Acute gynaecologic disorders are commonly encountered in daily clinical practice of emergency departments (ED) and predominantly occur in reproductive-age women. Since clinical presentation may be nonspecific and physical findings are often inconclusive, imaging is required for a timely and accurate diagnosis. Although ultrasound is the ideal non-invasive first-line technique, nowadays multidetector computed tomography (CT) is extensively used in the ED, particularly when a non-gynaecologic disorder is suspected and differential diagnosis from gastrointestinal and urologic diseases is needed. As a result, CT often provides the first diagnosis of female genital emergencies. If clinical conditions and scanner availability permit, magnetic resonance imaging (MRI) is superior to CT for further characterisation of gynaecologic abnormalities, due to the excellent soft-tissue contrast, intrinsic multiplanar capabilities and lack of ionising radiation.The purpose of this pictorial review is to provide radiologists with a thorough familiarity with gynaecologic emergencies by illustrating their cross-sectional imaging appearances. The present first section will review the CT and MRI findings of corpus luteum and haemorrhagic ovarian cysts, gynaecologic haemoperitoneum (from either ruptured corpus luteum or ectopic pregnancy) and adnexal torsion, with an emphasis on differential diagnosis. Additionally, comprehensive and time-efficient MRI acquisition protocols are provided. The CT and MRI hallmark of a corpus luteum is a cystic adnexal structure with crenulated, strongly enhancing peripheryKey differential diagnoses of haemorrhagic ovarian cysts include endometrioma, dermoids and mucinous cystic tumoursGynaecologic haemoperitoneum with or without active bleeding is most usually caused by ruptured corpus luteumPelvic haemoperitoneum, partly haemorrhagic adnexal mass and positive β-hCG test allow diagnosing ruptured ectopic pregnancyA benign ovarian mass  underlies over half of adnexal torsion casesAlthough less prevalent than obstetric issues, acute gynaecologic disorders in non-pregnant women are not uncommon in busy emergency departments (ED). Ultrasound (US) represents the ideal non-invasive technique for initial investigation of suspected genital diseases. However, due to fast acquisition and widespread availability on a 24/7 basis, multidetector computed tomography (CT) now represents the ‘workhorse’ imaging modality in the ED and quickly provides accurate and reproducible diagnosis of most acute abdominal and pelvic complaints. As a result, acute abnormalities of the female genital organs are increasingly detected on urgent CT studies, most usually when clinical manifestations are non-specific and a gynaecologic disorder is not prospectively considered as the primary origin of pelvic pain, or when differential diagnosis from gastrointestinal and urologic conditions  is required. Furthermore, CT provides the first diagnosis of an acute gynaecologic disorder when transvaginal US  is unavailable or inconclusive, and when genital abnormalities extend beyond the sonographic field of view or require further characterisation. In these situations, the role of the radiologist is to alert ED physicians about the possible or probable presence of an unsuspected genital disease that warrants immediate gynaecologic consultation –4.Magnetic resonance imaging (MRI) represents the ideal modality for characterisation of abnormal or inconclusive sonographic and CT findings. Advantages of MRI over CT include the lack of radiation exposure, native multiplanar acquisition, optimal soft-tissue contrast and tissue characterisation including the possibility of demonstrating the presence of fat and blood products. Although limited by scanner availability, exam duration and cost, the use of MRI in urgent settings has steadily grown over the last decade, particularly in paediatric patients and child-bearing age women, to avoid the risks of administering ionising radiation to the genital organs –7.This pictorial essay aims to provide radiologists with an increased familiarity with recognition and characterisation of acute gynaecologic disorders on CT and MRI, in order to allow timely diagnosis and appropriate treatment. The present instalment reviews the cross-sectional imaging appearances of corpus luteum and haemorrhagic ovarian cysts, gynaecologic haemoperitoneum (from either ruptured corpus luteum or ectopic pregnancy) and adnexal torsion, with an emphasis on differential diagnosis. In the second section, uterine emergencies and the spectrum of pelvic inflammatory disease (PID) will be presented , 9.To investigate patients with acute abdominal or pelvic pain, intravenous iodinated contrast agent is generally used, unless contraindicated by allergy or impaired renal function. Alternatively, gynaecologic abnormalities  may be encountered in unenhanced abdominal CT studies, such as those requested to investigate suspected urolithiasis and acute renal colic: in these situations, further investigation with contrast-enhanced CT or better MRI should be suggested after gynaecologic consultation , 4, 8.In postmenopausal women, most institutions perform a preliminary unenhanced acquisition followed by optional arterial and mandatory portal venous phase CT scanning. In patients with hip prosthesis or other metallic implants, metal artefact reduction algorithms decrease beam-hardening effects that impair the assessment of the pelvic organs .In reproductive age women with unspecific abdominal complaints, a limited CT protocol that includes a single acquisition in the portal venous phase is beneficial to limit the ionising radiation dose. If available, novel CT techniques such as iterative reconstruction should be used. When a gynaecologic disease is prospectively considered or sonography shows a non-trivial pelvic effusion, we suggest obtaining also a precontrast scanning at least of the pelvis, as it may be helpful to discriminate high attenuation reflecting the presence of blood or debris within a cyst or collection from contrast enhancement. Regardless of patient age, an additional arterial-phase (CT-angiography) acquisition using bolus tracking technique with the region-of-interest in the abdominal aorta is warranted when a concern for blood loss exists, such as in patients with vaginal bloody discharge, dropping haematocrit, hypotension or haemodynamic instability and after sonographic detection of haemoperitoneum , 4, 8.In state-of-the art multidetector scanners, CT generates almost isotropic sub-millimetre voxels that allow reconstruction of high-resolution images along arbitrary planes. Even if a gynaecologic disease is not prospectively suspected, in females, the routine reconstruction of 3- to 5-mm-thick sagittal and coronal CT images of the pelvis is recommended to depict the anatomy of normal and diseased genital organs. Sagittal viewing allows appropriate assessment of size, morphology and enhancement patterns of the uterus, the latter markedly varies according to age, menstrual phase and delay between contrast injection and CT acquisition. Additional MRI-like oblique-axial and oblique-coronal reconstructions  may be helpful to discriminate extra-uterine structures and to better assess topography of uterine abnormalities such as subserosal or pedunculated leiomyomas –4, 11.At CT, the fallopian tubes are generally not visible in normal conditions but become identifiable as tubular structures when dilated by intraluminal fluid, secretions or blood. The normal ovaries measure approximately 3 × 3 × 2 cm and show low attenuation with a faintly recognizable follicular structure on contrast-enhanced images. Identification of the ovaries relies on their location close to the external iliac vessels, posterior to the round ligament. Recognition of the suspensory ligament  as a linear or fan-shaped soft-tissue band allows diagnosis of ovarian versus non-ovarian masses. The ovaries are located anterior or antero-medial to the pelvic ureters, conversely iliac lymph nodes lie lateral or posterolaterally .Our comprehensive MRI acquisition protocol of the pelvis for acute pelvic pain or suspected acute gynaecologic disorders is performed on a closed-configuration 1.5-Tesla superconducting MRI scanner  using an eight-channel high-resolution phased-array torso coil with array spatial sensitivity technique (ASSET) parallel acquisition, with the patient lying in the supine position (entry position feet first). An antispasmodic agent such as glucagon or N-butyl-scopolamine, unless contraindicated  can be administered intravenously or intramuscularly to reduce bowel motion artefacts , 14.This state-of-the-art MRI protocol Table  relies oAlthough very complete, the abovementioned protocol Table  lasts apOn MRI pelvic examinations, orthopaedic metal implants such as hip prosthesis may determine signal loss, geometric distortion, and failure of fat suppression. Metal artefact reduction sequences (MARS) is a general definition which does not refer to a single specific technique, but rather encompasses a variety of sequences optimised to reduce artefacts from metal. As a general consideration, artefacts are less severe at 1.5 T than at 3 T, since the susceptibility-induced field inhomogeneity is doubled at 3 T MR scanners. As for conventional techniques, turbo spin echo are preferable to gradient echo sequences, and short tau inversion recovery (STIR) performs better than standard spectral fat saturation.Other tricks to optimise standard sequences, although often at the expense of acquisition time, include increasing receiver bandwidth, increasing matrix size, decreasing echo times and decreasing slice thickness. In recent years, new dedicated metal artefact reduction techniques have been developed including view angle tilting, the multispectral imaging techniques, multiacquisition variable-resonance image combination and slice-encoding for metal artefact correction. In patients with metal implants, these sequences, associated with advanced image acquisition techniques such as parallel imaging, allow to acquire MARS in a clinically feasible scan time .In reproductive age females, the normal ovary shows homogenous soft-tissue T1-weighted signal intensity and is often barely perceptible from adjacent bowel. On the other hand, on T2-weighted images, ovaries are almost always identifiable on the basis of characteristic zonal anatomy including a low signal intensity cortex and high signal intensity medulla with scattered well-circumscribed fluid foci corresponding to follicles. The normal, non-dilated fallopian tubes are usually hardly recognizable at MRI unless outlined by pelvic fluid. In the presence of peritoneal effusion, the fallopian tubes become visible as paired, thin and serpentine juxtauterine structures. When dilated by serous fluid, haemorrhage or pus, fallopian tubes appear as fluid-filled tubular structures that arise from the upper lateral margin of the uterine fundus, showing incomplete parietal plicae or folds that give them the characteristic convoluted appearance .In reproductive age females, the majority of ovarian cysts are physiological or functional in nature and encompass dominant follicles, follicular cysts and luteal cysts. During the follicular phase of menstrual cycle, under oestrogen stimulation, ovarian follicles grow until only one in each cycle reaches a size of approximately 20–25 mm, becoming the dominant follicle. Sometimes a dominant follicle fails to ovulate, but remains hormonally sensitive and continues to grow becoming a follicular cyst. During ovulation, the dominant follicle ruptures, releases the oocyte and then collapses; at this point, the luteinising hormone causes proliferation of granulosa cells in the inner lining of the remaining follicular bed, resulting in the formation of the corpus luteum.A functional structure which regresses spontaneously when pregnancy does not occur, the corpus luteum is frequently seen when cross-sectional imaging is performed in the second part  of the menstrual cycle. The CT hallmark of a corpus luteum is a well-circumscribed, unilocular (rarely bilocular) cystic structure located within the ovary, which measures up to 3 cm and is demarcated by a crenulated, briskly enhancing rim that reflects prominent blood flow within thickened walls Fig.  and corrWhen a corpus luteum fails to regress, a luteal cyst develops and may be filled with either fluid or blood, so the terms corpus luteum cyst and haemorrhagic corpus luteum are often used as synonyms . At CT, Unilateral acute pelvic pain and tenderness leading to ED admission may result from cyst enlargement, haemorrhage or rupture. Bleeding within luteal cyst or other functional cyst results in the formation of a haemorrhagic ovarian cyst. In the former case, bleeding is caused by the hypervascularity and fragile vessels of the granulosa layer within the wall of the cyst. At CT, the presence of blood leads to the appearance of increased attenuation  within the cyst. Haemorrhagic ovarian cysts are unilateral, show mixed (in the range of 25–100 HU) internal attenuation with common fluid-debris or fluid-fluid levels Fig.  and ofteTable The endometrioma represents the key differential diagnosis of haemorrhagic ovarian cysts. Affecting 10% of women of reproductive age, endometriosis is defined by growth of ectopic endometrial tissue outside the uterus, which bleeds synchronously with the endometrium. Clinically, endometriosis is suggested by cyclic perimenstrual pain and by other manifestations such as dysmenorrhoea, dyspareunia, infertility and pain during defecation. Although dysmenorrhoea and chronic pelvic pain represent the classic clinical presentation, a sudden onset with acute pelvic pain may also occur . BecauseMore common (80% of patients) compared to superficial peritoneal implants and deep pelvic endometriosis, endometrioma results from ectopic endometrial glands in the ovarian cortex that form haemorrhagic ‘pseudocysts’ which become detectable by imaging. Endometriomas have highly variable, often heterogeneous CT appearance and often mimic mixed solid/cystic or predominantly solid masses Fig. a, b. ComMRI is arguably the best imaging technique for the diagnosis of endometrioma, relying on high signal intensity on T1-weighted images and the pathognomonic T2 ‘shading’ sign  reflecting cyclic repeated bleeding over time Figs.  and 11. In this regard, it is important to remember the importance of post-processing images when facing a cystic mass that is hyperintense on unenhanced T1-weighted fat-suppressed sequences. Subtraction techniques that suppress the T1 hyperintense background of the cystic content allow to detect true gadolinium enhancement in suspicious solid mural nodules (a finding suggesting tumour arising from endometrioma), which may be hardly identifiable at visual assessment alone , 28.In women of reproductive age, the genital tract is the most common source of non-traumatic haemoperitoneum and bleeding corpus luteum represents the leading cause, secondary to the high vascularity of the luteal walls , 30. ThiAt CT, the ruptured corpus luteum is seen as a shrunken ovarian cyst with discontinuity of the thickened wall, often surrounded by hyperattenuating ‘sentinel clot’. Occasionally, active bleeding may be detected as serpiginous or jet-like contrast extravasation adjacent to the cyst, most usually in the portal phase reflecting intermittent or venous haemorrhage Figs.  and 13. At MRI, haemoperitoneum shows variable signal intensity depending on its age (haemoglobin degradation stages), the extent of the bleeding and blood clot formation. Within 48 h from bleeding, pelvic intraperitoneal haemorrhage generally shows intermediate signal intensity (higher than that of urine) on T1-weighted images and intermediate T2-weighted signal intensity Fig. . Later, Whereas in the past, the majority of cases were treated surgically, nowadays, in haemodynamically stable patients, the management approach is increasingly conservative with close observation and roughly 20% of patients ultimately require laparoscopic surgery , 32. SomThe second most frequent gynaecologic cause of haemoperitoneum, ruptured ectopic pregnancy (EP) accounts for 6% of pregnancy-related mortality. Mostly encountered in the fourth decade of life, EP occurs in approximately 1.3–2.4% of all pregnancies and refers to the implantation of a blastocyst at a different location from the endometrium of uterine cavity, most usually in the fallopian tube . The other uncommon implantation sites are listed in Table The key distinction between pregnant and non-pregnant patients is determined by β-hCG levels in correlation with menstrual history. Albeit acute appendicitis is the most frequent cause of acute abdominal or pelvic pain in pregnancy, EP represents a leading consideration in patients with positive β-hCG test and non-visualisation of intrauterine pregnancy at transvaginal US. Unfortunately, US has a high specificity (near to 100%) but low sensitivity about 15–20%) , 36. Occ–20% , 36In patients with equivocal clinical and sonographic findings, MRI represents an appropriate tool to make the correct diagnosis of EP. MRI findings include (a) haemoperitoneum in the pelvic cul-de-sac; (b) a heterogeneous, partly haemorrhagic, adnexal mass; and (c) tubal dilatation  with wall enhancement. The adnexal mass corresponding to the gestational sac appears as a thick-walled cystic structure which typically has high signal on T2-weighted sequences and frequently contains foci of acute blood . Unfortunately, the cystic component may not be visible, and in these cases, EP appears as a heterogeneous, mostly T2-hyperintense mass. Secondary to the invasion of the tubal wall by the trophoblasts, haematosalpinx appears as hyperintense fluid on T1-weighted sequences within the dilated fallopian tube Fig. . The bloRuptured EP is still considered the primary cause of death in the first trimester of pregnancy. It is due to the invasive growth of trophoblast into the wall of the fallopian tube; the risk of rupture increases with the enlargement of the EP .Imaging findings of ruptured EP include a poorly defined complex adnexal mass with the presence of pelvic fluid; the identification of bloody effusion (measuring 30–45 HU) in the Morrison pouch and in the cul-de-sac should raise concern for a ruptured EP. On contrast-enhanced images, active bleeding may be detected, especially in the case of a massive rupture. A specific CT finding is the sentinel clot sign: the presence of a hyperdense clot (> 60 HU) within the adnexum, thus confirming the source of bleeding , 40.At MRI, diagnostic features of tubal rupture include the disruption of tubal wall enhancement and the occurrence of acute haematoma . However, in pregnant patients, the detection of blood in the pelvic cul-de-sac is not specific for EP; the differential diagnosis includes haemorrhagic corpus luteum cyst and spontaneous abortion; however, the association of haemorrhagic fluid without an intrauterine pregnancy should raise suspicion for EP , 38, 39.Overall, due to its higher contrast resolution, MRI may detect EP more accurately than CT. The value of MRI in diagnosing EP has been evaluated in retrospective studies that included the presence of a direct sign  and indirect signs . The diagnostic accuracy of the direct sign was 92%, with a sensitivity of 91.3% and specificity of 100%, more accurate than that of any single indirect sign; accuracy increased up to 100% when diagnostic criteria required the presence of a direct sign or at least two indirect signs . FurtherLaparoscopic surgery is the gold standard treatment for EP. Indications include rupture, pain and haemodynamic instability. However, in selected patients, expectant management or pharmacotherapy (methotrexate) is being increasingly used , 36.The rare adnexal torsion (AT) accounts for 2–3% of gynaecologic emergencies, involves partial or complete rotation of the ovarian vascular pedicle in the suspensory ligament and preferentially occurs on the right side because of contralateral protection from the sigmoid colon. It may affect the ovary, the fallopian tube or both, concomitant ovarian and tubal torsion being the most common event (up to 67% of cases) . AT occuThe usual presentation is abrupt-onset lower abdominal pain radiating to the ipsilateral flank or groin, with adnexal tenderness. Alternatively, presentation is often nonspecific with intermittent pain, low-grade fever, nausea and vomiting. Recurrent attacks may be due to episodes of torsion and detorsion. In the early phase of AT, the low-pressure venous and lymphatic outflow is compromised, causing ovarian oedema, congestion and enlargement. If torsion persists, over time, the arterial circulation is impaired, thus resulting in ischaemia and haemorrhagic infarction. Therefore, the extent of imaging findings depends on the duration of torsion , 45.In this respect, it is important to remember that the ovary has a dual blood supply:( a) from the ovarian artery , contained in the infundibulopelvic ligament that extends from the pelvic sidewall to the ovary; and (b) from the ovarian branch of the uterine artery , contained in the utero-ovarian ligament that connects the ovary to the uterus. Ovarian torsion occurs when an ovary twists on its ligamentous supports (both the infundibulopelvic ligament and the utero-ovarian ligament) .US represents the primary imaging modality to assess ovarian torsion. During ovarian torsion, due to the abovementioned dual blood supply, colour Doppler US may still demonstrate some arterial vascular flow. Nevertheless, in the presence of suggestive clinical and imaging findings, this persistence of adnexal vascularisation does not rule out torsion .After inconclusive US (lesion not clearly depicted or ambiguous findings), in premenopausal women, MRI represents the better technique to assess suspected AT, but CT is often performed in patients with alternative presumptive diagnoses. At CT, the unilaterally enlarged ovary  is displaced from its expected site and often located on the midline, and the uterus is attracted towards the ipsilateral side by the shortened adnexal ligament. The oedematous ovary may show eccentric or concentric wall thickening or a ‘target-like’ pattern corresponding to peripheral displacement of follicles. Congested, enhancing ovarian blood vessels are generally seen; in this regard, it should be recalled that the pathognomonic twisted pedicle usually shows a spiral configuration, but may also present as a solid-like component adjacent to the ovarian mass. Pelvic free fluid and inflammatory fat stranding are generally present. In full-blown AT, haemorrhage appears as a hyperattenuating area and adnexal contrast enhancement is minimal or absent. When present, a mature cystic teratoma shows calcifications, foci of fat attenuation and enhancing mural nodules Fig.  9, 42, , 47–49.Also in acute and in subacute settings, MRI may depict more clearly both direct and indirect findings of AT. The twisted pedicle, containing the thickened fallopian tube and the ligamentous support with its vascular structures, presents as a beak-like protrusion adjacent to the ovarian mass or to the enlarged ovary. In the early stage, when vascular congestion and oedematous changes prevail, the thickened (diameter > 10 mm) fallopian tube appears hypointense on T1-weighted images and hyperintense on T2-weighted images. In this stage, contrast agent administration allows to demonstrate the characteristic thickened swirling configuration of vascular structures. When ischaemic alterations and haemorrhagic infarction occur, the thickened fallopian tube may show high signal intensity on T1-weighted images. Post-contrast MRI sequences with post-processing subtraction techniques can demonstrate minimal or absent enhancement of the mass presenting high signal intensity on unenhanced T1-weighted fat-saturated images. An enlarged oedematous ovary with central afollicular stroma hyperintense on T2-weighted images and peripherally displaced follicles in a string-like appearance (‘pearl string sign’) is a quite specific sign of ovarian torsion; corpus luteum may be seen in the second half of the menstrual cycle Fig. . In thisAt a later stage, haemorrhagic alterations develop, highly associated with infarction and secondary necrosis. Subacute haemorrhage shows high signal intensity on fat-saturated T1-weighted images and may involve the periphery of the enlarged ovary, as T1-hyperintense rim, or the central stroma. In this stage, heterogeneous, minimal or absent enhancement on gadolinium-enhanced subtraction of fat-saturated T1-weighted images confirm the evolution towards infarction , 47–50..Petkovska et al. described a perifollicular T2 hypointense rim correlating with perifollicular haemorrhage; the absence of this finding should be useful as a predictor of ovarian viability .DWI, with both visual assessment and quantitative ADC measurement, may be useful in the diagnosis of ovarian torsion. In patients with ovarian torsion, the ADC value of the torsed ovary is significantly lower than that of the nonaffected side . Kato etIn patients with torsed ovarian masses, at visual assessment, higher signal intensity of the wall of the ovarian lesion on DWI significantly correlates with haemorrhagic infarction. Moreover, the high-contrast resolution of DW images may be useful to detect the twisted pedicle, reflecting in some cases of fallopian tube necrosis . The conIsolated fallopian tube torsion without ovarian torsion is exceptionally rare and requires surgical intervention too. Predisposing factors are hydrosalpinx, tubal ligation, tubal or paratubal cystic and solid masses .CT, better if with multiplanar reformatted images, may show an adnexal structure distinct from the ovary. MRI usually shows a distended dilated fallopian tube with thickened walls; the tube may demonstrate a vortex-like appearance (due to more twists) distant from the ipsilateral ovary, and the latter appears normal. Thick and twisted pedicle can also be observed (whirlpool sign) , 50.Paraovarian or paratubal cysts are unilocular cystic structures located in the broad ligament, between the fallopian tube and the ovary. Isolated torsion of paraovarian/paratubal cysts rarely occurs and its prevalence is higher in children than in adults. MR imaging generally demonstrates a unilocular cyst distinct from the ipsilateral ovary, hyperintense on T2-weighted images and hypointense on T1-weighted images. The signal intensity of the cystic content may be slightly high on T1-weighted images due to haemorrhagic changes related to torsion .The differential diagnosis of AT includes massive ovarian oedema and ovarian hyperstimulation syndrome (OHSS). The former entity is a rare, benign condition thought to result from intermittent or partial torsion of the mesovary compromising the venous and lymphatic drainage but with preserved arterial supply .The imaging appearance of massive ovarian oedema is similar to that of AT: an enlarged ovary with oedematous stroma and multiple nonovulatory follicles pressed towards the periphery of the cortex Fig.  56]. Ho. Ho56]. OHSS is an iatrogenic complication of assisted reproduction techniques, characterised by bilaterally enlarged ovaries with several, peripherally located cysts accompanied by fluid shift from the intravascular to third space from increased capillary permeability that may cause the development of ascites, pleural effusions and oliguria in severe forms . TypicalOther features of OHSS include free fluid in the peritoneal, pleural and pericardial cavity; the free fluid seen in OHSS is generally simple ascites, although it may display slightly higher attenuation due to ruptured haemorrhagic cysts. The modified Golan classification categorises OHSS as mild, moderate or severe on the basis of symptoms severity and radiologic findings .Congested pelvic veins without AT are the hallmark of the pelvic congestion syndrome (PCS), an underdiagnosed condition that affects 9% of premenopausal women and may be exacerbated by intra-abdominal pressure. The multifactorial pathogenesis involves a family history of varicose veins, previous pelvic surgery, multiple pregnancies, hormonal influences and retroverted uterus. Although the key symptom is chronic dull pelvic pain that worsens in the premenstrual phase, acute and severe pain may also occur. Flow reduction, thrombosis and mass effect on nerves may lead to worsening pelvic pain. Other clinical manifestations may include dyspareunia, postcoital ache, dysmenorrhoea and perineal pain , 64.Similarly to male varicocele, dilatation of the gonadal veins and pelvic venous plexus more commonly occurs on the left side because of left ovarian vein draining into left renal vein before reaching the inferior vena cava. Mechanical extrinsic compression of drainage veins such as in retroaortic left renal vein, in May-Thurner syndrome (left common iliac vein compression against the lumbar spine by the right common iliac artery) or in nutcracker syndrome , may lead to venous congestion and engorgement of the ovarian vein . In thisAt CT, tortuous and dilated (> 4 mm) tubular structures nearby the ovary and uterus show slow blood flow and enhancement synchronous with veins. The diagnostic criteria for PCS require at least four ipsilateral parauterine veins of varying calibre, at least one measuring more than 4 mm in diameter, or an ovarian vein diameter greater than 8 mm Fig. . SimilarAdditionally, up to 50% of women with PVI present cystic formations in the ovary, ranging from scant cysts to classic polycystic ovary aspect Fig.  64]. Th. Th64]. Although liberal use of CT should be discouraged in childbearing age women, radiologists performing urgent CT studies may encounter an unsuspected acute female genital disorder that warrants immediate gynaecologic examination, further workup or prompt intervention. Therefore, familiarity with these conditions and their imaging appearances is crucial to avoid missing or misinterpreting clinically important entities which may require surgical treatment. If clinical conditions and scanner availability allow, MRI is superior to CT for further characterisation of acute disorders such as adnexal torsion, corpus luteum and haemorrhagic cysts , 22.The following second instalment will present acute uterine disorders causing abnormal vaginal bleeding in non-pregnant women  and the imaging spectrum of PID and atypical genital infections.


In the era of precision oncology and publicly available datasets, the amount of information available for each patient case has dramatically increased. From clinical variables and PET-CT radiomics measures to DNA-variant and RNA expression profiles, such a wide variety of data presents a multitude of challenges. Large clinical datasets are subject to sparsely and/or inconsistently populated fields. Corresponding sequencing profiles can suffer from the problem of high-dimensionality, where making useful inferences can be difficult without correspondingly large numbers of instances. In this paper we report a novel deployment of machine learning techniques to handle data sparsity and high dimensionality, while evaluating potential biomarkers in the form of unsupervised transformations of RNA data. We apply preprocessing, MICE imputation, and sparse principal component analysis (SPCA) to improve the usability of more than 500 patient cases from the TCGA-HNSC dataset for enhancing future oncological decision support for Head and Neck Squamous Cell Carcinoma (HNSCC).Imputation was shown to improve prognostic ability of sparse clinical treatment variables. SPCA transformation of RNA expression variables reduced runtime for RNA-based models, though changes to classifier performance were not significant. Gene ontology enrichment analysis of gene sets associated with individual sparse principal components (SPCs) are also reported, showing that both high- and low-importance SPCs were associated with cell death pathways, though the high-importance gene sets were found to be associated with a wider variety of cancer-related biological processes.MICE imputation allowed us to impute missing values for clinically informative features, improving their overall importance for predicting two-year recurrence-free survival by incorporating variance from other clinical variables. Dimensionality reduction of RNA expression profiles via SPCA reduced both computation cost and model training/evaluation time without affecting classifier performance, allowing researchers to obtain experimental results much more quickly. SPCA simultaneously provided a convenient avenue for consideration of biological context via gene ontology enrichment analysis. Data generated for standard clinical oncology care has expanded exponentially. In addition to well-known clinical variables like symptoms, stage and histology, tumor specimens are now routinely sequenced for a range of mutations that may be more or less well characterized. These molecular profiles may suggest sensitivity to a range of molecularly targeted agents. Furthermore, high resolution, functional and molecular imaging methods (like CT-PET and MR) create quantitative metrics described through radiomics features. These also suggest profiles that can guide intervention and response. To facilitate the development of novel clinical decision support tools for oncologists, we have used the publicly available data characterizing head and neck squamous cell carcinoma (HNSCC). These profiles present a large data analysis problem necessitating the use of machine learning, dimensionality reduction, and biological pathway analysis techniques. We utilize machine learning classifiers to predict patient two-year recurrence-free survival and evaluate a variety of feature sets to discover potential useful clinical biomarkers. Feature sets include combinations of patient clinical and molecular data. To improve utility of this dataset for oncological decision support, imputation and dimensionality reduction methods are used to transform feature sets to more usable, interpretable forms.Current HNSCC literature often focuses on association of regulation of specific genes with prognosis , 2. OtheThe Cancer Genome Atlas (TCGA) Research Network  is a cooLarge, multi-institutional datasets present a variety of challenges to the development of methods and tools for clinical decision support. Namely, several clinical data fields in TCGA-HNSC offered issues of sparsity and inconsistency. Out of 15 identified clinical characteristics relevant to treatment regimen, none were populated for every patient. More specifically, the number of cases  with missing or unavailable data for these fields ranged from 88 to 504, with a mean of 349.5 and median of 342 cases lacking data for each field. In addition to the problem of missing data, several fields were populated inconsistently, with responses varying both due to human error (e.g. leading zeros in numeric fields) and varying convention . Such complications required extensive preprocessing and an expert system built using domain-specific knowledge to determine whether each patient had received a specific type of therapy. Even after this preprocessing and condensing of treatment fields, issues of missing data persisted. Whether a patient had received radiotherapy and/or chemotherapy was unclear for 47 and 27% of cases, respectively. One possible technique for handling such problems is to exclude cases or variables with missing data, as was done previously with this dataset . Due to Molecular datatypes are often extremely high-dimensional. Feature selection and dimensionality reduction techniques are necessary steps when utilizing such data to best employ available computational resources. There are several strategies for selection and dimensionality reduction, including feature filtering, feature transformations, and wrapper methods such as sequential selection . In thisPerforming imputation on large datasets requires development of an expert model. Careful examination and correction of inconsistent and missing values was performed in collaboration with expert oncologist Dr. John Buatti . A rubric for consistent preprocessing and condensing of the 15 relevant treatment fields resulted in a much more concise and usable dataset. However, a significant fraction of the TCGA-HNSC patients still had uncertain status in their treatment regimens. To address this, Multivariate Imputation by Chained Equations (MICE) [MICE builds predictive models for each missing variable to realistically impute entries based on the remaining predictors. It takes into account uncertainty during imputation, allowing it to outperform single imputation methods , 17. ForNaïve Bayes and Random Forest (RF) classifiers were selected for this evaluation. The Bayesian model provides a pure conditional-statistical effort to predict survival, though it does not consider interaction effects. The Random Forest model, being a set of recursive partitionings, extensively leverages interaction effects. Earlier work suggests RF models are effective for this classification problem . The preIn the TCGA-HNSC dataset, solid-tumor expression was available for 520 of the 528 patients. With a feature set of 20,531 solid-tumor RNA expression variables, seven tumor grading variables, and the random forest-imputed treatment variables, several RF classifiers were trained to predict two-year recurrence-free survival. The classifiers varied in feature sampling and tree construction procedures: a standard RF, a weighted subspace RF (WSRF) , and a cWith the full set of RNA expression data, the feature set was first refined through two filters: a univariate, near-zero variance filter to remove uninformative features and a multivariate correlation filter to remove features with correlation greater than 0.9. These filters removed several thousand expression variables from the feature set.In addition, a dimensionality reduction was performed via Sparse Principal Component Analysis (SPCA)  which haAfter training, variable importance for the 10-component SPCA feature set was evaluated for each of the classifiers. The genes that comprise the most and least important variables were examined with a gene ontology enrichment analysis (GOEA) , 23. AnaA nested cross validation (CV) is used to tune hyperparameters and estimate out-of-sample performance, a measurement of how well a classifier would generalize if it were to be trained on the entire dataset. Data is split into ten folds (or partitions) for the outer CV. In each iteration, one fold acts as the testing data and the remaining nine folds act as the training data. Within each fold of the outer CV, a repeated grid search CV procedure  is carriFor the missing data imputation, models were trained and tested in Weka 3.9.1  with 10-In the RNA expression experiments, three different RF classification procedures are considered. Classifier training and evaluation was handled using the R package caret . ClassifTo evaluate classifier out-of-sample performance, the area under the receiver operating characteristic curve (commonly denoted AUC) is employed. AUC is a very popular and commonly used classifier metric in the literature with an intuitive probabilistic interpretation: AUC is the probability that the classifier will score positive observations higher than negative observations. Mathematically, an AUC of 0.5 is equivalent to random guesses and is the standard baseline for this metric.Feature importance is a measurement of how perturbations to variables affect classifier performance. A conditional variable importance procedure has been applied in this work. Conditional importance involves not only univariate perturbations, but conditional perturbation of variables and the variables with which they correlate . For theAs shown in Table The relative importance of treatment features doubled as a result of imputation  allow comparable classifier performance while drastically reducing runtime and necessary computation. Though not quantified here, memory requirements were also much lower for the dimensionality-reduced data. Additionally, this reduction allowed us to identify gene set candidates for GOEA. In both important and not-important SPCs, the GO term “cornification” is found, indicating that this biological process is related to high-variance genes in this dataset. Terms found only in the high-importance SPC gene sets are related to cell motility , immune response, cell growth, and blood vessel development. Activity of genes involved in these processes could be indicative of a cancer’s ability to survive, grow, and metastasize, suggesting that these SPCA transformed RNA data contain useful information about underlying relationships between solid- tumor expression and two-year recurrence-free survival.In modern oncological research, TCGA datasets present significant large data analysis challenges, from clinical parameter sparsity to high dimensionality. Facing these problems requires significant preprocessing and machine learning modeling to uncover new knowledge. A multivariate imputation method (MICE), SPCA dimensionality reduction, and an SPC-focused GOEA are presented in the context of TCGA-HNSC clinical and RNA expression variables to improve usability of data for future HNSCC decision support.vAs others , 31 haveUnsupervised transformation of RNA expression data via SPCA was extremely useful in improving interpretability of survival models and biomarker identification, by limiting the number of genes contributing to each principal component and allowing for a more nuanced examination of the underlying biological processes. The biological processes found to be associated with only high-importance SPCs may be useful in future feature vselection for biomarker discovery. Additionally, the SPCA functioned well as a dimensionality reduction technique, as the dimensionality reduced features allowed for significantly lower computation time without significantly affecting classifier performance. From the literature and these analyses, unsupervised transformations of RNA expression data seem a viable option for future integration of molecular data into HNSCC clinical predictive models.Future work will consider the effect of clinical imputation on models also utilizing molecular data, both with SPCA transformations and other unsupervised feature transformations methods such as denoising autoencoders. Additionally, biomarker evaluation will be expanded to directly consider right-censored survival.


Hemodynamic dysfunction mainly characterizes pathophysiology of peripheral arterial disease (PAD) leading to chronic ischemia. Hemodynamic dysfunction is the origin of intermittent claudication (chronic PAD) or of critical limb ischemia (very severe PAD). Notably, it is well known that oxidative stress (OxS) plays a pathophysiological role in PAD. The higher production of reactive oxygen species (ROS) from OxS and reduced redox capability are two crucial players in initiating and progressing PAD. A number of biomarkers highlight OxS and monitor it in PAD. The present review summarizes data on OxS, on biomarkers available to mark OxS occurrence and to monitor on PAD progression, as well as to evaluate the effects treatments in PAD patients. In conclusion, by detailing OxS and its biomarkers, we hope to encourage more studies to focus on drugs which combat OxS and inflammation. Peripheral arterial disease (PAD) is one clinical aspect of atherosclerotic disease, presenting impaired blood flow in the lower limbs and where intermittent claudication is the most significant symptom  1,2]. P,2. P1,2]The close relationship between OxS and the atherosclerotic process have been widely investigated ,22,23,24OxS has been closely associated with cardiovascular diseases, oxidative biomarkers correlating with disease progression. OxS biomarkers have proven helpful in improving the early diagnosis of PAD, particularly in asymptomatic patients . CurrentAntioxidants are classified into two groups according to the mechanism by which they prevent or retard oxidation. The primary (chain-breaking) antioxidant group  acts by interrupting oxidation and converting free radicals to stable species. The secondary antioxidant  group reacts with oxygen before the start of oxidation. The oxidation rate may be evaluated by generating hydroperoxides (per minute and per milligram of low density lipoprotein). Another surrogate biomarker of OxS is malondialdehyde concentration. MDA plasma levels differ between PAD and healthy controls as well as MDA levels being higher in PADs than in non-PAD individual before starting a treadmill test ,57. Maxi4-HNE  is generated by the peroxidation of n-6 polyunsaturated fatty acid. HNE has a number of biological properties mainly represented by HNE-adduct generation on free amino groups and thiol groups on proteins. HNE adduct accumulation initiates cellular dysfunction, such tissue damage being a huge factor in atherosclerosis and arterial atherosclerotic diseases. HNE contributes to the atherogenicity of oxidized LDL by deviating the LDL metabolism and leading it to generate foam cells through the scavenger receptor pathway of macrophage cells. HNE de-regulates cellular responses at higher concentrations causing inflammatory responses and apoptosis. HNE is involved in re-modelling atherosclerotic plaque through the progressive modification of smooth muscle cells, also effecting proliferation, angiogenesis, and cell apoptosis. HNE adducts in the core of atherosclerotic plaque contribute to macrophage and smooth muscle cell apoptosis which in turn lead to a high risk of athero–thrombotic events. There is data on the accumulation of biogenic toxic aldehydes in critical ischemic tissues such as in ischemia-reperfusion injury. Notably, 4-hydroxy-2-nonenal (4-HNE) is a highly reactive aldehyde with abundant adduct formation which negatively effects contractility and the mitochondrial energy of muscle cells. High levels of 4-HNE modified proteins were found in PAD patient muscles such that the accumulation of the derived aldehyde product (4-HNE) is now considered as a marker for skeletal muscle injury initiated by OxS ,62.While the peculiar role played by endothelial cells on correct function of circulation is clearly known, on the other hand, it is known that vascular smooth cells communicate with endothelial cells via mRNAs. Such mRNAs  promote cell-to-cell interplay, finally favoring specific effects. Transport of delivered mRNAs to endothelial cells has been shown by using a high resolution imaging method .mRNAs 143/145 play a key role for maintaining contractile phenotype of vascular smooth cells (VSMC) dysregulation of vascular tone and of arterial pressure were demonstrated in mRNAs-143/145−/−deficient mice. It is clear as the mRNAs show different capabilities both in contrasting or in maintaining functions of endothelial cells leading to equilibrium of vascular tone .Capability of endothelial cells on circulatory equilibrium must achieve aiming against haemodynamic dysfunction and its unbalance is directly favorable to provoking arterial damage. Such mRNAs as mRNA-126 released by the endothelial is internalized by white cells (monocytes) and vascular smooth cells leading them to play a role to against endothelial dysfunction. There is a clear explanation of the crucial protective capability of mRNAs 126 from study on diabetes having highly prevalence of arterial consequences also linked to endothelial cell dysfunction ,66,67,682α  as a dependable method for evaluating oxidative stress in PAD patients [Markers for the oxidation of LDL and eicosanoid formation  were among the first to be analyzed. Isoprostanes derive from the metabolism of arachidonic acid induced by free-radical catalyzed processes . Severalpatients ,74. The . Among the most significant biomarkers of oxidative stress with the role of antioxidants is Paraoxonase (PON): this family of enzymes protects lipoprotein from peroxidation . Particularly in PAD patients compared to no PAD individuals were demonstrated lower paroxonase-1 (PON-1) concentration whereas PON-1 activity was raised. As we know, PON-1 activity is strongly dependent on the oxidation of lipoproteins, and in PAD patients there is a compensatory mechanism in favoring protective of PON family to against peroxidation [xidation . PON is xidation ,76,77,78Transcriptional factor Nrf2 is one of the most important regulator molecules in the antioxidant system, appearing to protect against reperfusion injury. It promotes the transcriptional activation of several antioxidant (ARE) binding responses . Nrf2 isIt is known that OxS plays a role in atherosclerotic processes as well as in PAD, thus it is reasonable that OxS must be targeted by using multiple approaches. High levels of OxS biomarkers were found in PAD patients with chronic ischemia causing intermittent claudication and in critical limb ischemia patients showing more severe ischemia of the peripheral arteries. OxS and pro-inflammatory conditions increased in PADs both of which are crucial in determining endothelial dysfunction. It is widely accepted that all the aforementioned conditions accelerate the atherosclerotic process. To date, endothelial dysfunction is considered an independent risk factor for morbidity and mortality in PAD patients. It is worth remembering that PAD is a predictive clinical marker for extended atherosclerotic processes in other arteries. From a therapeutic point of view, physical and pharmacological options are mandatory to reduce or counteract OxS and inflammation. On the other hand, to date we note the positive effect originated by no pharmacological strategy for PAD as the supervised physical training. It reduces OxS as well as the plasma levels of inflammatory biomarkers in parallel with improved walking distances measured by the treadmill test. These findings are very intriguing because improved muscle and physical performance may be crucial issues for PAD patients. It is clearly accepted that a well-structured rehabilitation program, may improve both performance, quality of life furthermore walking capability effects on the functional balance of patients with PAD ,90,91,92


Following a traumatic spinal cord injury, a 53-year-old male developed a central cord syndrome with at-level neuropathic pain. Magnetic resonance imaging revealed a classical “snake eye” appearance myelopathy, with marked hyperintensities at C5-C7. Clinical examination revealed intact pinprick sensation coupled with lost or diminished thermal/heat sensation. This dissociation could be objectively confirmed through multi-modal neurophysiological assessments. Specifically, contact heat evoked potentials were lost at-level, while pinprick evoked potentials were preserved. This pattern corresponds with that seen after surgical commissural myelotomy. To our knowledge, this is the first time such a dissociation has been objectively documented, highlighting the diagnostic potential of multi-modal neurophysiological assessments. In future studies, a comprehensive assessment of different nociceptive modalities may help elucidate the pathophysiology of neuropathic pain. The spinothalamic system transmits noxious and innocuous thermal and noxious mechanical information. While the assessment of thermo-nociceptive pathways using contact heat evoked potentials (CHEPs) is well-established for the diagnosis of incomplete spinal cord lesions , 2, objeExamination of the different modalities of nociception is recommended for a comprehensive assessment of spinothalamic integrity . MoreoveThe specific aim was to compare segmental outcomes, neuroimaging, and clinical signs and symptoms.A 53-year-old male with a posttraumatic incomplete spinal cord injury  was examined during his neurological rehabilitation at the Spinal Cord Injury Center at Balgrist University Hospital, Switzerland. The study was approved by the “Kantonale Ethikkommission Zürich”  and written informed consent for publication was obtained from the patient.The patient presented clinically with a central cord syndrome, and at-level neuropathic pain with mechanical allodynia  on both forearms. Light touch was normal within the cervical segments, and sharp dull discrimination was preserved while the intensity of the pinprick stimulus was only mildly attenuated. The upper extremity motor score  showed a profound weakness of elbow extensors, and no voluntary muscle activity in the finger flexors and abductors bilaterally. The lower extremity motor score was normal . Biceps and brachioradialis reflexes, as well as knee-jerk and ankle-jerk reflexes were bilaterally exaggerated. The Babinski sign was negative. Muscle tone was normal without signs of spasticity. Coordination and gait showed discrete signs of ataxia. Magnetic resonance images revealed a multi-segmental (C5-C7), longitudinal “snake eye” appearance myelopathy Figure . Ulnar sThe CHEP recording was performed according to an established protocol , 2. NoxiCHEPs were normal at C4 and T4 with high pain ratings to the heat stimulation, but almost complete loss of painful heat sensation and absent CHEPs at the level of C6 Figure . In contThe patient presented with signs and symptoms of a posttraumatic central cord syndrome including at-level mechanical allodynia. MRI findings indicated a “snake eye” appearance myelopathy with bilateral high intensity signal changes in the central gray matter from below C5 down to C7. Corresponding with radiographic findings, the patient showed a clinical dissociation of normal light touch  but mildly impaired pinprick sensation. However, cervical CHEPs revealed a distinct impairment of noxious heat sensation, while mechanically evoked potentials  remained normal throughout all tested segments. Below the level of the focal central myelopathy heat sensation and CHEPs remained normal.Collectively, these observations confirm pathology in decussating spinothalamic afferents conveying heat sensation, coupled with preserved integrity in the antero-lateral quadrant and dorsal columns Figure . Unlike A previously published case determined that PEPs are conducted in the antero-lateral system, abolished alongside laser evoked potentials in a patient with a selective lesion of the contralateral anterolateral quadrant .Corresponding sensory deficits associated with antero-lateral cordotomy include the loss of both pinprick and temperature sensations . MidlineThe patient reported herein presented with sensory deficits consistent with midline spinal cord damage . These findings were confirmed by neuroimaging and through the application of multi-modal electrophysiological testing—the latter demonstrating objective signs of preserved pinprick and absent temperature sensation at C6. Based on damage localized to the centro-medullary cord in this patient, one potential explanation for the dissociation between temperature and pinprick sensation is a distinct topographical organization within the decussating spinothalamic pathways. Afferents conveying temperature sensation are positioned posterior to those afferents conveying pinprick sensation Figure . This woThe present case highlights the application of multi-modal neurophysiological assessments combined with conventional neuroimaging to improve the characterization of spinal cord damage beyond conventional clinical examination. Their combined application may also play an important role in elucidating novel mechanisms underlying the development of neuropathic pain.JR contributed substantially to the conception and design of the study, the data acquisition, analysis, and interpretation. Furthermore, he was chiefly involved in drafting the manuscript. MH contributed substantially to the data acquisition and interpretation, and revised the research article. PH was chiefly involved in creating the figure and contributed to data acquisition and analysis. CJ was involved in the data analysis and revising the research article. JK was involved in the data analysis and contributed substantially to the revision of the research article for important intellectual content. AC contributed substantially to the conception and design of the study, data analysis and interpretation, and was involved in revising the research article for important intellectual content.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.


In cases of avulsion fracture of the ischial tuberosity in which the bone fragments are substantially displaced, nonunion may cause pain in the ischial area. Various surgical procedures have been reported, but achieving sufficient fixation strength is difficult.We treated a 12-year-old male track-and-field athlete with avulsion fracture of the ischial tuberosity by suture anchor fixation using the suture bridge technique. The boy felt pain in the left gluteal area while running. Radiography showed a left avulsion fracture of the ischial tuberosity with approximately 20-mm displacement. Union was not achieved by conservative non-weight-bearing therapy, and muscle weakness persisted; therefore, surgery was performed. A subgluteal approach was taken via a longitudinal incision in the buttocks, and the avulsed fragment was fixed with five biodegradable suture anchors using the suture bridge technique.Although the majority of avulsion fractures of the ischial tuberosity can be treated conservatively, patients with excessive displacement require surgical treatment. The suture bridge technique provided secure fixation and enabled an early return to sports activities. Pelvic apophyseal avulsion fracture is a category that includes a few types of avulsion fractures. Avulsion fractures of the anterior inferior iliac spine, anterior superior iliac spine, and iliac crest are comparatively common, but avulsion fractures of the ischial tuberosity are rarely described in the literature . HoweverA 12-year-old boy presented with a left avulsion fracture of the ischial tuberosity. Informed consent was obtained from this patient and his family. The patient’s family history and previous medical history were unremarkable. The patient was a track-and-field athlete who felt severe pain in his left buttock while running. He visited a local hospital, where plain radiographs and computed tomography (CT) of the pelvis showed an avulsion fracture of the left ischium  was used to restrict knee movement to within 45° of flexion in postoperative week 3 and to within 10° of flexion in week 5, while passive assistive hamstring stretches were performed; then, active ROM exercises were started from week 6 (Fig. Since the first description by Berry in 1912 , avulsioConservative treatment is the standard primary treatment modality for avulsion fracture of the ischial tuberosity , 10, butWood et al. reported that a delay in surgical repair renders the repair more technically challenging, may increase the likelihood of sciatic nerve involvement, increases the need for postoperative bracing, and reduces postoperative outcomes in terms of hamstring strength and endurance . In thisThe surgical techniques reported previously include the use of a reconstruction plate, lag screws, and suture anchors , 19–21. The postoperative orthosis used in this case was a Snyder sling, a sling originally used to treat Perthes disease . The SnyIn conclusion, although the majority of avulsion fractures of the ischial tuberosity can be treated conservatively, patients with excessive displacement require surgical treatment. The suture bridge technique is a useful technique that provides sufficient strength for avulsion fracture of the ischial tuberosity.


M = 28.14, SD = 5.26), 12 non-circadian DSPD patients  and 51 healthy controls (M = 21.47 SD = 3.16) completing the protocol. All participants were free of central nervous system acting medications and abstained from caffeine and alcohol on the day of the assessment. Two pupillary light reflex measurements were completed by each participant, one with a 1s dim (~10 lux) light exposure, and one with a 1s bright (~1500 lux) light exposure. Circadian DSPD patients showed a significantly faster pupillary light reflex than both non-circadian DSPD patients and healthy controls. Non-circadian patients and healthy controls did not differ significantly. Receiver operating characteristic curves were generated to determine the utility of mean and maximum constriction velocity in differentiating the two DSPD phenotypes, and these demonstrated high levels of sensitivity (69.23–-100%) and specificity (66.67–91.67%) at their optimal cut offs. The strongest predictor of DSPD phenotype was the mean constriction velocity to bright light (AUC = 0.87). These results support the potential for the pupillary light reflex to clinically differentiate between DSPD patients with normal vs. delayed circadian timing relative to desired bedtime, without the need for costly and time-consuming circadian assessments.This study investigated the utility of the pupillary light reflex as a method of differentiating DSPD patients with delayed melatonin timing relative to desired/required sleep time (circadian type) and those with non-delayed melatonin timing (non-circadian type). All participants were young adults, with a total of 14 circadian DSPD patients ( Delayed sleep phase disorder (DSPD) is categorised as a circadian rhythm sleep disorder according to the International Classification of Sleep Disorders  describes the acute response of the pupil to a pulse of light. In mammals, the pupillary light reflex is mediated by retinal cells including rods, cones and melanopsin-containing intrinsically-photosensitive retinal-ganglion cells (ipRGCs) . MelanopEthical approval was obtained from the Monash University Human Research Ethics Committee (MUHREC). All participants provided written informed consent and were reimbursed for their time.A total of 26 patients completed the study, 14 circadian DSPD patients, 12 non-circadian DSPD patients. Circadian and non-circadian DSPD patients were categorised according to our previously developed criteria . TherefoM = 1.74 years, SD = 0.51) after their initial diagnosis. Thus, the clinical diagnoses and circadian assessments used to classify them were retrospective. We compared PLR results from DSPD patients with those of 51 healthy volunteers from a database of healthy sleepers. Healthy participants were not taking any medications and had no diagnosed sleep disorders or psychiatric conditions. Participant demographics are presented in At the time of the PLR assessment, participants were not taking central nervous system acting medications. We recruited participants from a previously established cohort of DSPD participants , approxiParticipants were instructed to refrain from drinking alcohol or consuming any caffeine for 24 hours prior to their PLR assessment. The assessment was ~12 minutes in duration and was conducted between 2 and 8 hours after habitual wake time. This time frame was selected as ipRGC mediated pupil responses are stable during this period . ParticiThe Neuroptics PLR-2000  was used to conduct all pupillometry assessments. This device is Therapeutic Goods Administration (TGA) approved, and allows customised measures of the PLR and steady-state pupil size using an infrared camera. The device takes monocular measurements, and all measurements in this study were conducted on the left eye. The in-built light source  was used to deliver two 1-second light stimuli, described here as dim (~10 lux) and bright (~1500 lux). The relative retinal impact for each light stimuli is presented in The two primary outcome metrics were mean constriction velocity (CV) and maximum CV. The mean CV is the average speed of the constriction in mm/s between the onset of constriction and peak constriction for the measurement. Maximum CV represents the fastest 33ms epoch during the constriction period (between onset and max constriction) in mm/s. Both of these metrics are automatically calculated by the device immediately following a successful, artefact free measurement.n = 1 circadian DSPD patient only dim mean CV could be determined; analyses for dim max CV, bright mean CV and bright max CV therefore include n = 13 circadian DSPD patients.One-way analyses of variance were used to examine differences in constriction velocity between circadian DSPDs, non-circadian DSPDs and healthy controls. Although our sample sizes were not equal, this did not result in heterogeneity of variances between groups. Post-hoc analyses were conducted using a Bonferroni-Holm correction . Receivep = .051). Post-hoc analysis for the three significant ANOVAs showed that circadian DSPD patients exhibited faster constriction velocity on all outcomes, compared to non-circadian DSPD patients. Circadian DSPD patients also exhibited a faster PLR than healthy controls for bright light exposures, while non-circadian DSPD patients did not differ from controls on any outcomes , and so was not considered a possible covariate.ANOVAs revealed significant between group differences for mean constriction to dim light, and mean and max constriction to bright light. The ANOVA for max constriction to dim light was approaching significance (omes see . AlthougEach of the ROC curves and the associated area under the curve (AUC) values are presented in This study investigated the utility of the PLR as a method of differentiating DSPD phenotypes, and distinguishing DSPD patients from healthy controls. We were able to successfully predict DSPD phenotype using a simple cut-off score for the mean and maximum constriction velocity of the PLR to both dim and bright light pulses. As hypothesised, circadian DSPD patients consistently demonstrated a faster PLR than non-circadian DSPD patients. Only circadian DSPD patients were distinguished from healthy controls using bright light PLR outcomes, exhibiting a significantly faster PLR than healthy controls, which may indicate a hypersensitivity to light. Non-circadian DSPD patients exhibited a ‘typical’ PLR, which was similar to that of our control participants. These findings suggest that PLR may be a useful clinical test in the diagnosis of DSPD phenotype.Although our DSPD patients and healthy controls differed in age, this is unlikely to explain the observed differences in the PLR between groups. Previous studies have demonstrated reductions in mean and max constriction velocity in elderly patients . Our groThe relationship between pupil responses and sleep-wake timing has been attributed to the direct stimulation of melanopsin containing ipRGCs , 14. PreIn clinical practice, the misdiagnosis of sleep disorders as a result of common symptomology is a prevalent issue . DSPD pawhen melatonin should be administered for the treatment of DSPD. Administration times which range from relative to DLMO (5 hours prior), relative to bedtime (20 minutes prior), to fixed clock times (ranging from 19:35–22:00 h) have been reported [Current guidelines for the treatment of DSPD recommend morning bright light therapy, and evening administration of melatonin . Both ofreported . Non-cirreported . The nonA potential strength of the PLR in identifying circadian and non-circadian phenotypes within a DSPD patient group is the apparently trait-like nature of our observed abnormality in constriction velocity. This is consistent with the previous finding that the circadian light response is largely genetic . Our catAlthough we had up to 87% accuracy in determining DSPD phenotype, not all circadian DSPD patients were captured by our test, and this is likely to be the case in larger samples as well. Some DSPD patients (and patients with other sleep disorders) may exhibit a delay in circadian phase which is not related to a difference in ipRGC function. For example, abnormal SCN activation in response to light, altered behavioural patterns in light exposure, or an abnormally short phase advance region of the phase response curve could all result in the circadian DSPD phenotype in the absence of abnormal pupil function. Therefore, a measure such as ours would serve best to confirm a likelihood that sleep disturbance is related to circadian function, rather than to rule out the role of circadian phase in producing sleep disturbance for an individual patient.In this study, we used a pupillary measure to better-characterise a clinical sleep disorder diagnosis. Of note, DSPD is associated with abnormally high circadian light sensitivity, and pupil responses are a measure of circadian light input. Although further validation of these pupil responses in relation to direct circadian outcomes are required, this study demonstrates the potential for the PLR to be used diagnostically in circadian medicine. Given the measure is brief and has no associated per-use cost, this could be easily adapted into clinical practice. Improved characterisation of these two distinct phenotypes of DSPD will allow for more targeted treatment recommendations, based on the specific aetiologies. This represents a critical step in the shift toward personalised sleep medicine.S1 DSPD PLR Dataset(XLSX)Click here for additional data file.


P = 0.038 and P = 0.034 at 30 and 60 min following butyrate ingestion [1.25 g/kg BW], respectively) and rabbits , while plasma GLP-1, insulin and glucose concentrations remained unaffected by butyrate in both species over time. These results are in contrast to butyrate’s stimulating effect on both incretin and insulin secretion in mice, indicating specific, species-dependent differences even among mammalian species. Further, based on the analyzed correlations between the measured endocrine parameters (regardless of the butyrate exposure), it can be assumed that incretins may regulate pancreatic insulin release in rabbits on a partly different way compared to mice, humans and chickens.The pancreatic secretion of insulin, a key endocrine regulator of metabolism and growth, can be greatly influenced by the gut-derived incretin hormones, namely by GIP (Glucose-dependent Insulinotropic Peptide) and GLP-1 (Glucagon-like Peptide 1). As insulin is a major stimulator of growth, affecting its producion may be of special importance in food-producing livestock. The aim of the present study was to investigate novel ways of modulating incretin and insulin homeostasis in chickens and rabbits by nutrition, e.g. by oral butyrate application, also studying the mechanisms of incretin action in both species as a comparative approach. Acute oral butyrate challenge significantly decreased plasma GIP levels by approx. 40% in both species: significant interactions of butyrate exposure and incubation time were found in both chickens ( Insulin plays pivotal role in the regulation of carbohydrate and lipid metabolism, while it is also considered as an indispensable stimulator of growth by increasing protein synthesis and affecting the expression of several growth-related genes . In bothin ovo application of novel synbiotics (composed of Lactobacillus spp. and oligosaccharides) significantly decreased intestinal GIP and GLP-1 production in 6-week-old broilers [Based on the anabolic properties of insulin, influencing its secretion by nutrition via the enteral incretin production may provide new ways to improve animal production. The short chain fatty acid (SCFA) butyrate, which is a commonly applied growth promoting feed additive, can stimulate incretin and insulin secretion  as well broilers .In association with the suggested species-dependent differences, investigating the molecular mechanisms of insulin and incretin homeostasis in various species can have great importance from comparative physiological approach. Therefore, the main goal of this study was to assess the effects of acute oral butyrate treatment on incretin and insulin secretion in chicken and rabbit. These two species were selected as being potential targets of butyrate administration as a feed additive and also serving as models for birds and mammals. Studying novel possibilities for nutritional modulation of the incretin homeostasis may also provide new data regarding the regulatory role of incretins on pancreatic insulin release in different species, and may highlight new data about the mechanisms of incretin action.All animal procedures reported herein were conducted in accordance with international and national laws as well as with institutional guidelines. Husbandry and experimental procedures were approved by the Government Office of Pest County, Food Chain Safety, Plant Protection and Soil Conservation Directorate, Budapest, Hungary (number of permission: PEI/001/1430-4/2015).ad libitum; the applied diet was formulated based on the requirements of the breed [Newly hatched male broiler chicks  of the Ross 308 strain, obtained from a commercial hatchery , were randomized into 3 experimental groups and were housed in floor pens on chopped wheat straw; housing and climatic circumstances were set according to the Ross technology over the entire trial . Chickenhe breed . Calculain vivo metabolic actions [On day 24, animals were treated following overnight feed deprivation with a single intra-ingluvial sodium butyrate bolus in two different doses, administered by a tube. A randomly selected group of chickens received a dose of 0.25 g sodium butyrate/kg BW , while a higher dose of sodium butyrate was given to another group  to test the dose-dependency of butyrate’s action. Physiological saline solution was applied for seven chicks under the same conditions as a control group . Butyrate administration in a daily oral bolus (following overnight feed deprivation) is an already approved treatment model, providing a fast, short-term butyrate exposure and being suitable for investigations on butyrate’s  actions .Blood samples were drawn from the brachial vein of chickens into heparinized tubes at the following time points: prior to the butyrate exposure (0 min); 10, 30 and 60 min following the bolus treatment. After immediate centrifugation , plasma samples were stored at -80°C until further processing.Six-week-old male rabbits  of the Pannonian Giant breed  were randomized into 3 experimental groups and were housed in floor pens under controlled environmental conditions according to the requirements of the breed. Similarly to chickens, rabbits were also allocated to groups randomly, but controlled for body weight. Animals had free access to the supplied pelleted diet , hay andAt the age of 7 weeks, after overnight fasting, rabbits were exposed to a single sodium butyrate bolus given by a gastric tube with the same dosage as administered in the chicken experiment , while control animals were treated with physiological saline solution .Blood samplings were conducted similarly to the chicken study; samples were taken from the marginal ear vein before butyrate ingestion (0 min) and 10, 30 and 60 min following the bolus exposure. Heparinized plasma samples were stored at -80°C until analysis.MBS2512151) with intra- and inter-assay variations below 15%, according to the manufacturer’s instructions . Blood glucose concentrations were measured by the colorimetric Fluitest Glucose Assay .The GIP, GLP-1 and insulin concentrations of blood plasma samples were assessed by chicken- and rabbit-specific sandwich ELISA kits, purchased from MyBioSource .Concentration of GIP in blood plasma of chickens was remarkably affected by butyrate exposure: significant interactions were detected between the higher dose (1.25 g/kg BW) of butyrate and incubation time . These interactions are reflected by the butyrate-associated decrease of plasma GIP levels at 30 and 60 min with approx. 40% compared to 0 min values . SimilarRegarding the GLP-1 and insulin concentrations, no significant interactions were observed between butyrate exposure and incubation time, so, according to our results, butyrate had no significant effects on GLP-1 and insulin levels either in chickens or in rabbits. Results obtained by GLP-1 measurements of chickens and rabbits are presented in Figs Blood glucose concentrations of chickens were not altered by time, and there were no significant interactions between any time points and treatments . In rabbConcerning the correlations between the measured endocrine parameters in chickens (regardless of the butyrate exposure), highly significant (P < 0.001) positive correlations were found between plasma GIP and GLP-1 values , GIP andIt has been described that orally applied butyrate as a potent effector of carbohydrate and lipid metabolism can increase both insulin secretion and insulin sensitivity in mice , mediateOur results regarding the interaction of butyrate exposure and incubation time showed that orally applied butyrate affected plasma GIP concentrations in both chickens and rabbits Figs . The dirThe observed butyrate-associated reductions in plasma GIP levels are in contrast with the findings of previous studies in mice, where similarly applied butyrate significantly increased the plasma concentrations of both incretins and insulin . NotwithAccording to the species-specific dietary requirements, rabbit diets always contain higher amount of dietary fibers than those of mice or chickens, providing more substrate for the microbial SCFA production in the large intestines. Therefore, a relevant amount of butyrate is produced in the caecum of rabbits , possiblThe effects of SCFAs on incretin homeostasis can be also diverse even in the same species. Continuous, chronic dietary exposure to SCFAs significantly decreased plasma incretin levels in mice , and feein vitro and in vivo studies. For instance, applying a daily oral bolus of butyrate for 5 days affected the acetylation rate of hepatic histones in chickens in a dose-dependent manner [Rhodopseum faecalis RLD-53, where butyrate supplied at lower levels increased hydrogen production, but not at elevated concentrations [When examining the effects of oral butyrate exposure on plasma GIP concentrations in chickens and rabbits, it can be seen that the higher butyrate dose (1.25 g/kg BW) was required to cause significant alterations in chickens, while only the lower butyrate dose (0.25 g/kg BW) was capable to similarly reduce GIP levels in rabbits. This difference might be explained by the mentioned intensive intestinal butyrate production of rabbits . The higt manner . The acet manner . Similart manner . The lactrations . Dose-astrations .The allocation of the animals to experimental groups was carried out randomly, but controlled for the body weight ensuring no significant differences between any groups. However, the baseline (0 min) plasma concentration of the measured parameters was not taken into account for the allocation, but no significant differences were observed between any groups at 0 min concerning all measures except one. Plasma glucose concentration of rabbits exposed to the lower dose of butyrate was significantly higher than that of controls at 0 min , but this difference cannot be considered as a relevant one and suggested to play no role in the observed endocrine alterations. The further observable numerical differences (e.g. GIP and insulin in rabbits) did not reach the level of statistical significance. Plasma glucose concentrations were increased with time in rabbits, independently of the butyrate exposure. This can be in association with the stress-sensitivity of rabbits, despite of the minimized pain and stress during repeated blood samplings.Concerning the correlations between the measured endocrine parameters, some major differences were found between chickens and rabbits. GLP-1 and insulin were positively correlated in both chickens and rabbits Figs , but thiThese findings highlight that there might be species-specific differences in the action of incretins on pancreatic insulin secretion, being an important point from comparative physiological approach. In chickens, the detected positive correlations confirm the presumed synergistic inducing action of both GIP and GLP-1 on β cell insulin release. However, it can be assumed that GIP may not play the same major insulinotropic role in rabbit, differing from chicken, mouse and human , 24.Concluding the present results, it has been justified that butyrate has a significant role in influencing insulin homeostasis in both chickens and rabbits, which is suggested to be partly mediated by incretins. It can be stated that butyrate may have different effects on incretin and insulin secretion in various species, presenting differences even among mammalian species. In contrast to butyrate’s stimulatory action on incretin production in mice, acute oral butyrate exposure significantly decreased plasma GIP levels in both chickens and rabbits, but had no effect on GLP-1, insulin and glucose concentrations of blood plasma. Therefore, it is suggested that the nutritional modulation of insulin secretion should be specifically investigated in each target species, and results from model studies may be extrapolated to other species only with strong limitations. In addition, it can be assumed based on the analyzed correlations that incretins may regulate pancreatic insulin release on a partly different way in rabbits compared to other examined mammals and chickens: the major stimulatory action of GIP on insulin secretion may be questionable in rabbits according to the lacking correlation of GIP and insulin and the negative correlation between the two incretin hormones.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift17 Besides its role as a classic model in developmental biology, the zebrafish embryo is being increasingly used in biomedical research to decipher disease-associated biological processes, with a high potential for subsequent drug discovery applications ,2,3. TheThe sample handling challenge generated by manipulating thousands of zebrafish specimens can usually be efficiently tackled even by smaller research laboratories. In contrast, classic zebrafish mounting procedures for microscopy are usually not compatible with large-sample volumes. Thus, zebrafish screening applications demand dedicated mounting techniques to avoid the acquisition of non-standardised image data of randomly oriented specimens . TechnicAutomated imaging and visualisation of biological events on the cell or tissue level in zebrafish is hindered by the impracticality of positioning these features precisely within the limited field-of-view (FOV) of higher magnification objectives while being mounted in microtiter plates ,19. ConsLarge-scale zebrafish screening employing automated multidimensional image acquisition leads to the generation of massive terabyte-scale image datasets . Tacklinintraflagellar transport 172 (ift172) gene was used to induce large glomerular cysts in GFP (green fluorescent protein) expressing pronephroi of the Tg(wt1b:EGFP) transgenic line . GF. GF18]. For each image the variance was calculated after background correction, and the images with variance values below 400 were classified as blurry and filtered out. Similarly, blank image detection was based on a variance check after Laplace filtering ,45. The To quantify the total kidney area, both cystic and pronephric areas were calculated for all the images. Pronephric areas were calculated by the “Analyze particles” function in Fiji, and areas of all the resulting particles were summed. The cystic area was analysed by inverting the binary images and drawing a convex hull around the objects. This separated the cystic area from the general background area. It was followed by the application of “Analyze particles”, leading to the formation of several bounded particles in the image, including those which represented cystic areas. All the particles were assigned numbers by the “Analyze Particles” plugin. The images here were processed so as to get the particles of interest within a count of 10. Therefore, cystic particles, among others, were selected by sequential investigation of all the particles and selecting those with a Feret diameter ≥70 and a particle number <11 per image. Areas of the selected particles were summed up to generate the final cystic area value. Finally, the images were checked for their assigned wild-type or cystic status from the classifications performed previously using the image quality control script. Based on these results, wild-type images were allocated pronephric area as the total kidney area, whereas, the cystic kidneys were attributed the sum of both pronephric and cystic areas as their total kidney area. Measurements of single microtiter plates were visualised as a heatmap using the Matplotlib library in Python.


The underlying biology of prostate cancer is complex and evolves from initial tumorigenesis to metastatic potentiation and castrate resistance. Metastases source directly from the primary tumor site but also can occur from an established metastatic site to another new metastatic site or even to the surgical bed, leading to local recurrence ,2. This oligometastatic disease as an intermediate step in cancer progression from a localized confined process to a disseminated state (spectrum theory of cancer)  ,4. The o cancer) ,6. The p cancer) –14. AlsoDiagnosis of oligometastatic disease relies fundamentally on the performance of diagnostic imaging tools, which are advancing rapidly –18. The P = 0.02). More recently, Ost et al. reported on the results of a prospective, randomized, multicenter phase II trial comparing surveillance with MDT for oligometastatic prostate cancer recurrence  . Interestingly, 35% of patients undergoing surveillance experienced relatively short-duration spontaneous prostate-specific antigen declines without any therapy, whereas 30% of patients treated with MDT progressed to polymetastatic disease within the first year. A similar phase II trial (ORIOLE) is also under way, which randomizes men with noncastrate oligometastatic prostate cancer (identified on prostate-specific membrane antigen ligand imaging with 18F-DCFPyl PET/CT) to either surveillance or selective ablative radiotherapy of metastatic lesions, with the primary clinical endpoint of progression at 6 mo from randomization (NCT02680587)  . Sixty-t2680587) . Safety,2680587) .In view of data cited in the studies above, several issues remain to be settled before the concept of oligometastases can take potential hold in the routine clinical management of patients with prostate cancer. A biologic anchor is needed to decipher whether oligometastatic lesions are indeed different from polymetastatic lesions and how oligo lesions evolve to poly lesions . Some woAlthough the concept of oligometastasis is interesting, there is still more basic research that needs to be done to establish it firmly as a distinct biologic entity along the natural history of prostate cancer. The current unsystematic approach in detection and management of oligometastatic prostate cancer will need to be standardized so that future clinical trials can be designed appropriately and, more importantly, compared properly. These critical prerequisites will elucidate whether detection and treatment of oligometastases should be incorporated into the routine clinical management of patients with prostate cancer.This study was supported by the National Institutes of Health grant numbers R21-EB017568 and P30-CA014089. No other potential conflict of interest relevant to this article was reported.