Melatonin application during cryopreservation improves the development and clinical outcomes of human vitrified-warmed oocytes.

In this clinical study, we investigated the potential of melatonin (MT) supplementation in the freeze-thaw medium used for cryopreserved human oocytes. In total, 152 patients who underwent in vitro fertilization between January 2020 and December 2022 were included and categorized into different groups as follows: the donor group, comprising 108 patients who donated their oocytes, with 34 patients using a vitrification and warming medium supplemented with MT (D-MT subgroup) and 74 patients using conventional medium without MT (D-0 subgroup); and the autologous group, comprising 38 patients who used their own oocytes, with 19 patients using medium supplemented with MT (A-MT subgroup) and 19 patients using medium without MT (A-0 subgroup). After thawing, the surviving oocytes in the D-MT and A-MT subgroups and D-0 and A-0 subgroups were cultured in a fertilization media with and without 10-9 MMT for 2.5 h, respectively, followed by intracytoplasmic sperm injection insemination, embryo culture, and transfer. The survival, cleavage, high-quality embryo, clinical pregnancy, ongoing pregnancy, and implantation rates were significantly higher in the D-MT subgroup than in the D-0 subgroup (all P < 0.05). Similarly, the survival, fertilization, high-quality embryo, and high-quality blastocyst rates were significantly higher in the A-MT subgroup than in the A-0 subgroup (all P < 0.05). These findings indicate that MT addition during cryopreservation can enhance the development of vitrified-warmed human oocytes and improve clinical outcomes.

[Homozygous Variant of FANCM of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism]. / 范科尼贫血通路FANCMåŸºå› çº¯åˆçªå˜å¯¼è‡´æ—©å‘æ€§åµå·¢åŠŸèƒ½ä¸å ¨çš„è‡´ç— æœºåˆ¶.

Objective: Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M (FANCM) is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including FANCA-FANCW. Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in FANCM. This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals. Methods: One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group, the EGFP FANCM-MUT group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody. Results: In a POI patient from a consanguineous family, we identified a homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's FANCM gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (P<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant. Conclusions: The homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.

Effect of melatonin on the clinical outcome of patients with repeated cycles after failed cycles of in vitro fertilization and intracytoplasmic sperm injection.

To explore whether embryo culture with melatonin (MT) can improve the embryonic development and clinical outcome of patients with repeated cycles after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure, immature oocytes from controlled ovarian superovulation cycles were collected for in vitro maturation (IVM) and ICSI. The obtained embryos were cultured in 0, 10-11, 10-9, 10-7 and 10-5 M MT medium respectively, and 10-9 M was screened out as the optimal concentration. Subsequently, 140 patients who underwent failed IVF/ICSI cycles received 140 cycles of embryo culture in vitro with a medium containing 10-9 M MT, these 140 MT culture cycles were designated as the experimental group (10-9 M group), and the control group was the previous failed cycles of patients (0 M group). The results showed that 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 (P < 0.01; P < 0.01; P < 0.0001; P < 0.0001; P < 0.0001). To date, in total, 50 vitrified-warmed cycle transfers have been performed in the 10-9 M group and the implantation rate, biochemical pregnancy rate and clinical pregnancy rate were significantly higher than those in the 0 M group (all P < 0.0001). Two healthy infants were delivered successfully and the other 18 women who achieved clinical pregnancy also had good examination indexes. Therefore the application of 10-9 M MT to embryo cultures in vitro improved embryonic development in patients with repeated cycles after failed IVF/ICSI cycles and had good clinical outcomes.Trial registration: ChiCTR2100045552.

Melatonin alleviates deoxynivalenol-induced apoptosis of human granulosa cells by reducing mutually accentuated FOXO1 and ER stress‡.

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins, which cause detrimental effects on human and animal reproductive systems by inducing oxidative stress. Increasing evidence has suggested the potential roles of melatonin in protecting granulosa cells from oxidative injury, but the underlying mechanisms remain largely elusive. Here, we demonstrated that suppression of FOXO1 and endoplasmic reticulum (ER) stress was engaged in melatonin-mediated protection against oxidative damage in human granulosa cells upon DON exposure in vitro. DON induced excess reactive oxygen species accumulation, cells viability loss, reduced estradiol-17ß, and progesterone production in human granulosa cells, whereas melatonin ameliorated these phenotypes. Next, we found that the protective effect of melatonin against apoptosis was via reducing ER stress because the inhibition of ER stress displayed similar protective effects during DON treatment. Moreover, melatonin provided no additional protection when ER stress was inhibited. We further found that FOXO1 is a pivotal downstream effector of melatonin and ER stress in regulating DON-induced apoptosis in human granulosa cells. Blocking of FOXO1 reduced DON-induced cells death and FOXO1 activation could be suppressed by melatonin or ER stress inhibitor. However, melatonin failed to further restore cells viability in the presence of FOXO1 inhibitor. Collectively, our results reveal a new mechanism of melatonin in protecting against DON-induced apoptosis and dysfunction by suppressing ER stress and FOXO1 in human granulosa cells.

Autotransplantation of the ovarian cortex after in-vitro activation for infertility treatment: a shortened procedure.

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.

Melatonin improves the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

Cryopreservation causes cryoinjury to oocytes and impairs their developmental competence. Melatonin (MLT) can improve the effect of cryopreservation in animal oocytes. However, no such studies on human oocytes have been reported. In this study, collected in vitro-matured human oocytes were randomly divided into the following groups: fresh group, MLT-treated cryopreservation (MC) group, and no-MLT-treated cryopreservation (NC) group. After vitrification and warming, viable oocytes from these three groups were assessed for their mitochondrial function, ultrastructure, permeability of oolemma, early apoptosis, developmental competence, and cryotolerance-related gene expression. First, fluorescence staining results revealed that oocytes from the 10-9  M subgroup showed the lowest intracellular reactive oxygen species and Ca2+ levels and highest mitochondrial membrane potential among the MC subgroups (10-11 , 10-9 , 10-7 , and 10-5  M). In subsequent experiments, oocytes from the 10-9  M-MC group were observed to maintain the normal ultrastructural features and the permeability of the oolemma. Compared with those of the oocytes in the NC group, the early apoptosis rate significantly decreased (P < .01), whereas both the high-quality cleavage embryo and blastocyst rates significantly increased (both P < .05) in the oocytes of the 10-9  M-MC group. Finally, single-cell RNA sequencing and immunofluorescence results revealed that aquaporin (AQP) 1/2/11 gene expression and AQP1 protein expression were upregulated in the MC group. Therefore, these results suggest that MLT can improve the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study.

RESEARCH QUESTION: Does calcium ionophore treatment of oocytes improve fertilization rate, embryo development and outcomes in specific groups of infertile couples? DESIGN: 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. RESULTS: 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 (P < 0.05 to P < 0.001) 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 P < 0.001) and high-quality blastocysts (P < 0.05 to P < 0.001) 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 (P < 0.05 to P < 0.001) 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. CONCLUSIONS: Artificial oocyte activation was able to 'rescue' the poor reproductive outcomes in certain types of infertile couples with history of failure to achieve pregnancy.

Melatonin promotes the development of immature oocytes from the COH cycle into healthy offspring by protecting mitochondrial function.

Melatonin (MT) regulates reproductive performance as a potent antioxidant; however, its beneficial effects on oocyte development remain largely unknown, especially in human oocytes. The collected 193 immature oocytes from the controlled ovarian hyperstimulation (COH) cycle underwent in vitro maturation (IVM) in IVM medium contained 10 µmol/L MT (n = 105, M group) and no MT (n = 88, NM group), followed by insemination and embryo culture. The results showed that the high-quality blastocyst formation rate in the M group (28.4%) was significantly higher than that in the NM group (2.0%) (P = .0001), and the aneuploidy rate was low (15.8%). In the subsequent clinical trials, three healthy infants were delivered. Next, single-cell RNA-seq data revealed 1026 differentially expressed genes (DEGs) between the two groups, KEGG enrichment analysis revealed that the majority of DEGs involved in oxidative phosphorylation pathway, which associated with ATP generation, was upregulated in the M group. Finally, confocal fluorescence staining results revealed that the mitochondrial membrane potential in the oocytes significantly increased and intracellular ROS and Ca2+ levels greatly decreased in the M group. Melatonin can promote the development of immature human oocytes retrieved from the COH cycle into healthy offspring by protecting mitochondrial function.

Effects of different polyvinylpyrrolidone concentrations on intracytoplasmic sperm injection.

To explore whether different polyvinylpyrrolidone (PVP) concentrations affect the results of intracytoplasmic sperm injection (ICSI), a prospective study was conducted for 194 couples undergoing 210 ICSI therapy cycles. These cycles were divided into three groups (10, 7 and 5% groups) using the corresponding concentration of PVP for sperm immobilization. The main outcome measures were analyzed. Results indicated that, with a decrease in PVP concentrations, all of the main outcome measures increased. In particular, the high-quality cleavage embryo rate in the 7% group was significantly lower than in the 5% group (P < 0.01), and the cleavage, high-quality cleavage embryo, and high-quality blastocyst rates in the 5% group were significantly higher than those in the 10% group (all P < 0.001). For high-/intermediate-quality semen, all of the main outcome measures were significantly increased with 5% PVP. For the poor-quality semen, only the high-quality cleavage embryo and high-quality blastocyst rates were significantly higher in the 5% group. Therefore, lowering PVP concentrations greatly promoted the development of embryos in ICSI cycles, with an optimal concentration of 5% for ICSI.

Cytotoxic properties of the alkaloid rutaecarpine and its oligocyclic derivatives and chemical modifications to enhance water-solubility.

The alkaloid rutaecarpine and its derivatives have been described as cytotoxic and hold potential as antitumor agents. Nevertheless, their synthesis is demanding and compounds display poor water solubility. Herein, we describe the synthesis of two sets of rutaecarpine derivatives with amine functions to improve solubility. Using a classic shake-flask experiment and a potentiometric titration platform, the water solubility of the compounds was determined. Solubility improved significantly with the amine functions connected over the indole-N atom. Reduction of metabolic activity and cell viability on HeLa cells was in the same range or better for these derivatives compared to the chemically unaltered parent compounds prepared in a new synthetic procedure established in our group.

Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Melatonin enhances the developmental potential of immature oocytes from older reproductive-aged women by improving mitochondrial function.

Aims: To evaluate whether melatonin (MT) supplementation during in vitro maturation (IVM) of human oocytes can reverse the age-related decline in oocyte quality. Main methods: We enrolled 172 patients aged ≥35 years (older reproductive-aged women) and 83 patients aged <35 years (young women) who underwent in vitro fertilization between 2019 and 2022. We conducted IVM with and without 10 µM MT in immature oocytes of different ages. Oocyte fertilization and embryo development were observed using a stereomicroscope. We assessed the immunofluorescence intensity of mitochondrial function, measured the copy number of mitochondrial DNA (mtDNA), and examined the spindle and chromosome composition in in vitro mature stage II (IVM-MII) oocytes using immunofluorescence and second-generation sequencing. Key findings: MT supplementation significantly improved the redox level in the IVM medium and IVM-MII oocytes in older reproductive-aged women. It also significantly increased the proportion of circular mtDNA and the adenosine triphosphate content in IVM-MII oocytes. In addition, the IVM-MII oocytes obtained with MT supplementation showed a significant improvement in the normal composition of the spindle and chromosomes. Thus, the aged immature oocytes also showed significantly improved maturation and blastocyst formation rates owing to the role of MT. Significance: Supplementation with 10 µM MT in the IVM medium reverses the age-related decline in oocyte quality. Our findings provide a viable solution for enhancing fertility in older reproductive-aged women.

HMGB1: a double-edged sword and therapeutic target in the female reproductive system.

HMGB1 that belongs to the High Mobility Group-box superfamily, is a nonhistone chromatin associated transcription factor. It is present in the nucleus of eukaryotes and can be actively secreted or passively released by kinds of cells. HMGB1 is important for maintaining DNA structure by binding to DNA and histones, protecting it from damage. It also regulates the interaction between histones and DNA, affecting chromatin packaging, and can influence gene expression by promoting nucleosome sliding. And as a DAMP, HMGB1 binding to RAGE and TLRs activates NF-κB, which triggers the expression of downstream genes like IL-18, IL-1ß, and TNF-α. HMGB1 is known to be involved in numerous physiological and pathological processes. Recent studies have demonstrated the significance of HMGB1 as DAMPs in the female reproductive system. These findings have shed light on the potential role of HMGB1 in the pathogenesis of diseases in female reproductive system and the possibilities of HMGB1-targeted therapies for treating them. Such therapies can help reduce inflammation and metabolic dysfunction and alleviate the symptoms of reproductive system diseases. Overall, the identification of HMGB1 as a key player in disease of the female reproductive system represents a significant breakthrough in our understanding of these conditions and presents exciting opportunities for the development of novel therapies.

Upregulated HMGB1 levels in maternal-fetal interface of patients with unexplained recurrent spontaneous abortion from different sources.

OBJECTIVE: To investigate the expression and sources of high mobility group box 1 (HMGB1) protein in the maternal-fetal interface of patients with unexplained recurrent spontaneous abortion (URSA), and further to verify the role of HMGB1 in the etiology of URSA. METHODS: 55 women at early pregnancy with URSA and 55 women undergoing selective termination of normal early pregnancy as control were included. The abortion tissues including villi and decidua were collected. The expression of HMGB1, CD45, CK7, and vimentin in abortion tissues was detected, and the localization and sources of HMGB1 were analyzed. RESULTS: Infiltrating immune cells and expression of HMGB1 were significantly increased in villi and decidua in URSA group compared with those in the control group. In the URSA group, HMGB1 was colocalized with the CD45-labeled immune cells, and it was more obvious in decidua than in villi; in addition, HMGB1 was colocalized with the vimentin-labeled decidual stromal cells, but not with the CK7- labeled villous epithelial cells. CONCLUSION: High expression of HMGB1 in the maternal-fetal interface in URSA patients was actively secreted by the infiltrating immune cells, and decidual stromal cells may passively release HMGB1 during necrosis.

Low-dose aspirin protects unexplained recurrent spontaneous abortion via downregulation of HMGB1 inflammation activation.

Background: High mobility group box protein 1 (HMGB1) is considered as a kind of sterile inflammatory mediators, which is an overexpression in patients with unexplained recurrent spontaneous abortion (URSA). Specific targeting effect of aspirin on HMGB1 has been revealed. Our previous studies have explored the application of HMGB1 as a therapeutic target of aspirin in URSA disease of mice model and human, but the dynamic process of aspirin downregulating HMGB1 concentration has not been demonstrated. Methods: From December 2018 to November 2020, women with URSA (n = 91) and control women (n = 90) with no history of recurrent abortion or adverse pregnancy were included in the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University. ELISA was applied to detect the concentrations of HMGB1 and IFN-γ in the peripheral blood. Thirty-one URSA patients were monitored for low-dose aspirin treatment (2 and 4 weeks), the changes of HMGB1 and IFN-γ concentrations in peripheral blood of URSA patients before and after using aspirin were compared, and pregnancy outcomes after aspirin treatment were followed up. Results: The levels of HMGB1 in peripheral blood were significantly higher in URSA patients compared with controls, decreasing trends of HMGB1 and IFN-γ concentrations in plasma of URSA patients were observed after treatment with low-dose aspirin continuously, and the expression of HMGB1 was positively correlated with IFN-γ. There were no birth abnormalities in the babies of the URSA patients treated with aspirin. Conclusions: High levels of HMGB1 may be one of the pathogenesis of URSA. Low-dose aspirin may provide protective effect on the HMGB1-triggered URSA.

Chirality and frequency measurement of longitudinal rolling of human sperm using optical trap.

Motility is one of the most critical features to evaluate sperm quality. As longitudinal rolling of human sperm has long been ignored until recently, its detailed dynamics and cellular biological mechanisms are still largely unknown. Here we report an optical-tweezers-based method to evaluate the chirality and frequency of sperm rotation. According to the intensity distribution patterns of off-focus micron-size particles, we established a method to judge the orientation of the sperm head along the optical axis in the optical trap. Together with the rotation direction of the projection of the sperm head, the chirality of longitudinal rolling of sperm can be measured without the application of three-dimensional tracking techniques or complex optical design. By video tracking optically trapped sperm cells from different patients, both rolling chirality and rolling frequency were analyzed. In this study, all the vertically trapped human sperm cells adopt a right-hand longitudinal rolling. The orientation and rolling frequency but not the rolling chirality of sperm in the optical trap are affected by the trap height. The rotation analysis method developed in this study may have clinical potential for sperm quality evaluation.

Melatonin Protects Mitochondrial Function and Inhibits Oxidative Damage against the Decline of Human Oocytes Development Caused by Prolonged Cryopreservation.

Melatonin (MT) can improve the effect of cryopreservation on oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma. In this study, MT was firstly applied to human oocytes' cryopreservation to explore the effect of prolonged cryopreservation on developmental competence and its role. Collected in vitro-matured human oocytes were cryopreserved in MT-containing or MT-free medium for 0 and 6 months; after warming, viable oocytes were assessed for developmental viability, intracellular protein expression, mitochondrial function, and oxidation-antioxidant system. Meanwhile, fresh oocytes were set as the control. The results showed that with the extension of cryopreservation time, the developmental competence of oocytes gradually declined, accompanied by the down-regulation of most mitochondrial function-related proteins, the reduction in ATP and GSH production, the increase in ROS accumulation, and the aggravation of the imbalance of ROS/GSH in oocytes. However, the participation of MT seemed to effectively mitigate these negative effects. Therefore, we speculate that melatonin may maintain normal ATP production and ROS/GSH balance in cryopreserved oocytes by protecting mitochondrial function and inhibiting oxidative damage, thereby effectively maintaining the developmental competence of human oocytes in prolonged cryopreservation.

Application of Two Blastocyst Biopsy Strategies in Preimplantation Genetic Testing Treatment and Assessment of Their Effects.

Background: Blastocyst biopsy has become the most mainstream biopsy method. Currently, there are two blastocyst biopsy strategies. Many studies have compared the advantages and disadvantages between blastomere and blastocyst biopsy, but fewer articles have compared the two blastocyst biopsy strategies. For the moment, no published studies have explored the entire set of information on embryo development, next-generation sequencing results, and clinical outcomes, including the baby's health status with the two blastocyst biopsy strategies. Methods: A total of 323 preimplantation genetic testing cycles from April 2018 to May 2020, including 178 cycles with Strategy A and 145 cycles with Strategy B. Strategy A was to create a laser-assisted zona pellucid opening for cleavage embryo on the third day after insemination, but Strategy B was not. Strategy A performed a biopsy for artificially assisted hatching blastocysts, while Strategy B performed a biopsy for expanded blastocysts on day 5 or 6. In this study, embryo development, next-generation sequencing results, pregnancy outcomes, and offspring health of the two strategies were compared and analyzed. Results: There were no statistical differences between the two groups in the rate of fertilization, blastocyst and abortion. The rate of cleavage from Strategy A was slightly higher than Strategy B, and the rate of high-quality cleavage embryo was lower than Strategy B, while the rate of high-quality blastocyst was higher than Strategy B. The rate of no-results blastocyst was significantly lower than Strategy B. In particular, the rate of biochemical pregnancy, clinical pregnancy, and live birth of Strategy A were significantly lower than those of Strategy B. The average Apgar scores of newborns were ≥8 in both groups, and there was no significant difference in average height and weight. In Strategy A, a baby was born with thumb syndactyly, and Strategy B had no congenital disabilities. Conclusions: Blastocyst biopsy strategy without laser-assisted zona pellucid drilling on day 3 achieves better clinical treatment effects. Therefore, Strategy B is an optimal treatment regime for PGT.

Comparison of IVF and IVM outcomes in the same patient treated with a modified IVM protocol along with an oocytes-maturing system containing melatonin: A pilot study.

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.

Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-ß1 Signaling.

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)-ß1 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-ß1. In addition, recombinant HMGB1 stimulation promoted release of active TGF-ß1 from cardiac fibroblasts and macrophages in vitro, and subsequent phosphorylation of Smad2 and Smad3 which were downstream of TGF-ß1 signaling. These data indicate that HMGB1 contributes to the CAV/fibrosis via promoting the activation of TGF-ß1/Smad signaling. Targeting HMGB1 might become a new therapeutic strategy for inhibiting cardiac allograft fibrosis and dysfunction.