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STUDY QUESTION Is it possible to establish a new in-vitro activation (IVA) protocol for infertility treatment? SUMMARY ANSWER A new IVA procedure is an efficient and easily performed approach for infertility treatment of patients with diminished ovarian reserve (DOR). WHAT IS KNOWN ALREADY IVA of primordial follicles with or without stimulators has been developed to treat patients with primary ovarian insufficiency (POI) successfully. However, the efficiency of the procedure is still very low. There is a requirement to optimize the protocol with increased efficiency for clinical application. STUDY DESIGN, SIZE, DURATION Newborn mouse ovaries were used to establish a new 1-h IVA protocol with the mechanistic target of rapamycin (mTOR) stimulator phosphatidic acid (PA, 200 µM) and the phosphatidylinositol-3-kinase (PI3K) stimulator 740Y-P (250 µg/ml); a prospective observational cohort study in POI patients was performed on 15 POI patients and 3 poor ovarian response (POR) patients in three different centers of reproductive medicine in China. PARTICIPANTS/MATERIALS, SETTING, METHODS One-third of ovarian cortex was removed and processed into bigger strips (1 × 1 cm2, 1–2 mm thickness). Strips were then sutured back after treatment. The new approach only requires one laparoscopic surgery. MAIN RESULTS AND THE ROLE OF CHANCE Follicular activation and development increased in cultured mouse and human ovarian tissues after 1 h of stimulator treatment. Compared with tiny ovarian cortex pieces (1 × 1 mm2), large ovarian strips (1 × 1 cm2) showed the lowest apoptotic signals after incubation. We applied the orthotropic transplantation procedure with large strips in the clinic, and 9 of 15 POI patients showed at least one-wave follicular growth during the monitoring period. One patient was reported with one healthy delivery after natural conception and another patient with a healthy singleton delivery after IVF. All the contacted patients (n = 13) responded with no side effects on their health 2–4 years after IVA procedure. LIMITATIONS, REASONS FOR CAUTION Further clinical trials with a large number of well-defined patients are required to compare different IVA protocols. A long-term follow-up system should be set up to monitor patient’s health in the future cohort study. WIDER IMPLICATIONS OF THE FINDINGS By using stimulators, the findings in the study provide a more efficient IVA protocol for the treatment of patients with DOR. It requires only one laparoscopic surgery and thus minimizes patients’ discomfort and costs. This strategy could be useful for patients diagnosed with POI and desire pregnancy as soon as possible after the operation. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Research and Development Program of China (2018YFC1003703 and 2018YFC1004203); the National Natural Science Foundation of China (81871221); Co-construction of Provincial Department (201601006). The authors have no conflict of interest to disclose. TRIAL REGISTRATION NUMBER ChiCTR2000030872.

Shunqing Su,1 Xiumei Ding,1 Huijuan Zou,1 Yuechun Lin,1 Jianmin Huang,1 Disheng Xiong,1 Jinan Kuan,1 Yanhong Zhang,2 Rurong Xie2 1Department of Burn&Plast, Dalang Hospital of Dongguan, Dongguang, Guangdong, 523780, People’s Republic of China; 2Department of Wound Repair, Hongmei Hospital of Dongguan, Dongguang, Guangdong, 523160, People’s Republic of ChinaCorrespondence: Xiumei DingDepartment of Burn&Plast, Dalang Hospital of Dongguan, No. 85 of Jinlangzhong Street, Dalang District, Dongguang, 523780, People’s Republic of ChinaTel +86 769-83009512Fax +86 769-83033122Email ding_xm123@outlook.comObjective: The present study aims to explore the individualized treatment options for multisite pressure ulcer (PU) at various stages in elderly patients with multiple medical conditions.Methods: Stages 1 and 2 PU at 146 sites were treated with closed negative pressure suction combined with continuous micro-oxygen perfusion and the local application of foam dressings, silver ion dressings, and moist burn cream. Stages 3 and 4 PU in the sacrococcygeal region were treated with skin or myocutaneous flap transplantation.Results: Stages 1 and 2 PU healed after treatment with closed negative pressure suction combined with continuous micro-oxygen perfusion and dressing changes. One case died during hospitalization due to an illness. Skin or myocutaneous flap repair was conducted in 34 cases of stage 3 or 4 PU in the sacrococcygeal area. Of these cases, 28 achieved primary healing, and 6 required two or three surgeries, 5 of which received micro-skin implantation. In addition, 10 small deep PU at other sites were repaired by direct excision and suturing or local flap repair. Seven cases were transferred to other departments or hospitals due to concomitant diseases or were discharged automatically without surgical treatment.Conclusion: Home care for geriatric patients is difficult. PU often occur at multiple sites because of the duration of various pressures, and different sites may demonstrate different stages because of varying degrees of pressure. When actively treating stages 3 and 4 PU, the trauma management of stages 1 and 2 PU should not be neglected.Keywords: oldest-old patients, pressure ulcer, wound management, flap transplantation, micro-skin implantation

Background: Melatonin (MT), a powerful antioxidant, can effectively ameliorate the in vitro development of animal embryos, but few studies have been performed on human embryos. Therefore, we investigated whether the application of MT in embryo cultures can improve embryonic development and clinical outcomes of patients with repeated cycles after failed in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.Methods: Human immature oocytes from controlled ovarian hyperstimulation cycles were collected for in vitro maturation culture and ICSI fertilization. The obtained embryos were cultured in vitro in medium containing 0, 10-11, 10-9, 10-7 or 10-5 M MT, and 10-9 M was determined to be the optimal concentration. Subsequently, 140 patients who experienced failed IVF/ICSI cycles underwent 140 cycles of embryo culture in vitro with medium containing 10-9 M MT. High-quality blastocysts were collected and cryopreserved for three months before vitrified-warmed embryo transfer. These culture cycles served as the experimental (10-9 M) group. The control (0 M) group comprised previous failed cycles. The patients were further divided into subgroups of 1, 2 or ≥3 failed cycles. The fertilization and embryo development statuses were compared.Results: The fertilization, cleavage, high-quality embryo, blastocyst, and high-quality blastocyst rates of the 10-9 M group were significantly higher than those of the 0 M group (87.7% vs. 83.6%, p p p -9 M group and the implantation rate, biochemical pregnancy rate and clinical pregnancy rate were significantly higher than those in the 0 M group (65.6% vs. 9.7%, p p p Conclusion: The application of MT to embryo cultures in vitro improved embryonic development in patients with repeated cycles after failed IVF/ICSI cycles and had good clinical outcomes. The optimal concentration of MT was 10-9 M.Trial registration: Name in the registry: Effect of melatonin on the clinical outcome of patients with repeated cycles after failed cycles of in vitro fertilization and intracytoplasmic sperm injection; registration number: ChiCTR2100045552; date of registration: April 19, 2021(retrospectively registered); URL of trial registry record: www.medresman.org.cn.

Cardiac allograft vasculopathy (CAV) charactered with aberrant remodeling and fibrosis usually leads to the loss of graft after heart transplantation. Our previous work has reported that extracellular high-mobility group box 1 (HMGB1) participated in the CAV progression via promoting inflammatory cells infiltration and immune damage. The aim of this study was to investigate the involvement of HMGB1 in the pathogenesis of CAV/fibrosis and potential mechanisms using a chronic cardiac rejection model in mice. We found high levels of transforming growth factor (TGF)-β1 in cardiac allografts after transplantation. Treatment with HMGB1 neutralizing antibody markedly prolonged the allograft survival accompanied by attenuated fibrosis of cardiac allograft, decreased fibroblasts-to-myofibroblasts conversion, and reduced synthesis and release of TGF-β1. In addition, recombinant HMGB1 stimulation promoted release of active TGF-β1 from cardiac fibroblasts and macrophages in vitro, and subsequent phosphorylation of Smad2 and Smad3 which were downstream of TGF-β1 signaling. These data indicate that HMGB1 contributes to the CAV/fibrosis via promoting the activation of TGF-β1/Smad signaling. Targeting HMGB1 might become a new therapeutic strategy for inhibiting cardiac allograft fibrosis and dysfunction.

Aim To compare embryonic developmental competence and clinical outcomes of oocytes matured in vivo (IVF oocytes) and those matured in vitro (IVM oocytes) from the same IVM/IVF cycles, and to analyze the clinical efficiency of a melatonin-supplemented in vitro maturation system combined with a modified IVM/IVF protocol. Main methods We randomly recruited 22 patients undergoing IVM/IVF treatment protocol in our medical centre. The fertilization, cleavage and blastocyst formation rates, as well as clinical pregnancy, implantation and live birth/ongoing pregnancy rates were analysed and compared between IVF and IVM oocytes. We evaluated mitochondrial function indicators by fluorescence staining and confocal microscopy, including mitochondrial membrane potential, reactive oxygen species and calcium (Ca2+) levels in 15 IVF and 15 IVM oocytes. Key findings There were no significant differences in fertilization or blastocyst formation rates between the IVF and IVM groups, whereas the cleavage rate was significantly higher in the IVF versus IVM group (100% vs 93.4 ± 10.9%, p = 0.03). There were no significant differences in the clinical pregnancy, implantation or live birth/ongoing pregnancy rates between the two groups. The cumulative clinical pregnancy and ongoing pregnancy/live birth rate per pick-up oocyte in the IVM/IVF treatment cycles were 68.2% (15/22) and 54.5% (12/22), respectively. The reactive oxygen species and Ca2+ levels were significantly increased, and mitochondrial membrane potential was significantly decreased, in IVM compared with IVF oocytes. Significance The modified IVM/IVF protocol can be effectively applied to the treatment of some indicated patients and achieve ideal clinical outcomes, even though the developmental potential of IVM oocytes may not be as high as IVF oocytes.

Does calcium ionophore treatment of oocytes improve fertilization rate, embryo development and outcomes in specific groups of infertile couples?This retrospective cohort study involved 796 couples undergoing oocyte activation with calcium ionophore (A23187) after intracytoplasmic sperm injection (ICSI) between 2016 and 2018. All metaphase II oocytes were exposed to 5 μmol/l ionophore for 15 min immediately after ICSI, cultured in vitro to the blastocyst stage, and transferred to the uteri of recipients on day 5 or cryopreserved for transfer in the next cycle. The previous cycles of the same patients formed the control group.Among 1261 ICSI cycles and 796 ICSI-artificial oocyte activation (ICSI-AOA) cycles, implantation, positive beta-HCG, clinical pregnancy and live birth rates were significantly (P0.05 to P0.001) improved for all groups, compared with previous cycles, except live birth rate in women with primary ovarian insufficiency (POI). Compared with previous cycles, rates of blastulation (all P0.001) and high-quality blastocysts (P0.05 to P0.001) were increased significantly for couples with male factor (oligoasthenoteratozoospermia [OAT]), unexplained infertility and couples with both factors in the ICSI-AOA cycles. High-quality blastocyst rate was increased in couples with polycystic ovary syndrome (PCOS) (P = 0.0453). Miscarriage rates were decreased significantly (P0.05 to P0.001) in couples with OAT, PCOS and unexplained infertility in the treatment cycles. No significant differences were found for fertilization rate, embryo development or live birth rate in patients with POI between both groups.Artificial oocyte activation was able to 'rescue' the poor reproductive outcomes in certain types of infertile couples with history of failure to achieve pregnancy.

A large number of recipients are in a compromised immune defense condition because of the routine application of immunosuppressive regimens after heart transplantation. Our previous work demonstrated that blockade of high-mobility group box 1 (HMGB1) prolongs the graft survival. Whether and how HMGB1 blockade impacts respiratory responses against pathogen-like challenge in organ transplant recipients when it improves cardiac graft are not elucidated. At the present study, after abdominal heterotopic heart transplantation, the recipient mice were treated with HMGB1 mAb, and then challenged with poly(I:C) or LPS intratracheally on day 7 post transplantation. We found that the level of bronchoalveolar lavage (BAL) HMGB1 was elevated after heart transplantation, and aggravated responses to respiratory tract poly(I:C)/LPS challenge were observed. HMGB1 neutralizing mAb treatment in poly(I:C)-challenged recipient mice alleviated pulmonary histopathological changes, neutrophil infiltration and inflammatory cytokine release, but unaffected the level of IFN-β, the distribution of CD11b(+)CD27(+)/CD11b(+)CD27(-) NK cell subsets, and CD8(+) T cell responses. In LPS-exposed recipient mice, HMGB1 mAb treatment ameliorated pulmonary inflammatory damage and enhanced the phagocytosis of phagocytic cells. Thus, this study may establish a basis for the application of HMGB1 blockade to improve the outcomes of heart transplant recipients because HMGB1 inhibition ameliorates pulmonary inflammation, but maintains defense-associated responses.

Aims HMGB1 has been reported to play a crucial role in the physiological and pathophysiological responses during pregnancy. However, it is still unknown whether excessively expressed HMGB1 at the maternal-fetal interface related to Unexplained Recurrent Spontaneous Abortion (URSA). This study was designed to investigate the local capability of HMGB1 in the pathology of URSA, determined the distributions and characteristics of HMGB1, its receptors (RAGE/TLR2/TLR4) and important signaling molecule NF-κB p65 expression at the maternal-fetal interface，as well as compared the differences of HMGB1 expression between the URSA group, control group and aspirin treatment group. Material and methods H&E staining, Western blot analysis, immunofluorescence assay and immunohistochemical staining were applied to determine the effect of HMGB1 and its receptors at the maternal-fetal interface. ELISA was utilized to detect the concentration of HMGB1 in plasma. Key findings Our study demonstrated that the activation of the HMGB1-RAGE/TLR2/TLR4-NF-κB pathway at the maternal-fetal interface may have occurred in the URSA group. HMGB1 concentration in plasma was higher in the URSA group than the control group. Furthermore, the levels of HMGB1 of subjects with URSA could be reduced by administrating low doses of aspirin (ASPL). Significance This is the first report indicating the roles of HMGB1 at the maternal-fetal interface of URSA patients and broadening the horizons for clinical treatment of URSA. HMGB1-RAGE/TLR2/TLR4-NF-κB signaling pathway may be activated at the maternal-fetal interface in URSA and account for its pathogenesis. HMGB1 have the potential to be promising biomarkers in prevention and therapy of URSA.

Blockade of extracellular high mobility group box 1 (HMGB1) can significantly prolong murine cardiac allograft survival. Here, we determined the role of HMGB1 in xenotransplantation. Sprague-Dawley rat hearts were transplanted heterotopically into BALB/c mice. Xenografts without any treatment developed predominant acute vascular rejection within 6 days. Both passively released HMGB1 from xenografts and actively secreted HMGB1 from infiltrated immune cells were significantly increased after xenotransplantation. HMGB1-neutralizing antibody treatment significantly prolonged xenograft survival and attenuated pathologic damage, immune cell infiltration, and HMGB1 expression and release in the xenografts. Compared to control IgG treatment evaluated at study endpoint, treatment with HMGB1-neutralizing antibody markedly suppressed xenoreactive B cell responses, as evidenced by the significant inhibition of anti-rat antibody production and deposition in xenografts at Day 6 posttransplant. Furthermore, treatment with anti-HMGB1 antibody suppressed B cell activation and reduced IFN-γ and IL-17A production after xenotransplantation. These results demonstrate for the first time that HMGB1 plays an important role in mediating acute xenograft rejection. Thus, we have shown that neutralization of extracellular HMGB1 can significantly inhibit xenoreactive B cell responses and delay xenograft rejection in a rat-to-mouse model of xenotransplantation, uncovering new insights in the role of HMGB1 in transplantation.

BACKGROUND High-temperatures in the cockpit environment can adversely influence pilot behavior and performance. OBJECTIVE To investigate the impact of high thermal environments on Chinese pilot performance in a simulated cockpit environment. METHODS Ten subjects volunteered to participate in the tests under 40°C and 45°C high-temperature simulations in an environmentally controlled chamber. Measures such as grip strength, perception, dexterity, somatic sense reaction, and analytical reasoning were taken. The results were compared to the Combined Index of Heat Stress (CIHS). RESULTS CIHS exceeded the heat stress safety limit after 45 min under 40°C, grip strength decreased by 12% and somatic perception became 2.89 times larger than the initial value. In the case of 45°C, CIHS exceeded the safety limit after only 20 min, while the grip strength decreased just by 3.2% and somatic perception increased to 4.36 times larger than the initial value. Reaction and finger dexterity were not statistically different from baseline measurements, but the error rate of analytical reasoning test rose remarkably. CONCLUSION Somatic perception was the most sensitive index to high-temperature, followed by grip strength. Results of this paper may help to improve environmental control design of new fighter cockpit and for pilot physiology and cockpit environment ergonomics research for Chinese pilots.

Chronic rejection that leads to diffuse narrowing and occlusion of graft vessels is the most important cause of morbidity and mortality following cardiac transplantation. The role and underlying mechanism of high-mobility group box 1 (HMGB1), as an established inflammatory mediator in acute rejection, remains poorly understood in chronic rejection. Here, we assessed the effects and mechanisms of HMGB1 on the chronic rejection using single MHC Class II-mismatched mouse cardiac transplantation model. It was found that HMGB1 was increased accompanying with the development of chronic rejection, while blockade of HMGB1 with specific neutralizing mAb substantially ameliorated chronic rejection-mediated vasculopathy and fibrosis of allograft, as well as markedly decreased T cell infiltration and production of IL-17A and interferon-gamma in allograft and recipient's spleen. Further, anti-HMGB1 antibody treatment significantly declined the number and frequency of mature dendritic cells (DCs) in allograft and recipient's spleen, especially CD11b(+) Ly6C(high) matured DCs that share the phenotypes with inflammatory-DCs. These findings indicate that HMGB1 contributes to chronic rejection, and HMGB1 blockade may be a novel mean to disrupt the proinflammatory loop after heart transplantation.