Acheta domesticus) flour. More specifically, the impact and optimal addition of hydrocolloids  and water content were studied. For all the responses, the model and model terms were highly significant and showed the different impact of the hydrocolloids on the rheological properties. To evaluate the predictive power of the models, two sets of optimal process settings were chosen: one based on dough properties, and another on baked chapatti. For both sets, the actual responses were in the range of predicted responses for almost all properties. In addition, it was shown that using the settings based on dough properties, the actual responses were not significantly different from the control chapatti, whereas for the settings based on baked chapatti, there were differences in terms of the extensibility of both dough and chapatti. Thus, the I-optimal design is suitable to optimize the dough properties and the baked chapatti when enriching chapatti with cricket flour.The fortification of food with edible insect flour can improve its nutrition profile, but also affect its techno-functional characteristics. In this study, an I-optimal design was applied to improve the rheological and textural properties of wheat flour chapatti containing 10% cricket ( Acheta domesticus) or mealworm larvae (Tenebrio molitor) powder increased the dough tenacity and reduced dough extensibility which resulted in a decreased bread volume [T. molitor) larvae flour were similar to that of control while the snacks enriched with 20% mealworm flour exhibited poor texture [Locusta migratoria) flour or mealworm (T. molitor) flour [Edible insects are gradually being explored as a potential alternative for traditional livestock and future food due to their high nutritional potential and environmental sustainability to cope with the increasing global protein demand ,2,3,4,5.d volume . Osimanid volume . In anot texture . Moreover) flour . Thus, tr) flour .To improve the gluten network properties different hydrocolloids have been widely used in wheat flour and gluten free baked products. For instance, hydroxypropyl methylcellulose (HPMC), xanthan gum and carboxymethyl cellulose (CMC) were reported to increase the specific volume and softness of potato flour based steamed bread . Azeem eA. domesticus) flour. The optimization was performed to obtain comparable rheological and textural properties of wheat-cricket flour dough and baked chapatti to that of control sample (100% wheat flour). After performing the experimental runs according to the design of experiment (DoE), two confirmation tests were conducted to test the prediction power of the developed models.As such, extensive research has been performed on the effect hydrocolloids on wheat flour breads or gluten free bread. However, research on utilizing hydrocolloids in chapatti (an unleavened flatbread) dough and/or baked chapatti is still limited. Chapatti is a staple diet in the Asian subcontinent that act as a major source of nutrition. A research on increasing nutrition of chapatti by the addition of crickets flour showed that 5% addition was possible without altering the techno-functional properties, while an increased hardness and reduced extensibility of chapatti at higher content of cricket flour (10–15%) were observed . Whereas1 to X5  hydroxypropyl methylcellulose (HPMC), guar gum, and xanthan gum, respectively) and two coded levels for each factor (low (−1) and high (+1)) were selected based on a preliminary screening test (results not shown), as can be seen in For the design of this study five experimental factors, from X0 is the intercept of the function, βi is the linear coefficient of Xi, βii is the quadratic coefficient Xi, βij is the interaction coefficient of Xi and Xj, and ϵ represents the error term.The regression model was used to calculate the predicted response as shown in Equation (1), where Y is the predicted response, βAcheta domesticus) were purchased from Nusect . Cricket flour was prepared according to the procedure described by Khatun et al. (2021) [Wheat flour (AllBak) was purchased from a wholesaler (Jan Gevers BVBA) in Mol, Belgium. House crickets (. (2021) . BrieflyAll the doughs were prepared according to the proportion of hydrocolloids and water as suggested by the DoE , while wChapatti was prepared according to the method described by Khatun et al. (2021) . A pastaThe dynamic rheology of the formulated dough was measured by a rheometer  using parallel serrated plates (25 mm diameter) with a strain sweep from 0.01 to 100% at a frequency of 1 Hz as described by Khatun et al. (2021) . When apThe biaxial extensibility of dough was measured using a texture analyzer  with a tortilla/pastry rig (HPD/TPB) as described by Khatun et al. (2021) . BrieflyUniaxial extensibility of baked chapatti was measured by a texture analyzer  using a tensile grip (A/TG) as described by Khatun et al. (2021) . Brieflyp ≤ 0.05) terms, which facilitates effect hierarchy. After removing non-significant terms, the models were fitted by accounting the coefficient of determination (R2) and adjusted coefficient of determination (Adj. R2) value. Afterwards, a prediction was used to choose optimal process setting for the desired response variable. Two sets of optimal process settings were chosen using the prediction profiler (JMP), one based on dough properties in terms of biaxial extensibility and viscoelastic properties, and another based on uniaxial extensional properties from the baked chapatti. Thereafter, a one-way ANOVA test was used to check the significant (p < 0.05) difference between the results of all parameters of control and experimental dough and chapatti during the confirmation tests.JMP software  was used to implement the I-optimal design and response surface analysis. Multiple regression analysis was conducted to model the variation of each response and to investigate the significant effects of all independent variables to the response. Analysis of variance (ANOVA) was performed at 5% level of significance to assess the model fitness. A backward elimination procedure was employed to select the significant (p < 0.05) main effects, interaction, and quadratic effect of water and four hydrocolloids level were retained. The resulting model and model terms were highly significant (p < 0.0001) while the coefficient of determination (R2) and adjusted coefficient of determination (Adj. R2) were higher than 0.80  and textural properties of baked chapatti . For all the response variables, the significant  of dough and baked chapatti prepared from wheat-cricket flour properties by 27 runs is presented in PC1 has been shown to be largely characterized by dough properties while PC2 was characterized by baked chapatti properties. Thus, PCA revealed the disparities among the wheat-cricket flour dough and baked chapatti properties as affected by different proportion of water and hydrocolloids type. Furthermore, a significant positive correlation was found between dough hardness and viscoelastic properties G′ and G″ (r = 0.88 and r = 0.71). As expected, dough hardness and dough extensibility showed a strong negative correlation (r = −0.78) which is clearly shown by PCA plot. Moreover, with the increase of chapatti hardness, its extensibility was reduced.p < 0.05) linear effect on the dough hardness.The most important characteristics of dough, force to extend the dough (hardness of dough) and extensibility of the dough at deformation have been extracted from the biaxial extensibility test. The prediction expression produced by the design is shown in Equation (2). From this expression, it can be seen that all the factors applied in the formulation of chapatti dough had a significant (5), followed by guar gum (X4), HPMC (X3) and CMC (X2), while water had a negative linear effect on the dough hardness. Although CMC had a positive linear effect on dough hardness, it showed a significantly negative interactive effect when combined with water. The increase of dough hardness by xanthan gum could be attributed to its electrostatic interaction with gluten proteins. On the other hand, guar gum can increase dough hardness by interacting with starch and gluten through hydrogen bonds [The most pronounced positive linear effect was shown by xanthan gum  and the coefficient of determination (R2) and adjusted coefficient of determination (Adj. R2) value were 0.88 and 0.85 respectively (1) exerted a significant (p < 0.05) positive linear effect while guar gum (X4) and xanthan gum (X5) had a significant (p < 0.05) negative linear effect on dough extensibility, with xanthan gum (X5) being more pronounced.For dough extensibility, the model and model terms were highly significant  found a reduced extensibility at 0.5% but increasing extensibility at 1.0% concentration of xanthan gum on wheat-cassava composite dough. However, such quadratic effect on dough extensibility by xanthan was absent in our study (Equation (3)) . It can The surface plot a clearlyp < 0.05) main effects on storage modulus (G′) and loss modulus (G″)  sheet . In our lus (G″)  but no i5) and guar gum (X4) indicating stronger and less extensible dough with more elastic interaction which is in line with dough hardness and extensibility  main effects on chapatti hardness, while quadratic effects of water (X1), guar (X4) and xanthan gum (X5) were also significant. Further, significant (p < 0.05) interaction effects of water with HPMC (X3), of guar gum with HPMC and xanthan were found on chapatti hardness.From the fitted model (Equation (6)), it was derived that all the factors had significant  and (7)) while such interaction between biopolymers was absent in the dough system (Equations (2) and (3)). It can be hypothesized that these interactions were developed due to the heat treatment during the baking stage where intermolecular interactions could occur between the heat-disordered segment of xanthan gum and the backbone of guar gum. This is supported by Khouryieh et al. (2006), who observed a strong interaction effect of guar and xanthan gum at 80 °C in an aqueous system, while cooling down to 25 °C left xanthan gum in a more disordered conformation . Furtherp < 0.0001) while 2R and adjusted 2R were 0.816 and 0.735 respectively indicating good fitting of the model (p < 0.05) positive main effect on chapatti extensibility. While water (X1) showed a negative quadratic effect, a positive quadratic effect was exerted by HPMC (X3).The coefficient of estimation for chapatti extensibility shows that the model terms are highly significant (he model . From th5) with HPMC (X3) and guar gum (X4) were also observed on chapatti extensibility. The quadratic effect of water , who reported that guar gum had a pronounced reducing effect on chapatti hardness followed by CMC and HPMC up to a content of 0.75% . The chaAnother confirmatory test based on the texture of baked chapatti was performed by excluding CMC and including xanthan gum . The resIncluding xanthan gum in the mixture design of HPMC/guar/xanthan gum resulted in higher dough strength (hardness and G′) than the ternary mixture of CMC/HPMC/guar gum . StrengtAcheta domesticus) using four hydrocolloids, namely carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), guar gum, and xanthan gum, and a variable water content. The obtained models showed that all the different hydrocolloids and water affected differently on the dough and chapatti rheology. Results showed that all the hydrocolloids had a positive influence on dough strength (dough hardness and G’). For dough extensibility, guar and xanthan gum showed a negative effect when used singly, while their interaction with water had a positive effect on dough extensibility, indicating a crucial function of water. In contrast, CMC and HPMC did not show any effect on dough extensibility. The quadratic effect of guar gum and xanthan gum could exert a softening effect on baked chapatti at low level while CMC and HPMC showed a hardening effect on baked chapatti with an increasing concentration. An I-optimal mixture design was employed to optimize the rheological and textural properties of wheat flour chapatti supplemented with 10% cricket flour , and the other on the baked chapatti properties (ternary mixture HPMC/guar gum/xanthan gum). In both cases, water content was predicted by the models. Results revealed that for the settings based on dough properties, the properties of the obtained dough and of the baked chapatti were within the predicted range, and not significantly different from the control (100% wheat flour). The measured responses of the second confirmation test, with settings based on baked chapatti, were also well in agreement with the predicted responses, but with some deviations, especially in terms of dough extensibility and chapatti extensibility. Therefore, it can be concluded that the design of experiments approach proved to be a strong tool to optimize the dough properties and the baked chapatti when nutritionally enriching chapatti with cricket flour.


Pseudomonas aeruginosa, that can go on to establish chronic infection of the lungs in people with cystic fibrosis (CF). Despite the importance of upper airway environments in facilitating early adaptation to host environments, currently availablein vitro models for study of respiratory infection in CF focus exclusively on the lungs. Furthermore, animal models, widely used to bridge the gap betweenin vitro systems and the clinical scenario, do not allow the upper and lower airways to be studied in isolation. We have developed a suite of culture media reproducing key features of the upper and lower airways, for the study of bacterial adaptation and evolution in different respiratory environments. For both upper and lower airway-mimicking media, we have developed formulations that reflect airway conditions in health and those that reflect the altered environment of the CF respiratory tract. Here, we describe the development and validation of these media and their use for study of genetic and phenotypic adaptations inP. aeruginosa during growth under upper or lower airway conditions in health and in CF.The respiratory tract is a compartmentalised and heterogenous environment. The nasopharynx and sinuses of the upper airways have distinct properties from the lungs and these differences may shape bacterial adaptation and evolution. Upper airway niches act as early colonisation sites for respiratory bacterial pathogens, including those, such as Research highlightsScientific benefit(s):•Study of bacterial pathogenicity in infection relevant conditions of compartmentalised airway niches•Study of bacterial host adaptation in cystic fibrosis (CF) and other respiratory diseasesAllows:3Rs benefit(s):•Reduces the need for vertebrates in respiratory microbiology by providing a pre-screening platform for bacterial pathogenicity and host-pathogen interaction related research questionsPractical benefit(s):•Inexpensive, simple to operate, no training required•Chemically defined and easy to manipulate the content of the media to study different research questions, with several respiratory pathogensCurrent applications:•Suitable for studying phenotypes including bacterial growth, antimicrobial resistance, biofilm formation,•Suitable for studying bacterial gene expression in health and CF under relevant host stressesPotential applications:•Studying long term evolution of bacteria in health and infection relevant media•Studying different pathogen combinations and how they affect virulence and resistance•Combining with cell or tissue-based models to fully capture the chemical and cellular characteristics of different airway niches in the lab environment•Assessing efficacy of drugs and therapeutics under relevant conditionsOffers a platform for:CFTR) gene, located on the long arm of chromosome 7. CFTR protein functions as an apical ion channel. CF has a range of severities, with airway disease being the main cause of morbidity and mortality. Defects in CFTR affect its ion transporting functions, leading to accumulation of sticky mucus on epithelial surfaces in affected organs, including the lungs, sinuses, pancreas, gastrointestinal tract, sweat glands and reproductive tract.Cystic Fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator .P. aeruginosa is a Gram-negative aerobic/facultative anaerobic bacterium found in high abundance in external environments.P. aeruginosa colonization and infection of the airways is a significant contributor to morbidity and mortality in PwCF. WhenP. aeruginosa enters into the airways from an environmental source, it not only must adapt to the changes in nutritional and physiochemical status associated with the airway environment, but also to stresses exerted from intense antimicrobial pressure and the actions of immune responses, competition from resident microbiota and viscous mucus-related osmotic stresses. All of these factors influence the adaptation and evolution ofP. aeruginosa within respiratory niches.The warm, humid and nutrient rich environment of CF airways supports growth of various bacteria.P. aeruginosa has a >6 Mb plastic genome that facilitates rapid adaptation to environmental stimuli. Environmental versatility is achieved through the action of regulatory systems, which collectively make up ~8% of theP. aeruginosa genome. Transition from colonization to acute infection and subsequent biofilm-associated chronic infection takes place as a result of changes in these regulatory systems that are key for controlling the expression of virulence factors in response to host environmental cues.P. aeruginosa during early infection. Colonisation of this environment is thought to precede chronic lung infection.,P. aeruginosa has been identified in sinusitis samples and a high level of similarity has been observed between paranasal and lung isolates. The presence of a low number of immune cells and reduced exposure to antibiotics in the upper airways, relative to lungs, provides a transitional niche within which the bacteria can adapt to host conditions before establishing chronic lung infection. Early infection of lungs with environmentalP. aeruginosa may be cleared by a combination of host inflammatory responses and antibiotic treatment, but the persistence of the pathogen within the upper airways offers opportunity for later downward reseeding of a host-adapted bacterial population. Armbusteret al. documented the role of the sinuses inP. aeruginosa within-host adaptation in 106 adult CF patients. They found that the sinuses harboured isolates with pathoadaptive characteristics that were previously seen in isolates from sputum and that have been associated with persistence in the CF lung. The continuous cycle of bacterial clearance from lungs and recolonization from sinuses may form an important part of the intermittent colonization stage that precedes chronic lung infection.The upper respiratory tract  has been suggested to be a protective niche for environmentally-acquiredP. aeruginosa, where persistence of bacteria in the upper airways was observed over the course of 28 days of infection. Initial colonisation of lungs was cleared within two weeks of infection, in the absence of antibiotic treatment, butP. aeruginosa was isolated from sinuses throughout infection and reappeared in the lungs of all mice at day 28 post-infection, suggesting that long-term upper airway persistence may allow bacterial migration and reseeding to the lower airways. In later studies, this within-host adaptation was linked to loss-of-function mutations in the two component regulatory system genepmrB, thought to have been acquired during upper airway infection. Isolates carryingpmrB mutations, recovered from the murine airways, showed enhanced potential for establishment and maintenance of lung infection that was linked to increased resistance to lysozyme and increased attachment to host surfaces. Similar loss-of-function mutations were identified inpmrB in clinical isolates from CF. As the experiments undertaken in mice did not involve antibiotic treatment, the selective pressure for the observed changes was hypothesised to be the result of the action of host-derived antimicrobials.This proposed route to lung infection was recapitulated in a mouse natural inhalation infection model with– Changes in colony morphologies, towards a so-called ‘wrinkly’ phenotype, were indicated to be the result of redox-drive adaptations to maximize oxygen accessibility in biofilm by increasing the surface area exposed to air. A slow growing phenotype has also been suggested to be a common characteristic of long term CF airway colonization and is thought to develop as a result of continuous activation of stress responses driven by exposure to cells and molecules of the immune system.Other pathoadaptive characteristics of strains from chronic infections have been identified and include overproduction of alginate, reduction in growth rate, loss of motility, decreased susceptibility to antimicrobials and diversification in colony morphology.P. aeruginosa adaptive evolution in different ways. A range ofin vitro andin vivo models are available for the study of respiratory infection in CF. The most basic of these involvein vitro culture in nutrient rich laboratory media that readily supports bacterial growth. These models are often a poor reflection of the airway environment, as they contain sugars and proteins at concentrations that are not physiologically relevant and they lack important host-derived factors that exert stress on pathogens or that are recognised by microbial environmental sensing systems. These limitations encouraged the development of chemically-defined liquid culture media that better reflect the conditions of the CF airways. Artificial sputum media (ASM), synthetic cystic fibrosis media (SCFM) and their derivatives– capture important chemical aspects of the CF airway environment, including extracellular DNA (eDNA), amino acids and mucins., These media support infection-relevant biofilm formation and have been of significant value to the research community. Furthermore, they can be combined with host tissue to provide a physical scaffold for biofilm formation. However, these models still lack key molecules of the lung, including host-derived antimicrobials,, polyamines, and a number of factors associated with CF comorbidities, including glucose, which is elevated in lungs in CF-related diabetes compared to healthy individuals, and bile salts, which can be inhaled into lungs during CF-associated gastro-oesophageal reflux.The respiratory tract is a heterogenous, compartmentalised environment, with the biological, chemical and physical properties of each niche shapingin vitro andin vivo models focus on capturing conditions of the lung environment and therefore do not reflect the heterogeneity of the airways and are unsuitable for studying early infection processes that may take place in the sinuses., Whilstin vivo infection models – most often performed in mice or rats - allow for a more holistic view of infection processes, they are costly, hard to maintain and there are important differences in host-derived antimicrobial structure and function between rodents and man. Furthermore, CF rodent models don’t reproduce the disease phenotypes and severity of human CF. In particular, they poorly replicate the severe pathology of CF lung disease, with the major disease phenotype instead manifesting in the gastrointestinal tract. The generation of gut-corrected CFTR transgenic mice has addressed this issue, but establishing chronic lung infection in these mice remains a challenge. Embedding bacteria within agarose beads and instilling these into the trachea of mice allows for establishment of long-term infection, albeit by an artificial route that requires surgical procedures and which is hard to standardise. Natural inhalation of CF-adaptedP. aeruginosa suspended in saline offers an alternative that recapitulates the sinus to lung infection route and allows for long-term infection studies, but this model has other drawbacks, including a relatively low density of infection in lungs. Study of major pathophysiological CF characteristics, such as chronic respiratory infection, inflammation, and mucus plugging, have been hampered by the limitations of existingin vivo models.Most currently available More than half of these procedures were carried out in mice (54%) and 51% of overall procedures were for basic research while 27% was for applied research including human infectious disorders. Moreover, in the field of respiratory microbiology, 10,540 publications report the use of mice for infection (Pubmed search “respiratory infection mouse” 2015-2022), more than half of these being for bacterial infections . Development of well-characterised respiratory mimicking media would offer opportunities for both replacement and reduction in such studies.According to Home Office Report from 2021, 3.06 million procedures were performed involving living animals for scientific purposes.in vivo andin vitro models to study human infections, Cornforth et al developed a computational framework that uses transcriptomic data fromP. aeruginosa grown under different environmental conditions and benchmarks it against the gene expression patterns observed in clinical CF sputum samples. This approach has been used to assess the clinical relevance ofin vitro andin vivo models, with results suggesting thatin vitro models are as good as or better than mouse lung infection models at mimicking theP. aeruginosa transcriptome in human CF sputum.With the aim of understanding the applicability of available2/CO2 levels, temperature, lysozyme, lactoferrin, polyamines,, mucins, amino acids, carbon source content and salt concentrations. These media were designed to be cheap, accessible and readily modifiable, according to the research question being asked. Development of validated culture media that are more relevant than using nutrient broth and more cost effective and ethical than animal models can offer a platform to understand bacterial within-host adaptation, to use for isolate virulence or antimicrobial susceptibility testing, for efficacy testing of new therapeutics, or for study of bacterial growth, gene expression and biofilm formation under relevant environmental conditions.Given the limitations of the available animal models for study of CF infection and bacterial within-host adaptation, we sought to develop novel culture media that mimics the conditions of compartmentalised airway regions. To enable study of early infection processes taking place in the upper airways, we designed sinus-mimicking media reflective of conditions in health or in CF . In parallel, we developed media reflective of the lungs in health or CF . The developed CF media will be of use for the study of CF pathogens under infection-relevant conditions, whilst the healthy media equivalents can be used for study of airway infection in other contexts, such as non-CF bronchiectasis, community-acquired or ventilator-acquired pneumonia, that occur without the underlying altered environment that is specific to CF. Many of the differences between airway niches and between health and CF that we aim to capture have been described previously, including in OMicrobial strains, base media and culture conditionsP. aeruginosa isolates used in this work are shown in2PO4, 64 g/l Na2HPO4, 2.5 g/l NaCl, 5.0 g/l NH4Cl) (Sigma-Aldrich) (autoclaved), 2 ml MgSO4 (autoclaved) (Sigma-Aldrich) and 20 ml 20% glycerol (Sigma-Aldrich) (filter-sterilized), in order. Sterilized water was then used to make up the volume to 1 L.AllSingle chemical growth rate assays® LUX, Thermo Scientific) . 2 μl of bacteria from overnight cultures were added to 198 μl of M9 media supplemented with the appropriate chemical. The bacteria were then incubated in sterile 96-well plates (U-bottom) (Greiner) for 20 hours and the growth rates assessed at OD600nm at 10 minutes intervals. Incubation conditions were 37°C/5% CO2 for lung conditions and 34°C/0% CO2 for sinus conditions. Three biological replicates were performed, per bacterial isolate, per condition. A single working concentration to be used in the final pooled media was then chosen for each chemical, per condition and growth rate assays were performed again with the chosen concentration before the chemicals were combined into a single pooled media. The ‘ideal’ concentration was chosen for the final media unless found to cause complete inhibition of bacterial growth.Each chemical under consideration for inclusion in the respiratory tract-mimicking media was first assessed by supplementation individually into M9 media at ‘low’, ‘ideal’ or ‘high’ concentrations. Ideal concentrations represent those estimated to most closely reflect the physiological concentration found in each niche, in either health or disease. The relevance of the final list of chemicals included in the final media, in the context of bacterial adaptation to airway environments, is summarised inMedia synonyms:Healthy sinus media (HSM),Healthy lung media (HLM),CF sinus media (CFSM),CF lung media (CFLM).4). Stock solutions of all the media components except glucose, succinate, N-acetyl glucosamine (Glc-NAc), bile, NaCl, albumin and amino acids were prepared and added to the media in solution form. Aforementioned chemicals were then added in powder form, slowly, under constant stirring at room temperature. Once all the components were fully mixed, the solution was sterilized by filtration using Vacuubrand ME 2 diaphragm vacuum pump and Millipore Steritop filter units with a pore and neck size of 0.22 μm. Sterile eDNA and mucin was then added to the filtered media and the pH of media was adjusted to 6.8-6.9 using sodium hydroxide (NaOH) (Sigma-Aldrich). The volume of the solution was brought to 1 litre by the addition of autoclaved distilled water. The media was then aliquoted to 50 ml falcon tubes and stored at -80°C until further use.Media components were pooled together at concentrations given inM9 media was prepared without the addition of M9 salts because exogenous salts were added in the form of NaCl and bile salts, as defined inViscosity of media was measured before and after allowing bacteria to form biofilms for three days, to mimic chronic infection. Viscosity measurements (mPa.s) were performed using a rheometer (Anton-Paar) with cone plate . 50-100 1/s range was used for all sterile media and healthy media during infection, and 0.01-1000 1/s range was used for CF media during infection. Changes in media viscosity was observed under each shear rate point.Stability of developed media was tested over a 30-day period by performing crystal violet stain assays (as described below) using media stored at 4°C for 30 days. This experiment was designed to determine whether media would continue to support biofilm formation after prolonged storage.2. Anaerobic jars were used to increase the CO2 concentration for lung conditions. CFU was measured at pre-determined time intervals by serial-dilution onto agar. Briefly, 10-fold serial dilutions were prepared in 96 well microplates (Greiner U-bottom) using PBS as the diluent. 20 ul of bacterial dilution was then plated to TSA plates covering the dilutions from 10-2 to 10-9. The plates were air dried and then incubated at 37°C until the colonies were clearly visible (18 hours for PAO1 and 24 hours for LESB65).Colony forming unit (CFU) assays were performed to observe the growth of PAO1 and LESB65 in the pooled media. Bacterial cultures grown in LB for 18 hours were centrifuged for 12 minutes at 3220rcf at room temperature. After the LB suspension was removed, bacterial pellets were resuspended in 5 ml of sterile phosphate buffer saline (PBS) (Sigma-Aldrich). The suspension was homogenized by vortexing and bacterial optical density was adjusted to OD 0.05-0.06 in PBS using an optical spectrometer (HANNA instruments). 50 ul of this suspension was then added to 4950 ul HSM, HLM, CFSM or CFLM and cultures were incubated under niche-appropriate conditions: sinus media at 34°C with no CO2, lung media at 37°C with 5% COMeasurement of attached biofilm biomass by crystal violet stainingStarting from an overnight liquid culture in LB (incubated for 18 hours), bacterial cultures were diluted 1:100 in the developed media, or in LB, or M9, two widely used laboratory bacterial culture media. To minimise edge-effect, non-perimeter wells of U-bottomed polystyrene 96-well microtiter plate (Greiner U-bottomed) were inoculated with 180 μl of these dilutions and perimeter wells were used as a negative control (sterile medium). Following 3 days of growth at 37°C, the supernatant (containing non-adhered cells) was removed from each well and plates were rinsed using 200 μl sterile PBS, twice. Subsequently, 200 μl of Crystal Violet (CV) was added to non-perimeter wells and plates were incubated at room temperature for minimum 20 minutes. After 20 minutes, the excess CV was removed by washing the plates under running tap water. Finally, bound CV was released by addition of 200 μl of 95-100% ethanol (Sigma-Aldrich) to the wells with bacteria. After incubating with ethanol for 30 minutes, the absorbance was measured at 600 nm using a BMG plate reader. 95-100% ethanol was used as a blank.Measurement of cell viability and free-floating biofilm formation by resazurin assays2O (VWR)] in water and pH to 4.6 with NaOH) and further incubated under aerobic conditions at 37 °C, while shaking at 150 rpm for 1 h. After 30 minutes incubation, biofilms were further disrupted by manual pipetting to ensure complete disruption of biofilms. A portion of disrupted biofilms were then transferred to 96-well plates (Greiner U-bottomed) and to determine the metabolic activity of the bacterial cells released from the disrupted biofilms, 10 μl of 0.02 % (v/v) resazurin (Sigma-Aldrich) (diluted in distilled water) was added to each well of the 96-well plates and incubated for 1-2 hours at niche specific temperature, while shaking at 150 rpm. Following incubation with resazurin, the fluorescence of each well was measured using an excitation wavelength of 540 nm and an emission wavelength of 590 nm in a Fluostar Omega microplate reader and the MARS Data Analysis Software.Overnight cultures of PAO1 and LESB65 were diluted in LB to an OD 600 of 0.05 (±0.01). These cultures were then further diluted in the developed media (1:100) to a total volume of 10 ml in glass universal tubes. Free floating biofilm formation in respiratory tract-mimicking media was compared to that observed in LB and M9. Diluted developed media cultures were incubated under conditions appropriate for each respiratory niche, as described above. Cultures were incubated for 3 days while shaking at 75 rpm. Three glass universal tubes containing bacteria-free respiratory media, LB or M9 were used as a negative control. After the incubation, biofilms were disrupted using 250 μl of 100 mg/ml cellulase . To extract RNA, TRI reagent (ZYMO Research) was added and mixtures were incubated for 5–10 min at room temperature. Bacteria were harvested by centrifugation for 30 min at 13,000 rpm and 4 °C. Supernatant was removed and bacterial pellets were used for subsequent RNA isolation or stored at −80 °C. RNA from bacterial cultures was isolated using the Direct Zol RNA Microprep kit (ZYMO Research: R2061) according to the manufacturer’s instructions. DNase digestion treatment was performed using DNAse 1 (ZYMO Research) as per the manufacturer’s protocol. Isolation was performed in an RNase-free environment using RNase-free consumables and reagents. Purified RNA was eluted with RNase-free water (ZYMO Research). Quantification of RNA was performed by measuring the absorbance at 260 nm wavelength using the NanoDrop8000 UV–vis Spectrophotometer (Thermo Scientific). and purity of RNA was analysed by determination of the 260/280 nm ratio. Nuclease free water (Invitrogen) was used as a blank. The 260/280 nm ratio obtained for all samples was between 1.8 and 2.0.First-strand cDNA synthesis was performed using the iScript cDNA synthesis kit (BIO-RAD: 1708891) according to the manufacturer’s instructions in an RNase-free environment using RNase-free consumables and reagents. For each sample, 2.5 ng RNA was added and incubated in a thermocycler (Applied Biosystem) under the following protocol: 5 min at 25 °C, 30 min at 42 °C and then 5 min at 85 °C. The cDNA was then stored at −20 °C until further use.® qPCR Master Mix (Promega: A6001) as per the manufacturer’s instructions. Each reaction contained 2 μl of cDNA (or nuclease free water for the no template control) and 0.2 μM of forward and reverse primers (Eurofins). Primer sequences are provided incDNA was used for quantitative real-time PCR (qPCR) in duplicate reactions using the GoTaqrpoD, encoding sigma factor RpoD, was used as a reference gene. cDNA obtained from cultures grown in LB was used as a control. Analysis of relative gene expression in each developed media and SCFM2 versus (vs) LB was performed using the 2−ΔΔCt relative quantification method as described previously.Each qPCR was performed with three biological replicates and in duplicate on each run. Gene-4, 10-6) every ten days. Plates were incubated at 37°C for 24 hours and for a further 48 hours at room temperature to allow the colonies to uptake the two dyes in the media.PAO1 was cultured in the developed media for forty days, sub-cultured to fresh media every 2 days and plated on Tryptone Congo red/Coomassie blue Agar (TCCA) prepared by mixing 20 mg/l Coomassie blue (Sigma-Aldrich), 40 mg/l Congo red (Sigma-Aldrich), 10 g/l Tryptone (Sigma-Aldrich) and 12 g/l Bacto agar (Fisher-Scientific) in distilled water and autoclaving at 121°C for 15 minutes. 100 μl of bacterial cultures were spread on to the plates in serial dilutions . All statistical analysis was done using Graphpad Prism versions 8 and 9 . For single chemical growth rate assays, R studio version 3.6.2 and GrowthCurver (version 0.3.1) was used to plot the area under the curve (AUC_e) values.Here we describe, step-by step, the procedure used to prepare 1L of each of the four developed media. Reagents and equipment used in this study are listed inPreparation of the base (same for both CF media)1.4.In a 1 litre Duran bottle, add 300 ml distilled water, 20 ml 20% glucose and 2 ml 1 M MgSOPreparation of CF sinus media1.Add 4.8 g eDNA in 300 ml water under continuous stirring with a magnetic stirrer at room temperature. Add the eDNA very slowly as it can form clumps in the water.2.Add 6 g mucin from porcine stomach (Type II) in 150 ml water under continuous stirring with a magnetic stirrer at room temperature. Add mucin powder slowly to water to prevent the formation of clumps. eDNA and mucin can be stirred overnight at 4°C to fully dissolve the powder.3.Once the eDNA and mucin solutions are fully dissolved, autoclave at 121°C for 15 minutes to sterilise.4.2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given inPrepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl5.Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in6.Add 35 μl of neat spermidine solution (9.296 g/l) to the base media.7.r 56.11 g/mol) and L-tyrosine in 2.95 ml sterile water, separately before adding them to the base media. (Amino acid concentrations are same for both CF sinuses and CF lung media.)Weigh the amounts of L-amino acids given in8.Weigh 7 g of albumin, 6.3 g of NaCl, 4.4 g of N-acetyl glucosamine, 250 mg of glucose and 2.95 g of succinate and add to the media one at a time. Wait for each chemical to dissolve completely before adding the next one. Succinate can take time to dissolve in water. Incubate the succinate at 37 °C while shaking at 180 rpm for 10-15 minutes to quicken dissolving.9.Sterilise the media by filtration using a Vacuubrand ME 2 diaphragm vacuum pump and Millipore Steritop filter units with a pore and neck size of 0.22 μm.10.Under sterile conditions, add 250 ml of autoclaved eDNA and 125 ml of autoclaved mucin to the mixture.11.Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.12.Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80°C (suitable for longer term use).Preparation of CF lung media1.Follow steps 1-3 of CFSM protocol for eDNA and mucin stock solution preparation.2.2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given inPrepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl3.Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in4.Add 37.6 μl of neat spermidine solution (9.296 g/l) to the base media.5.Add amino acids in the same way as for preparation of CF sinuses media See.6.Add 7 g albumin, 6.3 g NaCl, 4.4 g of N-acetyl glucosamine, 250 mg of glucose, 2.95 g of succinate, 300 mg bile salts, one at a time.7.Sterilise the media in the same way to CF sinuses media (see above protocol).8.Under sterile conditions, add 250 ml of autoclaved eDNA and 125 ml of autoclaved mucin to the mixture.9.Adjust pH to 6.8-6.9 with 5M NaOH and bring the volume to 1 litre with sterile water.10.Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80°C (suitable for longer term use).Healthy sinuses mediaPreparation of the base media 1.4.In a 1 litre Duran bottle, add 250 ml distilled water, 20 ml 20% glucose and 2 ml 1 M MgSOPreparation of healthy sinuses media1.Add 1 g eDNA in 500 ml water under continuous stirring with a magnetic stirrer at room temperature. Add the eDNA very slowly as it can form clumps in the water.2.Add 1.4 g mucin from porcine stomach (Type II) in 140 ml water under continuous stirring with a magnetic stirrer at room temperature. Add mucin powder slowly to water to prevent the formation of clumps. eDNA and mucin can be stirred overnight at 4 °C to fully dissolve the powder.3.2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given inPrepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl4.Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in5.Add 21.5 μl of neat spermidine solution (9.296 g/l) to the base media.6..Add amino acids in the same way as for preparation of the CF media, see7.Add 500 mg albumin, 1g NaCl, 1.28 g of N-acetyl glucosamine, one at a time.8.Sterilise the media in the same way as for CF sinuses media (see above protocol)9.Under sterile conditions, add 480 ml of autoclaved eDNA and 120 ml of autoclaved mucin to the mixture.10.Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.11.Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80 °C (suitable for longer term use).Healthy lungs mediaPreparation of healthy lungs media1.Follow steps 1-3 of HSM protocol for eDNA and mucin stock solution preparation.2.2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given inPrepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl3.Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in4.Add 26.8 μl of neat spermidine solution (9.296 g/l) to the base media.5.Add amino acids in the same way of preparation of the CF media, see6.Add 1.5 g albumin, 1 g NaCl, 1.28 g of N-acetyl glucosamine, one at a time.7.Sterilise the media in the same way to CF sinuses media (see protocol for CF sinuses).8.Under sterile conditions, add 480 ml of autoclaved eDNA and 120 ml of autoclaved mucin to the mixture.9.Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.10.Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80 °C (suitable for longer term use).Incubation conditions for the media2 can be achieved by incubating the media in GasPak Jars with candles, using Campygen packs or microaerobic incubators.Incubation conditions differ between lung- and sinus media. The incubation conditions for each media are given inNotes on handling of media components and media preparation•Break bovine serum albumin into smaller pieces before adding to media, as it can take time to fully dissolve.•Add eDNA and mucin to minimise clumping. Make sure that each addition is dissolved before adding more.•Succinic acid may not fully dissolve in water immediately. Leave it at 37 °C for 30 minutes if needed.•Polyamines should be handled in a fume hood due to toxicity at high concentrations. Read supplier instructions carefully.•If a sterile pH probe cannot be used, we recommend aliquoting a small volume of media (2 ml) before each measurement, to maintain media sterility. Discard the aliquoted media after taking a measurement.•As CF media is rich in chemicals, it is advised to use more than one filter unit, to prevent saturation.Testing the effect of individual chemicals on bacterial growthP. aeruginosa growth was determined at three different concentrations. Experiments were performed over 20 hours of growth for PAO1 and LESB65, using M9 media as a base to support minimal bacterial growth . PAO1 was chosen as a non-CF laboratory reference strain, to act as a comparator to the CF-adapted strain LESB65. Chosen concentrations of all chemicals were higher in CF conditions vs health conditions. Most chemicals were included in both health and CF conditions, with the exception of bile salts and glucose, which were only tested in CF conditions. Airway glucose is significantly elevated in CF related diabetes and bile in the lower airways is associated with CF, as a co-morbidity of gastroesophageal reflux disease.,For each chemical under consideration for inclusion in respiratory-mimicking media, the effect onResults from these experiments showed that, when used individually, most of the tested chemicals enhanced growth of both PAO1 and LESB65 . The highest concentrations of bile salts, succinate and extracellular DNA (eDNA) used in CF conditions were toxic to bacteria ). The antimicrobial activity of lysozyme and lactoferrin was also observed to be both niche and concentration dependent, potentially due to decreased antimicrobial activity in lower temperatures, charge interference at high concentrations or induction of bacterial defence mechanism.PAO1 and LESB65 can be maintained in respiratory-mimicking media for at least three days8 CFU/ml  values were determined for both strains, in all media, using Growthcurver, and demonstrated that PAO1 performed better under CF than health conditions  staining assays showed that there are niche, disease state and strain specific influences on biofilm formation  genes were selected as they are virulence genes ofP. aeruginosa known to contribute to chronic infection., PA2911 and PA2382 genes were also selected, as expression of these genes in clinical isolates in CF sputum is observed but expression has not previously been captured inin vitro CF models.algU is responsible for alginate overproduction, causing mucoidy and contributing to chronic infection in PwCF. MexB is responsible for exporting biological metabolites and antimicrobial compounds, playing a crucial role in antimicrobial resistance in the airways. PA2911 is predicted to be a Ton-B dependent receptor located in the outer membrane and responsible for sideophore transport. PA2382 (lldA) is a L-lactate dehydrogenase, important for pyruvate metabolism. Two-way ANOVA analysis was performed for each gene of interest, to assess whether strain background or media type influenced gene expression. Strain was found to be a significant variable formexB andPA2382 expression (p < 0.0001 for both), whilst media type was a significant variable formexB (p = 0.0308) andPA2911 (p = 0.0462) expression. In Dunnett’s multiple comparison test, with LB set as the control group for each strain, significant differences in gene expression were identified formexB with LESB65 grown in HSM (p = 0.0249) or CFSM (p = 0.0201) and forPA2382 with PAO1 grown in CFSM (p = 0.0243) or CFLM (p = 0.0273). In contrast to the other genes tested, expression ofalgU appeared to be only minimally affected by media type and strain background  and healthy lung (HLM) media will allow the research community to study other aspects of respiratory microbiology, such as bacterial sinusitis, or pneumonia.To date, different liquid culture media have been developed with the aim of capturing the conditions of CF sputum. However, these media reflect the properties of lower airway regions. The growing evidence for the importance of the upper airways in shaping bacterial within-host adaptation makes liquid culture media reflecting sinus conditions a useful addition to the CF microbiologist’s toolkit. Having bespoke media for individual airway compartments allows airway niches to be studied in isolation, something impossible to do– In one example study, researchers competed different pneumococcal serotypes in a mouse model of pneumococcal nasopharyngeal carriage. The study used >200 mice, but many experiments could have been performed in sinus mimicking media, had it been available, with only the neutrophil-depletion studies necessitating animal usage. Thus, sinus mimicking media could have enabled 60-75% reduction in usage in the study. Carriage protocols are mild-severity, but similar studies to compare virulence in moderate or severe pneumonia models could utilise lung mimicking media.In respiratory microbiology a common experimental approach is to screen panels of bacterial isolates in mouse models to determine virulence.in vitro orin vivo systems, potentially leading to a significant reduction in the number of animals used to study bacterial pathogenicity in the host environment.The culture media could be used to examine how bacterial communities develop in the face of host pressures, enabling study of biofilm formation in relevant environments. They offer a platform for assessment of bacterial gene expression in response to stress and to assess efficacy of drugs and therapeutics in a system more relevant than pathogen growth in broth, but more cost effective, less labour-intensive and ethical than animal models. The media can also be used as a pre-screening tool, to identify the most phenotypically interesting isolates to take forward into studies in more complexP. aeruginosa biofilm formation, although we would recommend storage at -80°C for continued long term use. Definitive confirmation of long-term stability of the media will require LCMS, for detection of individual media components, coupled with detailed metabolic phenotyping of bacteria grown therein.The developed media were designed to be cost effective for the purposes of long-term experiments . The med,– Iron and zinc are essential metals for bacteria. Bacteria must actively acquire them through high affinity transport mechanisms. In addition to these potential nutrient sources, the host derived antimicrobials lysozyme and lactoferrin were able to partially inhibit the growth of PAO1 at concentrations associated with health, however they did not cause significant growth inhibition at the higher concentrations of CF sinuses.At concentrations associated with conditions of the healthy sinuses , PAO1 showed increased growth in the presence of only two of the chemicals (eDNA and FeCl2), relative to the M9-only control. By contrast, at concentrations associated with CF sinuses , significant increases in growth were observed for six of the chemicals , relative to the M9-only control. Chemicals that induced higher growth rates at one or more of the concentrations tested for healthy and CF sinuses were albumin, eDNA, amino acids, iron, magnesium, copper, zinc and N-acetyl glucosamine (GlcNac). Many of these factors are well known to promote bacterial survival and may act as nutrient sources in these niches.Similar to PAO1, increased growth of LESB65 was observed at CF-relevant concentrations for the majority of the chemicals. Growth was higher than the M9 control for at least one tested concentration of albumin, amino acids, mucin, spermine, zinc, lysozyme and lactoferrin. Relative to PAO1, the airway adapted isolate LESB65 appeared better able to grow in the face of high concentrations of host-derived antimicrobials and airway-abundant chemicals such as polyamines. The enzymatic activity of lactoferrin will not function optimally at 34°C in sinuses media (SLM), nullifying its growth inhibiting activity.One of the key differences between CFSM and CFLM conditions was that the host-derived antimicrobial lactoferrin decreased the growth of PAO1 in CFLM while it increased the growth at all tested concentration in CFSM, where it may be acting as a nutrient source, as it is heavily glycosylated.P. aeruginosa in CFLM but, in CFSM, all concentrations stimulated higher rates of growth. This may be a result of lysozyme-dependent changes in bacterial phenotype. Previously, it has also been suggested that the effectiveness of lysozyme is reduced in chronically infected airways due to electrostatic sequestration of the enzyme by anionic biopolymers., Over production of inhibitor of vertebrate lysozyme (ivy) protein may also explain success in lysozyme-rich environments, asivy knockout mutants show growth defects in the presence of lysozyme. eDNA elevated bacterial growth in CFLM while it showed a growth inhibiting effect in HLM, where the concentrations tested were lower, further highlighting the positive effect of the chemically rich CF airways on bacterial survival.Lysozyme had no significant positive or negative effect on growth ofYang et al. for PAO1 in CF sputum (66 minutes). The doubling time of PAO1 was ~85 minutes in healthy sinus and lung media. Doubling time of LESB65 was ~105 min in CFSM and ~92 minutes in CFL and ~86 minutes in both healthy media. This finding can be supported by the findings ofYang et al. who found two to threefold slower generation times in isolates from PwCF patients, compared to a laboratory strain. This observed reduced growth was also seen in other CF isolates, compared to non-CF isolates. One possible explanation for this could be that the slow-growing phenotype develops as result of the stressful conditions associated with continuous exposure to host-derived antimicrobials and other molecules of the immune system. PwCF also undergo intensive antibiotic treatment, which acts as a further stress signal. Chromosomal mutations associated with antibiotic tolerance often also have growth rate-reducing effects.Growth curves of PAO1 and LESB65 in airway media suggest a doubling time for PAO1 in CF media of 74 minutes (in both CFSM and CFLM), similar to the findings ofin P. aeruginosa that differ considerably from common laboratory and currently available liquid culture media (such as SCFM2), as evidenced by differences in attached and free-floating biofilm formation and expression of infection-relevant genes, including those that are highly expressed in CF sputum but poorly expressed in available liquid culture models. For a robust assessment of the ability of respiratory mimicking media to induce appropriate gene expression inP. aeruginosa, we await the outcome of RNAseq experiments, in which transcriptomes from LESB65 grown in CFSM and CFLM will be benchmarked against those from CF sputum, using a validated quantitative framework.Results from phenotypic assays show that airway media induce responses whereas HSM, HLM, CFSM and CFLM have been developed with the aim of not only capturing nutritional components but also the host derived antimicrobial content of different airway niches as well as stimulators of bacterial signalling systems, such as polyamines and bile salts. We observed emergence of new colony morphologies inP. aeruginosa PAO1 cultured long-term in respiratory mimicking media. Similar observations have been made following bacterial culture in SCFM and SCFM2,, but also in nutrient rich media, suggesting this phenotype may be a generalised adaptation to growth in biofilm. For the media described here, as well as for other respiratory mimicking media andin vitro andin vivo models of CF airway infection, there is a need to better understand those phenotypes that are specifically niche-adaptive, as distinct from those that confer benefits for a particular lifestyle, such as growth in biofilm.SCFM was developed to mimic the nutritional components of CF sputum and contains amino acids, ions, glucose and lactatein vitro mimics is that they fail to capture the host cellular characteristics of different airway niches. The presence of immune cells significantly affects the pathophysiology of CF as a result of both the inflammatory processes and the direct effects of immune cells on bacteria. Experiments combining the developed media with cell lines or primary cells  could provide additional information about host-pathogen interactions within the airways.An obvious limitation of theseP. aeruginosa, a well-known CF pathogen, in health and disease by recapitulating different areas of the respiratory tract. With these compartmentalised airway-mimicking media, we aim to answer questions relating to how each airway niche affects bacterial behaviour and adaptation in CF and non-CF conditions, without the need for invasive whole animal studies for preliminary studies. We hope these media will become a resource for clinical microbiologists, respiratory clinicians, evolutionary biologists, pharmacologists and immunologists, as a tool to understand bacterial physiology, evolution, virulence and resistance to novel therapeutics, and as a means of reducing or replacing animal use.In summary, we present here a collection of culture media replicating different airway niches in health and CF. Importantly, our study showed that these media can be used to study different phenotypic characteristics of bacteria or study of host-pathogen interactions. We show that the media can be used to studyhttps://doi.org/10.6084/m9.figshare.20463159.v6.Figshare: Underlying data for “Development of liquid culture media mimicking the conditions of sinuses and lungs in cystic fibrosis and health”,-Biofilm assay OD values and fluorescent intensity.xlsx-F1000_qPCR_RawData.xlsx-Growth CFUs.xlsx-viscosity supplementary.xlsx-Colony E fig 7.png-colony D fig 7.png-colony C fig 7.png-viscosity supplementary.xlsxThis project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the Some additional statistical analyses have now been included in the revised version. I have no further comments.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:MicrobiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this article, the authors sought to develop four new culturing media—Healthy Sinus Media (HSM), Healthy Lung Media (HLM), Cystic Fibrosis Sinus Media (CFSM), andPseudomonas aeruginosa growth. Furthermore, there are yet no media that have been developed to capture the environments of healthy human lungs or sinuses, nor that of CF-impacted sinuses. Cystic Fibrosis Lung Media (CFLM)—to recapitulate the environment of human lungs and sinuses, both in healthy individuals and those with cystic fibrosis (CF). The authors justify the need for the development of novel media by emphasizing that Artificial Sputum Media (ASM) and Synthetic Cystic Fibrosis Media (SCFM and SCFM2) capture many key nutritional aspects of the CF lung, they lack critical host-derived antimicrobials, polyamines, glucose, and bile salts. All of which impactP. aeruginosa infection, including but not limited to: culturing strains in each chemical component independently before pooling, measuring growth and biofilm phenotypes, assessing long-term media stability, measuring gene expression, etc. This article is scientifically sound in developing and thoroughly testing four novel media to model human airways. The authors employ many different experimental tests to assay the viability of these novel media and assess their relevance to The authors use appropriate statistical tests as necessary and conduct all experiments with technical and biological replicates. In addition, the authors describe detailed methods for preparing each medium, including supplier information for all reagents, stock concentrations, volumes, final concentrations, sterilization procedures, and special notes on media preparation so that others may easily reproduce these procedures.in vivo, for example, altering growth temperatures between sinus and lung media. The article also provides sufficient rationale for the necessity of these media in the scientific community. The creation of these novel media will significantly add to the scientific community’s ability to better model CF infection dynamicsin vitro preclinical models. Including healthy lung and sinus media will no doubt serve as a useful control for these experiments than LB or M9. Having accurate models for upper airway infection is also key in understanding the intermittent colonization and persistence ofP. aeruginosa during initial colonization and antibiotic treatment. The authors provide a rationale for each component added with references to literature. The authors conduct all experiments in appropriate conditions to mimic The various components tested by the authors for the development of these novel media were: Lysozyme, Lactoferrin, Spermidine, Spermine, Putrescine, Mucin, eDNA, Albumin, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, NaCl, Sialic acid, Bile, N-acetyl glucosamine, Glucose, Succinate, Galactose, L-Methionine, L-Phenylalanine, LProline, L-Serine, L-Threonine, L-Tryptophan, L-Valine, L-Ornithine, L-Tyrosine, L(+)-Asparagine monohydrate, L-Alanine, L-Arginine, L(+)-Aspartic acid, L-Cysteine, LGlutamine,P. aeruginosa in their tested conditions. The authors first grew these strains in M9 media supplemented with each chemical component independently at three different concentrations. These concentrations were selected to most closely reflect the physiological concentration of each compound within each niche, based upon cited references in the literature or experimental testing of metabolites. Upon confirmation that these strains could grow sufficiently at the selected concentration of each metabolite, the authors then pooled the components at the appropriate ratios to yield four novel respiratory media: Healthy Sinus Media (HSM), Healthy Lung Media (HLM), Cystic Fibrosis Sinus Media (CFSM), and Cystic Fibrosis Lung Media (CFLM). L-Glycine, L-Histidine, L-Isoleucine, L-Leucine, and L-Lysine. The authors chose the PAO1 and LESB65 as representative laboratory and CF-adapted strains of The authors tested the viability and relevance of these media as preclinical models for human respiratory infections by assessing the: i) growth and biofilm formation of strains cultured continuously for 72 hours; ii) viscosity and shear rate in the media pre- and post-biofilm formation; iii) the stability of media after storage at 4° C for 40 days; iv) transcriptional changes of infection-relevant genes; and v) changes in colony morphology of strains evolved for 40 days; of PAO1 and LESB65 in each medium.P. aeruginosa in SCFM (authors may include the publications that used these media to study various aspects ofP. aeruginosa infection). The crystal violet assays, which included growth in M9 and Luria-Bertani (LB) as additional comparators against the newly described media, show niche, disease state, and strain-specific differences in surface-attached and free-floating biofilm formation, highlighting the importance of using clinically-relevant models for estimating biofilm formation. Although the aggregate and aggregate formation are not tested here, authors can discuss those future assessments for these media. The growth rate experiments show that PAO1 and LESB65 could be maintained for at least 72 hours, with similar dynamics to those previously published for The authors also show that the viscosity and shear rate measurements of their proposed media agree with those published previously for CF and healthy human sputum. The authors also show that each media could support the same level of biofilm formation in PAO1 after 0, 10, 20, and 30 days of storage at 4° C, implying that the media remains stable for this duration. qPCR analysis shows that expression of the four tested virulence genes—algU, mexB, PA2911, and PA2382—significantly increased in all four novel media compared to their transcription levels in LB and additionally were comparable to transcription levels of the same strains grown in SCFM2. Long-term culturing of PAO1 in these media shows the evolution of a wrinkly colony morphotype, a potential redox-driven adaptation to maximize oxygen availability in biofilms, and morphological adaptations specific to CF media.Some minor comments:P. aeruginosa in the stored media displays any altered metabolism, physiology, or spatial organization. The authors determined these novel media's viability over 30 days by measuring PAO1 biofilm formation at 0, 10, 20, and 30 days. While it is nice to show that the media sustain similar levels of biofilm growth regardless of whether the media is freshly prepared, this might not be the best indicator of media stability over long periods. Instead, it would have been nice to see the authors conduct LCMS analyses to determine the stability of each of the chemical components at 0 and 30 days and detect if any possible degradation had occurred. Also, it is important to determine whether the growth ofP. aeruginosa grown in new media (fresh and stored at 4° C) to the transcriptional profile of cells grown in SCFM2 andin vivo samples would confirm the similarity of these preclinical models to the infection and healthy environments. Additionally, instead of hand-picking a short list of four genes, it would have greatly strengthened the supporting case for these novel media by RNA-seq analysis. For example, comparing the entire transcriptional profile of I also understand that these experiments can be addressed in the future and are out of the scope of this study, which aimed to introduce these novel media for upper and lower respiratory tracts; however, I recommend that authors address this in the discussion section. I recommend authors include these studies and discuss the key benefits of using the novel HSM, CFSM, HLM, and CFLM media over the other versions available. In the section titled “Airway mimicking media is stable for at least 30 days at 4° C,” the wording of the first sentence leads to some confusion. At first glance, it appears to imply that PAO1 was cultured for 30 days, although the authors meant to say that PAO1 was cultured in media that had been stored for 30 days. The authors may want to re-write this sentence to avoid confusion. In Tables 3, 8, and 12, please use a consistent format for the concentration units μg/L or μg/l. In Figure 5, the legend below the chart is redundant, as it shows the same data as the labels on the X-axis. I would recommend removing this legend to simplify the readers' view. In Figure 6, standard deviation bars and significance labels would help the reader understand which relationships are significant without having to reference the text.P. aeruginosa in TSB, SCFM, and SCFM2 by Schick A, Kassen R (2018), Azimiet al., 2020, and Flynnet al., 2016, showing the emergence of various morphotypes in the populations that authors may want to include. There are studies on the evolution ofP. aeruginosa in other preclinical infection models are not unique to novel developed media. I suggest that authors include previous studies, discussing how future studies of genetic and phenotypic adaptation to the developed media are required. Thus, future analysis can confirm the unique adaptive traits observed in this study, similar to upper and lower CF respiratory infections. The emergence of the observed morphotypes and phenotypes in this study is similar to what is observed as a result of evolving In Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. I would consider re-titling this table to reflect the prevalence of each morphotype over the evolution period.Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?PartlyIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Chronic infections, Biogeography of Infection, Pseudomonas aeruginosa population dynamics.We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for the positive comments regarding the manuscript and for your helpful suggestions for improvements and discussion points. We have addressed each point raised, below and have submitted an updated manuscript version in which the changes have been made.The authors determined these novel media's viability over 30 days by measuring PAO1 biofilm formation at 0, 10, 20, and 30 days. While it is nice to show that the media sustain similar levels of biofilm growth regardless of whether the media is freshly prepared, this might not be the best indicator of media stability over long periods. Instead, it would have been nice to see the authors conduct LCMS analyses to determine the stability of each of the chemical components at 0 and 30 days and detect if any possible degradation had occurred. Also, it is important to determine whether the growth of P. aeruginosa in the stored media displays any altered metabolism, physiology, or spatial organization. We agree both that LCMS would offer a more complete determination of chemical stability and that investigation of bacterial metabolism, physiology and spatial organisation would be desirable. We have added a sentence to the discussion, highlighting the need for further studies into media stability. These studies are underway in our lab.Additionally, instead of hand-picking a short list of four genes, it would have greatly strengthened the supporting case for these novel media by RNA-seq analysis. For example, comparing the entire transcriptional profile of P. aeruginosa grown in new media (fresh and stored at 4° C) to the transcriptional profile of cells grown in SCFM2 and in vivo samples would confirm the similarity of these preclinical models to the infection and healthy environments. I also understand that these experiments can be addressed in the future and are out of the scope of this study, which aimed to introduce these novel media for upper and lower respiratory tracts; however, I recommend that authors address this in the discussion section. I recommend authors include these studies and discuss the key benefits of using the novel HSM, CFSM, HLM, and CFLM media over the other versions available. We completely agree and these studies have already begun. The large RNAseq dataset will require careful analysis. We feel that the space and word count needed to describe those data fully would make the current manuscript overly lengthy. As the reviewers suggest, we have instead mentioned the need for these investigations in the discussion.In the section titled “Airway mimicking media is stable for at least 30 days at 4° C,” the wording of the first sentence leads to some confusion. At first glance, it appears to imply that PAO1 was cultured for 30 days, although the authors meant to say that PAO1 was cultured in media that had been stored for 30 days. The authors may want to re-write this sentence to avoid confusion. Sorry for this confusion, we have now reworded the sentence.In Tables 3, 8, and 12, please use a consistent format for the concentration units μg/L or μg/l. We have now used μg/l throughout.In Figure 5, the legend below the chart is redundant, as it shows the same data as the labels on the X-axis. I would recommend removing this legend to simplify the readers' view. This figure has been reworked and the legend removed.In Figure 6, standard deviation bars and significance labels would help the reader understand which relationships are significant without having to reference the text. Standard deviation bars and significance labels have now been added to these graphs and we have described ANOVA summary statistics in the main text.1There are studies on the evolution of P. aeruginosa in TSB, SCFM, and SCFM2 by Schick A, Kassen R (2018)2, Azimi et al., 20203, and Flynn et al., 2016, showing the emergence of various morphotypes in the populations that authors may want to include. Thank you for highlighting this. Reference to these studies has been added to our discussion.The emergence of the observed morphotypes and phenotypes in this study is similar to what is observed as a result of evolving P. aeruginosa in other preclinical infection models are not unique to novel developed media. I suggest that authors include previous studies, discussing how future studies of genetic and phenotypic adaptation to the developed media are required. Thus, future analysis can confirm the unique adaptive traits observed in this study, similar to upper and lower CF respiratory infections. Thank you for this suggestion. We have added the three references mentioned in the previous comment, alongside some discussion of the need to deconvolute those phenotypes that are adaptive for different respiratory environments from those that are merely adaptive to different modes of growth.In Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. I would consider re-titling this table to reflect the prevalence of each morphotype over the evolution period. We have re-titled this table. Unfortunately, these data were captured as presence/absence for each morphotype, at each time point. We don’t, therefore, have information on relative or absolute morphotype abundance over time. This is a very nice manuscript describing four new synthetic media mimicking growth conditions in the human lung. The main advancement is the development of both CF and healthy lung media and the addition of host-derived antimicrobials and stimulators  of bacterial signalling systems that are not included in commonly used SCFM2 medium. To some extent, this comparison falls a bit short as not all measurements are performed in SCFM2 and developed media. Moreover, the gene expression analysis relies on the expression of a few marker genes instead of whole genome transcriptomics, which could have revealed further interesting differences and allowed linking data with available transcriptomics data from CF lungs. Nevertheless, these are small omissions and could perhaps just be discussed briefly in the discussion. My main concern is the statistical analysis that could be improved a bit. I would suggest using full factorial analysis to clearly show if growth in the media is different for the two studied strains and if there are any interesting interactions between strain identity and specific media. These statistics should be properly reported in the text or supplementary tables including test statistics, error, and degrees of freedom.Use Repeated Measures Anova or GLMMs for analysing time-dependent data in Figure 1.Include strain identity in the model in Figures 2-3 to statistically compare if strains grew differently in these media.Figure 4: Which bacteria were grown in these media before the measurements? Or is this data averaged over both bacteria? Needs a little bit more information.Figure 5: Could also analyse the effect of time for each media and across all media to show that no change in biofilm formation was observed. I would reconsider naming this measure as ‘Stability of biofilm formation’ as this is what you measure instead of media stability, which could be understood as a measure of degradation of its components. Just to avoid confusion.Figure 6: I think the results here are much more nuanced than described in the results text. For example, the expression of algU is clearly different from other marker genes and there is no clear increase in its expression relative to growth in LB media (in contrast to what is said in the results text). Here, factorial Anova (or GLMM) would be powerful to show significant interactions between different media, studied strains, and developed media.Figure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive.Out of interest, did you specifically test the effect of freezing (-80°C) for media composition? I would suggest:Is the rationale for developing the new method (or application) clearly explained?YesAre the 3Rs implications of the work described accurately?YesIs the description of the method technically sound?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?YesIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre a suitable application and appropriate end-users identified?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:MicrobiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for the helpful comments and suggestions for alternative data analysis. We have addressed each point raised, below and have submitted a revised manuscript in which the indicated changes have been made.To some extent, [the phenotypic] comparison falls a bit short as not all measurements are performed in SCFM2 and developed media. Moreover, the gene expression analysis relies on the expression of a few marker genes instead of whole genome transcriptomics, which could have revealed further interesting differences and allowed linking data with available transcriptomics data from CF lungs. Nevertheless, these are small omissions and could perhaps just be discussed briefly in the discussion. We agree that transcriptomic data would represent an important validation dataset for these media. We have now added some discussion of this point to the manuscript and hope to be able to report these data shortly. We agree, also, that it would be preferable to have SCFM2 data for all phenotypic comparisons. Where these are absent, we have now referenced studies conducted by others, in which SCFM2 was used.My main concern is the statistical analysis that could be improved a bit. I would suggest using full factorial analysis to clearly show if growth in the media is different for the two studied strains and if there are any interesting interactions between strain identity and specific media. These statistics should be properly reported in the text or supplementary tables including test statistics, error, and degrees of freedom. I would suggest:Use Repeated Measures Anova or GLMMs for analysing time-dependent data in Figure 1. We were cautious of over-interpreting these data, particularly when comparing between strains, as PAO1 and LESB65 have considerable differences in growth profile, even in standard laboratory media. However, we agree with the reviewer that comparisons of relative growth between media would be informative. We have now included empirical area under the curve (AUC_e) values for both strains, in all four media. These were determined from the original growth data in Figure 1. Two-way ANOVA analysis  of the AUC_e values shows that both strain and media type are significant variables . Sidak’s multiple comparison testing demonstrates the success of PAO1 in the CF media, relative to in healthy sinus and lung media. P values and a description of the findings have been added to the manuscript text and the AUC_e values are presented in Figure 1C.Include strain identity in the model in Figures 2-3 to statistically compare if strains grew differently in these media. We have done as suggested and provide updated figures, analysis and text in the revised manuscript. Strain was found to be a significant variable in both biofilm assays. We have reported summary statistics and pairwise comparisons in the text. Briefly, for crystal violet assays, PAO1 formed attached biofilms more readily than LESB65 in all airway niche media bar HLM . In resazurin assays, PAO1 biofilm formation differed significantly from that of LESB65 in HLM (p = 0.0001) and HSM (p < 0.0001) but not in the other media tested.Figure 4: Which bacteria were grown in these media before the measurements? Or is this data averaged over both bacteria? Needs a little bit more information. This was done with PAO1. We have added this info to the figure legend and the text in the results section.Figure 5: Could also analyse the effect of time for each media and across all media to show that no change in biofilm formation was observed. We have re-analysed with an ordinary two-way ANOVA. Media is a significant factor (P<0.0001), whilst media storage time is not (P=0.4174). No individual pairwise comparisons between timepoints proved significant for any of the four media. We have reported the ANOVA summary statistics in the text, as suggested.I would reconsider naming this measure as ‘Stability of biofilm formation’ as this is what you measure instead of media stability, which could be understood as a measure of degradation of its components. Just to avoid confusion.  The title has been changed, as suggested.Figure 6: I think the results here are much more nuanced than described in the results text. For example, the expression of algU is clearly different from other marker genes and there is no clear increase in its expression relative to growth in LB media (in contrast to what is said in the results text). Here, factorial Anova (or GLMM) would be powerful to show significant interactions between different media, studied strains, and developed media.            mexB andPA2382 expression (p < 0.0001 for both), whilst media type was a significant variable formexB (p = 0.0308) andPA2911 (p = 0.0462) expression. In Dunnett’s multiple comparison test, with LB set as the control group for each strain, significant differences in gene expression were identified formexB with LESB65 grown in HSM (p = 0.0249) or CFSM (p = 0.0201) and forPA2382 with PAO1 grown in CFSM (p = 0.0243) or CFLM (p = 0.0273). We agree thatalgU is clearly different from the other genes and have acknowledged this in the manuscript text. Apologies, our interpretation of these data was overly simplistic. We have now performed two-way ANOVA for each gene, to assess the contribution of strain and media type to observed differences. We have reported these in the manuscript, along with updated graphs and data descriptions. Briefly, strain was found to be a significant variable forFigure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive. Unfortunately not. We plated a proportion of the total growth at each transfer and then recorded the different colony morphotypes as present or absent, rather than a quantitative measure.Out of interest, did you specifically test the effect of freezing (-80°C) for media composition? Only in the same way as reported for the 4°C storage, i.e. we confirmed that the media would still support biofilm growth. We’ve added to the discussion that LCMS could provide additional useful data on media stability under different conditions.


We also found that q247 cfDNA levels were significantly higher in women with an associated female factor, such as endometriosis, PCOS and POF, compared with women with no specific cause of infertility (p = 0.033). The concentration of cfDNA did not vary significantly in each group of women with an associated female factor. The concentration of cfDNA was significantly higher in the FF of women that obtained embryos with a high fragmentation rate, compared to embryos with a low fragmentation rate (p = 0.007). Finally, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels (p = 0.043). The quantification of cfDNA could be an important biomarker of follicular micro-environment quality to predict embryo quality and the success of IVF, making them more specific and effective.Cell-free DNA fragments detected in blood and in other biological fluids are released from apoptotic/necrotic cells. In this study, we analyzed cfDNA levels in follicular fluid (FF) samples from patients with infertility. Samples were collected from 178 infertile women and cfDNA was extracted and quantified by qPCR, using ALU115 and ALU247 primers, and statistical correlations were performed. We found that cfDNA concentration was significantly higher in FF pools from women aged 35 and over than in women under 35 years of age ( Infertility is a disease characterized by the failure to establish a clinical pregnancy after 12 months of regular, unprotected sexual intercourse or due to an impairment of a person’s capacity to reproduce either as an individual or with his/her partner. Worldwide, about 1 in 10 couples develop primary or secondary infertility. Fertility interventions may be initiated in less than one year based on medical, sexual and reproductive history, age, physical findings and diagnostic testing. We often associate infertility with a female cause; however, infertility is a disease of the couple which may have female, male or mixed causes . Thus, iIn in-vitro fertilization (IVF) treatments, the selection of embryos with greater potential for implantation is limited, depending mainly on morphological criteria. Embryonic classification via embryo morphology is very subjective, despite being the most used nowadays. Some of these limitations include comparing, grouping and statistically stratifying embryos to determine the most viable embryo for implantation ,6. It isSeveral studies have shown that the follicular fluid (FF), which surrounds the oocyte, is directly involved in follicular maturation, oocyte growth and development ,8,9. In Cell-free DNA (cfDNA) is characterized by non-cell-bound, double-stranded DNA fragments present in human plasma or serum . The disPrevious studies that analyzed fetal circulating DNA in maternal plasma were essential for the development of non-invasive prenatal diagnostic tools . These fIn summary, cfDNA quantification is believed to be a non-invasive biomarker that could help to determine the chance of IVF success in the future. This technique could help patients who have had multiple in vitro fertilization failures, and who have already tried other specific techniques without success . As mentIn total, we collected 178 FF samples of women under 39 years of age that failed to establish a clinical pregnancy after 12 months of regular, unprotected sexual intercourse, followed at the Assisted Reproduction Unit from the Academic Hospital Center of Cova da Beira, Covilhã, Portugal. A total of 45 women had endometriosis, 60 were diagnosed with PCOS, 40 presented premature ovarian failure (POF) and 44 women were considered as controls with no specific cause of female infertility. Women were enrolled between October 2015 and July 2019 A. The inThe study was approved by the Ethical Committee of the Academic Hospital Center of Cova da Beira, Covilhã, Portugal . Informed consent was obtained from cases before participating in the study. Oocyte retrieval was performed by transvaginal ultrasound-guided aspiration 36 h after the injection of human chorionic gonadotrophin and each follicle was aspirated. To avoid any blood contamination, only clear fluid samples were included, whereas bloodstain and cloudy follicular fluid samples were excluded B.g for 15 min. Supernatants were filtered with 0.2 μm filters to eliminate cell debris and then stored −80 °C until cfDNA extraction. A total of 178 FF pools were collected for this study.All FF samples from the same patient were pooled and a volume of 15 mL was centrifuged at 3000× g for 5 min, supernatants were removed and stored at −80 °C for cfDNA quantification , using ALU115 and ALU247 primers. The ALU115 primer amplified both short (apoptotic) and long (non-apoptotic) DNA fragments, whereas the ALU247 primer amplified long non-apoptotic DNA fragments only. The ALU-qPCR result obtained with ALU115 primers represents the total amount of serum DNA. This allows the calculation of DNA integrity by using the q247/q115 ratio, which represents the proportion of cfDNA generated by necrosis over total cfDNA . The seq®. Results were considered significant when p ≤ 0.05  and categorical variables as numbers and percentages. Analysis of variance (ANOVA) or a Mann–Whitney test was used to compare cfDNA levels based on the homogeneity of variances by the Levene test and normality of the distribution assessed using the Shapiro–Wilk test. A multiple linear regression (GLM) as multivariable analysis was performed for the variables infertility factor, βhCG analysis result, age and embryo fragmentation. Statistical tests were performed using SPSSp ≤ 0.05 E.2 (range: 17 to 33 kg/m2). The mean age at menarche was 12 years ± 1.82 (range: 8 to 18 years) and the infertility length was 53 months (range: 14 to 192 months). This prospective study recruited 178 women enrolled in conventional IVF and ICSI. The clinical and pathological characteristics are shown in The calculation of DNA integrity by using the q247/q115 ratio represents the proportion of cfDNA generated by necrosis over total cfDNA. The mean of q247/q115 ratio was 0.99 (SD: 0.12) in follicular fluid samples (n = 178), suggesting that the cfDNA analyzed was mainly originated from cellular necrotic events. p = 0.017). Concerning BMI, 123 women presented less or equal 25 kg/m2 BMI and 55 had more than 25 kg/m2 BMI. FF cfDNA concentration was not significantly different between these two groups. Considering ethnicity, 174 women were Caucasian, 2 were Gypsy and 2 were Afro-Europeans. We did not find statistically significant differences between these groups. On what concerns age at menarche, 102 women had their first menstruation before 13 years, 52 between 13 and 14 years and 21 presented an age at menarche over 15 years. FF cfDNA concentration was not significantly higher in either group. Regarding smoking habits, 130 women never smoked, 21 previously smoked and in 27 smoking habits are present. When we compared these groups, in terms of cfDNA concentration, we did not find statistically significant differences. Considering infertility length, 9 women presented infertility for less than 24 months, 91 had an infertility time between 24 and 48 months and 78 had been infertile for more than 48 months. FF cfDNA concentration was not significantly different between the three groups. We performed statistical comparison for each ALU primer (q115 and q247) separately, in addition to the ratio of q247/q115 (cfDNA concentration) for all parameters. p = 0.033).Female factors and women with no specific cause of infertility are presented in AMH, AFC, days of stimulation and total dose of gonadotropins are described in p = 0.007). Lastly, 44 women had a positive βhCG result (pregnant women) and 84 had a negative result (non-pregnant women). When we compared the results of pregnant women with non-pregnant women, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels than women who had a confirmed pregnancy (p = 0.043).p = 0.044; R2 = 0.083]. However, in this model, only embryo fragmentation was considered as a predictor of cfDNA concentration values . Finally, we performed a multiple linear regression to verify whether the variables infertility factor, βhCG analysis result, age and embryo fragmentation can predict cfDNA concentration values. The analysis resulted in a statistically significant model . In this model, only embryo fragmentation was considered as a predictor of cfDNA concentration values . This result enforces our previous results and allows us to state that this predictive model could be used as a supplemental tool for determining the chance of IVF success. Finally, we performed a multiple linear regression to verify whether the variables infertility factor, βhCG analysis result, age and embryo fragmentation can predict cfDNA concentration values. To perform this regression, six presuppositions must be met, namely: the value of n must be greater than 20 for each independent variable; residual values must be independent; residuals must be normally distributed; homoscedasticity, absence of multicollinearity and absence of outliers. Our data meet all presuppositions. The analysis resulted in a statistically significant model [F = 4, 112) = 2.531; A limitation of our study is the use of exogenous gonadotropins in short and long PCOS protocols to increase the number of retrieved oocytes, because the use of exogenous gonadotropins interferes with follicular steroidogenesis at certain levels and might be one of the causes of higher concentration of cfDNA in FF. So, further research considering IVF populations during a non-stimulated menstrual cycle is required. It is also very important to include a control group in the study and use more primers to determine the apoptotic or necrotic origin of cfDNA. In the future, we also aim to evaluate cfDNA concentration in the serum of stimulated women, because another limitation of this study was not having performed real-time PCR in the serum of these women. In addition, future studies that include genetic sequencing of DNA fragments to determine the origin of the cfDNA would be helpful to recognize potential pathologies that affect fertility and prevent successful implantation after IVF treatments.In conclusion, this study indicates that cfDNA quantification is a non-invasive biomarker, can help to choose top-quality embryos to transfer and can be an aid when determining the chance of IVF success independently of the infertility cause. The identification of new biomarkers that have greater precision in the assessment of embryonic quality can contribute to an improvement in infertility treatments personalization and, therefore, to an increase in the success of IVF, where the assessment of cfDNA concentration in follicular fluid samples can then function as a non-invasive biomarker of embryo quality. It is extremely important to consolidate the optimization of the different existing procedures to make cfDNA analysis routine during the IVF process. Research in this area should continue, as this new test could be incorporated into the program of current practice and improve personalized patient care. Cell-free DNA shows great promise as a new biomarker; however, there are still many questions that are difficult to overcome. Even though it is considered an easy and quick technique to perform, it is also very costly. So, it is fundamental that the cost of analysis becomes more reasonable.