Objective: Individuals with autism spectrum disorder often present somatic and/or psychiatric co-morbid disorders. The DSM-5 allows for consideration of additional diagnoses besides ASD and may have impacted the prevalence of co-morbidities as well as being limited in capturing the true differences in prevalence observed between males and females. We describe the prevalence of ASD and frequently observed co-morbidities in children and adolescents (<18 years) in the United States and five European countries.Methods: Two systematic literature reviews were conducted in PubMed and Embase for the period 2014–2019 and focusing on the prevalence of ASD and nine co-morbidities of interest based on their frequency and/or severity: Attention Deficit Hyperactivity Disorder (ADHD), anxiety, depressive disorders, epilepsy, intellectual disability (ID), sleep disorders, sight/hearing impairment/loss, and gastro-intestinal syndromes (GI).Results: Thirteen studies on prevalence of ASD and 33 on prevalence of co-morbidities were included. Prevalence of ASD was 1.70 and 1.85% in US children aged 4 and 8 years respectively, while prevalence in Europe ranged between 0.38 and 1.55%. Additionally, current evidence is supportive of a global increase in ASD prevalence over the past years. Substantial heterogeneity in prevalence of co-morbidities was observed: ADHD (0.00–86.00%), anxiety (0.00–82.20%), depressive disorders (0.00–74.80%), epilepsy (2.80–77.50%), ID (0.00–91.70%), sleep disorders (2.08–72.50%), sight/hearing impairment/loss (0.00–14.90%/0.00–4.90%), and GI syndromes (0.00–67.80%). Studies were heterogeneous in terms of design and method to estimate prevalence. Gender appears to represent a risk factor for co-morbid ADHD  and epilepsy/seizure  while age is also associated with ADHD and anxiety (increasing until adolescence).Conclusion: Our results provide a descriptive review of the prevalence of ASD and its co-morbidities in children and adolescents. These insights can be valuable for clinicians and parents/guardians of autistic children. Prevalence of ASD has increased over time while co-morbidities bring additional heterogeneity to the clinical presentation, which further advocates for personalized approaches to treatment and support. Having a clear understanding of the prevalence of ASD and its co-morbidities is important to raise awareness among stakeholders. Autism Spectrum Disorder  is definFurthermore, individuals with ASD often present co-morbid psychiatric disorders. In a recent review, Hossain et al.  reportedA systematic literature review (SLR) was conducted to evaluate the prevalence of ASD in children and adolescents (from 2 to <18 years old) in EU-4  plus the UK, and the US. This manuscript presents studies reporting the latest prevalence data per country in order to understand the current epidemiological situation of ASD, as well as studies examining the prevalence of ASD overtime. Studies reporting on the prevalence of co-morbidities in ASD were retrieved from a second SLR on the clinical burden of ASD which considered the same population and geographic scope.A SLR was conducted on July 24th 2019 in PubMed and Embase for the period 2014–2019, and completed with a gray literature search in December 2019 focused on governmental institutions and research associations such as ASDEU (Autism Spectrum Disorders in the European Union). The search string included the following search terms: “ASD”; “epidemiology” [and related terms]; as MeSH or Emtree terms; or in the title and abstract of articles. The full search strategy for this SLR is presented in The search focused on retrieving the latest prevalence estimate per country as well as prevalence over time. Study designs were limited to observational studies. Studies with prevalence as primary outcome were included, regardless of the method. Only articles published in English language were considered for review. The second SLR was conducted on July 24th 2019 in PubMed and Embase for the period 2014–2019. This SLR included several search strings related to the clinical burden of ASD to inform several topics such as risk factors, mortality and prevalence of co-morbidities. The search string included the following search terms: “ASD” [and related terms]; “co-morbidity”; “mortality”; and “risk factor”; as MeSH or Emtree terms; or in the title and abstract of articles. The full search strategy for this SLR is presented in This SLR focused on nine co-morbidities of interest: ADHD, anxiety, depressive disorders, epilepsy, Intellectual Disability (ID), sleep disorders, sight/hearing impairment/loss and GI problems . These cTwo researchers independently screened the titles and abstracts of identified studies and then full texts, to assess eligibility. Disagreements were resolved by discussion between the two reviewers and decision was made by a third reviewer if no agreement could be found. Information from included articles was extracted into a predefined data extraction template which included study characteristics, target population details and study outcomes.Due to the variety of different study types included and the challenges in comparing study quality, studies were included without assessment of their methodological quality. As the study design, participant intervention and reported outcomes measured varied markedly, this research focused on describing the studies, their results, their applicability and their limitations and on qualitative synthesis.We retrieved 13 publications reporting on the latest prevalence data for each country (8 studies) at the time the study was conducted and the evolution of ASD prevalence overtime (6 studies). The PRISMA diagram for this review is presented in Prevalence point estimates were found for each country within our scope and ranged from 0.38% in Germany to 1.85% in the US. 95%: 1.61–1.80%) in children aged four year old (95%: 1.80–1.91%) in children aged eight year old (95%: 1.25–1.44%), 1.53% (CI95%: 1.43–1.63%) and 1.70% (CI95%: 1.61–1.80%), for the years 2010, 2012, and 2014 respectively. Prevalence over time in children aged eight year old was also found in Myers et al. (95%: 0.32–0.54%) in 2000 to 1.41% (CI95%: 1.09–1.82%) in 2008, which was in line with the estimates from the CDC who reported estimates of 0.67% (CI95%: 0.45–0.99%) and 1.13% (CI95%: 0.48–2.12%) for the same years  and 0.73% (CI95%: 0.60–0.87%) for the South-East and South-West region respectively, however the study mentions that 5% of the parents of potential ASD cases refused to participate which slightly impacts the previous estimates. The South-West cohort also reported a higher male/female (M/F) ratio compared to South-East region (5.4 vs. 4.0). In Italy, Narzisi et al. (95%: 0.62–0.97%) or 1 in 126 children. Prevalence for the second phase of the study was 0.2% (CI95%: 0.06–0.33%) and 0.3% (CI95%: 0.12–0.45%) when adjusting for non-response. Additionally, males had a five time higher prevalence of ASD compared to females. Prevalence over time for France was reported in van Bakel et al. (95%: 0.22–0.32%) in 1997 to 0.41% in 2003 (CI95%: 0.36–0.48%).Prevalence estimates for France and Italy were found in Delobel-Ayoub et al.  and Narzi et al.  reportedl et al.  whose stl et al.  and pres95%: 0.89–2.20%) in children aged 4–5 years and 1.00% (CI95%: 0.48–1.51%) in children aged 10–11 years, while direct prevalence was 1.06% (CI95%: 0.66–1.46%) and 0.78% (CI95%: 0.44–1.12%) respectively. Additionally, the authors mentioned that between 1.84 and 2.59% of the children exhibited subclinical diagnoses of ASD. Finally, in both cohorts the prevalence was four times higher in males compared to females.A similar study was conducted by Morales-Hidalgo et al.  in SpainPrevalence rates for the UK were obtained from the Survey of the mental health of children and young people 2017 available on National Health Service digital which aimed at assessing the prevalence of mental health conditions (including ASD) in children and young people aged 2 to 19 years . Case deBachmann and colleagues estimated the prevalence of ASD in Germany by conducting a claims database analysis using the Allgemeine Ortskrankenkassen database for the period 2006–2012 . Additionally, prevalence of co-morbidities was reported as a primary outcome in 19 (58%) studies and as a secondary outcome in the other 14 publications. Studies were heterogeneous in terms of study design, with a mix of cohort studies (n = 23), cross sectional studies (n = 4) and case-control studies (n = 6). Studies were also heterogeneous regarding the method considered to estimate the prevalence of co-morbidities: tools and scales  were used in 18 studies while eight considered database or registries and seven relied on clinical expertise or caregivers report only. All studies considered cohorts with more than 30 autistic individuals, the lowest sample size being retrieved in Mosner et al.  were conducted in the US while seven were conducted in a European country , anxiety (n = 13), depressive disorders (n = 12), epilepsy/seizure (n = 12), GI syndromes (n = 7), hearing impairment (n = 3), ID (n = 13), sleep disorder (n = 7), and vision impairment (n = 3). Study characteristics are presented in The number of studies per co-morbidity was as follows: ADHD (95%: 50.00–78.70%) in male children aged 4–8 y and 38.60% (CI95%: 23.90–53.40%) in female children. The authors also calculated a statistically significant odds ratio (OR) of 2.9 (CI95%: 1.2–6.9), confirming the disorder was more prevalent in male individuals. In a similar manner, Stacy et al. . Supekar et al. (95%: 3.40–6.73).Prevalence for ADHD in ASD children ranged from 0.00% in children aged 3–4y  to 86.00y et al.  also repy et al. . Prevaler et al.  assessedr et al.  who founr et al.  found AD95%: 0.90–6.00)]. However, an Intellectual Quotient (IQ) above 70 was identified as a risk factor for anxiety in ASD with a statistically significant OR of 2.90 (CI95%: 1.00–8.10). Salazar et al. (95%: 1.57–3.39)]. In their paper, Salazar et al. ]. Salazar et al. (95%: 0.8–22.4). Mild and moderate/severe depression was estimated at 7.2 and 3.6% respectively in females and 4.5 and 7.8% respectively in males in Stacy et al. . Superkar et al. ((CAQ) = 2.22 (CI95%: 1.77–2.79); RR(BDQ) = 2.18 (CI95%: 1.57–3.02)]. In the CAQ cohort, the authors also established that female sex was associated with 40% increased risk for epilepsy compared to males.Prevalence for epilepsy/seizure in ASD children ranged from 2.80% in children aged 3–11y  to 77.50r et al.  reportedr et al.  reportedr et al.  specific1/2-5 , and authors reported an overall prevalence of 37.40%  vs. 14.80% in typically developed children (p = 0.0001). “Constipated” and “not eat” were most prevalent subtypes observed in the ASD group with 15.70 and 27.00% respectively, and both rates were significantly higher compared to the typically developed group. Similarly, the research by Kang et al. (n = 37), constipation , bloating  and gastro-enteric reflux/vomiting . The authors also demonstrated that the presence of any GI disorder was associated with significantly higher rates of sleep disorders (p = 0.001) and food intolerance (p < 0.001).Prevalence of GI varied from 0.0% for inflammatory bowel disorders in female with ASD aged 0–18y  to 67.80g et al.  specificPrevalence of hearing impairment ranged from ~0%  in Polyak et al.  a study 95%: 13.40–18.50). Finally, Goldin  for sleep abnormalities in Soke et al. , insomnia (9.52%) and sleep disorder breathing (9.26%). The authors calculated a RR of receiving a sleep disorder diagnosis of 1.97 in children with ASD (CI95%: 1.91–2.02). Aldinger et al.  in children aged between 0 to 15 years old  for the outcome “blindness/partial sight loss”, and 3.80% in the 16 to 24 year old group .Prevalence for vision impairment ranged from ~0%  in Polyak et al.  study exThe objective of this study was to describe the clinical burden associated with ASD in children and adolescents based on recent prevalence data and trends over time, as well as understanding the prevalence of nine co-morbidities associated with ASD and potential differences by age and gender. Our research focused on children and adolescents with ASD (age <18y) and considered studies conducted in the US, France, Germany, Italy, Spain, and UK.Our first review found the latest prevalence data for each country, with 1.70 and 1.85% in the US in children aged 4y and 8y respectively, while prevalence of ASD in Europe ranged from 0.38% in Germany (0–24 years) to 1.55% (3–5 y) in Spain. Of note, Fuentes et al.  recentlyIt is possible that prevalence estimates in ASD are underestimated for multiple reasons, the first one lying in complexity of the disorder itself. ASD is a complex disorder associated with a wide array of phenotypes and numerous levels of severity. International guidelines on ASD suggest that each case should be considered individually and diagnosis made by a multidisciplinary team. However, availability of multidisciplinary teams and expert clinicians is rare, leading to extended waiting list and children escaping diagnosis. Underestimation of the prevalence of ASD could also be attributed to the multiplicity of diagnostic tools available. Such tools are commonly used to support diagnosis and complete clinical assessment, but no gold standard officially exists. Although, the combination of the ADOS and ADI-R is commonly reported, both tools require time and skills to administer which can lead to inaccurate evaluations. On the other hand, it is also possible for prevalence to be overestimated in case of misdiagnosis with other disorders and in situations where ASD is associated with services and care that would not be otherwise granted for other disorders. Evidence as to whether the switch from the DSM-IV to the DSM-5 algorithm of ASD has impacted on its prevalence is conflicting , 57.As for the prevalence of co-morbidities in ASD, our SLR included a total of 33 studies and highlighted wide prevalence ranges for each co-morbidity: 0.0–86% for ADHD (17/33 studies), 0.0–82.2% for anxiety (13/33 studies), 0.0–38.6% for depressive disorders (12/33 studies), 2.8–43.75% for epilepsy/seizures (12/33 studies), 0.0–49.0% for GI syndromes (7/33 studies), 0.0–87.8% for hearing impairment (3/33 studies), 0.0–91.7% for ID, 6.4–72.5% for sleep disorders (5/33 studies) and 0.0–15.3% for visual impairment (3/33 studies). Lower estimates of 0.0% for ADHD, anxiety, depressive disorders and ID originate from Houghton et al.  and corrThe literature suggests that the prevalence of some co-morbidities can be different with age and gender. Prevalence of ADHD and anxiety appear to increase until adolescence, with a decline observed when reaching adulthood , 42, 58.Similar to our findings for the prevalence of ASD, several factors should be considered when interpreting the heterogeneity in co-morbidity prevalence. First, the definition for a given co-morbidity differed across studies. This was observed particularly for anxiety disorders where claim database studies such as Houghton et al.  considerOur study presents several limitations. First, a broad search term for comorbidity was considered and not terms for specific comorbidities. This approach allowed us to identify the most frequently observed comorbidities in ASD whose relevance was further confirmed by clinical experts. The second limitation lies in the decision to include studies reporting prevalence as a secondary outcomes. While these studies did provide relevant information, the rates extracted from these studies might include a selection bias by selecting specific subpopulations of children with ASD. Third, although the purpose of this study was to provide a descriptive review of the prevalence of ASD and its co-morbidities, additional research could involve analysis focusing on a single co-morbidity to estimate an overall prevalence rate while accounting for the heterogeneity across studies highlighted in our research. Fourth, our review focused on the most common co-morbidities reported in the literature but other co-morbidities can be observed in association with ASD such as dementia or allergic/autoimmune diseases. Finally, as our search strategy focused on children and adolescents in the US and five European countries, this study did not capture the evidence generated in other geographic regions as well as in adults with ASD.Our research provides a descriptive review of the prevalence of ASD and its co-morbidities which can be valuable for clinicians as well as parents/guardians of children with ASD. Despite substantial heterogeneity in estimates, our results indicate that co-morbidities are highly prevalent in ASD, and should be kept under consideration when diagnosing and managing individuals with ASD. In addition, co-morbidities should be re-assessed regularly as evidence suggest that prevalence evolves over time, especially during transition ages such as entry to school or adolescence. Also, co-morbidities bring additional heterogeneity to the presentation of ASD, further advocating for personalized and tailored approaches to treatment and support. Finally, co-morbidities in ASD negatively impact the quality of life and life expectancy of individuals as well as representing a substantial economic burden. Results from Hirvikoski et al.  reportedOur findings have clear implications for the diagnostic process of young people referred for suspicion of ASD and for service organization. The relative high probabilities of psychiatric and somatic co-occurring conditions should be incorporated into clinical guidelines for assessing ASD. The assessment should not only be focused on identifying the core symptoms of ASD but also on the wider range of other psychiatric and somatic problems. To this end, systematic information from parents and teachers should be collected through validated broad band questionnaires that tap internalizing and externalizing behavior problems, such as the ASEBA Questionnaires , the ConThese findings are supportive of a global increase in ASD prevalence independent from regions and healthcare systems and call for stronger awareness within populations and healthcare policies. Our review also provides prevalence estimates for nine co-morbidities frequently associated with ASD and highlighted the importance of age and gender in prevalence of both ASD and its co-morbidities. Across Europe, there is still a need for studies applying a similar methodology when estimating prevalence in order to allow comparison across countries. This review highlights the substantial clinical burden associated with ASD, with co-morbidities further complicating evaluation, management and prognosis of ASD. Finally reliable estimates for prevalence of ASD and associated co-morbidities would support economic analysis and further assessment of burden in ASD.The original contributions presented in the study are included in the article/All authors were involved in the conception and design of the study. Data collection and analysis was conducted by CB and RC. Writing of the manuscript was done by CB. All authors reviewed and approved the manuscript.This study was funded by Servier.CB and RC are employees of Syneos Health. Syneos Health was contracted by Institut de Recherche Servier to conduct this study. JB received honoraria from Institut de Recherche Servier for clinical expertise. FP-B and RS are employees of Servier.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.


A nested retrospective cohort study including 112 mothers was also done to determine longitudinal effects of SCM on four pro-inflammatory cytokines; IL6, IL8, IP10 and RANTES. The cumulative incidence for any SCM (Na+/K+ ratio > 0.6) and severe SCM (Na+/K+ ratio > 1) were 58.6% (95%CI: 52.7 – 64.5) and 27.8% (95%CI: 22.5 – 33.1), respectively. In majority of affected mothers (51.4%) severe SCM was recurrent. Both breasts were involved in 11.1%, 33.3% and 70% of the mothers with a single episode, 2 and 3 episodes respectively. In affected breasts, an episode of severe SCM resulted in steep upregulation of the four cytokines considered  compared to: before and after the episode; contralateral unaffected breasts; and SCM negative control mothers. Recurrent severe SCM significantly increased the odds of shedding cell-free HIV-1 in breast milk  whereas single episode of severe SCM did not . A Na+/K+ ratio > 1 indicative of severe SCM is an excellent indicator of breast inflammation characterized by a steep, localized and temporal upregulation of several pro-inflammatory cytokines that favor HIV-1 shedding in mature breast milk and may facilitate postnatal HIV-1 transmission through breastfeeding.Subclinical mastitis (SCM) is an important risk factor of postnatal HIV-1 transmission that is still poorly understood. A longitudinal sub-study of the ANRS12174 trial including 270 breastfeeding mothers in Lusaka, Zambia measured sodium (Na The concept of subclinical mastitis (SCM) in humans is still poorly understood , 2 despiSCM may be caused by several factors. These include milk stasis arising from inefficient milk removal , 18. Hum+/K+) ratio. Na+/K+ ratio reflects increased permeability of the mucosal epithelial barrier allowing influx of interstitial Na+ into the mammary gland and subsequently into breast milk  and high (above 1) membrane permeability. Other markers previously used to determine SCM include raised sodium concentration  that followed 1273 HIV-1 exposed infants for up to 50 weeks in Zambia, Uganda, South Africa and Burkina Faso from November, 2009 to May, 2012. Trial protocol  and mainTo collect breast milk, mothers were asked to express breast milk manually, from each breast, in separate sterile 50 ml polypropylene conical centrifuge tubes after breastfeeding. Collected breast milk samples were immediately transferred to the trial’s research laboratory within the University Teaching Hospital. Up on reception, breast milk samples were centrifuged at 1,200 g for 15 minutes at 4°C to separate the acellular aqueous fraction (lipid layer and lactoserum) and breast milk cells (BMCs). Both the acellular fraction and the dry BMC pellets were aliquoted and stored at -80°C.+ and K+, the frozen acellular breast milk fraction was allowed to equilibrate to room temperature and centrifuged at 1,200 g for 15 minutes to separate lactoserum and the lipid layer. Clear lactoserum was diluted in 1:101 ratio with deionized water before testing. Na+ and K+ were measured in diluted lactoserum using a PFP 7 flame photometer  according to manufacturer’s recommendations. A standard curve was built at the beginning of each batch of measurements using commercially available standard solutions . Na+/K+ ratio was then calculated as a measure of SCM.To measure NaBoth HIV-1 RNA and DNA were measured in breast milk from both breasts as previously described .Four pro-inflammatory cytokines, namely IL6, IL8, IP10 and RANTES were measured using colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s recommendations .+, K+ and cytokine assay results were entered in an MS Excel worksheet and individually crosschecked for errors. The resulting clean worksheet was imported into SPSS version 25  for analysis. Concurrent blood test results and sociodemographic data were extracted from the trial database and merged with the dataset of breast milk results using participants’ individual identifiers and study visit as matching variables. The resulting database was checked for errors and prepared for analysis.Na+/K+ ratio less than or equal to 0.6 (Na+/K+ ≤ 0.6) were classified as negative while those with Na+/K+ ratio greater than 0.6 to 1 (0.6 < Na+/K+ ≤ 1) were classified as mild SCM and those with Na+/K+ ratio greater than 1 (Na+/K+ > 1) classified as severe SCM, as previously described ; mild SCM at least at one visit but no severe SCM at any visit (mild SCM); and severe SCM at least at one visit (severe SCM). From each group we randomly selected participants to include in the retrospective cohort study. We then determined normal ranges for cytokine concentration in mothers whose samples tested negative for SCM throughout the follow up period as 2.5 and 97.5 percentiles after removal of outliers (mean ± 3SD). We determined the proportion of samples with high cytokines in the different groups of SCM. Generalized linear mixed models were used directly or after log10 transformation to compare continuous variables between groups depending on whether the data was normally distributed or not. Fixed degrees of freedom were used with robust covariance and mean estimate tests were adjusted using sequential Bonferroni procedure with a significance level of 0.05. In a sensitivity analysis we compared the results within-breast and between breasts.Based on the NaMultinomial logistic regression model was used to determine the effect of recurrent severe SCM on HIV-1 shedding in breast milk. All variables associated with the dependent model (p < 0.200) were included in the model (method: entre). CFV, CAV and SCM status were determined based on both breasts as previously described . We usedAll participants, at inclusion, had provided informed consent for sample storage and use in future research. Authority to conduct this sub-study was obtained from the National Health Research authority (NHRA) in Zambia and the scientific committee of the ANRS12174 trial. Ethical approval was obtained from ERES CONVERGE Institution Review Board in Zambia , and the Regional Committees for Medical Research Ethics of Norway (REK Nord no 2008 02523-2).270 mothers met inclusion criteria and were all included in this sub-study . They practiced exclusive breastfeeding (100%) up to six months. Breast health problems were rare with only 7 (2.6%) mothers ever reporting. Only one of these (week 14 case) required antibiotic treatment for 5 days. The proportion of mothers with unsuppressed viral load (≥1000 copies/ml) increased over the duration of the study, from 50.2% at day 7 to 87.0% at week 38 postpartum. CD4 cell count did not change significantly over time.+ and K+ analysis (+ in lactoserum significantly reduced between week 14 and week 26 (p < 0.001) but remained stable between week 26 and week 38 while that of Na+ reduced significantly between week 14 and week 26 (p = 0.004) and tended to increase between week 26 and week 38 (p = 0.072). On the other hand, lactoserum Na+/K+ ratio remained stable throughout the follow up period.Of the 270 mothers included in the study, 1610 breast milk samples were available for Na+/K+ ratio > 0.6) at week 14, week 26 and week 38 was 35.6%, 36.8% and 41.9%, respectively, while that of severe SCM (Na+/K+ ratio > 1) was 16.5%, 15.2% and 17.4%, respectively. Bilateral severe SCM accounted for 4.5%, 7.3% and 21.3% of those with severe SCM at week 14, week 26 and week 38, respectively.The prevalence of any SCM  while that of severe SCM was 27.8% (95%CI: 22.5 – 33.1). The incidence of severe SCM was significantly higher (p = 0.002) in those who had mild SCM at week 14 (25.5%) compared to those who had no SCM at the same time point (9.4%). Of the 74 mothers that had severe SCM, 36 (48.6%) had a single episode, 18 (24.3%) had two episodes while the remaining 20 (27.0%) had three episodes. Both breasts were affected over time, but not necessarily at the same time, in 11.1%, 33.3% and 70.0% of those who had 1, 2 and 3 episodes of severe SCM.To study the effects of SCM on breast milk cytokines over time, 672 breast milk samples were retrieved, accounting for 38 SCM negative mothers, 37 mild SCM mothers and 37 severe SCM mothers  was observed in 3.3%, 7.0% and 62.3% of samples with no, mild and severe SCM, respectively. During an episode of either mild or severe SCM, IL8 concentration increased about 2- and 9-fold, respectively, compared to SCM negative mothers. After any type of SCM, IL8 concentration returned to pre SCM levels.The normal range for IP10 concentration in lactoserum was from 6 to 1010 pg/ml. High IP10 concentration (> 1010 pg/ml) was observed in 3.8%, 12.2% and 31.2% of samples with no, mild and severe SCM, respectively. There was a tendency for IP10 concentration to increase during mild SCM but the increase was not statistically significant. However, during an episode of severe SCM, the IP10 concentration increased about 4-fold compared to SCM negative mothers. After any type of SCM, the concentration of IP10 retuned to pre SCM levels.RANTES was more likely to be detected in samples with severe SCM (70.1%) compared to those with mild SCM (44.3%) and no SCM (38.0%). The normal range for RANTES concentration in lactoserum was from 0 to 39 pg/ml. High RANTES concentrations (> 39 pg/ml) was observed in 4.4%, 11.3% and 39.0% of samples with no, mild and severe SCM, respectively. RANTES concentration did not change significantly during mild SCM. However, during an episode of severe SCM, RANTES concentration increased about 4-fold compared to SCM negative mothers. After severe SCM, the concentration of RANTES retuned to pre SCM levels.IL6 was more likely to be detected in samples with severe SCM (57.1%) compared to those with mild SCM (20.9%) and no SCM (22.7%). The normal range for IL6 concentration in lactoserum was from 0 to 74 pg/ml. High IL6 concentration (> 74 pg/ml) was observed in 3.8%, 7.0% and 35.1% of samples with no, mild and severe SCM, respectively. IL6 concentration did not change significantly during mild SCM. However, during an episode of severe SCM, the concentration of IL6 increased about 5-fold compared to SCM negative mothers. After severe SCM, IL6 concentration retuned to pre SCM levels.For the four cytokines, their concentration among SCM negative mothers (controls) was similar to that of unaffected contralateral breasts in unilateral SCM. In affected breasts, cytokine concentrations before either mild or severe SCM was similar to that of SCM negative mothers too (Severe SCM increased CFV HIV-1 shedding in breast milk 7 folds from a median of 41 copies/mL (IQR: 0 – 179) mild SCM to a median of 309 copies/mL (IQR: 18 – 1940) in severe SCM. The median CFV-1 HIV shedding in SCM negative mothers was 0 copies/mL (IQR: 0 – 71). Similar trends were observed for CAV HIV-1 shedding and total breast milk cell count . Second, a NaThese results compare well with earlier similar studies. For instance, we report a 16.5% point prevalence of severe SCM at week 14 which is in line with similar studies in the same location  and in t+/K+ ratio and the severe SCM observed in the first week (day 3 to day 7) postpartum followed by a gradual reduction up to week 16  triggers production of pro-inflammatory cytokines , and the Regional Committees for Medical Research Ethics of Norway (REK Nord no 2008 02523-2). The patients/participants provided their written informed consent to participate in this study.All authors contributed substantially to the design, conduct, data analysis and results interpretation. DR drafted the paper. All authors reviewed and approved the final version of the manuscript.This work was supported by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS#12274), European & Developing Countries Clinical Trials Partnership (#CT.2006.33020.004) and the Research Council of Norway (GlobVac grant # 183600). DR is the beneficiary of doctoral scholarship from the Norwegian government (Quota scheme).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.


Parabens (PBs) and triclosan (TCS) are generally used as antimicrobials mostly in personal care products. Their wide prevalence in daily products raised an acute need for the biomonitoring of these contaminants and the investigation of possible health impacts.In this study we aimed to quantitatively determine PBs and TCS levels in urine and amniotic fluid samples using a liquid chromatography – mass spectrometry system (LC–MS). Ninety nine (99) pregnant women took part in this research. The samples were collected during the amniocentesis in the early second trimester of their pregnancy. Women of all ages, education, household income and profession were selected. The exposure and the burden of pregnant women and their infants were also evaluated.The most prevalent compound in urine, among the analyzed, was TCS with 74.7 % positive samples while in amniotic fluid methyl paraben (MePB) with 21.2 % positive samples. MePB was detected at higher concentrations in urine (mean: 378.5 ng/mL) followed by TCS (mean: 55.3 ng/mL), ethyl paraben (EtPB) (mean: 23.2 ng/mL) and butyl paraben (BuPB) (mean: 2.3 ng/mL) while benzyl paraben (BePB) was not detected in any urine sample. Concentrations in amniotic fluid samples were much lower. In particular, the mean concentrations were 6.6 ng/mL for MePB, 9.2 ng/mL for EtPB, 0.4 ng/mL for BuPB, 0.6 ng/mL for BePB and 1.8 ng/mL for TCS. The detected levels of all analytes in urine were correlated with those in amniotic fluid but no statistically significant results arose (p >n0.05). Negative associations were observed between amniotic fluid levels of MePB and maternal age (p = 0.05) while both urinary and amniotic levels of TCS were correlated with maternal BMI (p = 0.04). Somatometric characteristics of the infants showed no statistical significant associations with the detected levels of PBs and TCS.This study indicated a strong/possible association between exposure of pregnant women to TCS and higher/lower maternal body weight gain during pregnancy. The same trend was observed between amniotic fluid MePB levels and maternal age. However, no statistically significant associations were observed between neonatal somatometric characteristics or health status and PBs and TCS levels. The main route of human exposure is personal care products (PCPs), such as deodorants, shower gels, shampoos, body creams and lotions . PBs areEuropean Union has notified specific guidelines regarding the presence of PBs and TCS in consumer products. In particular, the maximum permissible concentration of TCS in cosmetics is 0.3 % , while tDespite the limitations, the widespread use of PBs and TCS has resulted in the long-termhuman exposure which has raised concerns about the possible health impacts in the human body. PBs and TCS have been characterized as endocrine disruptors (EDs) as several studies have indicated that they affect the proper action of hormones and therefore the proper function of the endocrine system, leading to several health problems. Until now, studies indicate that exposure to PBs is correlated with male (even to the descendants) and female reproductive problems, breast cancer, increased chance of developing obesity, potential genotoxicity, increased sensitivity to allergens and neurotoxic effects . SimilarThe burden of general population usually is estimated by hair analysis . The las22.1Escherichia coli from Roche Diagnostics . Ultrapure water was produced by Merck’s Direct-Q 3UVwater purification system .MePB, EtPB, BuPB, BePB, ethylacetate (High Pressure Liquid Chromatography (HPLC) grade), hydrochloric acid (≥37 %), ammonium acetate  and solid phase extraction (SPE) cartridges C18 (100 mg) were purchased by Sigma – Aldrich . TCS (100 %) was obtained by Honeywell–Fluka , methanol (LC-MSgrade) and acetonitrile (Liquid Chromatography - Mass Spectrometry (LC–MS) grade) by Honeywell–RiedeldeHaën  and phenobarbital-d5 used for internal standard by Isotec Inc. . Phosphate buffer saline was purchased from FlukaBiochemika  and β-glucuronidase enzyme from 2.2Sampling took place at the “Mitera” Maternity Hospital in Heraklion, Crete. Maternal urine and amniotic fluid samples were collected from ninety nine (99) pregnant women during the amniocentesis in early second trimester of their pregnancy. The amniocentesis procedure was scheduled by the doctor due to one (or more) of the following reasons; the maternal age, the maternal medical/obstetric history and the health condition of both mother and fetus. In specific, in Greece, it is almost obligatory to conduct amniocentesis on pregnant women over the age of 35. Also, in case that the mother, or fetus, had presented any health problem  either in previous or the current pregnancy, amniocentesis was necessary to ensure the health of both mother and fetus.Before sampling procedure, all participating women were informed about the research and asked to sign participation consent. Samples were collected in screw glass tubes and stored at −20 °C until analysis. This study was approved by the ethics committee of the University of Crete (43/22.11.2018).2.3During the sampling, the participants were asked to complete questionnaires about maternal demographic  and somatometric characteristics ) before and during pregnancy, nutritional  and lifestyle habits , medical and obstetrical history and the frequency of personal care products. Six to twelve months after childbirth, additional information was collected regarding infants’ development ) and health condition as well as birth type .2.41 mL of each amniotic fluid sample was placed in glass screw tubes, since the samples had been centrifuged. Subsequently, 10 μl of β-glucuronidase enzyme was added to each sample to hydrolyze the conjugated (glucuronidized) compounds and 250 μl of 0.1 M phosphate buffer pH 6.5 to create the appropriate environment for the enzyme to act. The samples were incubated in a water bath at 37 °C for 12 h. After incubation, 100 μl of 2 M hydrochloric acid were added, followed by extraction with 2 mL of ethyl acetate. The samples were shaken for 20 min and then the extract, that is the organic phase, was transferred to glass evaporation tubes. This process was repeated twice, followed by evaporation to dryness under nitrogen flow, reconstitution of the evaporation residue in 100 μl of methanol and transfer to 2 mL vials (inside inserts) for LC–MS analysis.2.5Similar procedure was followed for urine samples. 1 mL of each sample was placed (after centrifugation) in glass screw tubes and 10 μl of β-glucuronidase enzyme and 250 μl of 0.1 M phosphate buffer pH 6.5 were added. After overnight incubation at 37 °C, 100 μl of 2 M hydrochloric acid were added, followed by extraction with 2 mL of ethyl acetate three times. At this stage of the experimental procedure, an extra clean step was added using SPE cartridges. In particular, the extracts were evaporated to dryness and then reconstituted in 1 mL of buffer solution pH = 2. The cartridges were cleaned and activated using 2 mL of acetonitrile, 2 mL of acetorintrile:water 1:1 and 2 mL of water. Then, the samples were loaded and washed with 2 mL of water. After cartridges had been dried up, the analytes were extracted using 2 mL of acetorinitrile : ethyl acetate 1:1. Finally, the extracts were collected, evaporated to dryness under nitrogen flow, reconstituted in 100 μl of methanol and analyzed with LC–MS.2.6m/z of each analyte are given.A Shimadzu  liquid chromatography – mass spectrometry system (LC–MS 2010 EV model) equipped with an autosampler was used. Separation of the analytes was achieved using a Supelco Discovery column C18 (25 cm)  at 30 °C stable oven temperature. A gradient of 5 mM ammonium acetate (solvent A) and acetonitrile (solvent B) were chosen for the analysis of PBs and TCS. The flow of chromatography was 0.6 mL/min and the total duration of the run for each sample was 25 min. Atmospheric pressure chemical ionization (APCI) combined with a quadrupole mass filter in a selected ion monitoring (SIM) negative mode were used for monitoring the aforementioned substances. In 2.7Measures of central tendency  and measures of dispersion  were used according to the data normality for the continuous variables. Counts, percentages and proportions were applied for discrete data. The correlation of discrete variables was tested using Pearson's χ2 test, changes to paired measurements of discrete data were tested using McNemar for 2 × 2 tables or Mc Nemar-Bowker test for nxn tables. Pearson’s rho coefficient was used to estimate correlation of continuous and ordinal variables. Boxplots were used for representing ordinal and discrete data and scatterplots were used for examining correlation between continuous variables. IBM SPSS Statistics 24.0 was used for data analysis and a level of acceptance was set at p = 0.05 level.33.12) was >0.99 for all compounds in both matrices. Linearity obtained from spiked samples at concentrations 0, 5, 10, 25, 50 and 100 ng/mL was similar with that of standard solutions (R2>0.99).Analytical parameters of the applied methods were checked and evaluated for the target compounds. Standard solutions were prepared at concentrations 0, 25, 50, 100, 250 and 500 ng/mL and the obtained calibration curves showed that linearity (RRecovery (%), accuracy (%) and inter day precision (%) were also calculated for both urine  and amniotic fluid  and found to be within the accepted values.Limits of detection (LOD) were calculated using the signal to noise ratio (S/N); S/N > 3 for LOD. The calculated values were 0.07 ng/mL for MePB, 0.31 ng/mL for EtPB, 0.12 ng/mL for BuPB, 0.17 ng/mL for BePB and 0.73 ng/mL for TCS.3.2A total of 99 women participated in the study and the mean age was 35.2 ± 5.8 years ranging from 18.0 to 44.0 years. Reported BMI before pregnancy was 24.4 ± 5.6 Kg/m2 (range 16.0–45.4) and the corresponding at the time of amniocentesis was 25.9 ± 5.3 Kg/m2 (range 17.2–46.3) showing a statistically significant increase (p < 0.001) (mean DBMI: −1.5 ± 1.4).Medical history of the participating women showed that thyroid problems were the most common (32.3 %), followed of allergic disorders/diseases (29.3 %), gynecological (24.2 %) and respiratory problems (7.1 %). Eighty-five mothers accepted to give additional information about infants’ development (6months – one year after birth) and possible health issues. Female neonates were 44 (51.7 %) and only 7 female neonates were born pre-term (<37 gestation weeks). Frequency of pre-term gestation did not differ between male and female neonates (p = 0.663). Female neonates presented a mean weight at birth 3048 ± 559 g which do not differ significantly from male neonates 2981 ± 670 g (p = 0.621). Similar findings were found for neonates’ length vs neonates’ gender (p = 0.790) with a mean length of 50.2 ± 2.9 cm and for neonates’ head circumference vs gender (p = 0.949) with a mean HC of 34.7 ± 1.5 cm. A total of 12 newborns 14.1 % suffered from a disease/disorder. The most common reported health issues were allergic disorders (10.6 %) followed by respiratory problems (2.4 %) and genital abnormalities (1.2 %) .Table 2S3.3Descriptive statistics of measured PBs  and triclosan (TCS) are given in Urinary levels ranged from 5.3 to 3501.3 ng/mL (mean: 378.5 ng/mL) for MePB, from 0.8 to 81.7 ng/mL (mean: 23.2 ng/mL) for EtPB, from 1.2 to 7.6 (mean: 2.3 ng/mL) for BuPB and from 0.7 to 615.5. ng/mL (mean: 55.3 ng/mL) for TCS. The big range of MePB and TCS is attributed to the fact that these two substances are the most common used antimicrobials in personal care products. Similar ranges have been observed in previous studies and other biological matrices, too . In amniDue to the small prevalence of positive samples for PBs and TCS, correlation between them was examined only for MePB and the estimated rs was 0.711 (p < 0.001) .Fig. 1Co3.4Detected levels of PBs and TCS in urine and amniotic fluid were correlated with the somatometric characteristics of women. Correlation results are shown in 4In the current study we aimed to determine PBs and TCS levels in urine and amniotic fluid samples from pregnant women. Maternal and fetal exposure as evaluated from biomonitoring data in urine and amniotic fluid, respectively, were associated with maternal demographic and somatometric characteristics as well as daily habits and medical history. Infants’ somatometric characteristics and health condition were also correlated with the found levels.The target compounds were mostly present in urine samples with % positivity ranging from 8.1 to 74.7 % for urine samples and from 1.0 to 21.2 % for amniotic fluid samples. MePB had the higher detection frequency (21.2 %) and mean concentration level (6.6 ng/mL) among the detected compounds in amniotic fluid. MePB presented also high detection frequency (64.6 %) in urine samples but TCS frequency was even higher (74.7 %). The higher concentration levels in urine were observed for MePB with mean value 378.5 ng/mL followed by TCS with mean concentration 55.3 ng/mL. BePB was not detected in urine while in amniotic fluid there was only one positive sample (0.6 ng/mL). Additionally, EtPB and BuPB presented very low detection rates in amniotic fluid . This high difference of detection rates and concentration levels supports existing views that PBs and TCS has lower biotransformation/metabolism in amniotic fluid than in urine which leads to insignificant accumulation potential ,28.Until now there are only three published studies on biomonitoring of PBs and TCS in amniotic fluid. The detected concentrations in the present study are similar with the levels reported in the literature . On the Philippat and co-authors correlated the levels of PBs and TCS in maternal urine with those in amniotic fluids, and found that urine and amniotic fluid followed the same trend. For instance, women with high urinary MePB and TCS concentrations presented also high levels in amniotic fluid . AlthougAccording to the literature, urinary PBs levels generally show the following trend MePB > EtPB > BuPB > BePB. The same distribution/order is observed in our study, too, and the respective mean concentrations are 378.5 ng/mL for MePB, 23.2 ng/mL for EtPB, and 2.3 ng/mL for BuPB, while BePB was not detected in any sample. Shekhar and co-authors reported a similar trend for amniotic fluids, specifically MePB > TCS > EtPB > BuPB, which is also observed in our results . This faCorrelation of maternal demographic characteristics with the detected levels showed no statistically significant results except for maternal age which was negatively correlated with MePB levels in amniotic fluid. There is no such association reported in literature however Ashrap and co-authors found that urinary PBs levels were higher in elderly pregnant women . The samIn the current study, urinary TCS levels were higher when small BMI alteration during pregnancy was observed. Li and co-authors also indicated that BMI is possibly associated with TCS . AdditioInfants’ somatometric characteristics have been previously correlated with both TCS and PBs. Previous studies have indicated that birth length, weight and head circumference tend to be smaller when TCS levels are high ,41,42 whOne of the most debatable issues of PBs and TCS exposure is the risk that may arise for both the mother and the infant. There are animal studies that claim preterm birth or miscarriage after animals’ exposure to PBs and/or TCS ,47. AlthThere are several studies that correlate children exposure to PBs and TCS with allergies and asthma incidents. However, when it comes to maternal exposure and infants’ burden only PBs are correlated with increased asthma rates . The inc5To the best of our knowledge, the current study is one of the very few, that aimed to correlate maternal and fetal burden to PBs and TCS. Also, this is of the very few studies that tried to assess the exposure of Greek women to these substances. The importance of this study lies in the fact that PBs and TCS are endocrine disruptors strongly correlated with fetus developmental problems and subsequent postnatal problems in growth. The study population consisted of women residents of Crete; a big Greek island which lands away from the mainland. The participants originated from all social layers with different incomes, education and professions. This way, we tried to simulate the exposure of Greek women using a sample size of 99 women. Although urine and amniotic fluid levels of PBs and TCS were not associated, significant associations arose between the detected levels and both maternal age and BMI. The correlation between urine – amniotic fluid levels and neonatal somatometric characteristics as well as neonatal health status did not result in statistically significant conclusions. This fact may be attributed to the low prevalence of the target compounds in the analyzed samples. Nevertheless, the detection of these compounds in maternal urine and amniotic fluid raises concerns about the real risk to both mothers and fetuses; hence further studies must be conducted in order to elucidate it. One fact that must be taken into consideration is the combined exposure to these substances and their potential synergistic toxicity  as this Vasiliki Karzi: Investigation, Formal analysis, Writing-review & editing; Manolis M. Tzatzarakis: Conceptualization, Supervision, Methodology; Eleftheria Hatzidaki: Resources, Validation; Ioanna Katsikantami: Investigation; Athanasios Alegakis: Formal analysis, Data curation; Elena Vakonaki: Methodology; Alexandra Kalogeraki: Resources; Elisavet Kouvidi: Resources; Pelagia Xezonaki: Resources; Stavros Sifakis: Resources, Validation; Apostolos K. Rizos: Conceptualization, Supervision.The authors report no declarations of interest.


Left ventricular outflow tract obstruction (LVOTO) is a recognized complication after complete repair of atrioventricular septal defect (AVSD). This study reviewed the incidence and management of LVOTO following AVSD repair at a single institution.From 1975 to 2019, 24 patients  underwent reoperation due to LVOTO following partial AVSD (pAVSD) and complete AVSD (cAVSD) repair. The data were retrospectively reviewed.P = 0.7406) and subsequent LVOTO (P = 0.7153) following complete repair of pAVSD and cAVSD. Combined access to the left ventricular outflow tract was not protective regarding LVOTO reoccurrence. Survival for both groups after LVOTO reoperation at 15 years was 95.6% (95% CI 99.4–72.9).The incidence of LVOTO following pAVSD and cAVSD repair was 4.4% (12/275) and 2.6% (12/455). Freedom from LVOTO reoperation following pAVSD and cAVSD repair at 25 years was 94.3%  and 95% . The median time from complete repair of pAVSD and cAVSD to LVOTO reoperation was 4.4 years [interquartile range (IQR): 3.4–6.7] and 2.6 years (IQR: 2.2–4.7). Freedom from second LVOTO reoperation at 5, 10 and 15 years was 83.7% , 59.2%  and 39.5% . The median time between the first and the second LVOTO reoperation in the groups of pAVSD and cAVSD was 6.1 years (IQR: 3.4–8.9) and 8.6 years (IQR: 5.7–9.8). There was no significant difference regarding the first  and partial atrioventricular septal defect (pAVSD) can be repaired with excellent results, however, the burden of reintervention remains significant . In the current era, complete atrioventricular septal defect (cAVSD) and partial atrioventricular septal defect (pAVSD) can be repaired with excellent results, however, the burden of reintervention remains significant , 2. LeftOur institutional database of atrioventricular septal defect (AVSD) was reviewed for all reoperations on the LVOTO.Data were obtained retrospectively from the hospital patients records and follow-up was conducted via correspondence with the patients’ cardiologists and general practitioners. Follow-up was 100% complete.The study was approved by the Royal Children’s Hospital (RCH) Human Research Ethics Committee (HREC32047E). The requirement for individual patient consent was waived due to the retrospective nature of the study.Our surgical approach to complete repair of pAVSD and cAVSD has previously described in detail , 2. BrieThe indication for LVOTO reoperation was peak gradient across the LVOT of 30 mmHg or more. We rely on the combination of transthoracic and transoesophageal echocardiography for planning and immediate postoperative assessment in all cases. All reoperations for LVOTO were conducted via median sternotomy on cardiopulmonary bypass with mild hypothermia. Depending on the anatomical substrate of the LVOTO, the decision was made intraoperatively how it should be approached. The surgical approaches were as follows: transaortic, left atrial or combined approach. A fibromuscular membrane was present in the vast majority of cases and consequently, it was approached via the aorta. The technique of fibromuscular resection has been previously described . The lefP < 0.05.All data were analysed using STATA version 13 . Continuous data are expressed as median and interquartile range (IQR). Fischer exact test was used comparing discrete variables when group size was <5. Time-dependent endpoints, specifically survival and freedom from LVOTO operation, were analysed using the Kaplan–Meier method, with time commencing at the time of initial AVSD repair. Differences between groups were compared using the log-rank test. The threshold for statistical significance was taken as From 1975 to 2019, a total of 275 patients with pAVSD underwent complete repair at the RCH, Melbourne, Australia. From 1990 to 2019, a total of 455 patients with cAVSD underwent complete repair in the RCH. One patient with concomitant double outlet right ventricle and pulmonary atresia was also included to the study group. A total of 24 patients  underwent reoperation for LVOTO in the study period. Of the 730 patients who underwent repair for cAVSD and pAVSD, 3.2% (24/730) required reoperation on the LVOT during the study period.The freedom from LVOTO operation for the total group of 730 patients after cAVSD and pAVSD repair at 5, 15 and 30 years was 96.7% , 94.8% 95% CI; 92.1, 96.7) and 94.8% , respectively . All 12 patients with cAVSD had 2-patch technique. The median time from the complete repair to the first LVOTO reoperation was 2.6 years (IQR: 2.2–4.7). A total of 12 patients required 16 reoperations due to LVOTO. Out of them, 41.7% had initially Rastelli type A of cAVSD (5 out of 12); 25% (3 out of 12) had Rastelli type C of cAVSD; 8.3% had Rastelli type B of cAVSD and in the rest 3 (25%) patients the type of cAVSD was not recorded at the time of the complete repair. Overall, the freedom from LVOTO reoperation in the group of cAVSD repair at 5, 15 and 25 years was 96.7% , 95%  and 95 , respectively . The median time from pAVSD repair to the LVOTO reoperation was 4.4 years (IQR: 3.4–6.7). A total of 12 patients underwent 16 reoperations due to LVOTO; 4 patients (33.3%) out of 12 required second LVOTO resection due to LVOTO reoccurrence. At the time of the first LVOTO operation, 4 patients (33.3%) underwent concomitant LAVV repair. Freedom from LVOTO operation for partial AVSD at 5, 15 and 25 years was 96.4% , 94.3%  and 94.3% , respectively  and 65 mmHg (IQR : 43–78) and for the second LVOTO reoperation, they were 60 mmHg (IQR: 52–62) and 92 mmHg (IQR: 78–100), respectively.A total of 32 LVOTO reoperations were performed on 24 patients with complete and partial AVSD. The median cardiopulmonary bypass time was 73 min (IQR: 53–129) while the mean cross-clamp time was 37 min (IQR: 29–69), respectively. For patients undergoing a second LVOTO operation, the median bypass time was 120 min (IQR: 73–157) and the median cross-clamp time was 85 min (IQR: 54–112).The anatomical substrate for the LVOT obstruction is represented in Table The surgical approaches to LVOTO are presented in Fig. The summary of the surgical details is represented in Table Surgical details of 2 patients with complex and diffuse narrowing of the LVOT are presented below. One patient with cAVSD and associated double outlet right ventricle with pulmonary atresia required left ventricular rerouting (tunnelling) using a segment of the 12-mm Gore-Tex tube. Later, he developed LVOTO due to the outgrowth of the ventricular septal defect patch, so it was replaced with bigger piece of the Dacron conduit 10.7 years after complete repair. Another patient after partial cAVSD repair underwent LVOTO operation 16 months later which included augmentation of LVOT by means of superior bridging leaflet augmentation with autologous pericardium and ventriculoplasty in addition to conventional subaortic membrane resection and myotomy.The median follow-up time in the group of partial AVSD repair was 14.8 (IQR: 3.8–22.2) years. One patient died in the group of pAVSD due to bowel cancer at age of 18 years; 90.7 months after the LVOT reoperation. In the group of patients with complete AVSD, the median follow-up was 12.9 years (IQR: 4.3–4.7). There was no late death in this group.Overall, the survival for both groups after LVOTO reoperation at 5, 10 and 15 years was 95.6%   and subsequent LVOT (P = 0.7153) reoperations.The freedom from the second LVOTO reoperation for the total group of 24 patients at 5, 10 and 15 years was 83.7% , 59.2%  and 39.5% , respectively  required the second LVOTO reoperation due to its reoccurrence. The median time from the first LVOTO reoperation to the second LVOTO reoperation was 8.6 years (IQR 5.7–10.8). At the time of the first LVOTO reoperation, 16.7% of patients (2 out of 12) required concomitant LAVV repair. None of the patients needed LAVV repair at the time of the second LVOTO reoperation. The freedom from the second LVOTO operation after the first LVOTO reoperation at 5, 10 and 15 years was 90% , 75%  and 25% , respectively . The freedom from the second LVOTO operation after the first LVOTO reoperation at 5, 10 and 20 years was 80% , 48%  and 48% , respectively , glutaraldehyde treated autologous pericardium (n = 2) and Cardiocell patch (n = 2). All 6 patients have mild LAVV regurgitation at the time of presentation with LVOTO. After LVOTO operation in 2 of them, the amount of regurgitation remained unchanged and in another 4 it decreased to trivial in 3 and to no regurgitation in 1. However, after 1 year, 1 patient was reoperated for severe LAVV insufficiency due to tearing suture line on the superior bridging leaflet patch. After reoperation, the amount of the LAVV insufficiency became trivial as it was immediately after LVOTO repair.In total, there were 6 patients who underwent superior bridging leaflet detachment with subsequent patch augmentation of the resulting opening. There were 4 and 2 patients on whom this was performed as a first and second LVOTO procedure, respectively. The patch material we used for the closure of the opening was as follows: untreated autologous pericardium patch  and surgical approach (double patch technique for cAVSD versus single patch technique for pAVSD), the ‘anatomical’ substrate is similar after complete repair with identical time-related risks for developing LVOTO. This is further supported by the recent findings by Fong et al. [P = 0.64).Anatomical factors which provide the substrate for the development of LVOTO include reduced inlet and increased outlet portion of the left ventricle leading to an elongated and narrowed LVOT, abnormal anchoring of superior bridging leaflet contributing to the tightness of the subaortic area, and deficiency of inlet portion of the interventricular septumare present in up to 70% patients after complete AVSD repair . Interesg et al. . By doinet al. [et al. [P = 0.4). Malattachment of the superior bridging leaflet to the ‘scooped-out’ crest of the ventricular septum that leads to narrowing of proximal LVOT is another target for LVOTO relief [et al. [We observe unexpectedly low freedom from recurrent LVOTO reoperations at 25% at 15 years in the group of cAVSD and 48% at 20 years in the group of pAVSD giving an overall 39.5% freedom from recurrent LVOTO reoperation at 15 years. A similar trend was observed by Stulak et al.  who repo [et al.  observes [et al. . In our O relief . Anatomi [et al. , 23. TheThis study is limited by the retrospective nature and small number of patients. Due to the long study period, some echocardiographic data were not routinely reported.Incidence of LVOTO after AVSD repair is low but reoccurrence rate is high. Standard subaortic resection does not always provide definitive LVOTO relief. Thus, other anatomical targets of LVOTO should be addressed. Superior bridging leaflet augmentation should be considered in addition to conventional subaortic resection.P.N. is supported by a National Health and Medical Research Council Medical Research Postgraduate Scholarship (1150242). Y.d’U. is a Practitioner Fellow of the National Health and Medical Research Council of Australia (1082186).Conflict of interest: Prof Yves d’Udekem is a consultant for Actelion and MSD. A/Prof Christian Brizard serves on the advisory board of Admedus.All relevant data are within the manuscript and its supporting information files.Yaroslav Ivanov: Conceptualization; Data curation; Project administration; Writing—original draft; Writing—review & editing. Edward Buratto: Data curation; Formal analysis; Methodology; Writing—review & editing. Phillip Naimo: Data curation; Writing—review & editing. Adrienne Lui: Data curation. Thomas Hu: Data curation. Yves d’Udekem: Writing—review & editing. Christian P. Brizard: Project administration; Supervision; Visualization; Writing—review & editing. Igor E. Konstantinov: Conceptualization; Project administration; Supervision.Interactive CardioVascular and Thoracic Surgery thanks Mark H.D. Danton, Carl Lewis Backer and the other anonymous reviewers for their contribution to the peer review process of this article.


Introduction: Small cell carcinoma of the urinary tract (SCCUT) is a rare finding with poor clinical course. This study sheds light on the molecular subtype and identifies risk factors in patients diagnosed with SCCUT.Methods: Immunohistochemical expression of immunotherapy target programmed death ligand 1 (PD-L1) and luminal (GATA3), basal (p63), and p53 markers are assessed in patients diagnosed with SCCUT. Univariate analysis identified risk factors. Overall survival (OS) is computed using the Kaplan–Meier method.Results: Tissue was available for 70.2% (33/47). All showed a high PD-L1 expression phenotype. p53 is seen in 93.9% (31/33), mostly as overexpression, GATA3 in 45.5% (15/33), and p63 in 57.6% (19/33). For the entire cohort (n = 47), 1-year survival was 59.6%, and the median OS was 17 months. Univariate analysis shows that chemotherapy , radical surgery , and diagnosis of non-pure SCCUT  are favorable prognostic features. Metastasis had negative associations with survival .Conclusions: In this series, pure and mixed SCCUT are characterized by p53 overexpression and a high PD-L1 phenotype. Histology of non-pure SCCUT is a positive prognosticator, and radical cystectomy or chemotherapy can improve OS. These findings demonstrate that SCCUT may be eligible for PD-L1 immunotherapy. Bladder cancer is the fifth most common malignancy, with an estimated incidence of 81,400 new cases in 2020 , a morbid disease with an incidence rate of ~1% of all cases of bladder cancer . First dPrior work suggests that UC with a basal gene expression profile translates into poor clinical outcomes. Conversely, the luminal subtype correlates with better OS, whereas the p53 wild-type molecular phenotype is associated with resistance to chemotherapy –9. HereiPatients diagnosed with SCCUT between 1990 and 2019 with at least 1 year of follow-up were included in this institutional review board–approved retrospective study, and clinicopathologic and demographic features were recorded for these patients. All available pathology slides were reviewed by a fellowship-trained urologic pathologist (A.A.) to confirm the diagnosis based on the current World Health Organization classification system and identify a representative block for immunohistochemistry. All cases with any amount of small cell component were included in the study. Clinicopathologic features of patients are detailed in 4-μm-thick sections were cut and mounted onto positively charged glass slides . Slides were baked in an Isotemp (Thermo Fisher Scientific) oven overnight at 37°C. The next day, slides were loaded onto the Ventana Discovery XT  for detection of the antibody using the Discovery IHC DABMap kit .Antigen retrieval was performed using standard cell conditioning 1 (Tris/borate/EDTA), including head induced epitope retrieval in Tris-EDTA buffer pH 7.8 at 95°C for 44 min. Slides were incubated with Endogenous Blocking Kit  for 10 min. Slides were removed from the Ventana Discovery XT and put into soap and DI mixture followed by a 5-min wash using DI. Washed slides were put in hematoxylin for 30 s, followed by DI for 5 min, 0.2% ammonia water for 1 min, and then washed with DI for an additional 5 min. Washed slides were dehydrated and cover slipped.For PD-L1 expression, the primary antibody used was anti-PD-L1 . A dilution of 1:1,500 was manually titrated onto the slides and incubated for 30 min. The universal secondary antibody was automatically dispensed and incubated for 30 min. Staining on tumor cells (TCs) and infiltrating immune cells (ICs+) was evaluated for percent of cells expressing PD-L1. Lymph node and tonsil tissue were used as positive controls.Interpretation of positive PD-L1 staining was performed based on vendor recommendations. In brief, PD-L1 status was determined by the percentage of TCs with any membrane staining above background or by the percentage ICs+ at any intensity above background. The percent of tumor area occupied by any tumor-associated ICs [ICs present (ICPs)] was used to determine ICs+, which is the percent area of ICPs exhibiting PD-L1–positive IC staining. PD-L1 status was considered high if any of the following were met: (1) ≥25% of tumor cells exhibit membrane staining; (2) ICPs >1% and ICs+ ≥25%; or (3) ICPs = 1% and ICs+ = 100%.To categorize the tissue based on molecular subtype, the GATA3 antibody  was used to identify luminal subtype, the p63 antibody  applied to classify the specimens as basal, and the p53 antibody  was employed to evaluate p53 expression. Expression of nuclear p63 and GATA3 was assessed using the three-point immunoreactive scoring system looking for the extent  and strength  in each specimen. Staining of p53 nuclear protein expression was ranked using a three-point system .p< 0.05 was considered significant.Clinicopathologic and demographic features were summarized using frequency counts and percentages. These variables were correlated with expression of the aforementioned immunohistochemical stains. OS was estimated using the Kaplan–Meier method. Cox proportional hazards models were used to evaluate the correlation of clinicopathologic features and immunohistochemical markers on survival. A univariate Cox regression model was performed on all covariates. Statistical analyses were performed using RStudio 1.3.1073 . n = 47), and the minor cohort includes patients with tissue available (n = 33).Two cohorts were generated: the major cohort includes all patients diagnosed with SCCUT , pT3 in 36.1% (17/47), pT2 in 36.1% (17/47), and pT1 in 6.4% (3/47); in four cases (8.5%), the final stage was not available (pTx). Pathologic lymph node assessment revealed N3 in 2% (1/47), N2 in 11% (5/47), N1 in 23% (11/47), and N0 in 64% (30/47). Distant metastasis was found in 17% (8/47), and 83% (39/47) were metastasis-free. Platinum-based chemotherapy was administered to 76.6% (36/47), 2.1% (1/47) received gemcitabine only, and 21.3% (10/47) did not receive chemotherapy. Radical cystectomy and nephroureterectomy were performed on 34% (16/47) and 2.1% (1/47) of patients, respectively; 64% (30/47) had transurethral resection of tumor (TUR) only.Pure SCCUT was diagnosed in 38.3% (18/47) of cases, and the remaining 62% (29/47) revealed a mixture of SCCUT and conventional high-grade UC or other UC variants. The mixed SCCUT cases in this cohort contained between 30 and 90% of small cell component.n = 47) are shown in p < 0.0001). Patients undergoing radical surgery had longer OS with median of 54 months  than patients undergoing TUR only, with median of 10 months  (p < 0.005). Patients diagnosed with mixed SCCUT had median OS of 17 months  and a better outcome than pure SCCUT with median OS of 9 months  (p < 0.05).Survival curves for all patients , and mixed SCCUT histology  with improved survival, whereas the presence of metastatic disease was associated with worse survival   = 0.015, 95% CI = 0.0018–0.13,  = 0.03) .p = 0.03).Metastatic disease was confirmed by imaging studies in 17% (8/47) of patients: four female and four male patients. Pure SCCUT was found in five of eight cases. One patient had pT4, three were pT3, and three were pT2, and in one patient, the degree of invasion could not be assessed (pTx). The most common metastatic sites included bone involvement in seven of eight, six of whom showed metastases to the thoracic and cervical spines, one patient showing involvement of the right hemipelvis. Two patients presented or progressed with liver and bone metastasis. One patient showed liver and lung metastasis and cervical adenopathy. Maximum survival in all metastatic patients was 23 months. The median OS for patients with metastatic disease (M1+) was 9 months , and for patients with no signs of metastasis (M0), it was 17 months  (n = 33), the median OS was 17 months  (Tissue was available for 33 of 47 patients (70%) and immunohistochemical expression was evaluated. For patients with available tissue ( months) .Comparison of the means using analysis of variance (ANOVA) revealed that common clinicopathologic features are not different between patients with tissue available and the whole patient population. The pertinent clinicopathologic features are detailed in Among the cases, 72.7% (24/33) revealed overexpression of p53 (score 2+), 21.2% (7/33) had wild-type (score 1+) p53 expression, and total loss of p53 (score 0) was noted in 6.1% (2/33).Expression of p53 (2+) was noticed concurrently in 15.2% (5/33) with GATA3 surrogate for luminal phenotype, 27.3% (9/33) with p63 surrogate for basilar phenotype, and in 15.2% (5/33) with both GATA3 and p63 overexpression. The remaining 15.2% (5/33) revealed p53 (2+) expression with no expression of GATA3 or p63. Wild-type p53 expression (score 1+) was concomitantly present with p63 and GATA3 in 6.1% (2/33), with p63 expression alone in 3% (1/33), and with GATA3 alone in 3% (1/33). Loss of expression of p53 was seen in 6.1% (2/33) of patient samples expressing both p63 and GATA3. There was no meaningful difference between expressions of p53 in basal or luminal tumors.p < 0.001) and elevated PD-L1 expression in TCs . Age older than 70 years was an adverse risk . A diagnosis of mixed SCCUT has some protective effects on survival . There were no significant associations between p53, GATA3, or p63 and survival.Univariate Cox proportional hazards analysis  revealedSCCUT is a rare variant of UC with aggressive clinical course and poor prognosis. To our knowledge, this is the first study to evaluate PD-L1 expression specifically in SCCUT and present an immunohistochemical classification based on molecular subtype. The examined SCCUT tumors were characterized predominantly (97%) by a high PD-L1 phenotype regardless of histology, treatment history, or p53 status. Univariate analysis identified increased hazards for older patients (>70 years old) and metastatic disease. Improved OS was exhibited from chemotherapy, radical surgery, diagnosis of mixed SCCUT, and high PD-L1 expression in TCs.As an immune escape mechanism, it is counterintuitive that high PD-L1 expression in TCs improves survival. Thus, given the overall risk reduction was 2% and the CI (0.97–1.00), it is likely that the effect is not clinically significant. Moreover, these findings do not align with the available literature. A large meta-analysis by Zhao et al. including 3,674 patients from 18 studies found that PD-L1 expression in ICs+ and not TCs was, indeed, protective and imparted positive prognostic value . FurtherContrary to our study, the study by Pichler et al. on 48 pure UC and 13 variants suggested that elevated PD-L1 expression was associated with diminished chemotherapy response . HoweverThere are data from Yang et al. suggesting that targeting the adenosine receptor A2A (ADORA2A), involved in cytotoxic TCD8 anergy, may be a rational drug development approach for SCCUT, as this receptor was expressed in 34 samples of SCCUT . These 3Anti-PD-L1 immunotherapy with PD-1/PD-L1 inhibitors is approved for treatment of UC in cisplatin ineligible patients regardless of PD-L1 expression . The IMvPlimack et al. included a small cohort of UC variants in their study  and report significant antitumor activity and acceptable safety for pembrolizumab in advanced UC and its variants . The autThe median OS in SCCUT has been reported in a range from 5.3 to 34.9 months with a 5-year survival rate ranging from 0 to 40%. , 25–29. p = 0.011). All chemotherapy administered was platinum based and administered in combination with at least one other agent in every patient (p = 0.04). Overall, the median survival for SSCUT and mixed SSCUT were 13.5 and 17 months, respectively (p = 0.1). These diverse observations indicate that as a whole, chemotherapy was protective; however, no direct comparison can be made with surgery.It is not clear why a non-small cell component mixed to SCCUT is a good prognostic factor. In patients with tissue available, the differences in clinicopathologic features between the two major histological groups—SSCUT and mixed SSCUT—are summarized in  patient . FurtherThe three subtypes explored in this study include p53 wild-type, basal-like, and luminal-like. GATA3, a marker of luminal type, is correlated with better outcomes compared to basal-like and p53 wild-type tumors . ExpressTwo patients with mixed SCCUT did not express any p53, demonstrating a truncation or deletion of the gene. However, this was not a statistically significant finding. Overexpression of protein p53 was found in 64.3% of pure SCCUT and in 78.9% of mixed SCCUT patients, suggesting that an abnormal or upregulated p53 transcription is present in these patients. These findings are most consistent with the p53-like subtypes. Overexpression has been correlated with chemotherapy resistance, and intact non-mutated expression is associated with worse survival than a mutated type , 33.As there are more than 12 isoforms of p53 with distinct functions , analysis of p53 expression and the specific mutation might help identify patients who are likely to develop metastatic SCCUT . It may A recent publication by Kadosh et al. shows that mutant p53 constitutively suppresses the oncogenic Wnt/β-catenin pathway in the upper gastrointestinal tract; in the lower intestinal tract, however, bacteria carrying an enzyme named shikimate produce gallic acid, which regulates the Wnt gene promoter and nullifies the p53 effect on growth suppression, likely potentiating additional carcinogenic pathways . This prThe herein presented study is limited as it deals with a rare disease. Actually, the low incidence of SCCUT begs for a retrospective study investigating all historical accounts of this malignancy in order to improve guidance in prospective cases. However, the low incidence of SCCUT and the nature of a retrospective study imply that several valuable clinical questions remain unanswered. Accurate comparisons of treatment cannot be made from this cohort as there were new developments made during the 29-year period included in the search, including improved treatment modalities. Thus, matched and balanced comparisons as befit a dynamically randomized clinical trial are near impossible in retrospective studies, especially when the sample size is small. In this study, the small sample size influences the reliability of additional analyses including detailed comparisons of patients with recurrence, metastasis, and chemotherapy type. Retrospective studies combining information from multiple institutions can generate a dataset large enough to withstand balancing and more clinically significant analyses including stage and treatment-matched cancer-specific survival.One of the greatest pitfalls of a retrospective study, especially one that investigates patients prior to the current information age, before the adoption of electronic record keeping, is the inconsistencies in record keeping. These concerns are apparent in this study as 30% of patients did not have adequate specimens for immunohistochemical processing. Knowing these limitations, as we continue to explore new treatment modalities and improve outcomes, it is of utmost importance that record keeping continues to improve. The final limitation in this study was in antibody selection as there are several clones available; however, neither all are commercially available nor used in clinical practice. It would be interesting to see expressions of additional clones of antibodies targeting p63, GATA3, p53, or PD-L1 in future studies of SCCUT.Our group aims to circumvent some of these shortcomings by describing gene signatures in SCCUT, compare its gene expression profile to demographically and clinically matched patients with small cell carcinoma of the lung, as well as patients with UC, and continue to enroll advanced SCCUT into clinical trials with an immune checkpoint backbone.To our knowledge, this is the first cohort of SCCUT demonstrating high expression of PD-L1. Approximately two-thirds of SCCUT in our series revealed a phenotype of p53 overexpression, which would potentially result in poor response to chemotherapy; therefore, the finding of remarkably high PD-L1 phenotypes in this cohort highlights important clinical implications, suggesting that SCCUT may be targetable by anti-PD-L1 therapies. Further study of SCCUT will provide insight into prognostic markers and therapeutic targets.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Miriam Hospital Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.BGo and AA conceptualized the study. All authors aided in data collection, review, and analysis. All authors wrote and reviewed the manuscript.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.


Corn mashes have high-viscosity and high-sugar characteristics, which hinders yeast-fermentation efficiency and the ethanol yield increase. The excessive viscosity of corn mash is caused by the unutilized cellulose in corn kernel fiber. A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in about 12% increased ethanol concentration compared with the reference mash at different batch-fermentation scales. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash and residual sugars by 40.9% and 56.3%, respectively. Simultaneously, the application of lignocellulolytic enzymes increased the value of the dried distiller’s grain with solubles (DDGS) by increasing the protein content by 5.51%. The in situ conversion of cellulose can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 12%. A quarter of the ethanol yield increase was due to the further hydrolysis of starch, while three quarters to cellulose. Thus, this technology will increase the net revenue of bioethanol industrialization. Ethanol production through biotechnology is an attractive option for sustainable fuel production. Starch is the second most important and abundant source of carbon and energy in plants. Starch is an important feedstock in the fermentation industry and is widely fermented to produce ethanol, which can be used as a basis for beverages or as an alternative biofuel ,2. In ChIndustrial corn mash has high viscosity and contains coarse particles. A problem during the processing of corn starch is the excessive viscosity of mash, which can increase the complexity of the process of starch hydrolysis and mash fermentation . The excThe reported work showed lignocellulolytic enzymes could improve ethanol yield from starch due to excessive enzyme dosage. The costly process brings out an inappropriate ethanol yield increase, which is unacceptable and hinders cellulase application in the field of bioethanol. In this work, we develop a novel lignocellulolytic enzymes preparation that has strong substrate specificity for high-sugar and high-viscosity corn mash. The main objectives are to convert cellulose in situ during simultaneous saccharification and fermentation (SSF) and to investigate the effect of the novel lignocellulolytic enzymes addition on SSF performance, including ethanol yield, corn mash viscosity, and residual sugars. The application of the novel enzymes cocktail can enhance ethanol yield by more than 12%, which is beneficial to bioethanol.w/v). Corn mash preparation was provided by Jilin Fuel Alcohol Company Limited . Equal amounts of tap water and back-set stillage were mixed with ground corn to a final dry matter content of 27% (4)2SO4, 6 g/L KH2PO4, 1 g/L MgSO4∙7H2O, 2.5 g/L CaCO3, 2 mL/L tween-80, and 30 g/L inducers. It had an initial pH of 5.0. The method of lignocellulolytic enzyme fermentation was referring to the reported ,11. For T. reesei A2H  was a lignocellulolytic enzyme-hyperproducing mutant strain obtained by chemical mutagenesis in our laboratory [boratory . The medboratory .A. niger 60B-3DW  was a β-glucosidase hyper-producing mutant strain obtained by chemical mutagenesis in our laboratory. To clone the whole fragment of the FAE (GenBank accession number: KY449280) and GH61 (GenBank accession number: ATQ35955.1) encoding gene from the chromosomal DNA of Penicillium piceum 9-3, a pair of oligonucleotide primers was designed based on the sequenced genomic data of P. piceum 9-3. The FAE and GH61 gene were digested with PstI and SalI and then separately inserted into digested p19-Pptef-2Apeptide-hph-Tcbh1 to generate enzyme-expression plasmids. Transformation of A. niger protoplasts was performed according to the previously described procedure [rocedure . The traT. reesei and auxiliary enzyme from A. niger at the ratio of 8.5:1 (v/v). The novel lignocellulolytic enzymatic preparation was a cocktail with cellulase from S. cerevisiae; Angel, Yichang, China) was suspended in distilled water at 32 °C for 1 h. Then, 1.0 mL of yeast was added into 100 mL of corn mash with a certain quantity of lignocellulolytic enzymes.Approximately 1.0% of alcohol instant active dry yeast  according to the Schoorl and Regenbogen method . The dexThe starch contents of the maize mash were determined using the modified Megazyme assay. The method was based on the hydrolysis of starch with α-amylases and glucoamylase to produce glucose, which was subsequently calculated to the starch content of the sample. w/w) H2SO4 at 30 °C for 2.5 h and then stirred every 15 min with a glass stirring rod. The solutions were diluted with 181.7 mL of water and then autoclaved at 121 °C for 1 h. Glucose and xylose concentrations were determined through high-performance liquid chromatography (HPLC)  with a refractive index detector (Shimadzu) on an Aminex HPX-87H column  running at a flow rate of 0.6 mL/min at 60 °C, with 5 mM H2SO4 as the mobile phase. Glucan and xylan concentrations were calculated according to Equations (1) and (2), where the factors of 0.9 and 0.88 reflect the weight loss as a result of glucose-to-glucan and xylose-to-xylan conversion, respectively [The carbohydrate in biomass was quantitatively analyzed according to the NREL Laboratory Analytical Procedures  for biomass using a two-step acid method . Approxi2SO4. Each sample was injected three times, and the average results were calculated [All samples were diluted and passed through a 0.45 µm filter before HPLC analysis. Residual glucose, ethanol, and other fermentation minor products were determined using high-performance liquid chromatography  with a refractive index detector (Shimadzu) on an Aminex HPX-87H column . The flow rate was 0.6 mL/min at 55 °C, and the mobile phase was 5 mM Hlculated .The viscosity was determined using a Hoppler viscometer type Visco Ball, expressed in mPa s at 20 °C. The determination principle for the viscosity of liquids using the Hoppler viscosimeter involved the measurement of the ball descent time through a constant distance in the studied liquid contained in a glass tube.n content data (N × 6.25) [According to the Kjeldahl method, 1 g samples are placed in the digestion tube. Twenty milliliters of sulfuric acid and a tablet containing 0.48 g of mercury oxide as a catalyst and 4.52 g of potassium sulfate are added. Blanks containing all these reagents are simultaneously processed. The tubes are placed in the preheated digestion block at 420 °C for 2 h 30 min. The resulting solutions are cooled at room temperature and diluted by adding 30 mL of water. The tubes are placed in the distillation–titration unit. Then, 20 mL of sodium hydroxide solution are automatically added and the solutions are distilled 6 min). The ammonia collected in the receiving solution (30 mL) is automatically titrated against the standard 0.25 M hydrochloric acid with colorimetric endpoint detection. Ammonium dihydrogen phosphate standard (NIST SRM 194) is used to check the concentration of the titrating solution. The total N content was calculated according to ammonium dihydrogen phosphate content . Crude p min. The × 6.25) . p  <  0.01 [For comparison of DDGS composition with/without lignocellulolytic enzymes addition, ANOVA was used, followed by Tukey’s multiple-comparison posttest with a 95% confidence interval. Statistics were performed by using SigmaPlot (version 11) . The dif <  0.01 . T. reesei and the coenzyme system from A. niger. Although the commercial cellulase was produced from T. reesei, the enzyme system from T. reesei has some drawbacks [T. reesei was incomplete and unbalanced. That is, it had no genes encoding cellobiose dehydrogenases and feruloyl esterase and few monooxygenases and β-glucosidase expressed in the T. reesei extracellular protein [Penicillium piceum had unique enzymology characteristics in our previous work [The composition of the lignocellulolytic enzyme cocktail for high-viscosity corn mash would be screened in the lignocellulolytic enzyme library. The lignocellulolytic enzyme library contained the main enzyme system from rawbacks . The mai protein ,23. Thes protein . For higous work ,25. The ous work ,25. The Considering the cost of using enzymes, the lignocellulolytic enzyme was selected for further experiments to test at a lower enzyme dosage. Corn mash fermentation was performed at different enzyme loadings (2–200 FPU/L) to facilitate the screening of optimum enzyme dosage. The ethanol concentration, sugar reduction, and viscosity of corn mash corresponding to different enzyme dosages are presented in The application of lignocellulolytic enzymes preparation decreased the mash viscosity and increased the yeast-fermentation efficiency. This result was consistent with a previously published report. The results of Czarnecki and Nowak indicate that there are beneficial effects of lignocellulolytic enzymes  on rye mashes, such as a decreased viscosity and an enhanced starch saccharification, and an increased productivity of ethanol ,27. The novel lignocellulolytic enzymes were applied at different fermentation scales from 0.3–70 L. At the 0.3, 1, 5, and 70 L batch-fermentation scales. The reductions in corn mash viscosity were 46.3%, 31.6%, 35.5%, and 40.9% compared with the reference mash (33.5 ± 1.5 Pa·s), respectively. The treatment of corn mashes with the novel lignocellulolytic enzymes resulted in increasing concentrations of ethanol by 12.4%, 12.0%, 11.8%, and 12.9% compared with the reference mash. The highest yield of ethanol in the corn mash digested with the novel lignocellulolytic enzyme reached 117.0 ± 0.1 g/L at the 70 L batch fermentation, whereas that in the control reached only 103.6 ± 1.0 g/L. After the lignocellulolytic enzyme addition, the residual starch content decreased to 5.34 ± 0.26%, whereas that without lignocellulolytic enzyme was 6.58 ± 0.86% . The resTo clearly elucidate the promotion effect of lignocellulolytic enzymes on alcoholic fermentation, the lignocellulolytic enzymes were added into the residual broth in SSF. The 36 h SSF broth (containing unconverted solids) with the ethanol evaporated was used for another round of separate hydrolysis and fermentation (SHF), which reduced the ethanol inhibition on the cellulase activity. As shown in p < 0.01) on the composition of DDGS. The residual cellulose content of DDGS decreased (p < 0.01) from 33.12% to 21.20%, while the starch content of DDGS decreased (p < 0.01) from 14.07% to 11.26% (p < 0.01). No difference in DDGS color was observed . The estimated ethanol increase can improve by 10% (conservative computation) in alcoholic industrialization, which is equivalent to increasing the income by RMB 600 yuan (6000 yuan/ton ethanol) from producing 1 ton of ethanol. The net revenue can increase to RMB 525 Yuan (600-75) from producing 1 ton of ethanol. The fuel ethanol production in Jilin Province is 0.6 million tons per year, while the fuel ethanol production in China is approximately 10.0 million tons per year in the near future. This technology can increase the net revenue of fuel ethanol in Jilin Province by RMB 3.2 billion Yuan, while the net revenue of fuel ethanol in China by RMB 52.5 billion Yuan per year. Moreover, the co-product will add a certain revenue due to the protein content increase of DDGS.Cellulolytic enzymes are believed to have common practical applications with the development of fuel ethanol production from lignocellulolytic biomass . The appThe presence of non-starch polysaccharides, such as cellulose and hemicellulose in cereal grain, caused the excessive viscosity of the mash, which could make the process of starch hydrolysis and mash fermentation more complicated. In this work, we developed a novel lignocellulolytic enzymes preparation that had a strong substrate specificity for high-sugar and high-viscosity corn mash. These lignocellulolytic enzymes successfully promoted the ethanol yield from corn mash by more than 12% on the premise of the same material amount. Use of the novel lignocellulolytic enzyme preparation decreased mash viscosity, facilitating the technological process and leading to the release of certain amounts of glucose. This in turn provided an opportunity to increase ethanol efficiency. A quarter of the ethanol yield increase was due to the further hydrolysis of starch, while three quarters were to the hydrolysis of cellulose. The reported work showed lignocellulolytic enzymes could improve ethanol yield from starch due to excessive enzyme dosage. T. reesei was 95.7 g/L at the solid concentration of 28% (w/w), while Klosowski et al. reported that when corn mash is digested with a multi-enzyme complex from Aspergillus sp., the ethanol concentration is 85.7 g/L [CeluStar XL for corn starch and obtained a high fermentation yield of 104.9 g/L [Rhizopus sp. for the SSF of raw corn flour. The conversion efficiency of raw corn flour to ethanol was 94.5% of the theoretical ethanol yield [w/w). Overall, the ethanol yield from corn starch reported in this study was higher than that achieved by previous studies. The lignocellulolytic enzyme cocktail application produces lower enzyme dosage, shorter fermentation time, and more significant ethanol improvement, which is more economical than the previous report.This patent by Abbas et al. showed that the ethanol yield from corn after adding cellulolytic enzymes from 85.7 g/L . Sapińsk04.9 g/L . Wang etol yield . Li et aol yield , which wThis technology does not require any additional equipment or control system and does not modify the alcoholic fermentation technology. The lignocellulolytic enzymes with excellent properties can improve economical benefit through minimal enzyme input in alcohol industrialization. This technology will bring a better understanding in engineering bioethanol improvement. The results provide new ideas for promoting fuel ethanol industrialization and the technological progress of renewable energy in China. This technology has been patented in China (patent no. 201811277689.7).