Fractures are the most common orthopedic diseases. It is known that static magnetic fields (SMFs) can contribute to the maintenance of bone health. However, the effect and mechanism of SMFs on fracture is still unclear. This study is aim to investigate the effect of moderate static magnetic fields (MMFs) on bone structure and metabolism during fracture healing.Eight-week-old male C57BL/6J mice were subjected to a unilateral open transverse tibial fracture, and following treatment under geomagnetic field (GMF) or MMF. The micro-computed tomography (Micro-CT) and three-point bending were employed to evaluate the microarchitecture and mechanical properties. Endochondral ossification and bone remodeling were evaluated by bone histomorphometric and serum biochemical assay. In addition, the atomic absorption spectroscopy and ELISA were utilized to examine the influence of MMF exposure on iron metabolism in mice.MMF exposure increased bone mineral density (BMD), bone volume per tissue volume (BV/TV), mechanical properties, and proportion of mineralized bone matrix of the callus during fracture healing. MMF exposure reduced the proportion of cartilage in the callus area during fracture healing. Meanwhile, MMF exposure increased the number of osteoblasts in callus on the 14th day, and reduced the number of osteoclasts on the 28th day of fracture healing. Furthermore, MMF exposure increased PINP and OCN levels, and reduced the TRAP-5b and β-CTX levels in serum. It was also observed that MMF exposure reduced the iron content in the liver and callus, as well as serum ferritin levels while elevating the serum hepcidin concentration.MMF exposure could accelerate fracture healing via promote the endochondral ossification and bone formation while regulating systemic iron metabolism during fracture healing. This study suggests that MMF may have the potential to become a form of physical therapy for fractures. Fractures are the most important orthopedic diseases. Epidemiological investigation showed that more than 170 million new cases of fracture in 2019, with this annual figure continuing to rise [Bone fractures were seriously impeding the daily activities of patients and exerting a substantial economic burden. Consequently, the bone fractures have been recognized as a global public health issue. The process of bone metabolism during fracture healing encompasses various stages, including the formation of cartilage callus, the transformation from cartilage callus to a hard callus, and bone remodeling , 4. EnhaElectromagnetic fields have served as a valuable modality in fracture treatment for more than 70 years . In the According to magnetic flux density, SMFs could be divided into hypomagnetic field , weak static magnetic fields (< 1 mT), moderate static magnetic fields , and high static magnetic fields (HiSMFs) . StudiesBruce GK et al. found that 0.22 T–0.26 T SMF exposure could enhance the mechanical properties of fracture site in rabbits . In addiThe impact on metal ion transport and metabolism is one of the biological mechanisms of SMFs . The balIn this study, we examined the changes of histomorphology, mechanical properties and serum bone metabolism indexes at various stages of the fracture healing process. Aim to elucidate the impact of MMFs whole body exposure on fracture healing and the associated bone metabolism mechanisms. Meanwhile, the iron content in liver and fracture callus, and serum ferritin and hepcidin level have been detected to evaluate the effects of MMF exposure on iron metabolism during fracture healing.The moderate static magnetic field (MMF) environment was generated by 96 neodymium N42 permanent magnets, and each magnet with a diameter of 15 mm and a height of 20 mm , lymphocyte count (LYMPH), red blood cell count (RBC), hemoglobin (HGB), mean corpuscular hemoglobin (MCH) and platelet count (PLT). The results showed that MMF exposure reduced the WBC on day 14 postfracture. However, there were no significant changes observed in LYMPH, RBC, HGB, MCH, and PLT. Meanwhile, the blood parameters in mice subjected to 28 days of MMF exposure were not significantly different from those in control animals , and callus mineralized volume fraction  at fracture sites were significantly increased by MMF exposure on day 28 postfracture  with distinct parameters can exert varying effects on bone tissue and cells . In thisRoutine blood parameters can serve as indicators of the fundamental physiological status of an organism, including the presence of inflammation . It was Inflammation are normal reactions in the early stages of fracture healing, and involved in promoting angiogenesis and initiating the repair process . In contDuring the healing of fractures, there are a continuous improvement in the microstructures of the newly formed mineralized tissue in callus. In this study, MMF exposure significantly improved BMD and BV/TV, and accelerated the deposition of mineralized bone matrix in callus during fracture healing. As the fracture healing process advances to its final stages, there is a gradual return of mechanical properties along with an improved maturation and increased structural organization . In thisThe bone metabolism during fracture healing can be categorized into two major biological stages: endochondral ossification and coupled remodeling . SMFs exIn this study, the MMF exposure increased the mineralized area of the callus on the 14th and 28th day of fracture healing, accelerated the transition from cartilage callus to hard callus. Furthermore, the MMF exposure increased the number of osteoblasts in callus after 14 days of fracture healing. It is suggested that MMF exposure promoted fracture healing by accelerating the process of endochondral osteogenesis. During the stage of coupled remodeling, bone matrix is continually renewed through osteoblast-mediated bone formation and osteoclast-mediated bone resorption , 36. In Osteoclast could participate in multiple stages of fracture healing, and indispensable for the coupled remodeling in the final stage of fracture healing . MeanwhiIron metabolism is closely related to bone health . SMFs coChanges in systemic iron metabolism could lead to symptoms, such as anemia . FurtherPrevious studies found that MMF had no significant effect on the microstructure and mechanical properties of bones in normal mice while could suppressed bone loss in ovariectomized mice and HLU mice , 16, 45.In summary, this study indicated that MMF could promote fracture healing in mice by regulating the processes of endochondral osteogenesis and bone remodeling. These finding offers valuable insights into the biological mechanism of MMF exposure influences on fracture healing and provides a theoretical basis for its application in clinical bone fractures therapy.n = 8), two groups of mice were exposed to a MMF, and the rest of the mice were exposed to geomagnetic field as control groups. Day 14 and 28 postfracture, the mice were culled by cervical dislocation under anesthesia. Blood samples were collected and stored at 4 °C for subsequent analyses. The collected tibia was preserved in 4% paraformaldehyde or at – 80 °C for subsequent analyses, respectively. All animal protocols used in this study were approved by the Lab Animal Ethics and Welfare Committee of Northwestern Polytechnical University.The 8-week-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and utilized to construct the model of open tibial fracture. The fracture surgeries were performed by specialized laboratory personnel with over 2 years of experience. The mice were anesthetized with isoflurane, then cut the skin with a scalpel, and the proximal tibia was drilled a hole with a 27 G needle. Cutting the diaphysis of the tibia to create a fracture, and then fix the fracture site through the hole with a stainless steel insect pin, as the tibia stabilized fractures. Then, the fractured mice were divided into four equal groups .g for 10 min, after which time the supernatant was collected to obtain the serum. Serum markers for bone metabolism, including propeptide of type I procollagen (PINP), osteocalcin (OCN), beta-isomer of the C-terminal telopeptide of type I collagen (β-CTX), and tartrate-resistant acid phosphatase 5 b (TRAP-5b) were examined by respective mouse enzyme linked immunosorbent assay (ELISA) kits . Meanwhile, the serum ferritin and hepcidin content of mice was detected using an ELISA kit . All procedures were strictly performed in line with the instructions of the manufacturers.The blood samples were collected and kept standing still at 4 ℃ until the serum separated out before being centrifuged at 10,000The fractured tibia was collected and the intramedullary pins as well as the external soft tissues were carefully removed. The post fracture tibia at different healing timepoints were scanned using Micro-computed tomography (Micro-CT) . Scanning was performed at 70 kV, 114 μA, 250 ms without a filter. The parameters of the fracture site were three-dimensionally reconstructed using NRecon software . Carried out on the middle 2 mm slices (1 mm above and below the fracture line) as the region of fracture callus, and low-density tissue was distinguished from high-density tissue on the basis of different thresholds. The micro structure parameters of fracture area were analyzed using CTAn software . The following measures of fracture callus structure and composition were evaluated for each specimen: callus bone mineral density (BMD), callus mineralized volume fraction , trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were analyzed.The bone mechanical properties on day 28 postfracture was evaluated by three-point bending test using the Instron-5943 Universal Material Testing Machine . The span of two supports was 8 mm and loading rate was 1 mm/min until the callus fracture again. The load–displacement curve was obtained from the Instron software and used for calculate the parameters of structural properties, including ultimate load, ultimate displacement, stiffness, and bending energy absorption. The stress–strain curve that was normalized by the geometrical measurements was obtained from the MATLAB software , which were used for calculate the parameters of material properties, including ultimate stress, ultimate strain, elastic modulus, and toughness.Histochemical examination of the fracture callus was performed on the 14th and 28th days after the fracture. The fractured tibia was fixed in 4% paraformaldehyde for 48 h, and decalcified by 10% EDTA. Then, the fractured tibia was embedded in paraffin and sectioned in 5 µm thick slices. Safranin orange/fast green (SFO/FG) staining was used to detect the distribution of cartilage and mineralized area in callus, cartilage tissue was stained in red, and mineralized bone tissue was stained in green. Alcian blue staining was also used for cartilage staining at fracture callus sites.HE staining was used to detect the basic morphology of fracture callus and to count osteoblasts, and the osteoblasts adhere to the surface of bone trabeculae in a long spindle shape. Tartrate-resistant acid phosphatase staining  staining was used to evaluate osteoclast in fracture callus, the osteoclasts are stained red. The osteoblast number per bone surface (N.Ob/BS), osteoclast number per bone surface (N.Oc/BS), and osteoclast surface per bone surface (Oc.S/BS) have been analyzed via Fluorescence microscope and Image J software following established procedures by personnel with over 5 years of experience in organizational assessment .The Fe content in the liver and fractured tibia was determined by atomic absorption spectrometry . The liver and fractured tibia were collected from the sacrificed mice after the cardiac perfusion by normal saline solution. Dried the tissues under 120 ℃ for 6 h, and measured its dry weight. After that, heated the tissues at 600 ℃ for 6 h to ash the tissue, then dissolved in 1 ml 65% HNO3. Dilute the samples with ddH2O to a suitable concentration, then the Fe concentration was measured by flame AAS. The Fe content in tissues was calculated as the mass fraction between the mass of the Fe and tissue dry weight, respectively.P > 0.05. Two-tailed Student t tests was used to compare the differences between two groups, and one-way ANOVA was used to analyze the differences between multiple treatment groups. For all statistical tests, P < 0.05 was considered to indicate statistical differences.All statistical analyses were performed using GraphPad Prism statistical software for Windows . The data are expressed as the mean ± standard deviation. The normal distribution was tested by Shapiro–Wilk normality test with


TM ISAC 112 (Thermo Fisher Scientific) on the etiological diagnosis and SIT prescription compared to the conventional diagnostic methods in patients with allergic rhinitis/rhinoconjunctivitis and/or asthma.Recognition of specific allergens triggering immune response is key for the appropriate prescription of allergen‐specific immunotherapy (SIT). This study aimed at evaluating the impact of using the commercially available microarray ImmunoCAP300 patients with respiratory allergic disease, sensitized to three or more pollen aeroallergens from different species, as assessed by a skin prick test (SPT) and specific IgE assays (sIgE), were included in this multicentric, prospective observational study. SPT and a blood test were performed to all patients. Total serum IgE and sIgE (ImmunoCAPTM) for allergens found positive in the SPT and sIgE allergen components (ImmunoCAPTM ISAC 112) were measured.Olea europaea followed by grass, Platanus acerifolia and Parietaria judaica. The molecular diagnosis (MD) revealed Ole e 1 as the most prevalent pollen sensitizer, followed by Cup a 1, Phl p 1, Cyn d 1, Par j 2, Pla a 1, 2, and 3 and Phl p 5. Immunotherapy prescription changed, due to MD testing, in 51% of the cases, with an increase of prescription of SIT from 39% to 65%.According to SPT results, the most prevalent pollen sensitizers in our population were TM ISAC 112, can help clinicians to improve SIT prescription.The identification of the allergen eliciting the respiratory disease is essential for a correct immunotherapy prescription. The advances in allergen characterization using methods, such as the commercial microarray ImmunoCAP Other important allergenic pollens are weeds, such as Plantago spp., Artemisia vulgaris, Salsola Kali and Chenopodium album. At a local level, Betulaceae in the north of Spain and Platanus and Cupressus species in Madrid and Barcelona are also considered responsible for respiratory allergies.Catalonia is a region located in the northeast of the Iberian Peninsula (south of Europe) bathed by the Mediterranean Sea. According to the official pollen information website of the Spanish Society of Allergy and Clinical Immunology (SEAIC)  is based on therapeutic vaccination with specific allergens and is the only specific etiology‐based treatment for allergic respiratory diseases .The Component Resolved Diagnosis (CRD) or molecular diagnosis (MD) has been introduced more than 10 years ago in clinical practice and has changed the paradigm of allergy diagnosis especially in polysensitized patients.This paper aims to evaluate the epidemiology of allergy sensitization in Catalonia and to assess/confirm whether MD can modify immunotherapy prescription.22.1This is a multicentric, prospective observational study that recruited a total of 300 consecutive patients from 12 different hospitals across Catalonia, who were previously diagnosed with asthma and rhinitis, according to Spanish guidelines  from Vall D’Hebron Institut de Recerca (VHIR) (PR(AMI)244/2013). The study was also approved by the Clinical Research Ethics Committees corresponding to each participant Hospital. All patients (or their parents/guardians in case of children) gave their written informed consent to participate. The experiments were carried out in accordance with the principles of the Declaration of Helsinki.Both the collection and processing of personal data were performed in accordance with the Spanish Organic Law 3/2018 on the protection of personal data. All personal data and sample references were anonymized using independent codes maintained into a database under secure conditions.2.2‐Patients diagnosed with respiratory allergic disease within the previous 2 years: intermittent or persistent, moderate to severe, rhinitis/rhinoconjunctivitis and/or mild or moderate persistent asthma, according to ARIA‐Patients sensitized to three or more pollen aeroallergens from different species as confirmed by SPT. Sensitization to other aeroallergens or food allergens was not considered an exclusion criterion.‐Subjects living in the same geographical region for at least 2 years;‐Patients who agreed to sign an informed consent form. For patients under 18 years of age parents or guardians had to sign the informed consent.Inclusion criteria were as follows:Patients were excluded in case of uncontrolled severe asthma, severe atopic dermatitis, dermographism, or any other baseline disease for which diagnostic testing (SPT) is controversial. Patients who had received previous allergen immunotherapy, those for whom immunotherapy prescription was not indicated and those with neoplastic and/or autoimmunologic diseases were also excluded.Three patients aged 3, 4 and 82 years were included in the study although their age was outside the classical immunotherapy age range. They were not excluded from the analysis since for the purposes of this study the recommendation for SIT was purely hypothetical and proposed on the basis of detected sensitizations.Sample size was inferred based on the estimation that 80% of the pediatric, adolescent, and young adult population attending the allergology outpatient clinic has some type of respiratory pathology . Of these, between 30% and 65% are expected to have a positive SPT to three or more allergens.2.3Clinical, demographic, and anthropometric variables for each patient participating in the study were collected by each researcher.Parietaria judaica, Artemisia vulgaris, Salsola kali, Chenopodium album, Plantago lanceolata, Betula verrucosa, Corylus avellana, Platanus acerifolia, Cupressus arizonica, Olea europaea, Phleum pratense, Cynodon dactylon, and Phragmites australis. Peach lipid transfer protein (LTP) and palm tree profilin were also included in the SPT. All patients had been instructed not to take medications during the 7 days before the test.SPTs were performed with standardized inhalant allergen extracts used in routine testing in every hospital, including the following pollen aeroallergens2, which would equal a papule of 3 mm in diameter.Histamine hydrogen chloride 10 mg/mL was used as positive control and physiologic saline as negative control. All SPTs were conducted using injections in the volar surface of each forearm. SPTs were performed following EAACI recommendations. Papules were measured at 15 min. Papules were considered positive if they were greater than 7.1 mmTM ISAC 112; Thermo Fisher Scientific, see supplementary Table Blood samples were taken from all patients included in the study. The following parameters were measured: total serum IgE; specific IgE to whole allergens detected by SPT ; IgEs to specific molecular components . In both cases, ITA was recommended according to the main guidelines that were prevailing during the study period.All the researchers filled out a questionnaire for each patient including the hypothetical indication and the allergen composition of the SIT that they would (or would not) recommend based on data obtained from clinical history, SPT and specific IgE results before obtaining the results of MD. After obtaining the result of ImmunoCAP2.5t test was performed for the comparison between 2 groups and the ANOVA test for the comparison of 3 or more groups. The ISAC result was presented by subject using a heatmap (based on ISAC classes) and using boxplots by component and by age groups. SIT use before and after MD was presented using a heatmap (use vs. no use). Detailed SIT use of the specific target allergens was presented showing the percent of subjects whose SIT contained each specific allergen both before and after MD, also segmented by age. All calculations were performed using the SAS 9.4 statistical package with a significance limit of 0.05.A descriptive analysis of the study data has been carried out through the elaboration of frequency tables for the nominal type variables and measures of central tendency and dispersion for the continuous variables. The comparison between groups was carried out using Fisher's exact test for qualitative variables. In the case of quantitative variables, the Student's 33.1A total of 300 patients were included in this study. Table 3.2Olea europaea followed by grass  and Platanus acerifolia , Cynodon dactylon (16.0%), Parietaria judaica (12%), and Chenopodium album (10.6%). No discrepancies were found for Salsola kali and profilins , c) patients with negative SPT and sIgE values between 0.35 and 1 kUA/l, and d) patients with negative SPT and sIgE values above 1 kUA/l. In the first group (negative to both tests), Salsola kali and profilins were predominant. In the second group (0.1–0.35 kUA/l), Chenopodioum album, Plantago lanceolata, Betula verrucosa, Corylus avellana, Phleum pretense, Phragmites australis, and LTP were mostly found. In the third group Artemisia vulgaris, Platanus acerifolia, Cupressus arizonica, and Olea europaea were the predominant pollens. Finally, in the fourth group, Cynodon dactylon and Parietaria Judaica showed mean values above 1 kUA/l.Patients with a negative SPT sometimes showed positive sIgE levels. Quantitatively, we distinguished into four groups: a) patients with negative results to SPT and sIgE, b) patients with negative SPT and sIgE values between 0.1 and 0.35 kU3.5Immunotherapy prescription changed due to MD testing in 51% of the cases Figure . The num3.6The change in SIT prescription followed the same trend across all age groups with half of the prescriptions changing in all age brackets and a clear dominance of a no to yes change . Therefore, a clear increase in SIT prescription was observed after MD Figure . The incCupressus arizonica, grass species , Olea europaea, Parietaria judaica, and Platanus acerifolia. With the exception of Platanus acerifolia, which had the same prescription levels both before and after MD, all other pollens showed a clear increase in prescription after MD. Cupressus arizonica displayed the greatest amount of change (15.3% increase), followed by grass  (11.7%), Olea europaea (10.3%), and Parietaria judaica (26.2%). This trend was observed in all age ranges . A positive correlation was considered for p values less than 0.05. A statistically significant correlation was only found for Sal k 1 (p = 0.032) in patients suffering from rhinoconjunctivitis. Regarding asthma, no correlation was found in any of the pollen components analyzed in this study . Such discrepancies can be explained by the fact that whole extract sensitization values cannot be directly compared to values of sensitization to specific molecular components as other constituents of the whole extract can contribute to sensitization.To that end, we evaluated the patients' sensitization profiles using three different methods: SPT, ImmunoCAPTM) as it is considered the reference method. Discrepancies between tests have already been described.Cupressus arizonica were found positive in the ImmunoCAPTM test, highlighting an important degree of underdiagnosis for the fourth most important sensitizer in our population. A similar situation was found for Cynodon dactylon (16.0%), Parietaria judaica (12.0%), and Chenopodium album (10.6%). At a quantitative level, Artemisia vulgaris, Platanus acerifolia, Cupressus arizonica, and Olea europaea were positive in patients with negative SPTs. Moreover, Cynodon dactylon and Parietaria judaica showed a discrepancy between SPT and whole IgE testing in more than 10% of the patients with an average value above 1 kUA/l, indicating an important degree of misdiagnosis from SPT for these particular allergens.To determine the usefulness of SPT as a first‐line screening tool for polysensitized patients, we qualitatively and quantitatively compared the test results with the corresponding serum‐specific IgE to whole allergens , which are in agreement with other publications.These results disagree with data published by Saltabayeva et al. who studied 95 patients with allergic rhinitis and reported a decrease in the number of SIT prescription after MD (119 SIT treatments) as compared to SPT (275 treatments). However, an important limitation of this study was that only 6 molecules were analyzed: nArt v 1, rArt v 3, rAmb a 1, rPhl p 1, rPhl p 5, and rBet v 1.TM  and reported that MD led to prescription changes in 54% of the studied population. They found low agreement comparing SPT and ISACTM, especially regarding Platanus acerifolia, Pla a 1 and Pla a 2. These data highlight the usefulness of using multiparametric tests to overcome diagnostic limitations of the SPT.An earlier work published by Sastre et al.There are two main limitations of our study. On one hand, the lack of evaluation of the clinical outcomes of the new composition of SIT prescription following MD. Nevertheless, it has to be taken into account that in this study, we set out to evaluate the hypothetical prescription of SIT. The evaluation of the correlation with clinical outcomes of the SIT is the next step and a key point for the appropriate management of pollen polysensitized patients. On the other hand, we are evaluating a specific cohort that does not represent the entire allergic population in our area of study, as only patients sensitized to 3 or more pollens were considered and more than half of the included patients live in Barcelona and its metropolitan area.5Cupressus arizonica, a really important pollen in the Mediterranean region, which is clearly underdiagnosed and thus undervaccinated. This study highlights the importance of sIgE at all levels  for appropriate allergy diagnosis and treatment.The use of MD significantly impacts, probably by increasing accuracy, SIT prescription patterns by allergologists, not only by increasing the number of patients considered good candidates for vaccination but also by changing SIT composition in patients prescribed SIT prior to molecular diagnosis. Our study reveals that in our cohort, sensitization to some pollens is underdiagnosed by SPT, so although SPT is a good first‐line screening approach, if there are compatible clinical symptoms, the results should be complemented with the determination of sIgE to whole allergens. Also, for the same pollen, SIT is underprescribed if MD is not used for diagnosis. The clearest example in our cohort is allergy to Teresa Garriga‐Baraut: conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; supervision ; validation ; writing – original draft ; writing – review and editing . M. M. San Miguel Moncín: conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; supervision ; validation ; writing – original draft ; writing – review and editing . Mercè Tena: formal analysis ; writing – original draft ; writing – review and editing . Moisés Labrador‐Horrillo: conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; supervision ; validation ; writing – original draft ; writing – review and editing .Mercè Tena works as Scientific Liaison at Thermo Fisher Scientific. The remaining authors have no conflicts of interest to declare.Supporting Information S1Click here for additional data file.


Overall, the effects of providing different space allowances during summer transports on within-truck ambient conditions, post-transport pigs’ welfare, and carcass and meat quality are similar, regardless of the slaughter weight. In summer loads, increased slaughter weight is associated with more aggressive pigs under mixed group conditions in lairage and a higher risk of DFD-like pork meat resulting from fatigue at slaughter. Winter transport results may have been biased by the short journey and the truck condition not travelling at its full load capacity. Further research aiming for longer journeys (>3 h) is needed to validate the space allowances applied in this study for heavier pigs during summer and winter conditions.Increasing the slaughter weight of pigs can create challenges in handling and transport, affecting the welfare and the final meat quality. Heavier pigs have different space requirements to fit their physical and thermal needs. This research was conducted through two separate experiments with the aim of investigating the effects of two slaughter weights (120 and 140 kg) and two space allowances during summer and winter transports, i.e., 0.54 and 0.62 m2/pig in summer and 0.44 m2/pig and 0.50 m2/pig in winter) on the behavioural and physiological response, and meat quality of pigs of two slaughter weights (120 kg and 140 kg). In summer, higher blood haematocrit levels were found at slaughter in heavier pigs transported at a smaller space allowance (p = 0.04). During lairage, pigs transported at a smaller space allowance started fighting later (p = 0.04). Fighting behaviour was greater in heavier pigs (p ≤ 0.05), whilst their drinking activity was lower (p < 0.05). This resulted in greater exsanguination blood CK levels (p < 0.01) and drier hams (p = 0.05) in heavier pigs. In winter, only lower space allowance influenced some meat quality traits (p < 0.05), but these effects were minor. The effects of space allowance during summer transports on within-truck ambient conditions, post-transport pigs’ welfare, and meat quality are similar. Mixing heavier pigs may result in greater aggressiveness and more fatigue-related meat quality variation during summer. Overall, winter transport results may have been biased by the short journey and within-truck load distribution.This study was carried out through two separate experiments aiming at evaluating the effects of two space allowances (0.54 and 0.62 m Global pig meat production is expected to grow 13% over the coming decade, due to growth in developing countries, accounting for 33% of the growth in total global meat production , reducedHowever, the increased weight and size of these pigs also create challenges to handling and transportation systems. Transport represents a stressful stimulus for the animal. It is well known that, under stressful stimuli, the body must find a new dynamic equilibrium, and this requires several adaptive body responses. Transport can result in a disruption of homeostasis, with a direct effect on animal health status and the physical performance of the animals ,9. The d2/130 kg is the legal requirement [2/110 kg) to entirely lie down in a full lateral recumbent position on the truck floor [Based on the monitoring of more than 12,000 trailer loads, Fitzgerald et al.  concludeuirement  to ensurck floor . This ne2/pig [Due to their increased size, heat output, poor dissipation rate, large muscle mass, and lower cardiac output ,17,18, h2/pig , for the2/pig  and a gr2/pig .There is clear evidence that both warm and cold temperature conditions are challenging for pigs during transport as indicated by decreased ease of handling at loading ,22 and i2/pig during summer and 0.40 m2/pig to 0.61 m2/pig during winter [For these reasons, to meet their different physical and thermal needs, it is recommended to transport heavier pigs (from 114 kg to 182 kg) at increasingly greater minimum floor space from 0.46 to 0.65 mg winter . Howeverg winter  nor code2/pig and 0.44 m2/pig and 0.50 m2/pig, during summer and winter transport conditions, respectively, on the behavioural and physiological response, and carcass and meat quality of pigs weighing 120 and 140 kg on average.This study unfolded through two separate transport trials whose objective was to evaluate the effects of two space allowances, i.e., 0.54 and 0.62 mAll experimental procedures performed in these experiments were approved by the institutional animal care committee  at the Agriculture and Agri-Food Canada (AAFC) Sherbrooke Research and Development Centre  based on the current guidelines of the Canadian Council on Animal Care .®, Zoetis Canada, Kirkland, QC, Canada) boars and gilts of the same genetics (progeny of DNA 600 sires mated with Topigs TN70 dams) were shipped in six loads (five loads of 60 pigs and one load of 59 pigs) from a commercial finishing farm to a commercial abattoir (2 h travel) during the summer season . Pigs were transported in the early morning (03:00 h AM) using a double-decked passively ventilated truck, featuring a fully hydraulic top deck and four compartments  and the body weight (120 ± 5 kg and 140 ± 5 kg). These space allowances were applied with respect to the Canadian Transport Codes of Practice [The animals were distributed according to a 2 × 2 factorial arrangement, where the factors were the space allowance in the truck , totalling 144 focal immunocastrated boars through the whole experiment, were selected from different finishing pens, weighed, and tagged in both ears for identification during blood collection at slaughter and carcass meat quality measurements, and were returned to the finishing pen where they were sorted out from during loading.In preparation for transport, feed was withdrawn from all pigs for approximately 11 h before loading .On the day of transportation, pigs, including focal ones, were loaded in small mixed groups of 5–10 pigs using paddles and sorting boards only. The truck left the farm upon completion of the loading process  to avoid the effect of the lack of passive ventilation on internal ambient conditions ,32 and pDuring transport, the side panels were kept fully open to allow passive ventilation. The truck was also bedded with fresh wood shavings. Due to unforeseen circumstances, the assigned driver was replaced for the last two shipments.2/pig) in the lairage pen, the truck compartment group size was reduced to 13 pigs/group  in each lairage pen. Pigs were kept in lairage for 2.5 h, on average, with free access to water.Pigs were unloaded almost immediately upon arrival at the abattoir  in groups of 13 pigs, which included 6 focal pigs, using paddles, and were driven to separate lairage pens based on the treatment combination or truck compartment. No DOA or non-ambulatory pigs were reported at unloading. No mixing of pigs between compartments/treatment combinations occurred. As the size of the truck compartments and lairage pens differed, to keep a constant stocking density . Immediately upon exiting the stunner, pigs were shackled and exsanguinated in the vertical position.After lairage, pigs were driven using paddles along the alleys and an automatic push gate system in the last chute feeding the CO®, Zoetis Canada, Kirkland, QC, Canada) boars and gilts of the same genetics (progeny of PIC L42 sires mated with PIC L8000 dams) were shipped in four loads  from a commercial finishing farm to a commercial abattoir (1 h travel) during the winter season . Pigs were transported in the early morning (03:50 h AM) using a double-decked hydraulic passively ventilated truck, featuring six compartments, of which only four (two in the front and two in the rear of each deck) were used in the experiment. The two middle compartments were kept empty as the farm did not have enough volume to fill them with pigs  and the pig’s body weight (120 ± 5 kg and 140 ± 5 kg). These space allowances were applied in accordance with the Canadian Transport Codes recommendation [The animals were distributed according to a 2 × 2 factorial arrangement, where the factors were the space allowance in the truck , totalling 96 focal immunocastrated boars through the whole experiment, were selected from different finishing pens, weighed, and tagged in both ears for identification during blood sampling at slaughter and carcass meat quality measurements, and were returned to the finishing pen where they were sorted out from during loading. In preparation for transport, feed was withdrawn from all pigs for approximately 7 h before loading .On the day of transportation, pigs, including focal ones, were loaded in small mixed groups of five to ten pigs using paddles and sorting boards only. The truck left the farm upon completion of the loading process . During transport, only 10% of the side panels were open to protect pigs from cold while providing sufficient airflow for air exchange in the truck. The truck was also bedded with fresh wood shavings. Two drivers were rotated across shipments or replicates to randomise the effect of the driver on handling and driving on pigs’ response to transport .2/pig) in the lairage pen, the group size was reduced to 13 pigs/group  in each lairage pen. Pigs were kept in lairage for 2.5 h on average, with free access to water.Pigs were unloaded almost immediately upon arrival at the abattoir  in groups of 13 pigs, which included six focal pigs, using paddles, and were driven to separate lairage pens based on the treatment combination or truck compartment. No mixing of pigs between compartments/treatment combinations occurred. As the size of truck compartments and lairage pens was different, to keep a constant stocking density . Immediately upon exiting the stunner, pigs were shackled and exsanguinated in the vertical position.After lairage, pigs were driven using paddles along the alleys and an automatic push gate system in the last chute feeding the COAmbient air temperature and humidity data were collected using iButton data loggers  attached on the inside of the four compartments (five iButtons/compartment). The iButtons were placed approximately 8 cm from the ceiling, with one positioned in the centre of the compartment and the other four placed in each corner of the compartment. The iButtons were programmed to record temperature (T) and relative humidity (RH) data every minute from the beginning of loading to the end of unloading. The temperature range of the data logger was from −20 to +85 °C with a resolution of ±0.0625 °C and an accuracy of 0.5 °C, and a relative humidity range from 0 to 100% with a resolution of ±0.04% and an accuracy of 5%. The iButtons were programmed and data were downloaded after each transport using the ExpressThermo software .Behaviour during lairage was recorded by a video camera  installed on each of the lairage pen walls to survey the pigs. The recording started as soon as the pen was filled with pigs and the gate was closed.All treatment groups were observed for a minimum time of 72 min. Scan sampling (every 2 min) was used to record the number of pigs lying down, standing, sitting, or engaging in other postures, while a continuous sampling was performed to record drinking and fighting behaviours .For the postural behaviour, the average percentage of pigs standing, lying, sitting, and other behaviours was calculated. Due to its low percentage during lairage, sitting behaviour data were merged with other postures. Data were analysed for the overall 72 min and separately for the first and second half of the lairage observation time (36 min each). The latency to lie down was calculated as the time (min) from the start of lairage until at least 45% of pigs were lying down, which was the minimum proportion reached by all compartment groups within the observation period (72 min) in this study.Drinking behaviour during lairage was analysed for the number of drinking bouts, percentage of time spent drinking, and latency for the first pig to drink by treatment group. Fighting behaviour was analysed for the percentage of time spent fighting, the total number of fights, and latency for the first pig to fight by treatment group. Data for drinking and fighting behaviour were analysed for the total 72 min observation time in lairage.Behavioural observations were performed by one trained observer using The Observer XT software , and the average correlation coefficient for intra-observer agreement for all behaviours observed was 0.99.®, VWR International Ltd., Montreal, QC, Canada). Whole-blood lactate concentrations were immediately assessed in duplicate with a hand-held Lactate Scout Analyzer  by dipping a test strip  into a serum tube containing the collected blood. Another blood sample was also collected in a second serum tube for CK analysis. Serum was collected after centrifugation at 1400× g for 10 min at 4 °C and then stored at −80 °C until analysis. Serum CK concentration was analysed using a creatine kinase_SL kit  and determined with a spectrophotometer. The intra-assay coefficient of variation for log-transformed blood CK was 2.70% in summer blood samples and 2.58% in winter blood samples. A third blood sample was collected in an EDTA tube , refrigerated at 4 °C and subsequently analysed in duplicate for haematocrit determination according to the microhematocrit procedure described by Matte et al. [At exsanguination, blood was collected from the bleeding wound of the 24 focal pigs per replication  in serum tubes  was obtained from the grading slips, and carcass yield (%) was calculated. Skin damage was assessed on the whole carcass in the cooler by a single observer using a subjective five-point photographic scale from 0 = no to very minimal lesions to 5 = severe lesions .post-mortem in the longissimus muscle (LM), between the third and fourth last rib, and in the semimembranosus (SM) and adductor (AD) muscles. The pHu was measured using a portable pH-metre  fitted with a spear tip pH electrode  and an automatic temperature compensation (ATC) probe .All meat quality measurements were taken at 24 h Instrumental colour  was meaMeat exudation was measured in the LM (same location as pHu measurement) using the filter paper wetness (FPW) test described by Kauffman et al. . BrieflyIn both seasons, within-truck average T and RH values were calculated for each transport phase , where analysis was performed considering space allowance, slaughter weight, and their interaction as fixed factors in a 2 × 2 factorial design. Results are presented as least-squares means (LSM) ± SEM. Replicate was considered a random factor.In the summer study, temperature humidity index (THI), which is normally used as an indicator of ambient conditions in heat stress studies and livestock transport guidelines ,41, was p ≤ 0.05 was chosen as the limit for statistical significance in all tests. Observed probabilities of p ≤ 0.10 were considered tendencies.For physiological and meat quality data analyses, the group of six sentinel pigs per compartment was considered as the experimental unit. Basic ANOVA assumptions were verified using the studentised residuals. Usual correcting steps were applied, if necessary. A probability level of 2/120 kg treatment group were missing due to one camera recording failure. Furthermore, in total (summer and winter studies combined), 12 carcasses  were lost through the slaughter chain. Owing to the difficulty of keeping pace with the speed of the bleed line on the kill floor, or due to poor sample quality, a total of 21 blood lactate , 14 blood haematocrit , and 16 CK  samples were lost. Furthermore, it was not possible to assess the lesion score in a total of 8 carcasses (3% in both seasons) and record 13 HCW .As a result of unforeseen events, during summer, lairage behaviour data related to the 0.54 mDue to differences in pig genetics, farm of origin, and transport conditions between the summer and winter studies, results cannot be compared between seasons and are thus presented separately.p > 0.10).As shown in p > 0.10).The interaction of space allowance × slaughter weight did not affect postures during lairage (2/pig) stood more and lay less in the lairage pen . However, this effect was significant in the first half of the observation time (36 min), where pigs transported at a greater space allowance (0.62 m2/pig) stood in greater percentage (p = 0.04) and, consequently, lay down in lower percentage (p = 0.02) compared to pigs transported at a smaller space allowance (0.54 m2/pig). Space allowance neither had any impact on the behaviours observed during the second half of the observation time in lairage (p > 0.10) nor on the proportion of pigs in other postures during the whole observation time (p > 0.10). Slaughter weight did not affect the percentage of pigs in any posture during lairage (p > 0.10). Neither space allowance nor slaughter weight affected the latency to lie (p > 0.10).For the entire observation period (72 min), space allowance tended to affect postures, where a greater proportion of pigs that were provided with greater space allowance in the truck (0.62 mp > 0.10). When compared to pigs transported at a greater space allowance (0.62 m2/pig), pigs transported at the lower space allowance (0.54 m2/pig), started fighting later , and tended to fight less .As shown in p = 0.02) and having fewer drinking bouts  than lighter pigs.Slaughter weight also had an effect on drinking during lairage, with heavier pigs having a longer latency to start drinking  and fighting more  than lighter pigs.Slaughter weight also had an effect on fighting behaviour during lairage, with heavier pigs spending a greater percentage of time fighting , with higher blood haematocrit levels being found at slaughter in heavier pigs when transported at a smaller space allowance (0.54 m2/pig) compared to higher space allowance (0.62 m2/pig) (p < 0.02).As shown in p > 0.10). Meanwhile, slaughter weight affected serum CK concentrations at slaughter with greater CK levels found in heavier pigs compared to lighter pigs .Blood lactate concentration at slaughter was neither influenced by space allowance, slaughter weight, nor their interaction .Neither space allowance, slaughter weight, nor their interaction influenced carcass yield or lesion scores (p = 0.07) and SM muscle being yellower  in pigs transported at a greater space allowance. When compared to lighter pigs, heavier pigs presented a slightly drier (lower drip loss) SM muscle  and a trend for a higher pHu value in the AD muscle .Space allowance had an effect on Minolta b* value, with loins tending to be yellower .As shown in p = 0.08 and p = 0.07 for each period, respectively; p > 0.10).A greater proportion of heavier pigs tended to stand less during the first half and the whole observation time in lairage when compared with lighter pigs (p > 0.10). Furthermore, neither space allowance nor slaughter weight affected the latency to lie down (p > 0.10).Space allowance had no effect on postural behaviour during lairage  in lairage.The interaction space allowance × slaughter weight tended to affect the expression of sitting or other postures in lairage .As shown in p > 0.10; p < 0.001).Neither space allowance, slaughter weight, nor their interaction influenced carcass yield or lesion scores . When compared to lighter pigs, in the heavier pigs the pHu value tended to be slightly lower in the LM muscle  and was slightly lower in the SM muscle . The L* value of the SM muscle of heavier pigs was also slightly higher, indicative of a paler colour, compared with lighter pigs .Space allowance influenced the pHu value in the LM, with slightly higher pHu being found in pigs transported at 0.44 m2 in the compartment. This thermal discomfort may explain the increased risk of animal losses in pigs transported at greater load density or size [The thermal environment inside the trailer is considered a serious concern for the health and welfare of pigs during transport . Since p or size ,47.Overall, during summer, except for the wait-at-farm phase, the temperature inside the test compartments was within the upper threshold of the thermo-neutral zone for market-weight pigs . The siOverall, during winter, the temperature inside the test compartments was below the threshold of the lower critical temperature (LCT) of the thermoneutral zone, which is defined as the point in effective ambient temperature below which an animal must increase its rate of metabolic heat production to maintain homeothermy and that for the transport of 100 kg to 140 kg market-weight pigs ranges from 10 °C to 14 °C . These r2) and associated this behaviour with greater fatigue-related resting during lairage. However, the different space allowances during summer and winter transports had no effect on the number of drinking bouts and time spent drinking, but, similarly to Urrea et al. [2) in the truck during summer preferred resting during the first half of lairage observation time, which delayed activities, such as fighting unfamiliar pen-mates, compared to pigs transported at greater space allowance. Although it was not possible to make reliable on-truck behaviour observations due to poor recording conditions in this study (low deck ceiling and standing pigs blocking the view of the rest of the compartment group), based on the observations from previous studies [The behaviour of pigs in lairage can be explained by their physical state on arrival at the abattoir, which, in turn, is influenced by their behavioural response during transport . Urrea ea et al. , a highe studies ,55, the  studies . In summ studies .Signs of some sort of physical fatigue condition on arrival at the abattoir have been also recorded after winter transports, especially in heavier pigs provided with a larger space allowance in the truck, that tended to sit or kneel, among other postures, during rest in lairage. Kneeling and sitting are, in fact, defined as transition postures from standing to lying with some indication of fatigue as pigs spent a few seconds in these postures before lying down ,58. The post-mortem meat acidification [The above-mentioned signs of fatigue observed in pigs during winter lairage did not translate into a difference in the concentration of stress variables analysed in exsanguination blood. However, similarly to summer, the blood lactate concentrations revealed a general fatigued condition at slaughter in pigs transported during this season, regardless of the journey conditions and slaughter weight, as they were well above the reported whole blood rest concentrations  and thefication . This refication .Serum CK concentration is a good indicator of long-term muscular activity or tissue damage in pigs at slaughter . The lac2/pig) was provided to pigs during transport.Overall, the recorded skin lesion scores were within the range of the slight lesion class in both summer and winter loads . HoweverA recent study reported a higher proportion of severe carcass lesions in heavier-weight pigs , whereaBased on existing literature reports, both the provision of greater and lower floor space can result in muscle fatigue, as shown by the higher pHu values, an indicator of DFD  pork meat, due to muscle glycogen depletion ,49. In b2/pig during summer on within-truck ambient conditions, pigs’ post-transport animal welfare, and carcass and meat quality are similar, regardless of the slaughter weight.Based on the results obtained under the transport conditions of these studies, the effects of providing space allowances of 0.54 or 0.62 mThe summer results may confirm the relationship between reduced floor space to lie down under warmer transport conditions and the pigs’ fatigue condition on arrival at the abattoir. This physiological condition increased pig resting by reducing the time for fighting during lairage and fatigue at slaughter.No clear conclusions can be drawn based on the results of the winter study that were biased by the short journey and the condition of the truck not travelling at its full load capacity.Further research is needed to validate the space allowances applied in both studies for heavier pigs under longer transport conditions (>3 h) since pigs are more likely to stay standing during the first 2–3 h of the journey and cannot take advantage of the floor space provided to rest and recover after loading, and better cope with warm and cold temperature conditions.The results of the summer study highlight the effects of increased slaughter weight on the behaviour of pigs, which, alongside being more difficult to handle as reported in previous studies, are more aggressive under mixed group conditions during lairage, resulting in fatigue at slaughter and a greater risk of DFD-like pork meat production.


Polycystic ovary syndrome (PCOS) is a complex endocrine metabolic disorder that affects 5–10% of women of reproductive age. The endometrium of women with PCOS has altered immune cells resulting in chronic low-grade inflammation, which attribute to recurrent implantation failure (RIF). In this study, we obtained three PCOS and RIF datasets respectively from the Gene Expression Omnibus (GEO) database. By analyzing differentially expressed genes (DEGs) and module genes using weighted gene co-expression networks (WGCNA), functional enrichment analysis, and three machine learning algorithms, we identified twelve diseases shared genes, and two diagnostic genes, including GLIPR1 and MAMLD1. PCOS and RIF validation datasets were assessed using the receiver operating characteristic (ROC) curve, and ideal area under the curve (AUC) values were obtained for each disease. Besides, we collected granulosa cells from healthy and PCOS infertile women, and endometrial tissues of healthy and RIF patients. RT-PCR was used to validate the reliability of GLIPR1 and MAMLD1. Furthermore, we performed gene set enrichment analysis (GSEA) and immune infiltration to explore the underlying mechanism of PCOS and RIF cooccurrence. Through the functional enrichment of twelve shared genes and two diagnostic genes, we found that both PCOS and RIF patients had disturbances in metabolites related to the TCA cycle, which eventually led to the massive activation of immune cells. A vaRIF is defined as a lack of clinical pregnancy after at least four embryos have been transferred in a minimum of three fresh or frozen cycles in a couple under 40 years of age, with an incidence rate of about 15% . To dateFurthermore, the mechanism of impaired endometrial receptivity (ER) has attracted research interest in recent years , 18. NotIn this study, we aimed to explore the potential biomarkers and underlying pathways involved in the development of PCOS and RIF. To achieve this goal, we integrated transcriptomic data related to PCOS and RIF from the Gene Expression Omnibus (GEO) and applied “LIMMA” package and weighted gene co-expression network analysis (WGCNA) to identify differentially expressed genes and critical modules in each disease. Our analysis using the intersection and three machine learning methods led to the identification of two disease-shared diagnostic genes, GLIPR1 and MAMLD1, with good performance validated by external datasets. Further, we conducted gene set enrichment analysis (GSEA) on each of these two genes to identify common pathways associated with PCOS and RIF. Additionally, we investigated the role of immune cells in the co-pathogenesis of the two diseases through immune infiltration analysis. Our results suggested that the co-pathogenesis of PCOS and RIF might be linked to increased immune response levels arising from abnormal TCA cycle metabolism. In conclusion, this study provides valuable insights into the shared molecular mechanisms underlying PCOS and RIF and highlights the potential of GLIPR1 and MAMLD1 as diagnostic markers for these conditions.22.1http://www.ncbi.nlm.nih.gov/geo/) since both diseases were designed in this study. For PCOS, we used the keyword “PCOS” or “granulosa cells” to search gene expression profiles. Inclusion criteria were as follows (The data sets related to PCOS and RIF were screened in the Gene Expression Omnibus database (GEO) |>0.585. For RIF, the P value adjusted to 0.05 and |log2 FC|>1 were used to identify the DEGs. Next, the difference analysis results for each group were presented using the heatmap and volcano plot. In both plots, blue indicated low expression, and red indicated high.After preparing the data for each disease, we compared PCOS and RIF datasets using the Linear Models for Microarray (LIMMA) package in R (version 4.1.2). Differentially expressed genes (DEGs) were calculated between disease and control groups with it. For PCOS, the DEG threshold was set as 2.3Using microarray specimens, WGCNA represents one of the most important and widely applied systems bioinformatics methods to describe the correlation patterns among genes. Genes can be grouped into modules based on their co-expression similarities across samples using the “WGCNA” R package. Additionally, the WGCNA method can be used to connect modules to clinical elements outside the genome. In this way, relevant functional networks can be used to identify biomarkers and new molecules. As input files, normalized mRNA expression data  were used to perform WGCNA to identify gene coexpression and the correlation between gene modules and clinical characteristics (PCOS or RIF compared to control groups). For each disease group, the following steps are followed : by usin2.4P<0.05.By combining DEGs and module genes identified by WGCNA, we were able to identify the shared key genes that contributed to the pathogenesis of both PCOS and RIF. For identifying genes’ biological functions and signaling pathways, we used the “clusterProfier” package for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The bar plots showed the significant enrichment results of the functional enrichment with 2.5Further screening of co-existing genes between the two diseases was carried out using three well-established machine learning algorithms . To ensure the repeatability of these algorithms, we set the seed at 123 in both disease groups.Firstly, the 12 shared genes obtained previously were input into the LASSO algorithm in each disease group. We constructed a regression model using the R package “glmnet” with 10-fold cross-validation. In the “family” parameter, we set “binomial,” and we chose the best lambda value by “lambda.1min”. Logarithm (lambda) profiles of the LASSO coefficients were drawn for the 12 features. Then we drew the partial likelihood deviation  curve and the logarithm (λ) curve. Next, we calculated the best value for 1se (1-SE standard) of minimum standard.Following that, SVM-RFE was used to eliminate recursive features. Using the “e1071” and “MSVM-RFE” package for SVM modeling, SVM-RFE applied sequential backward feature elimination to determine the optimal hub gene. All of the 12 shared genes were used in our SVM model. The result of SVM-RFE was visualized, and by tenfold cross-validation, the red circle indicated maximum classification precision and the corresponding gene sets were the most accurate diagnostic markers at the lowest 5×CV error and the highest 5×CV accuracy.Finally, we used Random Forest to classify the significant genes with the R package “randomForest”. Using a decision tree algorithm, random forest analysis identified which variables were most important. As a result of this algorithm, we were able to filter the shared genes to find disease signature genes. Our first step was to construct a random forest model using 500 trees on the discovery cohorts and determine the optimal number of trees using cross-validation errors. Then, we ranked genes by importance and plotted the 10 most significant genes. For each disease group, the significance threshold was set at 0.9 to decide the final result.After screening the above three algorithms, we took the intersection of the results of each algorithm. There were five genes common to the PCOS group and seven common genes in the RIF group shown in a Venn plot. Furthermore, we took the intersection of the above common genes again and finally obtained two genes as the disease diagnostic target genes. The “pROC” package was used to construct ROC curves and displayed using “ggplot2” to assess the accuracy of the two diagnostic genes in discovery cohorts.2.6To further test the accuracy of the two diagnostic genes, we searched the GSE80432 for PCOS and GSE26787 of RIF for external validation. We first downloaded the raw data of these two data sets from the GEO database and normalized them with the “RMA” package. For GSE80432, we filtered out a deviation sample through PCA and finally adopted four healthy control samples and three PCOS patients. For GSE26787, five healthy controls and five patients with RIF were used for validation. The diagnostic gene expression pattern in validation cohorts was shown by boxplot, and AUC (area under the ROC curve) was also calculated.2.7After obtaining the diagnostic genes, we performed single-gene gene set enrichment analysis (GSEA) for each diagnostic gene in the two groups using the “clusterProfiler” package. Using GSEA, we compared the biological signaling pathways between the disease group and the healthy control group. MSigDB (c5.go.bp.v7.5.1.entrez.gmt) was used to download gene sets. Enrichplot was used to show the top 5 activating and inhibiting pathways for each gene in the two disease groups.2.8http://cibersort.stanford.edu/), LM22 contains 22 annotated gene signatures. With CIBERSORT and 1000 iterations, we quantified 22 types of immune cells based on the LM22 gene signature. In the following analysis, we selected samples with a CIBERSORT P value less than 0.05 for analysis. For each sample, the output estimates from CIBERSORT were normalized to sum to one to facilitate comparisons across immune cell types and datasets. R packages “corplot,” “vioplot” and “ggplot2” were used to visualize the results. Further, the correlation between immune infiltrated cells and diagnostic target biomarkers was determined by nonparametric correlations (Spearman).Each sample of disease was subjected to CIBERSORT analysis to determine the relative levels of immune cells. The CIBERSORT algorithm resolves immune cell composition by deconvolution based on gene expression data. According to CIBERSORT’s website  with 101 up-regulated and 100 down-regulated genes were obtained for PCOS. For RIF, there were 253 DEGs  with 167 up-regulated and 86 down-regulated genes. Volcano plots showed all DEGs of PCOS  were applied based on the above 12 shared genes. In the PCOS group, based on the LASSO coefficient profiles and the optimal tuning parameter selection map, λ was set at 0.06851194 (PCOS)  and higher in the PCOS groups (P<0.01). MAMLD1 expressed lower both in the PCOS (P<0.01) and RIF groups (P<0.0001) , and MAMLD1(AUC=0.774). The RIF group was also subjected to the same ROC analysis. Predictive performance was robust for each biomarker: GLIPR1(AUC=0.723), and MAMLD1(AUC=0.879) (GLIPR1 was decreased (P<0.05) in the RIF group and increased in the PCOS groups (P<0.05) (MAMLD1 was reduced both in the PCOS groups (P<0.01) and the RIF groups (P<0.05)  is a gene that encodes a protein with diverse biological functions, including cell apoptosis, cell cycle regulation, and DNA damage response. While the direct relationship between GLIPR1 and PCOS and RIF has not been fully established, several studies have suggested a potential involvement of GLIPR1 in the pathogenesis of these diseases. Notably, altered expression levels of GLIPR1 have been observed in PCOS patients, indicating a possible association between GLIPR1 and PCOS . StudiesMAMLD1 (NM_001177465) is a gene associated with disorders of sexual development in 46, XY individuals, and it has been reported that mutations in MAMLD1 resulted in male fetal sexual development  36, 37), 3736, 3To further explore the underlying pathogenic association and mechanisms between PCOS and RIF, we performed GSEA for the two diagnostic genes in two disease groups. As a result of GSEA analysis, both PCOS and RIF genes were enriched in metabolism pathways. In the PCOS group, both two genes were enriched in glycine, serine, and threonine metabolism. Similarly, GLIPR1 and MAMLD1 were found to involve in the metabolism of butanoate, propanoate, and thiamine in the RIF group. Interestingly, metabolites like glycine , serine Consistent with previous studies , 49, ourBased on the above findings, we hypothesize that activation of a large number of immune cells in cellular immunity caused by disturbances of metabolites associated with the TCA cycle accounts for the co-pathogenesis of the PCOS and RIF. As previously mentioned, activated DCs and macrophages showed a truncated TCA cycle that resulted in an accumulation of citrate. Of note, the TCA cycle intermediates may leak from impaired mitochondria, and increasing evidence indicated that these metabolites of the TCA cycle played a substantial role in immune regulation . In PCOSSome limitations need to be mentioned in the present study. First, only 3 datasets were selected for each disease, including 18 cases and 14 control samples in the PCOS group, and 27 cases in both the RIF disease and normal groups for discovery. For WGCNA and immune infiltration analysis, more sufficient samples are needed to ensure test accuracy. Second, we know little about the processing of the raw data, and the datasets used in our study were profiled by microarray which was far behind today’s advanced sequencing technology. These intrinsic traits of these public datasets pose a challenge to the accuracy and advancement of our analysis.In conclusion, diagnostic biomarkers GLIPR1 and MAMLD1 were identified as critical biomarkers responsible for regulating immune cell activation caused by an imbalance of TCA cycle metabolites. Our analysis reinforces the theoretical basis for the co-pathogenesis of recurrent pregnancy failure in PCOS patients.http://www.ncbi.nlm.nih.gov/geo/). The PCOS group includes three datasets with GSE10946, GSE34526, and GSE580432. GSE103465, GSE111974, and GSE26787 are involved in the RIF group. Further inquiries can be directed to the corresponding authors.The original contributions presented in the study are publicly available. Publicly available datasets used in this study can be downloaded from the GEO database (The studies involving human participants were reviewed and approved by the Ethics Committee of Scientific Research and Clinical Trial of the First Affiliated Hospital of Zhengzhou University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.WC and QY conceived the study idea. WC performed the data analysis and produced the results. YS, QY, and WC wrote and revised the manuscript. LH, WC, MW, ZY, and XZ were responsible for sample collection and experiment. All authors contributed to the article and approved the submitted version.


N-methyl-d-aspartate (NMDA) receptor antagonist, can reverse central sensitization associated with causation and propagation of CFP. This systematic review aims to assess the role of ketamine for CFP.Craniofacial pain (CFP) poses a burden on patients and health care systems. It is hypothesized that ketamine, an Databases were searched for studies published up to September 26, 2022, investigating the efficacy of ketamine for adults with CFP. Primary outcome was the change in pain intensity at 60 min postintervention. Two reviewers screened and extracted data. Registration with PROSPERO was performed (CRD42020178649).Twenty papers  including 670 patients were identified. Substantial heterogeneity in terms of study design, population, dose, route of administration, treatment duration, and follow-up was noted. Bolus dose ranged from 0.2–0.3 mg/kg (intravenous) to 0.4 mg/kg (intramuscular) to 0.25–0.75 mg/kg . Ketamine infusions (0.1–1 mg/kg/h) were given over various durations. Follow-up was short in RCTs (from 60 min to 72 h) but longer in observational studies (up to 18 months). Ketamine by bolus treatment failed to reduce migraine intensity but had an effect by reducing intensity of aura, cluster headache (CH), and trigeminal neuralgia. Prolonged ketamine infusions showed sustainable reduction of migraine intensity and frequency of CH attacks, but the quality of the evidence is low.Current evidence remains conflicting on the efficacy of ketamine for CFP owing to low quality and heterogeneity across studies. Ketamine infusions are suggested to provide sustained improvement, possibly because of prolonged duration and higher dosage of administration. RCTs should focus on the dose–response relationship of prolonged ketamine infusions on CFP. Central sensitization is characterized by an increase in neuronal excitability secondary to repetitive stimulation of the nociceptive C-fibers in the trigeminocervical complex and the brain, which is mediated by the activation of 12 Its primary mechanism of action is as a noncompetitive antagonist of the NMDA receptors residing in the central nervous system.13 Ketamine is a versatile drug that can be administered via many routes, including intravenous (IV) and intramuscular (IM) but also oral, intranasal, inhalation, topical, and rectal, rendering it an easy agent for out-of-hospital care. It can be given as a single bolus or infusion or a combination of both. Although its potency is comparable to that of opioids, ketamine has a much better safety profile and is less likely to lead to development of tolerance. Possible adverse effects of ketamine include hypertension and tachycardia, hallucinations, and hepatic toxicity with chronic exposure.14Ketamine is a chemical derivative of phencyclidine with analgesic, dissociative, and psychomimetic properties.11 but its therapeutic role in CFP has not been completely established. Therefore, the objective of this systematic review was to evaluate the efficacy of ketamine for the treatment of CFP and examine its effects on pain-associated domains.Ketamine has successfully been used in the treatment of complex chronic pain states such as complex regional pain syndrome and neuropathic pain,This systematic review was conducted according to the recommendations of the Cochrane Collaboration and was reported as per the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The protocol of this systematic review was registered with PROSPERO (ID CRD42020178649).We conducted a comprehensive search of the literature from inception to February 1, 2020. Updated searches were conducted over the same databases and clinical trial registries on November 3, 2020, and September 26, 2022, with the assistance of a medical information specialist (M.E.).The following databases were searched: Embase (1947–), MEDLINE (1946–), MEDLINE ePubs, In-Process and Other Non-Indexed Citations, Cochrane Database of Systematic Reviews (2005–), Cochrane Central Register of Controlled Trials (1991–), and PubMed-NOT-MEDLINE. We also searched Web of Science Core Collection  and Scopus . Clinical trial registries, ClinicalTrials.Gov, and the World Health Organization International Clinical Trials Registry Platform were searched to identify trials.We restricted our search to human subjects with moderate-to-severe pain. For Embase, MEDLINE, Cochrane CENTRAL, and Scopus, both controlled vocabulary terms  and text word searching were conducted for each of the following segments: (“ketamine” or related synonyms) AND ” or “short-lasting unilateral neuralgiform headache attacks with cranial autonomic symptoms (SUNA)” or “trigeminal neuralgia” or “trigeminal autonomic cephalalgia (TAC)” or “occipital neuralgia” or “cephalalgia” or other related terms). The most recent Ovid Medline search strategy and the MEDLINE search strategy are provided in Appendix 1.The studies were screened for eligibility based on title, abstract, and subsequently full manuscript. Studies were included if they met the selection criteria as listed below.International Classification of Headache Disorders third edition.15 Studies that investigated effects of ketamine on a mixed population of patients with pain for which data on CFP could not be extracted separately were excluded. No restrictions were put in terms of chronicity, frequency, or duration of the attack. An initial restriction of including participants with only moderate-to-severe pain was waived at the start of the title screening process because of fear of selection bias.This review included studies of human subjects ≥18 years of age with a CFP  diagnosis that fit the criteria of the The intervention was defined as bolus/infusion administration of ketamine of any dose or administration type . Studies in a perioperative setting and/or with mixed combinations of ketamine with another drug were excluded. There was no limit on the duration of treatment or number of treatments.Comparators included no treatment, placebo treatment, or conventional medical management, which could include pharmacological, physical, psychological, and/or interventional therapies.16 The initial restriction of 6 months’ follow-up time was extended to 18 months at the time of data collection.The primary outcome was the change in intensity of pain assessed on a numeric rating scale (NRS)/visual analog scale (VAS) at 60 min after the intervention. Secondary outcomes included (1) positive response (defined as a reduction in pain score by ≥30% from baseline at 60 min after the intervention) and effect of ketamine infusion on (2) pain intensity (NRS/VAS) at any time after the intervention up to 6 months posttreatment, (3) adverse effects, (4) functional outcome, (5) quality of life, (6) mood, and (7) patient satisfaction. The threshold of >30% pain relief has been demonstrated to constitute clinically meaningful improvement.17 was used as a systematic review management tool. Papers of interest were then full-text screened. Of the selected papers, data were independently extracted by two reviewers (T.M. and M.P.). Any disagreement was resolved through discussion with the senior author (P.K.).All citations were independently screened on title and abstract for eligibility by two reviewers (Y.H. and P.K.) as per the inclusion criteria. CovidenceThe reference data, populations, and outcomes were extracted from the articles into prespecified tables on a standardized data collection form in Word that was pilot tested before use. Extracted data for each study included general characteristics , patient characteristics , clinical information , details of intervention and comparator (dose and administration regimen), data on primary and secondary outcomes of interest, follow-up time points, and adverse effects.18 Any disagreement was resolved through discussion with the senior author (P.K.). The Robins-I was used to assess the risk of bias in observational studies.19 For case reports/case series, the Quality Appraisal Tool for Case Series was used.20Two review authors (Y.H. and M.P.) independently assessed the risk of bias for randomized controlled trials using the Cochrane Risk of Bias tool 2.0.We narratively synthesized the characteristics of all studies that met inclusion criteria. Study characteristics and treatment details were summarized. For continuous data, means (or medians) and standard deviations (or interquartile ranges or ranges) were extracted. No meta-analysis was performed because of the heterogeneity of data, low quality, and small sample sizes of included studies.A total of 2080 unique articles were retrieved from all searches, of which 1956 were excluded at the screening stage. During the initial search, 72 full texts were assessed for eligibility, of which 14 papers were deemed to meet all eligibility criteria for inclusion in this review . During 21–26 and 14 observational studies, of which 5 were prospective cohort studies,27–31 3 were retrospective cohort studies,32–34 3 were case series,35–37 and 3 were case reports, were included38–40 ded38–40 .Table 1.25 and “high risk of bias” for the remaining four RCTs.26 The risk of bias assessment of the included nonrandomized trials showed one study of low risk of bias,30 one study of moderate risk of bias,33 two studies deemed to have a serious risk for bias,34 and two studies that demonstrated a critical risk of bias31 (Appendix 4). The quality of the two case series was moderate36 and one was of high quality37 (Appendix 5).The results for assessment of the risk of bias for the included RCTs are listed in Appendix 3. The overall risk of bias was deemed to have “some concerns” for two of the RCTsn = 616), including 5 RCTs22–26 and 12 observational studies .35–40 Three studies (n = 54) reported the use of ketamine for treatment of neuropathic facial pain, including 1 RCT21 and 2 prospective observational studies.28Demographic and clinical characteristics of the 670 patients reported in the 20 included studies are summarized in 22–26 Baseline pain intensity ranged from VAS of 56/100 to 95/100.24–26We found five RCTs including 299 patients with primary headaches: migraine, tension-type headaches, and cluster headaches  .22–26 Ban = 222), cluster headache (CH)–episodic (n = 17) or chronic , short-lasting unilateral neuralgiform headache with conjunctival injection and tearing , medication overuse headache , and new daily persistent headache .29–40 Baseline pain intensity was moderate to severe in all observational studies. The duration of headache history was only provided in 3 studies.37We found 12 observational studies including 317 patients  with the following diagnoses: chronic migraine  looked into the effect of and intranasal bolus of ketamine compared to midazolam,23 diphenhydramine,22 ketorolac,26 or no comparator.35 Bolus dose ranged from 0.25 to 0.75 mg/kg. Ketamine was administered as an IV bolus in two RCTs.25 Bolus dose ranged from 0.2 to 0.3 mg/kg and comparators were placebo25 or prochlorperazine with diphenhydramine.24Treatment with a single40 Patient population in these studies was mixed and included patients with CM and CH. Infusions were given over dosages ranging from 0.1 to 1 mg/kg/h over various durations (ranging from a single 1-h infusion up to infusions for up to 6 h on nine consecutive days). Two studies had a comparator (lidocaine).34The effect of IV ketamine infusions was reported in ten observational studies (including 283 patients).26 to 72 h posttreatment,24 with one additional trial specifying only that follow-up occurred for six consecutive migraine attacks.23 The follow-up time for the observational studies was longer, ranging from immediately posttreatment,34–36 to 1 week,37 1 month,32 3 months,38–40 and 18 months29 after treatment.The follow-up time in the RCTs ranged from 60 min24–26 and five studies  investigated ketamine infusions.31–36Four studies  investigated the analgesic effect of ketamine bolus treatment on migraine intensity,26 The three other RCTs failed to demonstrate superiority of ketamine compared to placebo, metoclopramide, or midazolam25 , providing significant pain relief up to 2 h posttreatment.azolam25 .n = 233) reported significant pain improvement at the end of the infusion period.36 Infusions of solely ketamine were given for 8 h36 to 5 days in a row.33 In two studies, the effect of multiday ketamine infusion was compared to multiday lidocaine infusion.34 In the studies with lidocaine as a comparator, both groups were associated with significant pain reduction at the end of the infusion as compared to baseline, although the difference between groups was significant in favor of lidocaine in one study34 and in favor of ketamine in the other study.31In stark contrast, all of the five observational ketamine infusion studies  reported a resolution or reduction in the attack frequency after ketamine treatment. An immediate but short-lasting effect was noted after bolus treatment30 and an immediate to sustained effect was noted after ketamine infusion.37–40 Patients were reported to have more than a 50% reduction in attack frequency for 6 weeks38 to complete resolution of attacks for 6 weeks38 up to 3 months.40All seven studies including patients with TAC , measured 30 min posttreatment, between the IV ketamine bolus and placebo groups was identified.25 In only one study was a significant difference identified, in favor of prochlorperazine.24Only three papers reported on patient satisfaction and/or preference for ketamine versus the comparator using an NRS.25 Two case series mentioned a reduction in dose and/or discontinuation of analgesics following ketamine infusion, but further details were not provided.39A few studies commented on change in use of rescue medication during or following ketamine treatment. Although not statistically significant, two RCTs demonstrated that a smaller proportion of patients in the ketamine group needed rescue medication during the infusion compared to the comparator.32 and one patient preferred to stop ketamine because of the side effects.33 One additional patient developed an asymptomatic elevation in liver enzymes.32 None of the patients needed to be hospitalized because of intolerable side effects such as feelings of insobriety, confusion, nausea and vomiting, hallucinations, and tachycardia.Approximately 70% of patients across all studies experienced side effects related to ketamine infusions; however, most were mild and resolved with decreased rate or ending the ketamine infusion. Side effects included dizziness, sedation, and blurred vision’ One patient’s ketamine infusion was stopped because the patient experienced suicidal thoughtsn = 30)21 and one small observational study (n = 7)28 examined the use of ketamine for patients with trigeminal neuropathy. Another small observational study (n = 17) included patients with neuropathic orofacial pain (not specified).27 Baseline pain intensity was moderate to severe28 or not provided.27 The duration of pain ranged from 6 months to 28 years.Of three studies including 54 patients , one crossover RCT .27 It was noted that those responding to IM ketamine also reported pain relief with oral ketamine.27 The adverse events profile was similar as reported in the primary headache studies.In the three studies, all patients received a single bolus of ketamine 0.4 mg/kg. One study compared ketamine to IM pethidine 1 mg/kg.Despite recent evolutions in pharmacological and interventional management of patients with CFP, many patients continue to experience a significant reduction in quality of life, thereby imposing a significant burden on the health care system. We therefore conducted this systematic review to evaluate the evidence on the role of ketamine for patients with headache and/or facial pain.26 and the remaining RCTs failed to demonstrate a significant effect of ketamine on headache intensity compared to various comparators. On the other hand, all observational studies demonstrated a significant reduction in migraine pain intensity and duration, as well as decreased frequency and intensity of CH attacks immediately postintervention that lasted for up to 3 months postinfusion. The difference in these findings may be explained as follows. First, it may be attributed to the shorter administration times and/or lower dose of ketamine bolus treatment in the RCTs, because nearly all observational studies explored single-/multiple-day ketamine infusions. The typical doses for chronic pain IV ketamine treatment are 0.2 to 0.75 mg/kg (bolus), 0.5 to 2 mg/kg/h (infusion), 0.1 to 0.5 mg/kg for IM administration, and 0.2 to 1 mg/kg for intranasal administration, which is higher than what was administrated in the RCTs.10 A high dosage and extended administration (infusions) of ketamine have been associated with better pain relief in patients with chronic pain.41 Similar conclusions were made in the consensus guidelines on the use of IV ketamine infusions for chronic pain.10 Future research in patients with primary headache should therefore focus on investigating the effect of high-dose and repeated administration of ketamine in a randomized controlled setting. Second, most RCTs had a mixed patient population, including patients with acute and chronic headache of migraine, cluster, and MOH types, which increased study heterogeneity, potentially affecting the outcome. Lastly, the 5 papers that did show a positive outcome of ketamine bolus treatment investigated patients solely with TAC, and bolus treatment failed in studies on patients with chronic migraine. This difference in outcome could be explained by a difference in pathophysiology, where TAC is driven by changes in the sphenopalatine ganglion, which potentially is more susceptible for the ketamine effect.Although we evaluated 5 RCTs and 12 observational studies, evidence remains conflicting on the efficacy of ketamine for treatment of primary headaches. This is likely due to the limited study quality, moderate to high risk of study bias, confounding and substantial heterogeneity across studies in terms of study design, patient populations, details of ketamine treatment, and (lack of) follow-up. Only 2 of the 5 RCTs demonstrated a significant immediate effect of ketamine on the intensity of pain and aura of migraine relief in the emergency room,21 and a tendency for continued pain relief with oral ketamine after initial IV/IM treatment was also noted.27 This treatment option of oral ketamine should be further explored because this could be a valuable therapy for patients in remote areas with limited access to health care facilities.The evidence on ketamine for neuropathic facial pain is scarce but appears promising. Intramuscular ketamine was associated with significant improvement in two out of three studies on patients with trigeminal neuropathy,International Classification of Headache Disorders third edition diagnostic criteria for CFP are widely accepted and recommended,42 a majority of the papers did not mention use of this validated instrument for diagnosis. This is a weakness that needs to be addressed to avoid misclassification bias and improve generalizability of the study results. Though a handful of studies examined patient satisfaction/preference and the need for rescue medications, most papers in this review did not mention use of validated tools to evaluate the effect of ketamine on pain-associated domains  in addition to its analgesic efficacy. This is surprising because the importance of evaluating pain-related domains in clinical and research settings is established, and validated tools to evaluate these domains are available.43 Ketamine has been demonstrated to have an effect on sleep and mood and could therefore potentially improve patients’ quality of life even if pain intensity is unchanged.45Although the 10 The incidence of reported ketamine-induced side effects in the included papers was high regardless of type of administration or dose and only included psychomimetic effects. Most side effects were mild and resolved after decreasing the dose or ending treatment. Although central nervous system effects appear to be dose dependent when ketamine is used in anesthetic doses, the evidence is not as clear for subanesthetic regimens, beyond a yet-to-be-determined threshold.10 In our review, no clear difference was noted in the incidence of side effects when comparing high to low doses. Most patients experienced hallucinations, which resolved after cessation of treatment, and one patient experienced suicidal thoughts. Based on the America Psychiatric Association44 guidelines on the use of ketamine, a history of psychosis is a contraindication for administration of subanesthetic IV ketamine.It is widely accepted that ketamine is associated with adverse psychomimetic, cardiovascular, and gastrointestinal effects resulting from its activity on a variety of substrate receptors including NMDA, acetylcholine, opioid, monoamine, and histamine.46 The authors concluded that the benefit of ketamine for headache treatment is unclear but that long-term follow-up and different ketamine dosages in patients with chronic pain should be explored. Therefore, our systematic review attempted to take a closer look at the larger amount of evidence in observational trials that focused on ketamine infusions. Our review demonstrated that prolonged ketamine infusions can indeed significantly and sustainably reduce migraine intensity and frequency of CH attacks. This provides promising evidence, and this temporal and dose-dependent relationship should be further explored in new high-quality trials. To our knowledge, no attempt has been made to systematically gather evidence regarding the effects of ketamine on facial pain.We found one other systematic review on the efficacy of ketamine for headaches, with evidence found in RCTs that were primarily focused on bolus administration of ketamine for acute headache pain relief.This review has several limitations. Most of the studies included in this review reported observational data, with inherent high risk of confounding and bias. Further, the heterogeneity across papers was substantial, with large differences in dosing, duration and route of administration, and comparators, which made comparison challenging and precluded a meta-analysis. Lastly, this review did not assess the long-term benefits and harms of ketamine for the treatment of CFP owing to lack of data in the literature.10 However, adverse effects of ketamine are common and could limit dose escalation, and the potential long-term risks of repeated administration of high doses of ketamine need to be further investigated.A high-quality placebo-controlled RCT investigating the effect and safety of high-dose and/or prolonged ketamine infusion treatment on refractory headache pain and pain-associated domains would be the logical next step to investigate ketamine’s potential in the treatment of CFP. Recent systematic reviews and meta-analyses of this evidence show that infusions of ketamine 1 mg/kg/h are associated with greater and sustained pain relief compared to one-off bolus and low-dose infusion regimens.Current evidence on the efficacy of ketamine for treatment of CFP remains conflicting, precluding the ability to make any recommendations. This is likely due to the limited study quality, moderate to high risk of study bias, and substantial heterogeneity across studies in terms of study design, patient populations, details of ketamine treatment, and (lack of) follow-up. Ketamine bolus treatment showed significant reduction of migraine aura severity, TAC intensity and frequency, and trigeminal neuropathic pain but failed to reduce migraine intensity or duration. The included observational trials suggest that ketamine infusion treatment has an immediate and sustained benefit on headache pain intensity, possibly because of the prolonged duration of treatment and higher dose of administration. Most papers failed to evaluate the effect of ketamine on pain-associated domains. RCTs are required that focus on the dose–response relationship and immediate and long-term effects of prolonged ketamine treatment on CFP and pain-associated domains.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.