NF1 gene in a 55-year old Chinese male with the mosaic generalized phenotype. He reported noticing increasing numbers of skin-colored papules over his face, neck, back and abdomen when he was about 40 years old. From both next-generation and Sanger sequencing data, the variant appeared to be mosaic and present at about 24%. It is in exon 39 and has not been reported in any database or published literature.Neurofibromatosis 1 (NF1) is a neurocutaneous syndrome characterized by multiple café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal freckling. Mosaicism in NF1 can either present as a generalized disease, or in a localized  manner. Mosaic generalized NF1 may have presentations that are similar to generalized NF1 or have a milder phenotype and hence may be under-recognised in clinical practice. We report a nonsense mutation in the Other features include optic nerve gliomas, central nervous system tumours, and long bone deformities. The autosomal dominant condition is caused by mutations or deletions involving the neurofibromin 1 gene  at chromosome 17q11.2, one of the largest human genes (282 kb) with 57 exons and three alternatively spliced transcripts. Over 3600 clinically significantNF1 variants are documented in the Human Gene Mutation Database (HGMD). They are distributed over the whole gene from the first to the last exon.Neurofibromatosis 1  is a neurocutaneous syndrome characterized by café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal frecklingde novo mutations and the other half inherited from an affected parent. The disease is highly penetrant and although the offspring are at 50% risk of inheriting a mutation, the manifestations are extremely variable even for family members who share the same mutation. Non-germline or mosaic NF1 is rare at 1:36,000–40,000, although it is probably under-reported as most cases are diagnosed clinically and molecular confirmation is not always performed.The estimated incidence of germline NF1 is 1:2500–3000, with approximately half due toWe report a patient with adult-onset NF1 presentations. He is of Chinese ancestry and the parents are not consanguineous. The 55-year-old male engineer presented in 2015 with increasing numbers of skin-colored papules over the past 13 years. The patient reports the onset of numerous light-brown macules in his late teens, starting on his trunk and back. He did not recall having these lesions earlier in life. The neurofibromas were only noted to occur in his 30s. Clinically he has generalized small 1 – 5 mm light-brown macules on his trunk and back (not limited to a segment or blaschkoid distribution), the largest of which measured approximately 10 mm. Excision of one such representative lesion measuring 0.5 mm × 0.5 mm from the right lateral neck was consistent with a neurofibroma. He was referred to dermatology and neurology departments on suspicion of neurofibromatosis.Clinically the patient had more than 20 small, soft, flesh-coloured papules and small nodules (ranging between 4 and 10 mm) on his face, neck, trunk and limbs. There was no axillary or inguinal freckling. He also had multiple scattered café-au-lait macules on his trunk. There were no lesions to suggest plexiform neurofibromas and no clinical evidence of long bone deformities. Slit-lamp examination revealed the presence of Lisch nodules in both irises. He had three older siblings, none of whom had similar skin lesions. Neither his parents nor members of his extended family had any features of neurofibromatosis. His two daughters (aged 17 and 21 years) were well with no features of neurofibromatosis. His other medical history was only significant for hyperlipidaemia for which he was on simvastatin (20 mg nightly) and cervical spondylosis which did not require intervention and he is no longer on active follow-up with the orthopaedics department.NF1 exons andNF1-related genes) with the Agilent Haloplex Target Enrichment System  and the MiSeq System . Output reads were processed using the MiSeq Reporter pipeline and variants were annotated withANNOVAR (2019Oct24). A heterozygous single nucleotide substitution in theNF1 gene that is predicted to result in premature termination: NM_000267.3: 5612T>A (p.L1871X) was identified. The number of reads for the variant was 28 out of 117. The presence of the variant was confirmed in our laboratory by Sanger sequencing using BigDye Terminator v3.1 in a volume of 10 µl. Amplification conditions included initial denaturation at 96°C for 1 min, followed by 25 cycles of 96°C for 10 sec, 50°C for 5 sec; and 60°C for 4 min. Following ethanol purification, the products were resuspended in Hi-Di™ formamide and loaded onto the ABI 3130 Genetic Analyzer . The peak height of the variant was approximately one-quarter to one-third that of the wild-type for both forward and reverse primers, confirming the mosaicism . Venous blood was collected from the patient after written informed consent and genetic counselling. Next-generation sequencing on genomic DNA was performed using a customised targeted panel  manner. Mosaic generalized patients may be clinically indistinguishable from generalized NF1 patients or have a milder phenotype. In contrast, the cutaneous manifestations of mosaic localized NF1 reflect the pattern of skin mosaicism and can vary from a narrow strip, one-quadrant, or half the body, unilaterally or bilaterally, in a symmetrical or asymmetrical fashion. Mosaic-localized NF1 patients may develop only pigmentary changes or neurofibromas, have a combination of pigmentary and neurofibromas, or have plexiform neurofibromas only.Mosaic NF1 was previously known as segmental NF1. It was initially defined as clinical features of NF1 limited to one or more segments of the body. The mosaicism which is caused by postzygotic mutations in the. Our patient is cognitively normal and had noticed the development of neurofibromas while in his late thirties. He also had Lisch nodules, which were not frequently observed regardless of the patient's age in other cohorts. He is currently on annual follow-up for his cutaneous presentations.Like generalized NF1, mosaic NF1 presents with pigmentary features and plexiform neurofibroma arising in childhood, and dermal neurofibromas usually in adulthood. However, not all patients with mosaic NF1 with pigmentary changes develop neurofibromas. Patients may also develop learning difficulties, pseudoarthrosis and malignancy, despite having a milder phenotypeNF1 mutation, but genetic analysis was performed in a case series of eight patients with segmental NF1 from Canada. Sequencing of DNA from skin lesions revealed a pathogenic loss-of-function variant in theNF1 gene in all eight: six were single or multi-nucleotide variants, while the other two were microdeletions. Seven out of the eight patients had pigmentary changes (cafe-au-lait macules and axillary freckling) ˗ with three of them having non-localized lesions and one with lesions crossing the midline. The eighth patient had cafe-au-lait macules, a plexiform neurofibroma and sphenoid wing dysplasia but no freckling. Only one patient had Lisch nodules.Most cases of mosaic NF1 did not have molecular confirmation of the presence of anNF1 mutation may transmit the mutation to their offspring in such cases. Thus, patients with mosaic NF1 should be counselled regarding the possibility of gonadal NF1 mosaicism and the risk of transmission to the next generation, especially when the mutation is also present in non-dermal tissues. Our patient had 24% mosaicism based on the number of reads from next-generation sequencing results of DNA from his peripheral blood. He was not evaluated for somatic or gonadal mosaicism. His two daughters appear to have no features of NF1, although he is considering genetic testing for his children.The risk of passing on the full-blown or localized disease to future generations is 50% for those with germline mutations. Although gonadal mosaicism is rare, individuals with mosaicNF1 gene, resulting in a mosaic generalized phenotype of NF1. Studies have sought to examine the genotype-phenotype correlation in mosaic localized NF1, although there is little in the literature regarding mosaic generalized NF1, which may be under-recognized given the clinical similarities with classic NF1. Future genotype-phenotype correlation studies in mosaic generalized NF1 would lead to a better understanding of the disease development process and long-term outcomes. With better sequencing technology that can detect low-level mosaicism, molecular confirmation should be carried out to enable healthcare professionals to provide a more accurate prognosis and genetic counselling.In summary, we report a novel nonsense mutation in theClinVar accession number: SCV001450721.Written informed consent for publication of the clinical details was obtained from the patient. This is a well written article demonstrating a thorough genomic evaluation of the NF1 gene in an adult-onset NF1 patient. The NF1 mutation identified was present on both NGS (23.9%) and Sanger sequencing (1/3 to 1/4) at similar variant allele frequencies, supporting the mosaicism. Mosaic generalized NF1 is less commonly seen in the clinic compared to its well-known, germline NF1 counterpart. I agree with the authors that this could also be a result of an under-recognition of mosaic cases, given their clinical similarities. NGS diagnostics and recognizing the importance of variant allele frequencies, like those presented here, provides an opportunity to close this gap.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?YesIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:I am an Otolaryngology surgeon with a PhD in Cancer Biology, focusing on cancer genomics and molecular subtypes of disease.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In the introduction the authors state that that non-germline or mosaic NF-1 is very rare, however they report that it is about 10 times less common than germline NF-1, which is actually relatively common. I would agree it is rare, but not very rare. The statement that many cases of generalized mosaic NF1 are likely undiagnosed is likely true and would further decrease the scarcity of this disorder.In the description of the case, the authors state that the patient had café au lait macules (CALMs) on his trunk, but do not state the size, number, and especially the duration of these lesions. In germline NF-1 CALMs generally precede the development of neurofibromas by often a decade or more. I think this is important information to include for the readership.I feel that only gonadal mosaicism should be used in this discussion since the term  gonosomal is relating primarily to sex chromosomes, and ch17, the chromosome with the mutation of interest, is an autosome.While not clear from the discussion, have any studies evaluated the percent mutated cells in somatic tissue, skin versus blood, in patients with either generalized or segmental mosaic NF1. Since patients with cutaneous “segmental” NF1 are known to harbor the mutation in other tissues and pass on the disease to offspring, do these individuals have a form or generalized mosaic NF1 or is it tissue/area specific? I understand if this info is not available.This is an enlightening and well written case report that brings to light a variant of a common genetic syndrome.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?YesIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?PartlyReviewer Expertise:I am an academic pediatric dermatologist with a cursory understanding of the details regarding the methodology of genetic mutation analysis but am very familiar with the cutaneous manifestations of NF1 and segmental NF1.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We have deleted the word "very" from "very rare" in the Introduction. We also added more details about the patient, including his ethnicity and that his parents are not consanguineous, and the size and duration of his lesions. We also changed "gonosomal" to "gonadal" as suggested.


Inflammatory bowel disease (IBD) is associated with an increased risk of Colorectal cancer (CRC), and its most important risk factors are the duration and extent of the disease. Pediatric-onset inflammatory bowel disease has a tendency for a more extensive, more severe, and longer predicted disease duration than adult-onset inflammatory bowel disease. This study aimed to identify the clinical characteristics of patients with CRC related to pediatric-onset IBD and consider the appropriateness of current surveillance endoscopy recommendations for the detection of premalignant lesions and early-stage CRC.We searched a research platform based on the SUPREME electronic medical record data-mining system to identify cases of colorectal malignancy in patients with pediatric IBD that presented between 2000 and 2020.During the follow-up, 4 (1.29 per 1000 person years) out of 443 patients with PIBD was diagnosed with CRC. The median age at diagnosis of CRC was 18.5 (range: 15–24) years, and the median period from diagnosis of IBD to CRC was 9.42 (range: 0.44–11.96) years. The sigmoid colon was the most frequent location of CRC (in 3 of the 4 cases). Adenocarcinoma was the most common histological type (in 2 of the 4 cases).Patients with pediatric-onset IBD exhibited a much shorter disease duration than that of adult-onset IBD at the time of diagnosis of CRC, suggesting that surveillance endoscopy for the detection of precancerous lesions and early-stage cancer should be initiated earlier in pediatric patients than in adult patients. Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC). The most important and well-recognized risk factors for CRC in patients with IBD are the duration and extent of the disease , 2. The We reviewed the clinical data of patients diagnosed with CRC after being diagnosed with PIBD between 2000 and 2020 at Seoul National University Children’s Hospital in Korea, and the study was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2009–093-1157) with a waiver of informed consent.A research platform was utilized based on an electric medical record data-mining system (“SUPREME”) to search for patients. Multiple sets of key words including disease name , 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) codes for CRC (“C18-C21”), diagnosis date (“from 2000 to 2020”), diagnosis age (“from 0 to 17 years old at diagnosis”) and internal code for identifying Rare Intractable Disease (RID) registration  were used.In our institution, diagnoses of pediatric CD and UC were made in accordance with published guidelines , 11 and A total of 443 pediatric patients (aged < 18 years) were diagnosed with IBD between 2000 and 2020 at Seoul National University Children’s Hospital. The end of follow-up was the date of their last outpatient visit. The follow-up period for each patient varied significantly, from under 1 year to 20 years, and 199 (44.9%) patients had more than 7 years of follow-up. In 3102 person years of follow-up, we found 4 CRC in patients with PIBD (1.29 per 1000 person years).The characteristics and details of the four patients are shown in Table We analyzed four cases of PIBD-related CRC at a single center in Korea. The case of VEOIBD (patient 3) which the initial diagnosis was UC but revised to CD immediately after the diagnosis of cancer was previously published . A subseIn our study, 4 (1.29 per 1000 person years) patients was diagnosed with PIBD-related CRC during follow up. However in nationwide cohort study in Sweden, among 9405 patients with PIBD, 122 (0.8 per 1000 person years) people with PIBD had CRC with hazard ratio of 33 for UC and 5.8 for CD [All patients except one had a disease duration of at least 8 years at the time of diagnosis of CRC. The median disease duration was 9.42 (range: 0.44–11.96) years , which is much shorter than expected for adult patients with IBD , 17. TheA case-control study revealed that the risk of colorectal neoplasia in patients with UC exhibited a highly significant correlation with the degree of colonoscopic and histological inflammation . In the Children with IBD are anticipated to have a longer disease duration than adults, and numerous studies have reported that PIBD tends to be more severe and extensive than adult-onset IBD –7. In ouAs the risk of CRC in patients with PIBD is as high as that in adults, while the disease duration until the diagnosis of CRC tends to be shorter than that in adults, surveillance endoscopy should be initiated earlier than currently recommended for IBD. Further studies are required to determine the appropriate time to begin surveillance and the optimal surveillance interval.As this study had a retrospective design, we relied on electronic medical records to obtain all patient information. The main limitation of this study is that the number of patients with CRC was small, which made it unclear whether the median values obtained were representative of actual patients with PIBD who developed CRC. The inclusion of additional cases will lead to more convincing results. Furthermore, the small number of patients prevented us from performing subgroup analysis based on a diagnosis of CD or UC, and all patients were classified into a single IBD group. The lack of a control group also made it impossible to elucidate the extent to which CRC cases were linked to IBD.In our study, disease duration of PIBD until the diagnosis of CRC was shorter than that of adult-onset IBD. Thus, surveillance endoscopy for precancerous lesions (dysplasia) and early-stage cancer should be initiated earlier in patients with PIBD than in patients with adult-onset IBD. Further studies are required to determine the appropriate time to begin surveillance and the optimal surveillance interval.


Consequently, PLWHA who are on Anti-Retroviral Therapy (ART) or Highly Active Anti-Retroviral Therapy (HAART) are usually predisposed to coronary artery diseases due to abnormal weight gain (lipohypertrophy), though with improved and unstable Cluster of Differentiation 4 (CD4) counts.The most significant clinical challenges in people living with HIV/AIDS (PLWHA) are decline in CD4The main aim of this study was to compare the effects of 6 weeks progressive resistance exercise (PRE) and moderate intensity aerobic exercise (MIAE) on CD4 count, and weight of PLWHA in Alex-Ekwueme Federal University Teaching Hospital Ebonyi State.The study adopted quasi experimental research design. The population of the study was 60 Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome patients that attended HIV clinics at Alex-Ekwueme Federal University Teaching Hospital Abakaliki (AE-FUTHA) which formed 58 sample sizes for this study.Simple random sampling technique was adopted for the study and flow cytometry, Heel Densitometer (X-rite 331C), and Omron BF 400 were the instrument used for data collection of CD4 counts and weight respectively; and they are standardized hence not validated. Mean, standard deviation and Analysis of Co-variance (ANCOVA) were used to analyze the data obtained. The reliability coefficient obtained from the pilot study was 0.848 and 0.994 for CD4 and WEIGHT respectively.The major findings revealed a significant difference after 6 weeks’ PRE and MIAE on CD4 and Weight of PLWHA.Health promotion policy maker's arm of the government and Healthcare givers should integrate and enforce the use of exercises either as a single regimen or combined exercises into the management plan of PLWHA for greater boost in absolute CD4 count bearing in mind that both have positive effects. Physiotherapist should be integrated in the managements of PLWHA for appropriate prescription and education of therapeutic exercises for body weight. CD4+ T helper cell is the main immune system infection fighter that triggers the body's response to infections. The cells’ reduction weakens the immune system and exposes the body to opportunistic infections as well as wasting syndrome (lipoatrophy). Consequently, PLWHA who are on Anti-Retroviral Therapy (ART) or Highly Active Anti-Retroviral Therapy (HAART) are usually predisposed to coronary artery diseases due to abnormal weight gain (lipohypertrophy). Surprisingly, it was recorded that a short burst of physical exercise increases blood Cluster of Differentiation 4 (CD4) lymphocyte counts, which perhaps may be a shift in the number of cells in circulation pathways, reflecting in changes of blood CD4 lymphocyte numbers. Research suggests that HIV-infected individuals who are on antiretroviral drugs can get significant physiological and immunological benefits from both progressive resistance exercise (PRE) and moderate intensity aerobic exercise (MIAE) after some weeks of training especially if performed 3 times a week at moderate intensity; presenting both exercises as forms of therapeutic measures used in healthcare for management and improvement of health conditions of patients. According to Kisner and Colby (2012), PRE is a system of dynamic resistance training in which a constant external load is applied to the contracting muscles by some mechanical means and gradually increased using repetitive maximum as a basis for adaptation. PRE equally burns up glycogen stored in the muscles without oxygen, thus is a type of anaerobic exercise. Moreover, PRE is considered safe and beneficial for PLWHA and is said to be effective in improving their muscle strength, immune system and body compositions. Meanwhile, MIAE or endurance exercise which is defined as a regimen in moderate intensity form, containing aerobic interventions like cycling, stair stepping, rowing, and walking; usually meant to promote significant effects in cardiac endurance, aerobic capacity and weight; measured by maximal oxygen consumption is equally beneficial to PLWHA especially those on ART/HAART.,29,30The most significant clinical challenges in people living with HIV/AIDS (PLWHA) are decline in CD4 that at the end of 16 weeks of supervised home-based aerobic and progressive resistance training regimen in women infected with human immunodeficiency virus, no significant difference was observed in CD4 cell count between groups. While the combined PRE and MIAE group showed a significant increase in muscular strength and cardiorespiratory fitness as well as a significant decrease in waist circumference when compared to the controls. Another study indicated that after the intervention program, a significant difference in CD4 cell counts was found between the 2 groups (P 1/4 0.01). With respect to mental health, the same study recorded significant improvement in all subscales including anxiety disorder, social function, depression and mental health's total score were observed in the exercise group compared to the control groups (P < .001). It was equally found in a study that there was significant improvement and significant increase in CD4 cell counts and TG respectively between pre- and post- tests while total cholesterol (TChol), high density lipoprotein (HDL) and low density lipoprotein showed insignificant difference. The significant improvement in CD4 cell counts found in the experimental group agrees with the findings of some past studies, and relatively at variance with others which reported stability or insignificant improvements. reported insignificant increase in CD4 cell counts of HIV infected persons after 12 weeks aerobic exercise. However, other studies like,12–14 reported an increase in CD4 cell counts, while few like,14 found stability in CD4 cells. in their study demonstrated more stable CD4 cell counts from baseline of −3% in the experimental participants, while the control group experienced significant reduction of −16% after 12 weeks.However, it was found in a study by Dolan et al found in a study that there was no significant difference between both groups. This finding however did not support that of in which PRE and MIAE witnessed a statistical significant increase in weight of both groups (PRE and MIAE). In addition, progressive resistive exercise and aerobic exercise is proven to be safe and could be beneficial for adults living with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) in their weight increment. More so in their study reported that there exists no statistical significant difference for weight after participant undergone MIAE and PRE intervention. Concurrently, another study recorded no statistical significant difference for weight after participant undergone MIAE and PRE intervention. However, the finding is not in support of who reported in a study that both PRE and MIAE had a significant reduction in weight for PLWHA.Furthermore, the weight loss, physical and mental health condition of the experimental group was better than the control group cardiovascular weakness, immune reduction,,21 and sometimes resistivity of ART drugs have been very much recorded with complimentary effects on both CD4counts and body weight. This is quite disturbing and has motivated lots of scientific investigations in area of exercises. Presently, very little work has been done here locally, while Nigeria positions the second most noteworthy of individuals living with HIV/AIDS around the world. Shockingly, the impacts of MIAE and PRE on CD4 and Weight has no general scientific consensus yet, it is still under debate; while a few scientists announced an improvement in their review, others found an abatement after routine exercise program. Consequently, the requirement for more experiments for clarification on impacts of MIAE and PRE on CD4 counts and body weight of PLWHA.In fact, the multisystem clinical manifestations of PLWHA which includes gross energy shortage, platelets reduction,2The main purpose of this study is to compare the effects of progressive resistive exercises and MIAE on CD4 counts and Bone Mineral Density of PLWHA who are on ART/HAART  in Enugu State University Teaching Hospital. Specifically this study will determine:1.The comparative effects of PRE and MIAE on CD4 counts of PLWHA.2.The comparative effects of PRE and MIAE on WEIGHT of PLWHA.2.11.What are the comparative effects of PRE and MIAE on CD4 counts of PLWHA?2.What are the comparative effects of PRE and MIAE on WEGHT of PLWHA?2.2For this study, the following null hypothesis will be tested at alpha level of 0.05Ho1: There will be no significant difference after 6 weeks comparative effects of PRE and MIAE on CD4 counts of people living with HIV/AID.Ho2: There will be no significant difference after 6 weeks comparative effects of PRE and MIAE on WEIGHT of people living with HIV/AID.3This study adopted an experimental research design with an equivalent (randomized) pretest and posttest data, utilized to observe the response of the dependent variables (CD4 and Weight) of the treatment group (MIAE and PRE). The setting for this study was at Physiotherapy department of Alex-Ekwueme Federal University Teaching Hospital Abakaliki (AE-FUTHA) in Ebonyi State. This health facility is a teaching hospital in Abakaliki. It has about 2000 beds, and up to 20 departments. This hospital was chosen based on the information gotten from health record that they diagnose more than 20 HIV/AIDS patients weekly and has up to 25% of 0.8% population of those living with HIV/AIDS in Ebonyi state. These include all HIV/AIDS patients that attend HIV clinics at AE-FUTHA between December 2019 to February 2020. The target population was 60 volunteers who were on ART/HAART  for not less than 24 months. However, only 58 subjects completed the study due to drop out by 2 persons from the control group. Thus, the sample size became 58 for the study.Forty participants on ART/HAART who were willing to participate and met the inclusion criteria were randomly assigned to the 2 groups , using balloting by replacement.4A. Inclusion Criteria:Only male HIV/AIDS patients within the age range of 18 to 60 years and female between 18 to 50 years of age.Only HIV/AIDS patients that have started taking their ART/HAART for the duration of 24 months and above, prior to the study, and attend HIV/AIDS clinics.B. Exclusion Criteria:Patients that are not on Anti-Retroviral therapy/ Highly Active Anti-Retroviral Therapyup to this duration of 24 months and above prior to the studyAll subjects with previous history of cardiac and diabetic complications.All pregnant subjects to avoid interference on CD4 count and weight.The following instruments were used for data collection in this study:Flowcytometery (PartecCyflow counter), Germany.Heel Densitometer (X-rite 331C) Germany.Omron BF400 weighing scaleThese instruments  are standard and used worldwide. Hence, it does not need to be validated.The obtained data during trial testing was subjected to Pearson Product Moment Correlation Co-efficient and the results were 0.848 and 0.994 for CD4 and WEIGHT respectively. These are said to be reliable because they are greater or equal to 0.8 according to.5The procedure for data collection in this research was assisted by 4 field assistants including; a Physiotherapist, a Radiologist, a Nurse, a laboratory Scientist and a medical doctor. The subjects were recruited at AE-FUTHA and informed consent was issued explaining the purpose, procedure, and relevance of the study before the onset of the intervention. The intervention was supervised by Research and Ethics Committee (REC) of Alex-Ekwueme Federal University Teaching Hospital Abakaliki. All the willing participants were assessed for baseline data, which included age, weight, blood pressure (BP) and heart rate. Randomized control trial technique by balloting was used to divide the willing participants who met the inclusion criteria into 3 groups . None of the groups were blind. The control group was not allowed to participate in the exercise and they were also asked not do any active exercise program for a period of 6 weeks of the study. The activities of daily living of the participants in the control group were monitored through a checklist and it was confirmed that none of them participated in any form of active exercise program. The MIAE and PRE (duration and frequency) of group (A) and (B) respectively was divided in this order: 18 sessions of sub-maximal aerobic exercises on Marshal Fitness bicycle ergometer and PA Pro Acting treadmill which consists of 4 stages:1.3 minutes warm up2.3 to 4 minutes to reach the Target Heart Rate (THR)3.20 minutes holding of the range of the THR, and4.3 minutes cool down for 3 times per week.The intensity of the aerobic exercises were increased gradually in this order: 45% to 50% heart rate reserve during the first 2 weeks, 50% to 55% heart rate reserve during the second 2 weeks and 55% to 60% heart rate reserve during the last 2 weeks.5.1Blood samples were drawn venopunctually using syringe into a test-tube. Reagents used were brought to room temperature, 850 μL of the count check bead green analyzed to make sure that the cyflow machine was working effectively. The needed numbers of Rohren test tubes were labelled appropriately and placed in a test tube rack. 20 μL of CD4 easy count kits (CD4 Mab-PE) were pipetted into them for the assay. Thereafter, 20 μL of blood samples were also pipetted into each test tube and incubated in the dark for 15 minutes at room temperature after mixing properly followed by the addition of 850 μL easy count. Lyse buffer was not added to each test tube. In order to avoid air bubbles, this was mixed properly and analyzed on the PartecCyflow. The outcome was displayed and copied from the screen.5.2The steps in data collection with Omron BF400 weighing scale started by setting the machine to display kilograms by adjusting the switch at the base, followed by placing it on the tile floor. The center of the scale was then pressed lightly with the researcher's foot to turn on the scale. The subjects were then asked to step onto the scale without their foot wears and heavy objects on them, placing their both feet side-by side on the feet prints land mark. However, the readings were displayed on screen and the records taken with the subject still standing still and upright on the scale.5.3 on the protection of the right of subjects while conducting human experimental research was strictly observed.Ethical approval was sought and obtained from the Research and Ethics Committee (REC) of Alex-Ekwueme Federal University Teaching Hospital Abakaliki with Registration Number AE-FUTHA/REC/VOL 3/2020/008. Participants’ privacy and confidentiality were maintained using code numbers instead of names, and ensured that records were destroyed at the end of the study. Subjects’ informed consent were obtained from the subjects before commencing the study and the principle of the Nuremberg declarations code6The data obtained was analyzed using Statistical Package for Social Sciences (SPSS) version 25. The statistical tools used were mean, standard deviation, and analysis of covariance (ANCOVA).7Research Question 1: What is the comparative effect of PRE and MIAE on CD4 count of PLWHA?By using the mean difference, the result in the graph above shows that there is an ordinal interaction effect of MIAE and PRE on CD4 of PLWHA. However, MIAE is higher in ranking in the graph than that of PRE within the period of 6 weeks while that of Control group has the least estimated marginal means with differences of 65.85, 27.95, and −76.67 respectivelyThe result in the bar chart above shows that MIAE has more effect on CD4 counts than PRE while the Control group has the least estimated marginal means of CD4 of PLWHA.7.1What is the comparative effect of PRE and MIAE on Weight of PLWHA?By using the mean difference, the result in the graph above shows that there is an ordinal interaction effect of PRE and MIAE on Weight of PLWHA. However, PRE is higher in ranking in the graph than that of MIAE within the period of 6 weeks while that of Control group is lower than PRE but higher than MIAE based on estimated marginal means with differences of 2.07, −0.9 and 0.49respectively.The result in the bar chart above shows that PRE has more effect on Weight than MIAE. However, the Control group is higher than MIAE based on estimated marginal means of Weight of PLWHA.Hypothesis 1:There will be no significant difference after 6 weeks’ comparative effects of PRE and MIAE on CD4 counts of people living with HIV/AIDSThe result in the above table is on the effect of PRE, MIAE and Control on CD4 counts of participants in this study. The table shows a probability value  of 0.000 for PRE and MIAE and control group. The significant value in the table above for groups is less than the alpha level of 0.05. This means that the hypothesis earlier stated is not accepted. Thus, there is a significant difference after 6 weeks’ PRE, MIAE and Control on CD4 counts of PLWHA. The result in the table also shows that there is a positively high effect size of PRE, MIAE and Control on CD4 counts when the experimental groups are compared with the control group in the study.By checking for pairwise comparison, the result in the above table is on the effect of PRE, MIAE and Control comparison based on CD4 in this study. The table shows that MIAE pair with PRE shows no significant difference; while MIAE pair with Control shows significant difference. PRE pair with MIAE shows no significant difference; while PRE pair with Control shows significant difference. Finally, Control pair with MIAE, and Control pair with PRE all show significant difference based on their respective probability value in the table above. Thus, all these show that the result is not consistent with that of Tests of Between-Subjects Effects which shows in general that there is a significant difference after 6 weeks’ PRE and MIAE on CD4 counts of PLWHA.Hypothesis 2:There will be no significant difference after 6 weeks’ comparative effects of PRE and MIAE on WEIGHT of PLWHAThe result in the above table is on the comparative effect of PRE and MIAE on WEIGHT of people living with HIV/AIDS. The table shows a probability value  of 0.001 for the Group  on WEIGHT. The significant value from the table above is less than the alpha level of 0.05. The decision rule is that if the probability value  is less than the alpha level of 0.05, then the earlier stated null hypothesis will not be accepted. Thus, we did not accept the earlier stated null hypothesis. Therefore, there is a significant difference after 6 weeks’ comparative effects of PRE, MIAE and Control on WEIGHT of PLWHA. The result in the table shows that there is a low effect size of PRE, MIAE and Control on WEIGHT and it implies that the mean difference has only little importance.By checking for pairwise comparison, the result in the above table is on the effect of PRE, Bone Mineral Density and Control comparison based on WEIGHT in this study. The table shows that MIAE pair with PRE, MIAE pair with Control, PRE pair with MIAE, PRE pair with Control, Control pair with MIAE and Control pair with PRE all show not significant based on their respective probability value in the table above which are all greater than 0.05 alpha level. Thus, all these show that the result is not in line with that of Tests of Between-Subjects Effects that there is a significant difference after 6 weeks’ PRE, MIAE, and Control on WEIGHT of PLWHA.88.1 who recorded that PRE and MIAE had an increase in CD4 counts at the end of the interventions. This is also in line with who found in their study that there was a little increase in the CD4 counts of PLWHA after interventions. There is a significant difference after 6 weeks’ comparative effects of PRE and MIAE on CD4 counts of PLWHA. However, this finding does not support that of reported that MIAE and PRE have no significant effects on CD4 count on PLWHA  finding is that if PLWHA with lower CD4 counts will participate more in MIAE other than PRE. This means that Exercise Physiologists and Health Managers will educate PLWHA to opt more for MIAE whenever they want to improve their CD4 counts.2.The implication of research question 2 (2) results is that PLWHA who chose to take part in exercise for WEIGHT gain, will participate in PRE other than MIAE. Thus, the Exercise Physiologists and Health Managers will educate PLWHA and create awareness on the appropriate therapeutic exercise which in this case is PRE to help weight gain.10.1The followings are the recommendations based on the findings of the study:1.Physical exercises especially MIAEs should be integrated as an adjunct therapy in the management of PLWHA who are either on Highly Active Anti-Retroviral Therapy for greater boost in immune system, resulting to improvement in absolute CD4 count.2.Exercise Physiologists and Health Managers should educate and encourage PLWHA to engage in PRE which is the appropriate therapeutic exercise for weight gain.Conceptualization: Euharia Ijego Asogwa, Chinyere Ori Elom, Anamelechi Nwele Ogai, Chukwudum Collins Umoke.Data curation: Okechukwu Sunday Abonyi, Chinyere Ori Elom, Christian Ajim Oduma, Chukwudum Collins Umoke, Chidebe Chijioke Uwaleke, Ignatius Obilor Nwimo.Formal analysis: Euharia Ijego Asogwa, Okechukwu Sunday Abonyi, Chinyere Ori Elom, Anamelechi Nwele Ogai, Ignatius Obilor Nwimo.Funding acquisition: Christian Ajim Oduma, Anamelechi Nwele Ogai, Chidebe Chijioke Uwaleke, Ignatius Obilor Nwimo.Investigation: Euharia Ijego Asogwa, Chinyere Ori Elom, Chukwudum Collins Umoke, Chidebe Chijioke Uwaleke.Methodology: Okechukwu Sunday Abonyi, Chinyere Ori Elom.Project administration: Euharia Ijego Asogwa.Resources: Christian Ajim Oduma, Anamelechi Nwele Ogai.Software: Euharia Ijego Asogwa, Okechukwu Sunday Abonyi, Ignatius Obilor Nwimo.Supervision: Okechukwu Sunday Abonyi.Validation: Chinyere Ori Elom, Chukwudum Collins Umoke.Visualization: Euharia Ijego Asogwa.Writing – original draft: Euharia Ijego Asogwa, Okechukwu Sunday Abonyi, Anamelechi Nwele Ogai.Writing – review & editing: Okechukwu Sunday Abonyi, Chinyere Ori Elom.


CT) using computed tomography coronary angiography (CTCA) determines both the presence of coronary artery disease and vessel-specific ischaemia. We tested whether an evaluation strategy based on FFRCT would improve economic and clinical outcomes compared with standard care.Fractional flow reserve  or standard clinical care pathways (standard group). The primary endpoint was total cardiac costs at 9 months. Secondary endpoints were angina status, quality of life, major adverse cardiac and cerebrovascular events, and use of invasive coronary angiography. Randomized groups were similar at baseline. Most patients had an initial CTCA: 439 (63%) in the standard group vs. 674 (96%) in the experimental group, 254 of whom (38%) underwent FFRCT. Mean total cardiac costs were higher by £114 (+8%) in the experimental group, with a 95% confidence interval from −£112 (−8%) to +£337 (+23%), though the difference was not significant (P = 0.10). Major adverse cardiac and cerebrovascular events did not differ significantly  and angina and quality of life improved to a similar degree over follow-up in both randomized groups. Invasive angiography was reduced significantly in the experimental group .Overall, 1400 patients with stable chest pain in 11 centres were randomized to initial testing with CTCA with selective FFRCT in patients with stable angina did not differ significantly from standard clinical care pathways in cost or clinical outcomes, but did reduce the use of invasive coronary angiography. A strategy of CTCA with selective FFR  Furthermore, the PLATFORM study suggested that the use of CTCA with FFRCT reduces costs in the patients who would have undergone invasive coronary angiography, and is cost neutral in patients who would have had a non-invasive test.CT be considered as a frontline test in patients with chest pain with the expectation of large cost savings.The ideal test to assess patients with new onset chest pain might therefore simultaneously provide information about the extent of both coronary atheroma and myocardial ischaemia. Fractional flow reserve derived from computed tomography coronary angiography  it was conducted in a manner consistent with the protocol, (ii) the data were complete, and (iii) the analyses were performed according to the Statistical Analysis Plan.FORECAST was an open-label, multicentre, randomized, controlled clinical trial. The rationale and design have previously been described,All screened patients were at least 18 years old and were attending a Rapid Access Chest Pain Clinic for assessment of stable chest pain. A full list of exclusion criteria is available in the trial protocol . In the standard group, patients were assessed according to usual clinical care pathways at the Rapid Access Chest Pain Clinic, based upon the local implementation of the NICE CG95 Guidance for Chest Pain of Recent Onset.CT). In the experimental group, all patients were referred for CTCA as the initial test and selectively referred for FFRCT if the CTCA demonstrated a stenosis of ≥40% in a coronary artery segment of diameter suitable for revascularization by either a coronary stent or coronary artery bypass graft surgery. Prior to randomization, the clinical team declared which initial test would be used in the event the patient was randomized to standard care. Subsequent clinical management was determined by the supervising physician based on the results of initial testing and clinical judgement.Patients were randomized, using an independent computerized system with block sizes of two and four, to either the usual care strategy based on clinical pathways (standard group) or a strategy of CTCA with selective FFRThe primary endpoint was cardiovascular costs over 9 months of follow-up, calculated from the use of all cardiac-related invasive and non-invasive tests, revascularization procedures, hospital admissions and outpatient attendances due to a cardiovascular cause , and cardiac medications. Data were collected using direct patient contact by research staff at each centre, as well as from local healthcare records. The total costs were calculated for each patient as the sum, over all specified resources, of the numbers of each resource used multiplied by a standardized cost weight  quality of life, as assessed using the EQ-5D-5L questionnaireThe sample size calculation and statistical analysis plan have been described in detail previously.2 tests. The primary endpoint was compared using a two-sample t-test after a log transformation due to skew in the cost data. Confidence limits on mean costs were calculated by bootstrapping. A two-sided P-value of 0.05 or less was considered to constitute statistical significance for all analyses. All analyses of outcome data were conducted using an intention-to-treat framework.The Statistical Analysis Plan for the trial data was determined in advance . Baseline characteristics were well balanced between the arms (Table 1).Between December 2017 and July 2019, 2494 patients with stable chest pain attending one of the 11 participating Rapid Access Chest Pain Clinics were screened for study entry, and 1400 patients were randomized (Table 2). Nine patients in the standard group were erroneously referred for FFRCT analysis, but the test results were not used in clinical management.Among the 700 patients randomized to the standard group, 439 (63%) had CTCA as the initial test, 187 (27%) had an initial stress test, and 47 (7%) had direct invasive coronary angiography  scans could not be analysed due to technical issues. In the 220 patients who had FFRCT performed, 126 patients (59%) had at least one epicardial vessel with an FFRCT ≤ 0.8, which led to requests for invasive angiography in 98 patients, a non-invasive stress test in 16 patients, and no further testing in 12 patients. Invasive angiography was performed more often in patients with lower levels of FFRCT: in 26 of 29 patients (90%) with an FFRCT between 0.50 and 0.60, in 23 of 23 patients (100%) with an FFRCT between 0.61 and 0.70, in 39 of 56 patients (70%) with an FFRCT between 0.71 and 0.80, and in 4 of 94 patients (4%) with an FFRCT >0.80. The FFRCT value was not recorded in 18 patients , invasive angiography was performed in 14 (78%) of these patients.In the experimental group, 674 (96%) patients underwent CTCA and 254 (38%) were selected for FFRTable 3). The use of invasive coronary angiography was 22% lower in the experimental group (Table 3): 136 patients vs. 175 patients in standard care strategy (P = 0.01). The number of invasive angiograms showing no obstructive epicardial lesion was 52% lower in the experimental group: 30 patients vs. 62 patients in the standard care strategy. The use of invasive pressure wire assessment was also lower in the experimental group: 18 patients vs. 28 patients (P = 0.18).Over 9 months of follow-up, fewer stress tests were performed at the discretion of the supervising physician in the experimental group than in the standard group . A total of 88 percutaneous coronary intervention (PCI) procedures were undertaken in 74 patients (11%) in the experimental group, compared with 75 PCIs in 69 patients (10%) in the standard group, with 28 patients in each group undergoing CABG surgery (Table 3). In the experimental group, 90 of the 102 patients who underwent coronary revascularization had a functional study (a stress test or FFR), compared with 49 of the 97 patients who underwent coronary revascularization in the standard group (P < 0.001).The overall rate of coronary revascularization did not differ significantly between the groups: 15% in the experimental group vs. 14% in the standard group (P = 0.10). The distribution of costs  was skewed upward by a minority of patients with high costs, such that the median costs were £70 lower in the experimental group than the standard group (£600 vs. £670).The mean total cardiac costs at 9 months were slightly higher in the experimental group (£1605) than in the standard group (£1491) , though the difference in mean costs was not significant (Table 3), and while there was significantly lower use of invasive coronary angiography in the experimental group, the number of hospitalizations, visits to outpatient clinics and emergency departments, and medication use did not differ significantly (Table 3).The pattern of non-invasive test use varied significantly (by design) between the two randomized groups (P = 0.80). Individual components of MACCE did not differ significantly between groups (Table 4). There were two deaths in the experimental group due to non-cardiac causes (metastatic cancer and progressive lung fibrosis).The overall rate of MACCE  was 71 (10.2%) in the experimental group vs. 74 (10.6%) in the standard group . The same pattern was evident in the EQ-5D scores over follow-up: both groups showed the reduced quality of life at baseline  that improved over follow-up (to a median score of 0.8 at 9 months in both groups), with no significant difference in the change in scores .Seattle Angina Questionnaire scores showed impairment at baseline  that improved significantly over 9 months of follow-up (to a median of 95.8 in both randomized groups). Scores improved to a similar degree in the experimental group  and the standard group , with no significant difference in the change in scores from baseline to 9 months (Table 5). The experimental group had 6.5% lower costs in the 94 patients with planned invasive angiography, 6.8% lower costs in the 393 patients with planned stress testing, but 20% higher costs in the 912 patients with planned CTCA (Table 5). The rates of MACCE did not differ between groups in any stratum, and the changes in Seattle Angina Questionnaire and EQ-5D scores were similar in all three strata. The effect of the experimental strategy on the use of invasive angiography was significantly greater (interaction P = 0.042) in the planned invasive angiography stratum than in the strata of planned CTCA and planned stress testing (Table 5).Prior to randomization, the supervising clinician identified the test that would be performed in the event the patient was randomized to the standard care strategy. The pattern of costs varied depending on whether the planned test was an invasive angiogram, a stress test, or a CTCA , and the high prevalence of CTCA as the planned initial test . This shift in standard practice in the UK towards routine CTCA, which was stimualted by the NICE CG95 Guidance on Chest Pain of Recent Onset, may have limited the cost savings potential of the experimental strategy based on initial CTCA and selective FFRCT.FORECAST was designed using cost as the primary endpoint because we anticipated, based upon previous observational studies, that clinical outcomes would be similar in a well-managed population of stable patients with chest pain, irrespective of the initial testing strategy. We hypothesized that a strategy based on initial CTCA with selective FFRCT has been to reduce the use of invasive coronary angiography, particularly angiograms showing no obstructed coronary arteries. In the observational PLATFORM study, invasive angiography was reduced by 61% in the FFRCT cohort, and the clinical event rates at 1 year were equally low in both groups.CT in routine clinical practice found unobstructed coronaries at invasive angiography in 14% of patients with FFRCT ≤0.8, compared with 44% of patients with FFRCT >0.8.CT >0.80, vs. 14 (0.3%) in subjects with an FFRCT ≤0.80 (P = 0.039). We therefore anticipated that the experimental strategy in FORECAST would result in less invasive angiography, and no difference in clinical event rates, compared with standard clinical pathways. The results of FORECAST have confirmed these expectations, with equivalent rates of clinical events, 22% fewer invasive angiograms, and half the rate of unobstructed coronary arteries at invasive angiography in the experimental group, and are consistent with the previous observational studies.Previous studies have consistently shown that the major benefit of FFRTable 3). From a patient’s perspective, achieving similar quality of life and angina outcomes with fewer invasive procedures represents a potential advantage for the experimental strategy based on FFRCT.In FORECAST, quality of life and angina status improved to a similar degree in both groups by 9 months of follow-up. This result is consistent with the 1-year data from PLATFORM, in which the five-item EuroQOL score did not differ significantly between the groups overall.CT) vs. standard clinical care pathways, whatever tests that should include. With almost two-thirds of patients in the standard group having planned initial CTCA, the contrast between the randomized groups in FORECAST was diminished, along with the potential for cost savings with the experimental strategy based on the use of CTCA with selective FFRCT. A recent individual-based Markov microsimulation model for patients with low-risk stable chest pain, based upon the PROMISE population, suggested that an anatomical approach using CTCA was cost-effective compared with functional testing.There are some limitations of FORECAST. First, and most important, is that we could not anticipate the precise rate of use of CTCA in the standard group. The national guidelines were revised during planning of the trial, and while they recommended that CTCA become the default test for most patients attending Rapid Access Chest Pain Clinics, the infrastructure in many areas of the National Health Service at that time could not provide the test. The subsequent major expansion in CT facilities greatly improved access to CTCA in the last few years. The FORECAST trial, however, was based upon a pragmatic design: the experimental strategy (CTCA with selective FFRA second limitation of the trial is that the costs in this study were based on UK National Health Service cost tariffs, and may not be generalizable to other countries with different cost structures in their health delivery systems. In an attempt to address this, one pre-specified sensitivity analysis for this trial is to apply US-specific cost tariffs to the FORECAST data, and this is the subject of ongoing analysis. Third, we used cardiac costs, rather than total medical costs, as the primary endpoint. Cardiac costs are more likely to be affected by the alternative strategies and were simpler for the local research teams to document. While it seems unlikely that non-cardiac costs would be affected by the management strategies tested, we cannot exclude the possibility that total medical costs differed, even though the cardiac costs did not.CT in routine clinical practice when CTCA is the default approach? In light of the findings from SCOT-HEARTCT based on the burden of atheroma found on CTCA, FFRCT analysis could be reserved only for patients with insufficient symptomatic response to optimal medical therapy in whom revascularization is therefore being considered. This approach would be consistent with a sub-analysis of the PROMISE trialCT in the setting of stable symptoms may be after optimal medical therapy fails to control angina adequately, at which time FFRCT could be performed using the previously collected CTCA dataset, and thereby assess the need for revascularization as part of a shared decision-making process with the patient.The significant reduction in death from coronary heart disease and non-fatal MI seen at 5 years in the SCOT-HEART trialCT in patients presenting with stable angina did not significantly reduce costs compared with standard clinical evaluation pathways, and led to similar clinical outcomes, including major adverse cardiovascular events, anginal symptoms, and quality of life. The experimental strategy based on FFRCT did, however, reduce the use of invasive coronary angiography, without reducing the use of coronary revascularization.In conclusion, the experimental strategy of initial CTCA with selective FFREuropean Heart Journal online. ehab444_Supplementary_DataClick here for additional data file.


Intra-tumoral budding (ITB) has been well demonstrated to be an independent risk factor for adverse outcomes in colorectal carcinoma. This study investigated the prognostic significance of ITB in high-grade serous ovarian carcinomas (HGSOCs). The medical records and slides of 84 SOCs, including 13 with neoadjuvant chemotherapy (NAC), were retrospectively reviewed. The histopathologic examination with scoring of p53 expression showed them to be 80 HGSOCs and 4 low-grade serous ovarian carcinomas (LGSOCs). ITB was found in 64 (80.0%) of the 80 HGSOCs and 1 (25.0%) of 4 LGSOCs. The presence of ITB in HGSOC was significantly correlated with a higher level of CA125, an advanced 2014 FIGO stage, the presence of Lymph node metastasis, and the presence of lymphovascular space invasion (LVSI). The median progression-free survival (PFS) was 18 months in patients with HGSOC with ITB and 36 months in patients with HGSOC without ITB (P = 0.006), and their median overall survival (OS) was 50 months and 60 months (P = 0.060). The multivariate analysis revealed that ITB was not an independent prognostic factor. ITB is a cost-effective prognostic indicator for patients with HGSOC and ITB in ovarian tumor tissue is considered a useful histological biomarker of the progression of HGSOCs. Although Shimizu and Silverberg3 in 2004 and subsequently adopted in the 2014 WHO Classification of Tumours of Female Reproductive Organs4. Using this two-tier grading system for serous ovarian carcinomas (SOCs)3, cases assigned to the low-grade category were characterized by the presence of mild to moderate nuclear atypia. As a secondary feature, they tended to show up to 12 mitoses/10 high-power fields (HPFs), whereas those in the high-grade category had marked nuclear atypia and as a secondary feature more than 12 mitoses/10 HPFs. Recent molecular genetic studies have shown that LGSOCs demonstrate mutations in KRAS or BRAF and few chromosomal abnormalities. In contrast, HGSOCs harbor mutations in TP53 and are, chromosomally, highly unstable5. Immunohistochemical algorithms and prediction models have been proposed for the five major histologic types of epithelial ovarian carcinomas6. Optimized p53 immunohistochemistry was reported to be clinically useful as it can exclude the possibility of a LGSOC7.Ovarian high- and low-grade serous carcinoma, was defined by Malpica et al.8. TB is a morphological phenotype representing a destructive stromal invasion and was included as an additional prognostic factor for colorectal carcinoma in the 2019 WHO Classification of Digestive System Tumours9. The term intra-tumoral budding (ITB), which is TB found within the main tumor body, was introduced to distinguish this form of budding from the classic peritumoral budding10. The reported interobserver variability for assessing TB has ranged from moderate to very good, depending on the study11.Tumor budding (TB) and its association with disease progression in patients with various solid cancers was first described by Imai in the 1950s12. Such destructive invasion was suggested to be associated with a poor prognosis in patients with stage I endometrioid and mucinous ovarian carcinomas13. TB is an independent, unfavorable, prognostic factor for patients with early-stage cervical cancer following radical surgery14. However, ITB has not been well described in SOCs.The prognostic significance of destructive invasive implants in extra-ovarian tissues has been well studied in serous borderline tumors (SBTs) of the ovaryThe present study evaluated the prognostic significance of ITB in HGSOCs, describing the correlation between ITB and other conventional histologic parameters.Among 84 SOC cases, p53 expression was scored as overexpression in 52 cases, complete absence in 25 cases and wild type in 7 cases. A cytoplasmic pattern of p53 expression was not observed in this study. Among seven tumors with wild type of p53 expression, three tumors showing grade 3 nuclear atypia were morphologically indistinguishable from HGSOC with abnormal expression of p53. These tumors were included in HGSOCs. As a result, the 84 SOCs were divided into 80 HGSOCs and 4 LGSOCs.The clinicopathologic data of 80 HGSOCs and 4 LGSOCs are shown in Table Figure Table Among the 13 patients treated with NAC, the prognostic statuses were dead of disease in 12 patients and alive with disease in 1 with median follow-up of 46 (range 12 to 63) months.Among the four patients with LGSOC, one patient was dead of disease at 75 months, one patient was alive with disease at 68 months, and two patients were alive with no evidence of disease at 47 and 52 months.When we examined the presence of ITB as a maker of destructive stromal invasion of SOCs, it was found to be a significant poor prognostic indicator for the PFS of patients with HGSOC, but did not reach statistical significance for the OS of patients with HGSOC in the univariate analysis. In a multivariate analysis, ITB was not an independent prognostic indicator. The prognostic significance of ITB was considered to be strongly affected by the 2014 FIGO stage. The large size studies stratified by the 2014 FIGO stage may be recommended.8. EMT is characterized by cytoskeletal rearrangements, cell motility and invasion, increased cell-associated proteolytic activity and reprogramming of the gene expression15. ITB was significantly associated with the incidences of LVSI and lymph node metastasis in the present study. Increasing evidence has highlighted a close relationship between EMT and chemoresistance in epithelial ovarian carcinoma (EOC)16. In one study, the SKOV-3 EOC cells were shown to trigger both EMT and chemoresistance after treatment by carboplatin17. ITB was found in all HGSOCs after NAC, except for in two complete regression cases, and was associated with a low number of mitoses of tumors and poor prognosis of patients.It is well-known that TBs are part of the tumor microenvironment and are associated with epithelial-mesenchymal transition (EMT)18. In a review, LGSOC was defined as a serous neoplasm showing destructive invasion, mild to moderate cytologic atypia, and relatively low proliferative activity. LGSOC accounts for 4.7% of SOCs and has an excellent prognosis, but long-term follow-up indicates that the prognosis for patients with stage III–IV disease is poor19. In the present study, LGSOC accounted for 4.8% of SOCs. ITB was found in one of four LGSOCs. The case number of LGSOC was too small to analyze the prognostic significance.The stromal-epithelial patterns of invasion in SBTs of the ovary have been subclassified as destructive and nondestructive. By definition, well-differentiated serous tumors featuring destructive stromal invasion are classified as LGSOCs whereas those with either no stromal invasion or stromal microinvasion are classified as SBTs of the ovary20. Tumors with the mesenchymal phenotype had poor prognoses, whereas those with the immunoreactive type had favorable prognoses. Murakami et al.21 said that a feature of spindle and isolated cells with destructive stromal reaction was referred to as the mesenchymal phenotype. Tothill et al.22 reported that a novel subtype of HGSOCs reflected a mesenchymal cell type, characterized by the overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples.Analyses of gene expression microarray data from The Cancer Genome Atlas (TCGA) project revealed that HGSOC could be classified as one of four gene expression subtypes: mesenchymal, immunoreactive, proliferative, or differentiated7. Chui et al.23 said that the existence of TP53-wildtype HGSCs is rare and comprise a heterogenous group of tumors which may arise via distinct pathogenetic mechanisms.The presence of morphological HGSOC showing wild type of p53 expression is controversial. One study reported that wild type of p53expression was observed in nine (5.3%) HGSOCs of which two (1.2%) did not have a detectable TP53 mutationIn conclusion, ITB was shown to be a cost-effective prognostic indicator for patients with HGSOC and a histopathologic biomarker of the progression of HGSOC. We may need to pay closer attention to the progression of HGSOCs.24, were not included in the present study. The ovarian specimens were taken from 71 patients at primary surgery and 13 after neoadjuvant chemotherapy (NAC). All cases with available histopathologic slides were included in the present study, except for two patients with complete regression of SOC after NAC. Hematoxylin and Eosin (H&E)-stained slides of the SOCs were cut from a median of 6 (range 2–18) tissue blocks per case.After a review of the 318 malignant ovarian tumors found in the database of the Department of Pathology at the University of Occupational and Environmental Health Hospital between 2000 and 2017, 84 SOCs were selected. Twenty peritoneal carcinomas, defined according to the Gynecological Oncology Group inclusionary criteriaEthical approval for this study was granted by the Review Board of the University Hospital of Occupational and Environmental Health on Ethical Issues (H27-185). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants.25. The postsurgical assessment of the interval debulking surgery was available for the 2014 FIGO stage of patients who received platinum-based NAC. Adjuvant platinum-based chemotherapy was not performed in one patient with stage IA HGSOC without ITB, and six patients with severe medical complication and/or advanced age, (including two with HGSOC without ITB and four with HGSOC with ITB). These latter six patients were not included in the analysis of the patient prognosis.The following clinicopathological parameters were evaluated: age at the diagnosis, CA125 level , 2014 FIGO stage, architectural grade, nuclear grade, ITB, lymphovascular space invasion (LVSI), mitoses and lymph node metastasis while referring to previous studies11, ITB was defined as a single tumor cell or a cell cluster of up to four tumor cells at the invasive tumor front. ITB was evaluated in the surgical specimens of the ovarian tumors to be clinically the site of the greatest tumor volume/size.According to the statements of the international tumor budding consensus conference (ITBCC)26, there was no significant difference in the cumulative survival between women with SOC with focal and diffuse ITB. The SOCs were therefore ultimately divided into two groups: (those with and without ITB).While we initially attempted to use a three-tier grading system of ITB  with a reference of Silva system for invasive endocervical adenocarcinomas7, expression of p53 was scored as overexpression, complete absence. cytoplasmic or wild type.Representative formalin-fixed paraffin-embedded (FFPE) tissue blocks of 84 cases were selected for immunohistochemical analysis. Immunohistochemistry for p53 was performed on formalin-fixed paraffin-embedded tissue sections using a commercially available mouse monoclonal anti-human antibody  at a dilution of 1 in 50. The sections were stained by use of the universal immunoperoxidase polymer method , in accordance with the manufacturer’s instructions. They were subsequently subjected to microwave heating and pressure cooking for the purpose of antigen retrieval. Positive and negative controls were also established. According to the proposed immunohistochemical scoringU test. The progression-free survival (PFS) was defined as the time from the date of initial treatment to the date of objective disease progression or last follow-up. The overall survival (OS) was defined as the time from the date of initial treatment to the date of death or last follow-up. The PFS and OS curves were estimated using the Kaplan–Meier method and compared using the log rank test. Univariate and multivariate Cox proportional hazards models were used to determine the association between potential risk factors and progression as well as death from disease. Statistical significance was considered to exist at a value of P < 0.05.Statistical analyses were carried out using the IBM SPSS Statistics, version 27 . Data for the clinicopathological factors were evaluated using the Chi-square test or Mann–Whitney Ethical approval for this study was Granted by the Review Board of the University Hospital of Occupational and Environmental Health on Ethical Issues (H27-185).


However, the role of normoxia for critically ill trauma patients remains uncertain. The objective of this study is to describe the study protocol and statistical analysis plan for the Strategy to Avoid Excessive Oxygen for Critically Ill Trauma Patients (SAVE-O2) clinical trial.Targeted normoxia  and post-implementation (intervention) data. All sites will begin in the control phase with usual care. When sites reach their randomly assigned time to transition, there will be a one-month training period, which does not contribute to data collection. Following the 1-month training period, the site will remain in the intervention phase for the duration of the trial.Main outcome measures: The primary outcome will be supplemental oxygen-free days, defined as the number of days alive and not on supplemental oxygen. Secondary outcomes include in-hospital mortality to day 90, hospital-free days to day 90, ventilator-free days (VFD) to day 28, time to room air, Glasgow Outcome Score (GOS), and duration of time receiving supplemental oxygen.SAVE-O2 will determine if a multimodal intervention to improve compliance with targeted normoxia will safely reduce the need for concentrated oxygen for critically injured trauma patients. These data will inform military stakeholders regarding oxygen requirements for critically injured warfighters, while reducing logistical burden in prolonged combat casualty care.ClinicalTrials.govNCT04534959. Registered September 1, 2020.The online version contains supplementary material available at 10.1186/s13063-021-05688-6. Oxygen therapy has undisputed importance in the care of critically ill patients to prevent morbidity associated with hypoxia and enhance oxygen delivery , 2. Exce2), which determined optimal range of 90–96%, partial pressure of arterial oxygen (PaO2) range of 60–100 mmHg (when applicable), and fraction of inspired oxygen (FiO2) of 0.21 for mechanically ventilated patients or room air for non-mechanically ventilated patients [To address this uncertainty, we are conducting the Strategy to Avoid Excessive Oxygen (SAVE-O2) for Critically Ill Trauma Patients clinical trial. The purpose of this trial is to determine the effectiveness of a multimodal educational intervention to safely reduce supplemental oxygen use in critically injured patients. Investigators will also evaluate the clinical effectiveness of the more targeted use of oxygen therapy. The pilot study for the SAVE-O2 trial demonstrated that the multimodal intervention is feasible and effective in reducing use of supplemental oxygen for critically ill trauma patients . We convpatients . These rpatients .SAVE-O2 is a multicenter cluster randomized, stepped wedge implementation trial to evaluate the superiority of a multimodal educational intervention to improve adherence to consensus-based normoxia compared to usual care in critically ill trauma patients. This pragmatic design allows for optimal evaluation of an intervention where benefits of targeted normoxia outweigh the alternative of potential increased frequency of hypoxia and/or hyperoxia . Eight l2 90–96%, PaO2 60–100 mmHg) in critically ill trauma patients, but do not have existing protocols and/or resources to promote this oxygenation strategy. The target population includes adults aged 18 years or older who meet criteria for entry into state or national trauma registries and who are admitted to a surgical or trauma intensive care unit (ICU) within 24 h of arrival to a participating hospital. This includes patients who present directly to the emergency department of a participating hospital and those who are transferred into a participating hospital. Exclusion criteria include prisoners and known pregnancy. There is no selection for inclusion/exclusion based on mechanical ventilation status, injury severity, injury mechanism, or traumatic brain injury.SAVE-O2 includes critically ill trauma patients from eight level 1 trauma centers geographically distributed throughout the United States. These hospitals have endorsed the consensus-based recommendation for normoxia , which serves as the single IRB for this study. Each enrolling site has ceded review to COMIRB under reliance agreements. Prior to the design of this study, we conducted a systematic review, Delphi consensus process, and pilot study. Together, these processes demonstrated preliminary safety evidence of targeted normoxia, identified consensus-based oxygenation targets, and provided a foundation to conduct this multicenter clinical trial with a waiver of informed consent under the Common Rule (45 CFR 46) that governs the ethical conduct and oversight of human subjects’ research, section 116 (d). The COMIRB determined that this trial represents no more than minimal risk to participants – this multimodal educational intervention helps hospitals implement a consensus-based oxygenation target that is not binding and remains at the discretion of the treating physician to ensure optimal care of all critically ill trauma patients. The use of a waiver of consent does not adversely affect the rights and welfare of subjects—patient care remains at the discretion of the clinical team to act in the best interests of an individual patient and data collection occurs at a hospital unit-level to ensure that patient care and privacy is not compromised. Finally, the study conducts research that could not practicably be carried out without the waiver of consent—there is no interaction with patients or their surrogates as the intervention is conducted at the level of each hospital unit with subjects as a whole receiving either usual care in the pre-intervention period or education-enhanced usual care in the post-intervention phase. The full details of the ethical considerations of this trial are detailed in the full study protocol . In randomly chosen order, each cluster will cross over from the pre-intervention phase of usual care to post-intervention targeted normoxia. This incorporates a 1-month run-in period during which the hospital engages staff in educational activities and trainings designed to increase familiarity and compliance with targeted normoxia protocols. These standardized activities and associated materials are part of the “intervention” provided by the clinical coordinating center. During this run-in period, patient-level data collected will not be used for the primary analysis. Cluster crossover occurs every 3 months for a total study duration of 27 months  provide essential background information and stress the importance of avoiding both hypoxia and hyperoxia as a potential way to decrease patient morbidity and mortality, (2) give an overview of the SAVE-O2 trial, (3) provide potential protocols to down-titrate supplemental oxygen in hyperoxic patients, and (4) give instruction for reporting potential adverse events. In addition to these presentations, flyers will be placed strategically throughout sites reminding providers of the ongoing SAVE-O2 trial. Further education and reminders, led by local coordinators and investigators, will be conducted at staff meetings, floor rounds, and daily team meetings.We will also provide monthly newsletters and feedback highlighting the pre- and ongoing post-intervention oxygenation practices. These will help guide sites in evaluating their progress toward a minimum goal of 90% of patient-hours spent within the targeted normoxia thresholds.2 or PaO2 measurements. They will make recommendations based on on-site practice patterns for oxygen titration which can be overridden by clinical judgments. In cases where a recommendation for down-titration of supplementation oxygen is overridden by clinical judgment, that patient remains in the trial. The educational component of this intervention is expansive and targets physicians, advanced practice providers, nurses, and respiratory therapists. However, in many cases, nurses and respiratory therapists will be primarily responsible for oxygen titration. Each site is free to follow its own practices for specific oxygen titration and the exact time frame in which titrations should be made. However, SAVE-O2 recommends down titration of supplemental oxygen in patients with sustained hyperoxia (30 minutes or greater) by FiO2 increments of 0.1 until normoxia is reached, a mechanically ventilated patient is on an FiO2 of 0.21 or until a non-mechanically ventilated patient is no longer on supplemental oxygen. In some cases, oxygen titration may not be possible, for example, patients who are hyperoxic with an SpO2 > 96% but not on supplemental oxygen and those who are hypoxic but on an FiO2 of 1.0. Treating physicians are also allowed to override study recommendations when determined to be in the best interest of the patients. Potential situations where it is anticipated that treating physicians may temporarily favor hyperoxia include carbon monoxide poisoning, untreated pneumothorax, and cyanide poisoning.In addition to multiple educational activities, some sites will also include an electronic health record best practice alert Fig. . These aOxygen titration is encouraged from the time that a patient enters the hospital through to his/her discharge from the ICU. While SAVE-O2 outcomes focus on oxygenation in the ICU, the intervention will also target emergency department providers who often establish post-acute injury therapeutic momentum. To further increase the success of the intervention, leadership from within each site’s emergency department, trauma surgery teams, and the ICUs will work with study “champions” to reinforce intervention compliance. Oxygen titration is “encouraged” but not mandated because the primary study question focuses on the effectiveness of a multi-faceted educational intervention to target normoxia in critically ill trauma patients. Thus, we focus on educating ICU providers and assess the effect of the education intervention on the ICU oxygenation of these patients. After a patient is discharged from the ICU or transferred to a floor bed, the patient’s oxygenation status is no longer monitored for the purposes of this trial. Patients subsequently readmitted to the ICU from the floor after 24 h may still experience the effects of the educational intervention which is deployed at the level of the ICU unit, but no further oxygenation data is collected for that patient during the ICU readmission.The primary outcome of SAVE-O2 is supplemental oxygen-free days (SOFD). This is defined as the number of days a patient is alive and not on supplemental oxygen from the time of presentation to day 28. SOFD is censored at hospital discharge. The score ranges from − 1 days (death) to 28 days . Patients discharged from the hospital prior to day 28 are assumed to be maintained at the level of oxygenation they require on hospital discharge—i.e., if they are discharged on room air or the level of supplemental oxygen used prior to admission, it is assumed that they remained on no additional supplemental oxygen to day 28. When a patient receives the same level of supplemental oxygen as they require at home prior to admission, this will count as being free of supplemental oxygen. We will not count toward SOFD the amount of time patients are intubated and ventilated only for a surgical procedure and are immediately extubated upon completion of that procedure.There is no patient follow-up after hospital discharge in SAVE-O2 and all secondary outcomes are assessed at hospital discharge. However, variables will be censored at various intervals while hospitalized: hospital discharge, 28 days, or 90 days. SAVE-O2 will assess ventilator-free days to day 28 (VFD28) , 11, 15 Secondary outcomes censored at 90 days include hospital-free days (HFD90), in-hospital mortality, and time to mortality. Hospital-free days is defined as the number of days a patient is alive and not hospitalized. The scores will range for − 1 days  or zero  to 90 days . In-hospital mortality is defined as alive or dead on the day of hospital discharge or day 90, whichever is first. Time to mortality is defined as the number of days from admission to death. Time to mortality is also censored at hospital discharge or day 90, whichever is first.2 of 0.21 or is on room air, no further oxygenation adjustments can be made. This time will count toward compliance with the normoxia protocol. We will also assess the time to the first incidence of no supplemental oxygen use , censored at hospital discharge.To better characterize oxygenation practices, we will collect data on multiple additional secondary outcomes. This includes the frequency and duration of ICU time a patient spends in the various oxygenation categories  and the time spent compliant with targeted normoxia. For cases where a hyperoxic patient is ventilated on FiO2 > 0.40 or more than 4 liters per minute. The proportion of participants on high levels of supplemental oxygen for more than 2 h in the ICU and the total duration of time receiving high levels of supplemental oxygen will be calculated.To characterize the potential reduction in the amount of supplemental oxygen used we will determine the total amount of supplemental oxygen administered. We will specifically examine the use of high levels of supplemental oxygen, defined as an FiOThe final patient-centered clinical outcomes include the discharge disposition and Glasgow Outcome Score (GOS) , 17. Dis2, PaO2, FiO2, and oxygen volume measurements. Many sites will also collect raw, unvalidated continuous SpO2 measurements. Instances that require manual chart extraction by site coordinators  will be minimized and obtained using standardized operating procedures with flow charts and detailed procedures for extraction.SAVE-O2 will leverage the expertise of the Data Coordinating Center (DCC) at Vanderbilt University Medical Center and the on-site informatics specialists to implement protocols that automatically extract variables directly from the electronic health record and state trauma registry data Table , includiSAVE-O2 uses the resources available at the DCC to provide specialized REDCap database development and management. REDCap is a secure, encrypted, HIPAA-compliant web application specifically designed for research data management with the ability for data capture and validation and data audits, and de-identified data export to common statistical packages . AutomatUnder the guidance of the Department of Defense and COMIRB, an Independent Safety Officer was selected with expertise in acute and critical care to monitor participant safety, evaluate study progress, and suggest changes in design or conduct as needed to address potential safety issues. Adverse events and unanticipated problems will be reported to the Department of Defense Scientific Officer, the Independent Safety Monitor, COMIRB, and the Department of Defense Human Research Protection Office in accordance with their reporting policies. The CCC will provide both the Independent Safety Officer and the Department of Defense Scientific Officer quarterly reports to further monitor trial progress and safety events. No formal interim analyses are required.We estimate an approximate accrual rate of 30 patients per site per month for a total sample size of at least 6000 patients over the 27-month study duration. Based on pilot study data, we estimated a mean SOFD of 15.5 under control conditions with a standard deviation of 11.3 days and an interclass correlation coefficient of 0.04. The estimated sample size of 6000 patients will allow us to detect a difference of 1.42 days in the primary outcome SOFD at 80% power and 1.64 days at 90% power. This assumes that data is normally distributed and that each site contributes the same number of patients as all other sites. As pilot data suggested that some of these assumptions may be false , simulation studies were conducted to address the impact of their violation on power. The simulations, conducted under the less restrictive distributional assumption of SOFD as an ordinal outcome, included removing a fraction of patients during a site crossover period to account for run-in and using the relative trauma patient volume of each site (which varies between 500 and 1500 trauma ICU patients per year) to adjust its sample size relative to the total. These simulation studies demonstrated almost no difference in power estimates relative to traditional power calculations, so power calculations reported above reflect the simpler formula-based approach .We will use descriptive statistics to create summary tables for patient characteristics and outcome variables. We will report the sample size, mean, and standard deviation for all continuous variables stratified by treatment condition (pre- vs post-intervention) and site. Descriptive statistics will exclude missing data. All analysis will be performed on the basis of intention to treat. We define a two-sided threshold for statistical significance of 5%. With a single primary outcome, we will not adjust for multiple comparisons; appropriate caution will therefore be used in interpreting the results of hypothesis testing for secondary analyses.We will analyze the primary outcome, SOFD, using a linear mixed-effects modeling framework. To account for possible temporal trends associated with intervention implementation at different times, a fixed effect for time will be included. We will account for clustering of patients within sites by including a random intercept term in all models. We will also adjust the model for the following patient-level covariates: age, sex, race and ethnicity, insurance type, Elixhauser Comorbidity Index , mechaniContinuous secondary outcomes  will be analyzed using a linear mixed modeling approach, similarly to the primary outcome. For dichotomous outcomes , we will use a logistic mixed model. For time-to-event outcomes , we will use a Cox proportional hazards regression model with a gamma-distributed random intercept for the site. Time zero will be taken to be the time of arrival in hospital. Kaplan-Meier plots of the time-to-event outcomes will be created to graphically compare distributions between treatment conditions. For ordinal outcomes , we will use a mixed-effects ordinal logistic regression model. The proportional odds assumption will be checked to assess if the relationship between the consecutive outcome levels is the same. If violated, a multinomial logit or partially proportional odds mixed-effects model will be used.2 is maintained constantly until a patient is extubated or a new FiO2 is entered. In non-mechanically ventilated patients, we will also assume that the level of supplemental oxygen provided will remain constant until a new value is entered up to 12 h later. However, after 12 h without a new measurement recorded, the patient will be assumed to be on room air. We will not assume an FiO2 until the first recorded measurement in the first 12 h. If, after 12 h, a measurement is still not recorded, we will assume the patient is on room air. This will ensure that the primary outcome has no missing data.Based on pilot study data, some oxygen exposure and measurements are expected to be missing due to charting inconsistency. In mechanically ventilated patients, we assume that FiOTable SAVE-O2 is multicenter cluster randomized, stepped wedge implementation trial. This trial will determine if targeted normoxia will safely reduce the need for concentrated oxygen for critically ill trauma patients. The primary outcome will be supplemental oxygen-free days, defined as the number of days alive and not on supplemental oxygen. In addition to answering the primary scientific question, these data will inform military stakeholders on oxygen requirements for critically injured warfighters, while reducing logistical burden in prolonged combat casualty care. This protocol and statistical analysis plan article have been submitted for publication before recruitment was completed.This article is based on the SAVE-O2 Trauma protocol version 1.1 as of June 3, 2020. Pre-intervention data collection started on July 15, 2020. The first site began the 1-month run-in implementation period on October 15, 2020. The estimated study completion date is December 15, 2022.Additional file 1: Appendix 1: SPIRIT ChecklistAdditional file 2: Appendix 2: Final ProtocolAdditional file 3: Appendix 3: Statistical Analysis Plan


In humans, sex determination and differentiation is genetically controlled. Disorders of sex development (DSD) result in anomalies of the development of the external and internal genitalia. Variants in transcription factors such as SRY, NR5A1 and SOX9, can cause changes in gonadal development often associated with ambiguity of the external genitalia.This study has been conducted to determine the frequency, types and associated genetic alterations in patients with DSD in the Algerian population.Thirty patients were included. Based on their clinical presentation, thirteen patients presented with ambiguous external genitalia, thirteen patients presented with hypospadias and four patients presented with bilateral undescended testes. Karyotype analysis was performed on peripheral blood lymphocytes using standard R-banding. DNA was isolated from blood leukocytes for PCR reaction and mutational analysis of SRY and NR5A1 was done by direct sequencing.Most patients with ambiguous genitalia had a 46,XY karyotype. One patient had a deletion of SRY, otherwise no point mutations in SRY or NR5A1 genes were identified. However, a single NR5A1 polymorphism (p.Gly146Ala) in patient with 46,XX DSD has been detected.The absence of mutations in these genes suggests that there are others genes playing an important role in sex development and differentiation. Disorders of sex development DSD comprise a series of genetic diseases in which chromosomal, gonadal, or anatomical sex is atypicalError in any of these stages may lead to DSD, which can be classified into the following groups: 46,XX DSD , 46,XY DSD , Ovotesticular DSD, 46,XX Testicular DSD , and 46,XY Complete Gonadal Dysgenesis (CGD)Mutations in several genes are known to cause DSD. Most often the underlying cause of DSD is a variant in a gene or genes regulating gonadal/genital or steroidogenic pathways. The commonest known genetic condition that leads to 46,XX DSD is congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase deficiency. However Among the 46,XY DSD, the most common cause is androgen insensitivity syndrome (AIS) with mutations in the androgen receptor geneThe Sox (SRY-type HMG box) genes encode a group of proteins characterized by the presence of an HMG-box, a 79 amino acid motif which can bind and bend DNA, which is the only part of the testis-determining gene SRY that is conserved between speciesRecently, a recurrent NR5A1 p.Arg92Trp mutation was also identified in several patients with 46,XX testicular/ovotesticular DSD, highlighting the role of NR5A1 in the development of both testes and ovariesIn view of the importance of the SRY and NR5A1 genes in normal gonadal development, this first study in Algeria was undertaken with an aim to screen for mutations in these genes in DSD Algerian cases and to describe their clinical and cytogenetics featuresing.Informed consent was obtained from all subjects participating in the study. Thirteen patients with ambiguous external genitalia, thirteen patients with hypospadias and four patients with bilateral testicular ectopy were included. Twenty-eight were children aged 22 days-13 years and two were young adult aged 18-20 years. All patients were of Algerian origin and from different geographic locations. These patients came to our laboratory for karyotype analyses. The controls were normal adult males with 46,XY and females with 46,XX chosen from volunteers. A blood sample was obtained from each subject for karyotype analysis, hormonal analysis and DNA extraction.Peripheral blood lymphocyte cultures were set up using TC199 medium for human lymphocyte culture for 72 hours. Dividing cells were arrested at metaphase stage with Colcemide and fixed using Carnoy acetic solution after treatment with hypotonic solution according to standard procedures. The harvested cells were dropped on clean slides and the chromosomes were studied by RHG method (R-bands after heat denaturation and Giemsa). A total of 30 metaphases were analyzed for each sample and the karyotypic descriptions were according to the ISCN recommendations to look at any numerical or structural chromosomal aberrations Previously we have analyzed the presence of the SRY gene in leucocytes in all cases by PCR. The complete open reading frames of SRY and each exon of NR5A1 were amplified by polymerase-chain-reaction (PCR) according to a previous reportEthics approval was obtained from the Ethics Committee of the university hospital center Benbadis of Constantine.Age- and sex-dependent hormone levels in the normal population are taken as given previously The SRY gene had only one exon. One pair of primer was designed for this exon. The whole coding sequence of SRY gene was sequenced but no mutation or deletion was found in all patients whose SRY gene was positive.In the 30 cases with DSD we did not detect a mutation in the whole coding sequence of NR5A1 gene. We identified a single NR5A1 polymorphism. This patient presented with 46,XX DSD. The polymorphism consisted of a G to C transversion at nucleotide position 437 in exon 3 that is predicted to result in a p.Gly146Ala amino acid change.DSD ranging from minor genital abnormalities to complete sex-reversal are the most common birth defects with an incidence rate of almost 3%These conflicting results indicate that consanguinity may lead to an increase in the incidence rates of both 46,XY and 46,XX DSD, depending on the population studied.The main gene implicated in sex determination in mammals is the sex-determining region on the Y chromosome (SRY), which encodes a transcription factor that is a member of the high mobility group (HMG)-box family of DNA binding proteins and in mammals triggers the development of undifferentiated gonads towards a testicular phenotypeThe limitation of the current study is small sample size and the fact that only two genes involved in the sex determination were screened.We found in Algeria that the most common form of DSD associated with ambiguous external genitalia in the newborn is 46,XY DSD. The pathology of the majority of these cases is not explained by mutations in the two most common genes associated with DSD namely, SRY and NR5A1 suggesting that other genes are involved. Finally, we anticipate in the future sequencing a panel of genes and whole exome for genetic diagnosis of Algerian patients with DSD.