fetal lung, notably surfactant protein‐A (SP‐A), SP‐D and PAF, drive inflammation in fetal membranes and uterine tissues to precipitate the labour cascade.Escherichia coli and Fusobacterium species. Other common bacteria associated with preterm delivery are the genital mycoplasmas, Ureaplasma parvum and Mycoplasma hominis, and Gram‐positive organisms such as Streptococcus agalactiae, peptostreptococcus spp. and Gardnerella vaginalisIn preterm labour, TLR4 ligands accumulate prematurely as a result of infection and/or sterile stressors in maternal and/or fetal compartments in the gestational tissues. Intrauterine infection occurs in 25–40% of preterm birth cases, particularly in early preterm birth.Infection is associated with elevated accumulation of pro‐inflammatory cytokines in the amniotic fluid of preterm labouring women, with higher levels of IL‐1β, TNF and IL‐6 compared to women without infection.In preterm birth without infection, excessive DAMPs including HSP70, HMGB1, cell‐free DNA, uric acid and oxysterols released from stressed and dying gestational tissues are thought to provoke pro‐inflammatory activation.Tlr4 do not deliver preterm after intrauterine administration of heat‐killed E. coli. In contrast, C3HeB/FeJ mice that express Tlr4 exhibit 100% preterm delivery.Tlr4−/− mice are also resistant to preterm labour induced by LPS.Tlr4−/− mice and C3H/HeJ mice both exhibit a lower fetal death rate and decreased placental necro‐inflammatory response after administration of Fusobacterium nucleatum compared to wild‐type mice.Tlr4−/− null mutation does not protect wild‐type fetuses from inflammatory injury.Mice with genetic deficiency in TLR4 provide compelling evidence that TLR4 is critical for preterm labour induced by Gram‐negative bacteria. C3H/HeJ mice that carry a spontaneous mutation in Myd88−/− and Myd88/Trif−/− mice do not deliver preterm and exhibit normal fetal viability after administration of intrauterine heat‐killed E. coli.Trif−/− and wild‐type mice are susceptible to preterm delivery and fetal death in utero.Myd88−/− and Myd88/Trif−/− mice after E. coli delivery, accompanied by lower expression of Il1b and Tnf mRNA, compared to Trif−/− and wild‐type mice.In mice, MyD88 facilitates LPS‐induced preterm labour (in contrast to term labour), while TRIF appears dispensable at least for TLR4‐mediated triggers.E. coli administration.Ptgfr, Oxtr and Gja1Tlr4−/− mice, expression of Il1a, Il6, Il12b, Tnf and Il10 in fetal membranes, placenta and uterus is blunted after LPS administration, showing TLR4 is upstream and necessary for cytokine induction.Mouse models have been informative for defining the feed‐forward mechanisms that amplify TLR4 signals to promote uterine transformation and cause fetal injury. In late gestation pregnant mice, IL‐1α, IL‐1β, IL‐6 and TNF are synthesised in the uterus and fetal membranes after transcriptional activation as early as 3–4 h after LPS or Il1r1/Tnfrsf1a−/−) exhibit reduced preterm delivery in response to heat‐killed E. coli, compared to wild‐type mice.Il6 gene both protect mice from LPS‐induced preterm birth.Il6 nor Il1b null mutation fully abrogate susceptibility to preterm birth induced by intrauterine heat‐killed E. coli.IL‐1β signalling is critical, as demonstrated by the potent efficacy of IL‐1R antagonist to block preterm birth and fetal inflammatory injury after LPS exposure.Il10 null mutation have greater susceptibility to LPS‐induced preterm birth.Il10−/− mice from preterm delivery.IL‐10 counteracts the effects of pro‐inflammatory cytokines to protect mice from preterm birth. Mice with Mouse models also allow the biological roles of specific leucocyte lineages in preterm birth to be defined. After administration of a microbial stimulus, cytokine induction is rapidly followed by influx of inflammatory leucocytes into gestational tissues. This response resembles the immune cell changes that accompany term birth and are attributable to direct and indirect effects of TLR4 signalling. Innate immune cells are the first and largest component of the leucocyte infiltrate, with neutrophils arriving in the myometrium and decidua within hours after intraperitoneal LPS.Macrophages accumulate in the uterine decidua, but not myometrium, during LPS‐induced preterm birth.Cells of the adaptive immune response are also involved. LPS administration causes activation of T cells and NK cells in maternal blood and placenta in mice, while anti‐TLR4 antibody administration diminishes the T‐cell and NK cell changes.Rag1−/−) are more susceptible to LPS‐induced preterm delivery than wild‐type mice.+ T cells before LPS injection protected Rag1−/− mice from premature delivery.+FOXP3+ uterine Treg cells normally decrease after LPS treatment,A shift towards an immunogenic profile in T cells can reflect loss of immune tolerance, to increase susceptibility to preterm birth. Chronic chorioamnionitis characterised by excessive effector T cells is common in late preterm birth in women and is presumed to reflect excessive erosion of maternal allograft tolerance.Together, the data from genetic mouse models provide evidence of a specific causal sequence in the roles of cytokines and immune cells leading to preterm birth. The results point to TLR4 signalling as a key effector at the apex of the inflammatory cascade and show TLR4 ligation causes MAPK and NF‐κB activation that induces IL‐1β and IL‐6 to elicit recruitment of pro‐inflammatory leucocytes, shift the phenotypes of regulatory immune cells and ultimately induce expression of uterine activation and cervical remodelling genes , conferring greater susceptibility to preterm birth induced by heat‐killed E. coli.Tlr4−/− mice have substantially lower preterm birth rates and a reduced placental and decidual cytokine response to cPAF.Furthermore, PAF acts to amplify PAMP‐induced preterm birth. Elevated PAF secretion along with increased Surfactants are phospholipid‐rich proteins of the collectin family synthesised by pulmonary alveolar type II cells to reduce surface tension and enable mature lung function after birth.Spa mRNA commences on gd 15 in the fetal lung and progressively increases until delivery 3–4 days later.Spa/d−/− mice causes delayed parturition in the second pregnancy, but not the first pregnancy, implying an interaction with the immune response and/or tissue repair mechanisms affected by parity.Spa/d−/− mice have reduced myometrial expression of Il1b and Il6 and the UAGs, Gja and Oxtr, in late gestation compared to wild‐type controls.Experiments in mice show that expression of 2.in vivo. In contrast, SP‐A exerts anti‐inflammatory effects in human amnion explants, acting to downregulate expression of IL1B, CXCL2 and CXCL5.Tnf, Il1b and Ccl5 expression in placenta and fetus. Further work is thus required to define how SP‐A interacts with TLR signalling induced by PAMPs to regulate inflammation in gestational tissues and the timing of labour.The kinetics of SP‐A onset in the human fetal lung suggests a more complex role than in mice. SP‐A synthesis begins around the final 2 months of gestation, and amniotic fluid SP‐A accumulates progressively until term,ex utero,Collectively, these studies provide convincing evidence that TLR4 is a key mediator of microbial and endogenous pro‐inflammatory effectors originating in the uterus, fetus and placenta, with a pivotal role in provoking parturition in a wide range of preterm and term scenarios. As well as infection‐associated preterm birth, TLR4 is implicated in sensing and amplifying amniotic fluid PAF and SP‐A, critical fetal signals that trigger parturition to coincide with fetal maturation and ability to survive F. nucleatum, a Gram‐negative bacterium that causes preterm birth and premature rupture of membranes in women.This scheme highlights TLR4 as an attractive drug target for delay or prevention of preterm birth  can prevent cardiac pro‐inflammatory cytokine induction and left ventricular hypertrophy and dysfunction.To date, most studies have concentrated on preterm birth as an endpoint rather than fetal outcomes. However, TLR4 antagonism may also be effective in prevention of fetal inflammatory injury resulting from preterm birth. Studies in rats show that after treatment to induce neonatal hyperoxia, a TLR4 antagonist LPS from the photosynthetic bacterium Using mouse models, we have explored the utility of novel TLR4 antagonists of the (+)‐naloxone family as pharmacological interventions for preterm labour.As a first approach, we tested the effect of (+)‐naloxone on birth outcomes in wild‐type mice. When (+)‐naloxone was given in late gestation, there were no adverse effects on pup health despite parturition and labour being delayed, consistent with an essential role for TLR4 in normal on‐time birth.E. coli, which more closely mimics the clinical situation. Furthermore, the local induction by E. coli of inflammatory cytokine genes Il1b, Il6, Tnf and Il10 in fetal membranes was suppressed, and (+)‐naloxone similarly attenuated cytokine expression in the placenta, uterine myometrium and decidua.There are limitations of LPS models of preterm birth, given that clinically more than one TLR ligand would usually be involved. It is predictable that, as a TLR4 antagonist, (+)‐naloxone should be effective in blocking LPS actions. Therefore, it was important to evaluate the efficacy of (+)‐naloxone in other models. A similar protective effect of (+)‐naloxone was seen in preterm birth induced by intrauterine administration of heat‐killed We have also utilised (+)‐naltrexone, which is structurally and functionally similar to (+)‐naloxone,in utero, male but not female offspring exhibited elevated adipose tissue mass, reduced muscle mass, and elevated plasma leptin concentrations at 20 weeks of age. These effects were largely reversed by co‐administration of (+)‐naloxone. LPS‐induced expression of Il1a, Il1b, Il6, Tnf and Il10 in fetal brain, placental and uterine tissues, where (+)‐naloxone suppressed the LPS‐induced cytokine expression. Fetal sex‐specific regulation of cytokine expression was evident, with higher Il1a, Il1b, Il6 and Il10 induced by LPS in tissues associated with male fetuses and greater suppression by (+)‐naloxone of Il6 in females. These data demonstrate that modulating TLR4 signalling with (+)‐naloxone protects against inflammatory diversion of fetal developmental programming in utero, associated with attenuation of gestational tissue cytokine expression in a fetal sex‐specific manner. Male fetuses often suffer more extensive damage or higher rates of fetal loss, with surviving fetuses experiencing a more debilitating legacy of inflammatory injury.The high survival rates and lack of overt adverse impact of (+)‐naloxone treatment in pups are encouraging, although studies to investigate impact of (+)‐naloxone on fetal tissues susceptible to inflammatory injury are not yet completed. In a recent study, we showed that (+)‐naloxone can protect against adverse metabolic programming effects induced by fetal exposure to inflammatory mediators.These data imply that (+)‐naloxone protects the developing fetus from cytokines synthesised locally in fetal tissues after LPS accesses the fetal circulation.(+)‐Naloxone, (+)‐naltrexone and related drugs may have clinical advantages compared to neutralising antibodies and other TLR4 antagonist compounds. (+)‐Naloxone potently blocks LPS‐induced TLR4‐mediated signalling in several non‐pregnancy models, suppressing NF‐κB activation and inhibiting TNF and IL‐1β induction in immune cells.These data indicate further studies are warranted to investigate small‐molecule inhibition of TLR‐driven inflammation as a strategy for fetal protection and delaying preterm birth. Three different intervention scenarios can be envisaged. Firstly, there is an urgent need for treatments to effectively curtail threatened preterm birth at clinical presentation, to prolong gestation and protect the fetus against inflammatory injury, while allowing a window of time for antibiotic therapy and corticosteroid treatment to promote fetal lung maturation. In the event of infection, TLR4 inhibitors could be administered together with antibiotics. Even during successful antibiotic therapy, substantial amounts of PAMPS are generated. A TLR4 inhibitor would reasonably be a useful adjunct to antibiotics, acting to suppress inflammatory activation.TLR4 gene polymorphisms associated with an increased risk of spontaneous preterm delivery.Secondly, TLR4 antagonists might have value as prophylactic agents that target the initiating triggers, and feed‐forward signals, to dampen or arrest parturition before overt symptoms arise. Predictive tests would be required to identify at‐risk women in early gestation, and allow tailored interventions appropriate to individual clinical parameters. TLR4 inhibitors may be particularly helpful in pregnant women at high risk of excessive inflammation as a result of exposures to environmental toxins or stressors, which can elevate TLR4 expression in the absence of infection.in utero inflammatory insult include fetal and newborn brain white matter destruction, cerebral palsy, necrotising enterocolitis and chronic lung disease,in utero. These complications have lifelong consequences for the survivors of preterm birth, and treatments that safely and effectively reduce or resolve inflammation in the neonatal phase will deliver enormous benefit.Thirdly, there is potential for TLR4 inhibitor use in preterm neonates to suppress progression of fetal inflammatory response syndrome.TLR4 agonists, either of microbial origin or originating endogenously after tissue injury, are clearly implicated in the pathophysiological mechanisms of spontaneous preterm birth, and fetal inflammatory response syndrome. Compelling preclinical studies show that TLR4 has a master role in parturition‐associated inflammation and is involved in its initiation, progression and persistence. TLR4 antagonism has a clear pharmacological advantage over current therapeutic strategies for treating preterm labour, in targeting the apex of the inflammatory cascade. Treatment scenarios including prophylactic and therapeutic administration to at‐risk women, or to premature neonates, can be envisaged. These alternative administration scenarios are associated with different clinical, pharmacological, and ethical challenges and imperatives.A pharmacologic strategy targeting TLR4 would overcome an important limitation of existing tocolytic agents, such as prostaglandin inhibitors, that attempt to stem uterine maturation and contraction, or progesterone, which offers less effective anti‐inflammatory suppression and has limited efficacySmall‐molecule inhibitors of the (+)‐naloxone family are potential drug candidates that offer considerable promise and warrant further development. Amongst the benefits of this family of compounds is their relatively straightforward synthesis, stability during handling and transport, and potential suitability for use in low resource settings, where the majority of infant mortality occurs.in utero exposure to TLR4 inhibitors, and their protective effect in the fetus against the damaging actions of inflammatory mediators, and ongoing consequences of inflammatory injury after birth.in utero, and ensure the benefits outweigh the alternative strategy of delivery and neonatal intensive care. In this context, it is critical to appreciate the differences between acute treatment for women in suspected preterm labour and chronic treatment of women to prevent onset of inflammation‐associated complications, and to design studies accordingly.A key consideration is the response of the neonate to The authors declare no conflict of interest.


Background: Hydatid cyst is a zoonotic infection caused by Echinococcosis granulosus. Primary single-intramuscular hydatid disease is rare, even in endemic regions of the world. Here we report the case of exceptional thigh mass due to a hydatid cyst in an Iranian man.Case presentation: An 86-year-old man, initially presented to Velayat teaching hospital surgery clinic in May 2017 with a single right-thigh mass, but physical examinations of other organs were unremarkable. Based on sonographic findings, the differential diagnosis was hydatid cyst. He underwent surgical resection of the cyst. Histopathological results confirmed the diagnosis. There was no evidence of recurrence of the lesion during the 23-month follow-up. Conclusion: Increase thigh mass due to a hydatid cyst is a rare event. In endemic regions with the presence of hydatid cysts, especially physicians of surgical clinics have to consider differential diagnosis of hydatid cysts in unusual locations in case of such a lesion. Echinococcosis granulosus . Th. ThEchinanulosus . Dogs are the main host and humans are the incidental hosts , 4]. Wh. Wh4]. WSome occupations which work closely with animals have the highest risk of the infection, such as tanners, slaughterhouse workers, shepherds, butchers, stockbreeders and veterinarians . CE is eThis infection affects the liver and lungs in most cases (more than 90%), but all organs such as kidney, spleen, brain and other soft tissue can be affected . CE rareHydatid cysts may be asymptomatic in most cases, but there are variety of symptoms depending on the size, location, and complications. Cyst rupture and immunological reactions are the most frequent complications , 6], , . An intrThe diagnosis of this disease may be coincidental, or the patient may refer with a mass in the specific organ. Based on the radiological appearance, cyst viability and contents, hydatid cysts are classified into five stages. An active disease with clear contents and “water-lily” sign in radiology indicate the first and second stages. The third stage is characterized by the integrity of the cyst being compromised by the host immune system or drugs. The fourth and fifth stages include an inactive cyst and thick, calcified cyst walls . One of For a muscle cyst, MRI is the best imaging procedure, as it can show the location of the cyst and its relation to other structures. MRI can detect hydatid cysts in all parts of the body , 26]..26].The most effective treatment of hydatid cyst is radical surgery. The most dangerous complication during surgery is cyst rupture, because it causes anaphylactic shock and dissemination throughout the body. Using 0.5% cetrimide, 15% hypertonic saline, 1% silver nitrate and sodium hypochlorite solutions can kill the daughter cysts and decrease the risk of anaphylactic reactions . The hydatid cyst has a specific character, and a long incubation time from exposure to definite diagnosis. For a definite diagnosis of CE, in addition to a complete medical history and physical examination, travel history and contact with animals play an important role . The preOn the other hand, one of the rarest systems to be affected by hydatid cyst is the musculoskeletal system (about 0.5–4%). Most of the time, it is an incidental finding in lung or liver, or by imaging studies in endemic regions . HoweverThe current paper reports this case and an associated literature review.Prior to entering the study, informed consent was obtained from the patient. The patient was an 86-year-old man presenting with a 2-month history of mass increase in the anterior part of the right thigh,who wa sreferred to the surgical clinic of Velayat teaching hospital of Qazvin in 2017. In last few weeks prior to presenting at the hospital, the mass had increased in size and the patient was aware of it. It prompted the patient to seek out a doctor. He had ischemic heart disease (IHD) and hypertension. His history of medications included nitroglycerin, aspirin (80 mg) and methyldopa. He had no past surgical history and his family history of CE and other diseases was negative. He was a retired grocer living in Qazvin city, which is not endemic for hydatid cyst. The patient had no contact with dogs, sheep, or other animals. He had no recent travel history to an endemic region, and his job was not related to CE, so suspecting hydatid cyst in this patient was not obvious. In appearance, he was not ill and denied any weight loss in the previous six months. The patient’s physical examination revealed a soft, non-tender and mobile mass inside the quadriceps femoris muscle. There were no skin changes or inguinal lymphadenopathy in right thigh. His laboratory, hematology, biochemistry and serologic findings were normal and erythrocyte sedimentation rate (ESR) was 65 mm/h. The differential diagnoses were abscess, hematoma and benign tumors .Ultrasound findings demonstrated a hypoechoic encapsulated multi-cystic lesion (M: 9*8*6.5 cm) inside the anterior quadriceps femoris muscle. There was a smooth calcification in the cyst wall. The most important differential diagnosis based on imaging findings was hydatid cyst Figure 1 .To exclude hydatid cyst in other organs, such as the liver and the lung, a pre-surgical CT scan of the chest and abdomen was performed, which chest and intra- abdominal organs were normal limit. Before surgery, the patient was prescribed albendazole. The patient that was suspected to have a hydatid cyst, underwent surgical resection of cyst. The cystic lesion was completely removed without damaging the cyst Figure 2 . A revieThe patient’s post-surgical course was non-remarkable. There was no evidence of recurrence of the lesion during the 23-month follow-up. The patient continued treatment with albendazole for three months. The imaging and histopathologic findings of our case are consistent with hydatid cyst referred with thigh mass. Echinococcosis is a larval infestation characterized by metacestode (hydatid) cysts in the intermediate host. This cyst consists of an inner germinal layer and an outer laminated layer. These layers are the parasite-derived layer, the interior of which is nucleated with daughter cysts, and brood capsules with scolices, while the outer part is hyalinized and acellular. The third layer is a host-produced fibrous capsule which is surrounded mainly by an inflammatory infiltration of lymphocytes, plasma cells, scant neutrophils leukocytes, eosinophils and occasional histiocytic aggregation; foreign body granulomatous reaction is also involved . E. granulosus occurs in the small intestine within 4–5 weeks after the eggs enter via a gastrointestinal pathway. The larvae are released in the intestine and penetrate the epithelium to reach the lamina propria. The larvae spread in the body through the blood and lymph systems. Finally, in target organs, the larvae develop into a hydatid cyst , , . BecauseIn the case report by Tahir et al. , the patSome clues can guide physicians to diagnosing this disease, e.g., occupation, travel history, occupation, and contact with animals and living in endemic locations. But diagnosing this specific disease in our patient without a related occupation or living in an endemic area was challenging.The incidence of CE is not related to age, and occurs in all age groups. Case reports documented hydatid cysts in persons between 1 and 75 years of age . Our patHydatid cyst manifests as a slow-growing mass resembling a soft-tissue tumor and can mimic symptoms of myositis or calcified hematoma , making Serological tests are available for this infection which help reach a definite diagnosis. The size, location and clinical stages of CE can affect the accuracy of serological tests. The same serological tests performed in different laboratories can change the sensitivities . HoweverThe sensitivity of serological tests depends on the stage of disease; patients with inactive or early cystic stages have test sensitivity of 54.8%, but the sensitivity for patients with active cysts increases . Our patSize and location of CE are important for complete healing. Perioperative albendazole treatment (at least four days before surgery) reduces the relapse rates, parasitic burden and risk of anaphylactic shock . In our Because differential diagnosis and the radiological features were unclear, complete excision of the lesion was the best option for definite diagnosis . MedicatDespite the exact and repeated review of the patient's history, some questions remained unanswered: How did the patient become infected with CE and what caused CE to infect his thigh?Thigh mass due to hydatid cyst is rare. Physicians in surgery referral treatment centers, especially those in endemic hydatid cyst areas, are much more likely to be familiar with and aware of this lesion in uncommon sites. Imaging and histopathological examinations help to accurately distinguish between hydatic cyst and other mass lesions of the thigh, and determine the outcome.The authors declare that they have no competing interests.We give our special thanks to the Clinical Research Center of Kosar Hospital and Mrs. Simindokht Molaverdikhani for assisting us in this project.


The rate of preterm birth has been increasing worldwide. Most preterm babies are at an increased risk of central nervous system impairments as well as respiratory and gastrointestinal complications. The aim of this study was to investigate the epidemiologic characteristics of and associated factors contributing to preterm birth in Taiwan.Information on obstetric antecedents and risk factors for preterm birth in pregnant women was obtained from the National Health Insurance Research (NHIR) database provided by the Taiwan National Health Research Institute. All live births from 2004 to 2013 in Taiwan were included in this study.p-values< 0.05).A total of 130,362 live births from 2004 to 2013 were included in this study. Overall, the average annual rate of preterm births increased by 5.3% (from 3.33% in 2004 to 5.11% in 2013). Multiple logistic regression analyses showed that nulliparous women, multifetal pregnancies, advanced mother age, history of preterm birth, history of maternal drug abuse/dependence, and maternal medical complications were positively associated with an increased risk of preterm birth (all The overall proportion of preterm births increased from 2004 to 2013 in Taiwan. Babies born preterm had a higher risk of developing morbidities and mortalities. The development of a comprehensive program to identify the high-risk group is needed for effective interventions to prevent premature birth. The rrm birth –15.Preterm birth is an important cause of long-term morbidity and death in children under 5 years of age globally –18. CompThe study methods have been published previously , 21. BriAll babies alive starting at 20 weeks of gestation in Taiwan from 2004 to 2013 were included in the study. We excluded stillbirths (0.5%) and subjects with missing obstetric information (2.7%).This work was approved by the Ethics Board, National Cheng Kung University Hospital, Tainan, Taiwan.Preterm birth was defined as a baby born alive before 37 weeks of pregnancy are completed , 24.Spontaneous preterm birth was defined as babies born with spontaneous initiation of labor and prelabor rupture of membranes (PROM). Provider-initiated preterm birth was defined as babies born with provider-initiated mode of onset, either medical labor induction or cesarean delivery before the initiation of labor.Gestational age was calculated on the basis of the last menstrual period (LMP) confirmed by sonographic examination prior to 20-week gestation , 26. If A first pregnancy woman who had never given birth was classified as nulliparity, and a woman who had borne 2 or more children was classified as multiparity . Maternaplacenta abruptio) during the pregnancy period [Maternal medical disorders were defined as mother with existing medical diseases (e.g. hypertension), as well as obstetric complications  and their 95% confidence intervals (CIs) by maternal obstetric characteristics. An alpha value of 0.05 or less was considered as statistical significance. All statistical analyses were performed using SAS software version 9.4 .www1.stat.gov.tw/ct.asp?xItem=15409&ctNode=4693&mp=3). In this study, 130,362 (5.7%) live births at greater than 20-week gestation being collected from NHIR database in Taiwan were enrolled in the study. Compared to the mean age of total population of live births in Taiwan, the mean age of our study subjects had not significant difference . Table Between 2004 and 2013, a total of 2,279,720 lives birth at greater than 20 weeks gestation were registered in Taiwan (p <  0.0001). The overall average preterm proportion was 4.7%. The average annual preterm birth rate steadily increased by 5.3% during the study period (from 3.33% in 2004 to 5.11% in 2013). The lowest rate was in 2004 (3.33%) and the highest rate was in 2012 (5.84%). When we stratified preterm births in population by spontaneous preterm and provider-initiated preterm birth, the change in the preterm birth rate after the spontaneous onset of labor for the total population was 0.5% (1.69% in 2004 to 2.17% in 2013), and the change in the preterm birth rate after provider initiated preterm for the total population was 1.3% (1.64% in 2004 to 2.94 in 2013).Figure Table p <  0.0001), multi-fetal births , advanced maternal age (≥40 years) , history of preterm births , history of maternal drug abuse/dependence , and history of maternal medical disorders .Table Our study, using the NHIR database, provides epidemiologic characteristics and associated risk factors for preterm births in Taiwan. This study highlighted a rising trend for preterm birth rate in Taiwan from 2004 to 2013. This finding was consistent with those of previous studies , 30.We found that provider-initiated preterm delivery was positively associated with the occurrence of preterm birth in this study. This finding is similar to the findings in previous studies in the USA and Latin America –33. ThisAfter adjusting for potential confounders, nulliparous women had a positive association with a higher risk of preterm delivery, which is compatible with previous studies , 36. It Previous studies have described that multifetal pregnancies are an important risk factor for preterm delivery , 40. Mul> 40 years). Another study found that maternal obesity was significantly associated with the risk of preterm birth according to maternal age and race or ethnicity [Previous studies have also reported that advanced maternal age is positively associated with the risk of early preterm birth , 45. Clathnicity . These fIt was reported that more than 70% of all preterm births occurred at 32–36 weeks of gestation in previous study . We founConsistent with prior studies , 28, ourPrevious studies showed that pregnant drug users had a significantly higher risk of preterm birth –54. Our The strength of this study was its large sample size with medical records obtained from the NHIR database. Despite this strength, several limitations were noted in this study. First, because this dataset was collected through a passive reporting system, underreporting or misclassification of obstetric information of study subjects would have occurred. Second, the estimates of risk for preterm deliveries generated by logistic regression analysis of data in this retrospective study should be interpreted cautiously, especially when the effect sizes of some risk factors were small and the confidence interval was wide. Third, information on certain risk factors, for example, maternal smoking history, BMI, or education level were insufficient in this database.In conclusion, this study highlighted that the preterm birth rate was rising in Taiwan between 2004 and 2013. The main predictive factors for the risk of preterm birth are nulliparous women, multifetal pregnancies, advanced maternal age (≥40 years), history of preterm birth, and maternal medical disorders. Babies born prematurely have a higher risk for the development of morbidities and mortalities. Therefore, developing a comprehensive program for identifying pregnancies at high-risk for preterm birth is needed for effective interventions to prevent premature births.