Lymph node involvement has been shown to be one of the most relevant prognostic factors in a variety of malignancies; this is also true of endometrial cancer. The determination of the lymph node status is crucial in order to establish the tumor stage, and to consider adjuvant treatment. A wide range of surgical staging practices are currently used for the treatment of endometrial cancer. The necessity and extent of lymph node dissection is an ongoing controversial issue in gynecological oncology. Lymph node surgery in endometrial cancer is technically challenging, and can be time consuming because of the topographic complexity of lymphatic drainage as such, and the fact that the lymph nodes are directly adjacent to both blood vessels and nerves. Therefore, profound and exact knowledge of the anatomy is essential. Sentinel lymph node mapping was recently introduced in surgical staging with the aim of reducing morbidity, whilst also obtaining useful prognostic information from a patient’s lymph node status. The present review summarizes the current evidence on the role of lymph node surgery in endometrial cancer, focusing on the embryological, anatomical, and technical aspects. Endometrial cancer is the most common gynecological cancer in developed countries; more than 380,000 new cases are reported each year worldwide . In accoAccording to the ESMO–ESGO–ESTRO guidelines, minimally-invasive surgery is recommended for the surgical management of low-  and intermediate-risk endometrial cancer  .Histologically, endometrioid adenocarcinomas are the most common type of endometrial cancer. Other subtypes, including adenosquamous, clear-cell, and serous carcinomas are associated with a poorer prognosis. They are typically more aggressive, and in a more advanced FIGO stage. Endometrial cancer grows into the surrounding tissue, most frequently the myometrium and the cervix. Lymphatic spread also occurs, mainly to the pelvic lymph nodes and then to the para–aortic nodes. The probability of lymph node metastasis across all of the FIGO stages is 15% .Lymph node involvement has been shown to be one of the most relevant prognostic factors in a variety of malignancies. The determination of the lymph node status is crucial in order to establish the tumor stage, and to consider adjuvant treatment . In two The introduction of minimally-invasive surgery in routine gynecology and oncology has minimized the invasive nature of many operations in the female pelvis and retroperitoneum. As mentioned earlier, minimally-invasive surgery benefits patients in many ways. Robotically-assisted surgery is the most dynamic advancement of minimally-invasive surgery, and a significant step in terms of technical evolution. The better visualization of the field of surgery by means of 3D technology and the extension of surgical instruments to 7 degrees of freedom permit the use of minimally-invasive surgery even in complex situations such as obesity or severe adhesions.A wide range of surgical staging practices are currently used for the treatment of endometrial cancer. The spectrum of lymph node surgery includes sentinel lymph node mapping and systematic pelvic or pelvic and para-aortic lymph node dissection. Peritoneal mesometrial resection  involves therapeutic pelvic and para-aortic lymphadenectomy (tLNE).The aim of the present review is to summarize the current evidence on the role of lymph node surgery in endometrial cancer, focusing on the anatomical, embryological, surgical, and technical aspects. The technical challenge of MIS  is compared with conventional laparotomy.Lymphatic drainage follows the blood supply of the respective organs. As the arterial supply to the uterus and uterine appendages is derived from two different sources, lymphatic drainage is provided by the pelvic as well as the para-aortic pathways. Due to the peculiar ontogenetic anatomy of the female genital tract, the lymphatic drainage is multidirectional and complex, and has a direct impact on the surgical strategy for endometrial cancer . Lymph nAs shown in As shown in The lymphatic drainage in the lower part of the vagina and the external genital organs is achieved through the superficial inguinal lymph nodes flowing into the external iliac lymph nodes. The lymphatic drainage from the upper part of the vagina is achieved mainly through the internal iliac lymph nodes. From the uterine cervix, lymphatic pathways reach the lymph nodes in the region of the large pelvic vessels and their branches: the external and internal iliac lymph nodes, the obturator lymph nodes, and the sacral lymph nodes. The subsequent lymph node chain, namely the common iliac lymph nodes and the lumbar lymph nodes, is located around the common iliac artery and the abdominal aorta. Lymphatic pathways from the uterine fundus, the fallopian tubes, and the ovaries course in the suspensory ligament of the ovary  along the ovarian vessels to the lumbar lymph nodes. The lymphatic pathways from the tube and the uterine corpus extend from the round ligament of the uterus through the deep inguinal ring to the lymph nodes in the groin, namely the superficial inguinal lymph nodes, and further on to the external iliac lymph nodes ,20. FiguMalignancies of the uterus metastasize, as described earlier, by the lymphatic or hematogenic route, or by the direct invasion of neighboring structures. Since metastasis is mainly caused by lymphogenic tumor spread, and since the lymph drainage of the female genital tract (such as the uterus) is highly complex compared to other organs, the embryology of the lymphatic system will be considered in the following figure a,b. EmbrHistorically, the investigation of the lymphatic vessels started in the 17th century. The anatomy of the large part of the lymphatic system had been described by the beginning of the 19th century. In 2010, Ribatti et al. presented an historical review of the embryonic origins of lymphatic vessels .The lymphatic system develops in close association with the venous system from the fifth week of gestation a 22]. E. E22]. ELymphatic drainage is achieved in endometrial carcinoma, as shown in the anatomical overview: on the one hand, through the pelvic lymph nodes, and on the other hand, through lymphatic pathways which can drain directly into the para-aortic regions through the adnexa. For this reason, para-aortic lymph nodes may be positive even in the presence of unaffected pelvic lymph nodes ,20,22.The pelvic lymph node groups include those of the obturator fossa, the lymph nodes in the region of the internal iliac artery and vein, the lateral and medial lymph nodes in the region of the external iliac artery and vein, and in the region of the common iliac artery and vein. In addition to the previously-mentioned vessels, the area of surgery encompasses other structures which must be visualized and protected. These include the ureter, the obturator nerve, the genitofemoral nerve with its femoral and genital branches, the lumbosacral trunk, and the superior hypogastric plexus in the region of the presacral lymph nodes ,22. In tThe para-aortic lymph node groups include the cranial portions of the common iliac artery, the region of the caudal vena cava, including the aorta and the inferior mesenteric artery, and further cranially, the interaortocaval tissue extending to the renal pedicle, which becomes visible through the renal vein ,22. DuriThe challenge of cancer surgery is twofold: to strive for a maximum radical operation on the one hand, in order to ensure curative therapy, and to strive for the least loss of function on the other hand, in order to preserve the quality of life after surgery . In the But why exactly this area? The lymph nodes that can be accessed with the least collateral damage are removed. Examples of these are the lymph nodes to the obturator nerve, but not further dorsally. Another example is the resection to the cross-over point of the renal vein, but not further cranially.We only operate within accessible anatomical areas, and not in the corresponding compartments derived from anatomical or embryological studies.Lymphadenectomy has been referred to by various names in the published literature. As listed in The individual aspects are addressed here in detail.The surgical management of endometrial cancer has undergone significant changes in the last few decades, including a change from open to minimally-invasive surgery. When the findings of the GOG#33 trial were published in 1998, the International Federation of Gynecology and Obstetrics (FIGO) introduced a shift from clinical to surgical staging, including lymphadenectomy ,24. SincPreoperative imaging studies to identify positive lymph nodes with techniques including magnetic resonance imaging, positron emission tomography or computed tomography have been unsatisfactory because of their poor sensitivity . SurgicaThe revised FIGO staging system in 2009 divided stage IIIC endometrial cancer into IIIC1 (positive pelvic nodes) and IIIC2 (positive para-aortic nodes with or without positive pelvic nodes), reflecting the fact that the prognosis is worse when para-aortic lymph nodes are involved .We still lack a standardized definition of adequate complete lymphadenectomy . The curp < 0.001) of cases [The outcome of an international survey about the surgical management and adjuvant treatment of endometrial carcinoma throughout the world was published in 2015 . Six-hunof cases .The probability of lymph node metastasis across all FIGO stages is 15%.A longer period of overall survival and fewer deaths were noted in patients with endometrial cancer who received pelvic lymphadenectomy combined with para-aortic lymph node dissection compared to those who received pelvic lymphadenectomy alone ,29.Three percent of the patients had isolated positive para-aortic lymph nodes and no positive pelvic lymph nodes. Of the 3%, 67–100% had lymph node metastases in a high para-aortic location, i.e., between the renal vein and the inferior mesenteric artery .In a study performed by Mariani et al., 281 patients with endometrial cancer received lymphadenectomy. Twenty-two percent of patients with high-risk endometrial cancer had positive lymph nodes. Of the 22%, 51% had positive pelvic and para-aortic nodes, 33% only had positive pelvic lymph nodes, and 16% had isolated para-aortic nodes. Seventy-seven percent of patients with para-aortic nodes had metastases above the inferior mesenteric artery. These findings indicate that para-aortic lymphadenectomy up to the renal vein is advisable .In terms of surgical boundaries for pelvic lymph node dissection, the published literature recommends the removal of lymphatic tissue from the deep circumflex iliac vein to the midpoint of the common iliac artery . CompletIn a multitude of solid malignancies, the lymph node count has become a marker of the adequacy of lymph node dissection. The results of two retrospective studies showed that patients with endometrial cancer had improved survival when 10–12 lymph nodes were removed ,34. The Systematic pelvic and para-aortic lymph node dissection enables the clinician to provide tailored adjuvant therapy and reduce adjuvant therapy-related morbidity .According to the ESMO–ESGO–ESTRO guidelines, lymphadenectomy is not recommended in patients with low-risk endometrioid cancer. A systematic lymphadenectomy should be recommended to patients with major risk factors  because of the higher prevalence of nodal metastasis in this population. In cases of intermediate-risk patients , the data have shown no survival benefit; a lymphadenectomy may be considered in this group for staging purposes .Complete pelvic and para-aortic lymph node dissection is associated with major comorbidities, including lymphedema, lymphocysts, cellulitis, and damage to adjacent nerves. Furthermore, complete lymph node dissection is technically difficult in obese women, and the latter constitute a large part of patients with endometrial cancer .Historically, the first successful instance of SLN mapping was reported in 1977; the procedure was a lymphangiography of the penis . Since tAs shown in SLN mapping must possess a high sensitivity and negative predictive value in order to be an acceptable staging method . The FIRPelvic sentinel lymph nodes follow two consistent lymphatic pathways: the upper paracervical pathway drains the medial external and/or obturator lymph nodes, and the lower paracervical pathway drains the internal iliac and/or presacral nodes. Furthermore, a non-pelvic pathway courses along the infundibulopelvic ligament to the para-aortic lymph nodes. SLN mapping for endometrial cancer revealed metastases in areas not usually included in a standard lymph node dissection. Sentinel lymph nodes are usually seen as ‘colored nodes’ or ‘radioactive nodes’ without regard to lymphatic anatomy . In a prAn increasing body of evidence suggests the non-inferiority of sentinel lymph node mapping compared to systematic lymphadenectomy in endometrial cancer . In 2014p = 0.63). With regard to the detection of positive pelvic lymph nodes, the authors concluded that sentinel node biopsy may be considered superior to lymphadenectomy [Recent data suggest that, in endometrial cancer, sentinel lymph node mapping does not influence the oncologic outcome of disease. Based on six studies comprising a total of 3536 patients, the authors of this meta-analysis concluded that, in terms of recurrence rates  and the detection of positive para-aortic lymph nodes, sentinel node mapping is not inferior to standard lymphadenectomy. The overall recurrence rates revealed no significant difference  staining and deeper serial sections) enhances the detection of malignant cells. The clinical significance of the increased detection of isolated tumor cells and micrometastasis is currently uncertain. Furthermore, the strategies used for the pathological investigation of SLNs vary among institutions and within the published data .Future trials will have to address these questions and determine oncologic outcomes after the use of sentinel node mapping for endometrial cancer.Our extended knowledge of the embryological development of organ compartments, tissue boundary control, and their association with tumor spread and tumor progression has resulted in a new approach in cancer surgery: compartmental surgery in cancer, as established by M. Höckel in gynecological oncology .According to this concept, tumor spread is initially restricted to permissive ontogenetic compartments and their corresponding lymph node basins. The complete surgical removal of these embryologically-defined areas by whole compartment resection with intact margins following ontogenetic planes will result in optimal tumor control . EmbryolThe transfer of this concept to endometrial cancer led to peritoneal mesometrial resection (PMMR) combined with pelvic and para-aortic therapeutic lymphadenectomy (tLNE) . The lymFollowing TMMR and therapeutic lymphadenectomy without adjuvant radiation, Höckel et al. conducted a prospective single-center study comprising 305 cervical cancer patients with FIGO stage Ib to IIB disease, of whom 71 had positive lymph nodes; the authors noted recurrence-free and overall 5-year survival rates of 94% and 96%, respectively . The preAs described earlier, the lymphatic network of the uterus can be visualized by the injection of indocyanine green (ICG) as a guide in compartmental surgery. This is conceptually different from its use in sentinel lymph node detection. In a trial published in 2016, Kimmig et al.  investigSeveral trials over the last few years have shown that minimally-invasive surgery is equivalent to open surgery with regard to the adequacy of the surgical resection and lymph node counts . The addWithin the spectrum of surgical laparoscopy, pelvic and paraaortic lymphadenectomy remain difficult high-end procedures. As described earlier, lymph node surgery in endometrial cancer is technically challenging, and can be time consuming because of the topographic complexity of lymphatic drainage as such, and the fact that the lymph nodes are directly adjacent to both blood vessels and nerves. Therefore, profound and exact knowledge of the anatomy is essential.What is the most appropriate time to switch from the umbilical optical trocar to the suprasymphysiary trocar (perspective from above/below versus below/above)?What is the most suitable position for the working trocars with reference to the steps of surgery (perspective from above/below as well as below/above) so that the mutual angle of the instruments, as well as the mechanical actions of the surgeon, can be achieved smoothly and conventionally (i.e. not towards the surgeon but away from the surgeon)?The following questions are yet to be answered conclusively:Conclusion: the course of the operation should not be oriented to the technical access and its limitations. Rather, it should be oriented towards the underlying anatomy and the embryological–oncological aspects.Minimally-invasive surgery  has rapidly replaced the open approach in endometrial cancer staging. Within the spectrum of surgical laparoscopy, pelvic and paraaortic lymphadenectomy remain difficult high-end procedures. Profound and exact knowledge of anatomy, including uterine lymphatic drainage, is essential for every surgeon. Lymph node assessment has also undergone significant changes after the introduction of SLN mapping. As in other diseases, the concept of ‘less is more’ has pervaded the treatment of endometrial cancer.However, a number of significant questions remain unanswered, regarding the following: oncologic outcomes when sentinel lymph node mapping is used, the clinical management and impact of low-volume metastases in SLNs, and a consensus concerning the technique. These issues will have to be investigated in further large prospective trials.


A seamless thin-walled hollow metallic cylinder with array of micro-perforations is one of the key components for some products. Normally, these micro-perforations are formed by removing material from the given metallic hollow cylinder (pipe or tube) one by one or row by row. To efficiently and flexibly manufacture such a highly perforated hollow cylinder, this paper proposed a hybrid technique combining extrusion moulding process and electroforming process. In the hybrid technique, the extrusion moulding process was used to create polymer extrusion patterns on the outside surface of the given stainless steel (SS) pipe, and then the electroforming process was carried out using the SS pipe as the mandrel. The formation of the polymer extrusion patterns was simulated and extruding molding experiments were carried out to examine the feasibility of the various mandrels. Electroforming experiments were implemented to verify the achievement of the seamless perforated thin-walled hollow cylinder. It was found that five different types of polymer extrusion pattern were able to be obtained on the same extruding pipe just by adjusting some extruding conditions and parameters, and correspondingly four types of perforated hollow cylinder with different tapered orifices are produced after the electroforming process. The obtainable perforations are: perforation with double conic-orifices, perforation with hemispheric orifice and conic orifice, unidirectionally tapered perforation, and straight-walled perforation. The geometric profile of the extrusion patterns is highly dependent on the processing conditions and parameters. The proposed hybrid process represents a promising alternative process to fabricate seamless thin-walled perforated hollow metallic cylinder efficiently, flexibly, and with low cost. Thin-walled metallic articles and components featuring a variety of micro-perforations are frequently used for various industrial and consuming products, such as nickel rotary screen for textile printing, stencil for surface mounting technology (SMT), filter sieve, shaver foil, fine metal mask (FMM) for OLED, etc. ,2,3,4,5.Until now, most of the perforated components and articles are formed on the planar conductive substrate by reversely duplicating the shape of photoresist patterns using the electroforming process. The photoresist patterns are normally prepared by the photolithographic process. Flat perforated thin plate products such as SMT stencil, lead frame, and ultraprecision nebulization plate are generally fabricated by a photolithography-electroforming hybrid process, which is very tedious and costly. However, the standard photolithographic process makes it extremely difficult to prepare the required photoresist patterns on the curved substrate, since thick photoresist film is not able to be uniformly coated on the curved surface, and homogeneous radiation of the exposure energy on such a surface is not easy be realized. Therefore, to manufacture curved perforated plates and hollow cylinders, the preparation of the photoresist patterns on the metallic substrate used as the electroforming mandrel needs to be significantly innovated. Herein, we present a typical example of electroforming nickel rotary screen. The nickel rotary screen which is a thin-walled hollow cylinder with a vast number of significantly tapered micro-perforations is manufactured mainly with the over-growth deposition manner. Preparation of the photoresist patterns on the cylinder metallic mandrel for electroforming the nickel rotary screen are distinct from the standard photolithographic process. Preparation involves a series of operation steps: shaping the cylinder, pre-treating the cylinder surface, depositing nickel coating, depositing copper coating, precision-turning and polishing the cylinder, forming cavities, depositing chrome coating, filling photoresist material into the cavities and over-growth depositing nickel, etc. ,15,16. HThis paper proposes a novel technique to fabricate seamless thin-walled hollow metallic cylinders with a vast number of micro-perforations by using the combination of the electroforming process and extrusion molding process. In the proposed technique, the seamless perforated hollow metallic cylinder is created by such a two-step process: form extrusion polymer patterns via an extrusion molding operation on the outside surface of a perforated stainless steel (SS) pipe, and subsequent electrodeposition of metal on the SS pipe. The proposition of this technique results from such a concept: the geometric shape of the extrusion polymer pattern during polymer extrusion molding can be flexibly changed just by altering the operating conditions. Correspondingly perforations with versatile geometric profile are expected to be obtained with the inversely duplicating mechanism after electroforming operations are implemented. In the following sections, feasibility of this hybrid process is investigated numerically and experimentally after the working principle is introduced.As shown in The main procedures of the described hybrid technique are illustrated in According to electrodeposition theories, geometric shape of the electroformed perforation is highly dependent on the geometric shape of the polymer pattern on the mandrel because electroforming is actually a duplication process. To better understand the feasibility of obtaining the desired feature, the formation of the polymer pattern during the polymer extrusion molding are simulated numerically.7 is referred to as the axis of the model and Γ3 is referred to as the interface between the air and the melt polymer. Some assumptions are made as follows:(1)the melt polymer is viewed as a kind of viscous fluid and is homogeneous in its material properties;(2)temperature of the melt polymer keeps constant and is the same everywhere;(3)neglecting the influence of gravity during polymer extruding-out;(4)outlet pressure of the melt polymer is set to be zero;(5)feeding rate of the extruding rod keeps constant;(6)melt polymer is considered as an incompressible liquid.For the sake of simplicity, a 2D model is established shown in  in whichTo understand the shape evolution of the polymer extrusions, the level set method is adopted. The level set method, which was first proposed by Osher et al. 1988) , is wide88 , is wThe final geometric shape of the extruded polymer pattern is, of course, determined by the ultimate shape of the two-phase interface between the solid polymer and the air, and the geometric shape of the extrusion pattern can be presented by simulation using the level set function (see Equation (1)) which has been proven very effective in solving two-phase interface problem . Generalpolymer and ρair are the density of the air and the melt polymer respectively, while uair and upolymer are the kinematic viscosity of the air and the melt polymer respectively.The density ρ and kinematic viscosity u of the interface are given as follows:st is the surface tension of the interface. The surface tension, Fst, is determined by the following formula:The Navier–Stokes equation and continuity equation are employed to describe mass and momentum transfer of the interface which are presented as follows:  follows :δ = 6|Φ is used to simulate the geometric shape of the extrusion pattern. The required initial values of some parameters used for the numerical analysis are listed in In summary, as described above, four types of different extrusion polymer pattern are able to be obtained numerically only by changing some operating parameters during the polymer extrusion molding based on the same SS pipe. This verifies the feasibility of the proposed idea. In the following sections, the related verification experiments are depicted and their results are analyzed.A stainless steel (SUS 304) pipe  with its one end being seamed was used as the extruding mold. Circular micro-perforations with straight wall were fabricated on the stainless steel pipe. The parameters of the perforated pipe used as the substrate for the electroforming are shown in 2. The electroforming time was 4 h. The polymer extrusions were removed chemically after the electroforming process. Topologies and morphologies of the polymer extrusions and the formed micro-perforations were examined by digital camera, scanning electron microscope  and digital microscope .To comprehensively evaluate the practicability of the proposed hybrid technique, the experiments using the electroforming mandrels with five types of polymer pattern shown in It was found experimentally that gibbous extrusion patterns can be formed under such conditions: heating temperature of the polymer, 210 °C; feed rate of the extruding rod, 0.5 mm/s; feed time, 1.5 s. As shown in Compared with the former case described in the As described previously, there are two mechanisms to control the formation of the electroformed features: reversely duplicating the shape of the mandrel and over-growth of the deposit surrounding the insulating pattern. Correspondingly, there are two methods to produce the seamless hollow cylinder with unidirectionally tapered perforations: electrodeposition duplication of the extruded polymer patterns show in  and overBased on the polymer mandrels shown in As shown in In order to create a seamless thin-walled perforated hollow cylinder more conveniently and cost-effectively, a novel hybrid technique combining the extrusion molding process and the electroforming process was proposed. Both simulations and experiments were carried out to verify the feasibility of this proposal. Some conclusions are drawn as follows.(1) Five types of polymer extrusion mandrels with different geometric profile were able to be achieved from the same extruding metallic pipe only by changing some extruding molding conditions and parameters, which is verified by both simulation and experimentation. The extruding molding is a very cost-effective method to create various mandrels for the electroforming process.(2) The seamless thin-walled hollow cylinder with a great number of perforations is able to be achieved by electroforming metal on the extruding metal pipe with polymer mandrels. The orifice shape of the perforation can be changed in terms of the polymer mandrels used. Correspondingly, four kinds of perforations are created: perforation with double conic-orifices, perforation with hemispheric orifice and conic orifice, unidirectionally tapered perforation, and straight-walled perforation. The proposed hybrid technique facilitates the manufacture of seamless thin-walled hollow cylinder with various perforations.(3) The geometric profile of the formed perforations is controlled by two formation mechanisms: over-growth electrodeposition and through-mask electrodeposition.(4) The hybrid technique can produce thin wall cylinder component with changeable perforation in a single manner, conveniently and cost-effectively, comparing with other process. A drawback of the hybrid process is that it requires precision mode and special designed apparatus. However, this can’t overcome advantages of the technique.


There is an ever-increasing appreciation for our gut microbiota that plays a crucial role in the maintenance of health, as well as the development of disease. Probiotics are live bacteria that are consumed to increase the population of beneficial bacteria and prebiotics are dietary substrates intended to promote the propagation of beneficial bacteria. In order to optimize the use of probiotics and prebiotics, a more complete biochemical understanding of the impact that these treatments have on the community and functioning of the gut microbiota is required. Nucleic acid sequencing methods can provide highly detailed information on the composition of the microbial communities but provide less information on the actual function. As bacteria impart much of their influence on the host through the production of metabolites, there is much to be learned by the application of metabolomics. The focus of this review is on the use of metabolomics in the study of probiotic and prebiotic treatments in the context of human clinical trials. Assessment of the current state of this research will help guide the design of future studies to further elucidate the biochemical mechanism by which probiotics and prebiotics function and pave the way toward more personalized applications. Bacillus bulgaricus to individuals to mitigate the production of these harmful toxins. Another early pioneer in this area was Yale bacteriologist, Leo Rettger who reported in 1920 that the consumption of Lactobacillus acidophilus could successfully recolonize the human gut as a curative [Research in the area of probiotic and prebiotic interventions has increased exponentially in recent decades. As defined by the International Scientific Association for Probiotics and Prebiotics, probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts, whereas prebiotics are substrates that are selectively utilized by host microorganisms conferring a health benefit . This nocurative . In thisIn the early 1990s, with the development of nucleic acid sequencing methods, the interest in the microbiome was re-invigorated. New methods including the analysis of ribosomal small subunit RNA gene probes, e.g., 16S ribosomal gene analysis, quickly found their way into the study of the intestinal environment. Computational methods enabled the determination of the microbial community with an unprecedented level of detail. However, beyond composition, it must be recognized that much of the impact of the bacterial community on the host is transmitted through the metabolites that are produced. Microbes produce or transform a wide range of circulating chemicals including amino acids, fatty acids, bile acids, hormones and vitamins ,5.The link between microbial community composition and metabolic activity is indirect and has been shown to be strongly influenced by functional redundancy and metabolic plasticity ,7,8. FunLactobacillus paracasei and some of this group also received a prebiotic composed of galactooligosaccharides. Metabolomics analysis using 1H NMR revealed that the metabolome of the conventional and conventionalized quickly converged and were distinct from the HBM groups. Furthermore, the HBM group receiving only the probiotic was distinct from the group that received both the probiotic and prebiotic. This study leveraged the tremendous level of control available in mouse models to demonstrate how the fecal metabolome can provide unique information on nutrient-microbiota relationships. Experimental models on the use of probiotics and prebiotics have provided a detailed foundation on the physiological impact of these interventions. Murine studies have included investigations into diabetes, neurodevelopmental disorders, non-alcoholic steatohepatitis and more ,10,11. AThe ultimate goal of much of the research on probiotics and prebiotics is to aide in the design of more effective and personalized interventions to improve human health. Unfortunately, the exquisite level of control available in animal studies including rigorously controlled diets and environments, strictly defined probiotic and prebiotic dosing schedules and the opportunity for invasive sampling are not available in humans. The focus of this review is to evaluate the current state of the science in analyzing the effects of probiotics and prebiotics in the context of human clinical trials. These studies will be evaluated to understand the challenges in study design, analytical approaches and interpretation. Furthermore, the focus will only include those studies which employed a discovery-based metabolomics approach to more completely interrogate the action of prebiotics and probiotics on human health.The studies that met the focused criteria were found in PubMed using the search string (microbiome OR microbiota) AND (metabolomics OR “metabolite profiling” or “metabolic profiling”) AND (probiotics OR prebiotics). Careful review yielded 15 studies that reported results from some form of human clinical trial and included a discovery-based metabolomics approach. These studies were found to focus on five main areas of health and the results of these studies will be described below. Details of each of these studies are presented in One of the main questions regarding prebiotic and probiotic interventions is how much they can influence the community and function of a healthy gut. The study by Vandeputte et al. examined the effects of inulin-type fructans in a randomized, double-blind, placebo-controlled, crossover trial . In thisBifidobacteria and Anaerostipes, along with a decrease in Bilophilia. The increase in Bifidobacterium was expected as the prebiotic concept actually emerged from the observation of selective stimulation of Bifidobacterium with inulin fermentation [The prebiotic intervention was composed of daily consumption of chicory derived inulin. Microbial community analysis was carried out using 16S rDNA gene sequencing. The results showed that there were significant alterations in the microbiota with inulin consumption. In particular, there was an increase in entation . This prentation .The second study examined the prebiotic effects of wheat bran extracts (WBEs) on the microbial community and metabolome . In addiThe metabolomics analysis provided relative quantification on a total of 285 different metabolites with an average of 91 metabolites per sample. To provide more quantitative results of the short-chain fatty acid, 2-ethylbutyric acid, was used as an internal standard. Multivariate analysis using partial least squares discriminant analysis (PLS-DA) suggested that wheat bran intake was associated with higher levels of cycloalkenes, alcohols, and esters, but no individual metabolites met a threshold for significance.Although the fecal water cytotoxicity and genotoxicity assays did not reveal a statistically significant difference between the prebiotic intervention and placebo, a PLS-DA showed that metabolite profiles were clustered based on the degree of cytotoxicity or genotoxicity of the samples. The analysis showed that acids, alcohols, and esters were associated with greater cytotoxicity, while cycloalkanes and a set of terpines including α-pinene, α- and β-phellandrene, limonene, 3-carene and camphene were associated with low toxicity. Terpines are secondary metabolites produced by plants and have been shown to have antioxidant and antimicrobial effects . AnotherBifidobacteria and Bacteroides. The metabolomics analysis was carried out using 1H NMR and found that the increased levels of Bifidobacterium were correlated to an elevation in the fecal concentrations in lactate, glutamate, ornithine and caproic acid. As lactate is product of Bifidobacerium metabolism, this result supports the prebiotic simulation of that bacterium. The changes in glutamate and ornithine are likely associated with changes in the diet. Diet records did not indicate any differences in protein intake between the treatment groups, but when correlating Bifidobacterium levels with protein intake across all subjects, the levels of protein consumption were higher in those with higher Bifidobacterium levels. The metabolomics data in this study supported the use of prebiotics to alter both the level and function of Bifidobacterium and also demonstrated how metabolomics analysis can reveal additional insights into the effects of dietary patterns in the study.During the aging process, alterations in the immune system have been observed and recent studies have shown that the microbiota and the microbial metabolome can impact certain immunological functions . In the The first associations between the gut microbiota and obesity were reported by Turnbaugh et al. in studies using fecal transplants from obese humans into germ-free mice . When im1H NMR analysis of urine and serum revealed several interesting correlations between metabolite changes and gut bacteria. The levels of Propionibacterium and Bacteroides vulgatus were both decreased by the prebiotic treatment and correlated with decreased plasma levels of phosphatidylcholine and lactate. The levels of these bacteria and metabolites also appear to be associated with lower adiposity. An increase in the levels of Collinsella, a genus of Actinobacteria was observed in the prebiotic group and was correlated to the levels of urinary hippurate. In contrast to most studies which focused on the fecal metabolome, this study examined urine and serum and found several novel correlations between the gut microbiota and systemic metabolite levels.In a study of obese women, the effects of inulin-type fructans (ITFs) was examined to evaluate the effects on gut microbial composition and systemic metabolism . This do® polydextrose (LU), (3) Bifidobacterium animalis subsp. Lactis 420® (B420) and (4) LU + B420, with the intervention lasting six months. The fecal metabolome was analyzed by 1H NMR and serum bile acids were measured using UPLC/MS/MS as described by Han et al. [Christensenellaceae was highly elevated in the groups treated with LU alone and in combination with B420. These levels were also positively correlated with the fecal branched chain fatty acids (BCFAs), isobutyric acid, isovaleric acid, 2-methylbutyric acid and 3-methyl-2-oxovalerate and the plasma bile acids, lithocholic acid and hyodeoxycholic acid. The BCFAs are typically generated by microbial fermentation of undigested proteins in the colon, but it has been shown that these metabolites can be derived from polydextrose consumption [de novo lipogenesis [Hibbert et al. reported a study involving both probiotic and prebiotic interventions in a randomized, double-blind, placebo-controlled trial of 134 overweight adults . This stn et al. . The ressumption . A recenogenesis . In thisBifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium longum subspeces infantus or a control formula with no supplements. Breastfed controls were also included in the analysis. The cohort was composed of 49 infants in the control group and 48 infants in the intervention. Diversity and composition of the fecal microbiota were determined by 16S rRNA sequencing and the fecal metabolome was analyzed by UPLC/MS. Infants in the intervention group showed decreased numbers of Bacteroides and Blautia, along with alterations in lipids and unknown metabolites at one month. Over the remainder of the study period, the microbiota composition and metabolome converged. Significant differences were observed between breast fed and formula fed infants with the major discriminating metabolites assigned to sterol lipids, glycerophospholipids and fatty acids. A targeted short-chain fatty acid analysis could also distinguish breast from formula fed infants with breast fed infants having lower proportions of propionate, butyrate, valerate and isovalerate, while formula fed infants had higher amounts of pyruvic acid and lactic acid.The composition of the infant gut microbiota has been shown to be dramatically influenced by dietary factors ,29. Clea1H NMR was carried out on 8 subjects from the placebo group and 11 from the probiotics group. The probiotic group experienced a significant decrease in minutes of crying per day at days 14 and 21 of the study. Interestingly, no significant changes in the composition of the microbiota were found over the course of the study, but 16 metabolites were found to be significantly different at day 21 between the placebo and probiotics groups. An interesting set of metabolites including the amino acids alanine, leucine, and isoleucine, along with the organic acids, 2-hydroxyisovalerate and 2-oxoisocaproate, were altered, suggesting a significant change in branched chain amino acid metabolism with the probiotic.Infantile colic is a condition experienced by somewhere between 10% and 40% of infants and is characterized by extended periods of inconsolable crying . It is cBifidobacteria probiotic can reduce the morbidity of RRIs and alter the urinary metabolomics profiles in preschool children [Recurrent respiratory tract infections (RRIs) are a common occurrence in preschool age children. Pidotimod is an immunostimulant that is used to treat RRIs in children. A recent metabolomics study of children with RRIs taking Pidotimod showed that the drug can partially restore the metabolic alterations induced by RRIs, but some of the microbiota derived metabolites remained altered . In the children . A cohorLactobacillus acidophilus-NCIMB701748 and Bifidobacterium bifidum-ATCC15696 (marketed as Infloran®). Fecal samples were collected before, during, and after probiotic intake from 7 patients and 3 time-matched controls. Samples were also collected following discharge from the NICU. The bacterial community profiling showed greater abundance of Bifidobacterium and Lactobacillus during the probiotic treatment compared to controls. After the supplementation stopped the Lactobacillus levels went down, but the Bifidobacterium concentrations remained high indicating successful colonization. The biggest difference in the metabolomics profiles was between subject post-discharge compared with those in the NICU. This would suggest that exposure to the new environment has a greater impact on these infants than the probiotic. Unfortunately, specific metabolite assignments were not made in this study.Probiotics have been used in a number of studies of pre-term infants with much of the attention going toward the treatment of necrotizing enterocolitis and late onset sepsis . For theLactobacillus spp. levels were significantly increased after the eight-week study, while the levels of Bifidobacteria did not change.Irritable bowel syndrome is the most common functional bowel disorder and is characterized by the association of abdominal pain or discomfort, with changes in stool frequency or consistency without a clear disease etiology . In the NMR-based metabolomics analyses were carried out on both serum and fecal samples. For both types of samples, no filtration or extraction methods were used so that the samples contained all of high molecular weight components including proteins and lipoproteins in serum and proteins, fiber and mucopolysaccharides in feces. Pattern recognition methods including principal components analysis were carried out on binned spectra. Analysis of the fecal samples suggested no significant differences between the placebo and probiotic treatment groups. The authors suggest that this is due to the confounding presence of unidentified metabolite peaks related to individual diets. The biggest effect found in the serum metabolomics analysis was between males and females. A well-known difference can be seen in the lipoprotein signatures of males and females in serum NMR spectra . This leThis observation of lipoproteins enabled the detection of some potential sex related effects of the probiotics, but unfortunately the broad baseline features from these macromolecules interferes with the ability to quantitatively fit the NMR peaks. The spectral profiles were compared using integral binning which is less quantitative due to the effects of spectral overlap and interfering baseline components. This analysis approach was also used for the fecal samples. These added sources of variability may have limited ability to detect significant metabolite differences.L. plantarum and L. fermentum and contained xylooligosaccharides as a prebiotic. Fecal samples were cultured before and after the intervention. It was found that the number of viable fecal Lactobacilli from IBS-D patients was significantly lower at baseline than controls and increased significantly after the intervention. No differences were found in the control group before and after the intervention. Serum NMR showed several significant differences between the IBS-D and control groups before the intervention, including elevated levels of acetoacetate, myo-inositol and sarcosine, along with reduced levels of threonine and methionine. Comparing the serum metabolome of the IBS-D groups before and after the intervention revealed a decrease in acetone and an increase in the concentrations of choline, phenylalanine and the three branched chain amino acids, leucine, isoleucine and valine.In another study of IBS, Noorbakhsh et al. used NMR-based metabolomics to investigate the metabolic impact of a synbiotic yogurt treatment on a cohort of diarrhea predominant IBS patients (IBS-D) . This stSeveral of the metabolites that distinguish between the healthy and IBS-D subjects point to alterations in one-carbon metabolism including serum methionine and sarcosine and urinary taurine. This led the authors to utilize an ELISA based assay to measure another key intermediate in the one-carbon pathway, homocysteine. The serum levels of homocysteine were found to be increased levels in the IBS-D group. After the intervention, the levels were reduced further suggesting that one mechanism by which the synbiotic treatment improves IBS-D symptoms may be through modulation of one-carbon metabolism.Lactobacillus genus [L. rhamnosis GR-1 and L. reuteri RC-14 and seven received a placebo treatment. The women were evaluated using the Nugent scoring system which is used to assess bacterial vaginosis (BV). All women in the study had intermediate scores indicative of a potential risk of transition to BV. The three-day treatment did not impact the Nugent score, but some changes in the microbial community were observed. Analysis with 16S rRNA sequencing revealed an increase in the relative abundance of Lactobacillus. An untargeted GC/MS analysis was conducted, but no differences were found between subjects on the probiotic and placebo. Obtaining a sufficient amount of sample for metabolomics was noted as a challenge using vaginal swabs and samples from only four women who demonstrated an increase in Lactobacilli were analyzed. A significant positive correlation between the lactate levels and the amount of lactate producing bacteria was found. The authors recognized this might seem to be an intuitive finding, but not that to their knowledge, this correlation had not yet been reported.The vaginal microbiota is part of a complex antimicrobial defense system that protects women against disease . In the us genus . It is tus genus . In thisThe next study of the use of probiotics on the vaginal microbiota, also conducted by the Reid Lab, evaluated the use of oral probiotics use in pregnant women in Rwanda . The useLactobacillus crispatis and BV-associated anaerobes were significantly higher in subjects with a high Nugent score.The results showed the expected low bacterial diversity associated with a healthy vagina. No significant changes in the vaginal microbiota composition was observed between the placebo and probiotic treatment. Similarly, no significant changes were observed in the vaginal metabolome. Interestingly, women in the placebo group were significantly more likely to give birth preterm with three women in the placebo group compared with none in the probiotic group. Given the small size of this study, this intriguing finding will have to be validated in a larger cohort. Further analysis of the metabolomics data confirmed a previously observed set of biomarkers associated with bacterial diversity and bacterial vaginosis . High leLactobacillus, Bifidobacteria and Streptococcus twice daily for 2 months. The study demonstrated that the probiotic increased the abundance of several taxa known to be depleted in MS such as Lactobacillus. Other taxa previously associated with dysbiosis in MS including Akkermansia and Blautia were found to be decreased with the probiotic further demonstrating the beneficial effects. Peripheral blood mononuclear cells (PBMC) were collected and gene expression studies showed a decrease in expression of a number of proinflammatory genes following probiotic administration. Correlation of the metabolomics with 16S rRNA sequencing data revealed a positive association between the Lactobacillus OUT 304,724 and the fecal hypoxanthine level. This metabolite also demonstrated a negative association with the decreased expression of the MS risk alleles HLA.DPB1 and HLD.DPA1 in PBMCs.The search criteria found studies in two areas for which there was only one example. The first was a study by Tankou et al. on the effects of a probiotic supplementation on relapsing remitting multiple sclerosis (MS) . The gutB. animalis subsp lactis LKM512 or placebo daily for 8 weeks. The probiotic provided a significant benefit to the patients as evaluated by reduced itching and dermatology-specific quality of life scores. Real-time PCR confirmed that the patients on probiotics had increased fecal levels of B. animalis subsp lactis LKM512. The fecal metabolomics from three patients who experienced dramatic improvement in their symptoms with the probiotics revealed a significant increase in kynurenic acid. Increased levels of this metabolite were also shown to be negatively associated with itching symptoms.The use of a probiotic supplement in a study of atopic dermatitis in adults was reported by Matsumoto et al. . Atopic A previous study by this group on a similar cohort of adults with atopic dermatis found that consumption of a probiotic LKM512 yogurt for only four weeks lead a similar improvement in symptoms . In thisWith the increasing interest in the use of prebiotic and probiotic interventions for a wide range of diseases, it is becoming more and more important to understand the detailed biochemical mechanisms through which they function. As discussed earlier, analysis of the microbial community structure using gene sequencing methods may not provide information on the actual functioning of the bacteria. Despite a plethora of pre-clinical studies, there have been relatively few studies of the use of probiotics and prebiotics in the context of human clinical trials that have taken advantage of advanced metabolomics approaches. The examples presented here cover a range of features including pathological condition, cohort size, dosing frequency and duration. The intent of this review was to evaluate how these studies were designed and analyzed and consider ways in which future studies could be optimized.A review of the study designs of the clinical trials described here shows one important variable that was generally not interrogated. As stated in the definition of probiotics and prebiotics, these therapies need to be “administered in adequate amounts” in order to provide the beneficial effects to the host. In the studies described, the dose, frequency and duration of the interventions were set at the beginning of the study and no clear indications of how these parameters were derived was given. Defining the dosing for a pharmaceutical involves a dose–response study with the guiding principal of “start low, go slow”. As probiotics and prebiotics are generally regarded as safe, such dose–response studies would hold far less risk than with a pharmaceutical. The obvious challenge with dose-range studies is that they will require larger numbers of patients to evaluate the effects of dosing, frequency and duration, but at some point, these types of studies will have to be carried out. The most practical applications might be in well-defined cohorts, e.g., patients with a specified pathology. In this way, the outcome phenotype should be strong enough that smaller cohorts may still have the statistical power to determine the effective dosing strategy.As with any metabolomics analysis, there are a number of analytical sources of variation that need to be considered. The range of analytical platforms used in the studies discussed here are representative of the rest of the metabolomics field. The advantages and disadvantages of these platforms has been extensively reviewed and a summary of the different platforms used in fecal metabolome analysis is presented in the review by Karu et al. ,74. GiveA challenge that is specific to metabolomics of fecal samples is the stabilization of the fecal metabolome. In studying the serum metabolome, the protocol requires that the serum be quickly separated from the metabolically active red blood cells. Similarly, the fecal metabolites need to be protected from continued metabolism by the biologically active bacteria. Rapid freezing at −4 °C or lower as well as storage in 95% ethanol have been shown to have provide sufficient protection against continued metabolism . In humaAnother source of variation in fecal samples is the water content. Fecal samples typically contain 60%–85% water, depending upon fiber intake . This vaThe studies reviewed here demonstrate the potential of metabolomics to contribute to the understanding of how prebiotics and probiotics function in humans. Continued work on the development of standardized protocols for fecal metabolomics will help minimize both the biological and analytical sources of variation. These developments, along with the use of multi-platform approaches, will provide more detailed and comprehensive metabolomics data. Combining this data with advanced microbial sequencing analyses will help elucidate the detailed biochemical mechanisms by which probiotics and prebiotics can be optimally applied to improve human health.


Joint diseases are conditions with an often progressive and generally painful nature affecting the patient’s quality of life and, in some cases, requiring a prompt diagnosis in order to start the treatment urgently. Synovial fluid (SF) laboratory testing is an important part of a diagnostic evaluation of patients with joint diseases. Laboratory testing of SF can provide valuable information in establishing the diagnosis, be a part of a patient’s follow-up and treatment with the purpose of improving the patient’s health and quality of life. Synovial fluid laboratory testing is rarely performed in Croatian medical biochemistry laboratories. Consequently, procedures for SF laboratory testing are poorly harmonized. This document is the second in the series of recommendations prepared by the members of the Working group for extravascular body fluid samples of the Croatian Society of Medical Biochemistry and Laboratory Medicine. It addresses preanalytical, analytical, and postanalytical issues and the clinical significance of tests used in SF laboratory testing with the aim of improving the value of SF laboratory testing in the diagnosis of joint diseases and assisting in the achievement of national harmonization. It is intended for laboratory professionals and all medical personnel involved in synovial fluid collection and testing. Chronic rheumatic conditions comprise more than 150 different diseases (including joint disease) with an often progressive and generally painful nature. They are among the leading causes of morbidity and disability worldwide and represent an enormous burden on healthcare systems . Laboratory testing of the SF can contribute to the differential diagnosis of rheumatic conditions accompanied with joint effusions. Indications for SF laboratory testing include inflamed joints with a known or unknown aetiology, suspected acute prosthetic joint infection, as well as infection identification by microbiological analyses and diagnosing crystal-induced arthritis, The Working group for extravascular body fluid samples (WG EBFS) of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM) conducted an extensive survey aimed at mapping critical areas in the preanalytical, analytical, and postanalytical phases of extravascular body fluids analysis in Croatia. The main results demonstrated that SF laboratory testing is rarely performed in Croatian medical biochemistry laboratories, and since the procedures used in other investigated extravascular body fluid testing are not harmonized, the same level of harmonization might be expected in SF laboratory testing  joints are enclosed in synovial cavities called synovia, which are filled with a viscous synovial fluid, produced by the ultrafiltration of plasma through the synovial membrane (and its adjacent capillaries) with the incorporation of hyaluronic acid secreted by synoviocytes. Synovial fluid glucose and uric acid concentrations resemble that of plasma, while its protein concentration is much lower compared to plasma protein concentrations. Synovial fluid also contains high amounts of locally synthesized hyaluronic acid, which contributes to the fluid’s viscosity. The main functions of SF are to lessen the friction between joints facilitating their free movement, to provide nutrients to the metabolically active and vascular-deficient cartilage, and to remove waste metabolites.etc., are associated with the accumulation of SF in the joint cavity. Laboratory testing of SF effusions is an additional and helpful tool for the diagnosis and classification of disorders affecting the articular membrane (i.e. arthritides). The SF analyses with the highest clinical value are SF crystal detection in crystal-associated synovitis (gout and/or pseudogout), and SF total and differential cell count in the confirmation of inflammatory (septic) arthropathies. Other biochemical analyses are neither specific nor sensitive but might provide useful additional information and narrow down the differential diagnosis of conditions affecting the joint. Combining the results of SF laboratory testing with the patient’s medical history and physical examination, arthritic disorders can be classified into four groups: non-inflammatory, inflammatory, septic and haemorrhagic  is present in the synovium. Conditions including infection, inflammation, metabolic disorders, trauma, advanced age, 2.2.1Since SF constituents mirror plasma concentrations, the recommended fasting period is necessary to equilibrate plasma and joint fluid concentrations in order to obtain reliable results of the SF analysis (2.22.3Improperly identified (or unlabelled) sample containers should not be accepted for analysis (2.4i.e. aseptic needle aspiration from a joint) which is performed for diagnostic and therapeutic purposes . If anticoagulant containing tubes are used, they should be mixed gently according to the manufacturer’s instructions to ensure mixing of anticoagulants and SF sample. These steps should be done at the site of the collection before transportation to the laboratory (per minute (rpm), biochemical testing is performed from the supernatant  recommends the following collection order: the first aliquot of the SF sample should be intended for biochemical analysis, the second aliquot for microscopic examinations , while the third should be intended for microbiological analyses  or calcium oxalate crystals, causing false positive results  and crystals diagnostics are feasible from a few drops of the SF sample. If a recommended sample volume is not available, the clinician should prioritize the test requested according to the suspected diagnosis in collaboration with the laboratory (which acknowledges the sufficiency of the sample volume for the requested tests) , the determination of specific biochemical analyses, and microscopic examination .4.14.1.1e.g. inflammatory). However, small SF volumes do not exclude a joint condition. The presence of particles like inclusion bodies or fibrin can lead to difficulties in obtaining an SF sample, which might result in a falsely low volume collected , chyle droplets, etc. , it might suggest an ongoing inflammatory process in the joint. A string longer than 6 centimetres is found in cases of septic arthritis. Although accurate measurements of SF viscosity can be performed using s viscometer, this is rarely performed due to the low clinical value of this parameter  viscosity of the SF indicating the presence of an inflammatory process in the joint. The protein SF content, as well as the protein type, cells, temperature, and enzymes, have an impact on the SF viscosity  responsible for SF viscosity. It provides little diagnostic information, but it is still used by some laboratories because of its simplicity. Mucin test can be performed to differentiate the synovial fluid from other fluids of uncertain origin  after collection might cause the artefactual formation and/or dissolution of crystals and the alteration of their optical characteristics. Thus, delayed analysis of SF samples could lead to the misdiagnosis of the underlying joint condition   are present only in very low concentrations or completely absent. Total protein concentrations in normal SF are about 1/3 of the protein concentration in plasma. The increased protein concentrations are caused by increased permeability of the synovial membrane or by increased synthesis in the joint cavity. Albumin represents the main protein fraction in SF with concentrations of approximately 12 g/L. The presence of ankylosing spondylitis, arthritis, arthropathies that appear as secondary consequences of Crohn disease, gout, psoriasis, ulcerative colitis, etc., are accompanied by high total SF protein concentrations . In general, synovial fluid RF determination in RA is not considered to be diagnostically helpful because positive RF may simply reflect serum concentrations and might derive from other chronic inflammatory conditions  can be used for the diagnosis of PJI. Alpha-defensin has shown high diagnostic sensitivity and specificity for suspected PJI, especially after total knee and hip arthroplasty. The usefulness of AD has been demonstrated in ruling out PJI and it could be used as a confirmatory test for PJI (4.2.7i.e. above 280 U/L) are found in inflammatory effusions   , in general, do not affect the total cell count in SF samples, although NSAIDs reduce the function of white blood cells  are indicative of various conditions affecting the joint . Due to SF sample’s high viscosity, the cells should be allowed to settle in the chamber for an additional period of time before counting. If necessary, sample viscosity should be reduced by dilution with hyaluronidase buffer enabling a more homogeneous cell distribution in the counting chamber  which may cause pseudoleukocytosis or pseudoeosinophilia, and the presence of possible matrix effect which might affect proper sample aspiration. Therefore, unclear automated results, SF samples with low WBC count, suspected malignant samples, as well as suspected interferences should be verified by manual microscopy , is prone to substantial intra- and inter-observer variations and lacks standardization. Modern automated haematology analysers adapted for the SF counting analysis offer technical and clinical advantages. Compared to manual methods, automated counting is more reliable and practical, reduces TAT and enables longer cell stability. However, the limitations of automated cell counting methods should also be addressed, especially their inability to detect or correctly classify malignant cells and atypical leukocytes, their high imprecision , the interference of non-cellular particles  have pathologic significance in the diagnosis of crystalline joint conditions  it concentrates SF components in a monolayer increasing sensitivity and recovery; b) the slides prepared might be retained for education, training and competency assessment; and c) the slides might be examined under microscope stained or unstained (e.g. MSU needle-like shape) in each field of 10 randomly selected fields of view. Crystals description should include birefringence , shape , cell location  and quantity   followed by the identification of crystals using a high-power field (HPF) magnification (400x or higher). A crystal positive sample of the SF should contain at least two typical shapes (5.e.g. glucose) should be reported on the same test report (The results of SF biochemical analysis should be reported in the same units as those obtained in the serum/plasma. The values of simultaneous measurement of an analyte in the serum/plasma and synovial fluid (t report . SimilarThe test report should include interpretive comments addressing the preanalytical and analytical phases of SF analysis. If available, a laboratory information system should be used to generate standardized and clear comments (


Malassezia, which is by far the most abundant component of the skin mycobiota, is associated with a variety of skin disorders, of which some can be chronic and severe and have a significant impact on the quality of life of those affected. Understanding the causative relationship between Malassezia and the development of such skin disorders requires in-depth knowledge of the mechanism by which the immune system interacts with and responds to the fungus. In this review, we will discuss recent advances in our understanding of the immune response to Malassezia and how the implicated cells and cytokine pathways prevent uncontrolled fungal growth to maintain commensalism in the mammalian skin. We also review how the antifungal response is currently thought to affect the development and severity of inflammatory disorders of the skin and at distant sites.The microbiota plays an integral role in shaping physical and functional aspects of the skin. While a healthy microbiota contributes to the maintenance of immune homeostasis, dysbiosis can result in the development of diverse skin pathologies. This dichotomous feature of the skin microbiota holds true not only for bacteria, but also for fungi that colonize the skin. As such, the yeast  The skin, one of our body's largest organs, harbors a wide variety of microbial communities, including innocuous symbiotic organisms but also potential pathogens   skin disorders  mammalian skin.To understand the role of Constant exposure of the skin to commensal microbes results in a continuous activation of the cutaneous immune system. Active immunosurveillance of the microbiota is critical to prevent dysbiosis, microbial overgrowth and translocation across epithelial barriers as evidenced by the frequent occurrence of opportunistic infections in immunodeficient individuals  and C-type lectin receptors (CLRs) recognize carbohydrates in the fungal cell wall  constitute prominent additional sources of IL-17A and respond swiftly to cytokine stimulation in an antigen-independent manner , a ligand-dependent transcription factor, has gained attention because Malassezia-derived metabolites, in particular indoles, can trigger AhR signaling. Indole production was first reported for M. furfur  prevents While type 17 immunity is primarily host-beneficial and supports a protective state by controlling commensal fungi in barrier tissues, IL-17 can also bear pathological potential if dysregulated. The IL-17 pathway is a strong driver of inflammation in several immune-mediated diseases, the prime example of which is psoriasis  that accumulate during a critical developmental window in neonatal life were found important to balance the antimicrobial Th17 effector cells directed against the commensal . Fungal dysbiosis in IBD is characterized with an enrichment of fungal taxa belonging to the Basidiomycota phylum, and this effect was balanced by an equivalent decrease in taxa belonging to the Ascomycota phylum  tumors, Malassezia was markedly enriched (Aykut et al., Malassezia exerted a tumor-promoting effect via a mechanism involving mannose-binding lectin, a CLR and activator of complement and implicated in antifungal innate immunity (Aykut et al., Malassezia is likely a ligand of MBL, although direct binding has not been demonstrated. Whether fungal dysbiosis is cause or consequence of oncogenesis is not fully clear. The link between Malassezia and carcinogenesis may be more general, as fungal dysbiosis was also observed in colitis-associated cancer with an enrichment of Malassezia in the colonic mucosa associated fungal microbiota (Richard et al., Beyond its impact on the pathogenesis of inflammatory diseases, Malassezia in the skin and on the divergent consequences that the antifungal response can have on the host. IL-17-dependent immunity against Malassezia contributes to the maintenance of fungal commensalism and skin homeostasis. The same pathway however can also have host-adverse effects in predisposed individuals, and this effect is likely not limited to the skin but also affects extracutaneous sites. The factors determining these context-dependent outcomes remain largely unclear. Moreover, it remains unknown to what extent the effects of the antifungal immunity on disease pathogenesis is shared or distinct between different Malassezia-associated disorders. Beyond immunological pathways, non-immunological factors such as neurotransmitters and possibly hormones affect the antifungal response, and this is likely also the case with Malassezia. Consistently, neuroendocrine changes are known to impact the course and severity of Malassezia-associated skin disorders. Future research will inform about the potential of therapeutically targeting fungal communities or the antifungal response for improving patient outcome.Recent advances in the field have shed light on the immune response to BioRender.com. All authors reviewed the manuscript.FS and SL-L wrote the minireview. FR created the figure with The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Condylar neck and sub-condylar fractures of the mandible are a frequent occurrence in maxillofacial surgery. The indication for surgical treatment of these fractures has changed over time, and several techniques have been developed with different results in the attempt to avoid the most worrisome adverse event, i.e., facial nerve injury. In this study, we present a new technique that combines an intraoral and a cutaneous pre-auricular access, which allows for easy and safe access to the surgical site, preventing facial nerve injury and avoiding surgical scars in high-impact aesthetic areas of the neck. Five consecutive patients affected by condylar neck or sub-condylar fractures were treated at a single institution using a combined intraoral and pre-auricular access. Results were evaluated after three months from surgery in terms of mandibular mobility, occurrence of complications, and patient’s satisfaction. All five patients had good outcome, with complete healing of the fracture and no occurrence of complications, including no facial nerve palsy. A key point of the technique is the safe reduction of the two mandibular fragments realized by a combined intraoral and a cutaneous pre-auricular surgical access. The periosteal plan of the ramus can be widely and safely elevated with the intraoral approach and connected to the condylar bone plane by the pre-auricular cutaneous approach without any need for soft tissue dissection at the fracture rim, thereby avoiding facial nerve injuries. Wide ramus periosteum elevation creates an “optical space”, allowing fragment reduction and fixation under direct oblique view without any endoscopic need. Our results strongly suggest that with our technique it is possible to treat sub-condylar and condylar neck fractures safely, avoiding facial nerve injury, which is an unacceptable complication due to its heavy impact on a patient’s life. Condylar fractures of the mandible are a controversial topic in maxillofacial surgery due to the variety of features they manifest, and the wide range of surgical techniques commonly used to treat them. Common disadvantages of surgery for mandibular fractures are scars, salivary gland-associated complications, maxillary artery, or pterygoid plexus injuries, but the greatest concern is facial nerve injury (FNI).Several surgical approaches have been proposed to treat condylar fractures; however, there is not yet a consensus on the surgical technique to be used to avoid FNI. Minimally displaced fractures can be treated by closed reduction with maxilla–mandibular fixation (CR/MMF), whereas moderately or severely displaced fractures usually need to be treated with an open reduction (OR). Indications for surgery in fractures of the mandibular condyle have changed over time. Zide and Kent described a series of absolute and relative surgical indications that became the gold standard in 1980s  1,2]. H. H2]. HoSimply on the basis of anatomical difference for surgical treatment, fractures of the mandibular condyle were commonly distinct in two categories: intra-capsular and extra-capsular fractures. More recently, mandibular condylar fractures were classified on the basis of the site in head (over the polars connecting line), neck (between bipolar and sigmoid noch line), and sub-condylar (under sigmoid noch line), which has also been adopted by the AOCMF for its utility in surgical technique selection . Intra-cDeep techniques provide submandibular (Risdon) or retro-mandibular approaches whereas superficial techniques are based on trans-parotid or trans-masseter and antero-parotid (TMAP) accesses ,4.The submandibular technique starts with an incision following the inferior border of the mandible. The dissection is purely subcutaneous at first, and then the platysma muscle is dissected about 2cm above the mandibular angle, giving access to the masseter fascia. The masseter muscle is then incised in order to reach bone contact. A sub-periosteal dissection is performed upward in direction of the condyle until eventually the fracture area is reached ,4,5.The retro-parotid approach is performed through an incision right below the ear lobe towards the mandibular border. The condyle is exposed through a dissection of the subcutaneous tissues posteriorly around the parotid capsule and after retraction of the parotid gland . The traThe superficial technique (TMAP) starts with a retro-mandibular incision in the original form, which can be extended to the pre-auricular region in the modified form. Dissection of the superficial muscle-aponeurotic system is performed until reaching the anterior border of the parotid gland, then the parotid is retracted posteriorly, and the condyle is approached passing posteriorly to the masseter muscle. A high cervical TMAP can also be used. In this case, the incision is performed from 0.5 to 1 cm below the inferior mandibular border, and the masseter muscle is reached after a subcutaneous dissection on the platysma muscle and dissection and retraction of the platysma itself. The access to the mandibular ramus is obtained through cutting the muscle anteriorly to the parotid gland in a so-called “nerve-free window” . Recent For all these techniques, after exposing the fractures, occlusion is obtained at maximum intercuspation by maxilla–mandibular fixation and the fracture is reduced and fixed mostly by plates. After surgery, maintenance of fixation by elastic bands on skeletal screw anchorages is recommended. In this study, we propose a method that can be used for both intracapsular and extracapsular fractures using a combined cutaneous pre-auricular approach for upper screw positioning and an intraoral transmucosal approach with wide periosteal elevation of the ramous, achieving a large space for transcutaneous lower screw insertion under direct view, preserving the integrity of the facial nerve and avoiding surgical scars in high aesthetic impact areas of the neck.The main treatment difficulties are related to neck condylar fractures because of the lack of general consensus on the surgical approach that needs to be used in order to avoid the most devastating complication, i.e., facial nerve injury ,6.In the recent literature, several studies have been published in the attempt to correlate the incidence of FNI with the different surgical approaches.Five possible procedures have been described on the basis of the way they cross the division site of the marginalis mandibulae branches of the facial nerve (MMB), following the criteria of classifying surgical access by superficial and deep approaches.There is also a second classification that divides surgical approaches from most posterior to most anterior, and it has been described that the more posterior the access, the higher is the risk of facial nerve injury .A general increase of side effects from posterior to anterior and from superficial to deep approaches have been described, with the posterior and deep access classified as the riskier for FNI insurgence ,8. AnothIn order to limit FNI as little as possible, we proposed a new technique for condylar neck and sub-condylar fracture treatment that has the advantage of limiting the incidence of facial nerve complications also in the presence of dislocated condylar neck and sub-condylar fractures. The new technique is based on the combination of the preauricular approach with an intraoral approach. By using our dual surgical access, risks for FNI may also be limited for dislocated fracture, avoiding wide soft tissue dissections in the division area of the MMB as needed in most common techniques for the reduction and fixation of dislocated condylar neck fragments.Since the incidence of FNI described in deep approaches is up to 48.1% with submandibular approach and up to 40% with the retro-parotid approach, the superficial approaches are relatively riskless with FNI occurrence of up to 30% in the trans-parotid, 7.7% in TMAP, and 0.6% in high cervical TMAP [Five fractured condyles were operated upon since October 2016, with patients’ characteristics being illustrated in All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of University of Salerno .Surgical treatment was performed in patients with widely displaced fractures with lack of contact between the two fragments , or whenA-shaped miniplates and screws (Tekka France) were used in condylar neck fractures, while 2 miniplates were adopted in wide based sub-condylar fractures depending on the fracture rim shape in order to achieve maximum stability in fixation .A-shaped plates are suitable for condylar neck fractures as resulting from finite element analysis (FEA) in the literature .Because use of resorbable plates in maxillofacial fractures is still debated, we selected titanium fixation systems, even if conceptually resorbable plates and screws may represent an advantage particularly in growing patients avoiding permanent limits in district growth and second surgery stage in case of removal necessity ,12.The rationale of this modified surgical approach for condylar fractures was to obtain the periosteal elevation at the surgical site via a pre-auricular transcutaneous access for the condylar area, combined with a wide periosteal elevation by intraoral approach and ramous preparation for the sub-condylar area, thereby avoiding a submandibular or retro parotid access and in this way reducing the risks for FNI at the fracture rim periosteal elevation time.The multi steps techniques combining pre-auricular and intraoral approaches followed the surgical flowchart of (1) intraoral incision with periosteal elevation on the mandibular ramous similar to sagittal split surgery; (2) lower temporalis muscle detachment from the coronoid process; (3) pre-auricular incision with periosteal elevation from the lateral side of the condyle; and (4) screw insertion on the condylar head to proper manage the condylar head position without damaging the condylar fragment and avoiding periosteal elevation and pterygoid detachment from the inner side of the condyle, thereby preventing condylar head damaging and necrosis.Fragment reduction was performed under direct view, combining the pre-auricular and intra-oral access and obtaining a direct view by wide periosteal elevation of the ramous.Multi-layer closure was performed after fixation with Redon drainage insertion.By this technique, we performed upper screws insertion under direct view of the condylar area by pre-auricular access a, while In particular, the posterior sub-condylar area was visible by preauricular incision, while the anterior sub-condylar area was also visible through intraoral mucosa access.Moreover, surgical scars in the aesthetic areas of the neck following current techniques were avoided. Cutaneous incision starts on the upper end of the helix proceeding in the pre-auricular area, extending to the tragus and the surgical path proceeded by deeply reaching the auricular cartilaginous plane and the TMJ capsule. Following opening of the TMJ capsule, we could find the condylar fragment that usually was dragged medially and anteriorly by the insertion of the lateral pterygoid muscle. The surgical path followed the bone plane of the condylar fragment and stopped at the edge of the fracture.An intraoral incision was performed on the vestibular mucosa at the level of the inferior retromolar trigone running along the anterior border of the ramous, followed by a wide elevation of the entire periosteal plane of the mandibular ramus, including the posterior margin. Following the bone plane by sub-periosteal intraoral route until the fracture edge, we could connect both the preparation of the condylar fragment and the preparation of the mandibular ramus, avoiding FNI risks at the division level of the main facial nerve branches that cross the fracture rim, superficially passing the bone plane.In detail, once we obtained full control of the fractured area by wide periosteal elevation of the ramous, we could obtain miniplate and screw positioning under direct oblique vision, combining the pre-auricular and the trans-oral approaches by direct trans-cutaneous oblique insertion or via a trans-cutaneous trocar approach. Full control for bone fragment reduction with miniplates and screw positioning could be achieved by a pre-auricular incision for the condylar area (upper screws) and by a transcutaneous access after intraoral periosteal elevation to place sub-condylar lower screws. Even in the case of a sub-condylar fracture limited to the posterior border of the ramous, lower screw positioning for the ramous fragment with proper positioning of the orthogonal axis could be performed by combining a TMAP access, although limiting any parotid or masseter dissection because of the previous wide ramus preparation by intraoral approach, thereby limiting or avoiding FNI risks. In terms of obtaining condylar fragment repositioning and reduction at the fixation time, a digit pressure to the retromolar area of the inferior wisdom teeth or wire traction at the gonion angle by screw insertion is an important maneuver in order to increase the posterior vertical dimension, achieving easier fracture reduction and avoiding dangerous manipulation of the condyle. A long screw partial insertion in the condylar head can be a useful shrewdness for safe traction, positioning, and fixation of the dislocated condyle b.Additionally, to avoid FNI in the case of full dislocated condylar fractures, after elevation of both the condyle periosteal plane through a pre-auricular access and of the ramous periosteal plane through an intraoral access, we performed the connection of the two planes with a limited dissection at the emerging point of a periosteal elevator inserted from the intraoral access along the bone plane up to the fracture rim. In this way, a very limited dissection of the soft tissues that bridge the two-bone fragment planes at the fracture rim area was performed, avoiding dangerous dissecting maneuvers in the area of the facial nerve main branch division.The results of this dual surgical access technique were evaluated on the basis of dental occlusion, bone fragment alignment after reduction and after fixation, facial nerve functionality, skin scarring, temporomandibular joint functionality, temporomandibular joint symptomatology, and patient satisfaction.Occlusion functionality was evaluated through checking precise intercuspation and group function guides in protrusive and bilateral jaw movements. Bone fragment alignment was evaluated by panoramic X-ray of the jaws and a computed tomography scan (CT scan) of the facial skeleton on axial and frontal view, with volumetric reconstruction. All the exams were taken pre-operatively and immediately post-op, and an X-ray panoramic alone was taken at 3 months follow-up. Bone fragment reduction and fixation was graded in a scale from 1 to 5, where 1 was worst and 5 was best alignment.For evaluation of facial nerve function post-operatively, we used the House–Brackmann Facial Nerve Grading System that assesses facial nerve palsy by considering two parameters: the extension of the elevation movements of the middle portion of the eyebrow and the extension of the lateral movements of the angle of the lip. The House–Brackmann system rates 1 point for each 0.25 cm of movement, and the scores are added to reach a maximum of 8 points.Evaluation of skin scarring was based on the Vancouver Scar System of Baryza, which considers 4 parameters: pliability, vascularity, pigmentation, and scar height. We obtained a rate of the global look of the scar, assigning a score to each parameter and adding the scores up to 13 points. The lower the score, the better the scar will look.Postoperative TMJ functionality and postoperative TMJ symptomatology were evaluated on the basis of post-operative mandibular motion values, maximum mouth opening, maximum deviation on left side, maximum deviation on right side, and maximum protrusion .Patients’ general satisfaction was evaluated by asking them to grade their satisfaction from 1 to 10 . We alsoAll the five cases were evaluated at three months and showed good aesthetic and functional results with proper alignment of the bone fragments. All the five patients showed good mouth opening, with three of five patients opening their mouth more than 40 mm, with measurements taken from the edge of the upper incisor teeth to the edge of the lower incisor teeth . Three pNo FNI were detected . Only inSurgical indications for condylar neck and sub-condylar fractures are widely debated in the literature because of the significant risk of FNI complications during surgery. No general consensus has been reached on the selection of the surgical technique to be used to avoid FNI, and FNI incidence is still the most important complication, even after selecting the most recently developed surgical techniques.A review of the literature using keywords for surgical technique selection and complication incidences related to FNI following condylar neck and sub-condylar fractures, as well as in relation to the plane of dissection, mainly showed five techniques. In relation to the surgical approach and incidence of FNI, surgical techniques can be divided into deep and superficial, posterior and anterior, depending on the site of dissection and the amount of dislocation, with increased FNI incidence being correlated to increased dislocation ,14.The main reason for FNI after condylar fracture treatment is related to the marginalis mandibulae branch damage of the VII cranial nerve because of its terminal pattern of innervation and division site, which runs close to the surgical field.A possible explanation of these negative events can also be correlated to the amount of soft tissue traction and dissection during surgery , as well as the site and amount of dissection , in comparison to posterior dissections.Regarding the higher FNI risk observed in condylar fractures with dislocated fragments, the correlation can be explained by the necessity of soft tissue dissection at the fracture site, where the main branches of the facial nerve run across the fracture rim. Using our technique, the dissection of soft tissue can be limited by a periosteal elevator insertion and use as a guide at the condylar and ramous fragments fracture rims.After both a pre-auricular incision and a mucosa intraoral access, both fields were connected by periosteal elevation running along the ramous and the condyle bone planes, without any need for further soft tissue dissection at the condylar neck fracture site.A key point of the technique we described is the safe connection of the sub-periosteal planes of the two mandibular fragments: condyle and ramus. Connection of the two periosteal elevation planes at the condyle area by pre-auricular approach and at the ramus fragment by intraoral approach after wide ramus periosteal elevation could be safely achieved without any wide soft tissue dissection in the sub-condylar area through simply connecting the bone surfaces of the two mandibular fragments by a periosteum elevator. In this way, selective and limited soft tissue dissection under elevator tip guide was possible in the sub-condylar area to prepare ramous stump, limiting dangerous maneuvers for main branch integrity of the facial nerve.Fracture reduction and fixation can be achieved under direct oblique view with the aid of a head light at the condyle level by the pre-auricular access for upper screw insertion on the condylar upper fragment and by intraoral access after the mentioned wide periosteal elevation by trans-oral mucosa view for lower screw insertion by trocar trans-cutaneous cheek access. Following our technique, lower screw area view could be achieved by preauricular trans-cutaneous (sub-condylar rims) or intraoral (condylar neck) access after wide ramous periosteal elevation, creating an optical view space. Moreover, in this way, reduction and precise alignment can be checked by finger tactile control through the oral access, avoiding endoscopic difficulties.In our opinion, periosteal plane elevation for ramus fragment identification by pre-auricular, submandibular, or retro-parotid cutaneous approaches are dangerous for FNI because of the soft tissue dissection in the division area of the facial nerve branches when connecting the two different sub-periosteum dissection planes of the two dislocated mandibular fragments at reduction and fixation time.The condylar fragment is commonly dislocated anteriorly and medially because of the external pterygoid muscle traction, while the mandibular ramus fragment is normally located on a more superficial plane in condylar neck fractures . ApproacAdopting our technique, the periosteal plane of the mandibular ramus can be widely and safely elevated by the intraoral approach, thus allowing a good view of the sub-condylar rim by the combination of the pre-auricular access with the trans-oral access, avoiding the need for an endoscopic device with related difficulties. Lower screws in the posterior ramus area can be inserted by the pre-auricular approach, while lower screws in the anterior sub-condylar area can be inserted through a cheek trans-cutaneous access by trocar under intraoral vision after wide temporalis muscle insertion detachment on the coronoid process, avoiding any risk for FNI, which is the main complication in condylar neck and sub condylar fractures.Even in the case of difficulties in fixing sub-condylar fractures at the posterior mandibular border by our technique, the combination with a trans-parotid or trans-masseter and antero-parotid (TMAP) approach can be performed, but through reducing the extent of soft tissue dissection because of the wide periosteal elevation already performed at the ramus area by the intraoral approach with less risks for FNI.A possible evolution of the technique will consist of digital planning of the operation through also using custom made fixation plates, reducing in this way the incidence of fragment malposition .FNI is a detrimental drawback for condylar neck and sub-condylar fracture treatment, which is unacceptable due to its heavy consequences on social life because of the importance of face expressivity in human relations. Because of the risk/benefit balance, only safe techniques for condylar fractures treatment should be considered.Our results suggest that a combined pre-auricular and intraoral approach for surgical treatment of sub-condylar and condylar neck fractures can be used to avoid facial nerve injuries.(1)Increased safety for FNI.(2)Less scarring, with the possibility to make a cutaneous incision only in the pre-auricular area where scars are almost invisible and aesthetically well tolerated.(3)Precision in final results with the ability to reduce and fix the fractured mandibular fragments under direct view by combination of multiple approaches.(4)Expansion in indication for open surgery in neck and sub-condylar fractures because of improved functional and aesthetic results, while reducing risks of complications.The main advantages of the technique we described areThe main disadvantage is the complexity of the technique, with related time extension needed at surgery.