Schistosoma haematobium infections of different intensities.Efforts to control and eliminate schistosomiasis have accelerated over the past decade. As parasite burden, associated morbidity and egg excretion decrease, diagnosis with standard parasitological methods becomes harder. We assessed the robustness and performance of a real-time PCR (qPCR) approach in comparison with urine filtration microscopy and reagent strip testing for the diagnosis of S. haematobium egg counts was assessed and Ct-value percentiles for infection intensity categories determined.The robustness of DNA isolation and qPCR was validated in eight laboratories from Europe and Africa. Subsequently, 792 urine samples collected during cross-sectional surveys of the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project in 2012–2017 were examined with qPCR in 2018. Diagnostic sensitivity of the qPCR was calculated at different infection intensity categories, using urine filtration microscopy as reference test. Spearman’s rank correlation between Ct-values and S. haematobium Dra1 DNA-positive samples were identified correctly in all eight laboratories. Examination of urine samples from Zanzibar revealed Dra1 DNA in 26.8% (212/792) by qPCR, S. haematobium eggs in 13.3% (105/792) by urine filtration, and microhaematuria in 13.8% (109/792) by reagent strips. Sensitivity of the qPCR increased with augmenting egg counts: 80.6% (29/36) for counts between 1 and 4 eggs, 83.3% (15/18) for counts between 5 and 9 eggs, 100% (23/23) for counts between 10 and 49 eggs, and 96.4% (27/28) for counts of 50+ eggs. There was a significant negative correlation between Ct-values and egg counts . Seventy-five percent of the Ct-values were ≥ 33 in the egg-negative category, < 31 in the light intensity category, and < 24 in the heavy intensity category.Dra1 based qPCR assay detected twice as many S. haematobium infections compared with classical parasitological tests. The qPCR is hence a sensitive, urine-based approach for S. haematobium diagnosis that can be used for impact assessment of schistosomiasis elimination programmes, individual diagnosis, and in improved format also for verification and certification of elimination.While the sensitivity of the qPCR was ~ 80% for very light intensity infections (egg counts < 10), in general, the ISRCTN48837681. Registered 05 September 2012 - Retrospectively registered.ISRCTN,  Schistosoma by 2020, to achieve elimination as a public health problem by 2025, and to interrupt transmission in selected countries by 2025 [Schistosomiasis is a debilitating disease that particularly affects poor and economically deprived populations in 78 endemic countries worldwide . In 2017 by 2025 , 4. OverS. haematobium) or stool (other Schistosoma species). Lighter and asymptomatic infections are more difficult to diagnose and classical parasitological tests such as the reagent strip method to detect blood in urine as a proxy for S. haematobium infection or microscopy to detect Schistosoma eggs in urine or stool start to miss infections [There are several countries considered endemic for schistosomiasis that have successfully controlled morbidity, eliminated the disease as a public health problem, and even have interrupted transmission . Most offections , 8. Morefections –11.Zanzibar is among the first settings in sub-Saharan Africa where elimination of urogenital schistosomiasis seems feasible and where elimination efforts implemented since 2012 have successfully reduced the prevalence and infection intensity to very low levels .S. haematobium specific Dra1 DNA repeat in urine samples from Zanzibar. First, the robustness of DNA isolation from urine samples and the qPCR assay system were verified in a ring trial including a total of eight hospital, industry, and academic research laboratories from Europe and Africa. Subsequently, a selection of 792 urine samples from participants of the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project [Here, we assessed the robustness and performance of a standardized real-time polymerase chain reaction (qPCR) approach for the detection of the  project  that hadUnguja and Pemba islands are the largest islands comprised in the Zanzibar archipelago offshore mainland Tanzania. The islands’ projected population for 2019 was 1.6 million inhabitants .S. haematobium was highly endemic in the last century, with more than half of the school-aged population being infected in high-risk areas [S. haematobium prevalence to < 2% and heavy infection intensities to < 1% in schoolchildren and adults in 2017 [Urogenital schistosomiasis caused by sk areas –18. Intesk areas , 19–21,  in 2017 , 13.S. haematobium.Efforts to eliminate urogenital schistosomiasis from Zanzibar started in 2011 with the formation of the ZEST alliance and strong political commitment from the Zanzibar government. The ZEST alliance included the Neglected Diseases Programme of the Zanzibar Ministry of Health (MoH), the Public Health Laboratory-Ivo de Carneri (PHL-IdC), the WHO, the Schistosomiasis Control Initiative, the Bill & Melinda Gates Foundation, the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE), the Natural History Museum in London, the Swiss Tropical and Public Health Institute and other partners and stakeholders . The ZanS. haematobium infections as described elsewhere in detail [Annual cross-sectional parasitological surveys were conducted in 91 study schools and 91 study communities, respectively, in Zanzibar (Pemba and Unguja islands), from November 2011 to May 2017. Every year and in each study location, urine samples were collected from ~100 randomly selected students aged 9–12 years and from ~50 randomly selected adults aged 20–55 years and examined for n detail , 21. In Similarly, the sheha (community head) of each study community (shehia) was informed about the study and subsequently the community was visited by the study team. Households were selected by a simple randomization procedure and a present adult household member was invited to participate in the study . Once wrAll urine samples were collected between 10:00 AM and 2:00 PM and examined in the laboratory of the Zanzibar Neglected Diseases Programme in Unguja or at PHL-IdC in Pemba, respectively, during the later afternoon of the day of collection.Siemens Healthcare Diagnostics Ltd., Camberley, United Kingdom). Microhaematuria was coded semi-quantitatively in line with the colour reaction of the reagent strips and the manufacturer’s instructions as negative; trace; +; ++; and +++. Subsequently, urine samples with sufficient volume were vigorously shaken, and 10 ml were filtered through a filterholder containing a polycarbonate filter with a pore size of 20 μm  using a standard 10 ml plastic syringe. The filter was then carefully transferred to a microscope slide, which was labeled with the participant ID, covered with hydrophilic cellophane soaked in glycerol, stained with Lugol’s iodine and examined under the microscope for the presence and number of S. haematobium eggs by trained laboratory technicians [Upon arrival in the laboratory, the urine samples were sorted by increasing participant ID and examined for microhaematuria using reagent strips  of collection. Finally, we randomly selected 585 samples to reach a total of 800 samples for examination with qPCR.A total of 1864 urine samples collected in Zanzibar between 2011 and 2017, were shipped on dry ice from Zanzibar to Basel in 2016 and 2017. Among them, 800 samples were selected by the study statistician for qPCR based examination at Biolytix AG in 2018, following these criteria: first, all samples from 2016 and 2017 were selected because there were only few samples available from those years; second, we selected all Dra1 based qPCR, eight laboratories based in Europe and Africa were invited to test urine samples for S. haematobium Dra1 DNA in 2017. Two laboratories were based in sub-Saharan Africa (one in Tanzania and one in South Africa), and six in Europe . Among them, two were hospital laboratories, three were industry laboratories, and three were academic research laboratories. The aim of the ring trial was to explore how the DNA isolation and qPCR performed in different laboratories with different technicians and thermocyclers. Urine samples, the DNA Isolation Kit material, and the PCR Kit, respectively, were pooled and aliquoted at Biolytix AG before sending them to the ring trial participants to provide the same conditions for all participants. The urine samples (U1–U8) were sent in duplicates: one egg-negative (U1 ≙ U3) and three S. haematobium egg-positive samples . Hence, in total, eight samples were included in the test material. The participant laboratories were blinded to the infection status. In addition to the samples, one negative control (water as non-template control) and one positive control (sample containing Dra1 DNA), the Kit material and a detailed protocol for DNA isolation and examination were provided to the participants. Once the test samples and material were received, ring trial participants isolated DNA from the urine samples using the provided Quick-DNA Urine Kit (D3061) and protocol as described below. The isolated DNA was analyzed by the multiplex qPCR system described below. Each sample was run on a thermocycler in 1:10 dilution and undiluted. Results were assessed qualitatively for the presence or absence of DNA of the target organism, S. haematobium, and the reference gene 18S rRNA. All participants submitted their qPCR results to Biolytix AG within three to 16 weeks. The results included the cycle threshold (Ct)-values of the qPCR analysis and the type of thermocycler used.For proficiency testing of the robustness of DNA isolation from urine and the Quick-DNA Urine Kit (D3061) from Zymo Research according to the manufacturer’s protocol for cellular and cell-free DNA precipitation, but using an elongated protein digestion time and using the Zymo-Spin I-96 Plate (C2004) instead of the Zymo-Spin IC-S Column.DNA was extracted from the urine samples using the commercially available g in a Hermle Z366 Centrifuge .First, for DNA precipitation, 4 ml of each urine sample were mixed with 280 μl of urine conditioning buffer and vortexed. Subsequently, 10 μl of clearing beads were added and vortexed well. Finally, samples were spun for 15 min at 3000×g in a Heraeus Pico 17 Centrifuge , and the cleared supernatant was transferred into a new 2 ml microcentrifuge tube. Then, the samples were transferred into a Zymo-Spin I-96 plate  on top of a collection box and spun for 2 min at 5796×g in a Sigma 4–15C plate centrifuge . The Zymo-Spin I-96 plate was placed onto a new collection block, 200 μl of urine DNA prep buffer were added to each well with a multichannel pipette, and the plate was spun for 2 min at 5796×g. Subsequently, the Zymo-Spin I-96 plate was placed onto a new collection block, 700 μl of urine DNA wash buffer were added in each well with the multichannel pipette and the plate was spun for two minutes at 5796×g. This step was repeated once, using 200 μl of the same urine DNA wash buffer. Then, the plate was transferred on top of a 96-well elution tube rack and DNA was eluted with 100 μl of DNA elution buffer prewarmed at 60 °C. Finally, the plate was spun for 2 min at 5796×g and the elution plate containing extracted DNA was covered with an adhesive aluminum sticker cover and labelled with a unique barcode number.Second, for protein digestion, the supernatant was carefully discarded at low speed (leaving around 100 to 200 μl liquid on top of the pellet). Subsequently, one volume of urine pellet digestion buffer was added to the mix. The pellet was then resuspended, and the mix suspended. Finally, 20 μl of Proteinase K were added to the suspension and incubated for 1 h at 55 °C in a shaking water bath. Third, for DNA purification, one volume of Genomic Lysis buffer was added to the digested mix (420 μl) and vortexed. Subsequently, the mix was transferred in a new 2 ml microcentrifuge tube, the sample was spun for 1 min at 3000×Dra1 repeat unit in S. haematobium were selected based on the publication of Hamburger et al. 2001 [S. haematobium detection were as follows: Sh_F; 5′-GATCTCACCTATCAGACGAAAC-3′, Sh_R 5′-TCACAACGATACGACCAAC-3′, and Sh_P; 5′-(FAM)-TGTTGGTGGAAGTGCCTGTTTCGCAA-BHQ1–3′. The sequences for the 18S rRNA detection were as follows: 18S_F; 5′-CGGCTACCACATCCAAGGAA-3′, 18S_R; 5′-CTATTGGAGCTGGAATTACCGC-3′, and 18S_P; 5′-(YY)-TGCTGGCACCAGACTTGCCCTCC-BHQ1–3′. The reaction was carried out in 20 μl containing 1× Takyon ROX Probe MasterMix UNG , 160 nmol/L of Sh_F and Sh_R as well as 80 nmol/L of Sh_P, 40 nM of 18S-F and 18S_R as well as 20 nmol/L 18S_P, and 4 μl of target DNA. The participants of the ring trial used the thermocyclers available in their laboratory for the proficiency testing of the PCR system. The multiplex qPCR to examine the individual samples from Zanzibar for the second part of this study was carried out at Biolytix AG with an ABI 7900HT thermocycler  with a 384 well block module using the thermal profile including 2 min at 50 °C for the UNG activity, 10 min at 95 °C followed by 50 cycles of 15 s at 95 °C, and 60 s at 60 °C.The primers used for the qPCR for detection of the al. 2001 . To incral. 2001 . MoreoveS. haematobium eggs counted on the polycarbonate filter in 10 ml urine were recorded on paper sheets by the laboratory technicians and entered into a Microsoft Excel (Microsoft Corporation 2010) database by data entry clerks in Zanzibar. The results of the qPCR examination were directly obtained in electronic format in Switzerland. Data were analysed using STATA version 14.0 . Only data from urine samples with complete microhaematuria, urine filtration, and qPCR result were included in the analyses.The results of the macro- and microhaematuria grading and the number of S. haematobium egg per 10 ml urine were considered as S. haematobium egg-positive. In line with WHO recommendations, S. haematobium infections were classified into light (1–49 eggs per 10 ml urine) and heavy (50+ eggs per 10 ml urine) intensity [Dra1 DNA-positive. The sensitivity of the qPCR was calculated as the proportion of Dra1 DNA-positives that were correctly identified when compared with the results of the reference test. Reference tests, though imperfect since they lack sensitivity to detect very light intensity infection [S. haematobium eggs per 10 ml urine were assessed with the Spearman’s rank correlation test.Urine samples with a reagent strip colour reaction indicating trace, +, ++ or +++ were defined as microhaematuria-positive. Samples, where the single urine filtration microscopy revealed at least one nfection , were i)All eight laboratories that were invited did participate in the ring trial. The eight laboratories used five different types of thermocyclers for the qPCR .S. haematobium egg-positive urine samples as Dra1 DNA-positive. Considering Ct-values of ≥33 as negative, the two egg-negative samples were correctly identified by seven among the eight laboratories. One laboratory obtained Ct-values of 30.0 and 31.9, respectively, for these negative samples when diluted 1:10 and Ct-values of 26.7 and 28.1 for the undiluted samples, which we considered as an incorrectly positive result.As indicated in Table S. haematobium eggs were identified by single urine filtration microscopy in 13.3%, microhaematuria by reagent strips in 13.8%, and S. haematobium DNA by qPCR in 26.8% of the urine samples.Urine samples from a total of 800 individuals from Zanzibar were selected for examination with qPCR in 2018  and the reagent strips (109/792), respectively. Among the 212 Dra1 DNA-positive samples, 44.3% (94/212) were egg-positive, 40.6% (86/212) were microhaematuria-positive, and 48.1% (102/212) were either egg-positive or microhaematuria-positive.As indicated in Table Dra1 DNA in 89.5% (94/105) of S. haematobium egg-positive samples. As shown in Fig. Dra1 DNA result increased with increasing egg counts. The sensitivity of the qPCR was 80.6% (29/36) for counts between 1 and 5 eggs, 83.3% (15/18) for counts between 6 and 10 eggs, 100% (23/23) for counts between 11 and 49 eggs and 96.4% (27/28) for counts of 50+ eggs. The one egg-positive sample which was not identified as positive in the last group had a count of 721 eggs. With regard to egg-count infection intensity categorizations defined by the WHO, the sensitivity of qPCR to detect light and heavy infection intensities was 87.0% (67/77) and 96.4% (27/28), respectively.When the urine filtration was used as reference method, the qPCR identified Dra1 DNA in 78.9% (86/109) of microhaematuria-positive samples. The sensitivity for Dra1 DNA detection was higher when the microhaematuria grading was ++  and +++ , compared with trace  and + .When reagent strips were used as reference method, the qPCR identified Compared with the combined results of urine filtration and reagent strips as reference method, the qPCR had a sensitivity of 79.1% and a specificity of 83.4%.S. haematobium egg counts in the urine filtration microscopy or microhaematuria-positive reagent strip results, but were negative for Dra1 DNA in the qPCR. Among them, 11 samples were identified as S. haematobium egg-positive by urine filtration but negative for Dra1 DNA by qPCR. Among the 11 samples, seven had egg counts ranging between 1 and 4. When the microscope slides were read a second time for five among those seven slides, four had zero egg counts, but one among those four was microhaematuria-positive. Among these 11 samples, four had egg counts ranging between six and 721 eggs, mostly confirmed by a second reading of the microscope slides and all were microhaematuria-positive.Tab. Dra1 DNA-negative, 13 were from females .Among the 16 samples that were microhaematuria-positive but egg-negative and S. haematobium eggs counted under the microscope .There was a significant negative correlation between Ct-values measured by qPCR and S. haematobium egg counts is illustrated in Fig. The median and first and third quartiles of Ct-values of samples when stratified by WHO defined infection intensity categories based on S. haematobium prevalence for Zanzibar in general or any of the study locations in particular. Figure Dra1 DNA-positive for each study site in Pemba and Unguja, respectively. A total of 223 egg-positive and/or Dra1 DNA-positive urine samples were identified in 34/46 locations in Pemba and in 30/45 locations in Unguja.The selection procedure for the 792 samples examined with qPCR was not designed to reveal a S. haematobium-positive by both urine filtration and qPCR, 118 samples were diagnosed as positive by qPCR only, and 11 samples were identified by urine filtration only  and nine locations in Pemba (n = 10), where no egg-positive samples were identified by urine filtration. Among the 11 egg-positive samples that were only identified by urine filtration but not qPCR, nine were from seven sites, where also additional egg-positive or Dra1 DNA-positive samples were identified and two samples were from one site in Pemba and Unguja each, where no other positives were found.Among those 223 samples, 94 samples were recognized as Schistosoma prevalence and infection intensities [Schistosoma infections will become harder with standard classical parasitological methods [S. haematobium diagnosis. Here, we assessed the performance of a standardized qPCR approach for S. haematobium diagnosis in a multi-laboratory ring trial and by examination of 792 urine samples collected in Zanzibar, one of the first settings in sub-Saharan Africa targeted for schistosomiasis elimination.The WHO envisions a world free of schistosomiasis . Mass drensities , 27–29, S. haematobium egg-positive samples were correctly identified by qPCR in all participating laboratories. Importantly, the results were comparable by running the qPCR on five different brands and types of thermocyclers. One laboratory obtained false-positive results in the egg-negative urine samples. Hence, while the standardized qPCR approach can be considered as accurately detecting S. haematobium-positive samples, the occurrence of false-positive samples could not be ruled out.The ring-trial involving a total of eight research, hospital or biotech laboratories from Europe and Africa showed that all S. haematobium diagnosis in urine samples from Zanzibar, Dra1 DNA detection by qPCR clearly outperformed egg detection by urine filtration microscopy and microhaematuria assessment by reagent strips. The qPCR identified twice as many samples as S. haematobium-positive compared with the classical diagnostic methods. Most of the samples (108/118) that were Dra1 DNA-positive and S. haematobium egg-negative had Ct-values < 33 and > 22 and might hence be regarded as light intensity infections. While it is known that particularly very light intensity infections are often missed by reagent strips or urine filtration microscopy [S. haematobium [Schistosoma DNA have been reported to be more stable over multiple days than egg counts [Applying the qPCR for croscopy , it mighmatobium –32. HencDra1 DNA-positive. Most of these false-negative samples (7/11) had extremely low egg counts (below five eggs per 10 ml urine) and some of them were not confirmed by quality control readings of the same slide. Hence, as suggested for false-negative results in other studies, the amount of DNA in these very lightly infected samples might have been below the detectable limit [Dra1 DNA-negative urine samples were from females, and the explanation that the microhaematuria was caused by menstruation blood or other infections of the urogenital tract is self-evident. Clearly, not all microhaematuria is caused by schistosomiasis [However, not all egg-positive and/or microhaematuria-positive urine samples were identified as le limit , 33. How samples /11 had esomiasis .S. haematobium [Using urine filtration microscopy as reference method, the qPCR had a sensitivity of 89.5%. This value is greatly in line with previous assessments of qPCR systems for matobium , 35, 36.matobium . Going bS. haematobium-positive individuals in locations, where also the urine filtration microscopy identified cases. Hence, in these places the S. haematobium prevalence is likely underestimated when urine filtration is used as diagnostic approach, and might be twice as high as assumed. Moreover, the qPCR identified some few cases in sites where all urine filtration examinations were negative. Both observations are in line with an earlier study using the highly sensitive up-converting phosphor lateral flow circulating anodic antigen (UCP-LF CAA) assay in Pemba, which showed that there are far more individuals infected with S. haematobium than estimated by the urine filtration [S. haematobium prevalence might be justified for an extended period, at least in areas where urine filtration shows a reasonably high prevalence . If, as suggested elsewhere [The examination of urine samples from 91 study sites in Zanzibar showed that the qPCR mostly identified additional ltration . The reslsewhere , 13, thelsewhere , 38 and lsewhere  assays. S. haematobium diagnosis that can be used for impact assessment of schistosomiasis elimination programmes, individual diagnosis of S. haematobium infection, and in improved format also for verification and certification of elimination.The qPCR approach presented here is a standardized and sensitive approach for Additional file 1: Table S1. showing the multivariate frequency distribution of S. haematobium infection measured by urine filtration (egg counts), reagent strips (microhaematuria), and qPCR (Dra1 DNA).Additional file 2: Table S2. showing the characteristics of 27 urine samples that had S. haematobium egg counts in the urine filtration (UF) microscopy or microhaematuria-positive reagent strip results, but were negative for Dra1 DNA in the qPCR.


The aims of this study were (1) to determine the accuracy, sensitivity, and specificity of panoramic and peri-apical radiographs in diagnosing furcation involvement, as well as (2) to evaluate the possible impact of clinical experience on these diagnostic parameters.An existing radiographic dataset of periodontitis patients requiring implant surgery was retrospectively examined for furcation involvement. Criteria for inclusion were the presence of a CBCT, panoramic and peri-apical radiograph of the site of interest within a one-year time frame. All furcation sites were classified using the CBCT, which was considered as the gold standard, according to Hamp’s index (1975). Ten experienced examiners and 10 trainees were asked to assess furcation involvement for the same defects using only the corresponding panoramic and peri-apical radiographs. Absolute agreement, Cohen’s weighted kappa, sensitivity, specificity and ROC-curves were analyzed.P = 0.903). When recategorizing FI Grades into ‘no to limited FI’ (FI Grade 0 and I) and ‘advanced FI’ (FI Grade II and III), the panoramic and peri-apical radiography showed low sensitivity , yet high specificity  for identifying advanced FI. The ROC-curves for the panoramic and peri-apical radiographs were 0.79 and 0.69 respectively. No significant difference was found between experienced periodontists and trainees (P = 0.257 versus P = 0.880).The study sample included 60 furcation sites in 29 multi-rooted teeth from 17 patients. On average, 20/60 furcations were correctly classified according to the panoramic radiographs, corresponding to a weighted kappa score of 0.209, indicating slight agreement. Similarly, an average of 19/60 furcations were correctly classified according to the peri-apical radiographs, corresponding to a weighted kappa score of 0.211, also indicating slight agreement. No significant difference between panoramic and peri-apical radiography was found (Panoramic and peri-apical radiography are relevant tools in the diagnosis of FI and provide high specificity. Ideally, they are best used in combination with furcation probing, which shows high sensitivity. Furthermore, clinical experience does not seem to improve the accuracy of a radiological diagnosis of furcation sites.Patient radiographic datasets were retrospectively analyzed. The diagnosis of periodontitis is based on clinical as well as radiographic examination , 2. Two-Panoramic radiographs may occasionally be combined with peri-apical radiographs as an alternative to a full-mouth series of peri-apical radiographs to reduce the total radiation dose . UnfortuA detailed diagnosis of periodontitis is more challenging in the posterior dentition because of furcation and root proximities. In the case of periodontal attachment loss around multi-rooted teeth, bone resorbs stepwise and the furcation may become involved . Such fuThe degree of attachment loss in the furcation of multi-rooted teeth may be categorized into four grades based on horizontal measurements as described by Hamp et al. . Grade 0In treatment planning, FI is an important criterion to consider when making a prognosis for a tooth being possibly secure, doubtful, or irrational to treat. This is based on the fact that FI is considered a locus minoris resistentiae increasing the risk for tooth loss. In this respect, McGuire and Nunn  reportedCone beam computed tomography (CBCT) is a relatively new 3D approach in oral imaging , 18. CBCTwo-dimensional radiographic examination is limited in assessing marginal bone levels and incipient inter-radicular bone loss because of overlap of bone and surrounding anatomic structures . UnderesPatient demographics and radiographs were retrospectively selected from Ghent University Hospital records of patients who had been treated at the Department of Periodontology and Oral Implantology. Patients demonstrated varying severity and extent of periodontal disease, had at least one molar in situ without merged roots and had one or more implants placed following periodontal therapy. Given that, CBCTs were available from all patients. Additional inclusion criteria were the presence of a panoramic and peri-apical radiograph of the site of interest within a one-year time frame. The study was conducted in accordance with the Helsinki declaration of 1975 as revised in 2000. The protocol was approved by the ethical committee of the Ghent University Hospital (B670201523577). Consent to participate was not applicable for all individual participants included in the study.Ten staff members who had been working for at least 5 years as a periodontist and 10 postgraduate trainees in Periodontology and Oral Implantology participated in the evaluation of the radiographs. As it was our intention to assess the diagnostic value of panoramic and peri-apical radiograph for assessing advanced FI in daily practice, the clinicians were deliberately not trained and calibrated beforehand.For every furcation site the degree of horizontal alveolar bone loss was established on CBCT  by two experienced clinicians . The CBCTs were taken with a standardized protocol for three-dimensional implant planning . Mean mA was 5.6 and ranged between 4.5 and 7.1 depending on the skull size and weight of the patient. All CBCT volumes were available in DICOM format, analyzed with Planmeca Romexis© software  and carefully assessed in all different planes on a 24-inch monitor  with dimmed surrounded light. The data on horizontal alveolar bone loss were transformed to the Hamp et al. [Every clinician scored the same sites using the Hamp et al.  classifiPanoramic images had been taken with 66 kV tube voltage and 8 mA. Digital peri-apical radiographs had been taken with Rinn XCP® Paralleling holders . The active exposure time ranged between 0.08 and 0.12 s with 70 kV tube voltage and 7 mA. Peri-apical radiographs were developed with an image plate scanner .All two-dimensional radiographic images were viewed in full-screen modus with specialized imaging software  on a 24-inch monitor .Data analysis was performed in IBM SPSS® Statistics 25 with the furcation site as the statistical unit. Descriptive statistics included frequency distributions for categorical variables  and mean values and standard deviations for continuous variables (age). The FI Grade as registered on CBCT was considered the true FI Grade (= gold standard). Absolute agreement and Cohen’s weighted kappa was calculated to determine the accuracy of panoramic and peri-apical radiography in assessing the FI Grade. FI Grades were recategorized into ‘no to limited FI’ (FI Grade 0 and I) and ‘advanced FI’ (FI Grade II and III) in order to determine the diagnostic sensitivity (SENS), specificity (SPEC), positive predictive value (PPV) and negative predictive value (NPV) of panoramic and peri-apical radiography in identifying advanced FI. For all diagnostic parameters 95% confidence intervals were calculated. To explore the possible impact of clinical experience, diagnostic parameters and area under ROC-curves were compared between experienced periodontists and postgraduate trainees using the Mann Whitney U-test. Finally, composite Receiver Operating Characteristic (ROC)-curves [In total, 60 furcation sites of 29 multi-rooted teeth from 17 patients  could be included in this controlled clinical study. The original sample included 75 furcation sites, yet FI could not be assessed for 15 sites due to restoration materials causing CBCT artifacts. Fourteen of the 29 teeth were maxillary molars. Of those, 5 were first molars, 8 s molars and 1 third molar. Fifteen of the 29 teeth were mandibular molars. Of those, 5 were first molars, 8 s molars and 2 third molars. Twelve of the included furcation sites were mesiopalatal, 24 were buccal, 12 were distopalatal and 12 were lingual sites.Periodontists had a mean age of 43 years (SD 10.1) and included 6 males and 4 females. They had on average 16 years (SD 9.6) of clinical experience in private practice. Trainees had a mean age of 29 years (SD 1.9) and included 9 males and 1 female.Based on detailed CBCT analysis, the degree of horizontal alveolar bone loss at the level of the furcation was classified as Grade 0 for 19 sites, Grade I for 25 sites, Grade II for 9 sites, Grade III for 7 sites.P > 0.257).Details on all diagnostic parameters of panoramic radiography for assessing FI are shown in Table P = 0.049). There was no significant difference between experienced clinicians and trainees for the other diagnostic parameters (P ≥ 0.082).When recategorizing FI into ‘no to limited FI’ (FI Grade 0 and I) and ‘advanced FI’ (FI Grade II and III), SENS of panoramic radiography amounted to 0.558 (95% CI 0.490– 0.622) and SPEC was 0.791 (95% CI 0.742–0.840). The results on PPV and NPV were 0.511 (95% CI 0.409–0.612) and 0.804 (95% CI 0.742–0.865), respectively. SENS was significantly lower for experienced clinicians than for trainees (P = 0.289).Composite ROC analysis showed an area under the curve of 0.79 for all examiners Fig. . For theP = 0.903). On average, 9/19 (SD 3.4) furcation sites with FI Grade 0 were correctly identified. For FI Grade I, II and III, the proportion of correct assessments amounted to 5/25 (SD 3.6), 1/9 (SD 0.9) and 3/7 (SD 1.6), respectively (Table P > 0.461).Details on all diagnostic parameters of peri-apical radiography for assessing FI are shown in Table P ≥ 0.425).When recategorizing FI into ‘no to limited FI’ (FI Grade 0 and I) and ‘advanced FI’ (FI Grade II and III), SENS of peri-apical radiography was 0.441 (95% CI 0.386–0.497), while SPEC was 0.790 (95% CI 0.768–0.812). The results on PPV and NPV were 0.439 (95% CI 0.413–0.846) and 0.780 (95% CI 0.748–0.835), respectively. There was no significant difference between experienced clinicians and trainees for any of the diagnostic parameters (P = 0.759). Figure Composite ROC analysis showed an area under the curve of 0.69 for all examiners Fig. . For theThe main objective of this study was to compare the accuracy of panoramic and peri-apical radiography with CBCT in detecting FI. Since panoramic and peri-apical radiographs are still taken regularly in daily general dental practice, data on their accuracy is of pivotal relevance . Proper FI is considered a locus minoris resistentiae in periodontal disease progression with high prevalence in periodontitis patients. Svärdström and Wennstrom  studied Measuring clinical attachment level by horizontal probing is a standard tool in clinical periodontology. However, access to furcations is not always easy and furcations may be difficult to predict in architecture, number of walls and extent. An ideal method to identify FI is by reflecting a mucoperiosteal flap. Evidently, this is impossible for ethical reasons when there is no pathology or clear clinical indication for surgery. Intra-surgical registrations have been often used as a gold standard in the diagnosis of periodontal defects indicated for surgery , 26, 28.These findings explains that inter-rater reliability is insufficient to conclude that one radiographic method is superior towards another for finding FI compared with CBCT. On the other hand, all diagnostic parameters need to be evaluated in detail before clinical recommendations can be made. Diagnostic parameters such as SENS, SPEC, PPV and NPV can only be calculated for dichotomous variables. For this purpose, FI Grades were recategorized into ‘no to limited FI’ (FI Grade 0 and I) and ‘advanced FI’ (FI Grade II and III). This recategorization makes sense from a clinical point of view since FI Grade 0 and I require no or limited non-surgical therapy, whereas FI Grade II and III need advanced surgical intervention. Panoramic and peri-apical radiography showed low SENS , yet high SPEC  for identifying advanced FI. These findings imply relatively high false negative ratings and low false positive ratings. In other words, advanced FI is frequently overlooked, but when it is identified on the basis of panoramic radiography or peri-apical radiography, it is most likely present. This can be explained by the fact that panoramic and peri-apical radiographs are two-dimensional images of a three-dimensional anatomy, hence a certain overlap is not unexpected. Even advanced lesions may be masked due to superimposition of bone, roots and restorative materials. On the other hand, peri-apical radiographs score superiorly for image quality  and bone details  , 32. PanThe PPV and NPV results are more difficult to interpret than SENS and SPEC since both are affected by the prevalence of advanced FI within the study sample. In this study, 16/60 furcations were Grade II or III. Only when this proportion resembles the proportion of advanced FI in the population, the data on PPV and NPV are valid. Clearly, the present study was based on a convenient sample of patients seeking implant therapy, which may not adequately represent the population. In addition, substantial regional differences may exist in the prevalence of advanced FI among periodontitis patients.In a retrospective study of Darby et al. , the diaWhen making surgical decisions, it seems particularly attractive to take a three-dimensional radiograph . Indeed,An interesting finding was that diagnostic parameters for panoramic and peri-apical radiography were not affected by the experience of the clinician. Hence, gaining clinical experience does not seem to improve the accuracy of a radiological diagnosis of furcation sites.When interpreting the results of the present study, several limitations should be taken into account. First, this is a retrospective study based on a convenient sample. Second, we used the most common classification on FI because of its simplicity. Furthermore, FI also has a vertical component , which wIn conclusion, panoramic and peri-apical radiography are relevant in the diagnosis of FI given high specificity. However, when clinical examination indicates pathology, diagnostic accuracy may be improved when clinical measurements are combined with radiographic examination. In clinical applications where CBCT images are present for justified reasons, the available CBCT images are more accurate than 2D images to assess furcation involvement. These are preferably combined with furcation probing, which shows high sensitivity. Clinical experience does not seem to improve the accuracy of a radiological diagnosis of furcation sites.


Plasma malondialdehyde (MDA) and carbonyl groups (CG) were assessed as markers of oxidative damage. Muscle strength was also measured. Dynamic torque of knee extensors and flexors as well as maximal handgrip strength was significantly improved after the completion of the training program. Plasma levels of TAS and erythrocyte glutathione reductase (GR) activity were significantly increased. Conversely, MDA and CG levels were significantly reduced. It was concluded RT improved antioxidant defence system and reduced oxidative damage in adults with DS. Further, long-term studies are required to determine whether the increased antioxidant system may improve clinical outcomes of adults with DS.Previous studies have found aerobic training improved oxidative damage in people with Down syndrome (DS). However, there is a lack of information regarding the influence of resistance training on redox imbalance in this population. Accordingly, this study was conducted to determine the effect of resistance training (RT) on antioxidant defence system in sedentary adults with DS. Thirty-six male adults with DS were recruited through different community support groups. Eighteen were randomly assigned to perform a circuit RT program with 6 stations, 3 days/week for 12 weeks. Plasma total antioxidant status (TAS), reduced glutathione (GHS), ascorbate, serum  In the last decades, life expectancy of people with Down's syndrome (DS) has increased considerably, now exceeding 60 years . AccordiIn a recent study, Oppewal et al.  reportedConsequently, regular exercise should be encouraged as key part of care and support for this group. In this respect, previous studies have found that aerobic training programs reduced oxidative damage in DS by increasing antioxidant enzyme activities –9.However, there is a lack of information regarding the influence of resistance training on redox imbalance in people with DS. In this respect, muscle hypotonia has been traditionally considered a major barrier to RT for people with DS . It is aFor the reasons already mentioned, this study was conducted to ascertain the effects of a short-term circuit resistance training on antioxidant defence system and markers of oxidative damage in sedentary adults with DS.v7.12  with an accepted two-sided alpha risk of 0.05 and a beta risk of 0.2. Lastly, a loss to follow-up rate of 10% was also estimated.A total of thirty-six adults with DS (28.1 ± 3.3 years) were recruited for the current interventional study through different community support groups for people with ID. They had an intelligence quotient (IQ) range of 60–69, determined by Stanford-Binet Scale, being diagnosed as having mild ID. Participants were excluded from the study if they meet any of the following criteria: (1) atlantoaxial instability, (2) cardiovascular diseases , (3) metabolic disorders , (4) toxic habits , (5) nutritional supplements, (6) participation in a training program in the 6 months prior to study entry, and (7) not completing at least 90% of the training sessions. The adequacy of sample size was tested using the statistical software Granmo Eighteen were randomly assigned to the experimental group using a concealed method. The control group included 18 age, sex, and BMI matched adults with DS who did not take part in any training program .The intervention consisted of a supervised circuit resistance training, 3 days per week for 12 weeks .This training was circularly performed in 6 stations: arm curl, leg extension, seated row, leg curl, triceps extension, and leg press. Each training session started and finished with a warming-up and cooling-down period of 5-10 minutes during which muscle stretching exercises were performed . FurtherIt should be pointed out that before starting training program, participants included in the intervention group underwent a pretraining session to be familiar with resistance exercises as well as to perform the 8-repetition-maximum (8RM) test per each exercise .To control the potential confounding effect of diet, parents were carefully instructed to avoid quantitative or qualitative differences. Furthermore, they were asked to complete a food consumption frequency questionnaire for three days (2 weekdays and 1 weekend day). Energy and nutrient intake were calculated using a specific software  based on updated Spanish food composition tables .p = 0.41). Furthermore, mean daily vitamin intake showed no significant differences .No significant difference was found between the intervention and control groups when assessing energy intake  was determined spectrophotometrically on a Hitachi 902 Autoanalyzer  by commercial kits . Similarly, reduced glutathione (GSH) level was determined colorimetrically at 412 nm following reaction with DTNB ) according to the instructions of manufacturer.The erythrocytes remaining after the removal of the plasma were washed three times with 310 mM isotonic Tris-HCl buffer (pH 7.4). Hemolysis was carried out by pipetting out the washed erythrocyte suspension into polypropylene centrifuge tubes which contained 20 mM hypotonic Tris-HCl buffer (pH 7.2). Superoxide dismutase  activity was measured by using the xanthine oxidase-cytochrome c method according to McCord and Fridovich's method, with slight modifications . In thisμmol NADP (NADPH) formed min−1 l−1 haemolysate.Glutathione reductase  activity was measured by following the decrease in absorbance due to the oxidation of NADPH as described by Goldberg and Spooner . In thisα-tocopherol was assessed by reversed-phase HPLC, using a C18 column and a photodiode array detector [Finally, plasma ascorbate was analyzed by paired-ion, reversed-phase HPLC as previously described . And serdetector .μmol/l.Similarly, levels of MDA were determined, based on the hydrolysis of lipoperoxides in plasma, using HPLC in reverse phase and quantified at 532 nm as previously described . ResultsPlasma levels of carbonyl groups were performed using an ab126287 Protein Carbonyl Content Assay kit . Absorbance was measured in an ELISA plate reader at 375 nm according to the instructions of manufacturer. Results were expressed as nmol of protein carbonyl group formation per mg of total protein. In this respect, the total protein content of plasma samples was also determined using commercial kits . Lastly, it should be pointed out all samples were analyzed in triplicate.The Jamar handgrip electronic dynamometer  was used to assess maximal handgrip strength of the dominant hand, defined as the one preferred for daily activities. It should be pointed out the one handle position was used as recommended for people with small hand size . The staThe measurements of dynamic torques produced by knee flexors and extensors at an angular velocity of 90°/s were conducted using the motor-driven dynamometer Technogym-REV 9000 . All tests, conducted by the same investigator, were performed in a sitting position with hip flexed at 90°. During trials, restraining belts were placed around the chest and abdomen to stabilize the body and, again, to minimize contractions with other muscles. Furthermore, the lever arm was attached to the midline, and its axis of rotation aligned with the anatomic axis of knee rotation. Prior to every testing, participants performed a standardized warm-up on a stationary bicycle at a comfortable pace followed by light stretching leg exercises. Participants were asked to exert the maximal force over the full range of motion.n = 36) underwent a preliminary session to be familiar with the correct use of both dynamometers [For both, the Jamar and Technogym-Rev 9000 dynamometers, three maximal attempts, separated each one by 90-second  and 20 minute (peak torque) resting periods, were given by each subject. The highest value was considered for further analysis. Verbal encouragement was afforded to ensure maximal efforts. Furthermore, all participants (mometers .It should be pointed out that the current protocol complied with the Declaration of Helsinki (2008). Written informed consent was obtained from all their parents or legal representatives. Further, the current protocol was approved by an Institutional Ethics Committee (Protocol number 29-076/2018).d statistics were used for determining mean effect sizes as follows: small d ≥ 0.2 and <0.5; medium d ≥ 0.5 and <0.8; large d ≥0.8. Mann–Whitney U test was used for significance of difference between intervention and control groups when the data were not normally distributed. Pearson's correlation coefficient (r) was used to determine potential associations among tested parameters. For all tests, statistical significance was set at an alpha level of 0.05.The results were expressed as a mean and standard deviation (SD) or median and interquartile range (IQR). The Shapiro-Wilk test was used to assess whether data were normally distributed. To compare the mean values, a one-way analysis of variance (ANOVA) with post hoc Bonferroni correction to account for multiple tests was used. Lastly, Cohen's p = 0.22) and flexors  were significantly improved in the intervention group. Angles at peak torque value for knee extensors and flexors were 57.8 ± 5.2° and 42.0 ± 4.1°, respectively. Furthermore, maximal handgrip strength was also significantly greater .When compared to baseline results, peak torques at an angular velocity of 90°/s of knee extensors . In a more detailed way, erythrocyte GR activity  and plasma levels of reduced glutathione  were significantly increased after the completion of the resistance circuit training. Conversely, no significant changes were found in plasma ascorbate (p > 0.05) and serum α-tocopherol (p > 0.05).Regarding antioxidants, resistance training significantly increased plasma TAS  and carbonyl groups , were significantly reduced in the intervention group.It was also found that both markers of oxidative damage, MDA  in the intervention group. No changes were found in any assessed outcome in the control group. These results are summarized in Finally, a weak but significant correlation was found between GR activity and maximal handgrip strength  groups in order to increase sample size with the aim of strengthening research design , 37. In In conclusion, resistance training improved antioxidant defence system in male adults with DS by increasing antioxidant enzyme activity. In addition, it reduced plasma levels of oxidative damage to lipids and proteins. Further, long-term follow-up studies are required to determine whether the increased antioxidant system induced by regular resistance training may improve clinical outcomes of adults with DS.


Premature ovarian insufficiency (POI) is an ovarian dysfunction that seriously affects a woman’s physiological health and reproduction. Mesenchymal stem cell (MSC) transplantation offers a promising treatment option for ovarian restoration in rodent POI models. However, the efficacy and mechanism of it remain unclear.POI mice model was generated by cyclophosphamide and busulfan, followed with the treatment of tail-vein injection of the human umbilical cord mesenchymal stem cells (hUCMSCs). Maternal physiological changes and offspring behavior were detected. To reveal the pathogenesis and therapeutic mechanisms of POI, we first compared the metabolite profiles of healthy and POI ovarian tissues using untargeted metabolomics analyses. After stem cell therapy, we then collected the ovaries from control, POI, and hUCMSC-treated POI groups for lipid metabolomics and pseudotargeted metabolomics analysis.Our results revealed remarkable changes of multiple metabolites, especially lipids, in ovarian tissues after POI generation. Following the transplantation of clinical-grade hUCMSCs, POI mice exhibited significant improvements in body weight, sex hormone levels, estrous cycles, and reproductive capacity. Lipid metabolomics and pseudotargeted metabolomics analyses for the ovaries showed that the metabolite levels in the POI group, mainly lipids, glycerophospholipids, steroids, and amino acids changed significantly compared with the controls’, and most of them returned to near-healthy levels after receiving hUCMSC treatment. Meanwhile, we also observed an increase of monosaccharide levels in the ovaries from POI mice and a decrease after stem cell treatment.hUCMSCs restore ovarian function through activating the PI3K pathway by promoting the level of free amino acids, consequently improving lipid metabolism and reducing the concentration of monosaccharides. These findings provide potential targets for the clinical diagnosis and treatment of POI. Premature ovarian insufficiency (POI) is a defect characterized by the cessation of ovarian function before 40 years of age. In the general population, 1–2% of women experience POI  with conMesenchymal stem cell (MSC) transplantation has emerged as an validated and useful treatment option for regenerative medicine in recent years. Based on their differentiation potential and secretion capacity, MSCs have been widely used to treat multiple conditions, such as nerve diseases and cardiac diseases –16. ReceMetabolomics is a bioanalytical strategy that provides information on the metabolite profiles of biological processes. It can be applied to identify metabolites in samples under normal conditions compared with disease-induced altered states and is considered as a powerful phenotyping tool . Early sIn this study, we established a POI mouse model by intraperitoneally injecting cyclophosphamide and busulfan , 28, 29.All animal procedures were conducted in accordance with the guidelines of the Institute of Zoology, Chinese Academy of Sciences, for the care and use of laboratory animals. Six-week-old female ICR mice were used in all experiments described below. After being purchased from SPF (Beijing) Biotechnology Company, the mice were fed a standard pellet diet and given free access to water. Vaginal smears were obtained daily, and only those who showed at least two consecutive normal 4 to 5-day estrous cycles by vaginal smears were included in the experiments.To establish the chemotherapy-induced POI model, 6-week-old female ICR mice were administered 120 mg/kg cyclophosphamide  and 30 mg/kg Busulfan  via intraperitoneal injection.The hUCMSCs were kindly received from the National Stem Cell Resource Bank and cultured in serum-free medium as described in our previous study . All the6 hUCMSCs in a volume of 0.1 ml of 0.1 M DPBS  on day 10  for at least 12 h. After dehydration and paraffin embedding, the samples were serially sectioned at 5 μm and then mounted on every fifth section. Routine hematoxylin  and eosin  (H&E) staining were performed for further histologic examination. Primordial, primary, secondary, and antral follicles were classified and counted. To avoid counting any follicle repeatedly, only those with an oocyte were included for further analysis.GFP-positive hUCMSCs were kindly received from the National Stem Cell Resource Bank. For cell-tracking studies, flow cytometry, animal imaging, and the detection of GFP signals were performed as follows. For flow cytometry, the venous blood was collected from the endocanthion of each mouse at 1, 4, 24, and 48 h after hUCMSC transplantation. After incubation with a whole-blood red blood cell lysis reagent for 30 min at room temperature, the cell suspension was washed with PBS and then applied for flow cytometric analysis. For GFP signal detection, the mice were sacrificed 7 days after hUCMSC transplantation. Ovarian samples were paraffin-embedded and sectioned as described above. The sections were then observed for GFP signals with a fluorescence microscope.The venous blood was collected from the endocanthion when mice were at the diestrous stage. The blood samples were then left at room temperature for 60 min. Upon coagulation, the samples were centrifuged at 4000 rpm/min for 15 min at 4 °C. The supernatant was then collected and sent to the Beijing North Institute of Biological Technology  for serum FSH and E2 measurements.After the vaginal introitus of the mouse was washed several times, physiological saline  was collected and smeared onto glass slides. The slides were fixed with 95% ethanol , washed with distilled water, and then subjected to H&E staining. Cell types and their proportions were quantified under an optical microscope.Three groups of mice were superovulated 10 days after hUCMSC transplantation. All these mice were intraperitoneally injected with 7.5 IU pregnant mare serum gonadotropin , followed by intraperitoneal injection of 7.5 IU human chorionic gonadotropin  48 h later. The oocytes were collected 12 to 16 h after HCG injection.g for 15 min at 4 °C. Each 400 μL of supernatant was transferred into a new tube, lyophilized, and stored at − 20 °C.Ovarian tissues were collected and rapidly washed in cold PBS to remove as much blood as possible. Washed samples were then transferred to QSP tubes and ground for 4 min in 400 μL methanol at 4 °C at 50 Hz. Then, the samples were vortex-mixed and centrifuged at 14,000 Each tube of lyophilized sample was dissolved in 100 μL of 20% methanol solution. Ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) was carried out. All measurements were collected by Xcalibur data acquisition software . One blank sample (20% methanol/aqueous solution) and two quality control (QC) samples  were also added to the plate for analysis.6 ion capacity, and the maximum injection time (IT) was 200 ms. For full MS/dd-MS2 analysis, the parameters of resolution, AGC target, maximum TT, and NCE were 17,500, 1 × 105, 50 ms, and 20 and 40 eV, respectively.Ionization of metabolites was carried out by using an electrospray ionization (ESI) source in both positive and negative modes, and data were collected simultaneously in the two modes. The mass spectrometry parameters were as follows: the mass range (m/z) was from 75 to 1100; the spray voltages were 3.50 and 2.50 kV in the two modes, respectively; the capillary temperature was 320 °C; and the flow rates of the sheath and auxiliary gas were 40 and 10 arbitrary units (Arb), respectively. The resolution was 70,000 in full-scan mode, the automatic gain control (AGC) target was a 3 × 10The same method was used for nontarget metabolomics. Selection of the target ion pair of samples by the method described above and then the model of mass spectrometry multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) were used for analysis, based on the information of these ion pairs, to collect the pseudotargeted data.Ionization of metabolites was carried out by using an ESI source in both positive and negative modes, and data were collected simultaneously in the two modes. The mass spectrometry parameters were as follows: the mass range (m/z) was from 100 to 1100; the spray voltages were 3.80 and 3.00 kV in the two modes, respectively; the capillary temperature was 320 °C; and the flow rates of the sheath and auxiliary gas were 35 and 8 Arb, respectively. The resolution was 70,000 in full-scan mode. For full MS/dd-MS2 analysis, the parameters of resolution and NCE were 17,500 and 20 and 40 eV, respectively.g for 15 min at 4 °C. Each 400 μL of supernatant was transferred into a new tube, lyophilized, and stored at − 20 °C.Ovarian tissues were collected and rapidly washed in cold PBS to remove as much blood as possible. Washed samples were then transferred to QSP tubes and ground for 4 min in 400 μL of methanol at 4 °C at 50 Hz. Then, 1 mL of methyltert-butyl ether (MTBE) was added, the sample was mixed by vortex for 1 min, and the sample was shaken for 1 h at room temperature. Then, 250 μL of water was added, the sample was vortexed for 1 min, and the sample was allowed to stand for 1 h at room temperature. Samples were centrifuged at 14000 The same as we described in nontarget metabolomics.Ionization of metabolites was carried out by using an ESI source in both positive and negative modes, and data were collected simultaneously in the two modes. The mass spectrometry parameters were as follows: the mass range (m/z) was from 100 to 1500; the spray voltages were 3.50 and 3.00 kV in the two modes, respectively; the capillary temperature was 350 °C, and the flow rates of the sheath and auxiliary gas were 45 and 10 Arb, respectively. The resolution was 70,000 in full-scan mode. For full MS/dd-MS2 analysis, the parameters of resolution and NCE were 17,500 and 25, 35, and 45 eV, respectively.P value less than 0.05 was considered to be statistically significant. All data are presented as the mean ± SD.The statistical analyses were performed using SPSS 21.0. One-way ANOVA with LSD tests was used to determine significant differences among the three groups. A We first created a chemically induced POI mouse model by injecting cyclophosphamide and busulfan. To ensure the successful modeling of POI, we assessed several specific physiological indexes 10 days after the treatment of chemotherapeutics  and serum samples  and compared their metabolic profiles using liquid chromatography-mass spectrometry (LC-MS). The total ion chromatogram (TIC) and basic peak ion chromatogram (BPI) revealed that the majority of metabolites were eluted during the first 25 min, indicating that these data qualified for further analyses Figs. SA, S2A. AR2 = 0.70, Q2 = 0.64; serum samples, R2 = 0.84, Q2 = 0.74) , 1190 features were determined. In further qualitative analysis, 180 different metabolites were identified in ovarian samples, among which 99 were increased and 81 were decreased  Figs. SC, S2D. BClinical grade hUCMSCs were established according to the good manufacturing practice (GMP) of medical product standards. The hUCMSCs resembled fibroblasts morphologically; had the ability to differentiate into chondroblasts, lipoblasts, and osteoblasts Fig. a; and exTo evaluate the efficacy of hUCMSC treatment, we systematically evaluated their physiological indices 20 days after transplantation, including body weight, ovarian weight, hormone levels, estrous cycles, ovarian structure, and follicular reserves Fig. a. In theAfter the validation of POI modeling and hUCMSC treatment, we collected ovarian tissues from the control, POI, and hUCMSC-treated POI mice and tried to clarify the mechanisms by which hUCMSCs promoted ovarian restoration.According to previous metabolic analyses, POI was highly associated with abnormal lipid metabolism in ovarian tissues Fig. . Therefol-tryptophan were elevated by chemotherapy and then declined after hUCMSC therapy  based on previous data Fig. . Heat maapy Fig. B-C, E-F.ism Fig. d.Fig. 6We then tried to further summarize the mechanisms by which hUCMSCs promoted ovarian functional recovery in POI mice. According to untargeted lipidomic analysis, the major lipid pathways influenced by POI modeling included glycerophospholipid metabolism, sphingolipid metabolism, the PI3K-MAPK signaling pathway, lipid transport and related pathways , and hormonal regulation (steroid hormone metabolism). After stem cell treatment, many of them, most notably glycerophospholipid metabolism and steroid hormone metabolism, recovered to near-normal levels, Fig. A-B. AddiAs a potential therapeutic agent, the stem cells are limited in widely clinical application because of the security concern. In 2019, Zhao et al. assessed and reported the tumorigenicity of hUCMSCs at various passages. They found hUCMSCs have multiple mutation spectrum but without tumor formation capacity in vivo . In thisTo date, some studies have indicated that MSCS transplantation can promote ovarian functional restoration in POI mice, as indicated by the recovery of body weight, ovarian weight, hormone levels, and ovarian reserve after stem cell therapy , 32–43. As reported by Maidarti and colleagues, the PI3K/Akt pathway is critical for ovarian development, follicular growth, and the proliferation and differentiation of granulosa cells . SphingoIn addition, intravenously injected stem cells may have other influences on the whole body. In the current study, we also compared metabolite changes in serum samples. Although their fold changes were not as notable as those in the ovarian tissues, some of them might also play a role in other important metabolic processes . Further analysis of the serum data may help us unveil more details of how hUCMSCs promote ovarian restoration by regulating metabolism in the whole body.Moreover, there are no previous studies of POI treatment focusing on the health of offspring. Therefore, we examined the behavior of the offspring of the control, POI, and hUCMSC-treated groups. The results of physiological tests demonstrated that the behaviors of the offspring from the hUCMSC-treated group were identical to the normal behaviors of the offspring from the control group, indicating that stem cell therapy has no adverse effect on offspring. We also found that there was no significant difference between the POI group and control group in terms of offspring behavior, which demonstrated that even though chemotherapy can weaken the fertility of the females, it does not influence the viability of their offspring.In summary, mesenchymal stem cell transplantation is effective in treating POI mice. Metabolomics studies manifested that hUCMSCs restore ovarian function through activating the PI3K pathway by promoting the level of free amino acids, consequently improving lipid metabolism and reducing the concentration of monosaccharides. To the best of our knowledge, this is the first study to reveal metabolite diversity among healthy, POI, and clinical grade hUCMSC-treated POI mouse ovaries. Our findings provide us with better knowledge of POI pathogenic mechanisms and may shed light on potential targets for its clinical diagnosis and the possible clinical translation of MSCs.Additional file 1: Supplementary figure 1. Metabolomics-data quality analysis of tissue samples from the control and POI modeling groups. A Total ion chromatograms (TICs) of all ovarian samples , which reflect the overall information of the samples. B Analysis of characteristic differences in quality control (QC) samples by metabolic mass spectrometry. The abscissa represents the relative standard deviation (RSD) of metabolic characteristics in QC samples, and the ordinate represents the percentage of metabolic characteristics whose RSD falls within the corresponding range. C Orthogonal partial least squares discriminatory analysis (OPLS-DA) was used to establish a model of the relationship between the expression of metabolites and samples, and the goodness of fit of the model could be predicted by the values of R2 and Q2. R2=0.70, Q2=0.64.Additional file 2: Supplementary figure 2. Metabolomics analysis of blood samples from the control group and POI model group. A Total ion chromatograms (TICs) of all blood samples, the sum of the total number of ions and their time-varying curves for the various specific charges, which reflect the overall information of the samples. B Heat map analysis of differential metabolome changes induced by chemotherapy in blood samples (control and POI groups). C OPLS-DA score plot of blood samples from both the control and POI groups. D Orthogonal partial least squares discriminatory analysis (OPLS-DA) was used to establish a model of the relationship between the expression of metabolites and blood samples, and the goodness of fit of the model could be predicted by the values of R2 and Q2. R2=0.84, Q2=0.74. E Volcano plot of screened differential metabolites in both groups. Each spot represents a metabolite, and the scattered spots represent the final screening result. Significantly upregulated metabolites are shown in red, significantly downregulated metabolites are shown in blue, and non-significantly different metabolites are shown in black. F KEGG was used to analyze the metabolic pathways in blood samples, and metabolic pathways with P < 0.05 are shown.Additional file 3: Supplementary figure 3. hUCMSCs transplantation promotes ovarian health status. A Line chart of body weight changes in the three groups of mice within 4 weeks after hUCMSCs transplantation . B Photograph of ovaries removed from the three groups after the hUCMSCs treatment. C The weights of the ovaries among the three groups were compared after the hUCMSCs treatment. D Measurement of serum E2 levels after hUCMSCs transplantation (n=4). E Measurement of serum FSH levels after hUCMSCs transplantation (n=4). F Immunohistochemical analysis of AMH expression (red arrowhead) in mouse ovaries. Scale bar = 500 μm. G The line charts are representative of estrous cycles in the three groups detected after the treatment cycle. D: diestrus, M: metestrus, E: estrus, P: proestrus. H The percentage of mice whose estrous cycles returned to normal after the treatment cycle . All data were presented as the mean ± SD. One-way ANOVA with LSD and Tamhane’s T2 post hoc test were used for analysis. *p < 0.05; **p < 0.01; ***p < 0.001.Additional file 4: Supplementary figure 4. hUCMSCs transplantation promotes ovarian functional reserve. A Representative oocytes of three groups after superovulation. Scale bar = 400 μm. B H&E staining of ovarian morphological changes after hUCMSCs transplantation. Scale bar = 400 μm. C Quantitative analysis of different categories of ovarian follicles after hUCMSCs transplantation . All data were presented as the mean ± SD. One-way ANOVA with LSD and Tamhane’s T2 post hoc test were used for analysis. *p < 0.05; **p < 0.01; ***p < 0.001.Additional file 5: Supplementary figure 5. Nontarget metabolomics of lipids from ovarian samples. A Heat map showing that the expression levels of many lipid metabolites changed after POI modeling and that the patterns of the control and POI groups were significantly different. B Z-score plot of screened metabolites in the POI group with significant differences in the mean and standard deviation from that of the control group. Each spot represents a metabolite. The control group is shown in blue, and the POI group is shown in red. Z-scores were sorted in descending order, in which the magnitude of the change indicates the fold by which the mean of the control group is higher or lower than its standard deviation. C Volcano plot of the screened differential lipid metabolites measured in the control and POI groups. Each spot represents a metabolite, and the scattered spots represent the final screening result. Significantly upregulated metabolites are shown in red, significantly downregulated metabolites are shown in blue, and nonsignificant different metabolites are shown in black. D Heat map showing that the expression levels of a large number of metabolites from the POI+hUCMSCs group changed significantly compared with those from the POI group after transplantation. E Z-score plot of the metabolites screened in the samples of the POI+hUCMSCs group, with significant differences in the mean and standard deviation from those of the POI group. F Volcano plots of screened lipid metabolites measured in the POI and POI+hUCMSCs groups, with significant changes.Additional file 6: Supplementary figure 6. Pseudotargeted metabolomics of ovarian tissue samples. A Heat map results show that the expression levels of a large number of metabolites changed significantly after POI induction, and most decreased. B, C Z-score plot and volcano plot show that the expression levels of the metabolites unrelated to lipids were significantly changed after POI induction, and most decreased. D Heatmap, E z-score plot and F volcano plot of comparison between the POI+hUCMSCs group and POI group show that the expression of most target metabolites was restored, manifesting as their levels being upregulated.Additional file 7: Supplementary figure 7. Metabolomics analysis of the mechanisms of hUCMSCs treatment for POI. A Map of metabolic pathways of glycerolipid. B Pathway of cholesterol synthesis and steroid hormone biosynthesis. Significantly upregulated metabolites are shown in red, significantly downregulated metabolites are shown in blue, and nonsignificant different metabolites are shown in black. The names in bold are the relevant metabolites detected in the nontarget/target metabolomics analysis. The substance in the red ellipse represents the enzyme or its corresponding protein. The solid arrow indicates that metabolites (enzymes/proteins and metabolites) have a direct relationship with each other, while the dotted arrow indicates a distant relationship. Tissues or parts of metabolic processes (such as lysosomes) are represented by solid frames, and related pathways (such as glycerolipid pathways) are represented by dotted frames. The underlined metabolites represent the metabolites that recovered after hUCMSCs transplantation compared to the changes observed after POI induction. The green arrows represent the paths recovered.Additional file 8: Supplementary table 1. Differential metabolomic changes induced by chemotherapy in ovarian tissue samples. (XLS 108 kb)Additional file 9: Supplementary table 2. Differential metabolites screened by volcano map in serum samples after chemotherapy. (XLS 21 kb)Additional file 10: Supplementary table 3. Differential lipid metabolomic changes in ovarian tissue samples of 3 groups . (XLS 314 kb)Additional file 11: Supplementary table 4. Pseudotargeted metabolic differences of nonlipid metabolites in ovarian tissue samples of 3 groups . (XLS 292 kb)


Anthonomus santacruzi Hustache, 1924, weevils. The aims of this study were to assess if the variation in body size in A. santacruzi weevils is a result of sexual dimorphism and what features can be used to distinguish males from females. The weevils were collected from field sites in Mpumalanga, South Africa, where they were introduced as biocontrol agents of Solanum mauritianum Scopoli. Body structures and the presence/absence of the tergal notch was examined under an optical stereomicroscope and SEM to assess differences between sexes. The morphometric analysis of the body structures included rostrum length , elytral length and width, pronotum length and width, first tarsus length, first tibia length, funiculus length and total body length. Rostrum length, elytra length and width and total body length were significantly larger in females than in males. A tergal notch in the 8th abdominal tergite was present in males and absent in females. The body structure; of rostrum length, elytra length and width and total body length overlapped between sexes in some specimens. The abdominal tergal notch was found to be the most useful body structure to distinguish males from females in A. santacruzi.There is evident variation in body size amongst The online version of this article (10.1007/s13744-020-00795-6) contains supplementary material, which is available to authorized users. Anthonomus Germar, 1817 (Coleoptera: Curculionidae) is a weevil genus containing more than 500 species, distributed predominantly in the Neotropic (over 400 species) and in the Palearctic (73 species) regions, but also in Indomalaya (9 species), the Afrotropics (3 species) and in Australasia (2 species) , which are regarded as serious pests   rainfall regions of South Africa, negatively impacting agricultural lands, plantation forestry, riverine habitats and conservation areas  Fig  is nativ Olckers . The hab & Burke , Olckers & Burke  and Mkhi & Burke , while te  in November 2017 by A. Sasa and were immediately fixed in 75% ethanol.A total of 97 weevils were collected (by hand) from randomly selected A. santacruzi we assessed some of the features studied by Smreczyński , on A. pomorum by Duan et al  total length of the rostrum (from base to apex); (2) metarostrum (using the distance between the base of the rostrum and the insertion of the scape); (3) the length of the pronotum ; (4) width of pronotum (measured at the widest part); (5) length of elytra ; (6) width of elytra (measured in the widest place); (7) length of the first tarsus ; (8) length of the first tibia; and (9) length of the funicle, were examined and measured on all 97 weevil bodies under an optical stereomicroscope (Olympus SZ11). Measurements were made by using a calibrated ocular micrometre at × 60 magnification.We also examined selected non-morphometric characters such as the sculpturing on the rostrum and pronotum, shape and sculpturing of the elytra and shape of abdominal sternites to check their usefulness in sex determination.A. grandis was also observed, as well as the differences of the 7th and 8th abdominal segments recorded as being sex related in A. pomorum , differences in the 4th and 5th abdominal segments, tergal notch, antenna and olfactory sensillae), were taken with an Olympus BX63 microscope and processed by Olympus cellSens Dimension software. The specimens selected for pictures using SEM (scanning electron microscopy) were at first dried in absolute ethyl alcohol (99.8%), rinsed in acetone and then gold-plated. A TESCAN Vega 3 SEM was used for the examination of selected structures. All these specimens were deposited in the collection of the Department of Zoology and Nature Protection, Maria Curie–Skłodowska University .n = 32) than male body length  in all specimens (n = 97) (t(95) = 4.911, P < 0.001)  = 2.382, P = 0.022) and wider (1.111 mm) (t(95) = 2.903, P = 0.005) in females than in males (length = 1.422 mm and width = 1.043) (Table Elytra were both significantly longer mean = 1.511 mm) (.511 mm 3) Table  and were Table  a3) Table .Fig 2Antt(95) = 7.416, P < 0.001) and length of metarostrum (t(95) = 7.943, P < 0.001)  Table .Fig 5TheThe 4th and 5th abdominal segments were clearly separated in males Figs , whilst Anthonomus santacruzi males have eight visible dorsal abdominal segments, while females have seven visible segments. The notch, on the posterior edge of the 8th tergite was distinct in males  (length: t(95) = 0.431, P = 0.668, width: t(95) = 1.077, P = 0.284)  it appears to be slightly curved, being “pear-shaped”, whereas in the males it is straight, being almost parallel. Pronotum length and width Fig , showed 4) Table .Fig 10LeOn females , the eyes do not stand out from the dorsal outline of the head, whereas in males  the eyes protrude slightly from the outline of the head Figs .t(95) = 1.089, P = 0.279), first tarsus length (t(95) = 0.444, P = 0.658) and first tibia length (t(95) = 0.202, P = 0.841) showed there were no significant differences between A. santacruzi sexes  reported distinct similarities between males and females of Anthonomini as well as rather less visible differences, e.g., relative length of rostrum , the insertion of the scape  and the sculpture of rostrum .A. santacruzi males and females, as there is a broad overlap in these characters between the sexes. However, these disparities can be used for initial separation of sexes , length of metarostrum (antenna–apex), elytral length and width and total body length were significantly different between the sexes, they cannot be used to completely distinguish Anthonomus species in the last century, but regarded as subjective because the characters were deemed subtle, variable and relative rather than absolute  were successfully separated from all females (n = 32) using this notch. This supports Sappington & Spurgeon  . Further, the presence of an elongated depression on the abdominal sternites of males was noted on Hylobius radicis  by Wilson  by Öhrn et al  by Hassan  to two (n = 65) males) in the sampled weevils used in this study might be a result of protandry . The biased sex ratio has been found to be promoted by climate alterations in the timing of rainfall and/or extreme temperatures  (Coleoptera: Curculionidae)


It is important to assess the temporal reproducibility of circulating cytokines for their utility in epidemiological studies. However, existing evidence is limited and inconsistent, especially for the elderly population.Sixty-five elderly (mean age = 77.89 ± 6.14 years) subjects were randomly selected from an existing prospective cohort study. Levels of 41 cytokines in 195 serum samples, collected at three separate visits that were up to 15.26 years apart, were measured by the Luminex technology. The temporal reproducibility of cytokines was estimated by the intraclass correlation coefficient (ICC) calculated using a mixed-effects model. In addition, data analyses were stratified by the median (4.49 years) of time intervals across sample collection. Sensitivity analyses were performed when excluding subjects with undetectable samples.A total of 23 cytokines were detectable in more than 60% of samples. Fair to good (ICC = 0.40 to 0.75) and excellent (ICC > 0.75) reproducibility was found in 10  and 5  cytokines, respectively. The results were not changed dramatically in the stratification and sensitivity analyses.Serum levels of the selected 15 cytokines measured with Luminex technology displayed fair to excellent within-person temporal reproducibility among elderly population. Cytokines are secreted polypeptides or glycoproteins that regulate proliferation, apoptosis, and angiogenesis, and involved in the development and progression of various diseases . CytokinCirculating inflammatory molecules are widely used in epidemiological studies to investigate their association with chronic diseases –10. In sSeveral studies have explored the intra-individual temporal reproducibility of cytokines in humans  subjects, with 3 serum samples each. Results from this study will be useful to select appropriate cytokines as inflammatory biomarkers for elderly population in the future epidemiological studies.The Washington Heights-Inwood Community Aging Project (WHICAP) is an ongoing, prospective, population-based cohort study which is conducted to identify risk factors and biomarkers for aging and dementia. The sampling strategies, recruitment and examination methodology of WHICAP have been described previously , 25. ParFor the current study, we selected a total of 65 participants from the second and third waves of WHICAP who met the following criteria: (a) ≥ 3 blood samples donated at different visits, (b) large number of aliquots left, (c) without history of diagnosed dementia, and (d) complete information on ages at blood draw, sex, race/ethnicity and years of education. To ensure individuals with certain demographic characteristics to be included in this sample, we randomly selected subjects from each of the 24 strata, defined by sex , race/ethnicity , waves (1999 vs. 2009), and age groups (< 80 vs. ≥80 years). For each selected individual, we selected one serum sample from the baseline, the most recent, and a middle visit. With three repeated samples each for a total of 65 subjects, we had 80% power to detect acceptable ICC of ≥0.55 with narrow 95% confidence interval (CI) (< ±0.13), or to detect acceptable ICC of ≥0.40 with narrow 95% CI (< ±0.15).Peripheral venous blood samples were collected from the participants at the time of their health examination, and were stored at − 80 °C. Never-thawed serum samples were packed in dry-ice and sent to the laboratory for analysis. A panel of 41 inflammatory biomarkers were selected according to the results of previous literatures  were assigned a value of LLOD divided by the square root of two . ObservaDue to the skewed distribution, cytokine levels were natural logarithm (log)-transformed for approximately normal distribution. The characteristics of subjects were presented as mean [standard deviance (SD)] and number [proportion (%)] for continuous and categorical variables, respectively. The repeated measures correlation (95% CI) between different cytokines were estimated with the R package “rmcorr” which accounts for non-independence among measurements from the same subject .The intraclass correlation coefficient (ICC) was employed to quantify the temporal reproducibility of cytokines. The mixed-effects model, fitted by the R package “lme4” (version 1.1.21) , was est2) categories  at each visit, or by additionally adjusting for the time of day (ante meridiem or post meridiem) of blood sample collection. Sensitivity analyses were also conducted by the stratification of sex.To examine the impact of time intervals on the cytokine reproducibility, data analyses were performed by the stratification of median of time intervals between the 1st and 3rd measures. Besides, we also repeated analyses by excluding samples with undetected values (< LLOD or > ULOD), or by additionally adjusting for the baseline comorbidities including hypertension, diabetes and cardiovascular diseases, or by additionally adjusting for the body mass index .Participant characteristics are presented in Table p < 0.05)  of cytokines measured in all samples collected at three separate visits was ranked and presented in Fig. Stratification analyses were additionally conducted, dividing by the median (4.49 years) of time intervals between the 1st and 3rd blood samples Fig. . CompareAfter excluding the subjects with undetectable levels of cytokines, for the cytokines with detectable rates less than 100% Table , the resIn the present study of 65 elderly subjects from WHICAP study, we assessed the temporal reproducibility of serum cytokines in samples collected at three separate visits over a relatively long period of time. The results revealed that 15 out of 41 cytokines measured by the Luminex technology were detectable in over 60% of serum samples and had good reproducibility (ICC > 0.40), indicating that a single measurement of these biomarkers well captures the within-individual average level over a long period of time.By definition, ICC measures the inter-person variability relative to the total variability . The higThe temporal reproducibility of circulating cytokines measured by multiplex immunoassays or ELISA has been reported previously. Our findings are consistent with previous studies, supporting good to excellent ICC for IL-17 , G-CSF , 14, 17,The circulating levels of cytokines are reported to be influenced by various factors including demographics , life styles , medical treatment and circadian physiological variability , 34–36. n = 28) [An assessment of the long-term reproducibility of biomarkers is essential to explore the associations between biomarkers and diseases with long etiological windows, such as the neurodegenerative disease, cardiometabolic disease, or cancer. Majority of the previous studies evaluating the reproducibility of cytokines had used samples spanning a shorter period of time, such as 14 days , severaln = 28)  or restrn = 28) .With increasing time intervals, the temporal reproducibility of G-CSF kept good to excellent over 14 days (ICC = 0.50) , 7 monthIn the sensitivity analyses without undetectable values, we found similar, although slightly reduced, temporal reproducibility for most cytokines except for MCP-3 of which the ICC increased  and similar reproducibility of cytokines between older males and females in this study, which might be due to the depletion of sex steroid hormones in elderly subjects. Although it has been reported that BMI is significantly correlated with levels of cytokines in cerebrospinal fluid [Results from previous studies demonstrated sex differences in levels of some cytokines among young adults (median age = 22 years) , and in al fluid , and thaal fluid , we founThere were several advantages in this study. Few studies have examined the elderly population previously. Our study was performed among subjects with a mean age of 77.89 years, which provided useful information for the studies focusing on the health effects of cytokines among elderly individuals. In addition, a large panel of cytokines  were analyzed in the present study, while fewer (no more than 20) cytokines were investigated in some previous studies , 21, 37.Some limitations should be noted. Although this study represent one of the largest study in terms of number of cytokines measured, a total of 41 cytokines may still not give a comprehensive insight into the overall inflammatory profile, which may involve more cytokines , 45. BesIn conclusion, the present study demonstrated a good temporal reproducibility of 15 serum cytokines  which were measured by the multiplex technology. This suggests that a single measurement of the selected cytokines is likely to be suitable for characterizing the immune and inflammation status over a long period in the prospective epidemiological studies, especially for the elderly population.Additional file 1:Table S1. Assessments on the temporal reproducibility of circulating cytokines in previous studies. Fig. S1. Paired repeated measures correlation among different cytokines. Fig. S2. ICC (95% CI) of cytokines by excluding the subjects with undetectable values. Fig. S3. ICC (95% CI) of cytokines when additionally adjusting for baseline comorbidities. Fig. S4. ICC (95% CI) of cytokines when additionally adjusting for BMI categories. Fig. S5. ICC (95% CI) of cytokines when additionally adjusting for time of day of blood sample collection. Fig. S6. ICC (95% CI) of cytokines stratified by sex.


Design modifications in prostheses may cause alterations in gait kinematics, thus influencing functional restoration of knees after total knee arthroplasty (TKA). The aim of the study was to investigate the differences in gait kinematics and clinical outcomes after single radius (SR) versus multiple radius (MR) TKA.The present retrospective study included 38 unilateral TKA involving 20 knees using MR design implant and 18 knees using SR design implant. Thirty-six healthy volunteers were also recruited. The mean follow-up time was 16 ± 3 months. At the end of follow-up, the 6 degrees of freedom (DOF) kinematics of knees and range of motion (ROM) were measured with a portable optical tracking system. Knee society score (KSS) and knee injury, and osteoarthritis outcome score (KOOS) were also collected.P = 0.012; 50.0 ± 20.8, P = 0.027, respectively). Significant differences were detected between MR knees and SR knees , and MR knees and healthy knees  in adduction/abduction ROM. The proximal/distal translation was significantly smaller in MR knees (0.58 ± 0.54 cm) compared with SR knees  or healthy knees . SR knees (0.24 ± 0.40 cm) had smaller translation compared with the MR group  and control group . No significant difference was detected in the other DOFs during the gait cycle. Significant difference was detected in extension/flexion, internal/external rotation, adduction/abduction, proximal/distal and medial/lateral among MR, SR and healthy knees.Patients in the SR group had significantly higher scores in activities of daily living (84.7 ± 15.9) and sports and recreation (67.5 ± 25.2) KOOS sub-score than MR group (69.9 ± 17.6, After TKA, patients have altered gait kinematics compared with the control group. MR and SR design showed varied characteristics in 6 DOF gait kinematics, which could be the cause of the difference in functional outcome. The number of total knee arthroplasty (TKA) is expected to grow exponentially in the coming years, because this procedure is effective in relieving pain and improve function in patients with end-stage osteoarthritis of the knee –3. NonetIn the classical knee kinematics, there exist at least two transient rotating centers in the knee within the functional knee range of motion. When the femoral condyle moves downward and backward during flexion, the radius of the condylar curvature diminishes . Thus, mClinical studies and meta-analysis that compared the SR and MR femoral design based on the clinical scores revealed contradictory results , 10–15. This retrospective, comparative study design was approved by the Institutional Review Board of our hospital. We obtained signed informed consent for participation from all study patients.2; (4) American Society of Anesthesiologists (ASA) class 1 or 2; (5) follow-up period longer than 1 year. Exclusion criteria were as follows: (1) inflammatory arthritis, including rheumatoid arthritis, suppurative arthritis or gouty arthritis; (2) revision TKA; (3) previous tibial or femoral osteotomy; (4) flexion contracture or extension deficit more than 10°; (5) varus or valgus malalignment more than 10°; or (6) any other lower extremity disease, including tumor, infection, etc..Inclusion criteria were as follows: (1) primary symptomatic osteoarthritis of the knee, patient with asymptomatic but degenerative contralateral knee was also included; (2) 55 to 85 years old; (3) body mass index (BMI) lower than 35 kg/mKnee Society Score (KSS) and Knee Injury, and Osteoarthritis Outcome Score (KOOS) were collected to assess the subjective knee function. In order to compare gait kinematics between TKA knees and normal knees, we also recruited 36 healthy people. These volunteers met the following criteria: 1) no symptomatic osteoarthritis of the knee; (2) no lower extremity deformity; (3) no other lower extremity disease, including tumor, infection, injury or history of surgery. A portable optical marker-based motion analysis system  was utilized to measure the 6 DOF kinematics of the SR TKA knees, MR TKA knees and healthy knees during treadmill gait  performed the surgery. After general or spinal anesthesia and a standard pro-operative antibiotic prophylaxis, all TKAs were performed under tourniquet. The knee was exposed with a straight ventral skin incision and then a straight medial parapatellar capsular approach. The patella was not resurfaced. We applied posterior cruciate ligament (PCL)-substituting design for all cases. Measured resection technique was manually performed to achieve appropriate component alignment and patella tracking. A slight tibia posterior slope was set at 3°. For the SR group, the Stryker Triathlon TKA system  was used, and PFC sigma  was used in the MR group. On the second day, patients in both groups were encouraged to mobilize including full-weight bearing. Other post-operative exercises included continuous passive motion, active of passive knee extension.Gait kinematic data was collected following a previously published protocol , 19. TwoP < 0.05. The least-significant-difference (LSD) test was performed between groups when significant differences were detected. These statistical analyses were done using SPSS 26.0 . A post-hoc power calculation was determined by the statistical power analyses G Power 3.1 to eliminate type II error.The data was described as Means and standard deviations. Kolmogorov-Smirnov test was used for testing normality. Chi-square test and Fisher’s exact test were used for categorical variables. The range of motion (ROM) in 6 DOF, KSS, KOOS were analyzed by 1-way analysis of variance (ANOVA), and the level of statistical significance was set at n = 20; SR group, n = 18). The mean follow-up time was 16 ± 3 months. MR group and SR group had comparable demographic characteristics including sex, age, height, weight, body mass index, surgical side and follow-up time. The data of the two groups were considered homogeneous . There were no significant differences between the two groups in other clinical outcome categories  and sports / recreation 67.5 ± 25.2) KOOS sub-score post-operatively compared with MR group  compared with healthy knees  than SR knees. Similarly, MR knees rotated more externally during the response phase (0 ~ 8%) and terminal swing phase (90 ~ 100%) compared with normal knees. However, during the mid-swing phase 76 ~ 84%), MR knees exhibited an internally rotating trend, while SR knees rotated in an opposite direction. SR knees showed less external rotation than healthy knees during 68 ~ 80% and less internal rotation 94 ~ 100% of gait cycle , and MR knees and healthy knees . In the gait curve, MR knees presented more abducted mainly in stance phase than SR (8 ~ 52%) and normal (8 ~ 60%) knees  and SR group , no significant difference was detected. Moreover, no significant difference was found with regard to the gait curve  compared with the MR group , and MR knees and healthy knees . In the gait curve, MR knees showed significantly less stretched than SR (78 ~ 84%) and normal knees (78 ~ 88%) in the mid-swing phase . When looking in the gait curve, the significant difference presented between TKA knees and healthy knees. For SR knees, they were significantly more medial shifted during either mid-stance phase (32 ~ 40%) and mid-swing phase (72 ~ 84%) in comparison with healthy knees. MR knees showed significantly more medial shifted during 32 ~ 48 and 80% of the gait cycle than normal knees, while during the early swing phase (62 ~ 68%) phase, MR knees were found more lateral translated than normal (Fig. According to current literature, quadriceps strength and range of motion during gait decreased in patients undergoing TKAs, but other kinematic parameters such as axial rotation and anterior-posterior translation during gait were seldom analyzed because of limitations in traditional gait analysis techniques , 20–22. The movements in the sagittal plane, the predominant motion during gait cycle, have been studied previously, and the flexion angles after PCL substituting TKAs during gait were decreased compared with the flexion angle of the normal , 15. NonKinematic alterations were also identified in other DOFs in TKA knees. In the transverse plane, SR knees showed an external rotation trend within flexion. This result was in accordance with the results reported by Tamaki , 25. TheWith respect to the coronal plane, we found that SR knees adducted while flexion, which was close to the mode of healthy knees, but MR knees showed an opposite movement. We suspected that this phenomenon probably related to the changing radius of the femoral prostheses might change the tension on the collateral ligaments and other soft tissues at different points in the range of motion , 29. So,Overall, TKA knees did not manifest a laxity more than 5 mm during active movement. We inferred that the cam-post design of tibial insert constrained the anterior translation. In the stance phase, the tibia moved posteriorly relative to femur. In the swing phase, the tibial component of SR knees tended to move posteriorly relative to femur by gravity because the tibial resection was tilted posteriorly . HoweverAs far as proximal/distal and medial/lateral translation in TKA knees, there were no such reports in the current literature. In our research, the proximal/distal translation may reflect the change of the joint gap. The compressive loading of the knee during stance phase reduced proximal/distal translation, which was consistent in two designs. In the swing phase, the joint was stretched by gravity, so the translation increased, but it was not so prominent in MR knees. This may indicate a compensation strategy that a stronger co-contraction of muscles to stabilize in the presence of mid-flexion subluxation in MR while the curvature radius changes. The range of medial/lateral translation of SR knees is less fluctuated. We considered this as another implication of the stability and isometry of collateral ligaments. It is noticed that the range of both of MR and SR groups was smaller than the control group, and it might be attributed to the lower compliance of the soft tissue around the joint after TKA.In the current study, the difference of biomechanics parameters was in line with the difference in patient-reported outcome measures. As was discussed previously, SR knees showed better stability in the DOF of internal/external rotation, valgus/varus rotation and proximal/distal translation when comparing with MR knees. Therefore, SR knees showed higher ability in performing more exquisite activities, such as jumping, twisting and jogging. We inferred that this may be the reason for the difference in activities of daily living and sports / recreation KOOS sub-score. Similarly, Cook  comparedThis study had some limitations. First, we only included the posterior stabilized and fixed-bearing knee designs. Other designs, such as in cruciate retaining or mobilized platform ones, may mitigate the effect of radius change. Khasian  reportedAfter TKA, patients have altered gait kinematics compared with the control group. MR knees showed less adduction/abduction ROM and proximal/distal translation, while SR knees had smaller medial/lateral translation. With regard to the gait curve, both TKA knees showed more extension during stance phase. MR knees presented a varied rotating mode opposed to normal knees, and they also showed more medial/lateral displacement. SR knees partly restored the mode in internal/external rotation and proximal/distal translation when compared with normal knees, but they showed more abducted during the stance phase and more medial translated in mid-stance and mid-swing phase. These deviated characteristics in gait kinematics may be the reason for the difference in functional outcome.