Pasteurella multocida strains by testing in vitro susceptibility. Two high-colistin minimal inhibitory concentration (MIC) isolates (D18 and T5) and one low-colistin MIC isolate (WJ11) were used in time-kill tests and therapeutic effect experiments using a neutropenic murine pneumonia model over 24 h. Pharmacokinetics (PK) in plasma was calculated along with pharmacodynamics (PD) to determine the PK/PD index. Synergy between colistin and gamithromycin was observed using high-colistin MIC isolates, equating to a 128- or 256-fold and 4- or 8-fold reduction in colistin and gamithromycin concentration, respectively. Interestingly, no synergistic effect of the combination on low-colistin MIC isolates was observed. However, regardless of the MIC difference among isolates, each drug tended to reach the same concentration in all isolates subjected to combined treatments, which was verified by the time-kill tests presenting similar rates and extent of killing for isolates D18, T5, and WJ11. The AUC(0–24 h)/MIC index was used to evaluate the relationship between PK and PD, and the correlation was >0.89. The relevant gamithromycin doses for combined therapy were determined, and the value decreased from 6- to 35-fold compared with monotherapy. Combined colistin and gamithromycin therapy provides a more potent therapeutic regimen than monotherapy against P. multocida strains.We investigate the antimicrobial activity of combined colistin and gamithromycin against nine  Pasteurella multocida is believed to be a causative factor in many host diseases, including fowl cholera, porcine pneumonic, and bovine hemorrhagic sepsis, all of which result in significant economic losses. P. multocida strains are classified into five serotypes  based on capsule antigens or 16 serotypes (L1–16) according to differences in lipopolysaccharides  can induce serious resistance against gamithromycin, tulathromycin, tylosin, and tilmicosin in P. multocida at MICs ≥512 mg/L (erm(42) and msr(E) – mph(E) genes results in a significant increase in the MICs of gamithromycin and tildipirosin (Bovine respiratory disease (BRD) associated with per year . Gamithrparasuis , 2015. Eparasuis . Compareparasuis . However512 mg/L . Similaripirosin . It is, Colistin is considered a drug of last resort for preventing Gram-negative bacterial infections, and it has been approved for treatment of relevant diseases in food-producing and companion animals . HoweverP. multocida while reducing the dose-dependent side effects of colistin and extending the therapeutic life of gamithromycin, which is the newest macrolide drug. Several experiments, including in vitro susceptibility and time-kill tests and in vivo PK and pharmacodynamics (PDs) assays were carried out using a neutropenic murine lung infection model.The purpose of this study is to provide an alternative and more effective therapeutic regimen for infections caused by P. multocida, including a standard strain (CVCC444) obtained from the China Institute of Veterinary Drug Control and eight clinical strains isolated from the livers of dead chickens  and pigs (PmP2 and PmP17) were used in this work. A multiplex PCR assay for capsular serogroup identification was conducted based on a previously reported method  was used in all experiments.Colistin sulfate powder was purchased from Sigma-Aldrich, and gamithromycin powder was obtained from Quality Control Chemicals Inc. . Cefquinome was purchased from Qilu Animal Health Products Co., Ltd. . Enrofloxacin, tilmicosin, tylosin tartrate, and florfenicol were obtained from the China Institute of Veterinary Drug Control . Antibiotic stock solutions were prepared according to Clinical and Laboratory Standards Institute (CLSI) criteria before testing . MuellerP. multocida isolates and the quality control isolate (ATCC 25922) to colistin, gamithromycin, cefquinome, enrofloxacin, tilmicosin, tylosin tartrate, and florfenicol alone was determined using the microdilution method in broth (conducted in triplicate) according to the guiding principles of the CLSI. Based on the action mechanism of each drug, the antimicrobial activity of colistin or cefquinome (drug A) in combination with gamithromycin, enrofloxacin, tilmicosin, tylosin tartrate, or florfenicol (drug B) was determined with the checkerboard method, according to the minimal inhibitory concentration (MIC) of each drug. Briefly, 50 μL of MH II broth was added to each well, and 50 μL of the indicated concentration of colistin or cefquinome was added into the first well of each line. This was followed by multiproportion dilution. Next, 50-μL samples of different concentrations of gamithromycin or the other drugs mentioned above were added to the dilution medium, and a 100 μL inoculum with a cell density of ∼1 × 106 colony-forming units (CFU)/mL was added to each well. The plates were then incubated at 37°C for 24 h. According to the EUCAST definitive document E.Def 1.2  were tested. A P. multocida inoculum at ∼5 × 106 CFU/mL was treated with colistin alone at 0.25, 0.5, and 1 mg/L; gamithromycin alone at 0.125, 0.25, and 0.5 mg/L; and with colistin in combination with gamithromycin. At 0, 1, 2, 4, 6, 8, 12, and 24 h after incubation, 100-μL samples were taken from each tube for colony counting. A growth control without drug administration was included in all tests.g for 15 min and resuspended in fresh MH II broth to obtain a solution containing 3 × 1010 CFU/mL of P. multocida. The agar plates were produced by adding different concentrations of colistin and gamithromycin alone or in combination into the Mueller-Hinton agar medium supplemented with sterile defibrinated sheep blood. Aliquots of 100-μL inoculum were plated on four agar plates containing the same concentration of drug(s). Mutant prevention concentration (MPC) was defined as the lowest concentration that prevented the growth of bacteria after 72 h of incubation at 37°C.The tested bacterial strains  were incubated in MH II broth for 12 h. The bacteria-containing broth was centrifuged at 3500 8 CFU/mL inoculum of each isolate was used after overnight incubation in MH II broth. A 0.5-mL sample of the inoculum and 0.5 mL of colistin or gamithromycin alone at 1 × or 4 × MIC, was added to 4 mL of fresh broth and incubated in a 37°C water bath for 1 h. Meanwhile, the same operation was performed for combinations of colistin at 0.25–0.5 mg/L and gamithromycin at 0.125–0.25 mg/L. Controls without drugs were included. Each inoculum was diluted 1000-fold after incubation for 1 h, and this was the starting point for PAE testing (0 h). Aliquots of 0.1 mL were withdrawn to count the bacterial burden as described above in time-kill tests at 0, 1, 2, 4, and 6 h. PAE was calculated using the equation: PAE = T – C, where T and C represent the time taken for drug-exposed (T) and control (C) cultures to increase by 1 log10 after drugs were removed (For determination of the postantibiotic effect (PAE) of colistin, gamithromycin was tested on D18, T5, and WJ11 isolates alone and in combination with colistin, and a 1 × 10 removed .8–109 CFU/mL) into the lung through the larynges rimae using a preinserted tracheal tube  mice weighing 20–22 g obtained from the Laboratory Animal Center of the Southern Medical University  were used in this experiment. Animals were maintained in the Laboratory Animal Center of the South China Agricultural University  in accordance with the National Standards for Laboratory Animals in China (GB 14925–2010). Neutropenia was generated by intraperitoneal injection of cyclophosphamide [TCI (Shanghai) Development Co., Ltd., Shanghai, China) 4 days before the 150-mg/kg dose and 1 day before the 100-mg/kg dose prior to lung infection . Three seal tube . All expPharmacokinetics analysis of colistin (2.5 and 5 mg/kg), gamithromycin  alone and in combination  was performed in 176 neutropenic lung-infected ICR mice following subcutaneous administration (SC). Plasma and lung tissue samples were withdrawn at the specified time point (four mice at each time point).The concentration of colistin and gamithromycin in plasma and lung tissue was measured using a previously reported method . The limIn vitro plasma protein-binding experiments for gamithromycin were conducted by adopting an equilibrium dialysis method for 72 h at 4°C. The corresponding unbound plasma PK parameters were obtained and used for the integration of PK and PD data.n = 4 for each group). Animals were humanely sacrificed 24 h after drug administration. Lung tissues were then removed under sterile conditions with the help of scalpels on a clean bench and homogenized in 0.9% sterile saline water with a tissue lyser. An appropriate diluent of homogenate was plated on Mueller-Hinton agar supplemented with sterile defibrinated sheep blood. Control animals without any drug administration were sacrificed at the beginning (0 h) and end (24 h) of therapy.Pharmacodynamics analysis of combined colistin and gamithromycin treatment in 264 lung-infected neutropenic mice was performed at 2 h after administration of the inoculum. To this end, 2.5 or 5 mg/kg colistin was subcutaneously administered every 12 h in combination with 1.5, 3, 6, 12, or 24 mg/kg gamithromycin, and colistin and gamithromycin were also tested alone at 1.5, 3, 6, 12, 24, 48, and 96 mg/kg  × Ce/(Ce + EC50), where E is the antimicrobial effect , Emax is the change in log10 CFU/lung for control groups, E0 is the maximum effect, Ce is the PK/PD index under analysis, and EC50 is the Ce value at 50% of E0.The relationship between the t-test provided by SPSS statistical software.The statistical significance of the difference between results was analyzed with a P. multocida and are often used to treat infections caused by this pathogen. We determined the MICs of frequently used drugs in the veterinary clinic, including cefquinome, enrofloxacin, tilmicosin, tylosin, and florfenicol, against three strains . With the exception of the MICs of tilmicosin and florfenicol (2–4 mg/L), the MICs of the other drugs against the three strains were more than 64 mg/L. Based on the action mechanism of these drugs, the FIC index was also calculated for the combination of cefquinome and gamithromycin. Although cefquinome and gamithromycin were found to have a synergistic effect, the concentration of cefquinome in the combination administration still remained high at 8–16 mg/L, which is higher than the clinically available drug concentrations. Therefore, the combined effect of colistin and gamithromycin against P. multocida was further investigated.Many types of drugs, such as β-lactams, macrolides, and fluoroquinolones, are known to be effective against Minimal inhibitory concentrations of colistin and gamithromycin alone and in combination against the nine isolates in MH II broth are listed in For all strains, all the clinically available concentrations of colistin (0.25–1 mg/L) were incapable of controlling bacterial growth . HoweverP. multocida when the concentrations of colistin and gamithromycin were 1 and 0.25 mg/L, respectively  and gamithromycin  ranged from 0.29 to 0.98 h and 0.21 to 0.59 h, respectively . Meanwhimax) of gamithromycin at 1.5, 3, 6, 12, and 24 mg/kg body weight after SC was 0.11–2.52 mg/L  within 0.25–0.75 h, consistent with 2.5 and 5 mg/kg body weight for colistin. The area under the concentration-time curve (AUClast) of colistin and gamithromycin was 3.51–10.52 mg⋅h/L and 0.26–5.06 mg⋅h/L, respectively (R2 ≥ 0.988 for Cmax and AUClast). The T1/2 value was 0.67–0.83 h and 11.81–12.58 h for colistin and gamithromycin, respectively. In lung tissue, the AUClast of colistin and gamithromycin was 5.22–12.20 mg⋅h/kg and 32.55–511.72 mg⋅h/kg, respectively. Correspondingly, the Cmax was 3.37–5.54 mg/kg and 2.27–35.24 mg/kg, after 0.25–1 h.In total plasma, the maximum concentration (Cectively . A good last between plasma and lung tissue is shown in The results of plasma protein–binding experiments show that the protein binding extent of gamithromycin was 13.83% under the preparation method employed in this research. Based on these results and those of a previous report on colistin (unbound extent = 8.4%) , free pl10 CFU/lung, and none of the other monotherapy groups exhibited inhibition of bacterial growth 24 /MIC for gamithromycin in plasma and the antimicrobial effect in the pneumonia model mice. A correlation with AUC(0–h)24 /MIC > 0.89 indicated that this index may be suitable for predicting therapeutic outcomes ((target) = Dose(test) × AUC/MIC(target)/AUC/MIC(test)  , the tarIC(test) . The aveP. multocida, thereby increasing the content of gamithromycin in the cytoplasm. To further investigate the potential mechanism of the combined activity of colistin and gamithromycin against P. multocida strains, changes in the transcriptome and protein content of P. multocida following treatment with drugs should be examined in the future.Herein, five R isolates  and four S isolates  were subject to MIC tests. Interestingly, synergistic and additive antimicrobial effects were observed against R and S isolates, respectively, when colistin was combined with gamithromycin. With regard to the mechanism of action, colistin may have damaged the outer membrane of P. multocida in response to colistin was stronger than that for gamithromycin. It is worth noting that, although the MIC of colistin against WJ11 was 1 mg/L, the MPC was as high as 64 mg/L. This indicates that monotherapy with colistin is more likely to induce the development of drug-resistant mutants. Surprisingly, the combination of colistin and gamithromycin reduced the MPC of colistin considerably by 64-fold and closed the mutant selection window of colistin. This indicates that combination therapy is more reasonable and feasible. The in vitro PAE results also confirmed the combination of colistin and gamithromycin produced a more profound and lasting bactericidal activity in vitro even at lower concentrations of each drug. PAE should not be ignored from a therapeutic perspective.Even though the MIC of colistin against WJ11 was 32-fold lower than that against isolate D18 or T5, each drug tended to reach the same concentration in combination treatments for both R and S isolates. Similarly, the rate and extent of killing for colistin and gamithromycin in combination did not differ between isolates D18 or T5 and WJ11 based on time-kill tests. Additionally, the killing effect was relatively insensitive to increases in the concentrations of colistin when the effect reached a certain level. The MPC results showed that the first-step resistant mutation ability of last to free plasma AUClast averaged 123.3, and there was no obvious interaction between PK indices for colistin and gamithromycin. The dose recommended by the EMA for cattle and swine for SC administration of gamithromycin is 6 mg/kg, which is associated with an AUClast of 7.82  was achieved by 2.5 mg/kg colistin plus 24 mg/kg gamithromycin. Furthermore, even greater killing of bacteria  could be achieved by combination therapy with 5 mg/kg colistin and 12 mg/kg gamithromycin.The main PK characteristics of macrolides are good lung tissue permeability and low plasma content . Unsurpr of 7.82  and 3.48 of 7.82 . These v of 7.82 . However(0–h)24 /MIC was considered to be the best indicator of therapeutic outcomes, and the target values required to achieve the same antimicrobial activity for combination therapy were 6- to 35-fold lower than those for monotherapy. Subsequently, an equivalent dose based on a comparison of the body surface area of mice and other animals could be calculated to predict the appropriate clinical target dose  (P. multocida stains with gamithromycin MICs of 0.5 mg/L in this study. This finding indicates that gamithromycin resistance might not be conferred by the mef(B) gene. Despite this, gamithromycin-resistant strains generated by the active efflux of drugs associated with the erm(42) and msr(E) – mph(E) genes have not been obtained so far, and the addition of colistin to gamithromycin may be effective against resistant strains via a mechanism that potentially involves an efflux pump. Future studies should examine whether the enhanced antimicrobial activity is reproducible in gamithromycin-resistant strains.The newfound macrolide efflux gene mef(B)  was idenP. multocida strains in vitro were determined and verified by adopting a mouse pneumonic model. It is interesting to note that the concentrations of the combination of colistin and gamithromycin tended to reach similar levels against different P. multocida strains even though there was a considerable difference in the colistin sensitivity of these strains. Additionally, the administration regimen was optimized by assessing the relationship between gamithromycin PK data in plasma and the antimicrobial efficacy in lung tissue. The findings indicate that combining colistin with gamithromycin holds promise for treating pneumonia caused by P. multocida strains.Herein, the increased antimicrobial activities of colistin-based combinations with gamithromycin against The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Committee on the Ethics of Animals of South China Agricultural University.YL carried out the main experiments, data analysis, and wrote the manuscript. MX, JZ, and HL took part in the animal study. TX and LW participated in the isolation and identification of bacteria experiments. HD participated in the data analysis. BF conceived and designed the experiments. All authors contributed to the article and approved the submitted version.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.


Epigenetic factors have been suggested as mediators of early-life nutrition to future health. Prior studies focused on breastfeeding effects on DNA methylation (DNAm), ignoring other feeding modes. In this analysis of the Isle of Wight birth cohort, feeding modes were categorized as exclusive breastfeeding (EBF), exclusive formula feeding (EFF), and mixed feeding based on whether the respective feeding mode lasted for at least 3 months. In addition, in the past, infant feeding modes were assessed using DNAm at one time point in childhood, not changes of DNAm. In this paper, methylation differences (delta DNAm) were calculated by subtracting residual methylation values at birth from age 10 years (adjusting for cell types and season of blood collection at both ages). These deltas were estimated for all methylation sites where cytosine was followed by guanine (cytosine guanine dinucleotide (CpG) sites). Then, we performed an epigenome-wide association study contrasting EBF, EFF, and mixed feeding with delta DNAm that represents changes in methylation from birth to 10 years. A total of 87 CpGs  were identified using separate linear regression models adjusting for confounders and multiple testing. The sum of all changes in methylation from birth to age 10 years was significantly lower in the EFF group. Correspondingly, the number of CpGs with a methylation decline was 4.7% higher reflecting 13,683 CpGs. Lower methylation related to exclusive formula feeding and its adverse potential for the child’s development needs future research to reduce adverse health effects. Adequate nutrition throughout infancy and early childhood, especially during the first two years of life, is crucial for ensuring good health, optimal growth and development of newborns to their full potential ,2. InfanFTO and CPT1A) are significantly higher in infants that were formula or mixed fed in comparison to exclusive breastfed ), where c is used as a constant to prevent zero in the denominator. Finally, a total of 292,366 CpGs (present both at birth and age 10) were included in the statistical analyses.Methylation data were quantile normalized using the minfi R package and batch corrected using ComBat in R . Probes Variables known to potentially confound the association between duration of infant feeding and offspring DNAm levels were selected as covariates. These include categorical variables such as maternal smoking, birth order, socioeconomic status (SES), mode of delivery, and maternal asthma, and continuous variables such as birth weight and maternal age. Maternal age at delivery (in years) was calculated using child’s and maternal date of birth. Information about maternal smoking during pregnancy (question on active smoking) and maternal asthma (obtained from the questions of whether the mother suffered from asthma or wheezing attacks) were reported by the mothers at birth. Mode of delivery  and birthweight in kilograms were ascertained from the birth records. The birth order of the index child in the family (1st/2nd/3rd or more) was gathered by questionnaire. A composite family socioeconomic status (SES)-cluster variable that included a combination of parental occupation (reported at birth), number of children in a child’s bedroom (collected at age 4 years), and family income (at age 10) was used in our analyses. This composite variable has been described elsewhere .In addition to covariates primarily related to breastfeeding, we also adjusted for variables that may confound effects of DNAm. These include infant’s sex , estimatSeason of blood collection at birth (obtained from the date of birth) and 10 years (obtained from the date of interview at 10 years of follow up) were regrouped as December–February (winter) March–May (spring), June–August (summer), and September–November  and used winter as reference . To evaluate whether the analyzed sample (subjects with DNAm data at birth and 10 years) differed from the study cohort, we compared the sample with the population using one-sample proportion tests  and one-sample t-tests (for continuous variables). The analyses were restricted to paired data, i.e., CpGs and participants present both at birth and 10 years . In the first step, to achieve normal distributions of DNAm measurements at birth and 10 years, we logit-transformed beta values to M values ). We then regressed M values on cell-type proportions and season of blood collection for each age (birth and 10 years) separately. This provides the residuals of DNAm (residDNAm) at birth and 10 years representing DNAm after correcting for cell types and season of blood collection. To determine differences in DNAm between birth and 10 years ), the difference between the residuals at birth and 10 years was calculated (ΔDNAm = residDNAm at age 10–residDNAm at Birth). The delta-residuals (ΔDNAm) were treated as the outcome for further analysis.ttScreening R package, which was applied on delta-residuals to identify CpGs associated with each infant feeding variables separately using the following conceptual model: In the second step, we used delta-residuals, the changes in DNAm from birth to age 10 years, as an outcome to perform three separate EWASs for each infant feeding variable  with two categories as the exposure by linear regression models. We screened all CpGs using the i method . CpGs thTo adjust for potential confounding, linear regression models were used for those CpGs that passed the screening with delta-residual at each CpG site as a dependent variable. All potential confounders  were included in the model. Next, we compared the direction of significant regression coefficients across all three infant feeding modes using bar and volcano plots.Additionally, we tested whether the overall global DNAm of 292,366 CpGs changed from birth to age 10 years. To this end, we first calculated the mean of the delta-residuals for each of the 201 subjects across all the 292,366 CpGs (paired data at birth and ten years). We then fitted a linear regression model using the means of all individual ΔDNAm as outcomes and the three infant feeding modes as exposures controlling for confounders . Second, to investigate whether the overall DNAm was affected by infant feeding, we repeated the above equation with the average DNAm over all CpGs as outcome. Then, to test whether overall DNAm changes is due to minimal changes of all CpGs or higher changes in a limited proportion, we tested the proportion with positive (increase of DNAm) and negative changes (decrease in DNAm). To perform this, we first calculated the upper limit Q1 (lower quartile) and the lower limit Q3 (upper quartile) of all 292,366 CpGs for each of the 201 participants. Then, we determined the population medians of these Q1s and Q3s over all 201 children. For each child, we computed the percentage of CpGs below the median of the population-based Q1 (representing negative changes) and the corresponding percentage of CpGs above the median of the population-based Q3 (representing positive changes). Finally, we conducted two separate linear regression models using the percentages of CpGs  that are below the population-based Q1 and the percentage of CpG that are above the Q3. Again, in these models, the three infant feeding modes were the exposures and we adjusted for confounders.For all outcomes of the linear regression analyses, we checked the multi-variate normal distribution. The flow of the statistical analysis is shown in In addition to statistical analysis, we performed a follow-up investigation of our findings to examine if we could find agreement between CpGs or genes from our analysis with those reported previously. Reports that used breastfeeding as a confounder, e.g., analyzed in tumor samples or studies that did not provide CpG sites information were excluded. The final list includes a total of 4308 CpGs, i.e., 12 CpGs from the study of Odintsova et al. ,32, 7 CpThe scope of this paper does not allow for a systematic investigation of long-term health effects related to potential stable CpGs. However, within our study focusing on allergies and related factors, we tested the role of more stable CpGs for three outcomes with a continuous scale: one lung function test, serum IgE levels , and body mass index (BMI) at age 18. Measurements of these markers have been reported in prior papers ,52,53.p-value of 0.1 and no adjustment for multiple testing.Since health effects later in life require some stability of the DNAm changes, before linking Δ residuals of the DNAm to these three health outcomes, we tested how many of the discovered CpGs can be characterized as showing little dynamic in their methylation levels and thus could be classified as informative biomarkers in contrast to random effects. Using further information on DNAm at 18 years , we usedAmong the 1456 enrolled children in the original cohort, 1360 were followed to age 10, of which 796 and 330 participants had DNA methylation available at birth and 10 years, respectively. For our analyses, we included 201 participants with methylation data for both ages and with non-missing information for infant feeding variables. The descriptive statistics comparing analyzed samples with the whole cohort are summarized in The subsamples included in the study represented the complete cohort with respect to all exposure and confounding variables included in the analysis, except for season of heel prick blood collection at birth, maternal age at delivery, and age at the introduction of solids. Significant differences were observed in the proportion of children born during the spring (20% vs. 14%) and summer (15% vs. 18%) for season of blood collection at birth. The average maternal age (29.2 vs. 29.6) and age of solid food introduction in weeks (14.3 vs. 15.2) were slightly higher in the analytical sample than the whole cohort. All these variables were controlled for in our analyses as potential confounders. ttScreening) analyzing 292,366 delta-residuals detected 87 informative CpGs across all the three feeding variables  (The training-testing method (feeding) . The delfeeding) . Inspecting the direction of regression coefficients in each infant feeding mode using bar and volcano plots shows that more CpGs related to EFF have negative regression coefficients (representing decreased methylation from birth to 10 years) than EBF and mixed feeding . A linear regression model suggests that globally, over all 292,366 CpGs, the average DNAm declined more in exclusive formula-feed infants compared breastfed infants . The mixIn addition, the percentage of CpGs with a more pronounced reduction in DNAm (ΔDNAm in the lower quartile of the distribution) was 4.7%  higher in exclusive formula-feed compared to breast-fed infants controlling for confounders . No assoInterestingly the 87 CpGs discovered for exclusive breastfeeding (EBF), exclusive formula feeding (EFF), and mixed feeding were also statistically significantly differently distributed on locations on the genes . The 27 GHR, FBXO4) that was associated with both exclusive breastfeeding and mixed feeding  and cg08600430  and ANGPT2/MCPH1 for cg04017131  and cg25350011 .According to gene annotation, the 87 CpG sites having significantly different delta-residuals and associated with three feeding variables represent 117 genes. Of these 87 CpGs, we found only one common CpG  from our study that were also reported previously  was surprising. Among the 87 identified CpG, we found a clear tendency for reduction of DNAm from birth to age 10 years for formula-fed infants. This motivated us to test overall changes. Considering the mean of changes of all 292,366 CpGs, formula-fed infants had a statistically significant lower average change, suggesting a reduction of their methylation levels at 10 years. Testing how many CpGs show a stronger reduction  revealed that a statistically significant proportion of 4.7% of all CpGs  are lowered from birth to age 10 years in formula-fed compared to breastfed children. This suggests that children who were given formula exclusively for at least three months had significantly lower methylation  than children who were exclusively breastfed. In addition to the different direction of the methylation changes of different feeding modes, we also found that CpGs related to EFF and mixed feeding were more frequently found in the body of the gene, where CpGs related to EBF were often located in the transcription start site of the gene. It is possible that both the different direction of methylation changes and the location of the affected CpGs on the gene are associated. For instance, decreased in methylation and CpGs in the body of the gene may indicate a different genetic ‘programming’ . HoweverWe considered the possible effects of most practiced infant feeding modes on DNAm changes by categorizing infants into three feeding groups. This is essential as it not only takes out the confounding effects of other infant feeding modes on breastfeeding while studying its association with DNAm, but also allows us to compare the effects of exclusive formula feeding and mixed feedings with recommended exclusive breastfeeding. Also, the use of these feeding modes is consistent with other association studies of infant feeding and adverse health outcomes such as obesity , gastroeOur statistical analysis used DNAm differences between birth and age 10  as an outcome by removing the effect of cell-type proportions and season of blood collection at both the time points. Therefore, a positive coefficient between infant feeding and delta methylation implies that DNAm was lower at birth and increased at 10 years following exposure to the infant feeding (EBF/EFF/mixed). On the other hand, a negative coefficient indicates that infant feeding is related to a decrease in DNAm from birth to 10 years. Note, we did not investigate differential DNAm detected in one measurement during childhood, but we did investigate changes of DNAm between birth and age 10 years for different infant feeding modes effective after birth. The use of these delta residual DNAm—compared to a single methylation assessment—is key to interpreting changes in methylation at age 10 with respect to baseline or birth, thus allowing us to examine the real exposure effects of different infant feeding modes on DNAm. For instance, as seen for the mother , the matOf the 86 discovered CpGs, we believe that 43 CpGs can be considered as potential biomarkers of infant feeding, since they show a significant effect when comparing DNAm between birth and 10 year and are more stable between 10 and 18 years. A potential limitation is that we did not investigate differentially methylated regions (DMRs). There is a possibility that CpGs characterizing DMRs were not discovered by our approach. However, we also have to consider that DMR can be identified using the DNAm profiles in one assessment (at one age) focused either on exposures or diseases, but not for DNAm changes, which we addressed here. For instance, the literature does not suggest that changes in DNAm can be characterized by changes of methylation profiles covering specific genetic regions. In addition, there is a number of limitations involved in the detection and statistical modeling of DMRs . Hence, Another limitation is that, within the scope of this manuscript, we could not systematically cover the variety of all potential health outcomes that could be related to changes in DNAm. However, we showed potential associations with health outcomes later in childhood for a few continuous health indicators. The selection suggests that changes in DNAm between birth and age 10 may provide novel and interesting insights into later health effects and that changes in DNAm seems to constitute epigenetic biomarker that needs further evaluations. Interestingly, infant feeding mode was not only related to specific DNAm but showed an overall wide-spread effect related to a significant decrease in methylation at 292,366 CpGs in exclusively formula-fed infants compared to exclusive breastfeeding. These unspecific consequences are in agreement with a non-specific variety of health outcomes related to infant feeding such as otitis media, lower respiratory tract and gastrointestinal infections, asthma, atopic dermatitis, childhood obesity, type 1 and type 2 diabetes, leukemia, sudden infant death syndrome, and overall infectious morbidity ,8,9. FTO and CPT1A and a low expression of PPARA in formula-fed and mixed-fed groups compared to the exclusively breastfed fed group [Although the current study is important for highlighting epigenetic modifications associated with different infant feeding modes, the following factors limit the conclusion of this investigation. Firstly, due to the small sample size in each feeding group and lack of formal replication for generating reproducible results, the findings of the study need to be considered with care. Second, while defining the breastfeeding group, we could not consider other common breastfeeding modes, such as direct feeding at the breast vs. pumping and feeding that have been shown to have different effects on other outcomes studied . Furthered group . Hence, Future large-scale studies are warranted to evaluate the results of our study. It is crucial to investigate and determine etiologic mechanisms that mediate the observed associations between formula feeding and decreased methylation development up to ten years. Understanding the mechanisms related to this association will first help us to reduce the susceptibility of risks for adverse health outcomes in later life by optimizing infant nutrition. The mechanisms may include bioactive factors in breastmilk or the protective nurturing bond between mother and infant. Second, this information will guide us to develop novel interventions to improve children’s health.


Percutaneous puncture ethanol sclerotherapy is a simple, effective, minimally invasive, and inexpensive procedure to manage symptomatic simple renal cysts. We modified specific technical aspects to balance certain potential intraprocedural factors for ensuring minimal procedural pain and duration as well as maximal clinical therapeutic effects and evaluated the safety and efficacy of this modified technique. A total of 84 eligible patients underwent computed tomography-guided single-session ethanol sclerotherapy using the conventional (group A) or modified (group B) technique. In group B, the puncture route was modified from tansparenchymal to direct for reducing renal injury, and preinjection of low-dose intracystic lidocaine was used to control distending pain caused by ethanol injections; therefore, greater ethanol volumes could be injected for improving the resistance and contact of ethanol with the cyst wall, precluding the need for patient repositioning multiple times to decrease procedural duration. Visual analog scale score for pain and procedural time were significantly higher in group A than in group B. The complication rate was slightly higher in group A than in group B, but the success rate was comparable between the two groups. These results suggest that the modified technique is painless, time-saving, and injury-reducing and can thus improve medical care. Symptomatic SRCs present common clinical symptoms such as flank pain or mass, hypertension, and hydronephrosis. Percutaneous ethanol sclerotherapy is a simple, effective, minimally invasive, and inexpensive treatment for symptomatic SRCs2. Therefore, it is often the first-line procedure and is widely used in clinical practice; however, the procedure has not been standardized to date3. The conventional technique reported by Xu et al.4 is used in early stages of the disease, and its advantages include the use of three-way drainage tube, which prevents air from entering the cyst, and of high ethanol concentration over repeated cycles of injection–suction, which eliminate the step of ethanol dilution4. Moreover, fragments of protein-denatured substances are removed to allow a directly contact between ethanol and epithelial lining of the cyst wall. The computed tomography (CT) value is used to monitor ethanol concentration for improving treatment efficacy5, which is based on the linear correlation between ethanol concentration/density and CT attenuation; when the CT value is lower than −190 HU, ethanol concentration is considered to be greater than 90%.Simple renal cysts (SRCs) are one of the most commonly acquired renal cystic diseases and are frequent in the elderly given that their prevalence increases with age6. Second, patients complain of distending pain caused by ethanol injections7. Third, the patient’s position needs to be changed twice to increase the contact area between injected ethanol and cyst wall, which requires more time5. Pain and prolonged procedural time reduce the patient’s compliance and increase their discomfort. Therefore, these factors should be balanced to ensure minimal procedural pain or time and maximal clinical therapeutic effects.However, we have encountered a few problems in the course of our clinical practice. First, this puncture route can damage renal tissues, thus increasing the risk of intracystic hemorrhage and hematuriaTo this end, we sought to modify specific technical aspects of the conventional procedure, which resulted in positive effects. The modified procedure appeared to be more comfortable, faster, and safer. However, the data have not been fully evaluated. Therefore, this exploratory randomized trial was conducted to evaluate whether our modified sclerotherapy technique for SRCs is superior to the conventional one.This present study was approved by the ethical committee of Dianjiang County People’s Hospital of Chongqing . This was a registered clinical trial  performed according to the Declaration of Helsinki. Written informed consent was obtained from all patients prior to participation in this study. All procedures on patients were performed in accordance with the relevant guidelines and regulations  using CT examination prior to the procedure and met the following inclusion criteria: cyst diameter between 5–8 cm; presence of symptoms (n = 70) such as flank pain or discomfort, mass, or hypertension; and absence of overt clinical symptom reassurance due to increasing cyst size (n = 26). Patients with peripelvic cysts, previously treated cysts, or procedural contraindications were excluded.8.Between June 2016 and May 2017, sclerotherapy was performed in 49 patients (49 cysts in group A) using the conventional technique. Between June 2017 and March 2018, after obtaining approval from the institutional review board, sclerotherapy was performed in 47 patients (47 cysts in group B) using the modified techniqueCT-guided single-session 99.9% ethanol sclerotherapy was performed on patients in both groups by the same operator, who has an experience of over 500 interventional procedures. Before the procedure, cyst volume was estimated according to the following formula: V = length × width × height × π/6. The patients were placed in the prone position, and 2% lidocaine was administered for local infiltration anesthesia at the puncture site. A 20-gauge coaxial needle  was used to puncture into SRCs, and a three-way drainage tube was used to connect to the coaxial needle. A sample (20 mL) of cyst fluid obtained during initial aspiration was sent for bacteriological, cytological, and biochemical tests. Patients but the operator was blinded to pre-injecting of intracystic lidocaine to control pain because all the medicinal liquids must be checked by the operator before being injected. During the procedure, patients’ visual analog scale (VAS) score for pain and volumes of each aspiration and injection were recorded separately. Of note, it is necessary to steadily aspirate 1–2 mL of cyst liquid through the syringe without exerting negative pressure to confirm that the needle tip is within the cyst prior injecting fluid  into the cyst. This procedure also avoids injecting a small amount of gas that may be present in the three-way drainage tube into the cyst.For group A, the puncture route was designed to pass through a portion of the renal parenchyma, with the tip being located at a distance one-third to half of the cyst diameter. Diluted contrast agent  was then injected to ensure that the cyst was not connected with the collecting system of the kidney and that no contrast material was leaking out. After aspirating most of the cyst fluid, 99.9% ethanol was injected into the cyst at a volume equal to 25% of the volume of aspirated fluid. This aspiration and injection cycle was repeated was with 20 mL ethanol until the aspiration fluid appeared clear on visual inspection. CT was performed to confirm that the CT value was below −190 HU. The patient was placed in the prone and bilateral decubitus positions for approximately 5 min each. All fluid was then aspirated out of the cyst, and the coaxial needle was removed. The patients were escorted back to the ward by a nurse and were discharged the next day if they did not develop complications.For group B, the puncture route was designed to avoid the renal parenchyma, and the needle tip was located near the cyst base. After injecting diluted contrast agent to confirm normal conditions, 3–5 mL of 2% lidocaine was injected into the cyst and retained for 1 min. Next, based on the estimated cyst volume, most of the fluid within the cyst was aspirated, with retention of only 10–20 mL. This enabled the operator to confirm that the needle tip was within the cyst by steadily aspirating 1–2 mL of fluid. Then, 99.9% ethanol (not more than 200 mL) was injected into the cyst to occupy approximately 75% of the original volume, which was calculated based on the estimated cyst volume. This aspiration and injection cycle with the same volume was repeated. Typically, following completion of the second or third replacement, the ethanol concentration in the cyst cavity exceeded 90% and remained unchanged for 5 min under the same patient position . A value of p < 0.05 was considered statistically significant.Normality of distribution of continuous variables was assessed by the Shapiro–Wilk test. Continuous variables with normal distribution were reported as mean and standard deviation; the means of variables between the two groups were compared by independent sample Student’s test. Continuous variables with non-normal distribution were presented as median (interquartile range), and the means of variables between the two groups were compared by the Mann–Whitney U test. Pain score and efficacy were classified as ordered categorical variables and analyzed using the Mann–Whitney U test between groups. For other categorical variables, frequencies were compared using Pearson χp < 0.01) than that in group B. Patients in group A complained of different degrees of pain, whereas only one patient in group B reported mild swelling but no pain. In this case, the cyst had a volume of 210 mL and was in the early stages of our modified technique experience. Only 3 mL lidocaine was injected into that cyst, which might have been inadequate. In one case in group A, acute intracystic hemorrhage occurred following the puncture of an 88-mL cyst. One unit of hemocoagulase was injected into the cyst and retained for 5 min. Then, it was repeatedly replaced with normal saline until the liquid showed no obvious blood color. Procedural time for that case was prolonged to 49 min. Microscopic hematuria occurred in two patients in group A. Ethanol exposure and procedural times were higher in group A than in group B  were enrolled in the study. Demographic and SRCs characteristics of the patients are summarized in Table 9. Bryniarski et al.10 have reported that the prevalence of hypertension in patients with SRCs was 43.3% (91/210 patients), of which 61.7% (56/91 patients) were relieved of hypertension following cyst sclerotherapy or laparoscopic. Therefore, after the cyst was ablated, the associated clinical symptoms were relieved; residual cysts without symptoms do not require further intervention11. Furthermore, some patients without overt clinical symptoms opt to undergo treatment because their anxiety increases with increasing cyst volume13.SRCs are usually asymptomatic; however, flank pain is a relatively common symptom in individuals with symptomatic SRCs. It possibly occurs due to dilation of the renal capsule; furthermore, some symptoms may appear secondary to obstruction of the collecting system15. Large cysts (>8 cm) tend to fold after cavitary collapse. Sometimes, there is no contact between injected ethanol and cyst, and such contact, if any, is less effective; therefore, laparoscopy is the preferred option in such cases14. Residual cysts detected during early follow-up after sclerotherapy may be secondary to transient, reactive, or inflammatory fluid accumulation, which may take 6–12 months to completely resolve16. Therefore, cyst diameter in this comparative study was controlled at 5–8 cm, with a follow-up time of 12 months. Although peripelvic cyst is not a contraindication for sclerotherapy17, ethanol leakage at the ureteropelvic junction may lead to fibrosis and advanced stenosis19. Therefore, for the sake of safety, peripelvic cysts were excluded from this study.Presence of a cyst with a diameter of >4 cm is one of the inclusion criteria for SRC sclerotherapy13. CT is preferable in obese patients or when the cyst is unclearly visualized and a safe access route cannot be identified.Ultrasound and CT are commonly used as a guidance modality. Ultrasound has the advantages of being cost-effective, real-time, and non-radiative, although it is much more dependent upon operator skills than CT17. However, some operators still prefer to use the former, as shown in previous studies20, because it avoids direct puncture leading to cyst rupture, and the coaxial needle is passively shifted out of the cyst during cyst wall collapses as the fluid decreases. However, this path may damage renal parenchyma. In a study by Dell’atti et al.6, incidence of hematuria and intracapsular hemorrhage was 8.3% (3/36 cases) and 2.7% (1/36 cases), respectively. Data on complications obtained in the present study were insufficient to determine whether they were caused by transparenchymal puncture. Nonetheless, direct puncture avoids renal parenchymal injuries; thus, we recommend it for suitable cases. This notion is consistent with the view put forth by Lee et al. that a cyst should be punctured as atraumatically as possible to decrease bleeding into it9. In our experience, care should be exercised to control patient’s breathing and to minimize the risk of cyst rupture during direct puncture. During the procedure, as the fluid in the cyst decreases, the wall collapses toward the cyst base. Therefore, the needle tip should be near the cyst base.Transparenchymal and direct puncture are both acceptable methods in the literature and are usually left to the discretion of operator, primarily dictated by the cyst location15, which results in a higher VAS score. Yonguc T et al. reported that mean VAS scores of the overall and severe case were 4.26 ± 1.99 and 5.1 ± 2.1, respectively15. Moreover, analgesics may be applied6 in some cases, while the operator is forced to inject smaller ethanol volumes in others21. In severe cases, the procedure is often suspended or discontinued21. Although studies have recommended the use of ethanol, 20% hypertonic saline can also be used to avoid pain20. Lidocaine is a commonly used and readily available local anesthetic that is inexpensive, blocks painful stimuli, and works rapidly. Because lidocaine controls pain22, greater ethanol volume can be injected, improving the contact of ethanol with the cyst wall. This prevents the need for repositioning the patient multiple times, leading to decreased procedural duration.The most common complication of ethanol sclerotherapy is distending lumboabdominal pain7 (maximum injected ethanol volume of 200 mL)23 without adverse effects23. The exposure time was 5–40 min, and most cases achieved good results24. Use of high ethanol concentrations can lead to dehydration, protein degeneration, and necrosis of epithelial cells of the monolayer cyst lining within 1–3 min25. Additionally, ethanol induces aseptic inflammation of the cyst wall, and after the cystic fluid is exhausted, the wall collapses and the adhesion remains closed5. Ethanol gradually penetrates the fibrous capsule in approximately 4–12 h, leading to minimal local or systemic adverse reactions25. The outcome of the volume of ethanol (>90% V/V) and time of exposure to it were nearly the same groups A and B; however, the dose–time effect value was lower than the value reported in most previous sclerotherapy treatments24. Therefore, systemic reactions, which are usually caused by large or prolonged exposure9 to ethanol, did not occur in either study group. Moreover, no major complications occurred, perhaps because of the small sample size, meticulous procedure, and inherent safety of the procedure26.In previous reports on single-session sclerotherapy, 95–99.9% ethanol at a volume of 15–50% of the cyst volume was used as a sclerosing agentThe modified technique is painless, requires a short time, and minimizes renal parenchymal injury, thereby ensuring a rapid and smooth procedure. Likewise, it is also suitable for ultrasound-guided sclerotherapy, wherein only the guidance technique is different. Moreover, if certain operators are accustomed to transparenchymal puncture or in case the puncture path cannot avoid the renal parenchyma, injection of intracystic lidocaine and subsequent procedures can still be performed with reference to the modified technique to produce the same effect. Acquiring details of patient improvements enables to minimize renal parenchymal injury, pain, anxiety, and discomfort caused by prolonged procedures, thus achieving better medical care.Our research has several limitations that warrant further discussion. First, it is not a prospective randomized controlled trial, and there may be a selection bias. Second, the sample size of patients treated with the modified technique is small; thus, further randomized studies in a large patient cohort are warranted to confirm our results. Finally, ethanol sclerotherapy was performed even in asymptomatic patients with SRCs.In summary, we recommend direct puncture of the cyst to avoid renal parenchymal injury, preinjection of intracystic lidocaine to control pain, and increase in ethanol volume/exposure area without changing the patient position to shorten the procedural duration. Furthermore, the modified technique is safe and effective and can thus improve medical care.Supplementary informationSupplementary information2 Supplementary information3


Preterm birth (birth before 37 weeks of completed gestation) is the leading cause of neonatal death, and has an incidence of 5–13% which is believed to be on the rise. The objective of this study was to determine the rate of spontaneous preterm birth and investigate the relationship between preterm birth and cervical length in a pregnant Asian population.A prospective observational study between September 2010 and November 2013 was performed at KK Women’s and Children’s Hospital, Singapore. 1013 women with single viable pregnancies were recruited at less than 14 weeks of gestation between September 2010 and November 2013, excluding those with multiple gestation, pre-existing autoimmune or renal disease or those with current pregnancies complicated by aneuploidy or fetal anomalies. Participant characteristics were obtained from an interviewer-administered questionnaire at the first recruitment visit. Cervical length was measured using ultrasound at each of the 4 antenatal visits  using the Fetal Medicine Foundation protocol. Data on pregnancy outcomes were obtained from obstetric case notes and records. The main outcome measure examined in this study was that of spontaneous preterm birth and its relationship to cervical length.There was a significantly shorter cervical length both in the 2nd trimester (18 to 22 weeks) and the 3rd trimester (28 to 32 weeks) in the preterm birth group compared to the term birth group (p = 0.028 and p < 0.001 respectively). In the first trimester (11 to 14 weeks), there was no statistically significant difference in cervical length between the two groups (p = 0.425). ROC curve analysis for cervical length in the preterm birth group for 18 to 22 weeks and 28 to 32 weeks showed an AUC of 0.605 and 0.725 respectively. At 28 to 32 weeks of gestation, a cut-off level at 2.49 cm has a sensitivity of 54.8%, specificity of 82.5%, negative predictive value of 97.9% and positive predictive value of 11.1%.There is a significantly shorter cervical length in the 2nd and 3rd trimester in the preterm birth group. Cervical length is a moderate predictor of preterm birth with good negative predictive value and a relatively good specificity. Ultrasound cervical length screening for pregnant Asian women between 18 and 22 weeks of gestation with a cutoff of ≥ 2.48cm can help to identify a group of women who are at risk for preterm birth. Preterm birth, defined by WHO in 1977 as all births before 37 completed weeks of gestation or fewer than 259 days since the first day of a woman’s last menstrual period, is the single largest and most commonly cited adverse outcome in pregnancy  Of all tnd leading cause of death after pneumonia in children under the age of 5. In our tertiary centre, over a period of 10 years from 2001 to 2010, prematurity was found to be the largest contributor to neonatal mortality at 47.5%. Also, significant morbidities to the child can follow the event of a preterm birth. Babies born a few weeks early are 6 times more likely to die in their first week of life than full-term babies, and 3 times more likely to die before their first birthday . Written informed consent was obtained from all participants. The study was conducted as part of the Neonatal and Obstetrics Risk Assessment (NORA) prospective cohort study at KK Women’s and Children’s Hospital in Singapore and funded by the National Medical Research Council Programme Project Grant [NMRC/PPG/KKH/2010].Participants were women with single viable pregnancies recruited at less than 14-week gestation between September 2010 and November 2013. Exclusion criteria included that of multiple gestation, patients with pre-existing autoimmune or renal disease and current pregnancies complicated by aneuploidy or fetal anomalies. These women were followed up from recruitment until their postnatal discharge from the hospital. A total of 4 antenatal visits were included as part of this study, with Visit 1 at less than 14+ 0 week of gestation, Visit 2 at 18 + 0 to 22 + 0 week gestation, Visit 3 at 28 + 0 to 32 + 0 week gestation, and Visit 4 at 34 week gestation and above. Gestational age was determined based on a first trimester dating scan measuring the fetal crown-rump length. Measurements of cervical length were conducted at each of the 4 visits for all patients based on the Fetal Medicine Foundation protocol using a 5MHz transvaginal probe. All sonographers performing the scan had extensive training and accreditation by the Fetal Medicine Foundation for competency in performing this scan. Each examination was performed over a period of 3 minutes and the best shortest measurements of cervical length was recorded.Participant characteristics, including details of maternal age, race, height, weight, socio-economic status, smoking and alcohol status, medical and surgical history, previous and current obstetric history were obtained from an interviewer-administered questionnaire at the first recruitment visit and entered into a computer database.Data on pregnancy outcomes were collected from obstetric case notes and records. The primary outcome of interest was that of spontaneous preterm birth before 37 weeks of gestation. The obstetric records of all patients delivering preterm before 37 weeks (<259 days) were examined to determine if the preterm birth was iatrogenic or spontaneous. Spontaneous preterm birth included those with spontaneous onset of labour and those with preterm pre-labour rupture of membranes.Statistical analyses were performed using Microsoft Excel and SPSS (Version 19). Continuous variables were summarized by the median values, and comparison between groups were conducted using the Mann-Whitney U-test. Univariable comparisons of dichotomous data were performed using Chi-square test and Fisher’s test where appropriate. Statistical significance was set at a p-value of <0.05. Relationship of various variables such as maternal obstetric history and demographic characteristics to that of cervical length were examined for significance. Receiver operating curves were generated at various time points in the pregnancy, and sensitivity/specificity analyses conducted to determine the best cut-off value for cervical length as a predictor for preterm birth, based on the prevalence of preterm birth determined within this study.A total of 3271 patients were screened for eligibility for inclusion into the study between September 2010 to November 2013. Of these, 2820 patients were deemed eligible for inclusion, and 1013 of them were recruited into the study. 934 (92.2%) of patients completed all 4 antenatal follow-up visits as described above, with 66 (6.5%) patients being lost to follow up and 13 (1.3%) patients who miscarried before 24 weeks of gestation. Of the 934 patients, 17 delivered at other institutions, but delivery data for 9 of them were retrieved via phone call. There were 926 patients in total with available study outcomes that we based our analysis of the data on. The predictive statistics generated later in this paper relating to preterm birth were calculated only from the group of patients who had spontaneous preterm births. Patients who had iatrogenic preterm birth due to other factors such as pre-eclampsia or medical indications were analysed as part of the group without spontaneous preterm birth.Of the 926 patients with all 4 completed visits, 62 (6.7%) of patients delivered prematurely at less than 37-week gestation. Of these, 39 (62.9%) of them were spontaneous preterm birth, while 23 (37.1%) of them were prematurity secondary to iatrogenic interventions. Of the spontaneous preterm birth group, 26 (66.7%) of them underwent spontaneous onset of labour, while 13 (33.3%) were associated with premature pre-labour rupture of membranes (PPROM). The prevalence of spontaneous preterm birth in the study population was 4.2%.nd trimester onwards after 22 weeks.On ROC curve analysis, cervical length showed an area under the curve of 0.61 for the prediction of preterm birth < 37 weeks’ gestation at 18 to 22 weeks . After cPreterm birth is a condition that varies widely between different countries or ethnicities. Reported incidence for preterm births ranges from 5–13%, and this number is believed to be on the rise ,3. The nCurrently, although there have been several published studies looking at the relationship between cervical length and preterm birth, these studies have largely been conducted in Western/Caucasian populations. However, there is still generally a paucity of data in Asian populations with regards to the clinical question of cervical length and its relationship to preterm birth. We know from existing published studies that the natural history of cervical length across pregnancy is that it is normally distributed and remains relatively constant in pregnancy until the third trimester –12. ThisIt is widely known that cervical length can vary amongst different ethnicities. Perhaps the most common example would be that of African Americans having a significant shorter cervical length compared to Caucasians, with this being been reported in various published studies ,14. Stud.Cervical length is known to be a better predictor of preterm birth in women at increased risk, such as those with a history of spontaneous preterm birth than in asymptomatic women at low risk, and this has been illustrated in multiple studies –25. Thesnd and 3rd trimester in the preterm birth group, as consistent with other published studies, with a recommended cut-off value of 2.48cm at 18 to 22 weeks with an area under the curve of 0.61 and 2.49cm at 28 to 32 weeks with an area under the curve of 0.725 according to the ROC curve analysis. Similarly, our study observed that the positive predictive values when using cervical length as a predictor of preterm birth were relatively low at 12.0% and 11.1% at 18 to 22 weeks and 28 to 32 weeks respectively. However, the ROC curve analyses conducted showed cervical length to be a moderate predictor of preterm birth, with a specificity of 90.1% and good negative predictive value of 96.5% at 18 to 22 weeks. This suggests that while cervical length cannot be used to diagnose or confirm at-risk patients, it has a good negative predictive value and relatively good specificity to identify the lower risk patients for preterm birth. By performing cervical length screening at 18 to 22 weeks’ gestation to identify patients at lower risk of preterm birth, we may then be able to identify a separate group requiring closer follow-up or intervention for the rest of the pregnancy. However, the cost-effectiveness of this strategy, which varies with the prevalence of preterm birth in our population, needs to be examined in future studies to determine if this would be economically feasible, balanced against the degree of benefit of potential interventions for preterm birth.Although cervical length has been found to be inversely related to the risk of preterm birth in asymptomatic women , 26–31, Several limitations need to be acknowledged in our study. Firstly, our study was a prospective study and not a randomized control trial, hence individual clinicians were not blinded to the results of the cervical length measurements performed at each time point in the pregnancy. This resulted in some women receiving prophylactic treatment for short cervices in the form of either oral, vaginal or intramuscular progesterone. We acknowledge that this may have affected the results in the number of spontaneous preterm birth, however it would have been unethical to not allow clinicians to offer progesterone treatment on identification of a short cervix, especially when there is already existing evidence to show that this treatment may be effective in preventing spontaneous preterm birth. In any case, the number of women in our study receiving some form of progesterone therapy during their pregnancy for preterm birth prevention was small (n = 30). Secondly, our patient population was not restricted to that of a low-risk population although this still made up the majority of the group– 33 patients had a history of a previous preterm birth and would hence be classified under the high risk group for spontaneous preterm birth. Including these 33 high risk patients in the ROC curve analysis may affect the cut-off value generated in our study, but these numbers were small compared to the significantly larger majority of low-risk patients.nd trimester cervical length, using a cut-off value of 2.48cm (rounded up to 2.5cm), similar to that in other Western studies, can be useful to identify a group of women who are at risk for preterm birth. These women could potentially undergo increased monitoring and interventions to improve perinatal outcomes.In summary, this study shows that in asymptomatic Asian women with a singleton pregnancy, measurement of 2S1 Data(XLTX)Click here for additional data file.


The SP samples were grouped into two annual periods according to decreasing or increasing daylight and ambient temperature. Intra-male variability was higher than inter-male variability for all cytokines. All SP cytokines showed concentration differences between the two periods of the year, showing the highest concentration during the increasing daylength/temperature period, irrespective of the male. Similarly, some cytokines showed differences between daylength/temperature periods when focusing on their total amount in the ejaculate. No strong relationship  was found between annual fluctuations in SP-cytokine levels and semen parameters. In conclusion, the period of the year during which ejaculates were collected helps explain the intra-male variability of SP-cytokine levels in breeding boars.The concentrations of cytokines in seminal plasma (SP) fluctuate over time in healthy males, weakening their practical usefulness as diagnostic tools. This study evaluated the relevance of intra-male variability in SP cytokines and to what extent the period of the year when ejaculate is collected contributes to such variability. Thirteen cytokines  were measured using the Luminex xMAP Seminal plasma (SP), a complex fluid mainly secreted from the epididymis and the accessory sex glands, contains many bioactive components, particularly proteins and peptides ,2,3. TheThe SP proteome includes cytokines, small proteins that have acquired growing reproductive interest in recent years because some of them are directly involved in the regulation of the immune environment of the female genital tract and, thereby, they play a key role in development and embryo implantation ,11. MoreAll procedures involving animals were performed according to international guidelines (Directive 2010/63/EU), and were approved in advance by the Bioethics Committee of Murcia University (research code: 639/2012).6 spermatozoa per mL, more than 70% showing progressive motility, and more than 75% displaying normal morphology. Seminal plasma was harvested immediately after ejaculate collection by double centrifugation ; Rotofix 32A; Hettich Zentrifugen, Tuttlingen, Germany). The second supernatant was microscopically confirmed to be cell-free.Ejaculate donors were healthy, sexually mature Pietrain breeding boars aged between 18 and 22 months at the beginning of the experiment. The boars were housed in individual pens in an AI center belonging to Topics-Norsvin España and located in Calasparra, Murcia, Spain . The environmental conditions of this locality during the experimental timeframe were characterized by daylight varying from 9 h 30 min at the winter solstice to 14 h 50 min at the summer solstice, and the maximum and minimum temperatures averaged 32.5 °C in July (summer) and 3.6 °C in January (winter), respectively. The building where boars were housed was equipped with evaporative coolers to maintain air temperature within comfort zones and an artificial lighting regime of 16 h of light per day. The boars were provided with water ad libitum and fed with commercial feedstuff specially formulated to fulfill the nutritional requirements of AI boars. The boars followed regular semen collections (an ejaculate every 3–4 d) to prepare liquid semen AI doses. Volume (measured directly in the graduated collection vessel), sperm concentration (counted by photometry), sperm morphology, and sperm subjective motility (assessed using a phase-contrast microscope) of each ejaculate were recorded in the AI center. All ejaculates used in the experiment fulfilled standards of quantity and sperm quality thresholds for liquid semen AI doses, namely, more than 100 mL of volume, more than 200 × 10® technology and a porcine cytokine/chemokine pre-coated magnetic bead panel . This panel allows the simultaneous measurement of 13 cytokines, namely, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFNγ), interleukin (IL)-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and tumor necrosis factor-α (TNFα). The protocol recommended by the manufacturer was followed for the analysis. A standard curve with seven points was generated for each cytokine. Serum matrix was added to the standard, control, and blank wells to mimic the SP composition. Two controls were added in their corresponding wells. The microsphere solution was sonicated, vortexed, and added to each well of controls, standards, and SP samples. The plates, in continuous stirring, were kept in the dark at 4 °C for 16–18 h. Thereafter, the contents of each well were removed, and they were washed thrice with the washing solution. Then, detection antibodies were added to the wells and the plates were kept at RT in the dark for 2 h. Thereafter, streptavidin–phycoerythrin was added to the wells and the plates were incubated at RT for 30 min. Then, the wells were washed, and the plates were run in a MAGPIX R . Data were acquired with xPONENT software version 4.2 (Luminexcorp) and analyzed using MILLIPLEX R Analyst Version 5.1 (Merck Millipore). The SP concentration of each cytokine, expressed as pg/mL, was obtained by measuring the average fluorescent intensity in a 5-parameter logistic curve-fitting.The concentrations of cytokines in SP samples were measured using the Luminex xMAPThe experiment evaluated the variability among breeding boars (inter-boar variability) and within the same boar (intra-boar variability) in the concentrations of cytokines measured in SP samples throughout the year. The study also tested the hypothesis that variations in ambient daylight and temperature in the region where the animals were held could explain the intra-boar variability of cytokine levels in SP. Therefore, a total of 180 ejaculates were collected over one year from nine boars, at the rate of 20 ejaculates per boar with at least one ejaculate collected per boar per month. The SP samples were grouped according to the two dominating daylength and accompanying temperature periods of the year, characteristic of the geographical place where the animals were held. Namely, increasing, from January to June, and decreasing, from July to December. A total of 90 SP samples, 10 per boar, were analyzed per each ambient daylength/temperature period.p < 0.05. Data were showed as the means ± standard error of the mean (SEM). The Spearman rank correlation coefficient was used to evaluate possible relationships between SP cytokines  and the ejaculate parameters . Only the relationships that explained a substantial proportion of the variance in the SP cytokines were considered, specifically, those with a correlation coefficient  greater than 0.70, which is indicative of explaining more than 50% of the variance [The IBM SPSS Statistics package  version 24.0 was used for statistical analysis. The Shapiro–Wilks test of standardized residual data was done for checking the assumption of normality, and data not normally distributed were square root transformed. First, a one-way ANOVA was performed to assess the variability between boars (1 to 9). Then, the intraclass correlation coefficient (ICC), using a two-way mixed-effect model , was used for measuring the relevance of intra-boar variance relative to the total . ICC valvariance .p < 0.001) in SP concentrations among boars (p < 0.001), Wilks’ Lambda test revealed a significant effect of the chosen increasing or decreasing period for ejaculate collection  = 13.548, p = 0.001) and also a significant interaction between boar and period  = 2.537, p = 0.001). The nine boars experienced differences between the two daylength/temperature periods for at least one SP cytokine. The boars numbered 2, 5, and 7 showed differences in 8 or more SP cytokines, while those numbered as 1, 3, 6, and 9 showed differences only in two or less SP cytokines. The pattern of variation between the ambient daylength/temperature periods was similar, irrespective of boar or cytokine, and it was characterized by higher (p < 0.05) SP-cytokine concentrations in the increasing than in the decreasing period  during decreasing than increasing daylength/temperature periods in all nine boars  in the SP concentration of all measured cytokines, already known in pigs  and in mIn this context, this study explored whether the period of the year in which ejaculates were collected would be one of the causal factors that explain the substantial variability among male pigs in SP-cytokine levels. The study was conducted in European pigs, a species of mammal that is generally not considered reproductively seasonal, but still shows a tangible seasonal reproductive pattern . This seBy grouping the SP samples into these two annual periods, the present study demonstrated, for the first time in any livestock species, that the period of the year when ejaculates were collected influenced the SP levels of cytokines. The seminal concentration of all cytokines differed between the two periods of the year following the same pattern of variation in all breeding boars. The highest concentrations were measured in the increasing daylength/temperature period. The SP cytokines GM-CSF, IFNγ, IL-1ra, and IL-6 were the most influenced by the period of ejaculate collection because their concentrations differed between periods in most breeding boars. In contrast, IL-1α, IL-1β, IL-2, IL-8, and IL-12 were the least influenced. In addition to concentration, differences in the total amount of some SP cytokines were also observed between the daylength/temperature periods. This clear difference between male pigs in the sensitivity of SP cytokines to environmental conditions was consistent with previous studies in men showing that the range of intra-male variation in SP cytokines over time, differs between cytokines ,37. SimiTo the best of our knowledge, there are no studies that identify which factors mediate the effect of environmental conditions on SP-cytokine levels and, consequently, which could explain why the SP levels of many cytokines differed between the annual periods of ejaculate collection. Similar to the fluctuation we found with cytokines, other authors have also found annual fluctuations for other bioactive SP components in breeding boars used in AI programs, for example, in melatonin , proteinIn summary, the present study demonstrated that the variation in SP levels of cytokines within individual breeding boars is greater than that between breeding boars and that annual fluctuations in daylength and air temperature contribute in explaining this intra-male variability. Further studies are required to investigate the clinical and biological relevance of annual fluctuations for the reproductive health of male pigs and also to elucidate the molecular mechanisms that drive the environmental effects.