Maternal thyroid hormones and carnitine are reported to affect neonate birth weight during the second trimester, which is one of the most important markers for fetal growth and perinatal mortality and morbidity. Nevertheless, the effect of thyroid hormone and carnitine in the second trimester on birth weight has yet to be understood.This was a prospective cohort study with 844 subjects enrolled during the first trimester. Thyroid hormones, free carnitine (C0), neonate birth weight, as well as other related clinical and metabolic data were collected and assessed.Pre-pregnancy weight and body mass index (BMI) as well as neonate birth weight were significantly different among different free thyroxine (FT4) level groups. Maternal weight gain and neonate birth weight varied significantly when grouped by different thyroid-stimulating hormone (TSH) levels. There was a significantly positive correlation between C0 and TSH (r = 0.31), free triiodothyronine (FT3) (r = 0.37), and FT4 (r = 0.59) . In addition, a significantly negative influence was found between birth weight and TSH , so as C0  and FT4 . Further assessment detected a stronger combined effect of C0 and FT4 (P < 0.001) and of C0 and FT3 (P = 0.022) on birth weight.Maternal C0 and thyroid hormones are of great importance in neonate birth weight, and routine examination of C0 and thyroid hormones during the second trimester has a positive effect on the intervention of birth weight. The Carnitine exists as free carnitine (C0) and acylcarnitine fractions in blood and plays an important role in fatty acid oxidation during the gestational metabolism . Carnitiin utero, which reflects fetal adaptations to the intrauterine environment. SGA newborns have an increased risk of prenatal mortality , FT4, free triiodothyronine (FT3), and total thyroxine (TT4) concentration in the second trimester, and neonatal birth weight data were available. Women with multiple pregnancies, accompanied with pregnancy complications such as abortion, GDM, and hypertensive disorders, using medication known to interfere thyroid hormones, or had a history of thyroid diseases were excluded.Maternal clinical characteristics—including age, height, pre-pregnancy and prenatal weight and body mass index (BMI), gravity, parity, mode of delivery, educational level, and neonatal birth weight—were obtained from hospital information system and child care system.The blood samples were collected after overnight fasting, and samples were centrifuged within 6 h. The concentrations of TSH, FT4, FT3, and TT4 were determined according to the measurement instructions.2.2We aim to investigate the 31-carnitine-related plasma metabolite level of pregnant women at the second trimester. We also obtain their neonate blood plasma sample. The blood samples were stored at −20°. Furthermore, the samples were prepared by tandem mass spectrometry  to test the concentration. The method used in the present study was essentially a modification of the procedure described elsewhere. Amino acid (AA) and acylarnitine (AC) were quantified using appropriate isotope-labeled standards. Liquid Chromatogram (LC) separation was performed on an Acquity UPLC HSS T3 column  using water with 0.1% formic acid (0.1% methanol and 5 mM ammonium acetate) detected with a Xevo-G2-QTOF MS (Waters Corporation) operating in a positive mode. Raw data were processed using TargetLynx as described previously. Accuracy of quantification was below 6% for all quantified metabolites except glutamic acid (13.9%). Quantitative data were obtained using MetIDQTM software.2.3P < 0.05 was considered statistically significant.The data were expressed as mean ± standard deviation (SD). The baseline characteristics of the subjects were described, and the p-values are indicated. Binary variables were presented as frequency and percentage and were compared using the Chi-squared test. A nonparametric test or a t-test was used to compare the medians of continuous variables. The heatmap was available from the package “ggplot” as an enhanced version or its basic function stats in R. We used the liner regression model, as well as multiple regressions to investigate the association of C0, FT3, FT4, TSH, and TT4 with birth weight. We assessed the combined effects of C0 and FT4 on birth weight by adding a product interaction term of the C0 × FT4 to the model. The same analysis was done on the effect of other hormone on birth weight. A heatmap was constructed to display the differences in birth weight. All statistical analyses were performed using R statistical software version 3.4.1  or Statistical Package of Social Sciences version 20.0 for Windows . In all analyses, 33.1The maternal characteristics grouped by FT4 quartile are shown in 3.2Among 31 carnitine-related metabolites, we selected the metabolites with statistically significant differences and listed them in Carnitine-related metabolites grouped according to the TSH level are shown in 3.3We used a clustering heatmap to describe the relationship between THs and 31 carnitine-related metabolites Figure 13.4P < 0.001), FT3 , and FT4 . There was no significant correlation between C0 and TT4.The relationships between C0 and TSH, FT3, FT4, and TT4 of all pregnant women are shown in 3.5In the second trimester, a higher TSH  level was associated with a lower birth weight , by 29.203 g , and by 21.079 g  for every unit increase in C0, FT4, and TSH concentrations, respectively Table 5.3.6P < 0.001, We found that the level of TSH, FT3, FT4, and C0 in the second trimester would have greater effects on birth weight. Meanwhile, we found a relationship between C0 and thyroid function parameters. Therefore, we assessed the combined effects of C0 and THs, including TSH, FT3, FT4, and TT4, on birth weight. 4Our research showed that there was a significantly positive correlation between C0 and TSH, FT3, and FT4. In addition, C0, FT4, and TSH all had significantly negative influence on newborn birth weight. Therefore, we evaluated the co-effects of C0 and THs on birth weight and found that the combinations of FT4 and C0 and of FT3 and C0 were significantly associated with birth weight, whereas that of TSH and C0 and that of TT4 and C0 were not.in vivo is limited by enzymes, there is no significant relationship between TT4 and birth weight. FT3 is three to five times more active than FT4. It is unclear whether maternal T3 actually crosses the placenta. FT3 is a TH that plays a direct biological role. Activation of deiodinase leads to a high rate of conversion of FT4 to FT3, resulting in low FT4 levels and high FT3 levels. High FT3 levels increase fetal weight directly through anabolic effects on fetal metabolism and stimulation of fetal oxygen consumption  for the publication of any potentially identifiable images or data included in this article.MY contributed to the collection, analysis, and interpretation of data as well as manuscript preparation. MS and QW contributed to the data collection and analysis, and YJ contributed to the interpretation of data. CJ contributed to the language editing. JX and QL contributed to the study design, data interpretation, and manuscript preparation. QL is the guarantor of this work and, as such, has full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.


Ocln knockout mice as well as a combined omics analysis and immunofluorescent labelling. Our results showed that the epididymis of Ocln-null mice displayed a phenomenon resembling epididymal sperm granuloma, which occurred especially in the junctional region between caput and corpus epididymidis. Sperm motility and fertilisation capacity were also impaired in these Ocln-null mice, accompanied by enlarged tubules in the proximal regions and degeneration in the distal regions of epididymis. Cellular localization analysis showed that OCLN immunofluorescence was enriched only in the apical junction of epithelial principal cells in the proximal regions of epididymis. Integrative omics analysis revealed the downregulation of gene clusters enriched in acid secretion and fatty acid metabolism in the Ocln-null epididymis, especially the enzymes related to the unsaturated arachidonic acid pathway. The number of proton-pump V-ATPase-expression clear cells, a key player of luminal acidification in the epididymis, declined drastically from prepubertal age before sperm arrival but not in the early postnatal age. This was accompanied by programmed cell death of clear cells and increased pH in the epididymal fluid of OCLN-deficient mice. The lipidomics results showed significantly increased levels of specific DAGs conjugated to unsaturated fatty acids in the Ocln-mutant. Immunofluorescent labelling showed that the arachidonic acid converting enzyme PTGDS and phospholipase PLA2g12a were prominently altered in the principal cells and luminal contents of the Ocln-mutant epididymis. Whereas the carboxylate ester lipase CES1, originally enriched in the WT basal cells, was found upregulated in the Ocln-mutant principal cells. Overall, this study demonstrates that OCLN is essential for maintaining caput-to-corpus epithelial integrity, survival of acid-secreting clear cells, and unsaturated fatty acid catabolism in the mouse epididymis, thereby ensuring sperm maturation and male fertility.Occludin (OCLN) is a tight junction protein and Ocln deletion mutation causes male infertility in mice. However, the role of OCLN in male reproductive system remains unknown. In this study, we used an interdisciplinary approach to elucidate the underlying mechanism of male infertility in related to OCLN function, including  The epididymis is a post-testicular male reproductive organ to provide a region-specific and optimal luminal microenvironment for sperm maturation and storage before sperm transport to the vas deferens for ejaculation –3. Such citation –13, and citation . Thus, sertility , 15, 16.ertility –8, 17. Mertility . All theOcln knockout (Ocln-KO) male mice are infertile  in the transcriptomes and proteomes of Ocln-KO compared to WT mice. The cellular expression of the significantly downregulated DEGs, including proton-pump V-ATPase and fatty acid catabolic enzymes PTGDs and CES1, were found to be altered in specific epithelial cell types. Overall, our study demonstrated the important role of OCLN in acid-base balance and metabolism in the epididymis.Occludin (OCLN) is an epithelial tight junction protein and nfertile ; howeverOcln-KO and B1-V-ATPase-EGFP mice were provided by Prof. Eveline E. Schneeberger and Prof. Sylvie Breton from MGH/Harvard Medical School. The background of these mice has been published previously  mice (C57BL/6) for maintenance. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of ShanghaiTech University.eviously , 24. AllOcln-KO male mice aged 6, 9, 12, 20, 40 and 60 weeks were mated with two female wild-type mice. At least three pairs of mice of each age were mated for at least two weeks. To determine male mice mating behavior, the female mice (12-week-old) from the mating cage were examined for the plugs and the vaginal plugs were smeared, and the spermatozoa were observed under microscopy.WT and in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) assays, sperms were directly obtained from caput or cauda epididymis of WT and Ocln-KO males, the assays were performed as previously reported , embedded in O.C.T. compound (Sakura) and sectioned at −18°C or −20°C (Leica Microsystems GmbH), as described previously . Images were acquired using laser scanning confocal microscopy  and analyzed with the original software before imported into Adobe Photoshop for typesetting. Details of the antibodies used in this study are listed in The immunofluorescent labelling procedure was performed as described previously . BrieflyWestern blot was performed as published previously . Cell ly2 with Full-IMDM supplemented with 1 nM 5α-dihydrotestosterone and containing 10% (v/v) FBS, penicillin and streptomycin, as described previously (Ocln-KO cells were generated in DC2 cells by using the CRISPR/Cas9 system. The Ocln sequence in the second exon 5′-CGGCTGAGAGAGCATCGGC-3′ (Ocln–sgRNA) was targeted with pGL3-U6-sgRNA-PGK-Puro (Addgene). DC2 cells were then co-transfected with Ocln–sgRNA-expressing vector and Cas9 expressed vector pST1374-NLS-flag-linker-Cas9 (Addgene) by Lipofectamine 2000 and selected with puromycin and blasticidin for 24  h. Single cells were isolated by cell sorting. The single clones were validated by immunoblotting analysis and DNA sequencing. The two plasmids were gifts from Xingxu Huang  and recorded it by video. Data values were measured on captured images 10- second after the sample liquid touched the pH paper and the different standards in grey scale were used for calibration using Fiji software.For pH measurement in vas deferens of adult WT and Ocln-KO mice were pooled into one sample. The distal epididymidum was treated the same way. Three samples per group were used in this study. Lipids were extracted using a modified version of the Bligh and Dyer’s method as described previously (https://www.cloudtutu.com/).For lipid extraction, the proximal epididymides from three WT or eviously . Lipidomeviously . GlyceroOcln-KO mice whole epididymis by lysis buffer (50 mM (NH4)2CO3, 8 M urea, 1 mM DTT and protease inhibitor) separately and quantitation using BCA protein assay kit. After alkylated by iodoacetamide and digested by trypsin, the peptide was labeled by TMTs label regent following the manufacturer’s protocol. The resulting peptides were separated and analyzed on an Easy-nLC 1000 system coupled to a Q Exactive HF (Thermo Scientific). The LC-MS/MS procedure and data analysis were performed as reported previously  and kept at -80°C.The RNA with RIN >6.0 is about to undergo rRNA depletion. cDNA libraries were constructed for each pooled RNA sample by using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. The tagged cDNA libraries were pooled in equal ratio and used for 150 bp paired-end sequencing in a single lane of the Illumina HiSeqXTen. Before read mapping of pair-ends, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent of bases with qualities of<20. The clean reads were then aligned to mouse genome (version: GRCm38 NCBI) using hisat2. Differential gene and transcript expression analysis of RNA-seq experiments with HTseq  analysis were obtained from the following criteria  was used for all statistical analyses.Comparisons were performed using the 2-tailed paired or unpaired Student’s t-test for only two groups, or one-way or 2-way-ANOVA for the comparisons of more than two groups at one- or two-levels, respectively. Significance was defined as a Ocln-KO male mice were reported to be infertile , which confirmed their sterility after 9-week-old. However, at juvenile ages of about 8 weeks, 1 out 8 Ocln-KO male mice was able to fertilize female mice to give birth to live pups  mice, although no difference in proportional size of the epididymis or other organs was found  of principal cells of the initial segment (IS) and CPT epididymidis, as well as the lateral membranes of WT or Ocln-KO leads to the observed epididymal defected, we profiled the transcriptomes and proteomes of the whole epididymis from adult Ocln-KO mice in comparison to those of WT mice to find differentially expressed genes (DEGs). An integrative approach was applied to the analysis of DEGs obtained by combining RNAseq and proteomic datasets. Through the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the results showed that the gene clusters enriched in the acid secretion, and metabolic pathways, including lipid and amino acid metabolic pathways, were the main nodes of downregulation , such as arachidonic acid , a key player in luminal acidification of epididymis, keeping sperm in a quiescent stage . In the luminal contents, the apoptotic index was decreased from CPT to CD epididymidis, whereas the apoptotic index was increased in the epithelial cells of epithelium , but it obviously decreased at puberty and in adulthood  and their derivatives diacylglycerols (DAG) in the epididymis  and triacylglycerols (TAGs) did not differ between the two groups , energy (e.g. ATP) and water. In the epididymis, it has been known that both luminal acidification and regulation of metabolism play essential roles in sperm maturation, as illustrated in the proposed model in 2 hydrolyses spontaneously in water to become bicarbonate, and CO2-bicarbonate equilibrium is an essential process contributing to acid-base balance in the epididymis , was converted to other forms in the distal versus proximal epididymis. However, this conversion remained effective in the Ocln-null epididymis, suggesting that releasing unsaturated long fatty acid chains from TAG58:7(22:6) was independent of the loss of OCLN and/or there is a compensation conversion pathway upregulated. Consistent with both speculations, the content of TAG58:7(22:6) in the caput epididymidis was slightly decreased, indicating a net reduction in the synthesis of this TAG component, and triglyceride lipase Lpl expression was slightly upregulated in Ocln-null epididymis.Fatty acid chains conjugated to DAG and/or TAG are an important source of arachidonic acid and thus are important upstream precursors of lipid-mediated signaling pathways. Arachidonic acid is a 20-carbon unsaturated fatty acid chain containing four double-bonds. Our results showed that impaired release of free long-chain fatty acids, such as arachidonic acid, from DAG is one of the major defects in the OCLN-deficient epididymis. This notion is supported by our lipidomic data, in which DAG38:5(16:0/22:5) with long fatty acid unsaturated chains in the proximal epididymis and arachidonate-conjugated DAG38:4(18:0/20:4) in the distal epididymis were significantly accumulated in the OCLN-deficient epididymis. In addition, the enzymes known to be involved in this process were all downregulated, including Ocln-KO. Our immunofluorescent labelling results showed that, in the WT epididymis, CES1 is predominantly expressed in basal cells from proximal to distal regions, and only in principal cells in distal regions. However, CES1 was significantly upregulated in principal cells but decreased in basal cells of caput epididymidis of OCLN-deficient mice. A potential role for basal cells in epididymal lipid homeostasis has been reported . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository, with the dataset identifier PXD037690 .The original contributions presented in the study are publicly available. This data can be found here: The RNA-sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE166141  of ShanghaiTech University. Written informed consent was obtained from the owners for the participation of their animals in this study.WS conceptualized, experimental designed, data analysis, curation, and interpretation. WS and BL wrote the manuscript. BL and BZ performed most of the experiments. DG performed the animal phenotypic analysis as well as some omics and imaging experiments. XX performed some imaging experiments. QL participated in experiments of IVF and ICSI experiments. All authors contributed to the article and approved the submitted version.This work was financially supported by the National Natural Science Foundation of China , and ShanghaiTech University.The authors thank all the staff of ShanghaiTech core facilities for their technical support, especially Dr. Xiao-Ming Li of Imaging Core Facility Dr Pi-Liang Hao of Mass Spectrometry Core Facility and Dr Ying Xiong of Cell & Molecular Core Facility.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite a global decline in under-five deaths, the neonatal mortality rate remains slow in developing countries and birth asphyxia remains the third cause of neonatal deaths. Globally, neonatal deaths accounts for 45% of under-five deaths, birth asphyxia causes 23–40% of neonatal deaths in Ethiopia. There is limited data on risk factors of asphyxia in Ethiopia, particularly in the study area. Therefore, this study aimed to identify the risk factors of birth asphyxia among newborns.This research followed a hospital-based unmatched case-control study design at Debre Markos comprehensive specialized referral hospital, Northwest Ethiopia, among 372 newborns (124 cases and 248 controls). Data were collected by interviewing index mothers and chart review using a pre-tested questionnaire. Then it was entered in Epi-data version 3.1 and transferred to STATA version 14.0 for analysis. Bivariate and multiple variable logistic regression were carried out to the possible risk factors. Finally, statistical significance was declared using adjusted odds ratio with 95% CI and p-value <0.05.Prolonged labor >12, meconium-stained amniotic fluid, assisted vaginal delivery, gestational age < 37 weeks, noncephalic presentation, comorbidity, birthweight<2500grams were found to be significant factors of birth asphyxia.In this study, Prolonged labor >12 hours, meconium-stained amniotic fluid, assisted vaginal delivery, gestational age < 37 weeks, non-cephalic presentation comorbidity, fetal distress, birthweight<2500grams were found to be risk factors of birth asphyxia were risk factors of birth asphyxia. Therefore, to reduce neonatal mortality associated with birth asphyxia, attention should be given to holistic pregnancy, labor and delivery care, and post-natal care. Moreover, interventions aimed at reducing birth asphyxia should target the identified factors. Birth asphyxia is the difficulty to initiate and maintain breathing at birth which is distinguished by a marked impairment of gas exchange; if sustained leading to progressive hypoxemia and substantial metabolic acidosis ,2. GlobaCommonly known determinants of birth asphyxia vary across regions, depending on the context. In Ethiopia, where birth asphyxia is the second leading cause of neonatal mortality, early recognition and management of the risk factors of birth asphyxia are pillars to reduce neonatal deaths and improve neonatal quality of life. Therefore, it is necessary to identify the specific risk factors in a particular region to take appropriate interventions. Thus, this study aimed to identify the risk factors of birth asphyxia among newborns at Debre Markos referral hospital, Northwest Ethiopia. The findings of this study will be used as input for policymakers and program planners who work in the area to inform, plan, implement and evaluate health promotion policies and strategies on the reduction of under-five children mortality and improvement of child health care.Study design, period and setting: Institutional-based unmatched case-control study design was conducted considering neonates with asphyxia as cases and those without asphyxia as controls among newborns from August 1/2019 to October 2019 at Debre Markos referral hospital which is located in Debre Markos town, East Gojjam Zone, Amhara regional state. The town is found 299 km north west from Addis Ababa, capital city of Ethiopia. According to information obtained from the administrative offices of Debre Markos comprehensive specialized referral hospital, they provide different services in the outpatient department, inpatient department and operation room theatre department. The hospital serves for more than 3.5 million populations in the catchment area and it has more than 30 beds in NICU with annual admission of more than 2800 neonates of which, more than 770 neonates are by birth asphyxia. There are five pediatricians and 21 nurses in NICU. In obstetrics and gynecology ward there 7 gynecologists and 45 midwifery professionals with the annual delivery 6734 neonates.Study participants: The study population was both neonates with asphyxia and those without asphyxia admitted to neonatal intensive care units (NICU) in Debre Markos referral hospital. All live newborns who were born after 28 weeks of gestation were screened for eligibility. The study classified participants into cases and controls. Newborn babies with < 7 APGAR scores at 5 minutes were defined as having asphyxia at birth (cases), while newborn babies with ≥7 APGAR scores at 5 minutes were considered non-asphyxiated (controls). The research included newborns who met the case-control definition and whose mothers gave informed consent. The study excluded newborns who had met the case- control definition but whose mothers had not been interviewed voluntarily. Newborns with one or more life-threatening malformations, including congenital cyanotic heart defects and hydrops, and other severe birth defects also excluded. Besides, newborns with no recorded of 5th minute APGAR score and who were not diagnosed as birth asphyxia were excluded from the study.Sample size calculation and sampling techniques: The sample size was estimated using an unmatched case-control formula (Kelsey), which assumed a confidence level of 95%, power (80%) and case-control ratio of 2, and odds ratio (OR) of 2 to be defined as significant using Epi-info7. The proportion of non-asphyxia neonates (controls) (31.3%) and asphyxia cases (60.0%) with prolonged labor exposure (>12 hours) were obtained from a previously conducted study  at Lumame hospital to verify the questioner's accuracy. Clarification of question and time to complete the questionnaire was assessed. The supervisors and the principal investigator reviewed and updated the computed questionnaires every day, and the data collectors provided the necessary input for the next morning before the actual procedures began.Study variables: Birth asphyxia was the dependent variable. While the independent variables include maternal characteristics and variables related to obstetric history , gestational age; antepartum factors ; intrapartum factors , mode or type of delivery); fetal factors .Data analysis: Data was entered in Epi-data version 3.1 and transferred to STATA version 14.0 for analysis. Using descriptive statistics, socio-demographic factors, antepartum, intrapartum, and neonatal- related factors are presented using frequency tables, figures, and percentages. In the second stage, bivariate logistic regression was used to identify possible factors of candidate variables with a p-value <0.2 for the final model. The model fitness test was carried out using the Hosmer – Lemeshow test, which is a statistical test for fitness for logistic regression models. Finally, the multivariable logistic regression model was fitted to identify significant risk factors of birth asphyxia through a backward stepwise method, risk factors of birth asphyxia among newborns were determined using their adjusted odds ratio with 95% CI and p-value < 0.05.The following operational definitions are used Birth asphyxia: Neonate failure to start and sustain sufficient respiration within 5 minutes of birth with an Apgar score below 7 ( below 7 .Cases (asphyxiated newborns): all neonates diagnosed with asphyxia by the attending health professionals using an Apgar score of less than 7 at 5 minutes after birth were considered as cases. Controls (non-asphyxiated newborns) – all neonates diagnosed as non-asphyxiated by the attending health professionals using an APGAR score of more than 7 at 5 minutes were considered as controls.Ethics Approval and Consent to Participate: The ethical clearance letter has been received from the research and review committee from college of health sciences, Debre Marks University. Additionally, prior to beginning data collection permission was obtained from the hospital authority. Finally, informed written consent was received from each participant mothers after explaining the research objectives. The participants were briefed on the study's purpose, procedures, potential risks, and benefits. In addition, the participants were told that failure to agree or to withdraw from the study would not change or endanger their access to treatment.Socio-demographic characteristics: In this study, a total of 372 newborns (124 cases and 248 controls) were included in the analysis. Regarding the age of index mothers, majority (71.8 %) of cases and nearly half (49.6%) of controls were in the age group of 19 – 34 years. By residence of mothers, more than half (67.7%) of cases were from rural area. But, majority (75.5%) of controls were from urban residence. Most of the study participants were Orthodox Christian followers (88.7% cases and 89.9% controls). By marital status, majority of index mothers (84.7% cases and 78.6% controls) were married. Twenty-three (23.4%) of the cases mothers and 24.2% of the controls did not have formal education  of the mothers in the cases and (48.8 %) of the controls were multipara. There was a higher rate of > 4 antenatal care (ANC) visits among cases (52.4 %) than controls (41.1%), and the incidence of comorbidity among cases during pregnancy (38.7 %) was higher in proportion than controls (24.2 %). A duration of pregnancy < 37 weeks of gestation was more common among cases (32.3 %) than controls (11.2 %) ((11.2 %) .Intrapartum related characteristics and neonatal/ fetal related factors: The non-cephalic fetal presentation was relatively common among cases (29.0 %) compared with controls (14.5 %). There was also a disparity in proportion in terms of prolonged labor (>12 hours), where (69.4%) of cases were born after prolonged labor and only (33.5%) of controls were born after prolonged labor. Forty-six (37.1 %) cases and (14.9%) controls had meconium-stained liquor on pelvic examination. Forty (32.3%) of the cases and only (12.5 %) of the controls were preterm; (55.6 %) and (36.3 %) were low birth weight. There were (24.2 %) twin newborns among the cases and (17.3 %) among the controls . The likelihood of developing birth asphyxia among neonates born from prolonged-labor mothers (> 12 hours) was around three times higher than their counterparts . Neonates with intrapartum fetal distress were nearly five times more likely to experience asphyxia at birth than those born with normal fetal heart rate . Instrumental delivery (vacuum or forceps) was found to be significantly associate with the occurrence of birth asphyxia. Specifically, neonates born with the help of vacuum or forceps were around three times more likely to be asphyxiated compared to neonates born through spontaneous vaginal delivery . Neonates born with low birth weight were around four times more likely to have birth asphyxia compared with those born with normal weight . Concerning fetal presentations, newborns with the non-cephalic presentation were nearly five times more likely than those with a cephalic presentation to experience birth asphyxia . Also, neonates born from mothers with co-morbidity during pregnancy were around three times more likely than their counterpart to develop birth asphyxia . Furthermore, preterm babies were around three times more likely to develop birth asphyxia than the term  .Birth asphyxia is one of the leading cause of mortality for newborns; the effect of birth asphyxia is not limited only to death but also leads to physical, mental and social incapability in newborns due to severe hypoxic-ischemic organ damage . Hence, The odds of birth asphyxia were 4.25 times higher among neonates born from mothers with a history of meconium-stained amniotic fluid. This finding was similar to previous studies in Uganda, India, and Sweden –22. The Newborns born with a gestational age < 37 weeks 2.60 times were more likely to develop birth asphyxia compared to neonates with a gestational age of ≥ 37 weeks. This study is in line with a study conducted in Jordan and Brazil , 42. ThiIn Conclusion this study identified that, the main risk factors of birth asphyxia were fetal distress, instrumental delivery, low birth weight, non-cephalic presentation, preterm, prolonged labor, co-morbidity during pregnancy and meconium tainted amniotic fluid. Most of these factors are preventable through holistic care for pregnancy, labor and delivery. Therefore, to reduce neonatal mortality associated with birth asphyxia, attention should be given to holistic pregnancy care services, labor and delivery care. Furthermore, intervention strategies aimed at reducing birth asphyxia should target the identified factors.This was the first birth asphyxia study in the study area which shows risk factors of birth asphyxia. The case-control study design that was appropriate to address the research question and enabled the identification of possible risk factors birth asphyxia. This research is quantitative; if qualitative approach was also used it would be stronger to examine the extra risk factors of birth asphyxia in depth. This research may have maternal recall bias as they recalled their previous due to the retrospective nature the design. Using small samples, it's difficult to generalize to society at large. Therefore, larger studies will be needed to demonstrate true associations in the population.


At inclusion and after 12 weeks of treatment, the blood samples were collected, and the oxidative stress , superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid, advanced oxidation protein products (AOPP), advanced glycosylation end products ((AGE) and isoprostane), and inflammation (C-reactive protein (CRP) and interleukin-6 (IL-6)) parameters were determined. P < 0.01), decreased the levels of prooxidants , and decreased inflammatory parameters . Antioxidative action was associated with the improvement in arterial function (obtained in the previous study) in the empagliflozin-metformin combination group.  The empagliflozin-metformin combination increased levels of the antioxidants (TAS, SOD, and GPx up to 1.1-fold;  Empagliflozin-metformin combination has strong antioxidative and anti-inflammatory capacity, in adults with type 1 diabetes that is greater than that for the individual drugs. Its antioxidative activity at least partially explains the improvement in arterial function. Therefore, it appears that the combination provides the most powerful vascular protection. The mechanism behind these promising findings have not yet been completely understood [Chronic hyperglycaemia and glycaemic variability due to long-standing diabetes increase the risk of chronic complications. A major vascular complication in diabetes is accelerated atherosclerosis, preceded by impaired endothelial function and increased arterial stiffness as preclinical manifestations . Cardiovderstood .Hyperglycaemia accelerates atherosclerosis through several mechanisms, the most prominent being the increased oxidative stress and enhanced activation of inflammatory pathways . In atheBased on previous data, the beneficial anti-inflammatory and antioxidative pleiotropic effects of empagliflozin and metformin , 17, 18 http://clinicaltrials.gov (NCT03639545).This study was designed as a double-blind, randomized, interventional study. Forty individuals with type 1 diabetes from the outpatient clinic at University Medical Centre Ljubljana were recruited. They were equally randomized into four groups, as follows: (1) control (placebo), (2) empagliflozin (25 mg daily), (3) metformin (2000 mg daily), and (4) empagliflozin-metformin (empagliflozin 25 mg daily and metformin 2000 mg daily). The treatment period lasted 12 weeks. Participants took study medications in addition to their standard insulin (multiple daily insulin injections or insulin pump) therapy. All individuals participated voluntarily; signed informed consent was obtained from all participants. The study was performed following the ethical standards of the Declaration of Helsinki and was approved by the National Medical Ethics Committee of Slovenia. It is registered at 2. Exclusion criteria were HbA1c below 7.0%, age below 30 years or above 65 years, and body mass index (BMI) below 25 kg/m2. Additionally, patients with important comorbidities such as advanced heart failure , kidney (estimated glomerular filtration rate below 60 mL/min/1.73 m2) or liver failure , clinically important benign prostatic hyperplasia, prostatic carcinoma, or history of frequent urinary tract infections were also not included.Inclusion criteria consisted of confirmed type 1 diabetes diagnosis with glycated haemoglobin (HbA1c) above 7.0%, age 30-65 years, and body mass index (BMI) over 25 kg/mβ-stiffness) measurements, as described previously [The participants were assessed at inclusion and after 12 weeks of treatment; no further follow-up was performed. At inclusion, a complete medical history and full medical examination were performed. At these two time points, venous blood samples and second morning urine samples were also collected for additional analyses. In addition, ultrasonographic and morphological arterial function assessments were performed at both times. A single operator who was blinded for the study protocol and treatment performed the measurements. The methods were observer-independent; the results were generated automatically. Endothelial function was assessed ultrasonographically by performing the brachial artery flow-mediated dilation (FMD) and tonometrically measuring the reactive hyperaemia index (RHI). Arterial stiffness was also determined ultrasonographically and tonometrically through pulse wave velocity (PWV) and common carotid artery stiffness .Total antioxidant status (TAS) in serum was measured by suppression of 2,2′-azino-di-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS) production in the presence of hydrogen peroxide with antioxidants from the sample. The absorbance was measured at 600 nm , Randox Laboratories Limited, United Kingdom).The glutathione peroxidase (GPx) activity in erythrocytes was measured through a coupled reaction with glutathione reductase (GR). In the assay, cumene hydroperoxide was reduced and glutathione (GSH) oxidized to glutathione disulfide (GSSG) by GPx. Glutathione reductase then reduced the generated GSSG to GSH with the consumption of nicotinamide adenine dinucleotide phosphate (NADPH). The decrease in NADPH is proportional to GPx activity and was measured at 340 nm .For the superoxide dismutase (SOD) activity in erythrocytes, a colorimetric method was used. The inhibitory SOD activity in the formation of reduced red formazan dye with superoxide radical and xanthine oxidase from tetrazolium salt was measured at 520 nm .Uric acid was determined through an enzymatic reaction with uricase and Trinder-like endpoint product detection. The absorbance of the complex was measured at 545/694 nm.μg/L and CV within the series was under 10%. Serum aliquots used for the measuring of AOPP were diluted 200-fold before analysis. Interleukin-6 was measured with high sensitivity sandwich ELISA  with a detection limit of 0.8 ng/L and CV within series <5%.The concentrations of AGEs, advanced oxidation protein products (AOPP), and interleukin-6 (IL-6) were measured in serum samples. The aliquots for each analyte were analysed in one batch. The AOPP was measured using competitive ELISA ; the limit of detection was 26 μg/L and CV within series <6%  was used for the measurement of 8-OHdG. The concentrations of PAI-1 and AGEs were measured with sandwich-type ELISA  with a detection limit of 0.08 μg/L and 0.2 mg/L, respectively; CVs within series were <8% for both assays. Advanced oxidation protein product was measured using competitive ELISA ; the limit of detection was 26 μg/L and CV within series was under 10%. Serum aliquots used for the measurement of AOPP were diluted 200-fold before analysis. The measurement of IL-6 was performed using high sensitivity sandwich ELISA  with a detection limit of 0.8 ng/L and CV within series <5%.The concentrations of 8-hydroxydeoxyguanosine (8-OHdG), plasminogen activator inhibitor type 1 (PAI-1), AGEs, (AOPP), and IL-6 were measured in serum samples. The aliquots for each analyte were analysed in one batch. Competitive ELISA immunoassay with a detection limit of 2 Participants were identified by study ID; all blood and urine samples were also labelled by a study ID; the participants remained anonymous throughout the whole course of the study. Study data  were collected through paper case report forms that were stored securely. The data was also entered into the electronic database on an on-going basis. The results of arterial function measurements were backed up from the measurement devices. Data from laboratory analyses were provided and stored in paper form; they were additionally transferred to the electronic database system of the study. The electronic database was stored in secured hard drives with backup on password-protected computers that were only accessible to the research team.P < 0.05 was considered statistically significant. The possible associations between parameters of arterial function with the parameters of oxidative stress and inflammation were calculated using Pearson's correlation coefficients. The GraphPad Prism 5.0 software was used for statistical analysis performance.The values are expressed as mean ± SEM. The statistical analysis was performed by using a one-way analysis of variance (ANOVA). Where significant interactions were found, the Bonferroni posttest was performed. A All male participants were aged 44.7 ± 2.5 years with an average duration of type 1 diabetes of 22.6 ± 3.9 years and an average HbA1c of 7.8 ± 0.3%. Their average baseline systolic and diastolic blood pressure values were 131.4 ± 4.1 and 80.1 ± 2.9 mmHg, respectively. In each study group, 20% were smokers and 10% of the patients had suffered from a previous cardiovascular event. Additionally, 41% of the study participants had diabetic retinopathy, 12.5% had diabetic neuropathy, and 22% had diabetic kidney disease. Importantly, the age of the participants, duration of diabetes, glycaemic control, smoking status, and the occurrence of microvascular chronic complications or previous cardiovascular events did not differ between study groups. Baseline characteristics of the enrolled participants are summarized in P < 0.01). The increase of TAS in the combination group was also significant compared to both individual drugs (P < 0.05 for both). GPx activity increased in individuals treated with the empagliflozin-metformin combination and in the group treated with empagliflozin only  compared to controls. The increase in GPx activity in the combination group was also significant compared to the metformin group (P < 0.01). Only the empagliflozin-metformin combination significantly increased the levels of SOD up to 1.1-fold, compared to the control and the individual drugs . No significant differences in serum antioxidant levels were observed in the control group throughout the study (The empagliflozin-metformin combination significantly increased TAS up to 1.1-fold compared to the control group (he study .P < 0.01). The levels of isoprostanes and AGEs significantly decreased up to 1.2–fold and 1.5-fold, respectively, in the empagliflozin-metformin combination group and empagliflozin alone compared to only metformin or control . No significant differences in the prooxidant levels were observed with the controls (The empagliflozin-metformin combination significantly decreased the formation of AOPP by 1.2-fold compared to the control or only metformin (both controls .P < 0.01). The level of IL-6 significantly decreased up to 1.5-fold (P < 0.001) in the empagliflozin-metformin combination group and up to 1.3-fold (P < 0.05) in the group treated with empagliflozin only, compared to the controls. No significant differences in the proinflammatory parameter levels were observed in the control group  and negatively with AOPP  and isoprostane . Arterial stiffness parameters also correlated with prooxidative parameters. Pulse wave velocity (PWV) and carotid artery β-stiffness are also positively correlated with AGE  and isoprostane . Carotid artery β-stiffness was also positively associated with AGE .The associations between prooxidative and proinflammatory parameters were studied only in the group treated with an empagliflozin-metformin combination. Brachial artery flow-mediated dilation (FMD) positively correlated with TAS  leading to uniform vascular impairment. The participants were oral antidiabetic drug-naïve, thus enabling us to study the effect of the pure drug in a homogenous group. That being said, we believe that the results obtained can be extrapolated to individuals with type 2 diabetes, as vascular impairment mechanisms in these two populations have common denominators. Treatment with empagliflozin-metformin combination increased serum antioxidant levels  and decreased serum prooxidant levels . Furthermore, inflammatory parameters (CRP and IL-6) also decreased after 12 weeks of treatment. The empagliflozin-metformin combination provided the most powerful antioxidative and anti-inflammatory protection, in comparison to the individual drugs. Additionally, the beneficial antioxidative effect of this combination was significantly associated with an improvement in arterial function. Taking all the results into account, we can speculate that the antioxidative and anti-inflammatory action of empagliflozin-metformin combination represents one of the major mechanisms in the improvement of arterial function.β-stiffness [The present study represents a further analysis of the results obtained in our previous study which showed the beneficial effects of the empagliflozin-metformin combination on arterial function in adults with type 1 diabetes . Previoutiffness . HoweverAccording to preclinical studies, improvement of vascular function with SGLT-2 inhibitors can be mediated through reduction of inflammation, vascular oxidative stress, and an increase in membrane hyperpolarization , 7–9. AsSeveral previous studies have proved the antioxidative and anti-inflammatory action of SGLT-2 inhibitors in preclinical and clinical studies, but the effect of combining it with metformin has not been studied. Canet et al. found that empagliflozin 10 mg daily for 12 weeks showed antioxidative and anti-inflammatory properties in individuals with type 2 diabetes, and the results were in accordance with a decrease in adhesion molecules and a reduction in leukocyte-endothelium molecules interaction. They concluded that these actions might underlie the beneficial cardiovascular effects . All theIn accordance with the above-mentioned results in individuals with type 2 diabetes, we found that empagliflozin, particularly, the empagliflozin-metformin 12-week treatment, acted antioxidatively, the effect of the combination being enhanced to the level where all studied antioxidative parameters  significantly increased with a concomitant decrease in the studied prooxidant parameters (AOPP and isoprostane), compared to the individual drugs or the control group. This concludes in favour of the powerful antioxidant activity of this combination, addressing both sides of the redox spectrum favourably. The anti-inflammatory effect was also the most powerful with the empagliflozin-metformin combination, with a decrease in both CRP and IL-6. In addition to confirming the antioxidative and anti-inflammatory action of the empagliflozin-metformin combination, we also found that the beneficial effects were associated with an improvement in endothelial function and a decrease in arterial stiffness. Therefore, these findings can be proposed as important mechanisms for preventing atherogenesis.In diabetic patients, regardless of the type, the multifactorial treatment, i.e., treatment of hyperglycaemia as well as regulation of blood lipid levels and hypertension, in addition to smoking cessation and weight loss, leads to significant reduction in major adverse cardiovascular events (MACE) , 24. TheA limitation to this study is the relatively small number of enrolled participants and the short duration without any additional follow-up, which did not allow for the determination of MACE. In addition, regression analysis between the risk factors and delta values of FMD was not performed, due to the small number of participants and small number of risk factors per participant. Also, only one dose of empagliflozin, i.e., maximal dose of 25 mg daily, was used, not allowing for comparing the effects of the clinically used empagliflozin doses (10 and 25 mg daily). The study was also not designed to explore the minimal time to effect; thus, we could not determine how fast the beneficial effects of the drugs occurred. Nevertheless, an important strength of this study is that, for the first time, the complex connections between antioxidants, prooxidants, inflammatory parameters, and arterial function parameters were explored.In summary, the empagliflozin-metformin combination possesses powerful antioxidative and anti-inflammatory capacity in adults with type 1 diabetes that could, at least partially, underlie the important vascular benefits of this drug combination. These findings add to the already established cardiovascular pleiotropic benefits of these drugs in diabetes.


Globally, the preterm birth rate has tended to increase over time. Ultrasonography cervical-length assessment is considered to be the most effective screening method for preterm birth, but routine, universal cervical-length screening remains controversial because of its cost.We used obstetric data to analyze and assess the risk of preterm birth. A machine learning model based on time-series technology was used to analyze regular, repeated obstetric examination records during pregnancy to improve the performance of the preterm birth screening model.This study attempts to use continuous electronic medical record (EMR) data from pregnant women to construct a preterm birth prediction classifier based on long short-term memory (LSTM) networks. Clinical data were collected from 5187 pregnant Chinese women who gave birth with natural vaginal delivery. The data included more than 25,000 obstetric EMRs from the early trimester to 28 weeks of gestation. The area under the curve (AUC), accuracy, sensitivity, and specificity were used to assess the performance of the prediction model.Compared with a traditional cross-sectional study, the LSTM model in this time-series study had better overall prediction ability and a lower misdiagnosis rate at the same detection rate. Accuracy was 0.739, sensitivity was 0.407, specificity was 0.982, and the AUC was 0.651. Important-feature identification indicated that blood pressure, blood glucose, lipids, uric acid, and other metabolic factors were important factors related to preterm birth.The results of this study will be helpful to the formulation of guidelines for the prevention and treatment of preterm birth, and will help clinicians make correct decisions during obstetric examinations. The time-series model has advantages for preterm birth prediction. Preterm birth, defined as birth occurring before 37 weeks of completed gestation, is the primary cause of neonatal death and disability and affects the long-term health of newborns ,2. AccorDespite the serious clinical consequences, there are currently no effective early screening methods for preterm birth. It is generally considered that ultrasonography cervical-length assessment is the most effective screening method ,12, but There is not a single or combined screening method for preterm birth that has high sensitivity and can reliably identify women at risk for preterm birth . The etiIn the literature, various methods have been proposed to predict the risk of preterm birth with machine learning. These methods can be broadly categorized into 2 types, according to their data source: special examination data or routine clinical data. Special examination data include findings from the cervicovaginal fluid , electroAlthough the above prediction models have relatively reliable performance, they all use huge, complex data sets for analysis. It can be difficult to obtain complete data sets of this size and complexity because of privacy issues. More importantly, these models ignore the influence of time-related factors. Time-series analysis and prediction methods predict future developments according to tendencies in past changes and highlight the role of time factors in making predictions. In fact, obstetric examinations are continuous and repeated time-series records and are considered to be related to pregnancy risk . PreviouFew studies have described the interpretability of their models. Khatibi et al  used IraAlthough none of the above methods are suitable for time-series models, it is encouraging that there have been recent proposals for interpretable frameworks for time-series classification that can be used in different medical scenarios. In the field of medical signals, Ivatur et al  proposedThis previous research motivated the current study, which makes the following key contributions: (1) we designed and implemented a complete process for preterm birth screening and providing early warnings based on regular EMR data; (2) we used machine learning based on time-series technology to analyze the obstetric examination data and improve the performance of the prediction model; (3) we provide a preliminary explanation of the quantitative interpretability of the model and explore time-series predictors affecting preterm birth.The data were collected from Hangzhou Women’s Hospital , Hangzhou, Zhejiang Province, China, between 2017 and 2020. This study included >25,000 pregnant women who received antenatal care at Hangzhou Women’s Hospital and eventually gave birth naturally through the vagina. The exclusion criteria were as follows: presence of multiple pregnancies, assisted reproduction, severe cardio- or cerebrovascular complications or comorbidities, and performance of cervical cerclage during pregnancy. The inclusion criterion was a first pregnancy test taken before 12 gestational weeks. According to the Chinese guidelines for prenatal examination , pregnanDemographic data, physical examination data, ultrasound records, and laboratory data from the antenatal period were retrieved from EMRs. At registration for pregnancy, information on maternal demographic characteristics , anthropometrics , and clinical history  were recorded. As shown in Participants were asked to wear light clothing when their height and weight were measured. BMI was calculated as body weight in kilograms divided by body height in meters squared. Sitting blood pressure was examined after at least 10 minutes of rest using a standard mercury sphygmomanometer with the patient’s right arm held at heart level. Maternal venous blood samples were drawn in the morning after an overnight fast of ≥8 hours.Based on the above-mentioned features, 2 machine learning models were constructed to predict preterm birth. One was an early prediction model based on the data sources in t–1 represents the output of the LSTM at the previous time step, and ht represents the current output; I, f, and o, respectively, represent the input gate, forgetting gate, and output gate in the LSTM unit. Equation (4) represents the process of the state transition of the memory unit, where ct is the state of the memory unit at the current time step. The current state is calculated by the previous time step state, ct–1, and the result of the forgetting gate and the input gate of the current-time LSTM unit.LSTMs realize information protection and control through 3 control gates, namely the input gate, the forgetting gate, and the output gate. The key in LSTMs is the unit state. The LSTM unit judges whether the output of the previous time step is useful; only useful information is saved and the rest is forgotten at the forgetting gate. Equations (1) through (5) represent the parameter update process, where σ represents the sigmoid function, hThe parameters of these prediction models were determined by grid search. The models were validated with 5-fold cross-validation. The 5-fold cross-validation splits the training dataset into 2 sections, where 80% of the dataset is used for training and the remaining 20% is used for testing. Simultaneously, the incidence rate of preterm birth is about 5%, so in situations where there were imbalanced class data combined with unequal error costs, random oversampling was used to balance the dataset to get true performance values for the classifier. The random oversampling method makes the number of minority classes the same as the number of majority classes by randomly copying minority class samples to get new equilibrium data.Under the Python 3.6 environment (Python Software Foundation), the data analysis and visualization were completed by using NumPy, Pandas, Matplotlib, Seaborn, and other libraries ,42. The P values <.05 were considered statistically significant. All analyses were performed using the statistical software SPSS 22.0 (IBM).The characteristics were compared between the preterm birth and full-term birth groups. Statistical tests were 2-sided; The prediction performance was considered an important factor to evaluate the proposed model. In this paper, the receiver operating characteristic (ROC) curve and AUC were used to evaluate the model’s ability to predict preterm birth. In addition, the evaluation indicators of the confusion matrix, including accuracy, sensitivity, and specificity, were used to analyze the relationship between the actual values and the predicted values for the risk of preterm birth. Accuracy, sensitivity, and specificity were calculated as follows: accuracy = (TN + TP) / (TN + TP + FN + FP); sensitivity = TP / (TP + FN); and specificity = TN / (TN + FP), where TP indicates true positive, FP indicates false positive, TN indicates true negative, and FN indicates false negative.Feature importance reflects the contribution each variable makes in classifying preterm birth, which explains the results of the model decision. In this study, feature importance for the XGB model was calculated by the sum of the decrease in error when split by a variable . For theThe study design was approved by the local Ethical and Research Committee . All medical procedures were performed following the relevant guidelines and regulations. The informed consent requirement for this study was waived by the board because the researchers only accessed the database for analysis purposes and all patient data were deidentified. The data set used in this paper comes from a hospital in eastern China and is very extensive, including maternal ultrasound records, prenatal examination reports, and laboratory data. Of the 5187 pregnant women enrolled in the present study, 4966 gave birth at full term. The remaining 221 women gave birth preterm. The general characteristics of the participants are presented in Based on the above-mentioned features in Based on the validation result for the training data set, an independent testing data set was used for predicting preterm birth. The matrices and ROC curves for the predictive models in the testing data set are shown in The identification of important features by the XGB and LSTM models is shown in Premature birth is widely recognized as an increasingly serious problem. In this study, 5 pregnancy test records in the first and second trimesters of pregnancy were selected to construct a time-series model to predict preterm delivery. Compared with traditional machine learning models, the use of a time-series model improved prediction performance for preterm birth and allowed the identification of important variables for predicting preterm birth.The early prediction of preterm birth has always been challenging. The input index of traditional prediction model research has usually been a special test item or a combination of tests that aim to find new markers that have a high contribution to preterm birth prediction; most past studies have not been clinically verified ,17,46. MIn fact, earlier works have already reported very close or even higher accuracy than this study. Compared with the large national databases used in previous studies, the conventional data used in this paper is still relatively weak, especially in its lack of key information, such as obstetric and gynecological history and family history. However, we are excited that this paper significantly improves the performance of prediction models through a machine learning method based on time-series technology.This study reveals various new factors that affect the prediction of preterm birth. Additionally, parameters that have been traditionally reported to be related to delivery date, such as age, prepregnancy weight, history of preterm birth, and menstrual cycle, were confirmed to be influential factors in preterm birth prediction ,56. InteThis study has several limitations. First, the laboratory examinations of the pregnant women were completed in their respective communities before 20 weeks of gestation, precluding them from being included in the analysis due to differences in test standards. In addition, the prepregnancy characteristics were affected by recall bias; moreover, most of the included women were primipara. Thus, the contribution of preterm birth history to the model was limited. Second, the performance of the model still needs to be improved, although LSTM has great potential. Nonetheless, considering this prediction model is a baseline model based on conventional data, it can continue to add biochemical and biophysical markers to increase screening performance. In addition, advanced maternal age was a clear confounding factor , and strPreterm birth is the primary cause of neonatal death and disability, and early prediction of preterm birth has great potential to improve the survival rate of preterm infants. In this work, we analyzed obstetric medical data based on time-series machine learning and evaluated the risk of preterm birth. Our study can screen high-risk groups for preterm birth in the early and middle trimesters of pregnancy. Compared with a traditional cross-sectional study, the time-series LSTM model in this study had better overall prediction ability with a lower misdiagnosis rate and the same detection rate. In future work, we will further improve the data set, especially regarding some key characteristics of premature birth that have been reported by past relevant research, and build a more sophisticated post hoc explainability framework for the time series model.


Despite a declining neonatal mortality rate globally, Ethiopia has scored 29–30 deaths per 1,000 live births. Birth asphyxia is a major contributor to neonatal mortality, where 4–9 million newborns develop birth asphyxia each year. This study aimed to assess the prevalence of birth asphyxia, its determinants, and its management among neonates admitted to the NICU in Harari and Dire Dawa public hospitals.p-value <0.05.A facility-based cross-sectional study was conducted among 409 randomly selected neonates and their index mothers admitted to neonatal intensive care units of public hospitals in Harari and Dire Dawa from June 20 to August 20, 2021. Data were collected through card review and interviewer-administered questionnaires. The collected data were entered into Epi data version 3.1 and exported to SPSS version 20 for analysis. Logistic regression models were fitted to identify factors associated with birth asphyxia. Adjusted odds ratios along with 95% CIs were estimated to measure the strength of the association, and statistical significance was declared at One-fifth of neonates [20.8% ] had birth asphyxia. Neonates born by instrumental delivery  and neonates born to mother with PIH , PROM , and chorioamnionitis  were more likely to have birth asphyxia compared to their counterpart. Ventilation with a bag and mask 50(58.8), putting on free oxygen 19(22.4), and endotracheal intubation 15(17.6) were taken as management methods.One out of five neonates had birth asphyxia. This urges care providers to adhere to national guidelines of obstetrics and neonatal continuum care. They also need to decrease instrumental delivery and treat PIH, PROM, and chorioamnionitis. Despite a declining neonatal mortality rate globally, striking disparities exist across regions and countries . For insBirth asphyxia is related to the reduced availability of skilled care during pregnancy, delivery, and postpartum periods . Women wBirth asphyxia is preventable with strategies such as increasing ANC coverage, skilled birth attendance, and postnatal care . GuideliA facility-based cross-sectional study was conducted in Harari and Dire Dawa public hospitals in Eastern Ethiopia from June 20 to August 20, 2021. Dire Dawa is one of the administrative cities of Ethiopia and is located at a distance of 515 km from Addis Ababa . Dire Dad = 0.05):The sample size was determined using a single-population proportion formula with assumptions; the prevalence of neonatal sepsis among neonates admitted to NICU is 52.6% according to the study conducted in Jimma , with 95Therefore, a sample size of 421 was used for this study with a 10% nonrespondent rate.N = 968) at each hospital by the total sample size (n = 421). The samples from each hospital were allocated proportionally to the number of neonates admitted to the NICU at those hospitals. All participants were obtained every two intervals after the first participant (number 2) was obtained by the lottery method  were included conveniently. Accordingly, 968 neonates admitted to NICU in the last 3 months in both Harari and Dire Dawa public hospitals were included. Systematic sampling was used to obtain study subjects. The sampling interval was calculated by dividing the number of neonates admitted to the NICU  were used to determine the correlation between independent variables, and the multi-colinearity test was used to determine the correlation between independent variables using variable inflation factors. Descriptive statistics such as tables were used to present frequency distributions. Bivariable analysis was employed to identify factors associated with birth asphyxia. Variables with Ethical approval was obtained from the Institutional Review Board (IRB) of Haramaya University, College of Health and Medical Sciences (Ref. No. IHRERC/105/2021). Written permission was obtained from each of the hospital administrative bodies and unit heads. The participants were informed that their participation was entirely voluntary. Moreover, informed and written consent was obtained from each study participant (mother of a neonate).A total of 409 neonates and their index mothers admitted to NICUs were involved in the interview; this yields a response rate of 97.1%. More than half of the mothers of neonates [225 (55.0%)] were found between the ages of 25 and 34 years. The majority of mothers of neonates [373 (91.2%)] were married, and 228 (55.7%) mothers were Oromo by ethnicity. Six out of ten mothers [247 (60.4%)] were urban residents. Regarding the educational status of mothers of neonates, 127 (31.1%) did not attend formal education and 181 (44.3%) mothers of neonates were housewives by occupation. Similarly, more than half [222 (54.3%)] were female neonates .Eight out of ten mothers [325 (79.5%)] had ANC for a recent pregnancy, and three-fifths of mothers [239 (58.4%)] were multiparous. Regarding infections of the mother, 17 (4.2%) mothers had HIV infection, 94 (23%) had UTI, and 20 (4.9%) had chorioamnionitis. More than three-sevenths of mothers [190 (46.5%)] had anemia during their recent pregnancy, and 70 (17.1%) mothers had PROM. Also, 29 (7.1%) mothers had APH and 31 (7.6%) had PIH. Eight out of ten mothers [331 (80.9%)] gave birth by SVD, and the majority of neonates [284 (69.4%)] were born at term gestational age .Of the total neonates who partook in the study, one-fifth [20.8% ] had birth asphyxia. Ventilation with a bag and mask 50(58.8), putting on free oxygen 19(22.4), and endotracheal intubation 15(17.6) were utilized as management methods .In bivariable logistic regression, maternal age, residence, sex of the neonates, birth weight, prolonged labor, gestational age, mothers with PROM, mode of delivery, maternal anemia during pregnancy, chorioamnionitis, and PIH were factors associated with perinatal asphyxia.However, in multivariable logistic regression, neonates born by instrumental delivery were twofold ] more prone to have perinatal asphyxia compared to neonates born by spontaneous vaginal delivery. The odds of perinatal asphyxia among neonates born to mothers with chorioamnionitis during pregnancy were three times ] higher compared to neonates whose mothers did not have chorioamnionitis during pregnancy.Similarly, neonates who were born from a mother with PROM were two and half times ] more likely to develop perinatal asphyxia compared to neonates who were born from a mother without PROM. The odds of perinatal asphyxia were three times ] higher among neonates born from mothers with pregnancy-induced hypertension compared to their counterparts .Birth asphyxia can lead to fatal outcomes or life-long complications among neonates. Hence, information regarding the prevalence of asphyxia and its determinants is needed to develop contextual interventions that are crucial in reducing the overall newborns' morbidity and mortality. One-fifth of neonates had birth asphyxia. Similarly, instrumental delivery, PROM, PIH, and chorioamnionitis were found to be determinants of birth asphyxia.In this study, nearly one-fifth of neonates (20.8%) admitted to NICUs in Harari and Dire Dawa had birth asphyxia. This finding was in line with the studies finding in Tigray (22.1% and 18%) , 22, norPremature rupture of the membrane (PROM) was significantly associated with birth asphyxia. This is supported by a study conducted in Uganda  and GhanThe odds of birth asphyxia were higher among women with pregnancy-induced hypertension (PIH) compared to their counterparts. This is in agreement with the study in Nigeria . Also, iNeonates born by instrumental delivery were twofold more likely to have birth asphyxia compared to neonates born by spontaneous vaginal delivery. This is consonant with reports of Kune et al.  and DubiThe odds of birth asphyxia were three times more among mothers encountering chorioamnionitis during their pregnancy, similar to what was reported in southwest Ethiopia , India , and PakThe strength of this study was that it was representative because it covered a larger geographical area. Moreover, this study tried to identify almost all determinants of birth asphyxia, which were modifiable with quality prenatal, intrapartum, and postpartum care, and by adhering to national guidelines of obstetrics and neonatal care. On the other hand, since this study was cross-sectional, it could not identify causation, and the sampling method included only neonates admitted to NICUs. This study did not elaborate on the management and clinical outcomes of these asphyxia neonates.In this study, one in every five neonates was affected by birth asphyxia. Instrumental delivery, PIH, PROM, and chorioamnionitis were identified as determinants of perinatal asphyxia. In this study, almost all determinants of birth asphyxia were modifiable with quality prenatal, intrapartum, and postpartum care and by adhering to national guidelines of obstetrics and neonatal care.


Preliminary evidence from China and other countries has suggested that coronavirus disease 2019 (COVID-19) mitigation measures have caused a decline in preterm births, but evidence is conflicting. Utilising a national representative data of 11,714,947 pregnant women in China, we explored the immediate changes in preterm birth rates during the COVID-19 mitigation period using an interrupted-time-series analysis. We defined the period prior to February 1, 2020 as the baseline, followed by the COVID-19 mitigation stage. In the first month of the COVID-19 mitigation, a significant absolute decrease in preterm birth rates of 0.68% (95%CI:−1.10% to −0.26%) in singleton, and of 2.80% (95%CI:−4.51% to −1.09%) in multiple births was noted. This immediate decline in Wuhan was greater than that at the national level among singleton births [−2.21% (95%CI:−4.09% to −0.34% vs. −0.68%)]. Here we report an immediate impact of COVID-19 mitigation measures on preterm birth in China. The broader impacts of COVID-19 mitigation measures may include changes in the incidence of preterm birth. Here, the authors use data from ~11.7 million pregnancies in China and find evidence of a small decline in preterm birth rates immediately following the implementation of COVID-19 mitigation measures. Such deliveries occur in an estimated 10.6% of pregnancies globally, and the rate has increased over time3. The same trend is seen in China (from 5.9% in 2012 to 6.4% in 2018), especially after the universal two-child policy took effect4. Preterm births place an enormous burden on the families of preterm newborns with regard to their offspring’s mortality and morbidity7. Families of preterm newborns often experience considerable psychological and financial hardship7. Regarding the infant, complications of preterm birth was the leading cause of death in children younger than five years of age in 2016, accounting for approximately 16% of all deaths, and the leading cause of death (35%) among neonates  globally3. In addition, preterm birth accounts for 75% of perinatal mortality and more than half of long-term childhood morbidity6. Therefore, reducing the incidence of preterm birth is a global priority for infant health.Preterm birth is defined as any birth prior to 37 completed weeks of gestation, or fewer than 259 days from the first day of a woman’s last menstrual period, according to the World Health Organization8. However, its risk factors have been studied extensively, and the known factors can be roughly divided into non-modifiable and modifiable risk factors. Modifiable risk factors include infection, work-related stress, exposure to certain environmental pollutants, and a series of lifestyle factors9. In 2015, the United Nations Sustainable Development Goal 3 (SDG 3) proposed the elimination of preventable deaths of newborns and children aged under five years following the global failure to achieve Millennium Development Goal 4. Achieving SDG 3 targets at an earlier date than planned requires more attention to modifiable risk factors of preterm birth and greater efforts to explore strategies to prevent preterm birth. Recently, numerous studies conducted in multiple countries assessed the effect of coronavirus disease 2019 (COVID-19) lockdown measures on the incidence of preterm birth and obtained remarkably varied results20. Several studies from the Netherlands, Denmark, Ireland, Italy, and Japan reporting on reduction in preterm birth following the implementation of lockdown measures has raised hopes for effective preterm birth prevention in the future; however, these studies need more evidence-based support20. The link between preterm birth rate changes and COVID-19 mitigation measures have been identified in two studies carried out in China; however, the conclusions from these studies were contradictory22. Both studies were single-centre studies and have relatively small sample sizes. In addition, all studies were restricted to singleton births. Moreover, few studies have assessed the differential impact of lockdown measures on iatrogenic and spontaneous preterm deliveries or across the socioeconomic status (SES) strata19 due to a lack of case-specific demographic data. An assessment of this association has also not been conducted in the whole of China, one of the world’s most populous, diverse countries, and one that has implemented some of the strictest COVID-19 mitigation measures and best outbreak control strategies23. China’s National Maternal Near Miss Surveillance System (NMNMSS) provides an opportunity for further research. NMNMSS, established in October 2010 by the National Health Commission of China, was designed to monitor women’s health status based on case information obtained from 438 member hospitals across 326 urban districts and rural counties in 30 provinces, and represented 8‒10% of all births across China.Currently, primary and secondary preventive strategies for preterm birth are deficient owing to a lack of understanding of its pathophysiologyIn this work, we aimed to use data from the NMNMSS to assess the immediate changes in preterm birth rate during the COVID-19 mitigation period in China, including changes related to the various categories of births/pregnancies along with concomitant changes in stillbirth rates. Furthermore, we also explored whether the impacts on preterm births associated with mitigation measures varied by SES.During the study period , 12,294,471 women delivered at least one baby who was more than or equal to 28 weeks of gestation or weighed 1000 g or more. After excluding pregnancies lacking data, 11,714,947 women were included in the final analysis . A more significant proportion of births were monitored by NMNMSS in 2020 (9.44%) than in previous years, and the proportion of births captured in February 2020 (8.98%) and March 2020 (9.47%), using strict epidemic mitigation measures, was also higher than those in previous years.  and 11 months were from the intervention stage .The sociodemographic characteristics of the women who gave birth before and after the implementation of COVID-19 mitigation measures are presented in Table p < 0.001; multiple: p < 0.001) and stillbirth rate  were observed from 2012 to 2020, using the Cochran Armitage test.In both singleton and multiple births, linear trends in preterm birth rate (singletons: p = 0.002). In the first month of the intervention, there was a significant immediate absolute decrease in the singleton preterm birth rate by 0.68% , followed by a significant increase in the rate (relative to the pre-intervention trend) of 0.10% per month . The immediate decline following the COVID-19 mitigation measures was mainly due to a decrease in moderate and late preterm births ; however, an immediate absolute reduction of 2.80%  was subsequently observed in the first month of the COVID-19 mitigation measures, followed by a significant rate increase of 0.80%  per month (relative to the pre-intervention trend). The immediate decline following the COVID-19 mitigation measures was mainly attributed to late preterm births , whereas an immediate absolute decline by 2.21%  was observed after the implementation of COVID-19 mitigation measures, followed by a steady trend of 0.28% per month  based on the baseline trend. The immediate reduction in the preterm birth rate in Wuhan was greater than that at the national level among singleton births (−2.21% vs. −0.68%). However, the preterm birth rate remained stable after the implementation of COVID-19 mitigation measures among multiple pregnancies in Wuhan  as their datasets did not have individual-level information on relevant covariates25. As an emergency response, China implemented a range of behavioural and clinical interventions to mitigate the pandemic in China and worldwide27. Behavioural interventions included the following aspects: (1) the use of personal protective equipment—particularly the wearing of face mask-wearing; (2) regular hand-washing or use of an alcohol-based hand sanitizer; (3) reduction in physical contact advising against handshaking and physical touch; (4) maintaining social distance—advice against social interactions and visits; (5) closing schools, factories, and entertainment venues; (6) banning public gatherings, encouraging working from home, and reducing commuting to and from work/home; and (7) recommendations to stay at home when experiencing symptoms (fever or respiratory complaints), after coming in contact with someone who tested positive for COVID-19, or after having visited a high-risk area. Recently, Deng et al. reported that the preterm birth rate had slightly increased before the universal two-child policy took effect in China, while a steeper increase was observed after implementation of this policy4. However, we found a brief reversal in this upward trend, where an immediate decline in the preterm birth rate was observed among singleton and multiple pregnancies following the implementation of COVID-19 mitigation measures. These findings are based on controlling for possible seasonality on preterm birth rates, which are consistent with those of studies from the Netherlands, Denmark, Ireland, Italy, and Japan20. However, several studies have raised doubts regarding whether the observed reductions in preterm birth occurred at the expense of an increase in stillbirths19. Therefore, we further investigated the immediate fluctuations in stillbirth rates and found no significant changes after the implementation of mitigation measures, confirming that the immediate decline in preterm birth did not occur at the expense of an increase in stillbirths. Perinatal birth in China is defined as the birth of a foetus after 28 completed weeks of gestation, or at a birth weight of 1000 g or heavier, which is much later than that of 20 or 22 weeks used to define the term in most developed countries28. We reassessed the impact of COVID-19 mitigation on preterm births and stillbirths according to the definitions of preterm birth and stillbirth in some developed countries (≥22 w), and the results were similar  can reduce contact with pathogens and subsequent risk of infection. The parallel and unprecedented reduction in influenza and other respiratory viral infections that was observed in New Zealand during the COVID-19 pandemic serves as strong evidence for this hypothesis30. These indicate that hygiene measures and anticipatory behavioural changes may have contributed to the timing of the decrease in the preterm birth rate19. In addition, the closure of most businesses and working from home may have led to less physically demanding activities, reduced shift work, less work-related stress, optimisation of sleep duration, uptake of indoor and outdoor maternal exercise, and increased social and family support, all contributing to possible beneficial effects. Furthermore, the decrease in air pollution caused by the mitigation measures may have also played a key role in the observed reduction in premature birth31, given that air pollution—particularly anthropogenic PM2.5 which can remain airborne for extended periods and travel hundreds of miles—is associated with 18% of premature births globally11.In our study, the immediate decrease in the preterm birth rate was mainly due to a decrease in moderate and late preterm births among singleton pregnancies, and a decrease in late preterm births among multiple pregnancies. Our findings are consistent with those of a study from the Netherlands, which suggested that the decrease in preterm birth is fairly constant regardless of the gestational age, although the vast majority of preterm infants are born moderate or late preterm 34. In the United States, clinicians have been urged to reduce nonmedically indicated or elective deliveries prior to 39 weeks34. Hypertensive disorders in pregnancy, placental factors, foetal distress, multiple pregnancies, intrahepatic cholestasis during pregnancy, and pregnancy combined with medical and surgical diseases constitute the main causes of iatrogenic preterm deliveries in China35. Obstetricians should have a good understanding of the timing of delivery related to various complications. In cases where termination of pregnancy prior to 37 weeks is contraindicated, premature intervention should be avoided to reduce unnecessary iatrogenic preterm delivery. The observed immediate reduction in the iatrogenic preterm birth rate in singleton pregnancies may be due to the lack of obstetric intervention, in pregnancies that did not require emergency intervention, because of isolation and limited medical resources during the COVID-19 mitigation period. This suggests that some iatrogenic preterm births may be controlled. However, we did not find a similarly decreasing trend in iatrogenic preterm birth rates in multiple pregnancies. Multiple gestation, accounting for only 2–3% of pregnancies, carries a substantial risk of preterm birth, constituting 15–20% of all preterm births2. The causes of preterm birth due to a multiple pregnancy are complex and relatively uncontrollable; therefore, studies are often limited to singleton pregnancies. We included multiple pregnancies to explore whether COVID-19 mitigation measures have a beneficial effect on the preterm birth rate in these cases, despite their inherent increased risk of preterm delivery.An immediate decrease in the iatrogenic preterm birth rate was only observed among singleton pregnancies; however, this decrease was lower than that in spontaneous singleton preterm births. It should be noted that the increased preterm birth rate in recent years can be partly attributed to changes in the use of obstetric interventions36, while women with low SESs may face more financial pressure, forcing them to remain in contact with the outside world, which may increase their chances of infection leading to preterm birth. The pandemic and associated lockdown measures may have aggravated the existing health and socioeconomic inequalities within populations39. Nevertheless, studies from California and Philadelphia in the United States reported no significant change in the rate of preterm birth after lockdown17. The differences between our findings and those of the above-mentioned studies could be explained by the fact that multiple risk factors for preterm delivery that might be responsive to lockdown measures vary across populations and that mitigation measures may have been inadequately implemented. The decline in preterm births may have been even greater during the mitigation period if the population with a low SES behaved similarly to the population with a high SES. Therefore, there is an urgent need to prioritise accessible and equitable health services as a strategic response to this pandemic and future health crises.The change in preterm birth rates during the lockdown period differed between populations with different SESs. An immediate reduction in preterm birth rates was exclusively found in the advantaged group with a high SES. Compared with women with low SESs, those with high SESs have a stronger sense of self-preservation due to their advanced educational levels, social status, and stable financial income. This renders them more likely to obtain protective products, in addition to having more purchase channels and the financial capacity to acquire them42. Therefore, the effect of underestimation is assumed to be relatively small. Finally, since our monitoring hospitals do not include most isolated hospitals, there may be some underestimation of the incidence of preterm birth after the outbreak of COVID-19. We were only able to identify 10 women infected with the virus in the present study. A previous study from Wuhan, the area most affected by the pandemic in China, reported that from December 8, 2019 to March 20, 2020, a total of 68 pregnancies diagnosed with COVID-19 were delivered during the study period, accounting for 0.56% of all deliveries during the same time period43. Therefore, we believe this bias will not greatly impact the overall trend of preterm birth rate from a population perspective. In conclusion, our results show that the COVID-19 mitigation measures in China were associated with an immediate reduction in the preterm birth rate. These findings suggest that some preterm births are preventable, although its underlying the mechanism needs to be elucidated. We also call attention to the need for equitable access to health services. Further exploration of possible mechanisms is required to inform the development of novel preventive strategies aimed at mitigating the global burden of preterm birth.One major limitation of our retrospective study was the presence of residual confounding factors, especially in the analysis of multiple pregnancies. Some important risk factors, such as the use of assisted reproductive technology and foetal complications, were not included in the NMNMSS. Future analyses should consider these covariates. Another limitation was that we calculated the gestational age using the last menstrual period when early pregnancy ultrasound is now considered a gold standard. Previous studies have shown that use of the last menstrual date may overestimate gestational age by approximately 2 days (range: 0.2‒2.8 days) compared with ultrasound, but the sensitivity and positive prediction of the last menstrual date in identifying preterm birth value is relatively high45. The NMNMSS covers 438 member hospitals each assisting more than 1000 deliveries annually. The member hospitals are situated across 326 urban districts and rural counties in 30 provinces in mainland China. We restricted our study population to pregnancies greater than or equal to 28 weeks of gestation or a birthweight of 1000 g or more birthweight according to the definition of the perinatal period in China28.We extracted all available data from the NMNMSS for the period of January 1, 2012 to December 31, 2020. The NMNMSS collects the sociodemographic and obstetric information of pregnant and postpartum women visiting obstetric departments from the time of admission to discharge. The collected data included the patients’ names, hospital code, age, education level, marital status, number of antenatal visits, date of delivery, gestational age at delivery, birth status, mode of delivery, number of foetuses, and maternal complications . The detailed sampling method used has been described elsewhereAll member facilities are required to directly report maternal questionnaires via the online NMNMSS platform. The doctors responsible for pregnancy care are responsible for completing the maternal questionnaire according to the patient’s medical records. An associate director of the obstetric department verifies and reviews the data at the primary level, investigates missing reports, aggregates the data, and reports to the NMNMSS. The county-, prefecture-, and provincial-level maternal and child healthcare hospitals oversee and verify the quality of data using random samples at the secondary, tertiary, and quaternary level, respectively. Finally, the National Office for Maternal and Child Health Surveillance conducts a data quality check at the quinary level. For institutions that fail to meet the data quality requirements, we asked them to record supplementary data. All data provided to us were de-identified. This study was approved by the ethics committee of West China Second University Hospital (Protocol ID:2012008).25. Subsequently, China entered the implementation stage of strict epidemic mitigation measures after February 2020. Therefore, we used February 2020 as an important cut-off point in the present study. We divided the study period into two parts: a baseline stage  and the intervention stage .To control the COVID-19 outbreak, China banned travel to and from Wuhan City on January 23, 2020; all provinces, municipalities, and autonomous regions in mainland China subsequently activated a Level 1 response over the following week46, we defined preterm birth as a delivery prior to 37 completed weeks of gestation, and further categorised it into very preterm (28–31 weeks), moderate preterm (32–33 weeks), and late preterm (34–36 weeks) birth. Preterm birth rates were calculated for each month and for each of the two stages (baseline and the intervention stage). In addition, we categorised preterm birth into (1) spontaneous labour with intact membranes, (2) preterm premature rupture of the membranes, and (3) labour induction or caesarean delivery based on maternal or foetal indications2; subsequently, we combined (1) and (2) as spontaneous preterm birth, and treated (3) as iatrogenic preterm birth. The secondary outcome was the monthly stillbirth rate, defined as the number of stillbirths (≥28 weeks of gestation) per 100 births per month. We defined term births as a delivery that occurred between 37 and 42 completed weeks of gestation, while births that occurred after 42 completed weeks of gestation were defined as post-term births. Gestational age is estimated based on the last menstrual period or ultrasound examination when the date of the last menstrual period is unknown47. We selected confounding factors based on the factors shown to be associated with preterm births in the scientific literature, including sociodemographic characteristics  and obstetric factors (presence of eclampsia)2. We defined the types of pregnancy as singleton and multiple births. As detailed elsewhere, the customary definitions of maternal region  and maternal education level  were used44. The definition of city and rural areas is based on the hospital’s location.The primary outcome was the monthly preterm birth rate, defined as the number of preterm births per 100 live births per month. In accordance with the criteria recommended by the World Health OrganizationMoreover, women who were illiterate, or had only primary school education, who were unmarried, or who had fewer than five antenatal visits were defined as the disadvantaged group. On the contrary, women with middle school or above education, married, and had more than five antenatal visits were defined as the advantaged group.We displayed the number of monthly births in the NMNMSS databases from January 1, 2012 to December 31, 2020 and calculated the ratio of annual monitored births to the national births and the ratio of monthly monitored births to the annual monitored births.We examined whether there was a linear underlying time trend of preterm birth-/-stillbirth rates using the Cochran Armitage test by the PROC FREQ procedure in SAS.p = 0.002) was subsequently observed in the first month when implementing the COVID-19 mitigation measures when seasonal effects are not considered, while an immediate reduction of −0.68%  was observed when seasonality is included as a fixed effect. Finally, a categorical variable for month was included as fixed-effect to each ITSA model to adjust for any seasonality in the data, which based on that previous studies have also confirmed the scientific soundness of this correction method48. We specified the following model to estimate the trend in preterm birth and stillbirth rates following the implementation stage of COVID-19 mitigation measures:We aggregated NMNMSS data down to a monthly level, then we tested whether the COVID-19 mitigation measures would specifically impact preterm birth rates and stillbirth rates. Singleton and multiple pregnancies were analysed separately, considering the inherent increased risk of preterm birth in multiple pregnancies. Interrupted time series analysis (ITSA) was used to examine changes in these rates per month over the baseline period  and the intervention stage . Potential confounders included in the preterm birth rate model were maternal age, parity, education level, and the presence of eclampsia, while maternal age, parity, and the presence of scar uterus were included in stillbirth rate model. Further, after comparing the regression results with and without the adjustments for seasonality, we observed the confidence intervals overlap, and the slope values are very similar. Taking the rate of preterm birth in singletons for example, an immediate reduction of −0.64%  or after (intervention = 1) the cap. In this model, β0 estimates the baseline level of the outcome; β1 is interpreted as the change in outcome associated with a time unit increase; β2 estimates the level of change in the preterm birth rate/stillbirth rate immediately after the intervention; and β3 estimates the slope change of the mean monthly preterm birth rate/stillbirth rate after the intervention, compared with the trend before the intervention49. Considering the temporary impact of COVID-19 mitigation measures, we considered significant p-values in β2 to indicate an immediate intervention effect rather than in β3 which indicates an intervention effect over time53. We first fitted an ordinary least squares model with a specified lag (0) and subsequently tested for autocorrelation in the error distribution. Thereafter, we identified the corresponding lag according to the autocorrelation. For a more in-depth understanding as to whether COVID-19 mitigation measures have aggravated existing health inequalities within populations, we further estimated the change in the preterm birth rate over time as per SES. Furthermore, in order to observe the difference in preterm birth-/ -stillbirth rates between a theoretical assumption that mitigation measures had not occurred and actual lockdown circumstances, we developed counterfactual scenarios. A predicted rate of preterm birth/stillbirth were calculated using the Betas from primary model (this produces Newey-West standard errors for coefficients estimated by ordinary least-squares regression), but setting the lockdown dummy at ‘0’ for the entire study period. This counterfactual preterm birth/stillbirth rate represented the theoretical rate of preterm birth, had COVID-19 mitigation measures not been implemented.Here, p-values were two-sided, and statistical significance was set at p < 0.05.A range of sensitivity analyses were conducted to evaluate the robustness of the results from our primary analysis. First, we confirmed the changes in the preterm birth rates at 1-, 2-, 3-, and 6-month lags after the implementation of COVID-19 mitigation measures. Second, we restricted the analysis to pregnancies in Wuhan to confirm the changes in the preterm birth and stillbirth rates over the COVID-19 mitigation period. Third, we performed a sensitivity analysis using 2016–2020 data instead of 2012–2020 data, allowing us to assess whether the results differed when the balance between the time points changed. Finally, given the timing of the outbreak, we redefined December 2019 and January 2020 as the cut-off points of the interrupted time series analysis to explore the change in the preterm birth rate. The measures at this stage were not widespread or stringent. Analyses were performed using Stata Statistical Software , and SAS statistical software version 9.4 . All Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Software 1Reporting Summary