Evidence for the association of thyroid dysfunction and autoantibody positivity with preterm birth remains controversial. We aimed to study the association of maternal thyroid dysfunction and autoantibody positivity with the risk of preterm birth.A hospital-based cohort study of 40,214 women was conducted. Gestational age-specific percentiles of the FT4 and TSH concentrations were used for the definition of thyroid dysfunction. Autoantibody positivity was identified when the concentration > the threshold. The association of thyroid dysfunction and autoantibody positivity with the risk of preterm birth was estimated.No significant higher risk of preterm birth was found for women with variants of thyroid dysfunction or autoantibody positive than euthyroid women. Sensitivity and stratification analyses indicated that thyroperoxidase antibody (TPOAb) positivity in the first trimester  and overt hypothyroidism restricted to women negative for TPOAb  was associated with an increased risk of preterm birth. Modification effects of gestational age were found for women who had the test ≤18 and > 18 weeks. Continuous FT4 measurements tested ≤18 weeks of gestation were associated with a higher risk of preterm birth , while a negative relationship for FT4 concentrations tested > 18 weeks of gestation .Some specific thyroid function abnormalities were associated with an increased risk of preterm birth. Interaction between gestational age and FT4 concentration on the risk of preterm birth was identified, with a critical node of 18 weeks of gestation.The online version contains supplementary material available at 10.1186/s12884-022-04806-9. Preterm birth is the leading cause of morbidity and mortality in children < 5 years , 2 and iMaternal thyroid hormone is essential for placentation and fetal development , 6. GestPrevious studies in estimating these associations had certain limitations since a lack of a unified definition for thyroid dysfunction and a low statistical power . RecentlIdentification of the association of thyroid dysfunction and autoantibody positivity with the risk of preterm birth and whether these associations have specific characteristics  are crucial for guiding the necessity and specificity of clinical intervention for women with disorders. Thus, we conducted a cohort study to address these concerns.A hospital-based cohort study of pregnant women who received prenatal examinations between April 2012 and August 2016 was conducted in 2018 , 15. AllThe primary outcome was preterm birth, which was defined as infants born before 37 weeks of gestation. Secondary outcome was very preterm birth .P = 0.633). Since only 33.5% (13971) of the included women received the test twice and 14.2% (5929) had three measurements. These measurements were not included for analysis  in the present study.Thyroid function, including production of TSH, free and total triiodothyronine , FT4, TT4, thyroperoxidase (TPO), TSH receptor (TR), and thyroglobulin (Tg) antibodies were measured with an electrochemiluminescence immunoassay  at the first visit to the hospital  . The medGestational age-specific percentiles of the FT4 and TSH concentrations were used for the definition of thyroid function, including euthyroid (both FT4 and TSH within the range between 2.5th and 97.5th percentile), subclinical hypothyroidism , overt hypothyroidism , isolated hypothyroxinemia , isolated hyperthyroxinemia , subclinical hyperthyroidism , and hyperthyroidism . TPOAb, TgAb, and TRAb were regarded as positive if the concentrations were > 34 IU/mL, 115 IU/mL, and 1.75 IU/L, respectively .Covariates included maternal age , residence , parity (nulliparous or pluriparous), assisted conception (yes or no), fetal sex , preeclampsia (yes or no), and gestational diabetes mellitus (yes or no) . PreeclaPrevalence  of the outcomes was estimated. Difference between women with and without a preterm birth baby was compared by Chi-squared/Fisher’s exact test  or Mann-Whitney U test . Odds ratio (OR) for the risk of preterm birth among women with thyroid dysfunction was calculated compared with euthyroid women. Adjusted ORs were estimated after controlling for the covariates.The risk of outcomes in women positive for thyroid antibodies was also assessed relative to those negative for the marker. In these analyses, women without thyroid antibody tests were also included as an unknown antibody status group. Basic characteristics and outcomes (preterm and very preterm birth) were compared among women with and without antibody tests. We conducted sensitivity analyses restricting to those negative for thyroid antibodies. Preeclampsia is an obvious risk factor for preterm birth and women with a TSH concentration > the upper target of the reference might receive potential treatment. Therefore, we further performed sensitivity analyses limited to women without preeclampsia and those with a TSH concentration ≤ the upper targets to exclude these potential impacts. Stratification analyses were performed according to gestation age at the thyroid test .The associations of TSH and FT4 concentrations with the risk of preterm birth were further probed. Outliers (mean ± 4 SD) of the TSH or FT4 concentrations were removed before the analyses. Interaction term analyses were performed to estimate the potential modification of gestational age at the thyroid test on these associations. Stratification analyses were conducted according to the thyroid testing time  because that the fetal thyroid gland is not functionally mature until 18–20 weeks of gestation  and thatP < 0.05 was considered statistically significant.All analyses were conducted using Microsoft Office Excel, Stata . A two-sided value of A total of 40,214 (77.3%) women were included in the analysis after excluding women with multiple pregnancies, gestational thyroid disease, and who were lost to follow-up  in stratification analyses . Interaction term analyses showed that there were no modification effects of gestational age on the association of FT4 and TSH concentrations with the risk of preterm birth among the included women.Continuous FT4 concentrations were positive correlated to risk of preterm birth , while a negative association was found for continuous TSH measurements . However, these associations did not reach statistical significance  did not produce different results for the interaction between TSH and gestational week. Nevertheless, modification effects of gestational age were found for women who had the test ≤18 weeks and > 18 weeks, the corresponding OR for the interaction between FT4 concentrations and gestational age at test was 0.99 (95% CI: 0.98–1.00) and 1.02 (95% CI: 1.00–1.03), respectively . Figure The results in the present study did not support that thyroid dysfunction and autoantibody positivity were significantly associated with a higher risk for preterm birth or very preterm birth. However, some specific thyroid function abnormalities, such as TPOAb positive in the first trimester and overt hypothyroidism restricted to women negative for TPOAb, were related to an increased risk of preterm birth. In addition, a modification of gestational age was identified for the association between FT4 concentration and risk of preterm birth, with a critical cut-off value of 18 weeks of gestation.In comparison with overt hypothyroidism where there is clear evidence for adverse events, the association between mild thyroid dysfunction  and pregnancy outcomes is unclear, includiIn the present study, both isolated hypothyroxinemia and subclinical hypothyroidism did not show a significant relation to the risk of preterm birth. These findings were consistent with the pooled results of previous observational studies , 13 and Overt thyroid dysfunction  during pregnancy is a known risk factor for preterm birth , 13. TheFT4 was deemed a better marker of thyroid function than TSH in pregnancy , 25, 26.The findings in the present study may partly explain the heterogeneity of the results in the previous studies and might suggest new insights into the potential mechanisms for preterm birth related to thyroid dysfunction , 27, 28.The large sample size in the present study ensured a high statistical power to detect the association between thyroid dysfunction and preterm birth and further probe the specificity of the associations. However, there were some limitations. Firstly, the present study was a single-center study in Shanghai where the iodine status of pregnant women was iodine deficient . In addiIn this hospital-based cohort study, women with overt hypothyroidism and negative for TPOAb and those with TPOAb positivity in the first trimester were associated with an increased risk of preterm birth. Interaction between gestational age and FT4 concentration on the risk of preterm birth was identified, with a critical node of 18 weeks of gestation.Additional file 1.


Fractional anisotropy (FA), a metric of DTI, had significant correlation with number of immune cells  infiltrating into the synovium and cytokines  in all patients and also in each group of patients and adhesion molecule expressed on these cells in all patients . DCE parameters significantly correlated with CD34  in each group of patients, a marker of neo-angiogenesis. FA was the best predictor of infiltrating inflammatory cells, adhesion molecule and proinflammatory cytokines. Amongst the DCE parameters, blood volume, was best predictor of CD34. Conclusion: DTI and DCE metrics capture cellular and molecular markers of synovial inflammation in patients with chronic inflammatory arthritis.Objectives: It is difficult to capture the severity of synovial inflammation on imaging. Herein we hypothesize that diffusion tensor imaging (DTI) derived metrics may delineate the aggregation of the inflammatory cells and expression of inflammatory cytokines and dynamic contrast-enhanced (DCE) imaging may provide information regarding vascularity in the inflamed synovium. Patients and methods: Patients with knee arthritis (>3-months duration) underwent conventional (T2-weighted fast spin echo and spin echo T1-weighted images) as well as DTI and DCE MRI and thereafter arthroscopic guided synovial biopsy. DCE and DTI metrics were extracted from the masks of the segments of the inflamed synovium which enhanced on post-contrast T1-weighted MRI. These metrics were correlated with immunohistochemistry (IHC) parameters of inflammation on synovium. Statistical analysis: Pearson’s correlation was performed to study the relationship between DTI- and DCE-derived metrics, IHC parameters, and post-contrast signal intensity. Linear regression model was used to predict the values of IHC parameters using various DTI and DCE derived metrics as predictors. Results: There were 80 patients  with mean age 39.78 years and mean disease duration 19.82 months. Nineteen patients had tuberculosis and the rest had chronic undifferentiated monoarthritis ( Synovium is the primary site of inflammation in various chronic inflammatory arthritides. Chronic inflammation is characterized by the accumulation of various inflammatory cells in the synovium and increased vascularity as a result of neo-angiogenesis . SecretiPresently, the most definitive way to delineate the severity and type of inflammation is through histological examination of the inflamed synovium. However, it is an invasive process, and not all the joints are amenable to synovial biopsy . MoreoveDiffusion tensor imaging (DTI) is a non-invasive imaging technique that measures the diffusion of water molecules in vivo and provides microstructural information about the tissue ,16. ReceDiffusion anisotropy can be more completely modeled using three basic metrics; linear anisotropy (CL), when diffusion is mainly in the direction corresponding to the largest eigenvalues, planar anisotropy (CP), where diffusion is restricted to a plane spanned by the two eigenvectors corresponding to the two largest eigenvalues, spherical anisotropy (CS) when diffusion of water molecules is unrestricted or isotropic diffusion. Its utility has been explored in a number of clinical studies ,19,20.T1-weighted dynamic contrast-enhanced (DCE) MRI using simple analyses of changes in the signal enhancement curve, such as measuring the maximum enhancement and the rate of initial enhancement, had been used to quantify the extent of synovial inflammation and response to treatment . QuantitNewly diagnosed subjects, age >18 years with inflammatory monoarthritis of the knee of >3 months duration with mono/oligo-articular flare of the knee or to rule out infection in an already inflamed joint were enrolled in the study. “Flare” was defined as either involvement of the new joint or exacerbation of inflammation in the previously inflamed joint. The study was approved by the institutional review board and written informed consent was obtained from all study subjects. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the institute ethics committee of the Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, approval number 2011-19-SRF-58, dated 21 March 2012. The involved knee joints were subjected to MRI and DTI followed by arthroscopic guided biopsy of the synovium and aspiration of the synovial fluid (SF) from the same joint within 48 h of the MRI.Conventional MRI including DTI and DCE-MRI was performed using a standard 8-channel knee coil on a 3-T MR scanner . Conventional T2-weighted fast spin echo (FSE) images and spin echo (SE) T1-weighted images were acquired. T2-weighted images were acquired with following parameters; repetition time (TR) (ms)/echo time (TE) (ms)/echo train length (ETL)/no. of excitations (NEX) = 6000/85/16/4. T1 weighted images were acquired with TR/TE/NEX = 1000/14/2. Fat-saturated T1- and T2-weighted images were acquired from adjacent (overlaid), 3 mm thick axial sections with 240 × 240 mm field of view (FOV) and image matrix of 256 × 256.2. The acquisition parameters were: FOV = 240 × 240 mm, TR~8 s, TE~100 ms, and number of excitations = 8. The acquisition matrix was 128 × 80. K-space data (128 × 128 and zero-filled to generate an image matrix of 256 × 256) was constructed using homodyne algorithm. The DTI data were processed as described in detail elsewhere . Gadolinium contrast  was administered intravenously through a power injector  at 5 mL/s and a dose of 0.2 mmol/kg body weight, followed by 30 mL saline flush, at the start of the sixth acquisition. Serial 1120 images  were acquired over 40 time points for 28 slices. Prior to 3D SPGR, T1-weighted, FSE, and fast double spin-echo proton density (PD) weighted and T2-weighted imaging were performed to quantify voxel-wise the pre-contrast tissue longitudinal relaxation time Tep) and DTI  metrics from the segmented regions  was introduced into the knee. Synovial fluid was aspirated, and lavage of the joint cavity was performed to allow macroscopic evaluation of hyperemia and hypertrophy of the synovial membrane. Biopsies were taken at inflamed sites under direct visualization, followed by another lavage to remove blood and debris. A total of 8–10 biopsy specimens were obtained from the suprapatellar anterior, medial, and lateral compartments.The grading of the synovial membranes was carried out on routine hematoxylin and eosin (H&E)-stained slides. The histopathological findings were compared using the histological scoring system reported by Rooney, which consists of six parameters: degree of synovial hyperplasia, fibrosis, number of blood vessels, perivascular lymphocyte infiltration, focal aggregates of lymphocytes, and diffuse infiltrates of lymphocytes . PatientThe infiltration by various types of inflammatory cells, vascularity, and cytokine expression in the synovium was analyzed by immunohistochemistry using antibodies CD3,  1:200, CD4,  1:50; CD8,  Ready to use; CD20  1:200; CD34  1:50; CD54 (Leica) 1:25; CD68  1:200; CD138 , ready to use. TNFα R , 1:100; IL-1β,  1:100. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections. A polymer-based peroxidase method with diaminobenzidine  as chromogen was used to detect the bound antibodies.The sections were evaluated under a light microscope (Olympus DP70). Only nucleated cells with clear-cut cytoplasmic or surface staining were counted and three hot spots; areas of synovium containing highest number of cells, per section were identified. Cells that were positive for IHC markers were counted in 10 consecutive high-power fields (X400) per hot spot. Thereafter, an average number of positive cells per three hot spots was calculated and results were expressed as total number of positive cells per square millimeter ,29. Two The human soluble IL-6, IL-1β, and TNF-α in synovial fluid were quantitatively measured by enzyme-linked immunosorbent assay (ELISA)  in duplicate. ELISA for these cytokines was performed as per manufacturer’s guidelines. Number of inflammatory cytokines in samples was determined by standard curve.p < 0.0055) was performed to study the relationship between different DTI- and DCE-derived metrics, IHC parameters, inflammatory cytokines measured in SF, and post-contrast signal intensity. Linear regression model was used to predict the values of IHC parameters of inflammation by using various DTI and DCE-derived matrices as predictors.Spearman correlation with Bonferroni correction . β0 is constant term and β1 is the coefficient of determinant parameter. Multivariate linear regression was used to estimate the best DTI and DCE parameters to predict synovial inflammation based on Rooney score.Model: y = βAll the statistical computations were performed using the SPSS  statistical software.p < 0.05 and 95% power. The minimum sample size required for the study under these assumptions was 75. Considering few drop outs it was planned to take 83 subjects for the study.Sample size estimation was based on our previous study results where correlation coefficients between DTI parameters and synovial fluid pro-inflammatory cytokines varied between 0.4 to 0.6 . For then = 14), chronic undifferentiated monoarthritis (n = 31), rheumatoid arthritis (n = 6), osteoarthritis (n = 4), ankylosing spondylitis (n = 2), reactive arthritis (n = 2) and juvenile idiopathic arthritis (n = 1). All six patients with RA were receiving methotrexate (mean dose 17.5 mg/week) and NSAIDs. None of them were on oral or parenteral glucocorticoids or biological therapy. Rests of the patients were receiving NSAIDs only, at the time of inclusion in the study.There were 80 patients, mean age of 39.78 years (range 18–76) with 52 being male . The meaep significantly correlated with severe synovial inflammation (Rooney synovitis score 3.1–5) whereas FA only correlated with moderate synovial inflammation (Rooney synovitis score 1.6–3)  . The meae 1.6–3) . Amongste 1.6–3)  and in de 1.6–3)  and varie 1.6–3) .In the synovial fluid, DTI parameters; FA, MD, and CL significantly correlated with the synovial fluid proinflammatory cytokines  in all pep were able to predict CD34 , B cells , macrophages (CD68), adhesion molecule (CD54), the total number of cells infiltrating into the synovium and proinflammatory cytokines (TNF-α and IL-1β) a–c. Otheict CD34 .The results of the present study showed a significant correlation between DTI-derived metrics with IHC parameters of proinflammatory cellular aggregates, adhesion molecule, and proinflammatory cytokines and DCE parameters with markers of neo-angiogenesis, respectively. Thereby, almost all the components of chronic synovial inflammation are captured on imaging with high accuracy.On application of a high-power magnetic field to water hydrogen and hydroxy ions are created with a particular line-up according to the magnetic field. When the magnet is switched off, these ions are free to move (diffusion) in any direction. Fractional anisotropy (FA) measures interference in the movement of these ions due to the vectrality of the tissue structure and creates anisotropy or fractional anisotropy (FA). Its values vary between 0  or isotropic diffusion and a maximum value of 1, where movement is possible in only one direction true for thin fibers (anisotropic structures). White matter fibers in the brain and spinal cord are anisotropic structures and have high FA values (but less than 1). In the normal synovium, there is not much vectrality however, in the inflamed synovium it was reported to be high as compared to a healthy individual’s synovium. It was also reported that high FA values in the inflamed synovium correlated with synovial fluid TNF-α and IL-1β levels . EarlierMean diffusivity (MD) is the measurement of free diffusivity of the water molecules and is generally inversely related to FA ,31,32. ABoth CL and CP values are responsible for increased FA values; however, their relative values indicate the shape of the anisotropy . The braInflammation is characterized by cellular infiltration and increased vascularity ,37. In aInfections are likely to have more intense inflammation as compared to chronic inflammatory conditions ,39,40 anOur study is limited by the small number of subjects in each subgroup as per Rooney synovitis score and hence correlation in each subgroup with DTI and DCE parameters may be underpowered and not robust.To the best of our knowledge, this is the first study that shows a direct correlation between cellular and molecular markers of synovial inflammation. The variability in the DTI and DCE metrics with the intensity of synovial inflammation has the potential to replace synovial histology in the future. It may become a non-invasive tool to assess the degree of synovial inflammation in response to various newer therapeutic molecules across various rheumatic diseases.


New pregnancies were identified prospectively, dried blood spot samples were collected during the third trimester, and information was obtained on gestational age at delivery and offspring weight after birth. Genome-wide DNA methylation was assessed with the Infinium EPIC array. Using a suite of 15 epigenetic clocks, we only found one significant relationship: advanced age on the epigenetic clock trained on leptin predicted a significantly earlier gestational age at delivery . Of the other 29 relationships tested predicting gestational age and offspring birth weight, none were statistically significant. In this sample of Filipino women, epigenetic clocks capturing multiple dimensions of biology and health do not predict birth outcomes in offspring.Adverse birth outcomes, such as early gestational age and low birth weight, can have lasting effects on morbidity and mortality, with impacts that persist into adulthood. Identifying the maternal factors that contribute to adverse birth outcomes in the next generation is thus a priority. Epigenetic clocks, which have emerged as powerful tools for quantifying biological aging and various dimensions of physiological dysregulation, hold promise for clarifying relationships between maternal biology and infant health, including the maternal factors or states that predict birth outcomes. Nevertheless, studies exploring the relationship between maternal epigenetic age and birth outcomes remain few. Here, we attempt to replicate a series of analyses previously reported in a US-based sample, using a larger similarly aged sample (The online version contains supplementary material available at 10.1186/s13148-022-01296-6. Birth outcomes like birth weight, length, and gestational age predict both short- and long-term health. For example, early gestational age at birth predicts the two largest causes of death in premature infants: underdevelopment of mature organs and bronchopulmonary dysplasia, a chronic lung disease that damages alveolar tissue , 2. In aWhile nutrition has received broadest attention for its role in fetal growth, there is growing evidence that the mother’s physiology and metabolism, including systems like stress physiology and inflammation, can impact fetal growth and development operating through effects on gestational conditions like nutrient delivery, oxidative stress, or exposure to metabolic or other hormones . As a reA set of tools called epigenetic clocks have recently been shown to reflect various domains of physiology and metabolism, and thus could be useful for gauging the intergenerational impacts of chronic maternal physiological and metabolic dysregulation , 18. Epin = 77), evaluated 15 epigenetic clocks as predictors of gestational age at birth and birth weight, and found mixed evidence that advanced maternal epigenetic age predicted early gestational age at birth and low birthweight in offspring, suggesting to the authors that epigenetic age may be predictive of adverse fetal outcomes [Since epigenetic clocks can be trained on effectively any set of metabolic/physiological processes or states, they are powerful tools for characterizing these states . For theoutcomes .In the present paper, we seek to replicate this analysis by examining relationships between the same suite of 15 epigenetic clocks, measured in DNA obtained from blood during pregnancy, and prospectively measured birth outcomes in the offspring of those pregnancies. Data come from the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a cohort study that has followed a large, diverse sample of women and their offspring in metropolitan Cebu City, Philippines for nearly four decades . The preData come from the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a longitudinal survey of 3,080 infants and their mothers who were recruited during their pregnancies between 1983 and 1984 in Metropolitan Cebu, Philippines . Out of A composite score of socioeconomic status was measured as a combination of income, education, and assets. Participants reported their annual income from all sources, including in-kind services, and the sale of livestock or other products by household members during the prior year, which were summed to determine total household income. Incomes were log-transformed. Maternal education (in years) was also reported. Participants also reported on ten assets  that were selected to capture population-relevant aspects of social class, including electricity, refrigerators, air conditioners, color televisions, cable TV, tape recorders, electric fans, jeepneys, cars, trucks, and owning their residence. In addition, house construction type  was coded as 1, 2, or 3, respectively. Thus, asset scores ranged from 0 to 13. A principal components analysis was run on log income and assets, along with maternal education, at sample collection. The first dimension explained 70% of the variation in our composite SES-score, and individual scores for the top component of variation were used as our measure of SES.r = 0.84). Offspring gestation age was calculated using the time between the last reported menstrual period and infant birth date. Days pregnant at maternal blood sampling was determined by subtracting the time between the blood sample and infant birth date from gestation age. Descriptive statistics of anthropometric measurements and other covariates are provided in Table Because women were enrolled in the birth outcome sub-study after they were pregnant, we used height and weight measurements collected during prior surveys to estimate pre-pregnancy BMI. We used 2009 BMI when available, and then used 2007 and 2005 data as necessary. Under the assumption that women will tend to maintain a stable position within the population BMI distribution even as the population mean increases with age, we converted all BMIs to age-specific within-sample Z-scores before pooling into a single pre-pregnancy BMI variable. Supporting the validity of this approach, the correlation between Z-scores for BMI measured in 2005 and 2009 was very high . After all exclusions, the final sample with all necessary biological and questionnaire data included 296 women singleton births with complete information.DNA was extracted from 1 to 3 dried blood spots (DBS) using a standard protocol; purified DNA was concentrated using a Vacufuge Plus vacuum concentrator (Eppendorf), then treated with sodium bisulfite , and quantified by nanodrop, and 160 ng of converted DNA was applied to the Illumina Infinium MethylationEPIC BeadChip under standard conditions . Technicians were blind to any information regarding participant characteristics, and samples were randomly assigned to plate, chip, and row. Background subtraction and color correction were performed using Illumina Genome Studio with default parameters. Data were then exported into R for further analysis. Quality control involved first confirming participant sex and replicate status. This was followed by quantile normalization using lumi on all probes including SNP-associated and XY multiple binding probes. To maximize the number of sites available for the epigenetic age calculator, probes with detection p values above 0.01 were called NA for poor performing samples only and were otherwise retained .http://labs.genetics.ucla.edu/horvath/dnamage/). Background-corrected beta values were processed further using the calculator’s internal normalization algorithms. Clocks included were: Horvath’s epigenetic age (DNAmAge) [Epigenetic age for all clocks was calculated using the online calculator (DNAmAge) , intrinsDNAmAge) , 28, extDNAmAge) , 28, DNADNAmAge) , DNAmTL DNAmAge) , senesceDNAmAge) , and theDNAmAge) . DNAmIEADNAmAge) . DNAmGriDNAmAge) . DNAmTL We first ran descriptive statistics before running a sequence of multiple linear regression models designed to assess relationships between maternal epigenetic age acceleration and two fetal outcomes . Models predicting gestational age adjusted for offspring sex, a composite score of socioeconomic status, and pre-pregnancy body mass index (BMI) z-scores. Postnatal outcomes were adjusted for days after birth of anthropometry measurement and gestational age at birth and our composite socioeconomic status score. Since we are replicating prior work that did not correct for multiple testing, we did not correct for multiple comparisons in our main analysis. For transparency, however, we also point out results that differed using a Bonferroni adjusted alpha of 0.05/15 = 0.003. Furthermore, we ran sensitivity analyses that included an additional term for chronological age , pregnancy order, cell counts , and batch effects . All statistical analyses were conducted using R version 4.0.4.The women in our study ranged between 25 and 30.8 years at the time of the study (mean age = 27.8 years old). Blood spots for DNAm were taken between 160 and 288 days into pregnancy, with a mean gestational timing of 207 days. Education ranged from 2 to 22 years , and 17 women smoked. Over 16% of the women in the study had experienced 5 or more pregnancies, while 57% had experienced at least 3 pregnancies. Descriptive statistics of these and other maternal covariates are provided in Table Slightly more infants were categorized as male 52%), with a mean gestational age at birth of 277 days. Postnatal measurement occurred between 1 and 14 days after birth, with the mean age at measurement of roughly 4 days. Descriptive statistics of infant weight, length, and other anthropometric measures are provided in Table %, with ap = 0.009). This effect was not statistically significant using the Bonferroni adjusted threshold of 0.003. Our results were unchanged in sensitivity analyses, which included an additional term for age (in addition to that in the epigenetic age acceleration measure) and pregnancy order  or not significantly (gray) associated with birth outcomes in Ross et al. .Additional file 2. Full model estimates for the relationship between maternal epigenetic age and gestation age and offspring post-natal weight. Estimates for models including birth order, cell count estimates, and batch, chip, and row are also provided.


We performed in vivo DTI and histology in rats at 79 days after SE and control animals. The analyses focused on the corpus callosum, hippocampal subfield CA3b, and layers V and VI of the parietal cortex. Comparison between control and SE rats indicated that a combination of microstructural tissue changes occurring after SE, such as cellularity, organization of myelinated axons, and/or morphology of astrocytes, affect DTI parameters. Subsequently, we constructed a multivariate regression model for explaining and predicting histological parameters based on DTI. The model revealed that DTI predicted well the organization of myelinated axons  and astrocyte processes  and possessed a predictive value for cell density (CD) . However, the morphology of astrocytes  was not well predicted. The inclusion of parameters from CA3b was necessary for modeling histopathology. Moreover, the multivariate DTI model explained better histological parameters than any univariate model. In conclusion, we demonstrate that combining several analytical and statistical tools can help interpret imaging outcomes to microstructural tissue changes, opening new avenues to improve the non-invasive diagnosis and prognosis of brain tissue damage.Non-invasive magnetic resonance imaging (MRI) methods have proved useful in the diagnosis and prognosis of neurodegenerative diseases. However, the interpretation of imaging outcomes in terms of tissue pathology is still challenging. This study goes beyond the current interpretation of  In recent years, diffusion magnetic resonance imaging (dMRI) methods, such as diffusion tensor imaging (DTI), have become widely used tools to study the brain’s response to pathological insults in both clinical and research settings . The senChanges in DTI parameters in the diseased brain have been assessed with histology. Histological stainings and quantitative analyses are known to provide morphological information on the cellular components and their changes occurring during pathological conditions . Traditiin vivo DTI can explain and predict the underlying microstructural tissue changes after status epilepticus (SE) induced-brain damage. In vivo DTI was performed 79 days after SE, subsequently, the brains were prepared for histology. Previous studies have described brain histopathological changes after SE in rats, such as axonal sprouting , housed individually in cages in a climate-controlled room with an n = 14) or pilocarpine . The control group was treated with an injection of 0.9% NaCl (n = 4). The development of SE and its severity score were assessed within 3 h after the injections  with an actively decoupled quadrature volume transmitter coil and a quadrature rat receiver surface coil. In the acquisition of in vivo DTI data, we used a diffusion-weighted segmented spin-echo planar imaging pulse sequence with the following parameters: TE = 30 ms, TR = 2.5 s, number of averages = 32, number of segments = 4, 21 directions , FOV of 21.2 × 14.08 mm2 , number of slices = 14, slice thickness = 500 μm, and scan time = 2 h 20 min. These animals were part of a longitudinal study . al study , and in 1 After eddy current corrections, the diffusion tensors, and their respective eigenvalues  were determined using FSL. Then, we generated the fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) maps   in the FMD) maps . We alsoMD) maps , which pMD) maps .2/O2) followed by transcardial perfusion, first with 0.9% NaCl, and then, by 4% paraformaldehyde (PFA). The brains were removed from the skull and postfixed in 4% PFA for 4 h. Then, the brains were placed in a cryoprotective solution . The brains were frozen in dry ice and stored at −70°C until cutting. The brains were sectioned in a sliding microtome . We stored the first series of sections in 10% formalin, and the remaining series in cryoprotectant-tissue collecting solution  at −20°C until processing.After the scans, all the rats were deeply anesthetized by an i.p. injection (6 ml/kg) with an anesthetic cocktail containing sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg), magnesium sulfate (127.2 mg/kg), propylene glycol (42.8%), and absolute ethanol (11.6%). The deep level of anesthesia was kept by 5% isoflurane  in 0.02 M sodium phosphate buffer (pH 7.4) containing 0.09% NaCl for 3–4 h in dark at room temperature (RT). After washing in 0.02 M sodium phosphate buffer containing 0.09% NaCl, the sections were incubated in 2.5% sodium thiosulfate solution (5 min). Then, the sections were washed in the buffer solution, dehydrated in an ascending ethanol series, cleared in xylene, and cover-slipped with DePeX .The first series of sections were stained with Nissl (thionin) to assess the cytoarchitectonics and severity of tissue damage after SE. The second series of sections were stained for myelin using gold chloride to examine the myeloarchitecture . Briefly2O2 (15 min) to remove endogenous peroxidase activity. After washing in buffer, non-specific binding in the sections was blocked by placing them in 10% normal horse serum (NHS) solution  for 2 h. Then, the sections were incubated for 48 h (4°C) in the primary antibody  diluted in 1% NHS, 0.4% Triton X-100 in KPBS. Next, sections were washed and incubated for 2 h at RT in the secondary antibody  solution . After washing, the sections were incubated for 1 h at RT in 1% avidin-biotin in KPBS . Then, the sections were placed black into the secondary antibody solution (45 min), followed by immersion in the avidin-biotin solution (30 min). To visualize the secondary antibody, we used a solution containing 0.05% 3′,3′ -diaminobenzidine  and 0.04% H2O2 in KPBS. After washing in 0.1 M PB, we mounted the sections on gelatin-coated slides and dried them overnight (37°C). The sections were intensified with osmium  and thiocarbohydrazide . Finally, the sections were washed with the buffer, dehydrated in an ascending ethanol series, cleared in xylene, and cover-slipped with DePeX.From the third series of sections, we stained three consecutive sections immunohistochemically with an astrocyte marker, glial fibrillary acidic marker (GFAP), to assess the morphology of the astrocytes . Briefly2/pixel.We acquired high-resolution photomicrographs of the whole brain in three consecutive Nissl-, myelin-, and GFAP- stained sections with a ZeissAxioImager2 light microscope  equipped with a digital camera (Zeiss Axiocam color 506). The images were acquired as tiles with a resolution of 0.013 μm2 An increase in CD on Nissl-stained sections was indicative of gliosis as reported in previous studies  as developed by In Nissl-stained sections, we quantified cell density (CD) using an automated cell counting in-house MATLAB code as described in  studies . To asse studies . In myel4   . Similar States) .t-test . We applied the multiple comparison correction by using the Benjamini-Hochberg false discovery rate .In the statistical analyses, we aimed to assess the effect of SE in DTI and histological parameters, reporting the effect sizes of SE-induced brain damage in addition to null hypothesis significance testing and utilizing an estimation statistics-based approach. In the present study, we consider both kainic acid- and pilocarpine-treated animals as one SE animal group for statistical analyses, since both animal models exhibited similar histopathological and DTI profiles based on previous studies . We perf BH-FDR;  to the pWhen evaluating the relationship between DTI and histology in the selected brain regions, we performed a multiple linear regression in an attempt to explain histological parameters based on DTI parameters. The model that we used isykj is a histological parameter for animal k in the region j , xkj is the vector of DTI parameters  for animal k in the region j, b and c are the regression parameters, and ekj represents normally distributed independent and identically distributed errors. We concatenated parameters from all the regions and all the animals into a single model.where R2 using the SPSS code from p-values . Moreover, we also report the F statistics to support that the regression model provides a better fit than a model without independent variables. The 95% CI values were computed as accelerated bootstraps in SPSS .We excluded AD and CL from the DTI parameters to avoid a singular variance-covariance matrix as AD = 3*MD - 2*RD and CL = 1 – CP – CS. We further noted that all DTI parameters were non-linear functions of just three eigenvalues of the diffusion tensor. Therefore, the five remaining DTI parameters displayed a notable amount of structural collinearity, which means that the inferences concerning individual coefficients of the regression model were not reliable. However, the five-parameter model had a non-singular variance-covariance matrix, and the explanatory and predictive accuracy of the model could be evaluated. The choice of AD and CL as the parameters to be removed was arbitrary and had no influence on the evaluation of the model’s accuracy. We used SPSS to fit the regression model and estimated the 95% CI values for Q2) of the relationship between the two has been analyzed in detail in Q2 is defined by ykj is the true histological parameter, k in the region j and 5We assessed how predictive DTI would be with regard to histology by evaluating the performance of the regression model by running leave-one-animal out and leaving-one-region out CV. Since the regression model (incorrectly) assumes that the parameters from different regions of the same animal are independent, we evaluated its predictive performance using leave-one-animal out CV. We report the quality of validated predictions using a cross-validated correlation coefficient R and the coefficient of determination ; as small .Myelin ; Myelin ], and AIGFAP  ], AD , CL , CP  and CS  . It is n, 3.14)] . The eff, 3.14)] , but the, 3.14)] . Thus, tis study .Myelin was extremely well explained by DTI parameters [R2 = 0.822, R2 adj = 0.807, 95% CI ; R2 > 0.60, R > 0.80; Myelin, DTI parameters explained AIGFAP well [R2 = 0.855, R2 adj = 0.843, 95% CI ; R2 > 0.65, R > 0.80; R2 = 0.557, R2 adj = 0.521, 95% CI ; R2 > 0.20, R > 0.45; R2 = 0.659, R2 adj = 0.632, 95% CI ; R2 = 0.507, R2 adj = 0.467, 95% CI ; R2 > 0.40, R > 0.60; When using the multivariate DTI model, we found that AIMyelin  and AIGFAP , and moderate for CD  when applying the approach of leaving-one-animal out  and histology (0.114 × 0.114 μm2) might influence the delineation of ROIs and the outcomes when comparing both methodologies. Also, larger ROIs outlined in DTI maps might exhibit more microstructural heterogeneity than the representative ROIs in photomicrographs, affecting the effect sizes of SE and the relationship between DTI and histological parameters. Moreover, the effects of chemical fixation and staining procedures can affect the tissue microstructure, and thus, the comparisons between in vivo DTI and ex vivo histological parameters (in vivo DTI data. Thus, 3D quantitative histological analyses can provide a more reliable comparison with DTI (There are a few limitations in our study that should be considered. First, based on the CV of DTI and histology, future studies should increase the number of animals and brain regions. As we demonstrated that including CA3b achieved a better prediction of histological parameters based on DTI when compared to other brain regions. In this regard, the presence of wide confidence intervals is also an indication that increasing the sample size would be one way to improve the estimations about the impact of SE when assessing changes in both DTI and histology. Second, as only a limited number of diffusion directions were measured with relatively low-tructure . Therefonditions . Third, rameters . Fourth,with DTI . Furtherwith DTI . Also, fin vivo DTI can be predictive of quantitative tissue microstructure parameters estimated from cellularity, organization of myelinated axons, and morphology of astrocyte processes in white and gray matter areas after SE-induced brain damage. This study offers new insights to help in the interpretation of in vivo DTI as it incorporated an advanced quantitative histological assessment and CV of DTI and histology. Future studies combining imaging and histology with advanced analytical tools are needed to improve our understanding of imaging outcomes in terms of tissue microstructural mechanisms during pathological conditions and to open new perspectives in the diagnosis and prognosis of brain diseases.In conclusion, our findings suggest that The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All animal procedures were approved by the Animal Ethics Committee of the Provincial Government of Southern Finland and performed in accordance with the guidelines set by the European Community Council Directives 2010/63/EEC.ISMM, JT, and AS designed the experiment. ISMM, RAS, and JT analyzed the data. ISMM, RAS, JT, OG, and AS wrote the manuscript. All authors contributed to the article and approved the submitted version.


Background: Premature birth impacts millions of newborns annually. Sixty percent of the world’s preterm births occur in Sub-Saharan Africa and South Asia. Somalia’s premature birth rates and maternal risk factors are poorly studied; hence, this study aims to identify maternal risk factors related to premature births in Mogadishu, Somalia. Methods: This unmatched case-control study was conducted at four maternity hospitals in Mogadishu, Somalia. The cases were newborns with gestational ages of less than 37 weeks; controls were newborns with gestational ages of 37 to 42 weeks. All were live singletons. Cross-tabulation and binary logistic regression were used to analyze the data; a p-value of 0.05 was deemed statistically significant. Result: Of the total of 499 newborns, 70 were cases, and 429 were controls. Adequate prenatal care, maternal urine analysis, tetanus toxoid (TT) vaccination, hemoglobin (Hb) measurement, ultrasound monitoring, intake of iron + folic acid (IFA) supplement, blood pressure (BP) measurement during the current pregnancy, as well as partograph usage during labor all significantly decreased risk of having premature births. A prior history of preterm delivery and preeclampsia, obstetric complications, and female genital mutilation (FGM) significantly increased the risk of preterm births. Conclusion: The utilization of antenatal care services, use of a partograph, obstetric complications, and prior history of premature birth and preeclampsia had a substantial effect on preterm birth rates. This study identifies female genital mutilation (FGM) as a previously unidentified risk factor for preterm birth that needs additional investigation. Preterm birth (PTB) is a global health problem that affects millions of newborn babies each year ,2. PTB iEach year, over 130 million infants are born, almost 15 million of who are preterm, or more than one in every ten births globally. Over a million of these preterm infants die within the first few days of their lives. However, preterm birth rates vary substantially across the globe, with Northern Europe and Japan having the lowest rates while developing countries with low or middle incomes have the greatest rates ,5. Sub-SSeveral maternal-related risk factors have been associated with PTB, including lack of antenatal care (ANC) visits, obstetric complications, maternal age, nutritional status and maternal infection, gestational hypertension, a history of abortion, and low birth weight. All of these factors make pregnant women more susceptible to pregnancy-related disorders, which could contribute to PTB. Besides, urinary tract infection (UTI) and bacterial vaginitis are significantly associated with the risk of preterm delivery ,7,8. FurGlobally, screening and prevention services are implemented in several countries intended to recognize high-risk women for PTB. For example, in Western Australia, ultrasonography is used to assess the risk of preterm birth based on cervical shortening and then to establish preventative measures for these complications. However, in East Africa, many preterm births continue to go unnoticed, and the underlying causes have not been identified in order to prevent their occurrence and associated complications, particularly in Somalia, where they are most prevalent.In Somalia, the prevalence and risk factors of preterm births have not been well described. Thus, this study intends to investigate the maternal risk factors of preterm births in Mogadishu, Somalia, and, more importantly, identify target areas for future evidence-based interventions.The study employed an unmatched case-control study design adapted with modification . The stuA total of 518 women gave birth during the study period. Mothers that delivered twin babies were omitted from the study, and they comprised a total of 13 twin neonates, so the sample size became 505 singleton neonates. In addition to the elimination of post-term neonates, the exact sample size taken for this study was 499. In this study, only live singletons were included: those deliveries with GA less than 37 weeks were considered as cases with a total sample of 70 neonates, and all term deliveries with a GA of 37 to 42 weeks were considered as controls, being the total of 429 neonates. The data was collected using a structured survey questionnaire. The questionnaire was divided into sections: maternal generation information, maternal characteristics related to the uptake and utilization of ANC services, and maternal medical and obstetric information.p-value. A p-value is considered statistically significant if its (<0.05).The data was entered into a Microsoft Office Excel database and exported to SPSS 26 version to analyze the data. The study employed cross-tabulation analysis to compare the proportion of each variable. In addition, binary logistic regression was used in order to find the relationship between the maternal factors associated with preterm birth and were presented as odds ratio (OR), 95% confidence interval (CI), and The study obtained ethical approval from the Jamhuriya University institutional ethics committee in 2020. Prior to the interviews, verbal comments were taken from mothers after they were fully informed about the objectives of the project while ensuring privacy and the confidentiality of participants.Maternal variables that influence preterm birth outcomes were studied among women attending four Mogadishu maternity hospitals between February and April of 2021. All these findings are concluded in the tables below.n = 347/499, 69.5%) were between the ages of 21 and 39 years, representing 46 (9.2%) of the cases and 301 (60.3%) of the controls. In terms of marital status, 13.4% of cases and 84.8% of controls were married. Some 8.6% of cases and 40.3% of controls were of low socioeconomic status. Mothers’ education revealed that 12% of cases and 70.5% of controls had low literacy rates. Female genital mutilation (FGM), a type of female circumcision that involves the complete removal of the female genitalia for non-medical reasons, was performed in 12.8% of cases and 41.3% of controls. Close to 80% of pregnant women of multigravida status were 10.4% of cases and 69.1% of controls. However, the maternal parity numbers varied. For instance, 8.8% of cases and 57.1% of controls were multiparous, while 2.4% of cases and 15.8% of controls were primiparous, and 2.8% of cases and 13% of controls were nulliparous. The mean maternal age of cases was 25 years, while controls were 26 years. In terms of the child’s gender, male babies made up 7.4% of cases and 41.7% of controls, while female babies made up 6.6% of cases and 44.3% of controls. In terms of birth weight, 6.2% of cases and 4.8% of controls were under 2500 g, while 7.8% of cases and 81.2% of controls were over 2500 g. The majority of participants . Thirty-seven (7.4%) of the cases and 301 (60.3%) of the controls received blood pressure (BP) measurements. The BP measurement at ANC was 2 times less likely to be premature delivery . Regarding other ANC-provided services such as tetanus toxoid vaccination, 37 (7.4%) of the cases and 293 (58.7%) of the control did receive vaccination and had nearly two times less chance of developing preterm delivery. Furthermore, half of all preterm cases  and more than two-quarters  of controls had received hemoglobin measurements at ANC and 2.7 less likely to get preterm birth . Thirty-nine (7.8%) of the cases and  of the controls received no iron and folic acid (IFA) supplementation during their antenatal care visits, while those who received iron and folic acid were at 2.8 times less risk of preterm birth . The mothers who started iron and folic acid in the last trimester were almost four times less likely to result in preterm birth . Another significant variable was the availability of ultrasound checks during antenatal care visits. Thirty-nine (7.8%) of the cases and 119 (23.8%) of the controls had no ultrasound checks during their current pregnancy compared to 31(6.2%) of cases and 310 (62.1%) of controls who did have an ultrasound check. Subsequently, those with ultrasound checks had a remarkably three times lower chance of becoming preterm cases than their counterparts . A greater population of 241 (48.3%) pregnant women, which is nearly half of the participants, attended 1–2 ANC visits, representing 26 (5.2%) of the cases and 215 (43.1%) of the controls. We found that frequent ANC visits (>3 visits) were three times less likely to result in preterm birth . A history of preeclampsia was in 3% of cases and 4.8% of controls, which were more likely to result in preterm birth . Moreover, a history of miscarriage was found among 13(2.6%) of the cases and 67 (13.4%) of the controls. Maternal anemia was prevalent in this study. The majority of the mothers with anemia were controls, with 313 (62.7%) compared to 55 (11%) of the cases. According to the type of anemia, participants with mild anemia had 28 (5.6%) of the cases and 160 (32.1%) of the controls, while those with moderate anemia were 18 (3.6%) of the cases and 122 (24.4%) of the controls were diagnosed with moderate anemia, and 9 (1.8%) of the cases and 31 (6.2%) of the controls had severe anemia. Our study included medical illnesses such as hypertension and diabetes. For instance, hypertension was present in 0.8% of the cases and 2% of the controls, yet it was not significant for this study. However, a history of previous preterm birth was present in a total of 26.3%, with 5.2% of cases and 21% of controls, and had more likely to be premature birth . For FGM, 12.8% were cases while 41.3% were controls and more likely to risk to be premature delivery . Regarding the way of presentation, the majority of the participants (13.2% of the cases and 79.6% of the control groups) were in cephalic presentation, while 0.8% of the cases and 5.8% of the controls were in breech presentation. About 12% of preterm cases and 67.5% of controls were vaginal delivery, while 2% and 18.4% were cesarean sections. According to the type of delivery, spontaneous vaginal delivery accounted for the majority, 64.5% of the controls and 11.2% of the cases, respectively, while 0.8% of cases and 3% of controls were assisted delivery, respectively. Those with cesarean section, especially with an emergency cesarean, were the majority, with 1.6% of cases and 10.6% of controls. Obstetric complications in this study were significantly associated with preterm birth, being a total of 19.2%, of which 4.4% were cases and 14.8% controls, which were more likely to lead to preterm birth  Regarding the type of obstetrical complication, preterm cases accounted for 1% of those with antepartum and 1% of those with postpartum hemorrhages compared to 2.6% and 4% of the controls, respectively. We recorded 0.8% and 2.6% of maternal outcomes as deaths. Out of 57 deliveries (11.4%) that were partograph monitored, 19 babies (3.8%) were born prematurely, while 38 babies (7.6%) were born full term. Partograph-monitored labors were less likely to result in preterm birth  is a problem that affects nearly half of pregnant women in this study. Findings from our study show that women who have had FGM are more likely to give birth prematurely, an association that has not been identified in other studies. Complications of FGM are most likely to blame, such as extensive scarring and narrowing of the birth canal, along with ongoing colonization by germs that can cause infections of the bladder and other diseases ,21. MorePregnant women with obstetric complications and preeclampsia had a higher preterm birth risk. Of the 7% of the women with preeclampsia in our study, 3% of their births ended prematurely. Prior research from Yemen and Tanzania supported our findings that preeclampsic mothers had a four-fold and seven-fold increased risk of premature delivery, respectively ,23. AnotThe study’s strength was that it was conducted at four primary maternity hospitals. Two of the hospitals (Benadir and S.O.S) are considered referral hospitals for high-risk pregnancies and women with complications. In addition, to our knowledge, this is the first study from the study area which investigated the maternal risk factors for preterm birth in Mogadishu, Somalia. One of the study’s drawbacks is that it was carried out in a hospital environment, where hospital delivery rates are low and might not represent the general population. Future studies should address the link between FGM and prematurity, which is critical.This study showed that there is a high prevalence of prematurity. Understanding and recognizing maternal risk factors associated with preterm birth may aid in the prevention of preterm birth complications. This study emphasizes the importance of prenatal care, obstetric complications, and FGM on preterm birth. To prevent the occurrence and associated long-term complications of prematurity, the Ministry of Health should engage in community education to increase ANC service utilization, as well as improve health institutions’ capacity to identify and treat obstetric complications.


Concentrated animal-feeding operations (CAFOs) emit pollution into surrounding areas, and previous research has found associations with poor health outcomes. The objective of this study was to investigate if home proximity to poultry CAFOs during pregnancy is associated with adverse birth outcomes, including preterm birth (PTB) and low birth weight (LBW). This study includes births occurring on the Eastern Shore, Virginia, from 2002 to 2015 (N = 5768). A buffer model considering CAFOs within 1 km, 2 km, and 5 km of the maternal residence and an inverse distance weighted (IDW) approach were used to estimate proximity to CAFOs. Associations between proximity to poultry CAFOs and adverse birth outcomes were determined by using regression models, adjusting for available covariates. We found a −52.8 g  change in birthweight and a −1.51  change in gestational days for the highest tertile of inverse distance to CAFOs. Infants born with a maternal residence with at least one CAFO within a 5 km buffer weighed −47 g  less than infants with no CAFOs within a 5 km buffer of the maternal address. More specific measures of exposure pathways via air and water should be used in future studies to refine mediators of the association found in the present study. Concentrated animal-feeding operations (CAFOs) emit ammonia, hydrogen sulfide, odors, volatile organic compounds, and particulate matter into adjacent environments . PopulatAccording to the Environmental Protection Agency, there has been a 16% increase in CAFOs in the United States from 2011 to 2020  . In 2020The etiology and pathophysiology of preterm birth (delivery <37 weeks ) and lowPrevious epidemiological studies in North Carolina and Wisconsin have found associations between living in close proximity to hog and poultry CAFOs and newborn mortality and low birth weight (LBW) ,30. FurtHere, we aim to build upon the existing literature by exploring the potential maternal health implications of living near poultry animal-feeding operations on the Eastern Shore, Virginia. Specifically, we use the address of maternal residence on state birth records, capturing births in Accomack and Northampton counties, from 2002 to 2015, combined with the locations of active poultry farms during each year of the study period. We hypothesized that birthweight and gestational weeks were negatively associated with increasing proximity to active poultry CAFOs during gestation, with increasing odds of preterm birth and low birth weight. This study utilized birth records occurring within Virginia’s Eastern Shore (Accomack County and Northampton County), from 2002 to 2015, provided by the Virginia Department of Health. Each record consisted of maternal residential address; birth plurality; parity; newborn weight and gestation; self-reported tobacco use during pregnancy; type of payment used for birth services; and maternal race and ethnicity, age, and education level. Singleton births with no unknown or missing covariates were utilized in the analysis. Missingness accounted for less than 1% of records . MaternaInformation on poultry CAFO facilities was collected from the Department of Environment Quality of the Commonwealth of Virginia . The datsf package in R software, representing a radius of 1 km, 2 km, and 5 km surrounding each geocoded maternal address [For the street-level analysis, we constructed 3 buffer distances by using the  address . Previou address ,35,36. S address . BrieflyY is the dichotomous birth outcome being modeled; Birth weight, gestational weeks, preterm birth (PTB), and low birth weight (LBW) were dependent variables in separate logistic and linear regression models, using the maternal street address or ZIP-code-level exposure estimates following the general equation below.uld vary . Similaruld vary ,40,41. Preterm birth was defined as less than a gestational age of 37 weeks, while low birth weight was defined as less than 2500 g total birth weight. Logistic regression models were used to predict odds of PTB/LBW, while linear regression models were used to examine the association between continuous birthweight/gestational weeks and maternal residence proximity to poultry CAFOs. Covariates included in the models included child sex; mother’s reported race, age, ethnicity, and education; self-reported tobacco use during pregnancy, method of payment for birth services, and previous births. Mother’s education was classified as not completing high school, high school completed, or college completed. Mother’s age was classified as those under 18, 18–35, and those above 35 years of age. Categories are based on prior studies showing an increased risk of adverse birth outcomes in the younger and older categories of maternal age . Parity The study area and locations of poultry farms within the study area are illustrated in In the ZIP-code-level analysis, a small non-significant increase in the odds of preterm birth and low birthweight, as well as a small decrease in birthweight and gestational weeks, was seen . AlthougIn the street-level analysis, a decrease in birth weight was found when active poultry CAFOs were within 5 km of maternal residence . Specifip-value = 0.01) and gestational days , while the buffer model suggested a significant association for birthweight only . A decrease in birthweight and gestational weeks was found when making comparisons in the low-to-high-exposure tertile (IDW= 2.8–6.2) (p = 0.01) in birthweight and a decrease of 1.51 gestational days   . Compare = 0.01) . The odd = 0.01) . The hig = 0.01)  were conThis study assessed birth outcomes on the Eastern Shore, Virginia, and suggests that maternal residency near active poultry feeding operations may be associated with reduced gestation and birth weight. Previous research has found that residential proximity to CAFOs is associated with poor health outcomes, particularly causing adverse respiratory symptoms ,52,53,54While future studies should examine birth outcomes in other areas near poultry/other agricultural animal-feeding operations, such as in Maryland and in the Midwest, prior research has shown that CAFOs are located in areas with higher proportions of persons identifying as Black or Hispanic and in low-income areas ,56,57. ALimitations of the current analysis include potential confounding by variables that were not available in the dataset, or residual confounding due to imperfect covariate or exposure measurement. For example, we did not have information on the mother’s prenatal care, nutrition, disease status, nontobacco drug exposures, body mass index, proximity to other sources of pollutants, and employment status. As shown in Our study used an inverse distance weighted model, which examined the density and distance of CAFOs. Although this method allows for a more refined exposure metric than methods used in previous studies, it does not fully assess CAFO exposure. Air dispersion models, such as the Industrial Source Complex Short-Term Model, (ISC-ST3) and the California Puff Model (CALPUFF) model, have been used by other studies to model ammonia and hydrogen sulfide emissions from CAFOs and account for wind-direction and land-cover pattern ,63. FurtOur findings suggest that there is an association between maternal residency near active poultry feeding operations and reduced gestation and birth weight. We further found that the inverse distance weighted model, which weights increasing numbers of CAFOs closer to the maternal residence, is more strongly associated with adverse birth outcomes. This study adds to the growing body of works in the literature exploring proximity to CAFOs and adverse birth outcomes.


Event histories such as marriage and birth have been used to study fertility behavior of women. Understanding the timing of these events provide insight to reproductive patterns of the population. Thus, the aim of this study was to assess the timing of marriage and durations of birth intervals and their associated factors, and and to examine their effects on the current fertility among women in Dabat health and demographic surveillance system site, Northwest Ethiopia.A community based cross-sectional survey was carried out in the beginning of 2020 among 1649 women of reproductive age group. Data were collected using structured and interviewer administered questionnaire. The parametric survival analysis was employed to estimate the relationships among socioeconomic and demographic variables with outcome variables, the timing of age at first marriage and duration of birth intervals.This study confirmed that median age at first marriage was the lowest estimated at 15 years which was below the national and regional average. The result of the study also revealed that married women waited almost a median duration of three years for their first, second, third and fourth child which was increased to nearly four years for three years preceding the survey. The parametric survival analysis showed woman’s education, occupation, and current age were the predictors of age at first marriage. divorce experience, women empowerment and marriage cohort were the determinant factors of first birth interval; women education, child death, and ideal number of children were the predictors of second and third birth intervals; and media exposure and child death experience of women were predictors of fourth birth interval.The study indicated that median age at first marriage was the lowest though the successive birth intervals were longer. The survival analysis identified women’s education, occupation, child death and ideal number of children affected the timing of age at first marriage and duration of birth intervals. Hence, encouraging women for higher education and giving opportunity to women in employments may contribute for delaying age at first marriage and increasing the duration of birth intervals which in turn slowing down the fertility of women. Event histories such as marriage and birth have been used to study fertility behavior of women . MarriagEarly marriage is often associated with early childbearing that marks a woman’s transition into motherhood , 11. TheBirth interval, the time duration between two consecutive live births, is also one of the important indicators of fertility situation of a country as the reproductive preference for spacing instead of limiting is peculiar to sub-Saharan Africa , 15. A sBirth interval has also received attention in public health researches because of its implications for maternal and child health . GloballAs noted elsewhere, in a population where fertility often takes place within marriage and contraceptive practice is low, there is an inverse relationship between age at first marriage and fertility. However, total fertility rate of ANRS is the lowest in the country with low age at first marriage . It is aA community based cross-sectional survey was employed from January 10 to February 29, 2020 at Dabat HDSSs. The Dabat HDSSs is hosted by the University of Gondar located in Dabat district, Northwest Ethiopia, 75 Kms north of Gondar and 804 Kms northwest of Addis Ababa. Dabat district has a total of 26 rural and 4 urban kebeles . According to the 2007 census report, the district had an estimated total population of 145,458 which was projected to be 172, 836 in 2015 , 23.Dabat district was initially selected purposively as a surveillance site for its unique three agro-climatic zones  with altitude ranging from about 1000 to 2500 meters above sea level . CurrentThe study population included women of reproductive age who have been living in the surveillance site at the time of the interview. The sample size was determined using a single population proportion formula considering the following assumptions: the proportion of women with reproductive age who had a first birth by age greater than 20 (47%) ; the ageData were collected using structured, pretested, and interviewer administered questionnaire. The questionnaire was adopted from Ethiopian demographic and health survey and other literatures. It consists of sociodemographic and reproductive health characteristics of the respondents. To maintain consistency, the questionnaire was first translated from English to Amharic, the native language of the study area, and was retranslated back to English by professional translators and demographers. The tool was pretested on 6% (100) of the total sample out of the study area. During pretest, the acceptability and applicability of the procedures and tools were evaluated. Twenty data collectors, two in each Kebele, and five field supervisors, both working in DHDSS site, were recruited for the study. Two days training on the objective of the study, content of the questionnaire, confidentiality of information, how to use open data kit (ODK) software suites and techniques to conduct interview was given to data collectors and supervisors. Data were collected using ODK software suites which is one of the most well-known mobile data collection frameworks. It is capable of controlling data entry error with its skip rules, internal consistency checks and facilities such as supporting multiple languages and making calculations . Data coThe dependent variables were the timing of age at first marriage (measured in years) and durations of successive birth intervals (measured in months). The duration between first marriage and first live birth is considered first birth interval. Other intervals are calculated by the difference between two successive live births. The events for this study are the first union with a husband, first birth, second birth, third birth and fourth birth. For women who have never been married and did not give birth at any birth interval (censored), the time was measured till the date of the interview. The independent variables were the socioeconomic and demographic characteristics of women with reproductive age. The variables were categorized for analysis as shown in Tables Data on women aged 15 to 49 years were entered into Open Data Kit (ODK) software suites and exported to STATA version 14 for cleaning and analysis. Descriptive statistics including frequencies and proportions were used to summarize the variables of interest. Median age at first marriage and duration of birth intervals were also carried out along with the respondents’ characteristics. The survival analysis methods were carried out because the study contained censored data. Log rank test was used to test the significance of different categorical factors under this study. The model selection in this study was done based on the distributional assumption of the baseline hazard. Based on this assumption, if the baseline hazard had a specific distributional pattern, comparison will be made among parametric survival models. In this study the baseline hazard had a distributional pattern. Akaike’s Information Criterion (AIC) was used for model comparison or to select the models, and the parametric survival models with low AIC value were considered as a best-fitted model. So, five models containing variables of interest were fitted for this study to determine the effects of various factors on age at first marriage and birth intervals. Model I examined the effects of predictor variables on age at first marriage. Models II, III, IV and V assessed the effects of predictors on first, second, third and fourth birth intervals, respectively. Based on AIC, the parametric survival models such as Loglogistic regression (AIC = 842.6) model for model I, Lognormal regression (AIC = 1676.72) for model II, and Loglogistic regression for Models III, IV and IV have the smallest AIC value  among the models considered. In parametric models, we measure the direct effect of the explanatory variables on the survival time instead of hazard. In this study, the effect size for parametric model is the time ratio. For each covariate, a time ratio value greater than one can be interpreted as individual experiences the event at a later timing. On the other hand, a time ratio value of less than one implies that individuals will experience the event of interest faster. Adjusted time ratio (ATR) with 95% Confidence interval in the multivariable model was used to select variables that have a statistically significant association with age at first marriage and birth intervals. Few cases pertaining to premarital births and shorter birth intervals (less than 7 months) were dropped. In this study, p-value less than 0.05 is considered to be statistically significant.Out of 1683 women of reproductive age planned to be included in the study, 1649 have participated in the interview making respondent rate of 98%. The study revealed that 1062 (64.4%) respondents were from rural areas, and about 18% were born in urban areas. Women whose age less than 25, and greater than 34 years constituted 47% and 28.74% respectively. Among respondents, 35.4% had no formal education, and 47% of them were housewives in occupation. Among the participants, ever married, currently married and never married/single women accounted 924(56%), 812(49.2%) and 725(44%) respectively. Women who had media exposure and whose ideal number of children greater than four accounted 27.8% and 36% respectively. Among all women age 35–49, 94% have been married at least once which indicated that marriage is nearly universal in the study area .As mentioned elsewhere among participants (1649), 924 (56%) women have been married at least once in their lives. The results showed that there are significant variations in marriage by socioeconomic and demographic characteristics of women. Among ever married women, 303 (32.8%) were from urban areas, 22.9% had media exposure, and 15.7% & 48.3% women age less than 25 and greater than 34 years respectively. Among ever married women, 56.6% had no formal education, and 75.3% of them were housewives in occupation. Ever married women whose age at first marriage and birth less than 18 years accounted 71.6% and 41% respectively. Among husbands, 68.6% were a farmer in occupation and 58% had no formal education. The marriage cohort between 1990 and 1999, and 2000 and 2009 constituted 30.3% and 31.5% of ever married women respectively. The oldest marriage cohort (married before 1990) and the recent marriage cohort (married between 2010 and 2020) also accounted 13.74% and 24.46% respectively. Two hundred fifteen (23.3%) ever married women had divorce experience at least once in their lives, and of these, nearly two-third of them had divorced before they gave birth to their first child .Among ever married women (924), 836 (90.5%), 717 (77.6%) and 572 (61.9%) had at least first, second and third birth respectively. Among those, 783, 817, 695 and 554 women were included in the interval analyses by dropping 141, 19, 22 and 18 women who had shorter birth intervals (less than 7 months) respectively. Never married women who gave birth to a child accounted 5.6% (41) and majority of them (34) lived in urban areas. Those women who have a premarital birth were removed from the analysis because the duration between first marriage and first birth is considered first birth interval. Other intervals are calculated by the difference between two successive live births after marriage.The median interval between first marriage and first birth  was longer in rural areas (43 months) compared to those in urban areas (36 months). The median duration of first birth interval of women who married before 18 years (47 months) was greater than those who married at age 18 or more years (28 months). It was longer for women with no formal education (51.5 months) but shorter for those whose educational status was secondary or above (30 months). There was a consistent increase in first birth interval from younger (28 months) to older (51 months) age groups. First birth interval for women who had media exposure was shorter (32 months) than those who didn’t have (43 months). Housewives had longer (43 months) first birth interval than employed women (30 months). Women who had divorce experience before first birth had longer first birth interval (67 months) than those who didn’t have (37 months). Women who had child death experience, whose husbands were farmers and with no formal education had longer first birth intervals, and women with highest wealth status had shorter birth intervals. There was a steady decline in first birth interval from older (64 months) to younger (28 months) marriage cohorts .Contrary to first birth interval; women in urban areas, employed in occupation, had media exposure, with no child death experience and with secondary and above educational status had longer second, third and fourth birth intervals. In addition, women whose husbands were employed in occupation and with secondary or more education, and those women with highest wealth status had longer second, third and fourth birth intervals.In three years preceding the 2020 survey, 443 children were born to 394 mothers. Among 394 women, 88.6%, 10.4% and 1% gave one birth, two births and three births, respectively. On the other hand, from 443 births, 79 births didn’t have a birth interval for 78 children were first birth order and one twin second birth order. Thus, the birth interval analysis in three years preceding the survey was computed for 315 mothers. Duration of birth interval considered was the time between their most recent birth and the one preceding the last birth. Hence, among these women, 6.67%, 7.3%, 12.38% and 73.65% gave birth within an interval duration of 7–17 months, 18–23 months, 24–32 months, and 33 & above months, respectively. The mean and median birth intervals of women who gave birth in three years preceding the survey were 53.88 and 46 months, respectively .The average duration of exclusive breastfeeding and postpartum amenorrhea had shown a slight increase as the birth interval elongates beyond 17 months. While the average duration of amenorrhea remained stable for a birth interval of more than 18 months duration, the average duration of breastfeeding was relatively shorter for the longest birth interval category as compared to births of 18–32 months of birth interval. The mean and median duration of exclusive breastfeeding and postpartum amenorrhea of women who gave birth in three years preceding the survey were 7 and 6 months, and 11.6 and 12 months respectively .For time-to-marriage and birth interval data, multivariable parametric models of log-logistic and log-normal distribution were fitted. The Log-logistic regression analysis (model 1) shows that current age of women, educational level, occupation, ideal number of children, and wealth status of women have a statistically significant relationship with timing of first marriage. According to the result, younger women have slower transition to first union compared to older women. Woman’s educational attainment significantly delayed the timing of marriage. Women with primary  and secondary  education had slower transition to first marriage compared to those without any formal education. Compared to housewives in occupation, students , employed women  and job seekers  wait longer time before first marriage. Women who had less than or equal to four ideal number of children wait longer time before first marriage compared to those with five or more ideal number of children . Women with the lowest wealth status postponed marriage timing compared to those with middle status .Based on the Log-normal model (model 2), divorce experience before first birth, ideal number of children, women empowerment, and marriage cohort of women were significant predictors of the duration of first birth interval. Women who had divorce experience before first birth have longer first birth interval than those who didn’t have divorce experience . Women with five or more ideal number of children had faster transition to first birth after marriage compared to those with less than or equal to four ideal number of children . Women who hadn’t empowered to decide the households’ monthly expense have shorter first birth interval than those who had empowered . Compared to the middle wealth status, women with lower status have longer first birth intervals . Women with the oldest marriage cohort, married before 1990, have longer first birth interval than those women who got married between 1990 and 1999 . First birth interval was also shorter among women who got married between 2000 and 2009  and after 2009  compared to those who got married between 1990 and 1999. Women who are in the older marriage cohort waited longer to have their first child after first marriage.The Log-logistic model (model 3) shows that age at first marriage, women education, divorce experience, child death experience, and ideal number of children have significant effects in the second birth interval, duration between first and second births. Women whose age at first marriage was greater than 18, legal age, have faster transition to second birth . The interval between first birth and second birth was longer among women who spent some time in schooling  than those who didn’t have formal education. Women who had divorce experience in their lives have longer second birth interval (TR = 1.24: CI = 1.13–1.36) than who didn’t have, and it is shorter among women who had child death experience (TR = 0.79: CI = 0.71–0.89) compared to those who didn’t have the experience. Women who had five or more ideal number of children had shorter second birth interval compared to those with less than or equal to four ideal number of children .Model 4 in In the case of the fourth birth interval (model 5), Log-logistic regression model illustrates that media exposure, child death experience, ideal number of children and marriage cohort of women are correlated with the fourth birth interval. Compared to women who didn’t have media exposure, those who had media exposure have longer fourth birth interval . The fourth birth interval is shorter among women who had child death experience (TR = 0.81: CI = 0.69–0.94) compared to those who didn’t have the experience. Women who had less than or equal to four ideal number of children had longer fourth birth interval compared to those with five or more ideal number of children , and it is shorter among women who got married before 1990 compared to those who got married between 1990 and 1999 .This study is conducted to analyse the covariates of age at first marriage and birth spacing and to examine their effects on the current fertility. The result showed that the population in the study area was characterized by low median age at first marriage and longer successive birth intervals. The age at first marriage was estimated at 15 years which was below the national and regional average . The exiThe result of the study also revealed that married women waited almost a median duration of three years for their first child with median age at first birth of 18 years. The median interval between first marriage and first birth, first birth interval, is nearly equal to that of in Amhara region which was the highest among other regions in the country . BesidesOn the other hand, the result revealed that married women waited a median duration of three years for their second, third and fourth child. However, contrary to first birth interval, the median length of second, third and fourth birth intervals were longer among women of urban residents, high age at marriage, better educational attainment, wealth status, employment status and media exposure. Women with these background characteristics got married at later age but not waiting longer for the first baby after marriage but delaying the second, third and fourth births by spacing the consecutive birth intervals.The median birth interval of married women for three years preceding the survey was also estimated at 46 months 3.8 years) which was higher than the previous studies  years wh, 29, 30 Results of multivariate analyses indicate that current age of women, educational level, occupation, ideal number of children, and wealth status of women have a statistically significant relationship with timing of first marriage. Younger women took longer time to get married than those who are higher ages. It may be partly attributed to younger women may have the chance to have higher educational attainment compared with their older counterparts. In addition, since recent times, marriage among younger women is based on self-selection of a partner that may contribute for late marriage. This finding is consistent with those of earlier studies conducted on Ethiopian women , 5. EducThe types of occupations in which women were engaged also affected the age at first marriage. Employed women waited longer time before first marriage compared to housewives. This may be the fact that employed women were possibly educated and were likely to marry at later ages. Employment influences on women’s marital decisions as it tends to lead women to appreciating singlehood and cohabitation over marriage . On the Women with the lowest wealth status postponed marriage timing compared to those with middle status which is consistent with studies in Ethiopia , 34 and The parametric survival analysis also identified the variables that were statistically associated with the duration of first, second, third and fourth birth intervals. Women who had divorce experience before first birth have longer first birth interval. This could be the fact that a woman who already divorced before her first birth may take longer time to give birth to her first child after remarriage. The result also showed that women with five or more ideal number of children had shorter first, second and third birth intervals. This may be women who desire to have large number of children in the future need to give birth to a child immediately after marriage or a birth to achieve the desired number of children which is in line with the previous studies . Women wThe study also revealed that women with the oldest marriage cohort waited longer time to give birth to first child after marriage compared to those with recent marriage cohort, consistent with a study conducted in Amhara region  and ChinWomen’s higher educational attainment is associated with longer second and third birth intervals compared to women without formal education. This might be attribute to the fact that when the education status of women increased, knowledge and awareness of the women upon the consequences of short birth interval on maternal and child health and the benefit of small family size will also be enhanced. This is similar to other studies , 38. WomThe effect of contraceptive use was not controlled for, as the interval was computed for some times in the past whereas information on use of contraceptive referred to the survey period. On the other hand, some of the findings contradict the results of previous studies in developing countries. Women’s residence was not statistically significant with either of the dependent variables, timing of age at first marriage and duration of birth intervals. The role of health extension workers in rural areas may help to have similar information with urban residents about the benefit of having desired number of children in a household. Age at first birth was the other variable that didn’t show any effect on the duration of birth intervals. This may be the fact that women who gave first birth at later age may have successive births until they achieve the desired number of children.The current research result showed that the median age at first marriage was the lowest which was below the national and regional average. However, the population in the study area was characterized by longer successive birth intervals with almost a median duration of three years for the first, second, third and fourth child, and even the longest median birth interval for three years preceding the survey  that harmonizes the effect of low age at first marriage for fertility rate. Longer successive birth interval is likely to reduce the total fertility rate of a woman. Increasing duration of successive birth intervals directly affect completed fertility by reducing the number of years available for childbearing of a woman. Women who delay child bearing may consequently have low fertility.The parametric survival analysis also identified that women’s educational attainment and occupation contributed most for the timing of age at first marriage. In addition, it is also found that the decision to give birth to a child at a given interval depends on women’s educational attainment, child death experience and ideal number of children a woman would have. Hence, encouraging women for higher education and giving opportunity to women in employments rather than housewives, working at home, which may decrease the child mortality and shape the desired number of children, may be the most influential way of delaying age at first marriage and increasing the duration of successive birth intervals which in turn slowing down the fertility of women in the study area.S1 Data(DTA)Click here for additional data file.


Recent work has reported calpeptin’s capacity to inhibit entry of the virus into cells. Elsewhere, several drugs, including calpeptin, were found to be able to inhibit extracellular vesicle (EV) biogenesis. Unsurprisingly, because of similarities between viral and EV release mechanisms, calpeptin has also been shown to inhibit viral egress. This approach, identifying calpeptin, through large-scale screening studies as a candidate drug to treat COVID-19, however, has not considered the longer term likely benefits of calpain inhibition, post-COVID-19. This perspective will reflect on the capacity of calpeptin for treating long COVID by inhibiting the overproduction of neutrophil extracellular traps potentially damaging lung cells and promoting clotting, together with limiting associated chronic inflammation, tissue damage and pulmonary fibrosis. It will also reflect on the tolerated and detrimental  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on entering the upper respiratory tract and infecting type II alveolar cells, causes severe inflammation ,2). ThisThis perspective will discuss the future use of calpeptin, the cell permeant cathepsin/calpain inhibitor, as a possible anti-SARS-CoV-2 drug. It will focus on calpeptin’s capacity to inhibit: (i) viral entry and (ii) extracellular vesicle (EV) release and viral egress. However, these reports have not commented on additional benefits of calpain inhibition, especially important in post-COVID-19. This article will therefore also reflect on calpeptin’s inhibition of (iii) neutrophil extracellular trap (NET) formation  and (iv)+ (TMPRSS2+) cells, a rapid entry is achieved in a pH-independent manner, cells being activated rapidly at the cell surface. (ii) In cells lacking or with low-level expression of TMPRSS2, the virus is endocytosed and sorted to endolysosomes where activation is pH-dependent.SARS-CoV-2 has two possible entry mechanisms, thus broadening its tissue tropism: (i) in transmembrane serine protease 2− cells  within the S1 subunit has bound ACE2 on target cells, the conformationally altered S2 subunit mediates membrane fusion following proteolytic cleavage away of S1, at the S1/S2 boundary (S2′ site)  showed an EC50 of 174 and 163 nM, respectively  ; von Wil− [vWF−] ) or systvelop PF . This havelop PF . TargeteCalpains promote inflammation by a number of mechanisms leading to NF-κB activation and production of proinflammatory cytokines. They also result in recruitment of inflammatory cells and migration . FurtheCAPNS1 was tolerated.By way of example of tolerated side-effects of calpeptin therapy, inhibition of calpain in preclinical models revealed itself to be neuroprotective following cancer chemotherapy , with noCAPNS1 knockout in aged mice and calpain-1 and -2 deficiency in muscle, resulted in dystrophy [CAPN1 mutation) helped bring about ataxia [CAPN1 knockdown in mice affected platelet aggregation but with no adverse effect on bleeding times [In terms of detrimental side-effects, ystrophy . In mousystrophy . Calpaint ataxia . In otheng times .This article has described the inhibition of EV biogenesis as a way of limiting viral cell-to-cell transmission. Depending on the EV biogenesis pathway being targeted, there may be added benefits, especially in ameliorating PF in COVID-19, as mentioned above. Treating COVID-19 with calpain inhibitors such as calpeptin, a potent inhibitor or EV release ,30 will This perspective has summarized four significant roles of calpain inhibition in COVID-19, using the peptidomimetic calpain inhibitor, calpeptin. As a prospective drug, calpeptin has low toxicity having been tolerated in mice for up to 4 weeks . Althoug