Cannabis sativa) and its products received attention and much research has been conducted to evaluate the effects of Cannabis sativa compounds in animals. Among various substances of this plant, cannabidiol showed desirable effects such as relieving pain and inflammation reduction in some studies. Considering the importance of animal welfare, especially in poultry production, the use of cannabidiol can be effective here.Plant feed additives have been used in animal diets for improving animal health and welfare. Thus, hemp (In recent years, interest in hemp use has grown owing to its chemical and medicinal properties. Several parts of this plant, such as seeds, leaves, flowers, and stems are used in medicine, industry, and environmental preservation. Although there were legal restrictions on hemp exploitation in some countries due to the trace presence of THC as a psychoactive element, many countries have legalized it in recent years. Cannabidiol or CBD is a non-psychoactive phytocannabinoid that can activate the endocannabinoid system and its receptors in the central and peripheral nervous system in bodies of different species. Cannabidiol has anti-inflammatory, antioxidative, analgesic, and anti-depressant effects. This review investigates various aspects of cannabidiol use and its potential in animals and humans. Cannabis sativa, generally known as hemp, is an oleaginous angiosperm annual plant from the Cannabaceae family [e family . Its usee family . Cultivae family  and aftee family ,4. Everye family . This ple family . Phenolie family .Cannabidiol (CBD) is a phytocannabinoid derived from flowers, leaves and, to a lesser extent, stems. Cannabidiolic acid (CBDA) can change to CBD due to heat exposure . CBD oilSince Yamauchi et al. conducted the first scientific research on the extraction of the chemical components of Cannabis in 1968, interest in the use of industrial hemp and CBD in animal and human nutrition research has grown in recent years .Hemp classification, laws and regulations on its use;CBD structure, extraction, and the mechanism of action in the body;CBD anti-inflammatory, hepatic and antioxidative effects;Hemp in livestock feeding.The following subjects will be discussed in this review:Sativa and indica are divided into different varieties [Hemp is included in the Cannabaceae family with four subspecies: sativa, indica, ruderalis, and afghanica . The prearieties , as demoHemp is an angiosperm dioecious plant with a strong root that can enter deeply into the soil . The besAmong several recognized phytocannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD) are the two most essential components. THC is a psychoactive compound that is used for recreational purposes. On the other hand, CBD is a non-psychoactive compound known for antioxidant, anti-inflammatory, antipsychotic, anxiolytic, and anticonvulsant effects ,17. CBD Cannabis indica, mostly known as marijuana or medical Cannabis, is bushier [Cannabis indica contains a higher level of THC, specifically Δ9-tetrahydrocannabinol (Δ9-THC), whereas Cannabis sativa, known as hemp or Industrial Hemp, is taller. The importance of hemp is for the seed and fiber used in several products and their medicinal value [ bushier  and has  bushier . Cannabial value . This plal value . In a stal value . This caal value . In anotal value  analyzedal value .In the 1930s, hemp use was prohibited in Canada by the Narcotics Management Act ,24 due tThere is a narrow line for identifying Cannabis as a drug plant or a non-drug one. In the United States and most of European countries including Poland—the maximum THC level for industrial hemp should be 0.3% and 0.2%, respectively ,27.As mentioned before, the regulations would be different among the countries related to the therapeutic use of Cannabis (CBD oil extracted from dried flowers) in humans and animals. In Canada, some European countries, and some states of the USA, physicians prescribe many cannabinoid medicines for humans . Based oVarious hemp-containing products such as tea, oil, and beer can be used in the food industry . In receSeeds are excellent sources of polyunsaturated fatty acids (PUFAs), 20–25% digestible proteins such as albumin and essential amino acids in high levels , carbohydrates (25–35%), vitamins, for instance, γ-tocopherol at an amount of 60.85 mg/100 g dry matter, and minerals ,31,32.Hempseed oil is rich in PUFAs at an amount of 25–35%, up to 90% consisting of α-linoleic acid (ALA) 18:3 (ω-3) 16%, linoleic acid (LA), 18:2 (ω-6) average 56%, with a 3.5:1 ratio ω-6 to ω-3 . Moreove21H30O2 and a relative molecular mass of 314.464 g. mol−1 [(1)In the cyclohexene ring at C-1 with a methyl group,(2)In the phenolic ring at C-1ʹ and C-5ʹ with a hydroxyl group, and(3)In the phenolic ring at C-3ʹ with the pentyl chain.CBD is a phytocannabinoid with the formula Cg. mol−1 . The strg. mol−1 :(1)In thCannabidiol is produced in the glandular trichomes of the female hemp flowers . Figure CBD extraction and purification can be challenging processes due to the presence of THC. The appropriate method should be practical and have the most negligible CBD loss.There are different stages in Cannabis processing: selection of variety, cultivation, harvesting, and extraction . In the The highest concentration of cannabinoids is found on the flower trichomes surface; hence, mechanical methods such as pressing would not be appropriate for extraction . IntenseTo obtain a pure product, chromatography, crystallization or distillation can be exploited ,41. MechThis method has been widespread for plant oil extraction  and someSome researchers used the Soxhlet method to extract some compounds from hemp. Pandohee et al. derived cannabinoids from Cannabis with the Soxhlet method in consecutive batches of ethyl acetate. Before the operation, roots and leaves were grounded and treated under 368 µm. In the first batch, 0.5 g of the powder and 300 mL of ethyl acetate were made at 78 °C for 1.5 h. The second batch consisted of extraction repetition with 300 mL of solvent for 1 h, and finally, the combination of extractions vacuumed at 40 °C . Chang eThe substance is immersed in the solvent for a period of time. Generally, this term is used for conventional extraction methods, including OSE methods. The extraction method is named according to the used solvent. Ethanol and methanol solvents, for instance, are widespread for cannabinoid extraction due to their polar properties and boiling point. However, ethanol is preferable due to its lower toxicity .Soxhlet and Immersion methods caused some safety and environmental concerns; thus other methods are applied for effective extraction.2 is non-toxic, non-flammable, and affordable, it has been employed extensively in the SFE method [2 . Recovering and reusing carbon dioxide during the process is another advantage of the SC-CO2 method. Furthermore, co-solvents moderate the solute and solvent and produce more efficient extraction [Different fluids are applied in the SFE process, such as methanol, ethanol, carbon dioxide, water, sulfur hexafluoride, nitrous oxide, and n-pentane . Since CE method ,53. To otraction . Water, traction , esters,traction  are empl2 has a medium to low potential for extraction of cannabinoids and terpenes [The supercritical extraction method is expensive, so the OSE method is still more commonly in use. SC-COterpenes . Howeverterpenes .Grinding, ultrasonication, and high-pressure homogenization are applied as pretreatment methods  for both cannabinoids and lipids ,50,56.2 method, supercritical hot water can be more expensive, impose more energy on the system, and has safety problems due to the water supercritical condition  [In this method, pressurized hot water is employed as a solvent with equivalent solvability features to ethanol and methanol . It has 064 MPa) .C. sativa with reverse-phase HPLC [This technique can be used for the separation and identification of CBD. This technique consists of a column with a diameter of 2 ÷ 4.6 mm and a length of 20 ÷ 250 mm, with different modifications in the surface stationary phase . The detase HPLC . Moreovease HPLC . In anotase HPLC .The endocannabinoid system is a type of endogenous signaling system adjusted by sleep, stress levels, physical activity, and food which maintains homeostasis in the body. Endocannabinoids include amides, ethers, and esters from long-chain polyunsaturated fatty acids (PUFAs) . ECS canDue to the resemblance between prostaglandins and endocannabinoid structures, interaction between metabolic pathways occurs . THC is There are 76 types of molecular CBD targets . CBD canNumerous dose-dependent studies on the pain-controlling effects of hemp oil and CBD were conducted on some animal species and humans. Since pain and inflammation exist simultaneously in many conditions, various studies show that CBD has analgesic potential, and anti-nociceptive and anti-inflammatory effects on some painful diseases and disorders.In veterinary medicine, CBD has been used to alleviate cancer pain, osteoarthritis, neuropathic pain, and mood disorders in dogs and cats ,87. MoreThe results of research on CBD’s anti-nociceptive and anti-inflammatory effects in recent years are shown in Based on the research results depicted in In recent years, studies have been conducted to investigate the effects of different parts of the hemp plant  and CBD on liver function in humans and animals. The results of research investigations related to the effect of Cannabis on the liver are depicted in Since hemp is a source of unsaturated lipids, it is prone to oxidation . The livSamara et al. showed that CBD can inactivate cytochromes P4503A (CYP3A) and P4502C in hepatic drug metabolism . CytochrBased on the results presented in the previous paragraph, it seems that higher levels of CBD result in more side effects and the risk of liver damage. Since CBD is a dose-dependent substance, it has shown different physiological responses. Thus, it is essential to indicate the optimal dose in different species.Several studies showed that CBD has beneficial effects on nervous system diseases and mental health. Hypoxic-ischemic (HI) is a brain injury that occurs due to oxygen deprivation in brain cells and causes neurological impairment, e.g., decreased cognitive function and epilepsy . In a st2) produces a superoxide that is a precursor to many reactive oxygen species such as hydrogen peroxide (H2O2). Due to the high lipid content in the brain, it is sensitive to oxidation [In the nervous system, the brain consumes a high amount of oxygen . Oxygen xidation . It is pxidation .CBD affects the elements in the redox system. CBD adjusts the activity and level of oxidants and antioxidants ,129. In Lipid peroxidation is among the most prominent processes that occur in the body which causes polyunsaturated fatty acid (PUFA) oxidation . When ROSome molecular compounds have a role in the redox system. CBD can indirectly interact with these molecules and play a role in regulating redox balance. Anandamide (AEA) can be increased due to the effect of CBD on the activity of the endocannabinoid system . Since ACannabis sativa use in farm animal diets, especially poultry, due to its positive effects. It is important to investigate the effects of the different components of this plant on livestock and poultry in future research.Much research has been performed on adding hemp to animal feed. In recent years, attention has been paid to The results of studies on hemp use in laying hens show  that hemCannabis sativa and its metabolites increase tibia strength and decrease the deformation rate in broilers and laying hens.There are studies regarding Cannabis inclusion in other animal species, as shown in Based on the research results of Kleinhenz et al. , the bioThere is a gap in the literature on CBD use, specifically in poultry. Since CBD is an efficient substance in the Cannabis plant, its effects in further studies should be taken into account.Cannabis has been utilized widely in recent years. Hemp oil, hempseed oil, and hempseed cake improved performance and bone strength, enriched egg fatty acid profiles, and increased milk yield in livestock production. CBD use as a non-psychoactive compound showed promising results in alleviating and preventing pain, oxidation, inflammation, and anxiety in different species of animals and also in humans. An appropriate method of extraction for a high level of purity and correct dosage of this substance is important in terms of hepatic conditions. Even though CBD has been used in several animal studies, the absence of research on CBD use in poultry is noticeable. Since welfare is a major concern in the poultry industry, evaluating the effects of CBD in further research should be considered.


EO therapy had a slightly better analgesic effect than placebo one week after the intervention  and at the four-week follow-up as well . EO therapy had a beneficial effect on stiffness (a secondary outcome) compared to the no intervention group . This systematic review and meta-analysis showed that topical EOs are beneficial as an add-on treatment in reducing pain and stiffness in the investigated MSDs.Essential oils (EOs) are widely used topically in musculoskeletal disorders (MSDs); however, their clinical efficacy is controversial. Our aim was to find evidence that topical EOs are beneficial as an add-on treatment in MSDs. We performed a systematic review and meta-analysis to summarize the evidence on the available data of randomized controlled trials (RCTs). The protocol of this work was registered on PROSPERO. We used Web of Science, EMBASE, PubMed, Central Cochrane Library and Scopus electronic databases for systematic search. Eight RCTs were included in the quantitative analysis. In conclusion, EO therapy had a favorable effect on pain intensity (primary outcome) compared to placebo. The greatest pain-relieving effect of EO therapy was calculated immediately after the intervention (MD of pain intensity = −0.87;  Musculoskeletal disorders (MSDs) are major public health issues all over the world because they cause long-term pain and physical disabilities and reduce people’s ability to work . MSDs coPharmacological treatments of MSDs include topical or oral analgesics , paracetamol, tramadol, and opioids), chondroitin sulphate, glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) and other drugs . PainkilEssential oils (EOs) are complex secondary metabolites that are produced by aromatic plants; they are composed of many apolar or semi-polar volatile constituents with low molecular mass. The largest group of them are terpenoids, but phenolic compounds are also dominant. An EO generally consists of 10–50 compounds with various structures, which can be carbohydrates or oxygen-containing compounds such as alcohols, ketones, aldehydes, ethers and esters . The cheThe purpose of this systematic review and meta-analysis is to evaluate the efficacy of topically used EOs and to assess the hypothesis that topical EO therapy is beneficial as an add-on treatment in MSDs. Furthermore, based on our results, our aim is to provide evidence-based recommendations for healthcare professionals.With the searching process, 752 articles were collected. Duplication removal resulted in 518 records for the subsequent title and abstract selection phase. After full text screening, altogether 12 studies ,29,30,31Baseline characteristics of the randomized controlled trials (RCTs) included in this paper are presented in In the EO therapy group, the EOs were applied topically as a complementary treatment in addition to the conventional therapy of MSDs. In the Placebo group, a placebo product (a vegetable carrier oil or an ointment without any EOs) was used as an add-on treatment in MSDs. In the No intervention group, patients did not receive either EO therapy or other additional interventions, only the conventional therapy. The EO-containing products and placebo products were applied by massage in most of the trials. The length of the interventions differed in the trials; they were mostly three or four weeks long.Different EOs were used in the trials. Lavender EO was used in seven trials, and the applied concentrations were between 1.5% and 3%. In the case of other EOs, the applied concentrations were between 0.5% and 2.5%. In one case , an ointAll investigated trials concluded that EO therapy might be a beneficial treatment for pain intensity (primary outcomes). The following conditions were investigated in the trials: knee osteoarthritis (OA) and hand OA, rheumatoid arthritis, low back pain, carpal tunnel syndrome (CTS) and neck pain. Due to the high heterogeneity of secondary outcomes and measurements related to the functional state, only stiffness was included in the quantitative analysis. QoL was measured in two articles. Yip and Tam (2008) investigated the effect of ginger and orange EOs on QoL. The results showed that EO therapy was not effective to improve QoL . PehlivaFor the analysis of pain intensity, seven articles were considered ,27,28,292 statistics and confidence intervals (CIs), are shown in p-value = 0.0465), indicating that the time-points had an overall effect on the outcomes. The test of residual heterogeneity of the overall model was not significant . Model results indicate that the application of EOs was beneficial at all time points compared to placebo treatments, with significant results on week zero  and week four.Calculated mean differences (MDs), together with within-group I2 = 61%; p = 0.014)). The difference is statistically significant between the EO group and the Placebo group.Four trials ,24,25,262 = 40.3%; p = 0.077)).The results of four trials ,27,28,292 = 59.3%; p = 0.049)).This analysis was performed on four trials ,25,27,29For the analysis of stiffness, three articles were considered ,29,31 wi2 = 68.57%; CI: 6%−96%; τ2 = 0.3312; p = 0.061)) indicates a slight improvement in the functional state of the MSD compared to no intervention. The result is nearly significant.The result  assessment was performed, and the overall certainty of evidence is very low in the case of both outcomes. The reasons for this may be the lack of blinding and the heterogeneity see .p = 0.99).We used Egger’s test and the sunset funnel plot see  to assesp-value is also near significant , improve blinding and provide comparable results, i.e., to reach a consensus on measurement tools intended to be used.We report our systematic review and meta-analysis based on the recommendation of the PRISMA 2020 guideline  AND  AND . No filters were applied.The following PICO framework was applied to select the relevant clinical trials. Participants: adults with MSDs; Intervention: EOs applied by massage or EOs applied without massage; Comparisons: placebo product (with or without massage), or no intervention; Outcomes: pain intensity (primary outcome), quality of life (QoL) and functional state (secondary outcomes).Only RCTs that met the established PICO were considered.Articles were excluded based on the following criteria: animal studies; EOs administered by inhalation; no available full texts; patients studied were suffering from acute pain ; patients studied were suffering from pain associated with diabetes or dysmenorrhea; the use of inappropriate placebos.After duplicates were removed by using EndNote X9 , the selection process was continued by two independent review authors (EB and EEG). The articles were selected based on title, abstract and full texts and in accordance with the predetermined inclusion and exclusion criteria. Inter-rater reliability was assessed by the calculation of Cohen’s kappa. Results of Cohen’s kappa determination showed a strong consensus degree. Disagreements were resolved by a third author (FD).Data were extracted by EB and PF. Disagreements were resolved by a third reviewer (FD). Data extraction was carried out by either taking published values or, in the case of one trial , using aThere were not enough data in the literature to perform the quantitative evaluation of QoL (secondary outcome). According to the inclusion criteria, studies performed on adult patients should have been selected into the meta-analysis. In the trial of Kong et al. (2012), the inclusion criterion of age was 15–35 years. In that case, we considered the patients under 18 years as young adults.The risk of bias assessment was performed independently by EB and EEG using the Cochrane risk-of-bias tool for RCTs (RoB 2) . DisagreThe GRADE approach was used to evaluate the evidence of the included trials . Accordi2 statistic, we followed Jackson’s methodology [Mean before/after change difference in pain intensity measured on VAS as the primary outcome was pooled using multilevel mixed effect models ,46. The hodology . Resultshodology  where unhodology  using thhodology , meta [5hodology , dmetar hodology  metafor hodology  and metahodology .This systematic review and meta-analysis showed that topical EOs are effective in reducing pain and stiffness in chronic MSDs, and that they contribute well to conventional therapies. Based on our results, we suggest that topical EO therapy should be applied repeatedly to reach the most effective pain-relieving effect of EOs.However, due to the limitations of our study , further clinical investigations are needed to establish our conclusions on efficacy, to determine the most potent EOs and to understand their mode of action.


Emotional dysregulation (ED), defined by deficits in the ability to monitor and modulate the valence, intensity, and expression of emotions, is typically expressed with irritability, tantrums, mood fluctuations, and self-harm in young children with autism spectrum disorder (ASD). Although ED does not represent a diagnostic feature of ASD, its manifestations are an important contributor to functional impairment and clinical referral. This study aims to examine the relationship between ED and adaptive functioning in preschoolers clinically referred for ASD or other neurodevelopmental disorders.A sample of 100 children , consecutively referred to a university clinic for neurodevelopmental disorders, received clinical assessments of psychopathology with the CBCL and the Autism Diagnostic Interview-Revised, of ED- with the CBCL-Attention, Anxious/Depressed, and Aggression index (CBCL-AAA), of autism symptom severity with the ADOS-2 Calibrated Severity Score (ADOS-CSS), and of global developmental/cognitive delay (GDD) with the WPPSI-IV or other age-appropriate standardized scales. Adaptive functioning was measured with the ABAS-II. Sixty-five children met DSM-5 criteria for ASD. Multivariate regression models were applied to evaluate the relative contribution of ED, ASD severity and GDD to the ABAS-II general (GAC), conceptual (CAD), social (SAD), and practical (PAD) adaptive functioning domains.n = 100), lower adaptive functioning was associated with higher CBCL-AAA (p = 0.003), higher ADOS-CSS (p < 0.001), and presence of GDD (p = 0.023). In the ASD group (n = 65), worse CAD was predicted by GDD (p = 0.016), and worse SAD and PAD by higher ADOS-CSS (p = 0.032) and ED (p = 0.002). No sex differences were detected in the study variables.Overall (Together with the severity of global developmental delay and of autism symptoms, ED is a significant contributor to impairment in adaptive functioning among young children with a neurodevelopmental disorder and, in particular, with ASD. ED could represent a specific target for early interventions aimed at enhancing adaptive functioning in early childhood. Emotional dysregulation (ED), defined as a deficit in the ability to monitor and modulate the valence, intensity, and expression of emotions, is typically expressed with mood instability, irritability, tantrums, and self-harm in young children with autism spectrum disorder (ASD) –4. AlthoEmotional dysregulation represents a transdiagnostic risk factor for mental health disturbances both in the general population , 8 and iWhen emotional regulation is not effective, intense emotions interfere with social interaction. Children with such difficulties tend to engage in peer conflicts and, consequently, to be less accepted by others and to experience negative feed-back in relationships, with consequent feelings of exclusions . Indeed,The ability to regulate emotions grows with age, being less effective in preschoolers than in older children, is influenced by puberty with decreased regulation during adolescence , and becPeople with ASD seem to be at higher risk of having impaired emotional regulation because of poor emotional awareness and low competence in emotional language, poor flexibility, high sensitivity to change and to environmental stimulation . Due to As ED underlies several behavioral problems in ASD, therapeutic interventions aimed at empowering the ability to recognize and regulate emotions might be an effective way of addressing behavioral problems . The socMore in detail, the ABAS-II Social Adaptive Domain score is derived from the scores of the two subscales: play and social abilities. Since ABAS-II examines “real-world” functions, it provides an index of the child’s competency in everyday contexts, such as the ability to engage in leisure activities by its own, to interact with others while playing, to ask for adult’s involvement while playing, to share emotions, and to exhibit social engagement, stages of according to different developmental age.To our knowledge no study has analyzed the relative contribution of ED to adaptive functioning in ASD in very young children.This study aimed to examine the relationship between ED and adaptive functioning in preschoolers clinically referred for ASD or other neurodevelopmental disorders, while taking into account autism symptoms, sex, and presence of global developmental/cognitive delay.This was a cross-sectional study of 100 consecutively referred children undergoing a diagnostic assessment for developmental disorder. All children under 6 years of age referred to the University of Turin-Pediatric Hospital Regina Margherita Outpatient Service for Neurodevelopmental Disorders between 1 July 2018 and 31 October 2021 received a comprehensive diagnostic evaluation. The children were referred by primary care pediatricians or directly brought in by their parents for suspicion of a neurodevelopmental disorder because of difficulties in communication and social interaction or behavioral problems. Neuropsychological and psychopathological evaluations were conducted by trained developmental psychiatrists and neuropsychologists. Main caregiver psychometric questionnaire was used for research purposes. Parents gave informed permission to participate, and all the procedures were approved by the local ethics committee. All the children received the following assessments.The Child Behavior Checklist 1.5-5 (CBCL) , ItalianThe Autism Diagnostic Interview-Revised (ADI-R)  was admiThe diagnosis of ASD was made according to the DSM-5 criteria , using tThe severity of the autism symptoms was measured with the calibrated severity score (CSS) , 35 of tEmotional dysregulation was measured using the Attention, Aggression, and Anxious/Depressed scales of the CBCL (CBCL-AAA), which consider the affective, behavioral, and cognitive dimensions of ED. The CBCL-AAA construct was first used to define the Deficient Emotional Self-Regulation (DESR) with scores between 1 and 2 standard deviations (SD) above the mean , while aAccording to age, expressive language level, and the child’s ability to engage and cooperate with the examiner, standardized tests were used to assess cognitive and global development level. Instruments included the Griffiths III or Bayley-II developmental scales, Leiter International Performance Scale-II edition or the WPPSI-IV  –44. All Adaptive functioning, defined as the set of conceptual, social and practical skills learned and used in everyday life, was measured with the Adaptive Behavior Assessment System-Second Edition (ABAS-II) . On the p-value < 0.05 was considered statistically significant.Statistical analyses were performed using the statistical programming language R (version 4.0.5) . Descripp-values corrected using the Benjamini-Hochberg procedure.Because of multiple testing with correlated subscales, multivariate regressions were used to determine the association of ED with adaptive functioning. The multivariate models considered the three ABAS-II subscales as dependent variables. All models were also adjusted by ADOS-CSS and a dichotomous variable for global developmental delay/intellectual disability. Pillai’s trace statistic was used to evaluate significance of the multivariate models. The associated univariate multivariable effects were then analyzed and Since no significant difference between males and females in ED, CSS, and adaptive functioning were detected in the whole sample, we did not include sex in the models. Also, as the sample was homogeneously composed by preschoolers, we did not adjust the models by age.The same multivariate and univariate multivariable models were used for evaluating the impact of ED, ADOS-CSS, and GDD on adaptive functioning in the ASD group.The sample consisted of 100 patients, of whom 74 were males (74%). Mean age at first evaluation was 39.43 months  . Sixty-fOn the CBCL, mean scores were: 56.51 ± 11.01 on the internalizing scale and 54.20 ± 9.74 on the internalizing scale, with a total score of 54.92 ± 11.20. Detailed subscales of CBCL are reported in p = 0.054). No sex difference was found in our sample with regard to adaptive functioning, ADOS CSS, CBCL AAA, and GDD , with no statistically significant difference between the ASD (175.30 ± 19.32) and the non-ASD group (169.05 ± 20.47)  of the sample was 71.45 ± 18.66, conceptual adaptive domain (CAD) mean score was 69.40 ± 18.38, social adaptive domain (SAD) mean score was 78.56 ± 18.95, and practical adaptive domain mean score (PAD) was 76.82 ± 19.08.p < 0.0001). Scores for DAS  and DAP  were also lower in children with ASD. No significant difference was found between the ASD and non-ASD with regard to CAD  was lower that in patients without ASD .The mean ADOS CSS score of the whole sample was 4.95 ± 3.16). As expected, the mean ADOS CSS in ASD (6.75 ± 2.37) was higher than in non-ASD . Significant associations between ADOS CSS and adaptive functioning (p < 0.001) and GDD and adaptive functioning (p = 0.023) were also revealed  and by higher ED , while both higher ED  and ADOS CSS  were associated with lower SAD. Higher levels of ADOS CSS  and ED , and the presence of GDD  were all significant predictors of worse PAD.To study the impact of ED on the diverse domains of adaptive functioning, we analyzed a univariate multivariable model for each adaptive functioning domain . Lower Cp value = 0.016). ADOS CSS  and ED  were both found to be associated with worse SAD. ADOS CSS  and CBCL AAA  predicted lower PAD.Likewise, when considering only children with ASD, higher ED was associated with lower adaptive functioning levels. Also the ADOS CSS was associated with overall adaptive functioning. With regard to the three adaptive functioning domains , CAD wasPrior research has suggested that ED in children with ASD is a significant problem that is likely to underlie behavioral difficulties, such as tantrums, self-harm and irritability, and thus to contribute to functional impairment. The pathogenetic relevance and the mechanisms of the relationship between ED and clinical symptoms are still a matter of study, and investigation into ED in ASD children is ongoing. This study aimed to contribute to the research in this area by examining the relationship between ED and functional impairment, and by measuring different domains of adaptive functioning in preschoolers clinically referred for suspected ASD. To our knowledge, this study has been the first to evaluate autism symptoms, emotional dysregulation and adaptive functioning in preschoolers with ASD and other neurodevelopmental disorders. The clinical characteristics of the study sample are consistent with those expected in this population, with a predominance of the male sex and about two-third of the referred children meeting criteria for ASD or being at high risk for ASD .p = 0.05  had been reported to be distinctive of ASD compared with other neurodevelopmental disorders . In our In our study, considering both the overall sample and also ASD sample, higher ED was associated with lower adaptive functioning levels . Social We didn’t find any sex difference in ED or in other clinical variables. This could be due to the rather small representation of females in the study sample and/or to referral bias whereby females with high ED might have been more likely to be clinically referred. Few data on sex differences in emotional regulation among ASD people are available, but scarcely comparable to our population , 73. FurThe results of this study are clinically important because they document the relevance of ED to adaptive functioning in young children and thus provide support to efforts to develop emotional regulation strategies as a possible means of ultimately improving functioning. In typical development, good parental scaffolding helps children overcome frustration and improve emotion regulation. ASD children too can benefit from parental scaffolding but they seem to have difficulty in internalizing and generalizing co-regulatory support. This is consistent with the clinical experience that children with ASD benefit from the presence of one-to-one support for regulatory management in education settings well into middle childhood and beyond . Parent-This study has several limitations. First, the cross sectional design precludes the possibility of assessing prospectively the interplay between ED and adaptive functioning or to evaluate the developmental trajectories of these constructs over time. Second, the study sample included only clinically referred children without a comparison group of typically developing children. Third, the male preponderance, while being consistent with the epidemiology of the disorder, limits inferences to female patients. Fourth, both the ED and adaptive functioning ratings were provided by the same informant (main caregiver), thus limiting somewhat the external validity of the associations. Finally, no systematic information on the social context was available. Further studies with larger samples and prospective design are needed to confirm and better clarify our findings.Higher levels of ED were associated with lower adaptive functioning in all the domains evaluated, regardless of the severity of the autistic symptoms or the presence of GDD. Decreasing ED in preschoolers with ASD or other neurodevelopmental disorders may be an effective way of improving their overall adaptive functioning, including prosocial behavior, ability to focus on goal oriented activities, and academic achievement. In particular, since a child’s ability to regulate emotions is modeled by parental scaffolding, parent-mediated early interventions may be an effective approach to improving the developmental trajectory of children with high levels of ED.The raw data supporting the conclusions of this article can be made available by the authors upon qualified request.The study was approved by the institution review committee . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.CD and DM conceived and designed the study protocol with input from FA, FR, and BV. CF, AG, LA, and BS carried out literature searches. BS, CF, VFC, and LA collected the data. DM designed and carried out the statistical analysis. CD, AG, DM, and BV interpreted the data and drafted the manuscript. FR and FA supervised the writing of the manuscript. All authors critically reviewed and contributed to the final version of the manuscript.In the last 2 years, BV has received consultant fees or honoraria from Medice, Menarini, and Angelini Pharmaceuticals. FR has received sponsorship or fee for advisory board from Roche, Novartis, Biogen, PTC Therapeutics, Sanofi Genzyme, Sarepta/biogen and CD has received consultant fee from Roche and Lundbeck, and DM has received consultant fee from Ethos Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metabolic dysfunction-associated fatty liver disease (MAFLD) is a new term that no longer excludes patients that consume alcohol or present other liver diseases, unlike nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate the role of different biomarkers as predictors of MAFLD in patients with type 2 diabetes mellitus (T2DM). In this regard, a cross-sectional, non-interventional study was conducted over a period of 8 months in patients with T2DM. Liver steatosis displayed by abdominal ultrasound certified the MAFLD diagnosis. A percentage of 49.5% of the studied patients presented MAFLD. Through logistic regression adjusted for gender, age, T2DM duration, lipid-lowering therapy, smoking status, nutritional status, we demonstrated that elevated triglycerides (TG) levels, high non-high-density-lipoprotein (HDL)-cholesterol-to-HDL-cholesterol (non-HDL/HDL) ratio, high atherogenic index of plasma (AIP), and increased Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) had predictive value for MAFLD in patients with T2DM. Furthermore, we calculated the optimal cut-off values for these biomarkers  which can predict the presence of MAFLD in patients with T2DM. To our knowledge, this is the first study to assess the predictive value of the non-HDL/HDL ratio for MAFLD in patients with T2DM.  For many years, fatty liver disease was artificially classified according to the patient’s alcohol consumption into two categories: alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) , the latThe gold standard for the diagnosis of NAFLD is liver biopsy. However, due to the adverse effects that may be associated with this procedure  NAFLD is diagnosed in the clinical practice by the measurement of increased levels of liver enzymes and abdominal ultrasound that evidences liver steatosis ,11.Studies have demonstrated an association between NAFLD and metabolic disorders ), cardiovascular diseases, and gut microbiome disruptions ,12.In this regard, a meta-analysis evidenced that NAFLD  was associated with a twofold increased risk of T2DM development as well as metabolic syndrome development over a 5-year median follow-up . FurtherThis high frequency of metabolic disorders associated with NAFLD led to the newly defined term “metabolic dysfunction-associated fatty liver disease” (MAFLD) . It is iAn early diagnosis of MAFLD in patients with T2DM may lead to an important decrease of morbidity and mortality in these patients . The athThe aim of this study was to assess the usefulness of different blood biomarkers in patients with MAFLD and T2DM.-Group 1: 101 subjects without MAFLD-Group 2: 99 subjects with MAFLDA cross-sectional, non-interventional study was conducted over a period of 8 months (January to August 2022). The study enrolled 200 subjects with T2DM  who wereThe study inclusion criteria used in the study were: adult patients (age ≥18 years) diagnosed with T2DM treated with oral and/or injectable non-insulin antidiabetic agents, recruited from the Outpatient Department of Diabetes, Nutrition and Metabolic Diseases of the Municipal Clinical Hospital “Philanthropy” of Craiova, after reading and signing the study informed consent. The exclusion criteria were: subjects under the age of 18 years, patients diagnosed with type 1 diabetes mellitus and other types of diabetes, patients with T2DM treated with insulin, severe uncontrolled psychiatric pathology, severe comorbidities , patients who underwent bariatric surgery, as well as patients with other conditions that could influence the results of the investigations.All the patients enrolled in the study participated voluntarily and were enrolled only after the informed consent form was read and signed. We conducted the study according to the guidelines of the Declaration of Helsinki, respecting the right to integrity and confidentiality, in accordance with good clinical practice. The patients were given the option to withdraw from the study at any time. The study was performed with the approval of the Ethics Committee of the University of Medicine and Pharmacy of Craiova, Romania (162/19.08.2022).2 (in meters), and the nutritional status of the subjects was classified according to World Health Organization criteria [For all the patients, we recorded demographic data (age and gender), anthropometric measurements , hip circumference (HC)), as well as any relevant data from a physical examination. The body mass index (BMI) was calculated using the formula: BMI = weight (in kilograms)/heightcriteria . The meaVenous blood samples were collected from the study patients, and the following blood tests were performed: fasting plasma blood glucose (FPG), fasting insulinemia, glycated hemoglobin (HbA1c), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lipid profile -cholesterol, TG). AIP, non-HDL-cholesterol, TG/HDL ratio, non-HDL/HDL ratio, and ALT/AST ratio were also calculated. Lipid disorders were defined based on an abnormal lipid profile and/or a lipid lowering treatment.Insulin resistance, a condition associated with MAFLD, was assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) calculated using the formula: (FPG (mg/dL) × fasting insulinemia (mU/L))/405. The visceral adiposity index (VAI), another parameter associated with insulin resistance, was calculated using the following formulas : VAI = [MAFLD was diagnosed by the presence of liver steatosis on abdominal ultrasound. This examination was performed by the same 3 physicians in all the studied subjects.All the data were recorded in an EXCEL database and afterwards analyzed utilizing the Statistical Package for the Social Sciences (SPSS) version 26.0 . Using the Kolmogorov–Smirnov test, we assessed the normality of continuous variables, data with a normal distribution being reported as mean ± standard deviation (SD), while data with an abnormal distribution being reported as median and interquartile range (IQR).p < 0.05.The statistical significance of the differences between groups was evaluated using the Mann–Whitney U test to compare the medians  and the t-test and ANOVA test to compare the means . The area under the receiving operating curve and the logistic regression were used to assess the associations between the studied parameters and MAFLD. The statistical significance threshold was defined as In this study, we found a frequency of MAFLD of 49.5% in patients with T2DM with a median diabetes duration of 6 years. The mean age of the patients enrolled in the study was 56.55 ± 9.32 years, and the patients were almost equally divided according to gender . Given the association between obesity and MAFLD, we studied differences regarding anthropometric parameters in the two study groups. As it can be observed in Regarding the biological parameters , we obseTaking into account that the smoking status can influence lipid parameters, we intended to analyze the relationships between MAFLD and smoking. In our study population, the frequency of actively smoking patients was 18.5% (37 patients), while 23 subjects declared to be former smokers (11.5%). As it is shown in Through backward stepwise regression, adjusting for gender, age, T2DM duration, lipid-lowering therapy, smoking status, BMI, and WC, we demonstrated that only TG, non-HDL/HDL ratio, AIP, and HOMA-IR were independent predictors for MAFLD in patients with T2DM .Furthermore, we also analyzed the AUROC for the biomarkers independently associated with MAFLD and determined the cut-off points suggestive of MAFLD in patients with T2DM for the four independent predictors .Currently, NAFLD is the most frequent cause of chronic liver disease worldwide, being an important risk factor for the development of liver cirrhosis and carcinoma ,31,32. MRecently, a new concept was developed that led to the definition of MAFLD, a term that does not exclude alcohol consumption and other liver disorders associated with steatosis and better reflects the association of liver steatosis with an unhealthy lifestyle and metabolic disorders ,38. TherAnother study published in 2022, analyzing NAFLD defined by liver biopsy in patients with metabolic syndrome, reported a total frequency of liver steatosis of 92.03%, ranging from 71.43% in patients with metabolic syndrome who had a normal weight to 91.84% in overweight patients, to 100% in patients with obesity . InteresIn the last years, there was increasing interest in the search of different biomarkers that could not only non-invasively assess the liver function and establish a MAFLD diagnosis, but also be predictors for the unfavorable evolution of patients with MAFLD. In this regard, recently published studies demonstrated that the AIP is associated with NAFLD and MAFLD both in the general population ,26 and iAnother study conducted by Xie et al. in 7838 adults demonstrated that subjects with AIP in the higher quartile had a statistically higher risk of NAFLD (AUC = 0.810), especially females and at young ages .Although some studies found a better association of AIP with NAFLD in subjects with obesity , the stuIn a recent study including over 100,000 subjects, it was demonstrated that AIP is strongly correlated with NAFLD, with a cut-off value of 0.05 and an AUC of 0.82 . FurtherA recent meta-analysis including data from eight studies involving 81,178 subjects, which assessed the accuracy of AIP in predicting NAFLD, reported controversial results, the authors not finding statistically significant differences regarding the AIP levels between patients with NAFLD and the control subjects . HoweverA previous study conducted by Chen et al.  indicateAnother lipid biomarker that was predictive of MAFLD in the patients with T2DM enrolled in this study was the non-HDL/HDL ratio, which presented a cut-off value of 3.9, associated with 62.6% sensitivity and 32.7% specificity. Recent studies demonstrated that this biomarker is a predictor of NAFLD in the general population. In the prospective study conducted by Wang et al.  in the CIn interpreting these results, it should also be considered the fact that, in our study group, a significant number of patients (89%) presented lipid disorders, with a prevalence slightly higher than that reported for hypercholesterolemia (69.1%) in a study conducted in the Romanian population aged 40–79 years . In ordeThe biomarker with the highest AUROC (0.712) and the highest sensibility (74.7%) for the detection of MAFLD in patients with T2DM was HOMA-IR, with an optimal cut-off value of 2.01. This is not surprising, as insulin resistance is the underlying physiopathological mechanism of MAFLD, obesity, T2DM, dyslipidemia, metabolic syndrome . A recenThe results we presented must be also interpreted in the context of the study limitations. The main limitation of the study is its cross-sectional design, as it was an observational study, a fact that did not allow to define a cause–relationship effect. Therefore, we cannot draw pertinent conclusions regarding the predictive value of the biomarkers that were analyzed in relation to MAFLD and could only describe an association of these parameters with MAFLD at the time of observation. Furthermore, the relatively small number of subjects enrolled in the study represents another study limitation. The patients were randomly included in the study, which affected the mean age of the participants. Another important issue that must be taken into account is the fact that the study results cannot be applied to the general population, as the study was conducted in a limited geographical area.MAFLD is an important comorbidity in patients with T2DM with a high prevalence, which was also confirmed in our study. It is important to find biomarkers that can identify patients with T2DM at risk for MAFLD progression, especially as studies have suggested that NASH is the most frequent manifestation of fatty liver in patients with T2DM. AIP and HOMA-IR, known markers of insulin resistance and cardiovascular disease, were good predictors of MAFLD in patients with T2DM.


Skin image analysis using artificial intelligence (AI) has recently attracted significant research interest, particularly for analyzing skin images captured by mobile devices. Acne is one of the most common skin conditions with profound effects in severe cases. In this study, we developed an AI system called AcneDet for automatic acne object detection and acne severity grading using facial images captured by smartphones. AcneDet includes two models for two tasks: (1) a Faster R-CNN-based deep learning model for the detection of acne lesion objects of four types, including blackheads/whiteheads, papules/pustules, nodules/cysts, and acne scars; and (2) a LightGBM machine learning model for grading acne severity using the Investigator’s Global Assessment (IGA) scale. The output of the Faster R-CNN model, i.e., the counts of each acne type, were used as input for the LightGBM model for acne severity grading. A dataset consisting of 1572 labeled facial images captured by both iOS and Android smartphones was used for training. The results show that the Faster R-CNN model achieves a mAP of 0.54 for acne object detection. The mean accuracy of acne severity grading by the LightGBM model is 0.85. With this study, we hope to contribute to the development of artificial intelligent systems to help acne patients better understand their conditions and support doctors in acne diagnosis.  Acne is one of the most common skin conditions , with acTo receive an acne diagnosis, acne patients traditionally have to visit a doctor’s office, where the dermatologist often observes the affected areas by the naked eye or through a dermatoscope. In combination with other types of information, dermatologists give a diagnosis. This process is dependent on the expertise and experience of the dermatologist ,10. In aMany skin image analysis algorithms have been developed, including algorithms for acne analysis ,14,15. HIn 2018, Xiaolei Shen et al.  proposedIn 2019, Junayed et al.  used theAt the end of 2019, Seite et al. reported an artificial intelligence algorithm based on deep learning for facial acne analysis using smartphone images . The metIn 2021, Yin Yang et al.  developeIn this study, we developed an AI system called AcneDet based on deep learning and machine learning that can automatically analyze facial smartphones images for both tasks: (1) detecting acne lesion objects of four types: blackheads/whiteheads, papules/pustules, nodules/cysts, and acne scars; and (2) grading acne severity based on the IGA scale . Output Data were retrieved from a database of a mobile application called Skin Detective, developed by our team, that is available on both iOS and Android smartphones. Users agreed with the app’s terms and conditions before using the app. Only with user agreement, users’ facial images were stored in the app’s database for AI training. For each user that agreed to share facial images for AI training, three selfie facial images taken at three different angles, i.e., the front, left, and right angles, were collected. During image capturing, distance from the user’s face to smartphone camera and lighting condition were checked by the mobile application. When the distance was within ~20 cm and lighting condition was sufficient, the app would inform the user to capture an image. All captured images were later manually checked for quality. Low quality images were removed. The remaining, a total of 1572 de-identified images of people of various race and skin color, were included in this study. The average size of the images was 720 pixel × 1280 pixel. The images were labeled by four dermatologists (two juniors and two seniors). The dermatologists used LabelBox software to label images. There were two main labeling tasks. One was to draw rectangular bounding boxes to mark the location and type of acne lesions. Four types of acne were labeled, including blackheads/whiteheads, papules/pustules, nodules/cysts, and acne scars. In this task, each image was first labeled using a junior dermatologist. A senior dermatologist then reviewed and corrected the labeling if necessary. Another labeling task was to grade the acne severity for each image based on the results of the first labeling task. Acne severity was graded based on the IGA  scale: 0A total of 41,859 acne lesions were labeled among 1572 images. Among them, acne scars are the most common, with 23,214 (55.46%), and nodular lesions are the least common, with 282 (0.67%). The number and percentage of each acne lesion type are detailed in In terms of acne severity, grade 1 is the most prevalent, with 56.18%, and grade 4 is the least, with 2.16%. The distribution of acne severity grades is shown in The IGA scale was recommended by the FDA to be a static, qualitative evaluation of overall acne severity. It has five levels ranging from grade 0 to grade 4, with grade 0 being clear; grade 1, almost clear; grade 2, mild; grade 3, moderate; and grade 4, severe .Acne object detection model: determine the location and type of acne lesions.Acne severity grading model: grade the overall acne severity of the input image using the IGA scale.In terms of the overall architecture, the system includes two models for two different tasks: The architecture of the system is shown in Acne object detection modelWe chose the Faster R-CNN architecture  with theAcne severity grading modelWe built our acne severity grading model based on the LightGBM algorithm , a tree-We divided the dataset into two sets, as shown in Evaluation metricsWe measured the model performance using two main metrics: the mean Average Precision (mAP) for the Acne object detection model and the area under the receiver operating characteristic (ROC) curve (AUC) for the acne severity grading model.To evaluate the performance of the acne object detection model in detecting acne objects, we used the Average Precision (AP) for each acne type and the mean Average Precision (mAP) for all four acne types. To evaluate the performance of the acne severity grading model, we used the AUC. The ROC curve and AUC of each acne severity grade of 0–4 in the IGA scale area are shown in In addition to the AUC, we also calculated the accuracy, precision, recall, and F1 . The aveWe believe that skin image analysis algorithms will play an important role in future dermatology, where dermatologists will likely be supported by AI systems during the diagnosis and treatment processes. Patients will also benefit from information provided by highly accurate skin image analysis algorithms. Acne is one of the most common skin conditions, affecting 9.38% of the world population, and can cause serious effects, including psychological effects and effects on quality of life. In this study, we developed an AI system called AcneDet that can automatically detect acne lesion objects and grade acne severity with a high level of accuracy.For the acne lesion object detection task, we used a Faster R-CNN architecture to build our model trained on a dataset of 41,859 acne objects. The Faster R-CNN performed reasonably well, with a mAP for all four acne types of 0.54. This mAP is higher than the previous results of Kuladech et al.  and KyunIt is noteworthy that our AI system was trained on a dataset with a larger number of acne objects. Regarding the acne types, the number of acne types in our study is the same as in Kuladech’s study, i.e., four, and is higher than in Kyungseo Min’s study. Our high mAP in acne object detection, in combination with our two-model and two-stage approach, results in the high accuracy of our acne severity grading. Note that, although the number of nodule/cyst lesions in our dataset is extremely small , our model was able to detect nodule/cyst lesions with a mAP of 0.68. This can be explained by the fact that nodule/cyst lesions often have an extremely distinct color  and a substantially larger size. Thus, our model could learn to recognize them better. By contrast, whereas the number of acne scars accounted for 55.46% of the total number of acne objects, the mAP was only 0.44. We attribute this to the fact that most of the acne scars are small with a color not much different from the surrounding skin. Therefore, although the training examples of acne scars were abundant, the model had a hard time learning to recognize them. We believe the same applied to blackhead/whitehead lesions, which accounted for 37.47% of the total number of acne objects, but had the lowest mAP of 0.4.To grade the severity of the acne, we used the output of the acne object detection model, specifically the number of each acne type, as input for the LightGBM-based acne severity grading model. Using this approach, we achieved an average accuracy of 0.85 for all five grades. The accuracy of our acne severity grading model is compared with the accuracy of previous studies in Notably, the images used in our study were collected through smartphones. Therefore, they are lower in quality and less clinically informative than images obtained through digital cameras or a dermatoscope, making their analysis more challenging. However, we were able to achieve a mAP of 0.54 and an accuracy of 0.85, which are higher than those of previous studies, which is a good foundation for future studies. Given the high number of people with acne, the shortage of dermatologists in many parts of the world, and the popularity of smartphones, an AI-powered smartphone app that can quickly and accurately analyze smartphone-captured facial images and help users to understand their acne conditions better, e.g., severity and acne type, is potentially useful. In the case of high grade/severe acne, the app could recommend and connect acne patients with remote dermatologists for teledermatology. In the case of low grade/mild acne, the app could recommend skincare products, diet, and lifestyle adjustments, etc. so that acne patients can achieve an acne-clear or near acne-clear skin condition. Through this study, we hope to contribute to the development of acne analysis algorithms to help acne patients better understand their conditions and support doctors in acne diagnosis.In this study, we developed an AI system called AcneDet for acne object detection and acne severity grading, using facial images captured by smartphones. The AcneDet system consists of two models for two different tasks: an acne object detection model using Faster R-CNN-based deep learning and an acne severity grading model based on LightGBM machine learning. Four types of acne could be detected: blackheads/whiteheads, papules/pustules, nodules/cysts, and acne scars. The test results show that the acne object detection model achieved a mAP of 0.54. The output of the acne object detection model was used as input for the acne severity grading model, which achieved an accuracy of 0.85. In the future, we plan to collect more data to improve the mAP and accuracy of the system, and apply semi-supervised and unsupervised learning techniques to reduce the labeling workload. In addition, we are also interested in developing AIs that could perform other tasks such as identifying acne patients that need immediate initiation of treatment to avoid scarring. We believe that such AIs would need to consider other factors such as age and sex, etc., in addition to facial images, to make accurate predictions.


The impact of elevated resting heart rate on early-stage renal dysfunction, manifesting as microalbuminuria, in hypertension is unclear. This study aimed to analyze the association between resting heart rate and microalbuminuria in patients with hypertension according to their blood pressure status. In addition, the effect of antihypertensive agents on this relationship was evaluated.P < 0.001). However, the association between elevated resting heart rate and microalbuminuria was not significant in patients with uncontrolled hypertension . The OR of the indirect effect of β-blockers on the risk of microalbuminuria incidence through heart rate was 0.926 , while the direct effect was 1.374 . Similarly, dihydropyridine calcium channel blockers were associated with a higher prevalence of microalbuminuria , but the association between non-dihydropyridine calcium channel blockers and microalbuminuria was not significant .We searched the National Health and Nutrition Examination Survey for eligible participants from 2009 to 2018. Data on key parameters such as age, sex, blood pressure, heart rate, albumin creatinine ratio, and medication were collected for analysis. Subsequently, participants were classified according to the heart rate quartile and blood pressure status for subgroups assessment. A total of 5,692 participants were enrolled in this study. After adjusting the confounding factors, there was a linear association between resting heart rate and microalbuminuria in patients with hypertension (OR 1.184 [per 1 SD]; 95% CI: 1.101, 1.274; Elevated resting heart rate is associated with a high risk of microalbuminuria in untreated patients and patients with controlled hypertension. Although there is a linear association between heart rate and microalbuminuria, the use of β-blockers exhibits a significantly increase in the prevalence of microalbuminuria in hypertension. Likewise, dihydropyridine calcium channel blockers may increase the risk of microalbuminuria in hypertension. Resting heart rate (RHR) is considered a practical marker of health and longevity. The impact of RHR on pathophysiological processes has been widely discussed from clinical and fundamental perspectives over decades. High RHR increases the risk of cardiovascular diseases, cancers, and all-cause mortality . AutonomMAU is a potent indicator for early-stage glomerular diseases and is considered an early marker of cardiovascular diseases . The preWe used baseline information from the National Health and Nutrition Examination Survey (NHANES) from 2009 to 2018. The NHANES is a population-based cross-sectional survey conducted by the National Center for Health Statistics, a part of the Centers for Disease Control and Prevention. The program aims to evaluate the health condition of citizens in the United States and uses a stratified, multistage, and cluster sampling design for selecting the target population. Approximately 5,000 participants from all parts of the country are enrolled in this survey each year.A total of 48,981 participants were assessed from 2009 to 2018. Among them, 41,202 individuals without hypertension; 373 individuals with irregular pulse; 82 individuals aged <20 years; and 1,320 individuals with missing important data such as those on marital status, educational level, poverty income ratio, body mass index (BMI), albumin, and total protein were excluded. Finally, 5,692 individuals met the inclusion criteria and were eligible for this study .Health examination was performed at Mobile Examination Centers (MECs) or homes if the participants were ≥50 years old or unable to come to the MEC. Radial pulse was measured for 30 s, which could be translated to the RHR by doubling the value. A standard protocol was used by well-trained practitioners for blood pressure measurement. Briefly, at least 5 min were required before measuring the blood pressure of participants. Then, participants were seated quietly, and an appropriate blood pressure cuff was chosen based on their mid-arm circumference. Blood pressure was measured three times, and the average systolic blood pressure (SBP) and diastolic blood pressure (DBP) were calculated.Participants were diagnosed with hypertension if their average SBP was ≥140 mmHg, their average DBP was ≥90 mmHg, or they were undergoing treatment with antihypertensive agents. Moreover, urine samples were collected for measuring the urinary albumin level, creatinine level, and albumin creatinine ratio (ACR). MAU was defined as an ACR of 30–300 mg/g according to the Kidney Disease: Improving Global Outcomes . To evalIn addition, data on demographics, education, marital status, and wealth were collected from the computer-assisted personal interviewing system. Individuals who were married, widowed, or separated were considered married individuals. Questionnaires were designed for identifying lifestyles. Moreover, some serum biochemical indicators, including cholesterol and creatinine, were assessed. The CKD–EPI equation was used for calculating the estimated glomerular filtration rate (eGFR) .Continuous variables, expressed as means ± standard deviations (SDs) for normally distributed data or as medians (interquartile range), were compared using one-way analysis of variance or the Kruskal–Wallis H test, as appropriate. Categorical variables, expressed as numbers (percentages), were compared using the chi-square test.p = 0.05 and p = 0.10, respectively. Heart rate was included in the models as a continuous variable (per 1 SD) and a categorical variable (the lowest heart rate quartile as the reference), respectively. P for trend was tested by treating heart rate quartiles as ordinal variables in the analyses.The odds ratio (OR) for risk of MAU associated with heart rate was calculated using the logistic regression model. The unadjusted model was first used, followed by multivariate-adjusted model. The candidate confounding factors included established MAU risk factors and variables that were significantly different between heart rate quartiles, including age (continuous), sex , race , marital status (married/other), BMI (continuous), poverty income ratio (continuous), current drinking status (yes/no), SBP (continuous), total protein (continuous), eGFR (continuous), total cholesterol (continuous), high-density lipoprotein cholesterol (continuous), diabetes (yes/no), β-blockers (yes/no), and calcium channel blocker (CCB) (yes/no). Forward stepwise selection was performed with entry and exit criteria set at To investigate the dose–response relationship between risk of MAU and continuous heart rate, the restricted cubic splines in logistic regression was used with three knots  and 62 per minute (approximately the first knot) as the reference group.2 were excluded for sensitively analysis.Subgroups analyses were performed using antihypertensive therapy, including angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin-receptor blockers (ARBs) (yes/no), β-blockers (yes/no), CCB (yes/no), diuretics (yes/no). The effect of the interaction between antihypertensive therapy and heart rate on the risk of MAU was tested by comparing the ORs from different subgroups using Z statistic . ParticiP-value of < 0.05 on the two-sided test was considered statistically significant.Finally, mediation analysis was performed using PROCESS in SPSS to evaluated the effect of heart rate (mediator) on the association between β-blockers (independent variable) and MAU (dependent variable) . All staIn total, 5,692 participants were included in the final analyses. The mean age was 60.8 ± 14.0 years, and 46.1% participants were men. The distribution of heart rate was approximately normal . The meaP < 0.001). Compared to participants in the first heart rate quartile, those in the third  and fourth  heart rate quartile had a significant higher risk of MAU. The association between heart rate and MAU remained significant among participants with controlled and untreated hypertension, but not in those with uncontrolled hypertension. The dose–response relationship showed that the non-linear relationship between heart rate and MAU was not significant . Thus, we assessed the total effect of β-blockers on the risk of MAU, which indicated a detrimental role of β-blockers .Mediation analysis was further performed to explore the possible effect of heart rate on the association between β-blockers and MAU . A signiP = 0.454). On the contrary, DHP was related to an increased prevalence of MAU   and MAU as well . The ass= 0.013) .In the present study, the relationship between elevated RHR and increased risk of MAU in patients with hypertension was significant. Except in uncontrolled hypertension, the detrimental effect of RHR on the incidence of MAU was prominent in different blood pressure statuses, which was minimally affected by antihypertensive agents. Among the four types of antihypertensive agents, β-blockers could indirectly reduce the risk of MAU due to its negative chronotropic effect, while the total effect of β-blockers on the prevalence of MAU was detrimental. Similarly, dihydropyridine calcium channel blockers were associated with a higher prevalence of microalbuminuria, but the association between non-dihydropyridine calcium channel blockers and microalbuminuria was not significant.In the Framingham study, elevated RHR strongly predicted a high risk of all-cause and cardiovascular mortality in both men and women with hypertension . The oveCuspidi et al. pointed out the prevalence of MAU was not affected by the increase in office or 48-h heart rate . SimilarN = 916) should be taken considered.From our perspective, the prevalence of MAU in participants with uncontrolled hypertension was not affected by elevated RHR, possibly due to the change in the relative proportion of MAU and macroalbuminuria. In the uncontrolled group, the relative proportion of macroalbuminuria increased and the elevated RHR was further associated with a high risk of macroalbuminuria (data not shown). Furthermore, the small sample size of uncontrolled group , it still indicated a harmful role of β blockers in the MAU development. It was found that DHP calcium channel blockers increased the risk of MAU. Given that DHP calcium channel blockers can contribute to the elevation of RHR, it may make sense why MAU became more prevalent.In our study, individuals benefited from the heart rate-lowering treatment because of the linear relationship between MAU and RHR. However, β-blockers may indirectly reduce the high incidence of MAU by decreasing the RHR, although the direct effect of β blockers treatment was detrimental. Interestingly, another heart rate-lowering agents, non-DHP calcium channel blockers, had minimal effect on the association between RHR and MAU. Although the insignificant effect of non-DHP may be explained by the limited users of non-DHP  can be found below: https://www.cdc.gov/nchs/nhanes/about_nhanes.htm. The patients/participants provided their written informed consent to participate in this study.The studies involving human participants were reviewed and approved by XW and YL collected the data for analysis. XP evaluated the statistical significance of related data and wrote this manuscript. YR contributed to the manuscript revision. NL designed the study and interpreted results. All authors read and approved the final manuscript.This work was supported by the National Science Foundation of China (Grant Nos. 81870244 and 81770318) and the Natural Science Foundation of Beijing (Grant No. 7192051).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Infertility is a topic of growing interest, and female infertility is often treated with gonadotropins. Evidence regarding comparative safety and efficacy of different gonadotropin formulations is available from clinical studies, while real-world data are missing. The present study aims to investigate effectiveness and safety of treatment with different gonadotropin formulations in women undergoing medically assisted procreation treatments in Latium, a region in central Italy, through a real-world data approach.A retrospective population-based cohort study in women between the ages of 18 and 45 years who were prescribed with at least one gonadotropin between 2007 and 2019 was conducted. Women were enrolled from the regional drug dispense registry, and data on their clinical history, exposure to therapeutic cycles ), and maternal/infantile outcomes were linked from the regional healthcare administrative databases. Multivariate logistic regression models were applied to estimate the association between exposure and outcomes.REC adj = 1.06, 95% CI: 1.01–1.12; RRCBD adj = 1.17, 95% CI: 1.11–1.24). Maternal outcomes occurred in less than 5% of deliveries, and no significant differences between treatments were observed . Regarding infantile outcomes, similar results were obtained for different gonadotropin formulations .Overall, 90,292 therapeutic cycles prescribed to 35,899 women were linked to pregnancies. Overall, 15.8% of cycles successfully led to pregnancy. Compared to extractives, recombinant and combined treatments showed a stronger association with conception rate (RREfficacy and safety profiles of REC proved to be similar to those of EXT. Regarding the efficacy in terms of conception rate and birth rate, protocols using the combined approach performed slightly better. Outcomes related to maternal and infantile safety were generally very rare, and safety features were overlapping between gonadotropin formulations. According to the World Health Organisation, infertility affects up to 15% of reproductive-aged couples worldwide . Among cThe assisted reproductive technology (ART) was born in order to help couples with infertility issues in having a baby. The first treatments of in vitro fertilisation used the spontaneous cycle of the women, with the retrieval of only one oocyte. Further studies have shown that it is possible to induce ovulation by administrating gonadotropins (Gonas) during the menstrual cycle, in order to obtain a higher number of oocytes.The availability of different Gona formulations in terms of extractive (highly purified) urinary Gonas (EXT) and recombinant Gonas (REC), and the authorisation of follitropin alpha biosimilars make the choice of the individual treatment complex.Evidence from clinical studies regarding different efficacy and safety profiles of urinary versus recombinant formulations is controversial: some authors report findings which favour one formulation over the other: a paper by Out et al.  states fA recent review of the results of comparative clinical trials, Cochrane analyses, and meta-analyses concluded that the available evidence from the published literature does not show significant differences between urinary and recombinant gonadotropins in terms of safety and efficacy Patki et al. . In the Since their introduction, recombinant Gonas have steadily gained market, outranging the use of urinary formulations. While in the first years, this might have been partly explained by better patient acceptability due to a simpler administration (subcutaneous) with respect to the urinary formulations (intramuscular), and this does not hold true in recent years, as almost all urinary formulations are administered subcutaneously now.Initial disadvantages of recombinant formulations due to higher costs with respect to extractives have been mainly overcome after the introduction of the recombinant FSH biosimilar, which led to price reductions of the originator as well.To date, evidence from real-world settings is missing, but might add useful information for clinicians, when choosing the treatment protocol for their patients, and for policy makers, when deciding on recommendations and targeting policies with respect to refunding the different formulations.The present study tries to close the gap on evidence under real-world conditions and provide evidence through an observational population-based approach, aiming to investigate the use patterns of the different Gona formulations and perform a comparative evaluation of the risk–benefit profile of different formulations of Gonas used for ovarian stimulation in female infertility, to answer to the question if recombinant formulations present advantages over urinary formulations.We performed a retrospective observational population-based study.The present study used data from the administrative healthcare databases of the Lazio region, namely: the regional healthcare assistance file, which contains demographic and residence information about all residents alive and registered in the regional health service at a specific year’s date. The regional drug dispensing registry (Pharm). The registry is limited to drugs dispensed to outpatients and reimbursed by the healthcare system. Drugs are identified by the national drug register code, which refers to the international Anatomical Therapeutic Chemical Classification System (ATC). Individual patient data and the date of drug claim are reported for every prescription. The hospital information system (HIS), which provides, for every hospitalisation in a regional hospital, information on patients’ demographic characteristics, discharge diagnoses, and procedure codes according to the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The regional mortality information system (MIS), providing information on demographic characteristics, as well as date, place, and cause of death (codified by ICD-9 codes). The regional database of birth certificates , which cFrom administrative healthcare databases, all drug claims of Gonas to women enrolled in the regional healthcare system in the 24 months before each drug claim (characterisation period), and aged 18–45 years were retrieved for the period 2007–2019. Women treated with human chorionic gonadotropin (HCG) only or with more than 9 treatment cycles were excluded. Our unit of observation was the treatment cycle, which was defined as a 21-day mobile time window , referring to patients treated between 2016 and 2019, allowed to evaluate the concordance between cycles registered in the clinical dataset and those retrieved from administrative data, for the same woman, in particular in relation to the definition of the therapeutic cycle .For each treatment cycle, the observation started on the day of the first drug dispensing and ended at the first occurrence of study outcome, death, or loss to follow-up.From HIS and CeDAP, pregnancies, both ending in abortion and in childbirth, were retrieved and linked with cycles within the 9 months from the last Gona claim.From CeDAP, the following parameters were considered: number of births, caesarean birth, twin births, sex ratio, preterm births, weight/length/cranial circumference at birth, Apgar score, still birth, and admission to emergency neonatal therapy , a second cycle (categorised in the three exposure categories), or the end of treatment. The same was investigated for those women undergoing a second treatment cycle, and so on.The association between exposure to EXT, REC, CBD, and each outcome was investigated applying log-binomial regression models.K = 0.9017). On average, women underwent 2.51 cycles and each cycle was defined based on 2.65 prescriptions. Accounting for the availability of data from birth certificate up to the end of 2018, and allowing 12 months of observation, our cohort included a total of 90,292 cycles related to 35,899 women , reaches pregnancy followed by abortion (black category), or continues the treatment with further attempts (same therapy or switch). In the white category, “loss to follow-up,” we observe women for whom we have no more information. The same setting is repeated for subsequent observations.The 2nd observation shows that 15.2% of women successfully achieve a pregnancy at first attempt (whether it results in a delivery or an abortion), with differences between Gona types, i.e., 15.9% for women exposed to REC, 12.7% to EXT, and 17.0% to CBD.Considering the entire pathway cumulatively, even beyond the 4th event, 28.4% of women overall achieve a pregnancy with delivery (this accounts for 11.6% of therapeutic cycles).23.3% of women are lost to follow-up after the 1st observation. Most women who achieve pregnancy over time do not encounter additional information from information systems.Table The conception rate, i.e., successful achievement of pregnancy, independently from childbirth, was highest for CBD cycles (16.7%) and lowest for EXT (14.4%). On the contrary, the number of cycles needed to achieve pregnancy was higher in the CBD group (19.1%).Regarding the effectiveness of therapeutic treatment, REC cycles, compared to EXT, showed a higher association with pregnancy  and with childbirth . The association was stronger when we compared the effectiveness of CBD vs EXT cycles: conception rate RR adj 1.17, 95% CI: 1.11–1.24 and birth rate, RR adj 1.19, 95% CI: 1.12–1.27.The characteristics of deliveries related to treatment with different Gona formulations were similar. Generally, multiple births are considerably more frequent in medically assisted pregnancies compared to the overall population, independently of the type of Gona used (18.1% respect to 1.8% in the Lazio birth register (13CeDAP)). Caesarean section was applied in almost 64% of deliveries, about one-fifth of children were born at preterm, and 11% were treated in emergency neonatal therapy. We observed very few stillbirths (0.2%), and most children were in excellent health conditions at birth (97.1%). The three maternal outcomes considered, occurred rarely , with similar proportions across exposure categories.REC: 0.93, RRCBD: 0.94), for CBD treatments regarding caesarean section (RRCBD: 0.93), and for REC treatment with respect to admission to neonatal emergency therapy (RRREC: 0.87). For all maternal outcomes, point estimates were slightly higher for REC and CBD cycles, but no significant differences were observed. At the same time, CBD and REC cycles were associated with a slightly increased risk for preterm (RRREC/CBD: 1.03) and multiple births .Results of the regression models investigating maternal and infantile outcomes are presented in Figs. In the present study, efficacy and safety profiles of recombinant gonadotropins have proved to be like those of extractive gonadotropins. Regarding the efficacy in terms of conception rate and birth rate, protocols using the combination of both formulations were found to perform slightly better. Outcomes related to maternal and infantile safety were generally very rare, and safety features were very similar between Gona formulations.Our findings of overlapping safety and efficacy are in line with the available evidence provided by clinical studies, reviews, and meta-analyses. The higher efficacy of the mixed approach with respect to monotherapy confirms findings reported in the Italian study Pacchiarotti et al.  based onAs contextual information, the average number of therapeutic cycles per woman is in line with the refund policies in use in the Lazio region.The added value of our study stems from its nature, being, to our knowledge, the first observational approach performed in a real-world setting and comprising treatments offered at the level of the resident population over a long period, granting a robust number of observations. This was possible thanks to the availability of administrative healthcare data and the collaboration in a multidisciplinary approach, comprising epidemiologists, statisticians, and specialist clinicians.Still there are some limitations to be mentioned. Most importantly, our data do not comprise information on treatment cycles, and consequently, we had to develop an algorithm for its approximation. Fortunately, we had the opportunity to use data from a clinical setting which allowed to evaluate the performance of our algorithm, and this comparison proofed satisfactory concordance about the categorisation of the cycle.Another weakness of our data stems from the lack of information regarding clinical details, and lifestyle data. This may have multiple impacts: first, it limits the choice of the outcomes we can measure. For example, our information on spontaneous abortions is restricted to cases requiring access to emergency room or hospital. This probably implies an underestimation of the conception rate. Similarly, intermediate treatment results, such as oocyte maturity or embryo cleavage, cannot be considered. Second, outcomes may be sensitive to different doses of gonadotropins, an information which is not currently available from our administrative data. Third, we do not have the possibility to distinguish different levels of medically assisted procreation with the right accuracy level. Last, we cannot account for some potential confounders such as BMI, alcohol consumption, or smoking. Yet, we do not expect any differences in the prevalence of these latter variables between treatment regimens.To date, there are no agreed treatment guidelines which favour one gonadotropin formulation over the other, enhance combined treatment, or recommend specific doses. Treatment choices are rather made by the specialist physician upon a complex evaluation of the specific case, which accounts not only for clinical aspects of the woman to be treated but also for parameters referring to the partner. These considerations cannot be retrieved in our data and leave space for indication bias.Taken all these considerations together, we believe that the findings of our study add an important piece of information to the complex field of infertility treatment with gonadotropins, both for clinicians and health policy makers.Future research might investigate the cost–benefit profiles of the different Gona formulations, to complete the picture.