Hydrogel combination products displayed important efficacy-related characteristics  with significant (p < 0.05) protective effects of the cellular extracts in oxidative environments. Overall, the present study sets forth robust control methodologies  for stabilized cell-free progenitor tenocyte extracts. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development  allowed for the conservation of critical biological extract attributes.Cultured primary progenitor tenocytes in lyophilized form were previously shown to possess intrinsic antioxidant properties and hyaluronan-based hydrogel viscosity-modulating effects in vitro. The aim of this study was to prepare and functionally characterize several stabilized (lyophilized) cell-free progenitor tenocyte extracts for inclusion in cytotherapy-inspired complex injectable preparations. Fractionation and sterilization methods were included in specific biotechnological manufacturing workflows of such extracts. Comparative and functional-oriented characterizations of the various extracts were performed using several orthogonal descriptive, colorimetric, rheological, mechanical, and proteomic readouts. Specifically, an optimal sugar-based (saccharose/dextran) excipient formula was retained to produce sterilizable cytotherapeutic derivatives with appropriate functions. It was shown that extracts containing soluble cell-derived fractions possessed conserved and significant antioxidant properties (TEAC) compared to the freshly harvested cellular starting materials. Progenitor tenocyte extracts submitted to sub-micron filtration (0.22 µm) and  Cultured primary progenitor tenocytes  were studied  and previously proposed as candidates for tendon tissue bioengineering or alternative homologous cytotherapeutic management of tendinopathies ,2,3,4. DHyaluronic acid (HA) has been extensively studied and therapeutically applied for tendinous tissue affections, such as tennis elbow or rotator cuff injuries . NaturalWhile implementation of the latter is often impossible in classical cytotherapy, in order to preserve cell integrity and viability, alternative technical processing measures  may be implemented in the production processes of cell-derived exosomes, for example ,23. An oPrevious preclinical research on the cytotherapeutic use of viable primary progenitor tenocytes in HA-based hydrogels has shown encouraging results from technical, safety, and function viewpoints ,2,3. SubAlthough some key functional parameters were studied at that time from a manufacturing and translational point of view, aspects of paramount importance such as the influence of sterilization processing on the stability and function of the considered progenitor tenocyte extracts were not previously addressed . Consequ60Co gamma irradiation. The main null hypothesis was, therefore, that sterilizing processing does not significantly affect the considered samples with regard to function-, stability-, or efficacy-related parameters or attributes. Appropriate experiments were performed on the stabilized cellular extracts and the reconstituted HA-based hydrogel combination products. A secondary hypothesis was also tested thereby, namely, the possibility to obtain similar functions with cell-free extracts compared to the original whole cells. Importantly, whether the sensitive phases of cytotherapeutic derivative manufacturing process development  allowed for the conservation of critical functional extract attributes was investigated. Overall, the present study sets forth robust control methodologies  for stabilized, sterilizable, and functional cell-free progenitor tenocyte extract preparation.The presented experimental work was carried out to test the hypothesis that functional parameters of the considered progenitor tenocyte extracts could be partly conserved following submicron filtration and/or w/w ; DMEM cell culture medium ; Penicillin-streptomycin ; Millipore Stericup and Millex GS filter-sterilizing membranes with 0.22 µm pores ; nested 2R tubular glass lyophilization vials and bulk 6R clear glass vials ; laminated lyophilization stoppers ; and lyophilization bags .The reagents and consumables that were purchased and used in this study are summarized hereafter, along with the corresponding manufacturers: purified water, PBS buffer, and NaCl 0.9% solutions ; laboratory grade sodium hyaluronate of 1.2–1.5 MDa and 2.2–2.4 MDa molecular weight ; Ostenil Tendon ; Teosyal RHA2 ; Restylane Skinbooster ; European pharmacopoeia (Ph. Eur.)-grade saccharose ; Ph. Eur.-grade dextran 40,000 ; D(+)-glucose, D(+)-mannose, and bovine serum albumin ; mannitol, saccharose, lactose, and sorbitol ; D(+)-galactose, D(+)-mannose, and D(–)-fructose ; xilitol ; D(+)-trehalose ; Total Antioxidant Capacity (TEAC) Assay Kits, Ferric Reducing Antioxidant Power (FRAP) Assay Kits, 2-hydroxyethyl cellulose, and hydrogen peroxide at 30% Samples were lyophilized in a Telstar LyoBeta Mini freeze-dryer . For sample analysis, flat bottom 96-well microtitration plates and Eppendorf tubes were purchased from Greiner, Frickenhausen, Germany. Luer-Lok syringes were purchased from BD, Franklin Lakes, NJ, USA and from Schott, Mainz, Germany. Needles for injection with 27G gauge were provided by Needle Concept, Biarritz, France. Component weighing was performed on a laboratory scale . Reconstituted samples were processed on a Countess 3 automated cell counter . Reconstituted sample pH was determined using a SevenCompact Cond meter S230 . Reconstituted sample osmolality was determined using an OsmoTECH XT . Sample centrifugation was performed on a Rotina 420R centrifuge  for manufacturing purposes or on a Legend Micro 21R centrifuge  for sample preparation purposes. For preliminary sample proteomic analysis, a BCA assay kit, NuPAGE Bis-Tris 4–12% protein gel, MOPS buffer, loading buffer, DTT and antioxidant, page ruler protein ladders, and Coomassie staining reagents were purchased from Thermo Fisher Scientific, Waltham, MA, USA. SDS-Page analyses were performed using a Mini Gel Tank and PowerEase 90W . Gel imaging was performed on a Uvitec Mini HD9 gel imager . Colorimetric measurements were all performed on a Synergy Mx microplate reader . Rheological measurements were performed on a HAAKE Mars Rheometer . Sample incubation at 37 °C under mechanical agitation was performed on a TS-100 Thermo Shaker . Karl Fisher residual humidity measurements were performed on a Coulometric KF Titrator Compact C30SD . Injectability measurements, friction force measurements, and adhesivity assays were performed on a Texture Analyzer TA.XT. Plus instrument .Primary progenitor tenocytes  were procured and produced under the Swiss progenitor cell transplantation program and were made available for the present study in dry cell pellet form. The pellets were stored at −80 °C until use, as previously described elsewhere . All stam/v and dextran 40,000 at 2% m/v in a buffered aqueous solvent. The alternative excipient formulas  were not used further in the study. The retained lyopreservation solution was subsequently used to reconstitute, freeze, and lyophilize the four types of cellular bulks or cell-derived extract bulks presented hereafter. In order to establish an optimal cryoprotective and lyoprotective formula for the various cellular extracts described herein, a preliminary formulation study was conducted. Therefore, various sugar-based excipient combinations  were prepared, frozen, and lyophilized using the same technical specifications described previously . Based o7 cellular equivalents/mL in a PBS buffer or in lyopreservation solution  before being processed for thermal cell disruption . The resulting cell lysates  were either stored at −80 °C until further use or processed further to obtain additional cell fractions. In detail, parts of the cell lysates were fractionated by centrifugation at 290× g for 20 min at ambient temperature before the resulting supernatants were isolated and filtered on 0.22 µm porous membranes to form the “soluble cell fraction bulk”. The remaining pellets at this stage were isolated to form the “cell membrane bulk”.In order to obtain the various fractionated cellular bulks from the cellular starting materials, portions of the pooled dry pellets were resuspended at 106 cell equivalents for the standard dose. Additionally, high doses were prepared using incremental unitary quantities of whole cell bulk ranging from 1.5 × 106 to 7.5 × 106 cell equivalents per vial. Lyophilization processing and post-lyophilization controls were performed using the same technical specifications described previously, with some adaptations [The various cellular extract bulks were dispensed in lyophilization vials directly after preparation. The final filling volume before lyophilization was of 0.75 mL in each 2R vial. Corresponding “placebo” formulations were prepared and contained no cell-derived biological constituents. The final quantity of cell-derived biological materials in each vial was of 1.5 × 10ptations . Notably60Co gamma rays and a standard irradiation dose of 30 ± 5 kGy. Additionally, several lyophilizate samples were submitted to alternative irradiation doses of 10 ± 5 kGy and 50 ± 5 kGy. It should be noted that the term “terminal sterilization” is used for sample irradiation at doses of 30 ± 5 kGy but that a validation of the corresponding γ-irradiation cycle was not performed following the ISO 11137 norm for sterilization process validation in the context of this study.Terminal sterilization was performed on the appropriate lyophilizate groups by an independent contractor . The samples were packed in polymeric primary containers  that were packed in a cardboard box  for sterilization processing. Sample irradiation was performed at ambient temperature using Descriptive controls were performed by two-operator visual assessment of the produced lyophilizates post-drying following a standardized grading workflow to determine lyophilization processing efficacy and sample stability, as described previously . Residuag at ambient temperature for 3 min and the clear supernatant was transferred to 96-well microtitration plates . Volumes of 100 µL of the reaction mix were added to each well and the plates were incubated at ambient temperature for 10 min. Absorbance values were determined at a wavelength of 570 nm and the TEAC values were calculated based on an experimental Trolox standard curve. All assays were performed using six experimental replicates and the results were presented in absolute values of Trolox equivalents per lyophilizate unit .The Trolox equivalent antioxidant capacity (TEAC) of the various lyophilized samples was determined using a colorimetric Total Antioxidant Capacity Assay Kit, following the instructions of the manufacturer. Briefly, each sample vial was resuspended in 300 µL or 500 µL of purified water and homogenized manually. The samples were then centrifuged at 5400× g at ambient temperature for 3 min and the clear supernatant was transferred to 96-well microtitration plates . Volumes of 190 µL of the reaction mix were added to each well and the plates were incubated at 37 °C for 1 h. Absorbance values were determined at a wavelength of 594 nm and the FRAP values were calculated based on an experimental ferrous standard curve. All assays were performed using six experimental replicates and the results were presented in absolute values of mM ferrous equivalents per lyophilizate unit .The ferric reducing antioxidant power (FRAP) of the various lyophilized samples was determined using a colorimetric FRAP Assay Kit, following the instructions of the manufacturer. Briefly, each sample vial was resuspended in 300 µL of purified water and homogenized manually. The samples were then centrifuged at 5,400× 2O2-challenged combination products were then quantitatively determined using a Peltier cone-plate C35 2°/Ti rotor at a set temperature of 37 °C, a constant system oscillatory frequency of 1 Hz, and a shear stress of 3 N/m2. The complex viscosity (η*), storage modulus (G’), and loss modulus (G”) values of the samples were recorded  over four minutes and three experimental replicates were used for all the assays. In another setup, the samples were not incubated following the hydrogen peroxide challenge but were analyzed immediately using the same rheological technical specifications over a measurement period of 10 min. In another setup, the HA-based hydrogels contained various doses of BSA instead of the lyophilized cell-derived extracts, to quantitatively replace the biological samples based on total protein-determined amounts. Measurements were performed on the same day as lyophilized sample reconstitution in the linear HA-based hydrogels or regularly over the four weeks following sample reconstitution and combination with the HA-based hydrogel.In order to characterize the hyaluronan-based hydrogel indirect viscosity-modulating function of the various lyophilizates, several versions of an adapted hydrogen peroxide challenge assay were used. Briefly, the basic experimental setup comprised a standard volume of 400 µL of linear HA-based hydrogel in a 1.5 mL Eppendorf tube, with the addition of 100 µL of test item  and 100 µL of challenge item . Control samples were systematically prepared with PBS instead of hydrogen peroxide as the challenge item or with PBS instead of the test item. In most cases, the oxidative challenge was performed with incubation of the samples at 37 °C in the dark for 1 h under 1.5 Hz horizontal agitation. The endpoint rheological behaviors of the H7 cell equivalents/mL. The samples were then centrifuged at 13,000× g at ambient temperature for 5 min. The isolated supernatants were then frozen at −20 °C in low protein-binding tubes and were shipped on dry ice for proteomic analysis. Total protein contents and protein migration profiles in the samples before and after the centrifugation step were determined using a colorimetric BCA assay kit and an SDS-Page setup, following the manufacturer’s protocols and related technical specifications. Briefly, electrophoretic analysis was performed by mixing the sample supernatants with reducing and loading buffers, heating the samples at 95 °C for 5 min, and loading the samples onto a Bis-Tris 4–12% gel. The electrophoresis was performed using 140V before the gel was stained with Coomassie solution for a period of 30 min, followed by destaining and imaging.Soluble protein and growth factor contents were quantitatively determined in the considered lyophilizates  following analytical processing by an external contractor  using specific multiplex analyses. The analyses (Discovery Assay) comprised the human angiogenesis array and growth factor 17-plex array, the human cytokine/chemokine 71-plex panel, the human soluble cytokine receptor 14-plex array, and the human MMP and TIMP panel for cell cultures. Briefly, suspensions were prepared using frozen bulk cell lysate, lyophilized samples, and γ-irradiated lyophilized samples at a final concentration of 10v/v fetal bovine serum. The various test items and the appropriate controls were incorporated into the cell culture medium and were incubated in contact with the cells for 24 h and 72 h in a humidified cell culture incubator set at 37 °C and 5% CO2. At the end of the incubation period, the metabolic activity of the target cells was determined using a WST-1 cell proliferation kit , following the instructions of the manufacturer.For the assessment of in vitro sample cytotoxicity in a cell-based assay, adult primary tenocytes  were procured and cultured as described elsewhere . Conflue−1. Maximal applied pressures of 100 N were used as stopping points for the injectability assay.The injection force profiles of various commercially available hydrogels containing lyophilized whole-cell extracts were determined using syringes and needles adapted for clinical product administration in the management of tendinopathies. In order to exclude the potential buildup of aggregates in the syringes with homogenous sample volumes of 1 mL, the samples  were automatically extruded into air by a Texture Analyzer TA.XT. Plus instrument  set at a constant piston speed of 0.5 mm·s2) was composed of steel  and was connected to the instrument piston with a non-extensible nylon cord, resting on a pulley to form the 90° angle between the base plate plane and the piston axis. Before each measurement, the base plate surface and the sliding bloc surface were wiped clean using PBS  and acetone . Then, a volume of 400 µL of hydrogel sample was placed on the base plate. Hydrogel samples were prepared by reconstituting the contents of one lyophilizate vial with 1.5 mL of HA of 2.2–2.4 MDa MW at 1% m/v in a water-PBS solution. Control measurements were performed without any lubrication of the system and with 400 µL of PBS instead of the hydrogel samples. The sliding bloc was placed on top of the hydrogel samples and allowed to settle for 20 seconds, with the cord adjusted and without initial tension. The piston was then raised at a constant speed of 0.1 mm·s−1. A trigger force value of 0.05 N was set, and the measurements were performed over 50 s once the force threshold had been attained. The force profiles were recorded in triplicate for each sample during piston-bloc travel. For the determination of mean kinetic friction forces, values between the 10-second and 30-second timepoints of travel were considered for analysis.In order to assess the impact of cellular extract incorporation  on the friction modulation properties of the considered samples, a classical in vitro coefficient of friction setup was used. A sliding friction rig was mounted on a Texture Analyzer TA.XT. Plus instrument . The base plate was composed of stainless steel and was secured horizontally, forming a 90° angle with the instrument piston axis. The rectangular sliding bloc  of 0.5 N was then applied for 30 s. The mucoadhesion probe was then raised at a constant detachment speed of 2 mm·s−1. The detachment force profile  was recorded in triplicate for each hydrogel sample. A second run of measurements was then performed as described hereabove using H2O2-challenged samples to assess the bioadhesive properties of the samples in an oxidative environment. For the H2O2 challenge assays, the hydrogel samples were prepared as described hereabove and were challenged with 30% w/w hydrogen peroxide  for 15 min at 37 °C under mechanical agitation before analysis.In order to confirm that the considered combination products in hydrogel form were able to adhere to tendinous tissue, a bioadhesivity evaluation assay was used with ex vivo equine tendon tissue. For the needs of the study, equine whole tendons were procured . The tissue had been harvested, mechanically cleaned and conditioned in plastic bags, and frozen at −80 °C until further use. Following thawing, tendon samples were placed and secured in a vice designed for gel mucoadhesion evaluation on a Texture Analyzer TA.XT. Plus instrument . Tissues were placed horizontally in order to display a plane surface of the tendon sheath for analysis. Before the measurements were performed, the exposed tissue surface was gently rinsed with PBS . Hydrogel samples were prepared by reconstituting the contents of one lyophilizate vial with 1.5 mL of HA of 2.2–2.4 MDa MW at 1% p value < 0.05 was retained as a general base for the statistical significance determination. The statistical calculations and/or data presentation were performed using Microsoft Excel, Microsoft PowerPoint , and GraphPad Prism v. 8.0.2 .For the statistical comparison of average values from two datasets, an unpaired Stu- dent’s t-test was applied after the appropriate evaluation of the normal distribution of the data. For the statistical comparison of values from multiple quantitative datasets from experiments where multiple variables applied, a one-way ANOVA test or a two-way repeated measures ANOVA test  was performed and was followed  by a posthoc Tukey’s multiple comparison test. A Several graphical elements  are provided as 6 cell equivalents/vial  were prepared and were parallelly lyophilized for stabilization . However2 values of 0.97 and 0.96 for unfiltered and filtered samples, respectively. Furthermore, the TEAC measurement results for the γ-irradiated versions of the same LYO-WC samples indicated that the dose-dependent relationship was conserved after γ-irradiation processing at 31 kGy, with R2 values of 0.99 and 0.91 for unfiltered and filtered samples, respectively  were determined with or without γ-irradiation of the samples and with or without a 0.22 µm filtration step during sample preparation  when using a FRAP assay setup, where the experimental Rwas 0.98 . In addiwas 0.98 A,B. Therwas 0.98 A,B. 2+-based antioxidant assay kit , indicating that the γ-irradiation processing resulted in the creation of species reacting with the components of the CuA separate experiment aimed to compare the TEAC of freshly harvested progenitor tenocytes  to the TEAC of whole cell lyophilizates  of the same cell type. The results indicated that fresh cells possess significant antioxidant activity, which is not significantly impacted by 0.22 µm filtration . FurtherIn order to determine whether a functional dose-response existed between the cellular extract dose and the viscosity modulation capacities of the extracts, rheological measurements were performed. The hydrogel viscosity modulation capacity of the various doses of whole cell extract samples was assessed in a hydrogen peroxide challenge assay, before and after sample γ-irradiation , with H2O2-challenged samples presenting an important and positive effect amplitude , but was established for the H2O2-challenged groups , a linear and dose-dependent relationship was outlined for complex viscosity η* modulation by the whole cell extracts. Indeed, the samples containing non-irradiated cellular extracts produced linearly increasing complex viscosity values with both PBS  and Hmplitude C. As regp value < 0.0001 between cell concentration extremes) increase in complex viscosity of the unchallenged samples  may be partly attributed to the increasing number of particles in suspension, the higher amplitude of complex viscosity increase in the H2O2-challenged groups is indicative that the underlying mechanism is partly dependent on the presence of the oxidative challenge item itself , it was shown that HA hydrogel degradation occurred rapidly  when no lyophilizates were added to the hydrogel samples  was on average of 20.8 ± 7.0 mg. Subsequently, the experimental TEAC values  for the stabilized whole cell extracts were found to be about three orders of decimal magnitude lower in value (6 cells) was capable of effectively protecting the considered HA-based hydrogel system against strong oxidative challenges  put the interpretation of pure antioxidant capacity into perspective. Indeed, while intrinsic functional parameters/attributes of the stabilized cellular extracts are of paramount importance for the functional controls thereof , the functional parameters of the combined system are the most significant from a prototype developmental perspective. Overall consideration of the obtained TEAC values for the progenitor tenocyte stabilized extracts indicated a relatively low intrinsic antioxidant potency compared to, for example, vitamin C or the vitamin E derivative Trolox. Indeed, gravimetric measurements performed on the cellular starting materials  indicated that the mass of 1.5 × 10in value A. HoweveOverall consideration of the results presented in A comparative assessment of the antioxidant activity of the various considered cellular extracts  was performed to determine which cellular fraction exerted the most effects A. In a F2O2-challenged samples containing active biological materials  compared to, for example, the membrane fraction  did not adversely impact the considered hydrogel viscosity modulation effects compared to the non-irradiated samples B–D. Specon doses B–D. Otheadiation . As alreadiation . This coOverall consideration of the experimental results presented in 2O2-challenge, it is possible to conclude on the partial direct contribution thereof in the form of protection of the system against the oxidative challenge.Of note, the intrinsic antioxidant capacity of a considered cellular extract plays an important role in the observed hydrogel viscosity modulation function of the same extract in a hydrogel submitted to oxidative challenge . AlthougVarious types of stability studies  were performed on the cellular extracts of interest and on the reconstituted hydrogel combination products . The resFurthermore, it was determined that the presence of air at atmospheric pressure in the lyophilization vials at the time of sample γ-irradiation did not significantly impact the measured TEAC values of the considered samples compared to the same samples that contained a moderate vacuum at the time of γ-irradiation .2O2 challenge assays was maintained or enhanced for at least four weeks  and at all investigated timepoints , but not on the molecular weight of the HA polymer composing the hydrogel  produced on average more complex viscosity augmentation effects than extreme challenge item concentrations  occurs and favors the interactions between the biological constituents of the cellular extracts and the HA polymeric structures, which, in turn, results in increased complex viscosity values of the system. The presented experimental results indicate that an optimal H2O2 dose exists for each given system, as complex viscosity values increase along with the oxidative challenge item dose up to a maximal point  slightly increased along with increases in H2O2 concentrations , or by using various amounts of test item , by using various concentrations of the oxidative challenge item  comprised 98 detectable targets . Among those 98 targets, 23% to 34% of the considered analytes displayed a significant modification  in detection levels . Therefore, it was established that the majority of the considered analytes did not display significant modifications in detection levels . Among the analytes displaying important modifications in detection levels, several were excluded as they displayed relatively low detected concentrations  or because discrepancies were noted between the experimental repetitions. Therefore, 14 analytes were included in the final analysis, with recorded increases or decreases in detection levels after lyophilization and gamma irradiation, respectively .Further analysis of the protein migration profiles of the various samples in SDS-Page electrophoresis indicated that at γ-irradiation doses of 25 and 50 kGy, a shift in the protein weight distribution profile occurred compared to the non-irradiated or lightly γ-irradiated  sample groups . SpecifiFor assessment of the considered cellular extracts from a translational point of view, both the cytotoxicity and the injectability of the samples were studied . As rega2O2 and 1 h incubation at 37 °C) was used as a preliminary characterization experiment. The results indicate that the extracts exerted protective effects on all the samples containing HA polymers; yet, a net increase in complex viscosity values after the H2O2 oxidative challenge was not observed in the case of Ostenil Tendon  and resulted in higher mean force of adhesion values compared to H2O2-challenged HA and challenged samples containing the placebo formulation, respectively  at the injection site in order to deploy lasting local mechanical effects. The results of the bioadhesion assays performed on the sheath of ex vivo tendon tissue indicated that mean peak detachment force  and mean work of adhesion values were conserved or improved with the incorporation of non-irradiated and gamma-irradiated cellular extracts, respectively, compared to HA A,B. The ectively C,D. The extracts . OverallAs tendon affections represent important morbidity and complex therapeutic challenges, arrays of complementary approaches have been clinically investigated, often comprising a medical device or cell therapy product of some sort ,24,25,26The results presented herein have notably outlined that, when focusing on antioxidant and rheology modifying properties or functions of progenitor tenocyte derivatives, neither cell viability nor the presence of structurally integral cells was required. Specifically, it was shown that the TEAC of progenitor tenocytes was conserved between viable cells and their lyophilized equivalents and that 0.22 µm filtration allowed for the conservation of the majority of the detected antioxidant effects . Further2O2, but that said reaction was directly dependent on the presence of the biological material of interest . Indeed, the measurement of antioxidant activity or indirect viscosity/adhesivity modulation functions constitute functional parameters of prime choice when considering therapeutic agents for tendinopathies . In detail, the robustness of the retained methods was confirmed in several assays , and this aspect should be underlined, especially when working with inherently variable biological starting materials . A compaStarting with the selection of a cell-based starting material for therapeutic preparation design, functional assays may be of high value and efficiency for screening or comparative assessment of various cell types. Then, control assays must be tailored to each manufacturing process with appropriate specifications and acceptance criteria, which may be challenging to define when working with cellular active substances or cellular derivatives. Specifically, normalization of starting material quantities may be performed at the cellular level  or at protein or nucleic acid levels to obtain uniform quantities within the manufacturing process. However, in order to specify the quantitative targets for such controls, insights must first be gained on the dose-response relationship existing for the considered cells or cellular derivatives and the desired effect or function. Indeed, it is the latter that must be standardized and, in many instances, a desired level of biological function may not be reliably attained with a specific cell amount or a specific protein titer due to biological material variability. Therefore, it may be considered useful to implement several functional control assays within the manufacture of biological derivatives, oriented toward the desired function thereof, for sparing use of control resources and enhanced levels of quality assurance.The first technically challenging step in obtaining biological derivatives characterized by optimal stability and simplicity of use had already been performed successfully by two-phase lyophilization of progenitor tenocyte derivatives . Such woThe results presented herein have outlined that the considered cellular extracts of interest were able to withstand two sorts of processing methods commonly used for sterilization, namely, sub-micrometer filtration  and gamma irradiation . Indeed, the maintenance of the dose-dependent relationships between the observed TEAC or hydrogel viscosity modulation function under oxidative challenge has confirmed the conserved nature of both functions, albeit reduced in amplitude after γ-irradiation . FurtherWhile the focus of this study did not encompass the advanced qualitative investigation of the effects of processing on the considered samples, the most relevant aspects were covered. In addition to physical and functional parameters of the cellular extracts  and of the combination products , semi-quantitative assessments of protein sample levels along the processing workflow have yielded interesting results . SpecifiAs regards published research on the influence of gamma irradiation on proteins, several sample-dependent mechanisms have been outlined to explain variations in detected levels, such as protein modification, denaturation, degradation, or solubility modification ,41,42,43As regards the state of the sample during gamma irradiation  and sample conditioning , it has been established that all formulation means of reducing the formation of reactive oxygen species (ROS) are beneficial from a quality point of view ,48,53,54Within the use of γ-irradiation processing in the food industry, specific studies have been conducted to outline the link between gamma irradiation and the antioxidant properties and functions of various foodstuffs ,58,59,60Taken as a whole, the existing body of knowledge available regarding gamma irradiation of complex biological samples , the effects of gamma irradiation on such samples, and the existing use of such γ-irradiated samples in human food chains or medical applications  enhances the technical interest in the described process for stabilized progenitor tenocyte terminal sterilization. Specifically, reports on the neutral or positive influence of gamma irradiation on the antioxidant properties and effects of various biological samples are in line with the results presented herein  58,59,6,659,60. From a mechanistic point of view and without pursuing extensive radiolysis product characterization in the γ-irradiated progenitor tenocyte extracts, several mechanisms may be considered to explain the variations in detected protein quantities. While protein upregulation is excluded during lyophilization and gamma irradiation processing , it is probable that the detected increases in protein levels in the γ-irradiated samples are a result of analyte dissociation from the extracellular matrix (ECM) or membrane proteins . In partSpecifically, functional-oriented assays and characterization of the interactions between cellular extracts and clinically usable hydrogels contributed to augment the overall translational relevance of the presented work. From an in vitro safety point of view, it was demonstrated that non-irradiated cellular extracts developed no observable cytotoxicity over 72 h of contact with viable tenocytes . Further2O2 oxidative challenge and syringeability assessment, all HA-based considered combinations have proved to present improved stability and easy injection , the retained experimental setup was assessed as optimal for the comparative evaluation of ex vivo bioadhesion parameters for the various forms of hydrogel combination product samples ,73,74,75Overall, and, based on the known mechanisms of HA-based therapies for tendon affections , it may be assessed that efficacy-related parameters of the considered hydrogel combination products are conserved or enhanced with the inclusion of the stabilized cellular extracts of interest . Finally, it may be assessed that therapeutic hydrogel combination product cohesive properties represent additional attributes of interest for optimization in view of augmenting in situ residence times  and may, therefore, be the object of future characterization experiments  .60Co gamma irradiation process as “sterilizing” at a certain dose and for a specified product payload. This aspect was not yet investigated due to the scale of manufacturing batches, which did not reach the 103-vial batch size at the time of this study. However, the inclusion of an irradiation condition using 50 kGy  was performed to partly remedy this aspect and predict the behavior of the samples at doses > 30 ± 5 kGy, should those be necessary to validate sterilization on the considered materials. Furthermore, the retained proteomic analytical method  and related sample preparation workflow focused on the soluble fraction of the considered stabilized cellular extracts, potentially providing a partial overview of the protein contents along the manufacturing workflow, as previously discussed. However, the selection of an alternative proteomic method taking into account the non-soluble fraction of the samples is not warranted in the case where the development of a cell-free extract is a priority, as the insoluble fraction of the bulk biological materials is excluded by the successive processing steps, as designed. The main study limitations in terms of processing comprise the absence of a full sterilization cycle validation phase to be able to refer to the As regards the evaluation of hydrogel combination product efficacy-related parameters, multiple additional in vitro/ex vivo setups and specifications may be used to better understand the friction modulation and bioadhesivity properties of the samples of interest. Notably, surfaces other than metal may be used to better approximate the in vivo environment of sliding tendinous and adjacent tissues, and alternative traveling speeds may be used to explore a wide dynamic range . However, due to the high variability encountered when using biological tissue samples in mechanical setups, lengthy optimization phases must be planned before optimal conditions and specifications may be obtained.Classical experimental workflows aiming to study HA-based product functionality or effects in vivo during animal preclinical studies comprise numerous limitations. Specifically, only post-sacrifice endpoint readouts are usually used  . Such stBased on the original data presented herein, several future areas of focus have been identified, such as the further qualitative and functional investigation of cell-free preparations , the technical investigation into the effects of alternative terminal sterilization methods  on the considered samples, and further preclinical assessments of stabilized cellular extract safety and effects . In particular, further study of the efficacy of the considered combination products containing linear HA and stabilized cellular extracts may tangibly be considered within a human clinical trial, as most outcomes of interest in tendinopathy management are clinically observed or patient-reported. Therefore, provided that sufficient safety data are further accumulated for the considered cytotherapy-inspired hydrogel combination products, initial clinical trials may be warranted, as no manufacturing, technical, or product formulation critical problems have been identified to date.60Co gamma irradiation (5–50 kGy). Of note, a specific dose-dependent functional relationship was outlined for the stabilized biological materials of interest , and this relationship was partly conserved following gamma sterilization. In addition to the selected proteomic investigation of the respective impacts of lyophilization and gamma irradiation on the complex biological samples, key technical aspects of translational relevance were assessed, such as the syringeability of the cellular extracts in commercially available hydrogels. Furthermore, the considered hydrogel combination products displayed important efficacy-related characteristics , with significant protective effects of the cellular extracts in strongly oxidative environments. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development  allowed for the conservation of critical progenitor tenocyte extract functional attributes. Overall, the work presented herein extends the technical foundations of the cytotherapy-inspired rationale for easy-to-use, cell-free, and localized therapeutic management of tendinopathies.The aim of this study was to characterize various forms of stabilized and sterilized cell-free progenitor tenocyte extracts, for inclusion in cytotherapy-inspired complex injectable preparations as hydrogel functionalization agents. The experimental results enabled confirmation that functional parameters  of the considered extracts could be conserved or partly conserved following submicron filtration (0.22 µm) or terminal sterilization by


To analyse the clinical efficacy of biofeedback electrical stimulation combined with doxycycline in the treatment of type IIIA chronic prostatitis. Qmax) and mean urinary flow rate (Qave) were measured before and after treatment in both groups, and the National Institutes of Health chronic prostatitis symptom index (NIH-CPSI) was used to score the urinary symptom, pain or discomfort, and quality of life and determine the efficacy of the treatment in both groups.  Eighty patients who met the diagnostic criteria of type IIIA chronic prostatitis in our hospital between February 2020 and February 2022 were selected and equally divided into the drug group and electrical stimulation group according to the random number table method. The drug group was treated with medication alone for 4 weeks; the electrostimulation group was treated with biofeedback electrostimulation on top of medication for 12 weeks. The expressed prostatic secretious (EPS) routine  and the maximum urinary flow rate (P < 0.05). After treatment, the Qmax and Qave were significantly higher in both groups than before, and both the electrical stimulation groups were higher than the drug group (P < 0.05). After treatment, the urinary symptom scores, pain or discomfort scores, quality of life scores, and total NIH-CPSI scores were significantly lower in both groups than before, and all were lower in the electrical stimulation group than in the drug group (P < 0.05). After treatment, the overall efficiency of patients in the electrical stimulation group was significantly higher than that of the drug group (P < 0.05).  After treatment, the number of lecithin bodies and white blood cells in EPS improved significantly in both groups compared to before, and both the electrical stimulation group was better than the drug group ( Biofeedback electrical stimulation combined with doxycycline in the treatment of type IIIA chronic prostatitis can synergistically improve the patient's inflammation level, urinary dysfunction, relieve pelvic floor tension myalgia, and improve their quality of life, opening up new avenues for the rehabilitation of patients with type IIIA chronic prostatitis. Chronic prostatitis is a chronic inflammation of the prostate caused by specific or nonspecific infection and is a common clinical genitourinary disorder in men, of which type IIIA is the most common . StudiesAt present, there is no specific treatment drug for type III prostatitis, and the clinical treatment of this disease mainly focuses on anti-infection, anti-inflammatory and pain relief, relieving urination symptoms, and symptomatic treatment . Because2, and mean BMI (21.57 ± 1.78) kg/m2; Patients in the electrical stimulation group were aged 22 to 49 years, mean age (32.00 ± 5.71) years, duration of illness 4 to 14 months, mean duration of illness (8.25 ± 2.05) months, BMI 18∼25 kg/m2, and mean BMI (21.64 ± 1.84) kg/m2. The differences in general clinical data such as age duration of illness and BMI of the two groups were not statistically significant (P > 0.05) and were comparable.Eighty patients who met the diagnostic criteria for type IIIA chronic prostatitis in our hospital between February 2020 and February 2022 were selected and equally divided into a drug group and an electrical stimulation group according to the random number table method. Patients in the drug group were aged 20 to 49 years, with mean age (32.45 ± 6.69) years, duration of illness 3 to 14 months, mean duration of illness (8.08 ± 2.04) months, BMI 19∼25 kg/mIn line with the 2014 edition of the Chinese handbook of diagnostic and therapeutic guidelines for urological diseases  and the Inclusion criteria were as follows: (1) meet the diagnostic criteria for type IIIA chronic prostatitis; (2) duration of disease ≥ 3 months; (3) age between 18 and 50 years, male; (4) not using other drugs or treatments for prostatitis in the previous 2 weeks; (5) informed consent and voluntary participation; (6) those who did not take their medication regularly or withdrew on their own during treatment; (7) those who required discontinuation of the drug during treatment caused by gastrointestinal reactions or allergies.Exclusion criteria were as follows: (1) those with other prostate diseases such as benign prostatic hyperplasia and prostate cancer in combination; (2)those with nonprostatic conditions that can cause pain in the pelvic region, such as inguinal hernia, ureteral stones, bladder stones, and bladder tumours; (3) persons with mental illness or serious systemic diseases such as cardiovascular, cerebrovascular, liver or kidney diseases; (4) those with allergies or hypersensitivity to the drugs tested in this test; (5) previous history of pelvic-related surgery.μs, and stimulation intensity of stimulation without pain. The patient was asked to contract the anus for 5 s, relax for 20–30 s, and then repeat for 30 min each time. During the treatment, the patient was asked to note the changes in electromyography and abdominal pressure curve so that the abdominal pressure curve did not rise when the patient contracted the anus. Treatment was given once every other day, for 4 weeks as a course of treatment, with a total of 12 weeks of treatment. Both groups were observed for efficacy at the end of the 4th week of treatment and relevant indicators were measured and evaluated.The drug group used 2 tablets (200 mg) of doxycycline hyclate tablets  orally once a day for each dose; supplemented with tamsulosin hydrochloride sustained-release capsules  1 capsule (0.2 mg) taken orally once a day, for a total of 4 weeks of treatment. The electrical stimulation group was treated with a UROSTYMTM biofeedback electrical stimulation device in addition to medication. The method was: the patient was placed in the supine position, an anal plug electrode was placed to record electromyography and a rectal manometry tube was used to record abdominal pressure. The treatment parameters were: current 25–50 mA, frequency 50–100 Hz, wave width 200–500 Qmax) and mean urinary flow rate (Qave) were measured before and after treatment in both groups. The NIH-CPSI was used to score the urinary symptom, pain or discomfort, and quality of life and determine the efficacy of the treatment in both groups. The total NIH-CPSI score ranged from 0 to 43, with a total score of 0 to 10 for urinary symptoms, 0 to 21 for pain or discomfort, and 0 to 12 for quality of life, with higher scores indicating more severe symptoms. Efficacy determination: Healed: > 90% reduction in total NIH-CPSI score after treatment; Significantly valid: 60–89% reduction in total NIH-CPSI score after treatment; Valid: 30–59% reduction in total NIH-CPSI score after treatment; Invalid: <30% reduction in total NIH-CPSI score after treatment [The EPS routine  and the maximum urinary flow rate   .P < 0.05)  .Qmax and Qave were significantly higher in both groups compared to before, and both the electrical stimulation groups were higher than the drug group, the difference had a statistical significance (P < 0.05)  .P < 0.05)  .P < 0.05)  .Currently, the reported prevalence of chronic prostatitis ranges from approximately 6.0 to 32.9% in China, 9.0% in the United States, and 2.0 to 10.0% worldwide . AlthougThere are different theories on the aetiology and pathophysiology of chronic prostatitis, including occult infection, inflammation/autoimmunity, pelvic floor muscle dysfunction, voiding dysfunction, intraprostatic urinary reflux and elevated intraprostatic pressure, neuropsychological factors, adrenal axis abnormalities, genetic predisposition and oxidative stress –15. TypeIn recent years, researchers have begun to more accurately diagnose prostatitis-like symptoms as pelvic floor muscle dysfunction, usually associated with pain, spasm, and pressure in the pelvic muscles. Pelvic floor spasms may lead to voiding dysfunction and pain, which in turn can increase pressure and make the condition worse . Pelvic Research  shows thIn this study, we used biofeedback electrical stimulation combined with doxycycline to treat type IIIA chronic prostatitis. The results showed that both treatments with drugs and combined treatment with biofeedback electrical stimulation significantly improved lecithin bodies and white blood cells in the patients' EPS, and the improvement was greater in the electrical stimulation group, with a significant difference compared to the drug group. This suggests that the use of antibiotics combined with biofeedback electrostimulation can synergistically improve the level of inflammation in patients with type IIIA chronic prostatitis and facilitate their recovery. Antibiotics  have long been the first-line drugs used by many physicians to treat chronic prostatitis . In factQmax and Qave were significantly higher in the electrical stimulation group than in the drug group after treatment; the urinary symptom scores, pain or discomfort scores, quality of life scores, and total NIH-CPSI scores were significantly lower in the electrical stimulation group than in the drug group after treatment; and the overall effective rate was significantly higher in the electrical stimulation group than in the drug group. As seen above, the clinical efficacy of biofeedback electrical stimulation in combination with doxycycline in the treatment of type IIIA chronic prostatitis is significant compared to the use of medication alone, which is consistent with the report in the literature [The results also showed that the terature . In addiIn summary, biofeedback electrostimulation combined with doxycycline in the treatment of type IIIA chronic prostatitis can synergistically improve the patient's inflammation level, urinary dysfunction, relieve pelvic floor tension myalgia and improve their quality of life, opening up new avenues for the rehabilitation of patients with type IIIA chronic prostatitis.


Guillain–Barré syndrome (GBS) is the most common severe acute paralytic neuropathy, with a mortality rate of 5% and permanent sequelae rate of 10%. Currently, the cause of GBS remains unclear. Therefore, we sought to determine potential predictors for GBS and its severity.A case–control study was performed at Tiantan Hospital in Beijing from January 2017 to December 2021. Laboratory and clinical characteristics were assessed in recruited GBS patients and healthy control individuals (matched by sex and age). The potential risk factors for GBS and severe GBS were assessed using a logistic regression analysis. The mRNA levels of toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2) and nuclear factor κB (NF-κB) in GBS patients and control PBMCs were detected by fluorescence quantitative PCR. THP-1 cells were costimulated with LPS and free cholesterol to demonstrate the effect of free cholesterol on monocyte activation.A total of 147 GBS patients and 153 healthy individuals were included in the study. Logistic regression analyses showed that preceding infection, alcohol consumption, remnant cholesterol, homocysteine and the dyslipidemia index were correlated with a higher risk of GBS. In contrast, increased HDL cholesterol was correlated with a lower risk of GBS. Moreover, remnant cholesterol and the dyslipidemia index were significantly correlated with severe GBS. The mRNA levels of TLR4, TLR2 and NF-κB in the PBMCs of GBS patients were significantly higher than those of healthy individuals. LPS activated THP-1 cells, and free cholesterol treatment increased the expression of TLR4, TLR2, NF-κB and IL-1β mRNA in LPS-activated THP-1 cells.Dyslipidemia was correlated with the risk of GBS and severe GBS. Remnant cholesterol may promote the activation of monocytes in GBS patients. It may be valuable to control lipid levels in the prevention of GBS and severe GBS. Guillain–Barré syndrome (GBS) is an autoimmune demyelinating disease in the peripheral nervous system (PNS). The pathological features are infiltration of inflammatory cells into small blood vessels and demyelination of peripheral nerves and nerve roots . The incThere have been many studies on the clinical epidemiology of GBS, but its cause is not completely clear. A proteomics study of the cerebrospinal fluid of GBS patients showed that the differentially expressed proteins between the GBS and control groups were mainly enriched in pathways closely related to lipid metabolism . It is iThese results are valuable for basic and clinical studies of GBS but must be validated in as many studies as possible. We hypothesized that common lipid levels are associated with GBS. In this study, we performed an untargeted analysis in a case–control study to explore possible risk and protective factors associated with GBS. Remnant cholesterol and dyslipidemia were identified as potential risk factors for the onset and severity of GBS for the first time. In addition, we validated the effect of free cholesterol on monocyte activation at the cellular level.All patients with GBS were prospectively consecutively recruited at Beijing Tiantan Hospital, Capital Medical University from January 1, 2017, to December 31, 2021. The protocol was approved by the Ethics Committee of Beijing Tiantan Hospital (batch number: KY-2022-039-01). The data supporting the findings of this study can be obtained from the corresponding author upon reasonable request.GBS was diagnosed based on clinical symptoms, electrophysiological characteristics and cerebrospinal fluid characteristics, according to the guidelines published in 2014 ; albuminAfter resting in a seated position for 15 minutes, diastolic and systolic blood pressure in the right arm were measured with a standard mercury manometer. Peripheral blood samples were collected in the morning after fasting for 12 h. Body mass index (BMI) was calculated as weight (kg)/height (m2). Peripheral venous blood was used to measure the white blood cell count (WBC), lymphocyte count, monocyte count, albumin (ALB), immunoglobulin G (IgG), triglyceride, total cholesterol (CHO), HDL cholesterol, low density lipoprotein (LDL) cholesterol, APOA1, apolipoprotein B (APOB), and homocysteine (Hcy). Remnant cholesterol was calculated by subtracting HDL cholesterol and LDL cholesterol from CHO . DyslipiCerebrospinal fluid (CSF) samples were obtained by lumbar puncture. Lumbar puncture and peripheral blood collection were performed when the patient was in the acute phase and not receiving treatment. CSF samples were used to measure ALB, IgG, intrathecal immunoglobulin G synthesis rate of 24 h  and total protein.The GBS disability score indicated the severity of disease. The GBS disability score at entry and at discharge were used to assess the functional status of patients with GBS , 13. TheThe clinical features of GBS patients at admission included sex, age, preceding infection, symptoms of preceding infection, hypertension, smoking, alcohol consumption, diabetes, surgery, trauma, heart disease and hyperlipidemia.Approximately 5 mL of peripheral blood was collected from 8 GBS patients and 8 healthy controls (from the above cohort and matched by sex and age) into EDTA-anticoagulated vacuum tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte isolation agent .The human monocyte leukemia cell line THP-1 was routinely maintained in RPMI 1640  supplemented with 10% heat-inactivated (1 h at 56°C) FBS  and 1% β-mercaptoethanol  at 37°C in 5% CO2. THP-1 cells were cultured at a density of 5×105 cells/mL in 12-well culture dishes and treated for 24 h with 50 µg/mL free cholesterol (Sigma USA) or 100 ng/mL LPS in the presence or absence of 50 µg/mL free cholesterol.Total RNA was extracted from PBMCs and THP-1 cells using TRIzol reagent  and reverse transcribed to cDNA using a Takara Prime Script RT Reagent Kit . qRT–PCR was conducted using a Roche Light Cycler 480 Real-Time PCR System  with a TB Green PCR Kit . The β-actin gene was used as the endogenous control. We used the 2-ΔΔCT method to calculate the relative gene expression. Primers for qRT–PCR are listed in SPSS software (version 26.0) and GraphPad Prism software (version 8.0) were used for statistical analysis. Continuous data were analyzed using the independent t test or the Wilcoxon test. Categorical data were analyzed using the McNemar test. We performed a forward stepwise conditional logistic regression analysis to explore the independent risk factors for GBS and GBS with severe conditions. The Spearman rank correlation coefficient and Mantel–Haenszel test were performed to analyze the correlation between clinical characteristics and risk factor quartile.There were a total of 147 patients with GBS and 153 healthy controls consecutively included in our study. The characteristics of the GBS patients and univariate factors associated with the risk of GBS are presented in The adjustment for potential covariates was made Figure 2Of 147 patients with GBS, 91 (61.9%) had severe GBS, and 56 (38.1%) had mild GBS. The univariate logistic regression analysis showed that SBP , triglycerides , HDL cholesterol , remnant cholesterol  and the dyslipidemia index (P<0.001) were correlated with severe GBS Table 2.The clinical characteristics of GBS patients based on the remnant cholesterol quartiles are shown in Serum total cholesterol included conjugated cholesterol and free cholesterol. Depending on how remnant cholesterol was calculated, it mainly included free cholesterol and conjugated cholesterol from intermediate density lipoprotein cholesterol, VLDL cholesterol and chylomicrons. Free cholesterol is a key component of remnant cholesterol. To investigate the potential effect of remnant cholesterol on the activation of monocytes in PBMCs from patients with GBS, we measured the mRNA expression of TLR4, TLR2 and NF-κB in PBMCs and cultured cells. The results showed that the mRNA levels of TLR4, TLR2 and NF-κB associated with monocyte activation in the PBMCs of GBS patients were significantly higher than those of healthy controls Figure 4GBS is the most common severe acute paralytic neuropathy, with a mortality rate of 5% and permanent sequelae rate of 10%. In the context of the COVID-19 pandemic, GBS is one of the serious sequelae , 17. It The potential etiological factors of GBS were revealed in the results of our case–control study. Although many studies have been conducted on GBS, the cause of GBS is not fully understood. The majority of patients have had an event including preceding infection, vaccination and surgery in the four weeks before the onset of neurological symptoms of GBS , 22. GBSOur results suggest that alcohol consumption may be an independent risk factor for GBS. Repeated alcohol consumption can damage the central and peripheral nervous systems. The main consequences of long-term alcohol consumption are sleep disturbances, chronic pain, cognitive and behavioral disorders, stroke, and damage to peripheral nerves and muscles. The pathogenesis of alcoholic neuropathy remains controversial. It is currently primarily associated with nutritional deficiencies and the toxic effects of alcohol. Recent studies have suggested that alcohol consumption may lead to axonal degeneration resulting from oxidative stress, the release of proinflammatory cytokines and the activation of protein kinase C . FurtherThere is strong evidence that high Hcy may cause sensory and motor peripheral nerve dysfunction . AlthougMost importantly, our study suggested that various lipid levels were possible risk or protective factors for GBS. Compared with controls, patients with GBS had higher levels of triglycerides, LDL cholesterol, APOB and remnant cholesterol and lower HDL cholesterol and APOA1 levels. After considering the lipid levels as a dyslipidemia index, GBS patients had a higher dyslipidemia index. In addition, increased remnant cholesterol levels and an increased dyslipidemia index were associated with the incidence of severe GBS. Increased remnant cholesterol and dyslipidemia were associated with increased GBS disability scores. Furthermore, the proportion of patients with severe GBS increased with increasing remnant cholesterol and increasing dyslipidemia index. Some studies have shown a link between altered lipid metabolism and peripheral nerve dysfunction. The protective role of HDL cholesterol and apolipoproteins in established and early MS has been demonstrated. LDL cholesterol and serum total cholesterol were correlated with cognitive impairment in MS patients . In typeIn conclusion, preceding infection, alcohol consumption, remnant cholesterol, homocysteine and the dyslipidemia index were independent risk factors for GBS, and HDL-cholesterol was an independent protective factor for GBS. Furthermore, high remnant cholesterol levels and an elevated dyslipidemia index were associated with an increased risk of severe GBS. The mRNA levels of TLR2, TLR4, and NF-KB associated with monocyte activation were increased in GBS patients, and free cholesterol may promote this process.There are several limitations in this study. First, because GBS is a rare disease, an insufficient number of cases were included in the study; this study was a case–control study. Second, this was a single-center study, which could lead to bias in the results due to regional differences. Finally, the effect of dyslipidemia on the pathogenesis of GBS should be demonstrated by additional cell experiments and animal experiments.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the institutional review boards of Beijing Tiantan Hospital. The patients/participants provided their written informed consent to participate in this study.Study concept and design: GJZ. Acquisition of data: YD, LW, and JS. Analysis and interpretation of data: YD, LW, JS, YS, GL, and XL. Drafting of the manuscript: YD and LW. Statistical analysis: YD, LW, and JS. Critical revision of the manuscript for important intellectual content: GHZ and GJZ. All authors contributed to the article and approved the submitted version.This study was supported by Beijing Municipal Natural Science Foundation (code: NO.7222052), Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (code: ZYLX202108), Beijing Hospitals Authority’s Ascent Plan (DFL20220505) and Beijing High-level Public health technical Personnel Training program (2022-2-013).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rescue workers carry a boy from the rubble of a building in Idlib in the Syrian Arab Republic. The building collapsed on 6 February 2023, when a 7.8 magnitude earthquake followed by multiple aftershocks struck south-east Türkiye, close to the border with the Syrian Arab Republic. As of 13 February, some 34 000 people were reported to have died, with the numbers expected to rise in the following days. There was also significant damage to essential infrastructure, including health facilities.At the request of the Turkish Government, the World Health Organization (WHO) and other United Nations partners launched a humanitarian response to the earthquake that struck the Syrian Arab Republic and Türkiye on 6 February. WHO activated a network of emergency medical teams to provide essential health care for the injured and most vulnerable in the days following the earthquake, and flew 110 metric tonnes of medical supplies out of the WHO Global Logistics Hub located within the International Humanitarian City in Dubai, United Arab Emirates. Sufficient to cover the needs of around 400 000 people, the delivery included supplies for urgent surgical interventions and the treatment of anticipated pneumonia cases among survivors, many of whom were left outside in sub-zero temperatures after buildings collapsed.WHO also released more than 16 million United States dollars (US$) from the Contingency Fund for Emergencies, including US$3 million within hours of the disaster, and, in the Syrian Arab Republic, was able to release prepositioned medical and surgical supplies to 16 hospitals treating survivors of the earthquake in the north-west of the country on day one of the emergency. The emergency in the Syrian Arab Republic is being compounded by an ongoing crisis, dating back to 2011, that has left millions exposed to health risks including outbreaks of infectious diseases and inadequate health services including disrupted referral networks. http://bit.ly/3lp8CDThttp://bit.ly/3DYKX3hEquatorial Guinea reported its first-ever outbreak of Marburg virus disease. Preliminary tests carried out following the deaths of at least nine people in the country’s western Kie Ntem Province confirmed the presence of the viral haemorrhagic fever.As of 13 April, 16 other suspected cases with symptoms including fever, fatigue and blood-stained vomit and diarrhoea had also been reported.Teams were deployed in the affected districts to trace contacts, isolate and provide medical care to people showing symptoms of the disease. WHO also deployed health emergency experts in epidemiology, case management, infection prevention and control, laboratory and risk communication to support the national response efforts and secure community collaboration in controlling the outbreak.http://bit.ly/3xj2vU2Africa is witnessing a steep rise in cholera cases amid a global surge. An estimated 26 000 cases and 660 deaths were reported as of 29 January 2023 in the 10 African countries facing outbreaks since the beginning of the year.The bulk of the new cases and deaths have been recorded in Malawi, which is facing its worst cholera outbreak in two decades, reporting over 600 new cases per day with a continually high case fatality rate (>3%). The outbreak was declared a public health emergency by the Malawi government on 5 December 2022.  In addition, neighbouring Mozambique has registered a sharp increase in cases and alerts since mid-December 2022, with cases reported from five provinces, including provinces bordering Malawi. On 26 January 2023, Zambia notified WHO of a cholera outbreak in the eastern province bordering Malawi and Mozambique. There remains a high risk of spread to other countries in the region, including the United Republic of Tanzania and Zimbabwe. In East Africa, Ethiopia, Kenya and Somalia are responding to outbreaks amid a prolonged and harsh drought that has left millions of people in dire need of humanitarian assistance. http://bit.ly/3xcHKJKhttp://bit.ly/3YJRg2HBetween October 2022 and January 2023, seven WHO Member States reported incidents of over-the-counter cough syrups for children being sold with high levels of diethylene glycol.More than 300 children were reported to have died as a result of ingesting the syrup in three of the countries concerned . Most were young children under the age of five. On 23 January, WHO issued medical product alerts to the national health authorities of all 194 WHO Member States requesting the detection and removal of contaminated medicines from circulation, increased supply chain surveillance, immediate notification to WHO of substandard products found, and public information campaigns. Because these were not isolated incidents, WHO called on various stakeholders engaged in the medical supply chain to take immediate and coordinated action.http://bit.ly/3YEQYdkWHO Director-General, Tedros Adhanom Ghebreyesus, announced plans to establish a tuberculosis Vaccine Accelerator Council to facilitate the licensing and use of effective, novel tuberculosis vaccines, if and when they become available. Speaking at a high-level panel on tuberculosis at the World Economic Forum on 17 January, Director-General Tedros explained that the council will seek to catalyze high-level alignment between funders, global agencies, governments and end-users to make sure that innovative products reach the people who need them.“One of the most important lessons from the response to the COVID-19 pandemic is that innovative health interventions can be delivered fast if they are prioritized politically and financed adequately,” he said.http://bit.ly/3JV1UzBGermany’s Federal Ministry of Health announced a 130 million euros (US$140 million) contribution to WHO for 2023, reinforcing Germany’s role as a global health leader and one of WHO’s strongest supporters. The announcement was made on the sidelines of the 152nd session of WHO’s Executive Board which took place at WHO’s Geneva headquarters between 30 January and 7 February.Germany was WHO’s largest overall contributor in the 2020-2021 biennium, and second-largest in 2022. The country has also been the top donor to the ACT-Accelerator, a global collaboration designed to support the rapid development, production and equitable distribution of COVID-19 vaccines, tests and treatments. The contribution will support WHO in priority areas including antimicrobial resistance, health emergencies, health systems strengthening and WHO reforms. http://bit.ly/3DWXx3kFive billion people globally remain unprotected from harmful industrially produced trans fat despite a target to eliminate the product from human consumption by 2023, called for by WHO in 2018.Trans fat is commonly found in packaged foods, baked goods, cooking oils and spreads, and is estimated to be responsible for up to 500 000 premature deaths from coronary heart disease each year.According to a status report published by WHO on January 23 that assesses global progress towards the 2023 target, 43 countries have introduced best-practice anti-trans fat policies that are helping to protect some 2.8 billion people globally. While this clearly represents progress, some 5 billion people remain exposed to the product. “Trans fat has no known benefit, and huge health risks that incur huge costs for health systems,” said Director-General Tedros at the report’s launch, adding that it was time to get rid of the product, “once and for all.”http://bit.ly/3HOBT27A place of worship in Gaziantep in Türkiye that was devastated by the earthquake that hit the country and neighbouring Syrian Arab Republic on 6 February 2023.The number of people requiring neglected tropical disease (NTD) interventions fell by 80 million between 2020 and 2021, and eight countries were certified or validated as having eliminated one NTD in 2022 alone. As of December 2022, 47 countries had eliminated at least one NTD and more countries were in the process of achieving this target.Global report on neglected tropical diseases2023 that was released on 30 January, highlighting the progress made and challenges faced in delivering NTD care worldwide, against a backdrop of COVID-19-related disruptions.This is according to the The report notes that the advances made in 2021-2022 build on a decade of significant progress that includes one billion people being treated for NTDs each year between 2016 and 2019 through mass treatment interventions, and 25% fewer people requiring interventions against NTDs in 2021 than in 2010.http://bit.ly/3HQXAP3th Session of the United Nations Commission on the Status of Women. United Nations Headquarters, New York. http://bit.ly/3jQqtTJ6 – 17 March. 67http://bit.ly/3DLSAdm27 – 31 March. Global meeting on skin-related neglected tropical diseases. WHO Geneva HQ. http://bit.ly/3kjKQsm3 – 5 April 2023. Fifth Global Forum on Human Resources for Health. Geneva, Switzerland.


Afterwards, the oocytes were lysed for measuring the total antioxidant capacity (TAC), activity of peroxidase, catalase and glutathione reductase, and ROS formation. The impact of vitrification on mitochondrial and lysosomal activities was also examined. Since glutathione, added at 5 mmol L−1, significantly increased the TAC of warmed oocytes, in the next set of experiments this dose was applied for post–warm recovery of oocytes used for IVF. Glutathione in the recovery culture did not change the total blastocyst rate, while increased the proportion of faster developing blastocysts (Day 6–7), reduced the apoptotic cell ratio and reversed the harmful impact of vitrification on the actin cytoskeleton. These results suggest that even a short recovery culture with antioxidant(s) can improve the development of bovine devitrified oocytes.Vitrification of bovine oocytes can impair subsequent embryo development mostly due to elevated oxidative stress. This study was aimed at examining whether glutathione, a known antioxidant, can improve further embryo development when added to devitrified oocytes for a short recovery period. Bovine in vitro matured oocytes were vitrified using an ultra-rapid cooling technique on electron microscopy grids. Following warming, the oocytes were incubated in the recovery medium containing glutathione (0, 1.5, or 5 mmol L Vitrification of mammalian oocytes has wide possibilities of application in the field of assisted reproduction as well as preservation of the gene pool. However, the process of vitrification–warming is accompanied by considerable damage to the oocyte, which mostly results in oxidative stress ,2,3,4,5.Cryopreservation causes oxidative stress by different mechanisms, including osmotic stress, leading to increased oxidative metabolism within mitochondria  and mitoDespite the increasing amount of publication focusing on bovine oocyte vitrification, yields of transferable blastocysts after the in vitro fertilization of vitrified bovine oocytes remain low ,10. OxidGarcía-Martínez with colleagues  concludeVarious other antioxidants , similar to GSH, added to the culture have been shown to ameliorate the oxidative stress associated with oocyte vitrification in mouse ,16, sheeAll the chemicals used in this study were purchased from Sigma-Aldrich Inc.  unless otherwise indicated.−1 glutathione (GSH) or without GSH (30 oocytes per group). Subsequently, the oocytes were lysed in a lysis buffer for measuring the total protein concentration and following antioxidant parameters: total antioxidant capacity, activity of peroxidase, catalase and glutathione reductase. A group of fresh oocytes was also lysed and served as a control. Based on the results of these measurements, a proper dose of GSH for further experiments was selected.In the first series of experiments (three replicates), vitrified oocytes after thawing were cultured during a 3-h recovery period in the maturation medium with 1.5 or 5 mmol L−1) to quantify the formation of ROS.In the second series of experiments, both vitrified–warmed and fresh oocytes were stained to assess the activity of mitochondria (MitoTracker Green) and lysosomes (LysoTracker DeepRed). A part of the vitrified–warmed oocytes was cultured with or without GSH . Non-vitrified (fresh) oocytes served as a control. Subsequently, both groups were in vitro fertilized and cultured to the blastocyst stage. Cleavage rate and blastocyst development (day 6−8) were evaluated. Randomly selected blastocysts were fixed and stained to evaluate the total number of cells (DAPI), the proportion of apoptotic cells (TUNEL-assay), and the quality of actin cytoskeleton .In the third series (6 repetitions), vitrified–warmed oocytes during the recovery period were cultured without (VGSH− group) or with GSH (5 mmol L−1), gentamicin (50 mg·mL−1), 10% FBS, and FSH/LH . Oocyte vitrification followed IVM lasting 21 h, at 38.5 °C and 5% CO2 in the atmosphere. After several weeks, the COCs were warmed and cultured for 3 h either with or without glutathione for post-thaw recovery. For the control group, fresh oocytes were matured in the same IVM medium for 24 h.The source of the ovaries were cows from a local slaughterhouse. Immediately after slaughter, the ovaries were brought at a temperature of 22–25 °C. In the laboratory, cumulus-oocyte complexes (COCs) were aspirated from antral follicles (2–8 mm in size) with a sterile 5 mL syringe and needle. The obtained COCs were washed several times in the holding medium . Oocytes with a complete and well-adjacent cumulus cell layer and homogeneous medium brown ooplasm were selected for in vitro maturation (IVM). The medium used for maturation was E-199 (Gibco) with L-alanyl glutamine, to which we added sodium pyruvate (0.25 mmol·Lzona pellucida) were excluded from further experiments.Vitrification of oocytes was carried out according to the methodology previously described in Olexiková et al. . BrieflyThe activities of the studied enzymes , protein concentration, and total antioxidant capacity (TAC) were determined spectrophotometrically using a Synergy microplate reader . Samples of fresh or vitrified–warmed oocytes with or without GSH (30 oocytes for each group) were lysed in 50 μL of lysis buffer mixture  and stored at −20 °C until analyses. Prior to other analyses, the protein concentration in the samples was determined by Bradford’s method using bovine serum albumin as a standard.−1 of ABTS. After 25 min of incubation at room temperature, the decrease in absorbance at 734 nm was measured and the total antioxidant capacity was compared with the antioxidant capacity of Trolox, which served as a standard.The total antioxidant capacity (TAC) of the samples was determined by the ABTS ) method , which i−1). The decrease in absorbance caused by the decrease in the concentration of hydrogen peroxide in the reaction by the catalytic action of the enzyme was recorded at a wavelength of 240 nm at 30 °C for 5 min.The catalase activity in the samples was determined spectrophotometrically according to Rahiminejad . The rea−1). Color product formation was measured at absorbance of 436 nm, at 30 °C for 1 min.The peroxidase activity was determined spectrophotometrically according to Lyttle and DeSombre . The rea−1 of NADPH and 1 mmol L−1 of GSSG. The decrease in absorbance at 340 nm was recorded at 25 °C for 5 min.Glutathione reductase (GR) activity was determined by a spectrophotometric method, based on measuring the decrease in absorbance, caused by nicotinamide adenine dinucleotide phosphate (NADPH) oxidation . Two subTM Green  and LysoTrackerTM Deep Red , respectively, in order to evaluate fluorescent activity and distribution in fresh and vitrified oocytes. Intact (unfixed) oocytes were incubated in LysoTracker solution (50 nmol L−1) for 30 min at 37 °C, washed three times in a phosphate–buffered saline (PBS) with 0.6% of polyvinylpyrrolidone (PVP) and subsequently incubated in MitoTracker solution (200 nmol L−1) for 30 min at 37 °C, according to the procedure of Jeseta [Mitochondria and lysosomes in oocytes were detected by simultaneous staining with MitoTrackerf Jeseta . AfterwaStained oocytes were scanned by an LSM 700 Zeiss confocal laser scanning microscope  and the mitochondrial and lysosomal activities were evaluated based on fluorescence intensity using ImageJ software. The corrected total cell fluorescence (CTCF = integrated density − (area of selected cell x mean fluorescence of background readings) formula was used to determine the fluorescence value. Mitochondrial and lysosomal distribution was evaluated based on occurring patterns . In the case of an aggregated pattern, the numbers of aggregates per oocyte were counted and rates of co-localization of mitochondria and lysosomes were evaluated.The level of intracellular ROS was assessed by fluorescent staining using CellROX Green . Denuded oocytes were incubated for 30 min in a dye mixture (according to the producer´s guide), washed in PBS–PVP, fixed for 10 min in 4% of neutrally buffered formalin, covered with a drop of Vectashield anti-fade mounting medium with DAPI, and mounted onto glass slides with a coverslip. Stained oocytes were scanned by an LSM 700 Zeiss confocal laser scanning microscope, and the ROS formation was evaluated based on fluorescence intensity using ImageJ software and the formula described above.−1 heparin, 50 mg mL−1 gentamicin) and put into 100 µL droplets of IVF–TALP medium previously covered with a mineral oil into Nunc 4-well plates . Afterwards, the sperm (at 2 × 106 per mL) and PHE solution  were added and incubated at 38.5 °C with 5% CO2 in atmosphere. At 18 h from the start of IVF, oocytes (now presumptive zygotes) were cleared of adhered spermatozoa and cumulus cell debris by vortexing in a microtube containing 75 µL of holding medium for 30 s. The zygotes were subsequently placed in prepared 4-well culture dishes (NUNC) with a grown confluent monolayer of BRL feeder cells . A medium prepared according to composition of Menezo B2, supplemented with 10% FBS, was used for cultivation. The development of in vitro fertilized oocytes was subsequently observed up to the blastocyst stage. We evaluated cleavage rate on Day 2 after fertilization and blastocyst rate on Days 6, 7 and 8.Morphologically good-looking oocytes from both control and vitrified/warmed groups intended for IVF were washed in IVF–TALP medium  of their nuclei. In addition to the total number of cells, the incidence of apoptotic cells in blastocysts was monitored by the TUNEL method, as well as the quality of the actin cytoskeleton after Phalloidin-TRITC staining. For staining, blastocysts were briefly washed in PBS-PVP solution (0.6%). They were fixed with freshly prepared neutral buffered formalin (4%) for 10 min. The In Situ Cell Death Detection Kit  was used to mark apoptotic cells according to the manufacturer’s instructions. Briefly, the procedure was run after permeabilization in the reaction mixture  for one hour at a temperature of 37 °C in incubator. After completion, blastocysts were washed in cold PBS-PVP solution.Afterwards, blastocysts were incubated in phalloidin-TRITC solution at room temperature for 30 min to stain the actin cytoskeleton. Then, the embryos were washed in PBS–PVP solution and on a glass slide covered with a drop of Vectashield anti-fade medium containing DAPI fluorochrome, with the help of which microscopic preparations were made. They were wrapped in opaque packaging and stored in the refrigerator until analysis. Fluorescence analyses of blastocysts were performed using a Leica fluorescence microscope and an LSM 700 Zeiss confocal laser scanning microscope.The actin cytoskeleton was classified according to Makarevich  with slit-test. All pairwise multiple comparison procedures and one-way ANOVA were conducted following the Holm–Sidak method. The overall significance level used was 0.05. All statistical analyses were performed using SigmaPlot 11.0 (Systat) software.Data on embryo development rates of control and vitrified/warmed bovine oocytes are represented by 6 replicates. Distribution to stages according to cleavage and blastocyst formation and distribution according to actin quality were analyzed by a Pearson´s Chi-square statistic test with the Yates correction for continuity. The incidence of apoptotic cells was represented as a percentage value (index) from the total cell number. All indexes before statistical analysis were subjected to arcsine square root transformation. The normality of the data was tested by Shapiro–Wilk test and equal variance test. The differences in the mean value of experimental versus control group in the total number of cells, the incidence of apoptotic cells and mean values of total protein and enzymes activity were evaluated by Student’s −1) indicating the uniformity of all tested groups. Vitrification of oocytes led to an increase in peroxidase activity and a decrease in catalase activity (p < 0.05). The addition of GSH at both concentrations to devitrified oocytes significantly reduced catalase activity. Oppositely, the addition of 5 mmol L−1 GSH significantly increased the activity of peroxidases and, at the same time, the total antioxidant capacity was increased in this group (VGSH 5) compared to the other groups  distribution pattern of mitochondria C. MitochIn the case of lysosomes, the aggregated pattern of distribution prevailed in both control  and vitrified  oocytes . More th−1 to test the effectiveness of GSH in the next experiments. Vitrification caused a significant increase in the production of ROS in oocytes, which was visible as increased green fluorescence intensity . In our study, a total of 840 oocytes were used, of which 421 oocytes were vitrified. Devitrified oocytes after post–warming recovery (3 h) were fertilized in vitro. This experiment was performed in six replicates.In this experiment, the developmental potential of the vitrified–warmed oocytes was compared to those exposed to 5 mmol LThe cleavage rate in individual experiments varied: from 50.05% to 69.81% in vitrified oocytes, from 47.05% to 72% in the VGSH+ group, and from 59.09% to 78.18% in the control oocytes. Blastocyst rate varied from 13.04% to 29.41% for vitrified oocytes, from 9.52% to 24.44% for VGSH+, and from 27.08% to 49.09% for the control oocytes. Average values of cleavage and blastocyst rate of in vitro fertilized oocytes are presented in p > 0.05). However, both vitrified groups (irrespective of GSH) had significantly lower total blastocyst rates compared to the control group (p < 0.05). Taking into consideration fast developing blastocysts (Day 7) from total blastocyst rate (TBR), GSH addition significantly increased the D7 blastocyst rate (71.79%) compared to the GSH-free group  up to the level of non-vitrified control (74.75%).The cleavage rate of fertilized zygotes did not differ among vitrified and control groups irrespectively of GHS addition  and was comparable to control oocytes. The most of slow-developing (D8) blastocysts were recorded in the vitrified group without GSH supplementation compared to GSH-stimulated vitrified (p < 0.01) or control (p < 0.01) groups.Most of fast developing (D6 and D7) blastocysts were recorded in the vitrified group supplemented with GSH, which was significantly higher than in the vitrified group without GSH  cells, and the quality of actin cytoskeleton .p < 0.05; Vitrification significantly decreased the total cell number of blastocyst produced from devitrified oocytes (98.35) and GSH did not significantly improve this parameter (101.00) compared to control (112.56). Vitrification has no effect on the incidence of apoptotic cells (TUNEL–index) in the blastocysts. Nevertheless, the addition of GSH to the recovery culture even suppressed apoptosis in this group of blastocysts compared to control .Low fertilization rates of cryopreserved oocytes are reportedly associated with chilling and freezing injuries including membrane damage, zona hardening, due to premature release of cortical granules or abnormal increase of cytoplasmic free calcium ions , spindleIt was demonstrated that oocyte vitrification can disturb the reduction–oxidation (redox) status, reduce glutathione content and increase ROS levels, resulting in the damage of biomolecules such as DNA, proteins and membrane lipids and leading to mitochondrial dysfunction ,35. MitoIn our work, we investigated the response of some important cell organelles, such as mitochondria to damage caused by oocyte vitrification. Mitochondrial activity was significantly decreased in the vitrified group compared to the control, what indicates the mitochondrial deficiency of oocytes after vitrification. A similar decrease in mitochondrial activity was described in vitrified mouse oocytes ,38. The In lysosomes, the aggregated pattern of distribution prevailed in both vitrified and fresh groups (83.72–89.36%) and most of the mitochondrial and lysosomal aggregates were co-localized. High co-localization of mitochondrial lysosomal aggregates (more than 80%) indicates an interconnection of these two organelles and the importance of rapid removal of damaged mitochondria by organelles with lysosomal activity. The decrease in the fluorescence intensity of lysosomal organelles in vitrified/warmed oocytes is probably due to the increased involvement in macrophagic/autophagic processes within the repair of cryodamages. In this process, autophagosomes are directed towards lysosomes, where their outer membrane fuses with the surrounding membrane to release single-membrane bound structures (autophagic bodies) into the lumen. The acidic environment inside the lysosome promotes the digestion of macromolecules by hydrolysis . One of Interestingly, additional post-thaw incubation of oocytes resulted not only in the recovery of the meiotic spindle , but alsGlutathione is known as an effective substance, directly or indirectly, in many important biological processes including the synthesis of proteins and DNA, transport, enzyme activity, metabolism and protection of cells . GlutathAlthough low and controlled ROS level is adequate for normal cell function, the vitrification process substantially increases ROS level in oocytes. An imbalance between the production and neutralization of ROS causes oxidative stress in the oocytes with a consequent impact on developmental competence . In our −1 of GSH) was also associated with a significant increase in the total antioxidant capacity. However, the GSH induction was not observed equally for all enzymatic antioxidant systems.In vitrified oocytes, we observed also changes in the most important intracellular enzymatic antioxidant systems, which may be the consequences of oxidative stress. The enzyme activity of bovine oocytes in response to oxidative stress during in vitro maturation was previously studied by Cetica . In our 2O and O2. However, at a low concentration of hydrogen peroxide and in the presence of a suitable hydrogen donor, catalase is otherwise regulated. Catalase can act peroxidically, removing hydrogen peroxide but oxidizing its substrate [Particularly, catalase activity in oocytes was reduced after vitrification–warming and GSH was able to suppress it more. Catalase in organisms has two enzymatic activities depending on the concentration of hydrogen peroxide. If the concentration is high, catalase removes hydrogen peroxide by forming Hubstrate . It is nubstrate . Catalasubstrate . PossiblAlthough in our experiments, the addition of GSH to the recovery culture did not have a significant effect on the blastocyst rate compared to the vitrified group without GSH, it had an effect on the speed, at which the embryos developed into the blastocyst. In our previous study , we alreTotal cell and apoptotic number are important parameters of embryo development and viability. Several studies described lower cell number ,61 and aThe actin cytoskeleton plays an important role in development, as its ability is to coordinate cellular functions and integration of various signals . During −1, significantly increased the total antioxidant capacity of warmed oocytes. Although the addition of GSH into the recovery culture did not increase the blastocyst rate, it was able to induce faster blastocyst formation and improve their quality by reducing the incidence of apoptotic cells. These results expand previous knowledge about the mitigating effect of antioxidants on the harmful consequences of the cryopreservation of oocytes in various experimental designs.According to our results, it is obvious that GSH confirmed its protective role against damage induced by vitrification. Even a relatively short period of post-thaw recovery culture (3 h) represents a space for improving the development of embryos produced in vitro from vitrified/warmed bovine oocytes. GSH, added at 5 mmol L