The law in Lebanon allows new mothers to take up to 10 weeks paid maternity leave, and some private organizations choose to give fathers 2 days of paternity leave in the absence of clear legislation. This falls short of the 6 months recommended for mothers in the literature.The sample consisted of 97 Lebanese mothers with children between 6 and 24 months of age. First, we examined correlations between the length of maternity leave and measures of work productivity and maternal sense of competence in their new role as a parent. Second, we explored correlations between the length of maternity leave and mother–child adjustment measures . Finally, we looked for predictors of work productivity and maternal sense of competence in demographic variables, mother–child adjustment measures, and partner attachment scores.Results showed significant correlations between work productivity and maternal sense of competence, irrespective of the length of maternity leave. Regression analyses showed that working for pleasure, mother-infant healthy bonding, and positive subjective experience of being back to work were significant predictors of work productivity, and that maternal stress predicted maternal competence.These findings are discussed within the framework of providing suggestions facilitating mothers’ transition back to work and increasing work productivity after having a baby. Over the past few decades, economic and social demands have imposed longer working hours on parents, thus affecting the work-family balance . This reOne of the reasons for which the optimal length of parental leave is crucial is its effect on decreasing parental mental health problems and stress, with mothers taking at least 12 weeks maternity leave showing less depressive symptoms than those who taking less than 12 weeks off , 12. SchOther factors have been found to play a role in determining the quality of the early mother-infant bonding relationship. Indeed, whether or not employed, mothers tend to be responsible for most household and childcare responsibilities . It is tOne cannot discuss paid maternity leave without including the effect it has on the length of breastfeeding and its relationship with mother-infant bonding, with mothers benefiting from longer paid maternity leave being more likely to exclusively breastfeed their child for longer . This waFathers have rarely been included in studies investigating the impact of parental leave on early development and parental competence; however, they play an undeniable role as the stability and quality of the couple’s relationship have been found to have a crucial effect on adjustment to parenthood , 25. OneTo our knowledge, studies investigating the effects of the length of maternity leave on breastfeeding practices and parental stress are scarce in Lebanon and the Arab region, with little to no advocacy in relation to the effects of maternity leave length on the mental health of both mother and child . The incOur study is unique in that it is the first conducted in Lebanon that focuses not only on parental leave and parental mental health but also includes their effects on work productivity and parental self-rated competence in mothers, as well as on the perceived mother-infant bond, maternal guilt and stress. It also includes factors such as length of maternity leave, whether the mother was breastfeeding or not, getting help from other family members, working because of necessity or for pleasure, subjective experience of being back at work, which will be coined as demographic factors. This study therefore aims to (1) look at the correlation between length of maternity leave, work productivity, and maternal competence, (2) investigate the correlation between length of maternity leave, working because of necessity or for pleasure, subjective experience of being back at work, and measures of mother–child adjustment including mother-infant bonding, maternal stress, and parenting guilt, and (3) look for predictors of work productivity and maternal competence in demographic variables, as well as mother–child adjustment measures, and attachment between partners.This study was a cross-sectional design, with data collected over a period of 2 months.M = 32.52, SD = 3.61), with children (55 boys and 42 girls) aged between 6 and 24 months . If a mother had more than one child within this age range, she was asked to rate statements about one of them only. The inclusion criteria included being a Lebanese parent having a child between 6 and 24 months, residing and working in Lebanon. There were no exclusion criteria.We aimed to collect data from mothers and fathers and received 99 responses in total. However, given that only 2 male participants completed the survey, they were excluded from the analysis. Thus, our sample consisted of 97 mothers, all married, aged between 25 and 42 years  [α = 0.81 [α = 0.96 in our sample.The re (HWQ)  is a 24-Two questions were added to the demographics sheet “Would you say you work mainly for financial necessity or pleasure?” and “Please rate your experience of being back to work after parental leave” in order to have a more subjective rating of participants’ working experience. Both questions were rated on a 0–10 Likert scale with 0 reflecting “working as a necessity” and “finding it difficult being back at work” respectively and 10 reflecting “working for pleasure” and”finding it easy being back at work” respectively.Parenting Sense of Competence (PSOC) [α = 0.79 for the total parental competence score, α = 0.75 for the parental satisfaction subscale, and α = 0.76 for the parental efficacy [α = 0.82, α = 0.76, and α = 0.78 respectively in our sample.The e (PSOC)  is a 17-efficacy  and α = Postpartum Bonding Questionnaire (PBQ) [α = 0.97.The re (PBQ)  is a 25-Parental Stress Scale (PSS) [α = 0.83 [α = 0.85 in our sample.The le (PSS)  is an 18Guilt about Parenting Scale (GAPS) [α = 0.89 [α = 0.85 in our sample.The e (GAPS)  is a briα = 0.89  and α = Short Experiences in Close Relationships-Revised (ECR-R short) [α ranging between 0.77 and 0.86 for attachment anxiety and 0.78 and 0.88 for attachment avoidance [α = 0.72 and α = 0.79 for the attachment anxiety and attachment avoidance subscales respectively.The R short)  is a 12-R short)  with α rvoidance . In our The survey also included a demographics sheet including information with regards to participants’ age and gender, the child’s age and gender, marital status, employment status (working or not), availability of caretaking, length of maternity leave, and whether the mother breasted or not.After receiving ethical approval from the Lebanese American University Institutional Review Board, we proceeded to collecting data using an online survey disseminated to acquaintances and through social media platforms. All questionnaires were used in their original English form. Filling out the questionnaires took approximately 15 min. All data was automatically saved anonymously through the Blue online platform.Data was analyzed using SPSS. This first aim of this study was to look at the correlations between length of maternity leave, work productivity (HWQ), and maternal competence . We used Pearson correlations for this purpose. Second, we investigated the correlation between length of maternity leave, working because of necessity or for pleasure, subjective experience of being back at work, and measures of mother–child adjustment, including mother-infant bonding (PBQ), maternal stress (PSS), and parenting guilt (GAPS) using Pearson correlations. Third, we ran two multiple linear regression models, with work productivity  as the dependent variable in the first and maternal competence  as the dependent variable in the second. Predictors in both models included measures of mother–child adjustment , demographics factors , as well as attachment to partner (ECR-R).M = 75.70. SD = 40.81), but 91% of mothers reported wanting to have had longer maternity leave. Most women (86%) breastfed their child. Means and standard deviations of the main variables are presented in Table The sample consisted of 97 mothers. Almost all (97%) women were employed, and 59% reported receiving help with child care. Participants reported taking between 7 and 365 days for maternity leave (r (95) = 0.24, p = 0.20  = 0.22, p = 0.03  = − 0.19, p = 0.05, work productivity and bonding, r (95) = 0.23, p = 0.02, maternal competence and stress r (95) =  − 0.63, p = 0.001, and guilt r (95) =  − 0.27, p = 0.01, as well as between maternal stress and guilt, r (95) = 0.22, p = 0.03  = 0.32, p = 0.001, maternal competence, r (95) = 0.32, p = 0.001, maternal stress, r (95) = − 0.22, p = 0.03, and maternal guilt, r (95) = − 0.24, p = 0.02.Similarly, significant correlations were found with work productivity, R2 = 0.25, F  = 2.86, p = 0.004). It was found that working for pleasure significantly predicted work productivity , as did ratings of the experience of being back at work , and mother-infant bonding , significantly explained 47.8% of the variance in maternal competence  = 7.88, p = 0.001; Table Results of the first regression indicated that three predictors explained 24.9% of the variance in work productivity , which might not only be negative on the fathers’ physical and mental health but also possibly affect their capacity to support their wives, hence weakening the couples’ structure as a whole. Lastly, it is important to note that the significant correlations between maternal stress, maternal guilt, and work productivity became insignificant when entered in the regression model, but that mother-infant bonding was a significant predictor of work productivity. It would therefore be interesting for future studies to explore the mediating roles of maternal guilt and stress in the relationship between healthy mother-infant bonding and higher work productivity, in an attempt to devise support groups empowering women’s sense of identity as mothers and professionals.In conclusion, despite the small correlations found in this study, we could build on these preliminary findings to suggest directions to facilitate mothers’ transition back to work and increase work productivity after having a baby. First, support groups can be created for mothers targeting stress and guilt management, providing them with strategies to balance their roles as mothers and workers. Multidisciplinary practices are increasing in Lebanon and these groups could be run through a collaboration between health care professionals (such as pediatricians or gynecologists) and psychologists. Second, focus group discussions can be conducted to better understand mothers’ perceptions of a positive work environment, as this factor was found to be crucial in decreasing maternal guilt and increasing work productivity. Finally, it takes two to tango; even though pregnancy and breastfeeding are specific to mothers, the role of fathers should not be reduced to maternal support solely. Healthy father-child bonding could well be critical to the child’s physical and mental health and should be further investigated in this specific part of the world considered simultaneously collectivistic and individualistic.


NAGS. The 3‐MGA was present at the time of persistent lactic acidosis, but improved with normalization of serum lactate, suggesting that it may reflect secondary mitochondrial dysfunction. NAGS deficiency should therefore also be considered in patients with hyperammonaemia and 3‐MGA. Studies in larger numbers of patients are required to determine whether it could be a biomarker for severe decompensations.N‐acetylglutamate synthase (NAGS) deficiency is a rare autosomal recessive disorder, which results in the inability to activate the key urea cycle enzyme, carbamoylphosphate synthetase 1 (CPS1). Patients often suffer life‐threatening episodes of hyperammonaemia, both in the neonatal period and also at subsequent times of catabolic stress. Because NAGS generates the cofactor for CPS1, these two disorders are difficult to distinguish biochemically. However, there have now been numerous case reports of 3‐methylglutaconic aciduria (3‐MGA), a marker seen in mitochondrial disorders, occurring in CPS1 deficiency. Previously, this had not been reported in NAGS deficiency. We report a four‐day‐old neonate who was noted to have 3‐MGA at the time of significant hyperammonaemia and lactic acidosis. Low plasma citrulline and borderline orotic aciduria were additional findings that suggested a proximal urea cycle disorder. Subsequent molecular testing identified bi‐allelic pathogenic variants in  We present the first published case of a patient with N‐acetylglutamate synthase (NAGS) deficiency who had 3‐methylglutaconic aciduria at the time of presentation. Previous reports suggested that the presence of 3‐methylglutaconic acid helped distinguish CPS1 from NAGS.1N‐acetylglutamate synthase (NAGS) deficiency (OMIM #237310) is a rare urea cycle disorder, with only approximately 100 cases reported in the literature to date. The defect in the NAGS enzyme (EC 2.3.1.1) prevents production of the co‐factor NAG, which is required for optimal activity of a rate‐limiting enzyme of ureagenesis, carbamoylphosphate synthetase 1 . Deficiency of NAGS therefore results in hyperammonaemia, which can present in the neonatal period and be life‐threatening.Previously 3‐methylglutaconic aciduria (3‐MGA) had been reported in many cases of neonatal‐onset CPS1 deficiency,We report the first patient in the literature with NAGS deficiency and 3‐MGA at the time of diagnosis. This patient presented with severe neonatal‐onset hyperammonaemia, with associated persistent elevation in lactate, suggesting that the 3‐MGA is more indicative of secondary mitochondrial dysfunction, rather than a disease‐specific finding. Further study of other patients with confirmed NAGS deficiency is required to establish whether 3‐MGA may be useful as a biomarker of severe metabolic decompensation.2A four‐day‐old male infant was brought in by ambulance to the local hospital because of respiratory distress. This was the third child to a nonconsanguineous couple, with no family history of any genetic disorders.The baby had been born at 41 weeks gestation weighing 2.95 kg, with APGAR scores of 9 (at one and five minutes of age) and was discharged home within a day. Standard infant formula feeds had been commenced from birth, with no concerns in the first 24 hours of life. Subsequently the baby developed progressive lethargy with poor feeding and had not woken for feeds for over 12 hours at the time of presentation.On examination, the infant was poorly responsive. He had delayed central capillary refill of five seconds, with only partial improvement with two 20 ml/kg boluses of 0.9% NaCl. There was marked respiratory distress and tachypnoea, with posturing and hypertonicity of the upper limbs also noted. Pupils were equal (3 mm) and reactive to light, and the fontanelle was not bulging. Femoral pulses were difficult to palpate. Abdominal and cardiorespiratory examinations were otherwise normal.The initial blood gas demonstrated a metabolic acidosis without respiratory compensation , with blood glucose of 5.8 mmol/L  and significantly elevated lactate of 8.7 mmol/L (RR 0.5–2.0 mmol/L). The baby was intubated and ventilated, commenced on antibiotics and transferred to the nearest tertiary pediatric center. A prostaglandin infusion was also commenced, as well as intravenous fluids with 10% dextrose at maintenance rate; the former was ceased once an echocardiogram had ruled out a duct‐dependent cardiac lesion.A serum ammonium level was markedly elevated at 1712 umol/L (RR <100 umol/L). Intravenous benzoate and arginine loading infusions were commenced, as well as a carnitine infusion, shortly after which haemofiltration was initiated. Despite this, the ammonium level was 500 umol/L after 24 hours and only normalized after 48 hours.Plasma amino acid analysis showed a marked elevation in glutamine of 2700 umol/L (RR <960 umol/L) and alanine of 1800 umol/L (RR <410 umol/L), in keeping with the hyperammonaemia and lactic acidosis respectively. Citrulline was significantly decreased,  and ornithine elevated , suggesting a proximal urea cycle disorder.Urine organic acid analysis revealed marginal elevation in orotate  which was considered unlikely to be significant diagnostically, as well as increased 3‐methylglutaconic acid  and 3‐methylglutaric acid . Fumarate  and malate  were also mildly elevated. The 3‐MGA improved to 12 mmol/mol creatinine once the serum lactate had normalized. Brain magnetic resonance imaging demonstrated both acute changes  and evidence of right middle cerebral artery territory infarction. Given the presence of 3‐MGA, a provisional diagnosis of CPS1 deficiency was made, in keeping with the revised urea cycle disorder guidelines.NAGS (c.622C > T|p.Arg208* and c.1368_1369delinsT|p.Gly457Alafs*110), consistent with NAGS deficiency. The patient had sustained a substantial neurological insult during this time, resulting in persistent irritability, hypertonia, and seizures requiring multiple antiepileptics. The molecular diagnosis was identified one month after the initial neurological insult: hence, carglumic acid was not pursued as a treatment option in view of his severe acquired brain injury and subsequent refractory status epilepticus.Next generation sequencing of genes associated with urea cycle disorders identified bi‐allelic pathogenic variants in 33.1The urea cycle is the main pathway in humans for conversion of nitrogenous waste products into urea, which can be excreted in the urine. Defects in any of the enzymes or transporters in this pathway results in build‐up of ammonia and associated clinical features.The first rate‐limiting step in the pathway is CPS1, which combines bicarbonate, ammonia, and adenosine triphosphate to generate carbamoylphosphate. The cofactor, NAG, is crucial to CPS1 function and is synthesized by the NAGS enzyme from acetyl‐Coenzyme A and glutamate. As a result, deficiency in NAGS reduces the availability of NAG for use by CPS1, effectively limiting the ability of this enzyme to function and causing hyperammonaemia.NAGS deficiency is the rarest of the urea cycle disorders, with only approximately 100 cases reported to‐date.The biochemical picture of NAGS deficiency  is identical to CPS1 deficiency. Liver enzyme activity measurements, molecular testing, or a trial of carglumic acid offer ways of distinguishing between the two conditions. However, CPS1 liver enzyme activity can be low in patients with NAGS deficiency, hence molecular testing is often preferred for diagnosis.3.23‐MGA occurs in a variety of conditionsThis “secondary” 3‐MGA is thought to occur due to nonspecific mitochondrial dysfunction. Su and Ryan (2014) suggest that altered mitochondrial metabolism results in increased NADH in the inner mitochondrial space, inhibiting citric acid cycle enzymes and causing accumulation of acetyl‐CoA.As well as occurring in the primary and secondary 3‐MGAs, 3‐MGA can also occur in other inborn errors of metabolism. It is seen most commonly with deficiency of the enzyme hydroxymethylglutaryl‐CoA (HMG‐CoA) lyase, the next step in the leucine catabolic pathway following 3‐methylglutaconyl‐CoA hydratase.SERAC1 deficiency (OMIM #614739) or TMEM70 deficiency (OMIM #614052).In a similar vein, Rokicki et al. (2017) noted 3‐MGA at the time of diagnosis, in nine of ten neonates with CPS1 deficiency.In the current case, a diagnosis of CPS1 deficiency was initially considered, as suggested by the diagnostic algorithm in the most recent urea cycle disorder guidelines.4We present the first reported case of 3‐MGA in NAGS deficiency. Whilst the elevated lactate may be non‐specific in a critically unwell infant, the additional findings of increased malate and fumarate in our patient are consistent with previous literature suggesting that 3‐MGA is a secondary finding, which occurs due to mitochondrial dysfunction in a range of inborn errors of metabolism. Whilst we were not able to serially monitor the extent of the 3‐MGA in our patient over time, it may be an indicator of severity of metabolic decompensation. This may be useful in patients with other urea cycle disorders with more variable phenotypes such as OTC deficiency. NAGS deficiency should be considered within the differential diagnosis for patients with 3‐MGA where a proximal urea cycle disorder is suspected; a trial of carglumic acid should be initiated if active management is being pursued.Arthavan Selvanathan: conception and design, ethics submission, collecting data, writing manuscript, reviewing manuscript.Kalliope Demetriou: analysis/interpretation of data, drafting of article.Matthew Lynch: analysis/interpretation of data, drafting of article.Michelle Lipke: analysis/interpretation of data, drafting of article.Carolyn Bursle: analysis/interpretation of data, drafting of article.Aoife Elliott: analysis/interpretation of data, drafting of article.Anita Inwood: conception and design, ethics submission, analysis/interpretation of data, drafting of article, reviewing manuscript.Leeanne Foyn: analysis/interpretation of data, drafting of article.Brett McWhinney: analysis/interpretation of data, drafting of article.David Coman: conception and design, analysis/interpretation of data, drafting of article, reviewing manuscript.Jim McGill: conception and design, analysis/interpretation of data, drafting of article, reviewing manuscript.We declare no conflicts of interest.Approval for publication was obtained from the local Human Research Ethics Committee (Case Report #1338344).There were no sources of funding that were required for this project.


Janus Kinase (JAK) inhibitors target the JAK and signal transducer and activator of transcription (STAT) signaling pathway that modulates T helper (Th)1, Th2, Th17, and Th22 signaling and is involved in many immune‐mediated diseases. Baricitinib, an oral selective JAK‐1, and ‐2 inhibitor, has recently been approved for adults with moderate‐to‐severe atopic dermatitis (AD).A 52‐year‐old man, who works as a builder, was diagnosed with severe hyperkeratotic CHE for 6 years. He has a history of asthma in childhood (not classified), allergic rhino‐conjunctivitis, and no history of AD. Routine patch testing was performed and the results were negative. His CHE did not improve, despite avoidance of work‐related irritants. Treatment history included ultra‐potent topical corticosteroids, alitretinoin, acitretin, cyclosporine, apremilast , and methotrexate (discontinued due to side effects).A 55‐year‐old woman, who works as an administrator, had severe AD with concomitant severe atopic CHE for 5 years. She has a history of allergic asthma, allergic rhino‐conjunctivitis, food allergies, and kerato‐conjunctivitis. She had no relevant exposure to irritants, and there were no contact allergies with clinical relevance to the hands. Treatment history included ultra‐potent topical corticosteroids, topical calcineurin inhibitors, prednisolone, methotrexate, cyclosporine, omalizumab , and dupilumab (discontinued due to side effects).Both patients were treated with baricitinib (4 mg tablet once per day), because Case 1 had severe refractory CHE and Case 2 had AD with concomitant severe CHE, that had great impact on their quality of life.Case 1 presented at baseline with ‘severe’ CHE according to the photographic guideCase 2 presented at baseline with “severe” CHE and a HECSI score of 47 severe4. BaricStaphylococcus aureus, while the culture of her scleral lens was negative.  4, IL‐13, IL‐22 and IL‐31,Our hypothesis was strengthened by previous literature on three different JAK‐inhibitors that showed promising efficacy on CHE. Recently, two phase 3 trials with upadacitinib, an oral JAK‐1 inhibitor, presented significant improvements in mild‐to‐severe CHE in patients with moderate‐to‐severe AD compared to placebo at 16 weeks.In the near future, patients with moderate‐to‐severe refractory CHE, regardless of subtype, may benefit from treatment with JAK‐inhibitors. Further research is necessary to investigate the efficacy and safety profile on a larger scale.M.L.A. Schuttelaar received consultancy fees from Sanofi‐Genzyme and Regeneron Pharmaceuticals; and is an advisory board member for Sanofi‐Genzyme, Regeneron Pharmaceuticals, Pfizer, LEO Pharma, Lilly. The other authors have no other conflicts to declare.Marie L.A. Schuttelaar: Conceptualization (lead); investigation (supporting); methodology ; supervision (lead); writing – review and editing . Fieke M. Rosenberg: Conceptualization ; data curation (lead); formal analysis (lead); investigation (lead); methodology ; project administration (lead); visualization (lead); writing original draft (lead); writing ‐ review and editing (supporting). Laura Loman: Conceptualization, ; data curation (supporting); methodology ; supervision (supporting); writing – review and editing . All authors read and approved the final manuscript.Table S1 Patient characteristics and outcome measures.Click here for additional data file.


Lactobacillus paracasei N1115 (LC‐N1115) to attenuate obesity. Four‐week‐old male C57BL/6J mice were fed a HFD for 12 weeks to induce obesity and were then randomized to supplemented placebo or LC‐N1115 treatment group for another 12 weeks. LC‐N1115 treatment reduced weight gain and liver fat accumulation as well as triglyceride, total cholesterol, and low‐density lipoprotein cholesterol levels. The administration of LC‐N1115 suppressed the expression of fatty acid synthase, interleukin‐1 β, and toll‐like receptor 4. Notably, the operational taxonomic units that negatively and positively correlated with the obesity phenotypes were enriched and reduced, respectively, in the LC‐N1115 treatment group. These results indicate that LC‐N1115 attenuates obesity by modulating the gut microbiota and the expression of lipid synthesis and proinflammatory cytokine genes.Disruption of the microbial structure of intestinal bacteria due to a high‐fat diet (HFD) is closely associated with metabolic disorders, such as obesity and type 2 diabetes. Probiotics are known to modulate the gut microbiota; therefore, we demonstrated the capability of  Lactobacillus paracasei N1115 relieved high fat diet induced obesity. Lactobacillus paracasei N1115 decreased body weight, fat accumulation and hyperlipidemia by increasing the abundance of beneficial bacteria (Allobaculum and L. Zeae) and decreased harmful bacteria (Turicibacter). Worldwide, the prevalence of obesity has approximately tripled since 1975. Globally, approximately 1.9 billion adults were overweight, and of these, more than 650 million were obese in 2016 , a novel strain with probiotic properties, is isolated from traditional homemade dairy products in Inner Mongolia, China. As a probiotic strain, LC‐N1115 exhibits a high‐level resistance to acidic and bile stresses, and its fermented milk products have been patented  and is considered safe for consumption. Therefore, LC‐N1115 has a high commercial value. Although several Lactobacillus spp. can reduce obesity, different bacterial strains in the same species or genus may have contrasting responses to the same intervention. Thus, it is essential to investigate the modulatory effect of probiotics on the gut microbiota at the strain level  and the other group was fed HFD (n = 32) to induce obesity for 12 weeks : obese mice receiving HFD and placebo (HFD group); those receiving HFD and a daily high dose of LC‐N1115 ; those receiving HFD and a daily moderate dose of LC‐N1115 ; and those receiving HFD and a daily low dose of LC‐N1115  via oral gavage for 12 weeks.Four‐week‐old male C57BL/6J mice were purchased from Beijing HFK Bioscience Co. Ltd. Animals were housed under conditions of constant temperature and humidity , with a 12 h light/dark cycle. During adaptation (first week), all mice were fed a normal diet (ND). After 1 week of acclimatization, they were randomly categorized into two groups: one group was supplemented with ND as a control  analysis. Stool samples were collected 1 day before sacrifice. Before sacrifice, the mice were fasted for 12 h and anesthetized with diethyl ether.2.2g for 15 min to detect total cholesterol (TC), triglyceride (TG), and high‐/low‐density lipoprotein cholesterol (HDL‐c/LDL‐c). These blood indicators were detected using an automatic biochemical analyzer (Hitachi 7600‐020) at the core clinical laboratory of No. 3 Hospital of Beijing University.Serum samples were obtained by centrifuging the blood samples at 3170 2.3Liver and epididymal white adipose tissues were dissected and fixed by immersion in 4% w/v formaldehyde in phosphate buffer at room temperature for 24 h and were embedded in paraffin for staining with hematoxylin and eosin (H&E). A fraction of the main liver lobe was fixed frozen in Tissue‐tek in liquid nitrogen. Frozen sections were sliced and stained with oil red O at 60°C for 10 min using 0.5% oil red O dissolved in propylene glycol. The sliced sections were then counterstained, and images were obtained under a microscope at 200× magnification.2.4Genomic DNA was extracted from the fecal samples of all mice using the bead‐beating method. The extracted DNA was used as a template for the amplification of the V4 region of the 16S rRNA. Herein, specific primers with barcodes, i.e., primer sets containing Illumina adapters, were used. Subsequently, the DNA samples were sequenced using the Illumina MiSeq platform. Clean reads were clustered into 16S rRNA operational taxonomic units (OTUs) within the threshold of 97% identity using UCLUST. The α‐diversity index was calculated using Mothur. The results of the β‐diversity principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) were computed using Jaccard.2.5−ΔΔct method, and GAPDH was used to normalize the data. The primers used in this study are listed in Table Total RNA was extracted from the liver and adipose tissues using TRIzol reagent for quantitative reverse transcriptase PCR analysis. RNA purity was assessed using NanoDrop (Thermo Fisher). Total RNA was reverse transcribed into cDNA using a reverse transcription kit. SYBR Premix Ex Taq, (TaKaRa) was used for RT. All reactions were performed in a single 96‐well reaction plate. Data were calculated using the 22.6p < .05 was considered statistically significant.Data were analyzed using SPSS 21 and were represented as mean ± standard deviations. Statistical comparisons were performed using two‐way analysis of variance followed by Duncan's multiple range tests, and 33.1LC‐N1115 supplementation could counteract the weight gain induced by HFD . The obese mice with HFD were successfully established. After probiotic supplementation for 12 weeks, the body weight of the animals in the NH, NM, and NL groups was lower than those in the HFD group by approximately 9.6%, 12.2%, and 10.1% . These results suggest that D Figure .The HFD group also showed a significant increase in the white fat mass compared with the ND group. Probiotic supplementation decreased fat accumulation as the partial white fat mass of the probiotic group was lower than that of the HFD + placebo group Figure .3.2LC‐N1115 treatment decreased the TG levels by 20.14%, 8.63%, and 17.99% in the NH, NM, and NL groups, respectively. LDL‐c levels in the HFD group were significantly elevated compared with those in the ND group. In contrast, the plasma LDL‐c levels of mice supplemented with probiotics were reduced by approximately 9.3%, 20.48%, and 31.59% in the NH, NM, and NL groups, respectively. However, the plasma HDL‐c levels were similar between the HFD and probiotic groups, and plasma TG levels were not significantly different. HDL‐c/LDL‐c, HDL‐c/TC, and TG/HDL‐c ratios indicated the atherogenic indices. The higher the HDL‐c/LDL‐c and HDL‐c/TC ratios, the lower would be the atherosclerosis risk. Furthermore, lower TG/HDL‐c ratio indicated that the individuals were healthy. The HDL‐c/TC ratio in the HFD group was significantly decreased compared with that in the ND group. All doses of probiotic treatment increased the HDL‐c/TC ratio. Furthermore, the HDL‐c/LDL‐c ratio in the ND and HFD groups was 5.14 and 2.62, respectively; however, the HFD group receiving probiotics showed increased ratios of 3.39, 3.23, and 3.51 in the NH, NM, and NL groups, respectively. In addition, the TG/HDL‐c ratio in the probiotic groups was lower than that in the HFD group. In particular, this ratio in the NH group was 0.44, which was 43% lower than that in the HFD group. The other two groups also showed different levels of improvement. In summary, LC‐N1115 treatment attenuated hyperlipidemia, but no dose response was observed with the probiotic supplementation.HFD increased the TC, TG, and LDL‐c levels but decreased the HDL‐c levels Table . As show3.3LC‐N1115 on preventing the development of liver steatosis. As shown in Figure LC‐N1115 inhibited lipid droplet deposition in the liver, as shown in Figure LC‐N1115 decreased the stored fat accumulation significantly.We used H&E staining to verify the effect of 3.4To analyze the effect of probiotics on the microbiota structure, we performed sequencing of the V4 region of the bacterial 16S rRNA gene from the fecal samples. Sequencing reads were assigned to OTUs with 97% sequencing similarity for assessing alpha diversity and were represented using rarefaction curves. At this sequencing depth, the rarefaction curves appeared to plateau in the samples, as shown in Figure The differences among the gut microbial communities (β‐diversity) were examined using heatmap, PCA, and NMDS according to the abundance of OTUs, as shown in Figure LC‐N1115) and the obesity index. Significant correlations were obtained between the obesity‐related indices  and the abundance of the gut microbiota. Among these taxa, Allobaculum and Lactobacillus were strongly negatively correlated with body weight and white fat mass, suggesting that they play the most crucial role in preventing obesity. In addition, Turicibacter was positively correlated with the body weight, white fat mass, and LDL‐c level, indicating that it plays an essential role in the development of obesity. Notably, three doses of LC‐N1115 reversed the changes in 11 OTUs that were altered by HFD, as shown in Figure LC‐N1115 treatment reduced the OTUs that were positively correlated with the obesity phenotypes. These results suggest that the LC‐N1115 treatment altered the gut microbiota of obese mice in both composition and structure.HFD feeding altered the abundance of 264 OTUs. Supplementation with NH, NM, and NL enhanced or reduced the abundance of 131, 104, and 126 OTUs, respectively, resulting in changes in 204 distinct OTUs. Spearman correlation analysis was performed to detect the association between the gut microbiota  gene was not significantly altered compared with that in the HFD group. The cholesterol 7‐hydroxylase (CYP7A1) and LDL receptor (LDLR) mRNA expression in the NM and NL groups was enhanced compared with that in the HFD group. The expression of the proinflammatory cytokine gene, IL‐1β, was significantly reduced in the NH group and was also reduced in the NM and NL groups but had no significant difference.We measured the proinflammatory cytokine and adipogenesis gene expression in the epididymal adipose tissues and liver. As shown in Figure LC‐N11115 treatment groups compared with that in the HFD group. The mRNA expression of toll‐like receptor 4 (TLR4) was significantly enhanced in the HFD group compared with that in the ND group and was significantly reduced in mice receiving LC‐N1115 treatment. The expression of the IL‐1β gene was reduced in the probiotic groups compared with that in the HFD group; however, the difference was not significant.In epididymal adipose tissues, as shown in Figure 4LC‐N1115 treatment attenuates obesity in HFD‐induced obese mice and attempted to determine the underlying mechanism.Although obesity is not fatal, related metabolic diseases threaten the safety of human life. Structural disorders of the gut microbiota and low‐grade inflammation are significant characteristics of obesity. Studies have reported that feeding HFD to C57BL/6J mice results in obesity. The C57BL/6J mice strain used in this study was highly susceptible to diet‐induced obesity. Abundant scientific evidence supports the role of probiotics in the prevention of obesity. Probiotics have multiple and diverse effects on the host: enhancing barrier integrity, modulating the immune system, and fighting against pathogenic bacteria. Modifying the gut microbiota composition via probiotics is an essential role for modulating energy metabolism, and it acts as a critical modifier of obesity and its related metabolic parameters , and an increase in the abundance of pathogenic bacteria , such as butyrate and propionate  could regulate the inflammatory responses in mice with colon cancer by altering the microbiota structure. The abundance of Allobaculum increased significantly in the probiotic group  and decreased that of harmful bacteria (Turicibacter), possibly contributing to weight loss.Each dose of LC‐N1115 on reducing obesity. Our results suggest that LC‐N1115 reduced body weight gain and white fat tissues. The plasma cholesterol levels of the NM and NL groups were significantly lower than those of the HFD group. All doses of LC‐N1115 can reduce the plasma TC levels, which had a statistical significance in the NM and NL but not in NH groups, compared with the HFD group. We measured the plasma LDL‐c and HDL‐c levels to determine the cholesterols that contribute to the change in TC levels. Our results suggest that different doses of probiotics have a distinct effect on plasma cholesterol levels. Accordingly, several studies reported that B. bifidum PTCC1644 increased plasma HDL‐c levels and reduced LDL‐c levels, whereas L. acidophilus NS1 reduced plasma LDL‐c levels with no change in HDL‐c levels .Table S1Click here for additional data file.


Acanthamoeba keratitis is often caused when Acanthamoeba contaminate contact lenses and infect the cornea. Acanthamoeba is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied Acanthamoeba’s behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites.Acanthamoeba  were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of Acanthamoeba which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids.Six clinically and/or scientifically relevant strains of Acanthamoeba trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed.Acanthamoeba to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote Acanthamoeba aggregation and encystment, and Acanthamoeba spheroids obstruct multi-purpose solutions from disinfecting Acanthamoeba.Aggregation/encystment is a protective mechanism which may enable  Acanthamoeba keratitis (AK) is a serious ocular infection that is extremely difficult to treat and can lead to blindness . Strains used and their information can be found in 2PO4, 10 μg vitamin B12, and 15 mg L-methionine per liter of distilled deionized water). Media was adjusted to a pH of 6.6–6.95 with 1 M NaOH and autoclaved at 121°C for 20 min before storing at room temperature for use within 3 months. ¼ Ringer’s solution was used to harvest organisms. To create a homogenous population of Acanthamoeba trophozoites, Acanthamoeba were scaled up in fresh AC6 media 24 h to testing. Cells were then collected and centrifuged at 500 g for 5 minutes, followed by a wash and resuspension using ¼ Ringer’s solution. Count seeding was confirmed via manual counting using a hemocytometer.As previously described , trophozInformation about contact lenses and multi-purpose solutions used and their details can be found in Acanthamoeba were allowed to settle for 10 min and then each well was imaged using a 2×2 stitched large image (NIS Elements AR 3.2) at 4× magnification for a continuous period of 12 h, with each well being imaged every 3 min. Later timepoints at 24, 48, and 72 h were also conducted for 30 min of continuous imaging with each wellbeing imaged every 3 min. All videos were concatenated such that each contact lens had a single video file containing 274 images representing the entire 72-h period of observation. Seven contact lenses plus a no lens control were executed for each replicate. Six replicates were conducted for each strain of Acanthamoeba and six strains of Acanthamoeba were utilized.Contact lenses were trimmed to 12 mm utilizing a biopsy punch. In a 48-well plate, a silicone O-ring was placed at the bottom of each well . The conAcanthamoeba. Count and normalized particle size were graphed as a function of time for each strain.Timelapse videos were recorded in grayscale using bright-field microscopy. Using ImageJ (version 1.53q), videos were converted into a high-contrast, binary format for analysis . Brieflyn = 6) of identical conditions were then averaged by timepoint, and standard error of the mean calculated. Normality was assessed using a Shapiro–Wilk test, and size and count  at any timepoint, and within each condition over time) were analyzed via two-way repeat measure ANOVA with a post-hoc Tukey’s multiple comparisons test (GraphPad Prism 9.2.0). An alpha of 0.05 was used to assess significance in all comparisons.To allow the amoeba to settle onto the lens and compensate for size differences observed between strains, the size of each strain-lens condition was normalized to its own 0.5–1.0 h reference time . AdditioAcanthamoeba ATCC 30461 trophozoites were seeded into Biofloat plates  at a density of 8, 16, 32, 125, 250, 500, 1,000, or 2,000 cells/well in replicates of 8 per plate. The experiment was conducted across 4 independent 96-well plates per time period. Timelapse images were taken of each spheroid every 5 min for 3 h, then every 15 min for the subsequent 6 h, and then every 30 min from hours 9 through 24. Each well made one spheroid and the timelapse videos were converted to binary images in the same fashion as the contact lens videos. Each spheroid video was analyzed to determine the number of trophozoites per spheroid, as well as the area of each spheroid as a function of time. A standard curve was generated as a function of trophozoite count vs. spheroid size over time using spheroid area  plates or contact lenses as described above . Spheroiarvation ] were starvation . EthidiuAcanthamoeba trophozoites were passaged and collected to create a pellet of 2 × 107 cells. Pellets were resuspended in CLB containing 0.25% 2-mercaptoethanol and 0.7 mg/ml RNase A and incubated at 54°C–56°C for 60 min at 1200 RPM in an Eppendorf Thermomixer R. Proteinase K was added to 7 U/ml and incubated with shaking for an additional 20 min. Two sequential chloroform:isoamyl alcohol extractions were performed, followed by an isopropanol precipitation. Crude extracts were dissolved at 54–56°C in Qiagen G2 buffer containing 0.2 mg/ml RNase A and 15 U/ml Proteinase K and further purified by through a 20/G genomic tip according to the manufacturer’s instructions. DNA purity was assessed by agarose gel electrophoresis and quantified using a Take3 Micro-Volume Plate with a Synergy H4 plate reader and Gen5 Software . Illumina sequencing, Oxford Nanopore sequencing, and analysis were performed by Seqcenter . Quality control and adapter trimming was performed with Crude DNA extracts were prepared from ATCC 30461 with a cetyl trimethylammonium bromide-based procedure using Carlson Lysis Buffer  samples were collected from the lenses directly into TRIzol  and RNA was isolated immediately using the PureLink RNA Micro Scale Kit . RNA integrity was assessed by agarose gel electrophoresis and quantified using a Take3 Micro-Volume Plate with a Synergy H4 plate reader and Gen5 Software. For each condition and time point, six independent replicates were prepared on separate days. RNA sequencing and analysis was performed by Seqcenter. Samples were then DNase treated with Invitrogen DNase (RNase free). Library preparation was performed using Illumina’s Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10 bp IDT for Illumina indices. Sequencing was done on a NextSeq2000 giving 2x50bp reads. Quality control and adapter trimming was performed with via STAR  of RNA sequencing results was performed in Python version 3.8.8 using scikit-learn version 0.24.1. Parameters used were 0.5 damping, a maximum of 200 iterations, 15 unchanged iterations until convergence. The dimensional inputs for Euclidean distance-based affinity propagation were composed of the following gene expression comparisons: lehfilcon A vs. polystyrene control at 4, 12, or 24 h, samfilcon A vs. polystyrene control at 4, 12, or 24 h, and comfilcon A vs. polystyrene control at 4, 12, or 24 h. The average log2 fold changes in gene expression from six RNA sequencing replicates were used as value inputs for each dimension. Only significantly differentially expressed genes were included in analysis. The resulting gene clusters were further reduced into phenotypic clusters by correlational distance-based affinity propagation on the median expression change of all genes included in each primary cluster. Heatmaps of the resulting gene subsets were constructed in GraphPad Prism 9.2.0.Locus tags from the genomic database created by DNA sequencing  and the associated information with each tag was used to identify homologs and inferred gene function based on the known function of the ATCC 30010 strain. The amino acid sequence of each ATCC 30461 gene was used to search NCBI’s BLAST  to determine percent homology with known genes of all species. As homology with ATCC 30010 was most prevalent, this and the associated Neff strain locus tag were used to estimate gene function. Neff strain locus tags (ACA1_*) were searched in the AmoebaDB informatics resource repository to define the GO (Gene Ontology) terms for each gene. Neff strain locus tags were also used to identify the associated protein ID in either the UniProt or KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, and significant common pathways were identified using STRING .Acanthamoeba ATCC 30461.The disinfection efficacy of individual trophozoites, spheroids, and cysts were evaluated in a disinfection study. The disinfection study was conducted concurrently across conditions with three independent inoculums of Acanthamoeba trophozoites such that wells contained either 100, 375, or 1,000 cells per well.Spheroids: The wells of a 96-well Biofloat plate  were seeded with a serial dilution of Cysts: Cysts were generated by starvation on non-nutrient agar plates. Briefly, trophozoites were harvested into ¼ Ringer’s and plated on non-nutrient agar plates and incubated at 28°C for a minimum of 10 days. After incubation, cysts were rinsed from plates using ¼ Ringers and stored at 4°C until testing.Acanthamoeba cells such that wells contained either 100, 375, or 1,000 cells per well.Trophozoites and cysts: The wells off a 96-well flat bottom tissue culture plate were seeded with a serial dilution of Escherichia coli was added to each well and the 48-well plates taped and incubated for 21 days at 28°C.Spheroids, trophozoites, and cysts: Eight replicates of each concentration were conducted per multi-purpose solution and independent inoculum. Cells were incubated in the wells for either 12 or 24 h prior to being exposed to multi-purpose solutions. Excess ¼ Ringer’s was removed from each well, and 200 μl of the designated multi-purpose solution was added to the well. At disinfection time (4 or 6 h), the MPS was removed and 25 μl of Letheen broth was added to each well to neutralize any remaining biocide. The total contents of each well were transferred to a 48-well plate containing 500 μl of non-nutrient agar. Heat-killed via 2-way ANOVA, with post hoc Tukey’s test (GraphPad Prism 9.2.0). Significance was set at 0.05.After 21 days, all plates were scored for growth. Each multi-purpose solution/cell type/incubation length/inoculum was quantified as a % outgrowth for a particular condition. Comparisons between cell type, incubation length and multi-purpose solutions were conducted and analyzed Acanthamoeba behavior on popular lens materials, we investigated six different potentially keratitis-causing strains of Acanthamoeba for 72 h on seven different lens materials, as well as a polystyrene no lens control in which Acanthamoeba appeared to move independently and consistently similar to previously examined surfaces (Acanthamoeba on their own without conditions otherwise changing). To quantify behavior, we determined both the particle count  and the particle size. As spheroids form, an inverse relationship between particle count and average size is observed .To understand the differences in surfaces . An expep < 0.05) from timepoints 1.5–2.0 h to 9.0–9.5 h, with comfilcon A, senofilcon A, samfilcon A, fanfilcon A, and omafilcon A being significantly lower through at least 12 h. Similarly, with all strains data combined, all lens materials except etafilcon A demonstrated a significantly higher particle size from at least 3.0–3.5 h through 72 h than lehfilcon A (p < 0.05). In this analysis, etafilcon A demonstrated a significantly higher particle size than lehfilcon A from 6.0–6.5 h through 11.5–12 h. All lens materials were also analyzed for their change compared to their baseline (0.5–1.0 h) in both particle count and particle size. When all strains were combined it was noted that lehfilcon A, omafilcon A, and etafilcon A did not demonstrate a significant change compared to their particle count baseline, while comfilcon A, senofilcon A, samfilcon A, and fanfilcon A did, beginning by 1.5–2.0 h to 2.0–2.5 h (p < 0.05). Some lens materials  maintained this difference through 72 h, while others  demonstrated a relative return to their baseline before the end of the experiment. Overall, when combining the data from all six Acanthamoeba strains examined , although they were consistently observed to be evenly dispersed throughout the well both in this study and in previous ones . Furtherp < 0.05). The exceptions to this were etafilcon A, which demonstrated little aggregation in ATCC 30010 or ATCC 50703. The ATCC 50703 strain was the only strain tested where cell count continued to increase over time for senofilcon A, samfilcon A, and omafilcon A. Senofilcon A produced the highest particle size  in ATCC 30010 and ATCC 30461, while fanfilcon A and/or comfilcon produced the largest spheroid size in ATCC 50370, ATCC 50703, and ATCC PRA-115, and omafilcon A produced the largest spheroid size in ATCC 50702. The particle size of the no lens control and lehfilcon A were not statistically different from each other at any time point in any strain tested. It is noted that some strain-material combinations produced a pronounced peak in spheroid size in later timepoints, which may be slightly reduced before 72 h (such as ATCC 50703 comfilcon A) or may continue to grow through the 72 h timepoint (such as ATCC 30461 comfilcon A), while the majority of other strain-material combinations demonstrated a more consistent particle size from at least 3.0–3.5 h onwards. To note, ATCC 50703 had the least stable spheroids of all strains, with several lens materials showing susbtantial early aggregation followed by deaggregation at later timepoints.When examined individually , most stp < 0.05). Comfilcon A, senofilcon A, samfilcon A, and fanfilcon A maintained significantly larger spheroids than the No Lens Control and lehfilcon A at hours 6, 12, and 24. Omafilcon A showed signficant aggregation at hours 6 and 12, with a moderate dispersal of the spheroids by hour 24. Comfilcon A also demonstrated the largest spheroids by size  while aggregating materials such as senofilcon A and samfilcon A were more likely to produce several spheroids of more moderate size .Each spheroid (defined as more than four cells touching at once) was analyzed to determine the number of cells it contained from baseline (0.5 h) through hour 24 . Each leAcanthamoeba cysts within spheroids and evaluate if aggregation was a similar process regardless of material trigger, we utilized fluorescent confocal microscopy and three different spheroid-forming conditions: a BIOFLOAT™ spheroid plate, senofilcon A, and comfilcon A  indicates for the first time that Acanthamoeba spheroids may be forming an extracellular matrix as they age. Fluorescein diacetate was noted in spheroids at all timepoints and in both trophozoites and cysts, although it was more prominent in older spheroids, indicating mature spheroids are still viable, metabolically active, infectious cells  on lehfilcon A, comfilcon A, samfilcon A, and the no lens control  were identified  in lehfilcon A vs. comfilcon A and samfilcon A, including two tRNA genes that were removed from the detailed analysis. The resulting 21 genes were visualized entified . All 21 entified  and alteAcanthamoeba in different cellular formations  in plates for 12 or 24 h . The regulatory requirements for Acanthamoeba multi-purpose solution disinfection efficacy continue to lag despite calls for action  where most multi-purpose solutions offer some level of disinfecting ability.reatment . While sifecycle . Even nolineated . Equallyaffected . Most ouaffected . Most dir action . Howeverthamoeba  and effede based . HoweverAcanthamoeba keratitis with small outbreaks associated with poor MPS efficacy, little examination has been done to evaluate the role of the contact lens in Acanthamoeba pathogenesis. Here, we have described a novel finding that many strains of Acanthamoeba will aggregate in response to specific contact lens materials. This is a significant potential risk to patients as Acanthamoeba aggregation is a precursor to encystment  to many contact lens materials, aggregating and initiating encystment (< 4 h) despite no chemical induction beyond the contact lens surface. No nutrients are provided on the lens but this is not a long enough time period to be considered starvation. The properties of the aggregation-inducing contact lens materials that trigger this response are unknown and would require future investigation, but this response could potentially be due to diminished water content or surface topography of the contact lens materials. Acanthamoeba prefers to exist at the interfaces between soil and water, and surfaces indicating a strong water-aversion  and PRJNA905484 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA905484).The data presented in the study are deposited in the NCBI BioProject repository, accession numbers PRJNA903937 (AC, RP, RW, JK, CR, PS, BP, and MC were involved in conceptualization. AC, RP, RW, MT, EM, VH, JK, BP, and MC were involved in data curation and methodology. AC, RP, CR, BP, and MC were responsible for formal analysis. AC, RP, VH, JK, BP, and MC were involved in validation. AC, RP, RW, MT, EM, CR, BP, and MC conducted visualization. PS and MC were responsible for project administration, resources, and supervision. AC and MC conducted writing of the original draft. All authors participated in manuscript review and editing. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. All authors contributed to the article and approved the submitted version.This research was funded by Alcon, which provided all financial support. CR reports grants from Purdue University, outside of the scope of the submitted work.The funder was not involved in the study design, collection, analysis, interpretation of data, or the writing of this article. The funder approved the decision to submit for publication. All authors except CR are employees of Alcon Research.The remaining author declares 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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1089092/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Bone implant‐associated infections induced by bacteria frequently result in repair failure and threaten the health of patients. Although black phosphorus (BP) material with superior photothermal conversion ability is booming in the treatment of bone disease, the development of BP‐based bone scaffolds with excellent photothermal stability and antibacterial properties simultaneously remains a challenge. In nature, chloroplasts cannot only convert light into chemical energy, but also hold a protective and defensive envelope membrane. Inspired by this, a self‐defensive bone scaffold with stable photothermal property is developed for infected bone defect therapy. Similar to thylakoid and stroma lamella in chloroplasts, BP is integrated with chitosan and polycaprolactone fiber networks. The mussel‐inspired polydopamine multifunctional “envelope membrane” wrapped above not only strengthens the photothermal stability of BP‐based scaffolds, but also realizes the in situ anchoring of silver nanoparticles. Bacteria‐triggered infection of femur defects in vivo can be commendably inhibited at the early stage via these chloroplast‐inspired implants, which then effectively promotes endogenous repair of the defect area under mild hyperthermia induced by near‐infrared irradiation. This chloroplast‐inspired strategy shows outstanding performance for infected bone defect therapy and provides a reference for the functionality of other biomedical materials. + and enhanced photothermal stability can achieve bacterial inhibition and photothermal osteogenesis in vivo.For infected bone defect therapy, developing black phosphorus (BP)‐based scaffolds with acceptable photothermal stability and antibacterial properties remains a challenge. Inspired by chloroplasts, a polydopamine multifunctional “envelope membrane” is induced to stabilize BP into the scaffold and synchronously grow Ag nanoparticles. The self‐defensive platform with pH‐responsive release of Ag Nevertheless, antibiotic resistance, disquieting cytotoxicity of over‐released nanoparticles, and weak osteo‐integration and osteo‐inductivity of the scaffold are still limitations of these implantations. Hence, developing bioactive scaffolds with satisfactory osteogenic potential and antimicrobial ability is still a challenge for the treatment of bone implant‐associated infections.Implant‐associated infections remain a serious problem in bone defect therapy and frequently result in repair failure, potentially threatening the health of patients.1, 2 Over the last few years, advances have been made by integrating carbon nanotubes or graphene oxide with scaffolds to facilitate osteogenesis. Nevertheless, the biotoxicity and immune responses of these inorganic particles remain controversial. Recently, black phosphorus (BP) nanosheets, which are promising 2D nanomaterials, have been widely researched and applied in the field of photothermal therapy owing to their high photothermal conversion efficiency and biological activities. Besides, the degradation products of BP provide phosphorus sources for bone regeneration. Unfortunately, the interaction between BP and the scaffold matrix is weak, and BP is degraded easily under physiological conditions, which weakens the thermal properties and limits its application in long‐term photothermal therapy of bone repair. For this reason, it is necessary to stabilize BP in a scaffold to achieve persistent photothermal stability. However, stabilizing BP in bone repair scaffolds and simultaneously endowing the scaffold with good antimicrobial properties has not yet been achieved.Photothermal therapy has proven to be an efficient strategy to improve the osteogenesis of implants.8, 9 Therein, thylakoids, which are interconnected through a stroma lamella network, are responsible for light‐harvesting and energy‐transducing via photosynthetic pigment molecules on their surface. Strikingly, the envelope membrane (lipid layer) of chloroplasts has special physical and biological functions. The envelope membrane acts as a natural barrier that can protect the structural integrity inside the chloroplast. Furthermore, special proteins, such as EDS5, localized on the envelope membrane, are responsible for the biosynthesis and transportation of immunity‐related salicylic acid from chloroplasts to the cytoplasm. Therefore, the envelope membrane of plant chloroplasts can adaptively defend against pathogens, such as bacterial infections and viruses.The chloroplast provides hints for the design of therapeutic scaffolds with photothermal stability and antimicrobial ability. As a sunlight‐driven metabolic organelle in higher plants, chloroplasts maintain the normal life activities of all organisms on earth.20 The was induced to stabilize BP nanosheets into the scaffold and synchronously realize in situ growth and anchoring of the antimicrobial agent of silver (Ag) nanoparticles. Ultimately, a chloroplast‐inspired 3D CS/PCL/BP/PDA@Ag scaffold was successfully developed  light and convert it to heat, while 3D porous chitosan and polycaprolactone (CS/PCL) composite fiber networks similar to the interlinked stroma lamella are used to interconnect these BP nanosheets. To further endow the BP‐based scaffold with practical stability and self‐defensive functionality, a mussel‐inspired polydopamine (PDA) multifunctional “envelope membrane” with rich catechol groups24 wasd Scheme .22.1Figure  In addition, Ag nanoparticles grown in situ had a diameter of ≈100 nm (as shown in the inset with a rectangular dashed line). It could be seen from both fractured cross section and surface images , X‐ray diffraction (XRD), X‐ray photoelectron spectroscopy (XPS), and Raman spectra , the hydrophilicity of the obtained CS/PCL/BP/PDA@Ag scaffold was highly improved, which is typically beneficial for cell spreading , (221), (203), (104), (313), (422), and (215) crystallographic planes of hydroxyapatite (JCPDF#09‐0432), respectively  after soaking in air‐exposed water for different time intervals  showed a slight decline under the protection of PDA after 14 days (89.3% of the ∆T of newly prepared scaffolds). In the meantime, the attenuation of temperature was remarkably distinct in the scaffold without the PDA layer (51.8% of the ∆T of the newly prepared scaffolds).In this work, a PDA “envelope membrane” was skillfully utilized to protect and passivate the BP in the scaffold so as to enhance the durable photothermal stability of the BP‐based scaffold. Consequently, the photostability of the CS/PCL/BP and CS/PCL/BP/PDA scaffolds was studied upon NIR laser irradiation  for both CS/PCL/BP and CS/PCL/BP/PDA scaffolds after 7 days, whereas, the cumulative release amount of P from CS/PCL/BP scaffold reached 3.925 ± 0.035 µg mL−1 after 14 days and 5.08 ± 0.02 µg mL−1 after 28 days, which is significantly higher than that of the CS/PCL/BP/PDA scaffold (3.74 ± 0.05 µg mL−1 after 14 days and 4.25 ± 0.05 µg mL−1 after 28 days). Similarly, the cumulative release of P from the CS/PCL/BP and CS/PCL/BP/PDA scaffolds was consistent with the photostability results, further confirming the protective effect of PDA “envelope membrane”.Furthermore, the stability of BP in the scaffolds was evaluated by determining the cumulative release curves of P from the CS/PCL/BP scaffolds with and without PDA coating after 7,14, and 28 days of water immersion Figure . Initial+ concentrations were studied  increased significantly by 28.1 °C under 808 nm laser irradiation within 6 min , whereas the temperature of CS/PCL and CS/PCL/PDA scaffolds increased by only 5.7 and 15.5 °C, respectively. Additionally, there was no significant attenuation of photothermal conversion in the CS/PCL/BP/PDA@Ag‐6 mm scaffold during five repeated cycles with the laser on and off, highlighting that the as‐designed scaffolds could be subjected to periodic NIR irradiation after implantation  and CS/PCL/BP/PDA@Ag‐12 mm group  were high, whereas the bacterial inhibition ratio in the Ag‐free group was <20%. This result was further confirmed by the turbidity of bacterial suspensions co‐cultured with different scaffolds  also illustrated the superior antibacterial properties of the CS/PCL/BP/PDA@Ag‐6 mm and 12 mm scaffold groups  was studied as well. As shown in Figure + release when the pH values were changed from physiological microenvironment (pH 7.4) to an affected acidic microenvironment , exhibiting a pH‐responsive release behavior of Ag+ in the CS/PCL/BP/PDA@Ag scaffold. Comparatively, the cumulative amount and release rate of Ag+ were much higher at pH 4.5 than that at pH 5.5, and the lowest at pH 7.4, indicating that Ag+ liberation is accelerated from the CS/PCL/BP/PDA@Ag scaffold under a bacteria‐triggered acidic metabolic microenvironment. As illustrated in the inset, the in situ anchoring strategy enhances the interfacial bonding force between the Ag nanoparticles and PDA by Ag‐phenolic oxy‐group coordination. Under local acidic conditions, the Ag‐O coordination is broken, and the Ag‐phenolic complex is converted to phenolic hydroxyl. Subsequently, Ag+ ions quickly eluted from the CS/PCL/BP/PDA@Ag scaffold to kill the bacteria. Based on this, the CS/PCL/BP/PDA@Ag scaffold was endowed with self‐adaptive antibacterial characteristics triggered by the metabolism of bacteria. Particularly, a responsive antibacterial process could be intelligently implemented depending on the degree of infection, which mimics the self‐defensive functionality of chloroplasts well. Simultaneously, cellular assays of the chloroplast‐inspired CS/PCL/BP/PDA@Ag scaffolds in vitro also demonstrated satisfactory cytocompatibility  has been demonstrated favorable for bone regeneration,36 ands Figure . As timeBesides, the osteogenesis‐associated protein and gene expressions of rBMSCs were measured under periodic photothermal stimulation. The cell experiments were divided into eight groups, namely control ± NIR, CS/PCL ± NIR, CS/PCL/BP ± NIR, and CS/PCL/BP/PDA@Ag ± NIR groups . Collectively, the expression of the four kinds of osteogenic proteins and genes including bone morphogenetic protein 2 (BMP‐2), type I collagen (Col I), osteocalcin (OCN), and alkaline phosphatase (ALP) showed the similar variation at both 7 and 14 days  was performed to explore the osteogenic mechanism of photothermal stimulation. Here, rBMSCs were co‐cultured with CS/PCL/BP/PDA@Ag scaffold with/without NIR irradiation , and total RNA of cells was extracted. Differential expression genes (DEGs) analysis Figure 5a and voGO analysis of the RNA‐seq revealed that scaffold + NIR treatment altered the biological process (BP), cellular component (CC), and molecular function (MF). Most of changes were significantly involved in hyperthermia and osteogenesis, such as the cellular response to heat, extracellular matrix, heat shock protein binding, Hsp 70 protein binding and ATPase activator activity Figure . Meanwhi To further clarify the osteogenic effect of photothermal stimulation, hence, the expressed levels of Hsp 47 and Hsp 70 in both instantaneous (5 hours) and persistent (7 days) hyperthermal stress under NIR irradiation were verified by RT‐PCR  and Hsp 70 (associated with the thermo‐tolerance of osteoblasts) were closely related to osteogenesis.37 To R Figure . The rel2.5−2 was used to study the photothermal osteogenesis in vivo because its hyperthermal temperature range is satisfactory.The superior anti‐infection properties of bioinspired scaffolds had been confirmed in vitro and in vivo, respectively. The potential of NIR‐mediated photothermal osteogenesis for chloroplast‐inspired scaffolds was investigated further using an infected femoral defect rat model. In this section, rats were randomly assigned to four groups: the CS/PCL ± NIR and CS/PCL/BP/PDA@Ag ± NIR groups . Similar to the photothermal results in vitro, the photothermal performance of the CS/PCL/BP/PDA@Ag scaffolds also showed power density‐dependent characteristics in vivo , CS/PCL + NIR group , and CS/PCL – NIR group . Evidently, the chloroplast‐inspired CS/PCL/BP/PDA@Ag scaffold could effectively suppress the infection at an early stage of implantation and promote new bone formation thereafter. In particular, the bone repair effect of CS/PCL/BP/PDA@Ag scaffold can be further enhanced upon mild NIR irradiation.In addition, the parameters of osteogenesis, including BMD, BV/TV, Tb. Th, and local Tb. N in all the groups showed a similar trend, as described above Figure . From 8 + release. Consequently, the areas of new bone formation in the two CS/PCL/BP/PDA@Ag groups were larger than those in the CS/PCL groups at the same time points. Particularly in the CS/PCL/BP/PDA@Ag + NIR group, the fresh osseous tissue exhibited a compact and integrated structure. Furthermore, bone marrow space (shown by black arrows) could be detected, suggesting that new bone tissue turns to maturity.For the histological assessment of newly formed bone in defects, H&E and Masson staining were performed to trace the process of bone formation in vivo. As shown in Figure maturity.38Furthermore, Masson staining (blue represents type I collagen) Figure  revealedFigure To further investigate bone repair at the molecular and genetic levels, bone‐specific proteins and osteogenesis‐related gene expression of the peri‐implant tissues. The protein expression of COL I and OCN Figure 7a,b was Finally, the osteogenesis‐related genes of COL1A1, OCN, and BMP‐2 Figure  in newly Following the collapse of the scaffold in vivo, especially the degradation of BP nanosheets therein, many PO43− are released as the precursor ions of bone inorganic ingredients, and thus capture Ca2+ from physiological fluid to generate inorganic minerals in newborn osseous tissue beneficial for osteogenesis. Furthermore, osteogenesis‐related signaling pathways, including COL1A1, BMP‐2, and OCN, might be activated synergistically through mild thermal stimulation. Taken together, all of the tests in vivo prove that the chloroplast‐inspired scaffolds promote osteogenesis under infective conditions with a facile external control of NIR irradiation. This chloroplast‐inspired strategy provides a practical and effective platform for bone repairing.The possible mechanism of osteogenesis for chloroplast‐inspired scaffolds mainly originates from two points. For one thing, this may be due to the fact that mild thermal stimulation could facilitate the expression of heat shock proteins and finally mediate osteogenesis in vivo, as discussed in Figure 28 Fol3+ in the acidic microenvironment of bacterial infection. Furthermore, bacteria‐triggered infection can be completely inhibited by chloroplast‐inspired scaffold at an early stage and effectively promote endogenous repair of femur defects under mild NIR irradiation thereafter.In summary, by studying the unique light conversion and defense protection properties of chloroplasts, we successfully designed a novel CS/PCL/BP/PDA@Ag scaffold with prominent photothermal osteogenesis and self‐defensive ability for infected bone defect therapy. The biocompatible scaffold provided a favorable microenvironment for cell growth with a bone‐like 3D porous structure, outstanding biomineralization ability, suitable degradation rate, and water uptake/retention ratio. Particularly, the chloroplast‐inspired platform shows satisfying photothermal stability owing to the protection of the PDA “envelope membrane”, and hence, the incremental ratio of photothermal‐induced temperature in the PDA‐protected scaffold (89.3%) was higher than that of the non‐protected scaffold (51.8%) until 14 days of testing. In situ anchoring of Ag nanoparticles endowed scaffolds with robust antimicrobial ability and anti‐infection potential in vitro and in vivo on account of the rapid responsive release of free Ag4Detailed experimental materials and methods can be found in the Supporting Information. All animal studies were performed with the permission of the Animal Research Committee of Sichuan University .The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


We used unsupervised immunophenotyping of blood leukocytes and measured cytokine production by innate immune cell exposed to LPS and R848. We show that COVID‐19 induces a rapid, transient upregulation of myeloid‐derived suppressor cells (MDSCs) accompanied by a rapid, sustained (up to 3 months) hyporesponsiveness of dendritic cells and monocytes. Blood MDSCs may represent biomarkers and targets for intervention strategies in COVID‐19 patients. Inflammatory and danger signals stimulate hematopoiesis and the generation of myeloid‐derived suppressor cells (MDSCs) that suppress innate and adaptive immune responses . High len = 45) and severe  COVID‐19 were similar for age, gender, underlying diseases, and history of immunosuppressive therapy. Severe COVID‐19 patients had higher leukocyte counts and longer hospital stay than moderate COVID‐19 patients  and monocytic‐MDSCs (M‐MDSCs), the two main subgroups of MDSCs , in 56 C+: CD3, CD7, CD19 or CD56 positive) cells; DCs; and classical, intermediate, and nonclassical monocytes than healthy controls, but fourfold more PMN‐MDSCs and twofold more M‐MDSCs (p = 0.03 and 0.01)  Fig. . Interesρ = 0.43; p = 0.03). PMN‐MDSCs inversely correlated with lymphocyte counts  and hepatocyte growth factor . M‐MDSCs correlated with IL‐1β, IL‐7, PDGF‐BB, and vascular endothelial growth factor   were detected in the serum of patients Fig. . PMN‐MDS03) Fig. . InteresWhole blood was stimulated with LPS and R848. Intracellular cytokine staining followed by flow cytometry analysis was used to quantify the proportion of monocytes and DCs producing TNF and IL‐6 Fig. . In healUpon stimulation with LPS and R848, 17 of 24 and 13 of 24 of cytokines were detected at lower concentrations in blood from patients than healthy controls Fig. . InteresMDSCs represented 10%–15% of blood leukocytes, peaked in severe COVID‐19, and were associated with cytokine levels, lymphocytopenia, worse outcome, and impaired cytokine production by monocytes and DCs. Three months after inclusion, leukocyte counts were back to normal. Yet, blood, monocytes, and DCs still displayed reduced cytokine production, revealing long‐term immune disturbances. Likewise, MDSCs were normalized while cellular abnormalities were uncovered several weeks after SARS‐CoV‐2 infection [Our work has limitations. The number of patients was small, which may have limited the detection of differences or correlations. Additional markers could be used to trace MDSCs. However, we elected to minimize analytical variations by labeling whole blood quickly after drawing and analyzing flow cytometry data by unsupervised clustering. Finally, we have not assessed the immunosuppressive capacity of MDSCs. Yet, MDSCs of COVID‐19 patients were shown to inhibit the proliferation and cytokine production by T cells , 5, 6.To conclude, our data suggest that MDSCs in peripheral blood represent biomarkers to stratify COVID‐19 patients. Targeting MDSCs and/or immune dysfunctions might proof useful to counterbalance immunosuppression, reduce nosocomial and long‐term infections, and decrease late mortality in severe COVID‐19 patients.The authors declare no commercial or financial conflict of interest.https://publons.com/publon/10.1002/eji.202249827.The peer review history for this article is available at Supporting information.Click here for additional data file.


Multiple gestations represent a considerable proportion of pregnancies delivering in the late preterm (LP) period. Only 30% of LP twins are due to spontaneous preterm labor and 70% are medically indicated; among this literature described that 16–50% of indicated LP twin deliveries are non-evidence based. As non-evidence-based delivery indications account for iatrogenic morbidity that could be prevented, the objective of our observational study is to investigate first neonatal outcomes of LP twin pregnancies according to gestational age at delivery, chorionicity and delivery indication, then non evidence-based delivery indications.Prospective cohort study among twins infants born between 34 + 0 and 36 + 6 weeks, in Emilia Romagna, Italy, during 2013–2015. The primary outcome was a composite of adverse perinatal outcomes.Among 346 LP twins, 84 (23.4%) were monochorionic and 262 (75.7%) were dichorionic; spontaneous preterm labor accounted for 85 (24.6%) deliveries, preterm prelabor rupture of membranes for 66 (19.1%), evidence based indicated deliveries were 117 (33.8%), while non-evidence-based indications were 78 (22.5%).p <  0.01), higher gestational age at delivery (p <  0.01), Caucasian race (p 0.04), ART use (p <  0.01), gestational diabetes (p <  0.01), vaginal bleeding (p <  0.01), antenatal corticosteroids (p <  0.01), diagnosis of fetal growth restriction (FGR) (p <  0.01), and monochorionic (p <  0.01). Two hundred twenty-six pregnancies (65.3%) had at least one fetus experiencing one composite of adverse perinatal outcome. Multivariate analysis confirmed that delivery indication did not affect the composite of adverse perinatal outcomes; the only characteristic that affect the outcome after controlling for confounding was gestational age at delivery (p <  0.01). Moreover, there was at least one adverse neonatal outcome for 94% of babies born at 34 weeks, for 73% of those born at 35 weeks and for 46% of those born at 36 weeks (p <  0.01).When compared to spontaneous preterm labor or preterm prelabor rupture of membranes, pregnancies delivered due to maternal and/or fetal indications were associated with higher maternal age (Our study suggests that the decision to deliver or not twins in LP period should consider gestational age at delivery as the main determinant infants’ prognosis. Delivery indications should be accurately considered, to avoid iatrogenic early birth responsible of preventable complications. Late preterm (LP) birth, defined as delivery between 34 and 0/7 and 36–6/7 weeks of gestation, accounts for about 70% of overall prematurity in Europe . Compare.Multiple gestations represent a considerable proportion of pregnancies delivering in the LP period. Twin gestations account for approximately 3–4% of all live births , 9, withWhile 70% of all twin births are indicated, 30% are due to spontaneous preterm labor . DespiteWe conducted a prospective, multicenter, area-based cohort study of the LP deliveries from December 1st 2013, to December 1st 2015 in Emilia Romagna, a region in Northern Italy with nearly 4.5 million people organized in 9 counties. The results of the single pregnancy cohort study were just published .+ 0–36+ 6 weeks for written informed consent, and respectively completed the maternal and the neonatal data collection forms, as they interviewed the patients and consulted the medical records. Five trained research associates visited the study sites monthly basis to review the maternal and neonatal data collection forms with the physicians that had filled them. At the Data Managing Center  research associates used optical character recognition technology  to scan the data collection sheets, extract pertinent data, and organize it in a password protected database. If automated data extraction failed (approximately 5% of the times), research associates reviewed the forms and manually entered the missing data in the database. Authors have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. Approval from the Institution Review Board of the 9 Emilia Romagna Counties was obtained . The study was financed by the Emilia Romagna County Grant .The study steering committee  defined the information to collect on pregnancies resulting in LP deliveries, and on neonates’ hospital course. Standardized chart abstraction forms were designed to obtain anonymized data on mothers  and their newborns . In Emilia Romagna, inpatient obstetric and pediatric care was provided at the time of the study by 28 hospitals within the National Health System. Recruitment occurred only at the 21 hospitals with at least 500 deliveries/year, equipped with NICUs or Intermediate/Step Down Units, that could care for LP infants: one obstetrician and one neonatologist/pediatrician from each study site approached mothers with twin pregnancy delivering at 34+ 0- and 36 + 6-weeks’ gestation, who were not in labor within 24 hours after rupture of membranes and had no indication for immediate delivery, were expectantly managed as detailed in the PPROMEXIL trial [Women with a viable twin pregnancy were classified according to the indication for delivery as spontaneous preterm labor (sPTL), preterm prelabor rupture of membranes (pPROM) or indicated delivery. The diagnosis of preterm labor was based on clinical criteria of regular uterine contractions accompanied by progressive changes in cervical dilation and effacement . As pretIL trial . InsteadIL trial . The diaCurrently no consensus on whether RDS prophylaxis should be administered in twin pregnancies in the LP population, thus no specific recommendations were given concerning administration of antenatal corticosteroids (ANCS) in this period.+ 0–34+ 6 weeks. Gestational age was based either on first trimester ultrasound scan or, in women with a regular cycle, on the first day of the last menstrual period if the expected date of delivery differed less than 7 days from that estimated by ultrasound.The timing of delivery was determined in completed weeks of gestation such that 34 weeks (for example) included deliveries at 34Chorionicity was assigned in the 1st trimester of pregnancy by ultrasound assessment of the “twinpeak” or “lambda” sign . After dThe primary outcome is a composite adverse neonatal outcomes, stratified by GA, chorionicity and delivery indication, including neonatal death, respiratory distress syndrome (RDS), transient tachypnea of the newborn (TTN), mechanical ventilation, hypoglycemia, newborn sepsis, confirmed seizures, stroke, intraventricular hemorrhage (IVH), cardiopulmonary resuscitation, umbilical-cord-blood arterial pH < 7.0 or base excess < − 12.5, a 5-minute Apgar score ≤ 3, NICU length of stay ≥5 days. As indicated by previous studies, these outcomes are associated with significant risks of neonatal mortality or long-standing adverse health complications, including hypoxic ischemic encephalopathy . Neonata3 in the absence of corticosteroids and 3) definite purulent fluid from the cervical os.Non reassuring fetal status was defined as category III  or persiCPAP and oxygen administration represented non-invasive respiratory support, as opposed to mechanical ventilation. Neonatal sepsis was defined as a clinical syndrome prompting antibiotic treatment, with or without positive cultures . CerebraIndependent medical records reviews by 2 senior authors determined if LP deliveries were due to sPTL, pPROM, maternal and/or fetal indications. Reviewers also determined if indicated deliveries were EB or not EB, according to ACOG , as wellP ≤ 0.05 was considered.As we stratified neonatal outcomes according to delivery indications, we also compared how maternal, fetal, and neonatal characteristics varied in case of sPTL, pPROM or deliveries prompted by maternal and/or fetal conditions. Categorical variables were presented as n (%) and tested with Chi square test or Fisher’s exact test as appropriate. Normally distributed continuous variables were presented as mean ± SD and compared with One Way ANOVA. Non-normally distributed continuous variables were presented as median (IQR) and tested with One Way ANOVA on ranks. A level of statistical significance of Multivariate logistic regression analysis was used to investigate if delivery indications independently affected the risk of adverse neonatal outcomes. The following variables were tested as potential confounders: maternal age, parity, race, low education, smoking, maternal BMI, excessive weight gain, utilization of assisted reproductive technologies (ART), treatment with antenatal corticosteroids (ANCS), ASA, low molecular weight heparin (LMWH), or progesterone.The strength of the association between the covariates and the dependent variable was estimated as area under the curve of a receiver operating characteristic (ROC) curve plotted with the true-positive rate compared with the false positive rate. Statistical analyses were performed using Stata 15 .Among the 346 LP twin gestations included in our cohort, 84 (23.4%) were monochorionic pregnancies (of which only 3 were MCMA), while 262 (75.7%) were dichorionic; sPTL accounted for 85 (24.6%) deliveries, pPROM for 66 (19.1%), EB indicated deliveries were 117 (33.8%), while non EB indications were 78 (22.5%).p <  0.01), higher gestational age at delivery (p <  0.01), Caucasian race (p 0.04), ART use (p <  0.01), gestational diabetes (p <  0.01), vaginal bleeding (p <  0.01), prescription of antenatal corticosteroids (p <  0.01), diagnosis of FGR (p <  0.01), and were more frequently monochorionic (p <  0.01). Only 15.3% (53/546) of pregnancies had a vaginal delivery of both fetuses. Cesarean deliveries in non-laboring women were 204 (58.9%), induced labor 37 (10.7%), while sPTL were 105 (30.4%). Women experiencing sPTL had the highest rate of vaginal delivery (27.1%).In the univariate analysis shown in Table p <  0.01) and higher SGA rates (p <  0.01). Median NICU length of stay was similar among study groups, although stay ≥5 days was less common when indicated deliveries were non EB (p <  0.05).Table Table p <  0.01). Moreover, there was at least one adverse neonatal outcome for 94% of babies born at 34 weeks, for 73% of those born at 35 weeks and for 46% of those born at 36 weeks, with a significant difference (p <  0.01).As detailed in Table + 0–36+ 6 were shown to vary according to delivery indications [Although outcomes of singleton gestations at 34ications , our larications . FurtherThe high prevalence of non EB delivery indications in our twin population raises great concerns. Twin pregnancies represent a fragile obstetric population, having worse outcomes than singletons ; furtherOur results are consistent with previous reports on singleton gestations  showing The balance between the downsides of prematurity and the risks associated with pregnancy continuation, can be hard to strike in the LP period, especially among twin gestations  accountiAlthough women with EB deliveries were more likely to have babies with adverse perinatal outcome, this did not remain significant in the multivariate analysis, where the only factor affecting neonatal morbidity was the gestational age at delivery, as demonstrated by other studies . DeliverSix stillbirths were detected in our population: 5 among BC pregnancies, 1 among MC twins. Our stillbirth rate (1.7%) is consistent with the current literature; however, we did not find any difference according to chorionicity . FurtherThe joined 2020 NIH  and SMFMStrengths of this study include the large sample size, area based and multicenter nature of the cohort: both characteristics increase the generalizability of our findings. The prospective design of the survey, along with predefinition of standardized chart abstraction forms completed by obstetricians and pediatricians, and periodically audited by research associates, limit misclassification bias and assures data validity. We also acknowledge some limitations. As the study did not provide clinicians with prespecified delivery indications, it could be challenging at times for study personnel to identify the rationale behind an indicated delivery. However, such lack of standardization also allowed us to collect information about indications that were non EB. Although inconsistencies in ANCS administration may reflect different opinion leaders’ viewpoints, we considered betamethasone treatment among the potential confounders in our multivariate analyses. Moreover, our data are related to the years 2013–2015 and clinical practices might have slightly changed in these years. Finally, due to the exploratory nature of our study, a specific sample size calculation was not performed.To our knowledge, this is one of the largest prospective studies investigating delivery indications among twin gestations, suggesting that the decision to deliver or not twins in the LP period should consider gestational age at delivery as the main determinant infants’ prognosis. Delivery indications should therefore be accurately considered, to avoid iatrogenic early deliveries responsible of preventable complications. Our findings can also be helpful when counselling mothers at risk of LP delivery and can be used to plan interventions for their newborns.