CircRNAs acting as miRNA sponges play important roles in the growth process of animal individuals. The prolificacy trait of goats is involved in many pathways, however, the variation of circRNA expression profiles in the different phases of the estrus cycle at high and low fecundity groups is still unknown. Here, we analyzed the circRNA profiles of ovarian tissues among high and low fecundity groups in the follicular phase (HF vs LF), high and low fecundity groups in the luteal phase (HL vs LL), and high and low fecundity in the whole estrus cycle (HF vs HL and LF vs LL) using RNA-seq. A total of 283 (114 upregulated and 169 downregulated), 559 (299 upregulated and 260 downregulated), 449 (254 upregulated and 195 downregulated), and 314 (210 upregulated and 104 downregulated) differentially expressed (DE) circRNAs were screened in HF vs LF, HF vs HL, HL vs LL, and LF vs LL groups, respectively. Enrichment analysis suggested that the targeting of DE circRNAs was mainly enriched in oocyte meiosis, the GnRH signaling pathway, and estrogen signaling pathway. After integrating our previous study of miRNA-seq, there were 56 miRNAs that could target to 192 DE circRNAs, including the miR-133 family (including miR-133a-3p and miR-133b), miR-129-3p, and miR-21, which also had important influence on the prolificacy trait of goats. Then, 18 circRNAs with coding potential were obtained by four software predictions, and 9 circRNAs were validated by RT-qPCR. Together, circRNAs play a key role in the prolificacy trait and the transformation of the follicular phase to the luteal phase in the estrus cycle of goats. The crucial correlated miRNA-circRNA and circRNA-mRNA regulator networks and pathways in the prolificacy trait and the transition from the follicular phase to the luteal phase were identified. Furthermore, the function and mechanism of DE circRNAs were predicted by functional enrichment and coding potential analysis. These analyses will provide a basis for the molecular mechanism of follicular development and the transition from the follicular phase to the luteal phase in goats. Our purpose was to clarify the molecular mechanisms of follicular phase regulation of goat ovaries and to provide a research basis for further study on the interactions between circRNAs, miRNAs, host genes, and signaling pathways for prolific traits and the transition from the follicular phase to the luteal phase in goats. This study also provides basic data for Yunshang black goat prolificacy trait-related candidate gene screening and the conservation of native breed resources.n = 5, average kidding number ≥3) and low fecundity goats  based on their kidding number records. All individuals were fed in the same environment and had free access to water in a Yunnan Province goat farm. The ovaries at the follicular phase of the estrous cycle were collected from animals with high (≥3) and low (≤ 2) numbers of kids, frozen immediately in liquid nitrogen, and stored at −80°C for conservation.Twenty she-goats (2–3 years old), no significant differences in age and weight, were selected and grouped into four groups, including high fecundity goats in the follicular phase (HF) and in the luteal phase (HL)  was used to check the purity of the RNA samples. The Qubit® 2.0 Fluorometer  with the Qubit® RNA Assay Kit was used to measure the concentration of RNA. The integrity of RNA was assessed by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2,100 system .According to the manufacturer’s protocol, total RNA was isolated from ground ovarian tissue powder using TRIzol reagent . The degree of degradation and contamination of RNA samples was monitored by 1% agarose gels, and the NanoPhotometer® Ultra™ Directional RNA Library Prep Kit for Illumina®  was used to deplete RNA from rRNA and generate the sequencing libraries in accordance with the manufacturer. In brief, the divalent cations in NEB Next First Strand Synthesis Reaction Buffer (5X) under elevated temperature were used to perform fragmentation. The random hexamer primer and M-MuLV Reverse transcriptase (RNaseH-) were used to synthesize the first-strand cDNA. Subsequently, DNA polymerase I and rnase H were used to synthesize second-strand cDNA. The dUTP replaced the dNTPs with dTTP in the reaction buffer. The active exonuclease/polymerase converted the remaining overhangs into blunt ends. The 3′-ends of DNA fragment adenylation and the NEB Next Adaptor with hairpin loop structure prepared for hybridization were ligated. The AMPure XP system  was used to purify the library fragments and to select the cDNA fragments with different lengths. PCR was then performed by adding 3 μl of USER Enzyme  to size-selected, adaptor-ligated cDNA for 15 min at 37°C, followed by 5 min at 95°C. PCR was subsequently performed with Phusion High-Fidelity DNA polymerase, Index (X), and Primer universal PCR primers. Finally, the Agilent Bioanalyzer 2,100 system was used to assesses library quality, and the AMPure XP system was used to purify the products.The 3 μg of RNA per sample was used as the starting material for the RNA samples. Initially, the Epicentre Ribo-zero™ rRNA Removal Kit  was used to remove rRNA, and ethanol precipitation was used to purify the rRNA free samples. Secondly, the NEBNextCircRNAs were identified by find_circ and CIRI2 . The folpadj values of < 0.05 were considered to be significantly differentially expressed.Quantification was performed based on reads that mapped across the circularized junctions and were normalized using SRPBM (number of circular reads/number of mapped reads (units in billion)/read length) . Differehttps://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2go.gz. For KEGG annotation, the KOBAS software was used to test the statistical enrichment of host genes with DE circRNAs  was used to establish a standard curve. The following steps were applied for RT-qPCR: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. The data were analyzed using the 2-∆∆Ct method . We constructed the PPI network by extracting a list of host genes from the sheep database. In this study, the online tool STRING was used analyze the PPI of host genes, and PPIs with a score of >700 were selected as significant interactions.As the goat database is not complete, we used the database of sheep, which has a high homology with goats, as a reference. The protein-protein interaction (PPI) analysis of host genes of DE circRNAs was conducted according to the STRING database (Organism: Capra hircus genome was 90.80% (from 87.16 to 94.36%). After mapping the reference sequence, we identified 6,034 circRNAs. The spliced length distribution of circRNAs was primarily in the range from 200 bp to 10,000 bp , the high fecundity group in the luteal phase , the low fecundity group in the follicular phase , and the low fecundity group in the luteal phase . In 0,000 bp . CircRNA0,000 bp . The cir0,000 bp . In addi0,000 bp .p-value < 0.05, 283 DE circRNAs (114 upregulated and 169 downregulated), 559 DE circRNAs (299 upregulated and 260 downregulated), 449 DE circRNAs (254 upregulated and 195 downregulated), and 314 DE circRNAs (210 upregulated and 104 downregulated) were identified in the HF vs LF, HF vs HL, HL vs LL, and LF vs LL comparisons, respectively  subclasses. Moreover, in the HF vs LF comparison, terms were related to cell part and cell terms. There were 3,643 enriched terms in this comparison, with 811 terms significantly enriched. In the HL vs LL comparison, we found 4,377 terms were enriched, 571 of which were significantly enriched. Of which, a lot of host genes for DE circRNAs were enriched in the cell (GO:0005623), cell part (GO:0044464), and cellular process (GO:0009987) subclasses, along with other subclasses . Our resComparison of the host genes and KEGG pathway analysis showed that the host genes were annotated into 154, 175, 157, and 183 pathways for the HF vs LF, HL vs LL, LF vs LL, and HF vs HL comparisons, respectively . Of thesTo further identify the key circRNAs in the prolificacy trait of goats, the binding sites of miRNA-bound circRNAs were predicted by miRanda software. We found that 56 miRNAs bind to 192 DE circRNAs. Of note, our result showed that the relationship of miRNA-circRNA showed the one-to-many and many-to-one characteristic . miR-133Among the four groups, in total, 4083 DE circRNAs were calculated with scores greater than 0.5 by IRESfinder software. Furthermore, four software programs were used to predict the coding potential of the above circRNAs, and 3,257 circRNAs were found. Finally, 18 circRNAs were identified with coding potential by the four software programs mentioned above .The PPI network of the host genes was constructed by Cytoscape using the analysis result of host genes from the STRING database . For theThe process of reproduction is very complex. In particular, the study of the goat reproductive molecular mechanism is still in its infancy and there are still many unanswered questions. The reproduction traits in goats are governed by the precise regulation of genes, regulatory elements, and other factors, such as spatiotemporal expression, transcriptional regulation, and feedback mechanisms . InvestiHere, the high-throughput sequencing of circRNAs in the ovaries from goats with different kidding number records was performed in different estrus phases. The circRNAs were distributed widely among chromosomes 1–11 in the four comparisons, and which showed that circRNAs had complexity and functional diversity . The majIn the HF vs LF comparison, circIGF2R, circMAPK8, circMYH10, and circPTEN were associated with the prolificacy trait. The IGF2R locus contains a highly abundant set of circIGF2R circRNAs, that showed multiple forms of different exon combinations in mice and plays significant roles in mammalian reproduction . MAPK8 mAmong the DE circRNAs of the comparison between the estrus phases (HF vs HL and LF vs LL), we found that the host genes of circLDLR, circMAPK1, circSMAD1, and circSMAD2 were LDLR, MAPK1, SMAD1, and SMAD2, respectively. SMAD1 and SMAD2 are members of the SMAD family, which is related to signal transduction in the TGF-β superfamily and the inhibition of BMP activity, and they have been thought to control follicular selection by balancing luteinizing hormone receptor (LHR) transcription . In addiPrediction of circRNA-miRNA binding relationships was completed by the miRNA target gene prediction method. In total, binding was predicted for 56 miRNAs, including the miR-133 family (such as miR-133a-3p and miR-133b), miR-129-3p, and miR-21. Several of these miRNAs have been shown to be closely related to ovarian development and the prolificacy trait. Differential expression of miR-133 occurs during meiotic maturation of oocytes . MiR-129As circRNAs have a loop-like structure consisting of exons, some circRNAs consist of an open reading frame and IRES. Therefore, researchers hypothesized that numerous circRNAs could be translated into protein in certain conditions . IRESfinIn summary, 283 (114 upregulated and 169 downregulated), 559 (299 upregulated and 260 downregulated), 449 (254 upregulated and 195 downregulated), and 314 (210 upregulated and 104 downregulated) DE circRNAs were identified in HF vs LF, HF vs HL, HL vs LL, and LF vs LL comparisons, respectively. Enrichment analysis showed that a number of host genes with DE circRNAs were enriched in oocyte maturation, ovarian steroidogenesis, oocyte meiosis, and the GnRH signaling pathway, which have important impacts on follicular growth and development. In addition, 192 DE circRNAs were found to target 56 miRNAs, 18 circRNAs had coding potential, and 69 PPI pairs were found. These results provide fundamental data on the molecular mechanisms underlying the prolific trait in goats.


Energy drinks (EDs) are non-alcoholic beverages containing high amounts of caffeine and other psychoactive substances. EDs also contain herbal extract whose concentration is usually unknown. EDs can have several adverse effects on different organs and systems, but their effects on the gastrointestinal (GI) tract have been poorly investigated. To determine the acute effects of EDs on the GI tract, we administered EDs, coffee, soda cola, or water to Sprague–Dawley rats  for up to five days, and analyzed the histopathological changes in the GI tract. Data were compared among groups by Kruskal–Wallis or Mann–Whitney tests. We found that, while EDs did not cause any evident acute lesion to the GI tract, they triggered eosinophilic infiltration in the intestinal mucosa; treatment with caffeine alone at the same doses found in EDs leads to the same effects, suggesting that it is caffeine and not other substances present in the EDs that causes this infiltration. The interruption of caffeine administration leads to the complete resolution of eosinophilic infiltration. As no systemic changes in pro-inflammatory or immunomodulating molecules were observed, our data suggest that caffeine present in ED can cause a local, transient inflammatory status that recruits eosinophils.  Energy drinks (EDs) are non-alcoholic beverages containing a high amount of caffeine and several other psychoactive substances, including the amino acid taurine, the glucose derivative glucuronolactone, and herbal extracts such as ginseng and guaranà , often present in uncertain concentrations . EDs werAlthough multiple effects of caffeine or caffeine-containing beverages have been observed on the GI tract, little attention has been paid to the possible contribution of these beverages to inflammation or the activation of immune cells. Recently, a study reported that EDs consumption can exert an anti-inflammatory effect in vitro, by reducing the release of Interferon (IFN)-γ by endothelial cells, the secretion of Interleukin (IL)-6 and Tumor necrosis factor (TNF)-α by cell lines of monocytic origin, and can reduce experimental colitis in rats, suggesting that EDs can have an effect on inflammation and immune response . In thisTwo sets of experiments were performed in vivo on rats. In the first set of experiments, a total of twenty-eight male adult Sprague–Dawley rats, not genetically related, were used. Sprague–Dawley rats were chosen because of their human-like digestive physiology. The rats were fed a dry standard laboratory diet, and their weights ranged from 230 to 250 g. They were housed in cages for a 12 h day/night cycle with controlled humidity and temperature. The animals were randomly divided into 4 groups, each composed of 7 animals receiving different sweetened beverages containing caffeine and caffeine-like substances. Group A: control group, receiving water with a known composition. Group B: commercial ED for 5 days. Group C: commercial soda cola for 5 days. Group D: commercial sweetened coffee for 5 days. The amount of each beverage was calculated considering the intake of caffeine ingested by an adult man of 70 kg of body weight consuming two cans/day of ED or soda cola, or 4/5 cups of espresso. The animals were sacrificed at the end of the treatment using the hypercapnia technique. In the second set of experiments, adult male rats, weighing 230–250 g and fed with a standard laboratory diet, were randomly divided into 2 groups, each one consisting of 2 subgroups (N = 5), one supplemented with water , the other with coffee (subgroup B). Both the groups received the treatment for 5 days followed by 5 days of observation before the sacrifice for the first group (group 1) and 10 days for the second (group 2). The amount of coffee was calculated in order to provide the same amount of caffeine taken by the rats fed with ED in the first experiment. All of the animals received human care in compliance with the European rules of experiments in animals (European Directive 2010/63/EU). The study was conducted according to the guidelines of the Declaration of Helsinki. The animal protocols were approved by the the Ethics Committee of Italian Ministry of Health, according to Italian law protecting animals used for scientific purposes . At autopsy, the organs connected with beverage contact, absorption, and metabolism, namely the entire GI tract, including the esophagus, stomach, duodenum, small intestine and colon, liver and pancreas, the immunocompetent organs, such as the spleen, thymus, and retro-peritoneal lymph nodes, and the thoracic organs, i.e., heart and lungs, were macro- and microscopically examined, and seriated specimens were taken from the stomach and intestine.Besides hematoxylin/eosin (H&E), Pagoda red  staining was performed to quantify eosinophils, as described by Trani et al. . A porti® platform , using a “Luminex Performance Human High Sensitivity Cytokine Magnetic Panel A”. The following soluble factors were quantified: IL-1β, IL-2, IL-4, IL-6, IL-10, IL-33, TNF-α, IFN-γ, TIMP metallopeptidase inhibitor (TIMP) -1, Vascular endothelial growth factor (VEGF), ICAM, and L-selectin. Plasma was collected from whole blood samples at the end of the treatment. Soluble factors, markers of tissue damage and cytokines, were analyzed in plasma samples using a magnetic-bead-based multiplex assay for the LuminexDroplet digital PCR was used to measure the amount of circulating mitochondrial DNA (mtDNA). Total DNA was extracted from rat plasma samples using a QIAmp DNA Minikit, Qiagen , following the manufacturer’s instructions. An amount of 1 µL of total DNA was added to a 20 µL final volume mixture composed of: 10 µL of 2× ddPCR Supermix for Probes, 1 µL of ddPCR assay for ND2 , 1 µL of ddPCR assay for EIF2C1 , and 7 µL of nuclease-free water . The droplet generations and readings were performed using the Bio-Rad QX200 ddPCR droplet system (Bio-Rad laboratories) ,19. Circp value < 0.05 was considered significant.Statistical analyses were performed using Prism 8.0 . The results are expressed as the mean ± Standard Deviation (SD). Non-parametric Kruskal–Wallis or Mann–Whitney tests were used to compare quantitative variables. A In order to determine the effects of EDs on the GI tract, we administered EDs, soda cola, or sweetened coffee to Sprague–Dawley rats for five days. The rats tolerated treatments without general or digestive complications. However, a weight gain of 7 ± 4.5% and 5 ± 3% (mean ± SD) was observed in animals receiving ED and soda cola, respectively. Furthermore, the rats supplemented with ED also showed increased excitability and difficult handling.The macroscopic examination of abdominal and thoracic organs did not disclose evident lesions. All of the common metabolic parameters tested just before the sacrifice were always in a normal range, as were the blood counts, including leukocytes and eosinophils (not shown).Histological examination of the GI tract excluded secondary lesions ingroup A of control animals supplemented with water, whereas an eosinophilic infiltration was observed in the mucosa of the stomach and duodenum of the animals supplemented with coffee, EDs, and soda cola .This pathology involved superficial epithelial cells and in full-thickness mucosa, submucosa until the lamina propria, but not the muscularis and serosa layers. Eosinophils showed a mature feature, with a normally sized nucleus, often ring-shaped, and a cytoplasm densely packed with granules. The esophagus appeared free from any lesion (not shown), while evident eosinophilia also involved the spleen, with a perifollicular infiltration not distorting its basal architecture . We nextThe levels of pro-inflammatory cytokines did not reveal any condition of systemic inflammation. TNF-α and IL-6 were below the limits of detection in all conditions tested, while IL-1 β displayed a high degree of variability but was always in the range of physiological concentrations .Concerning Th1 response, all conditions tested showed plasma concentrations of IFN-γ similar to those observed in animals receiving water. IL-4 and IL-13, two typical Th2-response cytokines, were undetectable in all conditions analyzed. Taken together, these results suggested that EDs, similarly to other caffeine-containing beverages, did not cause any systemic inflammation or Th2 immune polarization, despite the activation of a local eosinophilic response. As the effects observed in the GI tract were present in rats treated with coffee, cola, and ED, we wondered if it could be attributed to caffeine. Thus, to rats, we administered a coffee solution containing caffeine at the same concentration taken by rats supplemented by ED in the previous experiment for up to 5 days, followed by a recovery time of 5 or 10 days without caffeine supplementation . The fivIn this study, we described the effects of the assumption of EDs on the GI tract. The analysis of rats from a phenotypic point of view showed weight gain and increased excitability. The same effects have been largely described in humans assuming EDs  and are Thus far, the only study describing the effect of EDs on inflammation limited its analysis to the GI tract . Our stuThe microscope analysis revealed eosinophilic infiltration in the GI tract and spleen. Eosinophils are proinflammatory leukocytes specialized in parasitic infections and tissue damage, also involved in different cell processes associated with body homeostasis and cancer immunology . The obsEosinophilic infiltration could be triggered by direct contact between an extraneous, but not tissue toxic, substance, the caffeine, and the gastrointestinal mucosa, which responded with a local allergic-type reaction and recruitment of eosinophils. The splenic eosinophilic infiltration, observed after 5 days from the interruption of caffeine supplementation, confirmed the exuberant production of these cells that found a compatible refugee-organ in the spleen, so confirming its good receptivity for myeloid-derived cells. This passive colonization was not followed by any secondary eosinophilic recirculation or splenic activation and resolved completely at the same time as the original gastrointestinal mucosal infiltration. The local gastrointestinal eosinophilopoiesis typically agreed with the absence of general mediators such as immunoglobulins and cytokines . MoreoveIn humans, eosinophilic stomach and small intestine infiltration is characterized by eosinophilic gastroenteritis (EGE). EGEs are eosinophil-associated diseases (EADs), rare pathologies characterized by an increase in the circulation or in the tissue number of eosinophils ,29. WhenOur results can have relevant implications when translated into clinics, particularly as far as the potential negative effects of EDs on human health, and especially on young people, are concerned. The intake of beverages with a high caffeine content can cause acute effects  and chronic effects on which little information is available. The synergy of the high caffeine dosage with other components of the drink such as taurine can enhance these negative effects on human health. Specifically, the effects detected on the GI tract with an enhancement of the pro-inflammatory  action can facilitate the onset of diseases. EDs also contain an important quantity of sugars. The consumption of sugar-sweetened beverages predicts poor health outcomes in the aging population, including cardiovascular disease, diabetes, and cancer ,35. EvidHowever, our study has some limitations. First, we did not have the possibility to dissect the effects of the single molecules of herbal extracts normally present in EDs, as their actual content and concentration are not disclosed. Second, our study is limited to short-term, acute effects, and did not explore the effects of long-term treatment with EDs. Finally, our study does not provide information concerning the functional consequences of eosinophilic infiltration on the GI tract. Thus, further studies are needed to determine if the eosinophilic infiltration we observed could, when prolonged over time, contribute to EGIDs. In summary, these results show that, despite eosinophilia, the acute assumption of EDs does not have deleterious consequences when compared with coffee. Moreover, our data show that acute caffeine treatment in rats could be a good model to study the origin and the development of acute eosinophilia in the stomach and intestine.


Over the last decade, technological growth has developed new devices for more precise surgery based on improved maneuverability, minimally invasive approaches, and magnification of the operating field. In this context, the exoscope has opened a new phase for more accurate and safer microsurgery, improving the perception of the volume of objects and the depth of structures for planning, targeting, and controlling fine movements. The exoscope could be used for middle ear, transcanal, transmastoid, and craniotomy procedures that require two-handed dissection, both to perform both totally VITOM-based techniques and coupled to traditional procedures with an operating microscope or endoscope. In addition, the VITOM 3D system allows the surgeon to work with high-definition images, which is essential in facial nerve surgery or submandibular salivary stone or tear surgery approaches, where magnification plays a fundamental role in surgical success and in reducing operating times. The 3D exoscope approach could also be included in traditional transoral procedures for oropharyngeal carcinoma. The exoscope may provide a relevant approach in teaching surgeons and nurses, allowing adequate training in non-oncological surgical procedures such as a tonsillectomy or lateral pharyngoplasty. During the last decade, the exoscope became an addition to the microsurgical armamentarium, which was designed to replace the operative microscope. The exoscope consists of a high-definition or 4K video camera with optical and digital zoom and a fiber optic-delivered or L.E.D. light source ,2. This http://www.researchgate.net/, accessed on 15 March 2022). Three authors analyzed the data from the literature . Any disagreements were resolved through discussion by our study team. The included studies were then analyzed to gather all available data and guarantee eligibility among enrolled subjects. In addition, the patient’s diseases, diagnostic procedures, treatment modalities, and main polymorphisms were collected. The following information was collected: author data, year, sample size , study design, statistical analysis, findings, and conclusions. We contacted the authors of the included studies if the required data were not complete, using the corresponding author’s email or ResearchGate  by three different authors, using MeSH, Entry Terms, and related keywords. The related search keywords used were as follows: “3D exoscope surgery; exoscope 4K assisted surgery”, “E.N.T. Microsurgery”, “VITOM assisted surgery”, and “Head and Neck 3D surgery”.®, Thomson Reuters, New York, NY, USA) to collect references and remove duplicates. The titles and abstracts of papers available in English were examined by the investigators . The identified full texts were then screened for original data, and the related references were retrieved and checked manually to identify other relevant studies.The ‘Related articles’ option on the PubMed homepage was also considered. We used reference manager software , Entry Terms, and keywords found in articles in this field. The following items were considered: Participants (E.N.T. patients); Intervention ; Control (applied); Outcome ; Study type . We imposed language, publication date, and publication status as restrictions. Thus, we considered surgical success of E.N.T. disorders treated with 4k exoscope-assisted surgery as the primary outcome. Other parameters assessed in the studies were considered secondary outcomes.Original articles;The article was published in English;The studies included clinically confirmed E.N.T. disorders treated with 4k exoscope-assisted surgery;The studies reported detailed information on 4k exoscope-assisted surgery as high-definition images of the surgical field, improved vision and depth perception, different treatment modalities, and patient’s comorbidities.We included all studies that met the following criteria:5.Editorials, letters to the editor, or reviews;6.Studies that included animal samples.The exclusion criteria were as follows:Given different E.N.T. disorders, surgical procedures performed, tissue samples, and the follow-up programs adopted, quantitative analysis was not performed because of the influence on the outcomes reported. Consequently, we performed a narrative synthesis following the synthesis guidelines, without meta-analysis reporting items .The research protocol was carried out according to the approved reporting items’ quality requirements for systematic review and meta-analysis protocol (PRISMA) declaration . We adopThe systematic review of the literature identified 239 potentially relevant studies . After rWe included 22 studies in the analysis ,29,30,31The quality of evidence evaluation conducted by the GRADE assessment was considered low. This was mainly because of the study design , heterogeneous methodology, and risk of bias in the included studies. The evidence appraisals are summarized in The patients’ average age was 54 ± 16.41 years, ranging from 11 to 82 years old. The major surgical procedures performed were otomastoid and skull base surgery in 101 cases (33.33%) ,18,19, sp < 0.05) [The application of the VITOM system to surgical procedures has not shown a significant increase in surgical time compared to traditional methods ,18. RubiBignami et al. (2021) reported an easy frontal fibro-osseous removal thanks to the easy transition from the assistant role to the first operator, with consequent advantages in terms of cost effectiveness . HoweverAmong the different techniques of otological surgery, post-aural approaches appear to be the most suitable for exoscopic surgery; therefore, the VITOM 3D system can be used in the treatment of chronic pathologies of the middle ear and several tumor types of the external and middle ear, and to perform cochlear implants ,16,17,18In 2018, Smith et al. stated that the 3D exoscope advantages could present a valid choice over the operating microscope for neurotological surgery . The autConsequently, in 2019, alternative middle ear cholesteatoma surgery was described by Minoda et al., including two cases of mastoid cavity involvement . The autIn 2021, Wierzbicka et al. described their initial experience with the high-definition three-dimensional exoscope for middle ear surgery by comparing it with the operating microscope . The resLarger studies with a more systematic evaluation of the 3D exoscope image quality, ergonomics, and impact on surgical eye fatigue and workflow could aid in evaluating the usefulness of the exoscope in E.N.T. surgery . p = 0.03) and no difference in manipulation or visualization of structures (p = 0.05).In 2019, Smith et al. performed 11 otological procedures with the VITOM 3D system together with or in place of the operating microscope, including cochlear implant surgery, two resections of the vestibular schwannoma eventually coupled with the operative microscope, and visualizing the tumor from the cerebellopontine angle . InsteadRecently, Colombo et al. evaluated the potential of the 3D exoscope in specific ear procedures; the innovative technology was retrospectively compared with surgeries treated with an operating microscope . The autFurther studies, including microscope comparison and cost-effectiveness investigations, with more representative samples and with real clinical scenarios, are necessary to understand its real potential in clinical practice . Rubini p > 0.05). Moreover, the facial and hearing function outcomes were similar [Surgical time, facial and hearing function outcomes, and intraoperative or postoperative complications were analyzed, demonstrating no intraoperative complications during all the procedures. In contrast, at follow-up, one complication occurred. The authors reported an average surgical time of 289 min in the exoscope group and 313 min in the microscope group, not revealing statistical differences  via a combined osteoplastic flap-endonasal approach . The patThe surgical approach to squamous cell carcinoma of the oral cavity and oropharynx plays a fundamental role in the management of the disease, both in the presence of early and advanced disease stages. However, the different techniques developed have always suffered from poor accessibility and visualization of the anatomical structures of the oral cavity, forcing surgeons to choose invasive approaches for the patient. The term 3D exoscopic surgery was coined to describe the technique used for transoral surgery with the exoscopic system as a visual tool . CrosettDue to anatomical conditions and characteristics of pathologies, laryngeal surgery requires direct visualization of the larynx both in diagnosis and in surgical treatment of benign and malignant lesions, allowing an enlargement and clear visualization of the larynx. The most recent technological developments have proposed the exoscope as a potential substitute for operating microscopes in microlaryngeal surgery, and it has been highlighted that this system has intrinsic advantages ,23,24. TThe VITOM 3D telescope was positioned 25 cm from the operating field in place of the operating microscope behind the patient’s head. The authors emphasized the excellent sense of depth and light transmission, providing an enlarged view of the vocal cords and details of lesions such as fine vascularization, nodules, and irregularities of the mucosa. A total of 12 patients were treated: 4 with Reinke’s edema, 2 with Reinke’s space cysts, 3 with vocal cord polyps, and 1 sulcus with double chordotomy. In all patients, the VITOM 3D system allowed an excellent surgical result, stable at the subsequent postoperative checks of the laryngoscopic voice and in narrow band imaging (N.B.I).De Virgilio et al. recently conducted a prospective pilot study to evaluate the feasibility of “exolaryngoscopic” surgery using the VITOM 3D exoscope and ARTip ™ robotic cruise system . Surgeonp < 0.05 for all) at 30 days after phonosurgery.The feasibility of the exoscopic system has been applied in both benign and malignant pathologies of the larynx ,24. Cant2 laser, which had initially presented some application difficulties to laryngeal surgery. The use of the VITOM 3D system coupled with a support arm with a laser micromanipulator was recently analyzed in a preliminary study [p > 0.05).Further technological advancements allowed the three-dimensional technology application to be added to the use of the COry study . The autThe VITOM 3D system was used to carry out a detailed surgical dissection of the parotid area, with a 3D magnification of the anatomical structures and a lower threat of iatrogenic damage to the facial nerve. p = 0.551), superficial parotidectomy performed , or bleeding , and no wound revision occurred in either group, a significantly higher rate of subjects in the exoscope group developed temporary facial nerve paralysis  [Bartkowiak et al. conducted a prospective study on patients with benign parotid gland tumors indicated for surgical resection, comparing the surgical results of the VITOM 3D system (n = 31) with the traditional system with an operating microscope n = 40). The authors randomized 71 patients to traditional or VITOM-assisted surgery and analyzed outcomes such as visualization quality , operative time, conversion rates, and surgical outcomes. Although no significant differences were found between the two approaches regarding duration of surgery  . Eight t 4; 10%) . Carta e 4; 10%) . All ninThe application of the 3D exoscope has found widespread use in salivary stone surgery. Previous studies found this approach to be effective, quick, and safe for the removal of large immobile stones from the hilum of the submandibular gland to the papilla. The literature shows a success rate of 87–99%, with symptom relief in 76–96% of patients. A 0–4% risk of permanent lingual nerve damage was demonstrated, and there was a low infection rate of 0–10% . The conThe exoscope can be considered to perform reconstructive head and neck free flap techniques, mostly during the execution of microvascular anastomoses. De Virgilio et al. (2020) studied the experience with the VITOM 3D system in a human clinical study of free flap harvesting and microvascular anastomosis in patients undergoing reconstruction after ablative surgery for head and neck carcinoma . They peBelykh et al. (2018) evaluated microvascular approaches via 3D exoscope or endoscope visualization . Using 3The rounded area scene via the oculars was bigger than the complementary rectangular one on the monitor. Nevertheless, the highest magnification permitted small-caliber vessel details (adventitia or intima) to be more distinctly and sharply visualized via eyepieces than a 3D 4K resolution monitor.Although promising, the literature exhibits several limitations, including study design, sample enrollment, and few outcomes adequately investigated. The surgical time, for instance, demonstrated debated outcomes, indicating that the exoscope system may be less compliant in certain surgical areas. From this viewpoint, further scientific proof is required to clarify whether benefits such as two-hand surgery could effectively lower surgical time and demonstrate the exoscopic procedure’s advantageous cost benefit. Indeed, it was appraised that such system features could be limited if a deeper surgical field or poor lighting due to small surgical corridors are needed. Although one paper reported that the system is more efficient and cheaper than the latest operating microscope systems, other evidence suggests a deterioration of the surgical images, with consequent pixilation and excessive visual fatigue due to the use of polarizing glasses, thus questioning the economic benefit. Moreover, it should be noted that although the results shown in different studies are promising and do not demonstrate significant differences between the control groups with the traditional surgical method, the data must be interpreted cautiously, as the number of patients enrolled is often insufficient.In this preliminary study, we show the great potential of this new technology, highlighting the advantages of the easy involvement of the surgical team and observers. In recent years, E.N.T. surgery has benefitted from new technologies and techniques. An increasing number of surgeons are using this technology, gaining scientific relevance every year. On the other hand, the VITOM 3D system has some limitations that can probably be overcome by advancing technology in the near future. However, today, these advantages are not enough to abandon the operating microscope. Overall, we believe that the improvements in this field of application are exponential, certainly leading to great results in the future.


Prehospital visits were associated with unfavorable health outcomes. This study aimed to determine whether prehospital visits to other medical institutions before admission are associated with prolonged hospital stay, readmission, or mortality rates in acute stroke patients. Using the claims data from the Korean Health Insurance Service, a cross-sectional study was conducted on 58,418 newly diagnosed stroke patients aged ≥ 20 years from 1 January 2019 to 31 December 2019. Extended hospital stay  following initial admission, readmission within 180 days after discharge, and all-cause mortality within 30 days were measured as health outcomes using multiple logistic regression analysis after adjusting for age, sex, income, residential area, and medical history. Stroke patients with a prehospital visit  had a higher risk of long hospitalization , readmission , and mortality  compared with patients without a prehospital visit. Female patients and those under 65 years of age had increased unfavorable outcomes ( Rapid and accurate detection of stroke is crucial for the timely initiation of treatment because strokes account for 11% of total deaths and 30% of disabilities requiring lifelong assistance . ProlongAs measured by symptom onset-to-door time, early hospital arrival is known to affect health outcomes ,10. HoweTo confirm the usefulness of prehospital visits as a measure of prehospital delay, this study was conducted to compare the health outcomes, such as extended hospital stays, readmission, and mortality, of newly diagnosed stroke patients according to the prehospital visits. A cross-sectional study was performed to evaluate health outcomes, such as length of hospital stay, readmission, and mortality, according to prehospital visit patterns in Korean stroke patients using the National Health Insurance Service (NHIS) claims data. The National Health Information Database is a public healthcare database maintained by the NHIS for health screening, sociodemographic variables, and mortality for the entire South Korean population  . Data fon = 101,033) were enrolled. After excluding participants who had a previous diagnosis of any stroke , 58,418 patients remained in the study  of secondary or tertiary hospitals and confirmed by brain computer tomography or magnetic resonance imaging from 1 January 2019 to 31 December 2019 (he study . n = 942) in 2019 were categorized according to the first code.A diagnosis of stroke involved at least one of the following ICD-10 codes in the NHIS data: I60 (subarachnoid hemorrhage), I61 , I62 , or I63  without a previous diagnosis in the three years prior. Hemorrhagic stroke was classified as I60, I61, and I62; ischemic stroke was classified as I63. Patients with ICD-10 codes for hemorrhagic and ischemic strokes , I65 , I66 , I67 (other cerebrovascular diseases), or I68 (cerebrovascular disorder in diseases classified elsewhere) prior to admission to the ED of a secondary or tertiary hospital. Age was calculated based on participants’ birth year. Income status involving six groups  was obtained based on each patient’s insurance coverage. The residential area was classified into “high urbanization” areas, including administrative districts with a tertiary hospital or a stroke care unit, and “low urbanization” areas. Comorbidities, such as hypertension (I10), ischemic heart disease , congestive heart failure , diabetes mellitus (E10), and arrhythmia (I49 and I802), were assessed using a physician diagnosis.An extended or “long” stay in a hospital was defined as any stay of more than seven days  during the admission for initial stroke treatment. Readmission was defined as admission within 180 days of discharge. We investigated mortality within 30 days of stroke diagnosis, and there were no restrictions on the cause of death. Continuous demographic variables were expressed as mean values and standard deviations, and categorical variables were expressed as frequencies and percentages. Age was presented as the mean with standard deviations, and categorical variables were used in the subgroup analysis. Univariate analysis was conducted between the prehospital-visit and no-prehospital-visit groups using t- and chi-square tests.p < 0.05.We calculated the odds ratio (OR) and 95% confidence interval (CI) of unhealthy outcomes, such as long stays in the hospital, readmission, and mortality, in the prehospital-visit group compared to the no-prehospital-visit group using multiple logistic regression analyses after adjusting for age, sex, income, residence area, and previous medical history . We calculated the OR and 95% CI of unhealthy outcomes at the subgroup level of sex and age (divided at 65 years). The time interval between the prehospital visit and the day of admission for treatment was obtained from the claims data. All analyses were performed using SAS version 9.4 , and statistical significance was set at p < 0.001). The low-income group with medical aid had a lower rate of prehospital visits than the group with medical insurance. Patients living in highly urbanized areas were more likely to be in the no-prehospital-visit group (p < 0.001). Patients with hypertension were more likely to be in the prehospital-visit group; however, patients with ischemic heart disease, congestive heart failure, diabetes mellitus, and arrhythmia were more likely to be in the no-prehospital-visit group (p < 0.001). The characteristics of the patients with ischemic and hemorrhagic strokes are presented in The characteristics of the 58,418 stroke patients, including those with ischemic and hemorrhagic strokes, are shown in p < 0.001). Of the participants, 30,571 (52.3%) were readmitted, and 4372 (7.48%) died, with the prehospital-visit group exhibiting higher readmission  and mortality rates . The number of stroke patients with extended hospital stay was 35,456 (60.7%) . The ratp < 0.001).After adjusting for age, sex, income, residence, and medical history , the ORs and 95% CIs for extended hospital stay , readmission , and mortality  in the prehospital-visit group were significantly higher than those in the no-prehospital-visit group . An extended hospital stay was defined as >7 days . Readmissions over 180 days were recorded. Mortality within 30 days was recorded.In participants younger than 65 years, a prehospital visit was associated with an increased risk of an extended stay in the hospital , readmission, and mortality  compared with participants aged 65 years and older .The associations between prehospital visits and poor health outcomes according to stroke type and patient characteristics, including income and urbanization, are presented in This study demonstrated that prehospital visit status was associated with an increased risk of extended hospital stay, readmission within 180 days, and 30-day mortality in newly diagnosed stroke patients. This association existed in patients with all types of strokes even after considering income, residence area, and comorbidities that may affect medical use behavior.Previous studies on prehospital delay focused on measuring onset-to-door time to identify the beneficial effects of early treatment, such as reperfusion therapy, anticoagulants, or antiplatelet agents in ischemic stroke patients ,17,18,19Presumptive onset based on symptoms, an indicator commonly used by many clinicians, may not accurately recognize the initial symptoms . Our stun = 10,992) in this study, 10,025 (91.2%) visited one medical institution and 967 (8.8%) visited two or more medical institutions. The proportion of patients who had made a prehospital visit on the same day as the final diagnosis was 8651 (78.7%). The number of patients who visited the stroke center one day later was 1198 (10.9%), and that for a visit two or more days later was 1143 . Prehospital delays of two days in 10% of cases is a relatively high rate, even with the limitation of counting one day after midnight in the claims data. Although the reason or required time for the prehospital visits is still being determined from these data, it may be that the prehospital delay was due to the number of medical institutions visited.Among the prehospital-visit group  symptom onset motivates a decision to seek medical attention, (2) time of the decision to seek medical attention to first medical contact, and (3) first medical contact to hospital arrival . Steps 1The prehospital visit rate was higher in males over 65 years and with comorbid diseases than in females under 65 years with no comorbid conditions . In thisIn Korea, a relatively low barrier to medical use may have influenced the rate of prehospital visits because of fewer financial obstacles. We found that high-income status was associated with an increased risk of extended hospital stay and readmission among ischemic stroke patients, as suggested in Compared with high urbanization areas, acute stroke patients living in low urbanization areas are expected to have a lower rate of direct visits to stroke units. Similarly, a higher rate of prehospital visits was associated with an increased mortality risk in patients with low urbanization. However, there was no significant difference in the subgroup analysis according to stroke type. Despite efforts to establish acute care hospitals across the country and certified stroke units in rural areas, we confirmed an association between poor prognosis and prehospital visits. Therefore, prehospital visits can be an indicator of health outcomes in the absence of external factors.This study has two main limitations. First, information about the utilization of an EMS; education levels that can influence stroke awareness; or lifestyle behavior data, such as smoking, exercise, or body mass index, could not be surveyed due to the nature of the claims data. Second, we used health outcomes focused on hospitalization duration, readmission, and mortality and did not include an individual’s functional status or disability. Third, the rate of prehospital visits may have been underestimated when a stroke was not suspected or another diagnosis, such as headache or dizziness, was made because we identified newly diagnosed stroke patients by ICD codes.Acute stroke patients with prehospital visits had increased risks of poor health outcomes, such as extended hospital stays, readmission within 180 days, and 30-day mortality, than patients without prehospital visits. The effects of prehospital visits on health outcomes were more pronounced in female patients and those younger than 65 years. Therefore, additional research and policy proposals are needed to clarify the effectiveness of visiting the hospital directly to treat patients with suspected strokes.


To better understand the causes of hypophosphatemia in children, we evaluated all serum phosphate tests performed in a tertiary hospital with unexpected but persistent temporary or isolated hypophosphatemia over an 18 year period. We collected 29,279 phosphate tests from 21,398 patients, of which 268 (1.2%) had at least one result showing hypophosphatemia. We found that endocrinopathies (n = 60), tumors (n = 10), and vitamin D deficiency (n = 3) were the medical conditions most commonly associated with mild hypophosphatemia, but in many patients the cause was unclear. Among patients with endocrinopathies, those with diabetes mellitus were found to have lower mean serum phosphate levels (mean 3.4 mg/dL) than those with short stature (3.7 mg/dL) or thyroid disorders (3.7 mg/dL). In addition, we found a correlation between glycemia and phosphatemia in patients with diabetes. However, despite the potential relevance of monitoring phosphate homeostasis and the underlying etiologic mechanisms, renal phosphate losses were estimated in less than 5% of patients with hypophosphatemia. In the pediatric age group, malignancies, hypovitaminosis D, and endocrine disorders, mostly diabetes, were the most common causes of hypophosphatemia. This real-world study also shows that hypophosphatemia is frequently neglected and inadequately evaluated by pediatricians, which emphasizes the need for more education and awareness about this condition to prevent its potentially deleterious consequences. Hypophosphatemia is a poorly recognized metabolic condition in clinical practice because serum phosphate is not part of routine biochemical screening in the primary care setting. In addition, phosphate results can be misinterpreted due to age-specific differences ,2,3. MorVarious endocrine disorders can be associated with persistent hypophosphatemia, which may have several consequences, particularly in the musculoskeletal system, including impaired bone mineralization, growth retardation, muscle weakness, and other manifestations of rickets . That isThe fact that mild hypophosphatemia frequently goes undetected, although it may have clinical consequences, motivated us to carry out a systematic search for cases of hypophosphatemia in children. The retrospective and prospective arms included 29,279 phosphate results in 21,398 patients , of whomAmong the 232 patients with hypophosphatemia included in the study, 107 (46.1%) were males and 125 (53.9%) were females. The mean age of the first analysis showing hypophosphatemia was 5.8 years (6.0 years in the retrospective arm and 5.2 years in the prospective arm). The mean age at which each subject showed the lowest serum phosphorus was 6.8 years, with a median of 8. Overall, we identified 777 lab results showing hypophosphatemia, with a mean of 3.3 analyses per patient and a median of 2 . In 77 patients (33.2%), serum phosphorus was determined only once, 61 of them in the retrospective analysis and 16 in the prospective arm. In 65.1% of patients with some phosphorus results below the reference range, hypophosphatemia was present in their first phosphate analysis available. Moreover, among patients with phosphorus analyzed twice or more, 44.5% presented hypophosphatemia in at least half of their samples. When assessing the lowest serum phosphate, the mean equivalent in the cohort was 3.6 mg/dL, with a median of 3.8 mg/dL (IQR 3.5–3.9) and a minimum value of 1.4 mg/dL. Other biochemical tests commonly obtained are shown in When we analyzed all serum phosphate levels of each patient as described in the Methods Section, hypophosphatemia was classified as intermittent in one-hundred and twenty children (51.7%), isolated in one-hundred and eight (46.5%), and persistent in four (1.7%). After reviewing the clinical records, we found that 18 children (7.8%) had an underlying disorder that could be associated with hypophosphatemia (“expected hypophosphatemia”). Two other subjects were considered to present Metabolic Bone Disease of the Prematurity (MBDP). Finally, 212 patients were considered to present unexpected hypophosphatemia. The distribution across the subgroups with different time courses is shown in The most frequent underlying conditions in patients with “expected” hypophosphatemia were malignant disease , vitamin D deficiency , drug therapy , and refeeding syndrome . p = 0.024). The pair-wise comparisons showed statistically significant lower phosphate values in patients with diabetes, with differences that were close to the significance limits in the unadjusted test , but nonsignificant after adjusting for multiple comparisons .A significant proportion of patients (25.4%) presented with endocrinopathies. The most common disorders were diabetes mellitus , short stature , thyroid disorders , and others . Although some of them are known to be associated with hypophosphatemia in some circumstances, their role as a major factor causing hypophosphatemia in each patient was unclear. Therefore, we decided to analyze them separately, describing serum phosphate results in the most frequent groups, irrespective of whether they were considered “expected” or “unexpected”. The mean phosphate levels in children with hypophosphatemia associated with thyroid disorders were 3.7 mg/dL, 3.7 mg/dL in those with short stature, and 3.4 mg/dL in those with diabetes . An ANOVp = 0.005), and there was a slight negative correlation between the serum levels of phosphorus and glucose  (p = 0.0004).In the diabetes mellitus cluster, we identified 21 patients with 112 phosphorus determinations, of which 29 (26%) were below the reference range. The mean phosphorus value was lower in patients with glycemia over 200 mg/dL than in those with glycemia < 200  b. When pp = 0.1 and p = 0.579, respectively).The group of thyroid disorders included one patient with congenital primary hypothyroidism, five with thyroiditis, four with subclinical hypothyroidism, and three with tertiary hypothyroidism. Among them, four patients had other associated endocrinopathies . Overall, in the group of 13 patients with thyroid disorders and hypophosphatemia, concomitant serum phosphate and thyroid hormones were measured 76 times; in 15 samples (19.7%), serum phosphorus was below the reference range. We did not find a statistically significant correlation between T4 or TSH and phosphorus levels . However, it is important to note that in only 61% of the tests, phosphate and IGF-one measured simultaneously, and in only 27% of the cases, phosphate and IGFBP3 were assessed at the same time. When evaluating the group of subjects who were treated with GH, the mean phosphate levels were higher in the post-treatment samples than in the pre-treatment samples .The cluster of patients with short stature included one subject with GH deficiency secondary to craniopharyngioma and another patient with eosinophilic granulomatosis involving the CNS. Among them, seven out of fourteen (50%) presented with GH deficiency, and six of them received GH therapy. A total of 100 phosphorus determinations were obtained in these 14 patients, and 29 of them (or 29%) were below the reference range. There was no significant correlation between IGF-1 or IGFBP3 and serum phosphate  of unknown cause who were invited to attend the clinic for a more comprehensive clinical and biochemical study. After further studies, we found that serum phosphorus had normalized in seven cases; in two children, we found significant hypovitaminosis D; and in one case, hypophosphatemia persisted due to an unknown cause (a DNA sequence is pending to exclude a genetic cause). Phosphate entails approximately 0.6% of the total weight of animals and plants . It is cIn adults, hypophosphatemia is relatively frequent in daily medical practice ,21,22,23For the purpose of this study, we used 4 mg/dL as the lower end of phosphate in patients under 12 years of age and 3 mg/dL in those aged 12–16 years as an adequate cut-off point to consider hypophosphatemia, according to what is promulgated. This is in line with the limits proposed in some reference textbooks  and is aIn more than 50% of cases, hypophosphatemia was an intermittent condition; in less than 2% of cases, it was persistent. Unfortunately, in more than 40% of patients with hypophosphatemia, we only have a single analysis of serum phosphate, and therefore the course of the abnormality cannot be determined. In most cases, there was not an underlying condition that would directly explain the hypophosphatemia.As phosphatemia can be altered by different mechanisms, including inappropriate urinary losses, the diagnostic work-up should include measuring the phosphate urinary excretion. Surprisingly, in our cohort, the renal phosphate losses were assessed in less than 5% of patients with mild but persistent plasma hypophosphatemia. Moreover, in the medical charts, there were no comments about the phosphatemic status. This reflects the fact that in clinical practice, pediatricians often neglect hypophosphatemia and do not adequately study its causes and consequences. This is somewhat disturbing in view of the adverse consequences of hypophosphatemia on the musculoskeletal system and other body organs ,25,26,27In our cohort, we identified a significant number of patients with different endocrine disorders associated with mild but intermittent hypophosphatemia. We do not know if this metabolic condition alters the endocrine-metabolic equilibrium and has subsequent clinical relevance or if it just reflects a different whole-body phosphate balance. Hypophosphatemia was particularly frequent in patients with diabetes, and serum phosphorus was inversely correlated with serum glucose and glycosylated hemoglobin. Although we did not have data about urine phosphate, hypophosphatemia is likely related to the phosphaturic effect associated with glycosuria more than the potential intracellular shift due to an excess of insulin. Furthermore, reduced tubular reabsorption of phosphate, as a direct effect of acidosis, likely contributes to low phosphate levels in patients with acutely decompensated diabetes ,30. In kDiabetic patients frequently present other electrolyte disorders, mainly hypo- and hypernatremia, hypopotassemia, and hypomagnesemia. These disturbances are more frequent in decompensated patients, especially in the context of diabetic ketoacidosis . DespiteApart from phosphate redistribution and urinary losses, low intestinal absorption may also contribute to phosphate depletion in diabetes. In poorly controlled patients, anorexia and malnutrition can be frequent , as dietSince phosphorus homeostasis in the pediatric diabetic patient is a marker for metabolic control of the disease, we recommend clinicians pay more attention to and study phosphorus metabolism more rigorously in patients with diabetes, with serial analyses of serum and urinary phosphate levels. By knowing the mechanisms underlying phosphorus deficiency, we can take specific measures, in some cases with dietary changes and phosphorus supplements, in others with vitamin D, and in some other cases by optimizing insulin treatment. Moreover, we must not forget the importance of phosphorus in multiple metabolic processes, including glycolysis which uses phosphate to synthesize ATP .Likewise, in children with growth retardation on growth hormone (GH) replacement therapy, plasma and urine phosphate values could also be useful biomarkers. On one hand, chronic hypophosphatemia is associated with growth retardation; on the other hand, exogenous GH has enteral and renal effects that notably influence calcium/phosphate homeostasis ,9,43,44.The major strength of our study is that we reviewed serum phosphate in a large number of pediatric patients with a variety of conditions. However, it also has important limitations related to its largely observational and retrospective design. Consequently, the clinical and biochemical options available were limited in many cases, including repeated phosphate analyses that would help to determine the time course of serum levels and the scarcity of urinary analyses that would help to clarify the causes of hypophosphatemia.The study center was the Hospital Universitario Marqués de Valdecilla. This is a tertiary care center in Cantabria, a region in Northern Spain with a population of 550,000. The hospital serves as the primary hospital for a population of about 320,000 and as the reference center for the rest of the region, which is also served by two community hospitals. The target population, those between 1 and 16 years of age, comprised roughly 82,000 individuals. The study included a retrospective and a prospective arm. In the retrospective arm, we performed a computerized search of the laboratory database between October 2002 and January 2021. We looked for patients with at least one determination of serum phosphorus. The prospective arm was carried out between January 2020 and March 2021. Within that time period, a phosphorus test was added to all lab requests from patients who met the above inclusion criteria. At this stage, all lab requests in the region of Cantabria were included. Thus, in addition to the Hospital Marqués de Valdecilla, the community hospitals in Sierrallana and Laredo also participated in the study. This was done by implementing an algorithm within the common system for ordering lab tests.Patients with hypophosphatemia, defined by serum phosphorus < 4 mg/dL in children aged 1–11 years and <3 mg/dL in those over 11 years (24), were reviewed and identified from both retrospective and prospective arms and their clinical records. Patients with persistent hypophosphatemia were invited to attend our outpatient clinic for clinical evaluation and additional biochemical or genetic tests.Serum samples were obtained in the morning after an overnight fast. Serum metabolic variables including creatinine, estimated glomerular filtration rate (2009 Schwartz-IDMS) (eGFR), total calcium, phosphorus, alkaline phosphatase, albumin, and intact parathyroid hormone (PTH) were analyzed using an Atellica CH&IM platform ; 25-hydroxycholecalciferol levels were measured using an automated competitive chemiluminescence assay .All pediatric patients with low serum phosphate were identified at least once. For the purposes of this study, we classified them into the following groups: (a) “persistent hypophosphatemia”, when phosphate levels remained < 4 mg/dL (or <3 mg/dL in subjects over 11 years) in at least two different samples separated by two months, with no subsequent normalization documented; (b) ”isolated hypophosphatemia”, included patients with a single analysis with low phosphate and no subsequent reassessment; and (c) ”intermittent hypophosphatemia”, in the case of patients with both low and normal values of serum phosphate. Next, in line with established clinical practice, hypophosphatemia was grouped according to its origin as “expected” (EH) in cases where an underlying disease known to cause hypophosphatemia was present; otherwise, it was considered “unexpected” (UH). Furthermore, the EH group was divided according to the potential pathophysiological mechanisms involved: decreased intestinal absorption, phosphate redistribution, or increased urinary losses.p < 0.05 was considered to indicate statistical significance.Data with a normal distribution were expressed as the mean SD; data with a non-normal distribution were expressed as the median IQR. When comparing different groups, the lowest serum phosphate level was used. A correlation and a linear regression model were used for the relationship analysis of the variables. An analysis of variance (ANOVA) test was used for group comparisons, followed by a t-test and Bonferroni adjustment for multiple comparisons. A value of The study was approved by the IRB . Written informed consent was obtained from the parents or legal guardians of patients evaluated at the outpatient clinic.In the pediatric age group, malignancies, hypovitaminosis D, and endocrine disorders, mostly diabetes, were the most common causes of hypophosphatemia. This real-world study also shows that hypophosphatemia is frequently neglected and inadequately evaluated by pediatricians, which emphasizes the need for more education and awareness about this condition to prevent its potentially deleterious consequences.


Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. They are crucial for sperm transport, and they emerge as contributors to the spermatogonial stem cell niche. They are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs), which had been isolated from testicular samples of donors with normal spermatogenesis. The significant overlap between our results and recently published ex vivo data indicates that HTPCs are a highly adequate cellular model to define and study these cells. Thus, based on the expression of several markers, HTPCs can be classified as testicular smooth muscle cells. Small differences between the in vivo/in vitro expressed genes may be due to cellular plasticity. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities, four cellular states were identified. Further analyses confirmed the presence of known stem cell niche-relevant factors  and identified unknown functions, e.g., the ability to produce retinoic acid. Therefore, HTPCs allow us to define the signature(s) and delineate the functions of human testicular peritubular cells. The data may also serve as a resource for future studies to better understand male (in)fertility. Understanding the cellular components of the human testis is of importance for the understanding of the testis, male fertility and infertility. Furthermore, with regard to male hypogonadism, such insights are required to guide regenerative medicine ,2.Recent studies have begun to unravel the cellular components of the human testis at the single-cell level ,4,5,6. TThey also provide some details about the least known cell type of the human testis, peritubular cells ,8, whichOf note, in states of male infertility, profound changes of human peritubular cells have been noted , and theAs these cells form a small compartment within the testis, the mentioned previous ex vivo analyses are based on a few hundred peritubular cells   closely https://www.proteinatlas.org; accessed on 1 April 2022), and transcriptomic information available . Based v/v) fetal calf serum (FCS)  and 1% (w/v) penicillin/streptomycin  at 37 °C, 5% CO2 and 95% humidity.We examined the HTPCs, which were derived from the small testicular samples of two donors  with obstructive azoospermia and normal spermatogenesis . Small p® Cell Counter, OMNI Life Science, Bremen, Germany). The cells were prepared to a concentration of ~1000 cells/µL in PBS 0.04% BSA for the single-cell sequencing and processed using the Single-Cell 3′ Reagent Kits v2 from 10x Genomics according to the manufacturer’s instructions, targeting 10,000 cells. This was followed by the GEM generation and barcoding, post-GEM-RT cleanup, cDNA amplification, library construction and quality assessment using the Bioanalyzer . Illumina sequencing libraries were sequenced using a NovaSeq 6000 (NovaSeq Flow cell Type S2 one lane), with an average read depth of 50,000 aligned reads per cell. The sequencing was performed in the genome analysis center of the Helmholtz Center Munich.For the single-cell RNA sequencing analysis, the HTPCs were cultured for 24 h with or without the supplementation of 10% FCS in the medium. The addition of FCS fosters proliferation and allows for the expansion and propagation of the HTPCs. The cells were trypsinized, washed with PBS, centrifuged, resuspended in PBS, filtered through a cell strainer (mesh size = 40 µm) and counted  of the mapped cells was performed using the recommendations by Luecken and Theis ; we seleTo generate the spliced/un-spliced expression matrices, we used the tool velocyto using the function run10x in order to directly accomplish the counting in the cellranger output folder . To defihttps://www.proteinatlas.org, accessed on 1 April 2022) to examine in situ expression. Because the human samples that had been used for the immunostaining and are depicted there do not necessarily represent healthy tissues, we used the images only when the signs of normal spermatogenesis were visible  to explore the ability of these cells to differentiate into osteoblasts and adipocytes. We used reagents from Miltenyi Biotec GmbH , following the instructions of the manufacturer . After a period of 14 or 21 days, respectively, alkaline phosphatase/oil red (SIGMA FASTTM BCIP®/NBT Buffered Substrate Tablet (Sigma #B5655); Oil Red O (Sigma #O9755)) was used to visualize the success of the differentiation. Furthermore, a qPCR for the typical osteoblast (ALPL/COMP) or adipocyte (PLIN1) markers was performed. The information about the primers that were used is given in the TCF21 in the FCS-treated HTPCs, we also addressed the question whether Leydig cells could be derived from HTPCs, following a recently published protocol [n = 2; patients were 29 and 31 years old). In brief, HTPCs were seeded on cell culture dishes and initially recovered for 18 h in DMEM + 10 % FCS. Subsequently, the cells were expanded for 3 days in DMEM/F12 which was supplemented with Normocin , 10 ng/mL PDGFAA Abcam, Cambridge, UK), 10 ng/mL PDGFBB (Abcam), 0.5 μM SAG  and 10 ng/mL FGF2 (Biomol). After 3 days, the expansion media were replaced with differentiation media: DMEM/F12 which was supplemented with penicillin/streptomycin, 10 ng/mL PDGFAA, 10 ng/mL PDGFBB, 0.5 μM SAG, 10 ng/mL FGF2, 5 mM LiCl2  and 10 μM DAPT (Biomol). After 10 days in differentiation media, the cells were analyzed for their Leydig cell properties by qPCR. The expressions of INSL3, LHR and steroidogenic enzymes were used as readout  which were cultured with and without FCS. The detection of retinoic acid was performed using an ELISA  following the instructions of the manufacturer.Because we found components of the retinoic acid synthetic pathway in the transcriptomic data, we examined whether the HTPCs produce retinoic acid, employing additional HTPCs . We focused on the sample derived from the HTPC-1 group  because it appeared to be most comparable to the samples of the recent ex vivo studies ,17. FurtWe compared the data of HTPC-1 (no FCS) with the ex vivo data ,17. To cThe addition of FCS induced cell proliferation, as expected. Four distinct states of cellular differentiation were identified upon the analysis, indicating the plasticity of these cells . Some diThe data obtained provide a thorough molecular signature of the human testicular peritubular cells. Here, we focus on the key results. We show in the following sections, the results that were obtained from the study of cells of HTPC-1, unless they are indicated otherwise.ACTA2, VIM, SM22A (TAGLN), calponin (CNN1), caldesmon (CALD1) and smoothelin (SMTN) were not or only marginally affected by the absence/presence of FCS, and this reveals the SMC character of the HTPCs , desmin (DES), myocardin (MYOCD), and serum response factor (SRF) or (not shown) CSPG4, RGS5, EZH2 and LGR5, were expressed only by a few subtypes of HTPCs, and they did not notably change upon FCS addition to the medium. PDGFRB, which is found in vascular SMCs, but not in airway or intestinal SMCs [Additional SMC markers , namely,nal SMCs , was prenal SMCs , and it nal SMCs  and Nie nal SMCs .BGN and DCN expression levels are increased in the absence of FCS in the culture medium. The corresponding proteins are represented in the peritubular cells in situ (Human Protein Atlas). These ECM components were also previously readily found, specifically, in the secretome of HTPCs [Known ECM components of the human peritubular wall compartment include DCN, BGN and COL1A1, among others ,35,38. Fof HTPCs . Hence, of HTPCs .AR and NR3C1 were homogeneously expressed, as were EGFR [PDGFRA [PGR and ESR1 were found, as well as the ion channels TRPV2 [P2X4/7 [The known receptors involved in the regulation of HTPCs are AR and NR3C1, which mediate the actions of androgens and glucocorticoids, respectively ,40. Figuere EGFR  and PDGF [PDGFRA . Furtherls TRPV2  and P2X4 [P2X4/7 . The cor [P2X4/7 .GDNF, CXCL12, LIF and NGF, as well as for ALDH1A1 and ALDH1A3 (n = 2). Higher levels were found in the supernatant of the cells without FCS in the culture medium, i.e., a condition, in which also the levels of ALDH1A1 were elevated. The factors that are produced by murine testicular peritubular cells and HTPCs can influence the SSC  and spermatogenesis ,44. In t ALDH1A3 . GDNF, CCD34, CD14, CD79A or CD19. Of note, the expression of ENG, NT5E and THY1 was increased when FCS was added to the culture medium  and ARX were not readily found. The available images of the immunostained peritubular cells that are provided in the Human Protein Atlas support this expression . A number of markers for LSCs had been previously suggested in human and nonhuman species. They include THY1 (CD90), CD51 (ITGAV), COUP-TFII (NR2F2), PDGFRA, TCF21 and Endoglein (ENG) ,47. Thespression .TCF21 expression was higher in the FCS-treated cultures, and the testicular cells with this marker have recently been identified to contribute to the somatic cells of the rodent testis, and they can regenerate adult Leydig cells in mice [INSL3 nor LHR, i.e., the prototype markers of the LSCs, became detectable . The corresponding proteins were not readily found in the images that were provided by the Human Protein Atlas collection, but the transcripts have been described by Di Persio et al. [A group of HTPCs is characterized by the expression of enzymes involved in steroid production, i.e., o et al.  and Nie o et al. , indicato et al.  and Nie o et al. .We compared the data from ex vivo-derived HTPCs, which recently became available ,17 with While the overall difference between the ex vivo-derived HTPCs and the in vitro-derived cells were small, it cannot be ruled out that they could be due to isolation and cell culture. Yet, importantly, the direct comparison between the data from the two studies of the ex vivo-derived HTPCs ,17 indicPhenotype switching is an intrinsic and not-fully-understood ability of SMCs , which cTo explore the cellular plasticity of HTPCs, we exposed them to FCS. The addition of FCS caused cell proliferation, and as expected, the transcripts associated with cell cycle were altered. The treatment also revealed four distinct states of differentiation which were characterized by the expression of genes related to, e.g., “collagens”, “extracellular region”, “ECM”, “ER to Golgi transport” and others, thereby providing a proof of principle for cellular plasticity and functional heterogeneity of HTPCs.Of note, while they are almost identical in their transcript expression, upon the commencement of the velocity analyses, the two samples (HTPC-1/HTPC-2) that have been examined here were not completely identical in this respect. The samples were from individual men, and it seems possible that differences are patient-related. The age differences between the donors (41 versus 48 years old) may also be involved. The fact that HTPCs and non-human primate testes-derived TPCs can age has been previously demonstrated, and insights into testicular and peritubular cell senescence have been gained ,51. It iACTA2, TAGLN and SMTN provide support for the SMC character [AR, STAR and CYP11A1, which to our knowledge are not known in this combination in other human SMC types [AR, NR3C1 or EGFR, were not or not strongly affected by FCS.HTPCs were originally named SMCs, and later, they were also named myofibroblasts ,53. Our haracter ,54,55. TMC types . Of noteMC types . This rePDGFRA and CD34, but of note not ACTA2, are thought to be typical for telocytes, and they have been reported in the human testis ,58. The BGN and DCN upon the addition of FCS indicate that HTPCs respond to clues with altered ECM production. ECM is present between the layers of peritubular cells in the human testis, normally, and its composition and ECM amounts change in idiopathic male infertility when, for example, the DCN and BGN levels increase ,40. ThisTHY1 (CD90), ENG (CD105) and NT5E (CD73)) were also observed [The expression of the MSC markers  in situ.While HTPCs are known to be important for sperm transport , HTPCs aogenesis ,70 and/oogenesis , and/or ogenesis  are possIn conclusion, the results show that our model of cultured HTPCs is a unique and highly adequate cellular model for the study of the plasticity and function of human testicular peritubular cells, which allow one to gain insights into the otherwise inaccessible human male gonad. While the scRNAseq method-derived data do not allow us to deduce to the level of gene expression in individual cells, they provide a molecular signature of HTPCs. We anticipate that this signature will stimulate future studies and promote research to better understand human testicular function.


There were no significant differences between DEL-1 and DEL-2. Scoliosis was more prevalent among adults with a DEL . Psychotic episodes were more prevalent among adults with an mUPD . In conclusion, there were no significant differences in physical health outcomes between the genetic subtypes, apart from scoliosis and BMI. The differences in health problems, therefore, mainly apply to the psychological domain.Prader–Willi syndrome (PWS) is a complex, rare genetic disorder caused by a loss of expression of paternally expressed genes on chromosome 15q11.2-q13. The most common underlying genotypes are paternal deletion (DEL) and maternal uniparental disomy (mUPD). DELs can be subdivided into type 1 (DEL-1) and  type 2 deletions (DEL-2). Most research has focused on behavioral, cognitive and psychological differences between the different genotypes. However, little is known about physical health problems in relation to genetic subtypes. In this cross-sectional study, we compare physical health problems and other clinical features among adults with PWS caused by DEL  and mUPD (N = 65). A meta-analysis, including our own data, showed that BMI was 2.79 kg/m Prader–Willi syndrome (PWS) is a complex, rare genetic disorder with an incidence of 1:16.000–1:20.000 live births ,4, whichMKRN3, MAGEL2, NDN, NPAP1, SNURF-SNRPN and clusters of small nucleolar RNA genes (snoRNAs) (6). The AS region contains the maternally expressed gene UBE3A [PWS is caused by lack of expression of a cluster of paternally expressed genes in the PWS-region on chromosome 15q11.2-q13 . The gensnoRNAs) . The AS The primary genetic mechanisms leading to PWS are paternal deletion DEL; 65–75%), maternal uniparental disomy mUPD; 20–30%) and imprinting center defects  [5–75%, ma0% and imSeveral differences between individuals with an mUPD and DEL have been described. Individuals with a DEL tend to have more compulsive behavior , better In this article, we describe the clinical characteristics of 93 adults with PWS according to genotype (mUPD or DEL). Furthermore, we provide a concise overview of the current knowledge on genetic subtype differences in health problems among adults with PWS.Approval for this study was waived by the local medical ethics committee of the Erasmus University Medical Center Rotterdam because participants were not subject to procedures or required to follow rules of behavior. In this cross-sectional study, we retrospectively reviewed the medical files of all adults with genetically confirmed PWS who visited the outpatient clinic of the Center for Adults with Complex Rare Genetic Syndromes at the Erasmus University Medical Center, Rotterdam, the Netherlands between January 2015 and June 2021. Adults with multiple genetic syndromes, such as a combination of Klinefelter and Prader–Willi syndrome, were excluded. All patients underwent a systematic health screening as part of regular patient care, including a structured interview, a medical questionnaire, a complete physical examination, biochemical measurements and a review of the medical records, as described previously . The iteUntil 2018, the genetic diagnosis was made with a methylation sensitive PCR test, based on allelic methylation differences at the SNRPN locus . In caseIn the absence of a DEL, the DNA of the index and the parents was tested with a set of highly polymorphic chromosome 15q markers to demonstrate the presence of an mUPD. As of 2018, this marker-test was replaced by SNP array . If no mUPD was identified as an underlying molecular cause, we concluded that hypomethylation was caused by an imprinting defect .Literature was searched in the databases Embase, MEDLINE (via ALL OVID), Web of Science Core Collection, Cochrane Central Register of Controlled Trials and Google Scholar. The full search strategy is included in the Statistical Package for the Social Sciences Statistics version 25.0 and R version 3.6.0. Descriptive statistics are presented as median and interquartile range (IQR) for continuous data and as count and percentage (%) for categorical data. The chi-square test  and Wilcoxon rank sum test (for continuous variables) were used to detect any differences in clinical features between the genotypes (mUPD vs. DEL and DEL-1 vs. DEL-2). Tests were only performed when at least 10 people had the genetic subtype. Logistic regression analyses were performed to detect differences in clinical features between mUPD and DEL, independent of (past or current) growth hormone (GH) treatment. Additionally, a stratified analysis for differences in health problems between the genetic subtypes was performed to visualize the impact of GH treatment. Further, a meta-analysis for BMI was performed. The result is shown as the pooled mean difference with a 95% confidence interval (CI). Data were pooled with inverse variance weighting on the number of subjects, mean and standard deviation of the studies. A fixed effect model was used when heterogeneity (assessed with I2 statistic) was <50%. p-values < 0.05 were considered statistically significant.Data were analyzed with IBM 2. Of the patients, 28 had an mUPD, 65 a DEL  and 3 an ICD. In 15 patients, genetic material from the parents was not available, making it impossible to distinguish between an mUPD and ICD. These patients were excluded from further analyses. The patient characteristics are described in A total of 111 patients  had a genetically confirmed diagnosis of PWS. The median age was 28.5 (IQR: 21.1–38.7) years and median body mass index (BMI) was 29.1 (IQR: 26.3–37.3) kg/mGender, age and BMI did not differ significantly between adults with a DEL or mUPD . The prop = 0.03), even after correcting for GH treatment (p = 0.04). Osteopenia was present in 47% of adults with a DEL (N = 21 out of 45) and 33% of adults with an mUPD (N = 7 out of 21), but this did not reach statistical significance (p > 0.05). Likewise, hypertension was twice as prevalent among adults with an mUPD  than among adults with a DEL , but this was not significant either. There were no differences in diagnoses of osteoporosis, epilepsy, hypercholesterolemia, diabetes mellitus type 2 (DM2) or hypothyroidism between the genotypes.Scoliosis was more often present in adults with a DEL  than in adults with an mUPD .Physical complaints and behavioral challenges reported by patients and/or caregivers did not differ significantly between adults with an mUPD and DEL. In both groups, complaints of constipation and daytime sleepiness were frequent. Although not significant, abdominal pain and fatigue were present approximately three times more often in adults with a DEL. Abdominal pain was present in 17% of patients with a DEL (N = 9 out of 53) versus 5% of adults with an mUPD (N = 1 out of 22). Fatigue was present in 26% of patients with a DEL (N = 13 out of 50) versus 10% of adults with an mUPD (N = 2 out of 21). Complaints of the feet, urinary incontinence, cold intolerance and swollen legs, on the other hand, were more often reported among adults with an mUPD compared to DEL . However, these differences were not statistically significant (p < 0.001). Temper outbursts, food-seeking behavior and skin picking also seemed slightly more prevalent in adults with an mUPD than in adults with a DEL . However, these differences were not significant. Psychotropic drugs were significantly more often used by adults with an mUPD than by adults with a DEL  out of 27 adults with an mUPD had a psychotic episode compared to 6 (9%) out of 65 adults with a DEL .Gender, age and BMI did not differ significantly between adults with a DEL-1 or DEL-2 . The prop = 0.18). There were no differences in diagnoses of osteoporosis, psychotic episodes, epilepsy, hypercholesterolemia, DM2, hypertension and hypothyroidism between the genetic subgroups.None of the health problems differed significantly between adults with a DEL-1 and DEL-2. Scoliosis was present in more than 80% of the adults with a DEL-1 or DEL-2. Osteopenia seemed slightly more prevalent among adults with a DEL-2  compared to DEL-1 . On the other hand, fatigue, cold intolerance, foot complaints, swollen legs and food-seeking behavior were more often reported in adults with a DEL-1 compared to DEL-2 , but these differences did not reach statistical significance. There was also no significant difference in the use of psychotropic drugs between the two groups.The stratified analysis on GH treatment shows that osteoporosis/osteopenia and obesity rates seem higher in adults who never received GH treatment . HoweverThe original search yielded 7762 articles. After deduplication, the titles and abstracts of 4174 articles were screened. Of 444 articles, we assessed the full text article, after which we included 30 articles in the literature review . The stuThere was no difference in hyperphagia between adults with an mUPD and DEL ,34,37,432 higher in adults with a DEL compared to adults with an mUPD . The cause of death was unknown in one patient and of cardiovascular origin in the other patients.Smith et al.  reportedUBE3A. The expression of UBE3A is higher in persons with an mUPD than in DEL and higher than in persons without PWS [UBE3A plays a role in the development and function of the nervous system and may indirectly alter the GABA and glutamatergic system [UBE3A can disrupt these systems, which disturbs the excitatory–inhibitory balance in the brain, possibly leading to psychosis [UBE3A can result in psychosis is supported by studies about patients with a 15q11-q13 duplication [UBE3A only [UBE3A might also contribute to the increased prevalence of autistic characteristics in patients with an mUPD [GABRG3, NDN, CYFIP1 and SNORD115 [GABRG3, NDN, CYFIP1 and the snoRNA cluster SNORD115 may act as a first hit [UBE3A may be the second hit, ultimately leading to psychosis in adults with an mUPD.Similar to previous literature, we found that psychotic episodes were more prevalent in adults with an mUPD ,51. In ohout PWS . UBE3A pc system ,57. Overlication ,59. Islelication . Additio an mUPD ,61. OtheSNORD115 . This moUBE3A may contribute to the decreased performance capacities in adults with an mUPD. This is supported by a study of Baker et al. who showed that higher UBE3A mRNA levels were associated with lower PIQ scores [NIPA1, NIPA2, CYFIP1 and TUBGCP5 are deleted, showed that verbal IQ was impaired in these patients [Several studies reported better verbal capacities and lower PIQ in adults with an mUPD compared to DEL ,17,42,48Q scores . Howeverpatients . This alpatients . Howeverpatients ,17.TUBGCP5 and SNRPN had a remarkable speech, speaking fluently but placing the words in the sentences in a random order. Language disorders in individuals with PWS were previously described by Dimitropoulos et al. [As a post-hoc analysis, we studied the occurrence of mutism or other speech/language abnormalities in relation to the genetic subtype in our cohort. In total, five adults  had impaired speech, of which three had complete mutism. What is remarkable is that all adults with complete mutism had an mUPD. The patient with an atypical microdeletion encompassing the genes between s et al. . They shNIPA1, NIPA2, CYFIP1, TUBGCP5) might play a significant role. In addition, Bittel et al. [NIPA2 and CYFIP1 mRNA levels especially were associated with several sub-items of compulsive behavior. Moreover, variants in TUBGCP5 are known to be related to obsessive-compulsive disorders [UBE3A, but also other genes within the chromosome 15q11.2-q13 region are probably involved.The studies on behavior were inconclusive. Most studies agree that compulsive behavior is more prevalent in adults with a DEL than in mUPD ,17, but l et al.  showed tisorders . Contrarisorders ,36,38. WNowadays, most people with PWS receive GH treatment during childhood, which is often continued into adulthood. Previous research showed that the positive effects of GH treatment are seen in both adults with a DEL and mUPD . CoupayeLike every study, our study has strengths and limitations. A strength of our study is the relatively large cohort of adults with a confirmed genetic diagnosis of PWS. Furthermore, we used a systematic health screening to evaluate the health of all adults with PWS attending our clinic, which improved the quality of our data. Moreover, we provide a thorough overview of the current knowledge, including a meta-analysis, and relate the previous findings to our own results. However, some limitations remain. Fifteen patients were excluded from analyses because it was impossible to distinguish between mUPD and ICD due to unavailable parental genetic material. Since this group generally resembles an older population with less involved parents, this might have influenced the results. However, the age difference between adults with an mUPD and DEL is small, which suggests that the groups are still comparable. Furthermore, since additional tests for osteoporosis were only performed on indication, we had, relatively, many missing values for osteoporosis/osteopenia. We had no data on the chronicity of the psychosis, i.e., whether it was a single episode of psychotic behavior or a recurrent form of psychosis. Furthermore, the questionnaire on physical complaints, symptoms of disease and behavioral challenges was not filled out by all participants, which reduced our statistical power. Therefore, more  differences may exist between the genetic subtypes, and our results should be interpreted with caution.To further explore the differences in health problems between adults with an mUPD, DEL-1 and DEL-2, more research is needed. Further research should not only focus on the differences in behavior and cognition, but also on physical aspects, such as BMI and scoliosis. Moreover, differences in language abilities between the genetic subtypes should be studied. In addition, more research on atypical microdeletions is needed to further unravel the influence of the individual genes in the chromosome 15q11.2-q13 region on specific components of the PWS phenotype.Differences in health problems between adults with an mUPD and DEL seem mostly present in the psychological domain. Especially, psychotic episodes were more frequent in adults with an mUPD. Apart from scoliosis and the meta-analysis on BMI, there were no significant differences in physical health outcomes between the genetic subtypes. More research is needed to further explore the differences in health problems between adults with different genetic subtypes. In addition, more data on individuals with an atypical microdeletion are needed to unravel the influence of the genes in the chromosome 15q11.2-q13 region on the PWS phenotype.