Klinefelter syndrome: Etiology and clinical considerations in male infertility.

Klinefelter syndrome (KS) is the most prevalent chromosomal disorder occurring in males. It is defined by an additional X chromosome, 47,XXY, resulting from errors in chromosomal segregation during parental gametogenesis. A major phenotype is impaired reproductive function, in the form of low testosterone and infertility. This review comprehensively examines the genetic and physiological factors contributing to infertility in KS, in addition to emergent assisted reproductive technologies, and the unique ethical challenges KS patients face when seeking infertility treatment. The pathology underlying KS is increased susceptibility for meiotic errors during spermatogenesis, resulting in aneuploid or even polyploid gametes. Specific genetic elements potentiating this susceptibility include polymorphisms in checkpoint genes regulating chromosomal synapsis and segregation. Physiologically, the additional sex chromosome also alters testicular endocrinology and metabolism by dysregulating interstitial and Sertoli cell function, collectively impairing normal sperm development. Additionally, epigenetic modifications like aberrant DNA methylation are being increasingly implicated in these disruptions. We also discuss assisted reproductive approaches leveraged in infertility management for KS patients. Application of assisted reproductive approaches, along with deep comprehension of the meiotic and endocrine disturbances precipitated by supernumerary X chromosomes, shows promise in enabling biological parenthood for KS individuals. This will require continued multidisciplinary collaboration between experts with background of genetics, physiology, ethics and clinical reproductive medicine.

[The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction]. / N6-ç”²åŸºè ºå˜Œå‘¤ä¿®é¥°åŠå ¶è°ƒæŽ§å› å­åœ¨ç”·æ€§ç”Ÿæ®–ä¸­çš„ä½œç”¨.

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/ß-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model. METHODS: The study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens. RESULTS: The results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αß T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment. CONCLUSIONS: Vγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e.,  Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.

Identification of a heterozygous variant of ZP2 as a novel cause of empty follicle syndrome in humans and mice.

STUDY QUESTION: Is a recurrent heterozygous mutation in ZP2, c.1925G>A (p.R642Q), associated with the Empty follicle syndrome (EFS)? SUMMARY ANSWER: ZP2, c.1925G>A (p.R642Q), led to female infertility related to EFS in humans and mice and resulted in ZP2 accumulation in the cytoplasm of oocytes. WHAT IS KNOWN ALREADY: EFS is a complex disease defined as a complete failure of oocyte retrieval after ovarian stimulation and after repeated aspirations and flushing of mature ovarian follicles. Furin-mediated cleavage is a post-translational modification (PTM) involved in various physiological processes, but the clear role of PTM mediated by furin cleavage of ZP2 protein on female fertility needs to be further explored. PTM is required for proteins to function in physiological conditions, and its perturbation has been linked to a growing number of human pathologies. Zona pellucida (ZP) proteins, which are important for oocyte development, are regulated post-translationally by well-characterized glycosylation events, as well as by furin-mediated cleavage. However, knowledge of the relevance of the consensus furin cleavage site of ZP proteins in female reproduction remains lacking. STUDY DESIGN, SIZE, DURATION: This was a basic medical research project to assess the pathogenicity of a heterozygous mutation in the ZP2 gene in EFS. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 3 families with EFS and a control group 2213 women with proven fertility. Whole-exome sequencing detected a heterozygous mutation in the ZP2 gene in all EFS patients. The mouse strain Zp2Arg635Gln/+ (ZP2R642Q) was generated by CRISPR-Cas9-mediated genome editing. RNA-sequencing was applied to investigate transcriptional changes in the ovaries of heterozygous ZP2R642Q knock-in (KI) mice compared to WT mice. MAIN RESULTS AND THE ROLE OF CHANCE: We found a heterozygous mutation of ZP2, c.1925G>A (p.R642Q), in unrelated females with EFS, which was inherited in an autosomal-dominant manner. We used CRISPR-Cas9 to generate a mouse model encoding the orthologous variant of ZP2R642Q detected in humans, and the female ZP2R642Q KI mice recapitulated the human EFS phenotype. We further found the decreased expression of key genes involved in oocyte maturation in ZP2R642Q KI mice compared to WT mice by RNA-sequencing analysis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only three families affected by EFS with the mutation were available because of its rare incidence. Although we have found different expressions of the several indispensable genes related to oocyte development between WT mice and ZP2R642Q KI mice through RNA-sequencing analysis, the specific regulatory mechanisms of the oocyte apoptosis in ZP2R642Q KI mice need to be studied further. WIDER IMPLICATIONS OF THE FINDINGS: These results are expected to open new avenues for researchers in the exploration of potential therapeutic strategies in treating EFS. STUDY FUNDING/COMPETING INTEREST(S): This project is funded by the National Key Research and Development Program of China (2018YFC1002804, 2017YFC1001500 and 2016YFC1000200). All authors declared no competing interests. TRIAL REGISTRATION NUMBER: N/A.

B7-H4 expression is upregulated by PKCδ activation and contributes to PKCδ-induced cell motility in colorectal cancer.

INTRODUCTION: B7-H4 is overexpressed in colorectal cancer (CRC) and plays an important role in tumor growth and immunosuppression. However, the exact mechanism that regulates B7-H4 expression remains largely unknown. Here, we investigated whether protein kinase C δ (PKCδ) regulates the expression of B7-H4 in CRC. METHODS: By using immunohistochemical (IHC) and immunofluorescence (IF) staining, we analyzed the expression of B7-H4 and phospho-PKCδ (p-PKCδ) in 225 colorectal tumor samples and determined the clinical significance of the expression patterns. In vitro experiments were performed with the CRC cell lines HCT116 and SW620 to detect the effect of PKCδ activation on B7-H4 expression, and xenograft-bearing mice were treated with rottlerin to monitor the expression of B7-H4 and tumor metastasis. RESULTS: The B7-H4 expression level was significantly correlated with the p-PKCδ level (r = 0.378, P < 0.001) in tumor tissues. Coexpression of p-PKCδ and B7-H4 was significantly associated with moderate/poor differentiation (P = 0.024), lymph node metastasis (P = 0.001) and advanced Dukes' stage (P = 0.002). Western blot analysis showed that Phorbol-12-Myristate-13-Acetate (TPA) increased B7-H4 expression in a concentration-dependent manner and that rottlerin abrogated the TPA-induced increase in B7-H4 expression. The protein levels of B7-H4 and p-STAT3 were significantly reduced by a PKCδ-specific siRNA. Moreover, the STAT3 inhibitor cryptotanshinone significantly decreased the B7-H4 protein level in CRC cells. Knockdown of B7-H4 or PKCδ suppressed cell migration and motility. Rottlerin also inhibited B7-H4 expression and tumor metastasis in vivo. CONCLUSION: The B7-H4 expression level is significantly correlated with the p-PKCδ level and tumor metastasis in CRC samples. B7-H4 expression is upregulated by STAT3 activation via PKCδ and plays roles in PKCδ-induced cancer cell motility and metastasis, suggesting that the PKCδ/STAT3/B7-H4 axis may be a potential therapeutic target for CRC.

A loss-of-function variant in SSFA2 causes male infertility with globozoospermia and failed oocyte activation.

Globozoospermia (OMIM: 102530) is a rare type of teratozoospermia (< 0.1%). The etiology of globozoospermia is complicated and has not been fully revealed. Here, we report an infertile patient with globozoospermia. Variational analysis revealed a homozygous missense variant in the SSFA2 gene (NM_001130445.3: c.3671G > A; p.R1224Q) in the patient. This variant significantly reduced the protein expression of SSFA2. Immunofluorescence staining showed positive SSFA2 expression in the acrosome of human sperm. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and Coimmunoprecipitation (Co-IP) analyses identified that GSTM3 and Actin interact with SSFA2. Further investigation revealed that for the patient, regular intracytoplasmic sperm injection (ICSI) treatment had a poor prognosis. However, Artificial oocyte activation (AOA) by a calcium ionophore (A23187) after ICSI successfully rescued the oocyte activation failure for the patient with the SSFA2 variant, and the couple achieved a live birth. This study revealed that SSFA2 plays an important role in acrosome formation, and the homozygous c.3671G > A loss-of-function variant in SSFA2 caused globozoospermia. SSFA2 may represent a new gene in the genetic diagnosis of globozoospermia, especially the successful outcome of AOA-ICSI treatment for couples, which has potential value for clinicians in their treatment regimen selections.

PRSS55 is a novel potential causative gene for human male infertility.

RESEARCH QUESTION: Testis-specific PRSS55 is a chymotrypsin-like serine protease that is highly conserved among mammalian species. The essential role of Prss55 in mouse male fertility has been established. What is the role of PRSS55 in human reproduction? DESIGN: Whole exome sequencing was used to identify the genetic cause in an infertile male with teratozoospermia. Papanicolaou staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to explore morphological defects in the patient's spermatozoa. Immunofluorescence staining and western blot analysis were conducted to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist the patient with fertilization. RESULTS: Sanger sequencing of the pedigree demonstrated that the infertile man carried a novel homozygous mutation in PRSS55 (c.575C>T [p.A192V]). Morphological defects in the sperm head, neck, midpiece and tail were demonstrated by Papanicolaou staining, SEM and TEM. Immunofluorescence staining and western blotting of the patient's spermatozoa showed that the point mutation changed the conformation of PRSS55 and caused a sharp decrease in the PRSS55 protein concentration. The expression and subcellular localization of PRSS55 in the testis and spermatozoa of mice and humans showed that PRSS55 was expressed in the head and flagella of spermatids and epididymal spermatozoa. Moreover, ICSI treatment for this kind of infertile patient was shown to be effective. CONCLUSIONS: These findings revealed a novel mutation in PRSS55 in an infertile patient, suggesting for the first time the crucial role of PRSS55 in human fertility. This study provides new insight into genetic counselling diagnoses and subsequent treatment for male infertility.

Minimal change disease associated with gastrointestinal stromal tumor accompanied by significantly elevated serum IgE level: a case report.

BACKGROUND: Minimal change disease (MCD) is a common cause of the nephrotic syndrome. Several studies have shown an increased incidence of cancer in patients with MCD. However, there are no reports on the association between MCD and gastrointestinal stromal tumor (GIST). CASE PRESENTATION: We report a case of a 66-year-old female with severe nephrotic syndrome and concomitant duodenal GIST. Immunoglobulin test showed a significant increase of IgE levels. The diagnosis of renal histopathology was MCD with subacute tubulointerstitial injury. The combination of preoperative Imatinib mesylate chemotherapy and tumor excision was accompanied by significant remission of proteinuria, and IgE level decreasing, without immunosuppressivetherapy. CONCLUSIONS: It is the first case report that MCD was associated with GIST and elevated IgE level. Clinically, in patients with elevated IgE level associated with nephrotic syndrome, the possibility of tumor must be taken into account when allergic factors are excluded.

B7-H3 confers resistance to Vγ9Vδ2 T cell-mediated cytotoxicity in human colon cancer cells via the STAT3/ULBP2 axis.

Immunotherapy based on γδT cells has limited efficiency in solid tumors, including colon cancer (CC). The immune evasion of tumor cells may be the main cause of the difficulties of γδT cell-based treatment. In the present study, we explored whether and how B7-H3 regulates the resistance of CC cells to the cytotoxicity of Vγ9Vδ2 (Vδ2) T cells. We observed that B7-H3 overexpression promoted, while B7-H3 knockdown inhibited, CC cell resistance to the killing effect of Vδ2 T cells in vitro and in vivo. Mechanistically, we showed that B7-H3-mediated CC cell resistance to the cytotoxicity of Vδ2 T cells involved a molecular pathway comprising STAT3 activation and decreased ULBP2 expression. ULBP2 blockade or knockdown abolished the B7-H3 silencing-induced increase in the cytotoxicity of Vδ2 T cells to CC cells. Furthermore, cryptotanshinone, a STAT3 phosphorylation inhibitor, reversed the B7-H3 overexpression-induced decrease in ULBP2 expression and attenuated the killing effect of Vδ2 T cells on CC cells. Moreover, there was a negative correlation between the expression of B7-H3 and ULBP2 in the tumor tissues of CC patients. Our results suggest that the B7-H3-mediated STAT3/ULBP2 axis may be a potential candidate target for improving the efficiency of γδT cell-based immunotherapy in CC.

Establishment of a novel double-monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA): tool for human B7-H4 detection in autoimmune diseases.

B7-H4, one of the immunoregulatory proteins, plays an inhibitory role by inhibiting T cell proliferation and cytokine production. Nevertheless, the significance of soluble B7-H4 (sB7-H4) in autoimmune diseases is unclear. In our study, we developed two novel mouse anti-human B7-H4 monoclonal antibodies (mAbs) (clones 8D4 and 7E1) with utilities for flow cytometry, immunoblotting and immunofluorescence. We characterized 7E1 as a functional antibody with antagonistic activity, which could promote T cell proliferation and regulate cytokine production. Furthermore, based on the different epitope specificities, we established a novel enzyme-linked immunosorbent assay (ELISA) which could detect sB7-H4 sensitively and specifically. Using this ELISA kit, sB7-H4 was observed in a high proportion of autoimmune diseases patients. We found that the levels of sB7-H4 were significantly higher in patients with systemic lupus erythematosus (SLE), type I diabetes (T1D) and Graves' disease (GD). Together, sB7-H4 in human serum is regarded not only as a regulator of T cell activation but may also be a diagnostic marker of autoimmune diseases.

Circular RNA hsa_circ_0003288 induces EMT and invasion by regulating hsa_circ_0003288/miR-145/PD-L1 axis in hepatocellular carcinoma.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has been associated with wound healing, tumorigenesis, and metastasis. Circular RNAs (circRNAs) are functional non-coding RNAs involved in multiple human cancers. However, whether and how circRNAs contribute to the EMT in hepatocellular carcinomas (HCC) remains to be deciphered. In this study, we investigated the regulation and function of hsa_circ_0003288 on programmed death-1 ligand 1 (PD-L1) during EMT and HCC invasiveness. METHODS: Hsa_circ_0003288 expression was measured by real-time quantitative reverse transcriptase PCR (qRT-PCR). Luciferase reporter assays, RNA pull-down assay and fluorescence in situ hybridization (FISH) were used to determine the correlation between hsa_circ_0003288 and miR-145 and between miR-145 and PD-L1. Furthermore, ectopic overexpression and siRNA-mediated downregulation of hsa_circ_0003288, transwell assays, and in vivo studies were used to determine the function of hsa_circ_0003288 on the EMT and invasiveness of L02 and HCC cells. RESULTS: miR-145 directly targeted the PD-L1 3'-untranslated region (UTR) region, and hsa_circ_0003288 acted as a miR-145 sponge to regulate PD-L1 expression. Overexpression of hsa_circ_0003288 increased PD-L1 levels and promoted EMT, migration, and invasiveness of L02 cells. These observations were reversed after knockdown of hsa_circ_0003288 in HepG2 and Huh7 cells. Overexpression of PD-L1 rescued EMT, migration, and invasiveness of HepG2 and Huh7 cells after knockdown of hsa_circ_0003288. Furthermore, hsa_circ_0003288 knockdown reduced EMT in in vivo studies. Hsa_circ_0003288/PD-L1 axis was found to mediate the metastatic phenotypes via the PI3K/Akt pathway in HCC. Additionally, expression levels of hsa_circ_0003288 were increased and positively correlated with PD-L1 expression in HCC tissues. CONCLUSION: Our findings demonstrated that hsa_circ_0003288 promoted EMT and invasion of HCC via the hsa_circ_0003288/miR-145/PD-L1 axis through the PI3K/Akt pathway. Targeting hsa_circ_0003288 may be a therapeutic strategy for the treatment of HCC.

Novel biallelic loss-of-function variants in CEP290 cause Joubert syndrome in two siblings.

BACKGROUND: Joubert syndrome (JS) is a rare genetic disorder, which can be defined by brain stem malformation, cerebellar vermis hypoplasia, and consequent "molar tooth sign" (MTS). JS always shares variety of phenotypes in development defects. With the development of next-generation sequencing, dozens of causative genes have been identified to JS so far. Here, we investigated two male siblings with JS and uncovered a novel pathogenesis through combined methods. RESULTS: The siblings shared similar features of nystagmus, disorders of intellectual development, typical MTS, and abnormal morphology in fourth ventricle. Whole-exome sequencing (WES) and chromosome comparative genomic hybridization (CGH) were then performed on the proband. Strikingly, a maternal inherited nonsense variant (NM_025114.3: c.5953G>T [p.E1985*]) in CEP290 gene and a paternal inherited deletion in 12q21.32 including exons 1 to 10 of CEP290 gene were identified in the two affected siblings. We further confirmed the two variants by in vitro experiments: quantitative PCR and PCR sequencing. CONCLUSIONS: In this study, we first reported a novel causative mechanism of Joubert syndrome: a copy number variation (CNV) combined with a single-nucleotide variant in CEP290 gene, which can be helpful in the genetic diagnosis of this disease.

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient.

RESEARCH QUESTION: Male infertility is a widespread symptom in patients with primary ciliary dyskinesia (PCD). PCD-related male infertility is often caused by asthenozoospermia, with barely normal sperm morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenozoospermia, characterized by various malformed morphologies of sperm flagella. To date, a limited number of genes have been suggested to be involved in the pathogenesis of both PCD and MMAF. What other genes associated with both PCD and MMAF are waiting to be discovered? DESIGN: Whole-exome sequencing (WES) was performed to identify the pathogenic mutation associated with MMAF in a PCD patient. Peripheral venous blood and semen samples were collected from the PCD patient. Transmission electron microscopy (TEM), immunofluorescence staining and western blotting were conducted to confirm the pathogenicity of the identified mutation. RESULTS: A novel homozygous mutation in CCDC39, c.983 T>C (p. Leu328Pro), was identified in two PCD-affected siblings of a consanguineous family showing a typical PCD phenotype, while the proband was infertile, which is associated with characterized MMAF. Furthermore, TEM revealed the abnormal ultrastructure of the patient's sperm flagella. Moreover, immunofluorescence staining revealed that CCDC39 was almost undetectable in the spermatozoa, which was further confirmed by western blotting. The outcome of intracytoplasmic sperm injection (ICSI) in the patient with the CCDC39 mutation was also favourable. CONCLUSION: This study demonstrates that a novel loss-of-function mutation of CCDC39 is involved in the pathogenesis of PCD and MMAF and initially reported that ICSI treatment has a good outcome. Therefore, the novel variant of CCDC39 contributes to the genetic diagnosis, counselling and treatment of male infertility in PCD patients with MMAF phenotype.

Tim-3 suppresses the killing effect of Vγ9Vδ2 T cells on colon cancer cells by reducing perforin and granzyme B expression.

Gamma delta (γδ) T cell-based tumor immunotherapy has been one of the most promising cancer immunotherapeutic strategies. However, the key regulators of the Vγ9Vδ2 T cell-mediated antitumor response remain unclear. Recently, mounting reports have indicated that Tim-3 performs critical roles in the regulation of the activities of immune cells, including Vγ9Vδ2 T cells. However, the roles of Tim-3 in Vγ9Vδ2 T cell-mediated killing of colon cancer cells and the underlying mechanism remain largely unknown. Here, the proportion of Tim-3+ γδ T cells was significantly increased in both the peripheral blood and colon cancer tissue of patients and was significantly associated with TNM staging and tumor volume. Additionally, the activation of Tim-3 signaling significantly inhibited the killing efficiency of Vγ9Vδ2 T cells against colon cancer cells. In addition, Tim-3 signaling reduced the expression of perforin and granzyme B in Vγ9Vδ2 T cells. Blocking the perforin/granzyme B pathway also decreased the cytotoxicity of Vγ9Vδ2 T cells to colon cancer cells. Moreover, Tim-3 signaling reduced the perforin and granzyme B expression of Vγ9Vδ2 T cells in an ERK1/2 signaling pathway-dependent manner. This knowledge reveals that Tim-3 may be a promising therapeutic target to improve Vγ9Vδ2 T cell-based adoptive immunotherapy for colon cancer.

The effect of a novel LRRC6 mutation on the flagellar ultrastructure in a primary ciliary dyskinesia patient.

PURPOSE: There are limited genes known to cause primary ciliary dyskinesia (PCD)-associated asthenozoospermia. In the present study, we aimed to expand the spectrum of mutations in PCD and to provide new information for genetic counseling diagnoses and the treatment of male infertility in PCD. METHODS: One sterile patient with typical situs inversus was recruited to our center, and semen sample was collected. We performed whole-exome sequencing (WES) on the patient to identify the pathogenic mutations associated with PCD and used transmission electron microscopy to investigate spermatozoal ultrastructure. In addition, western blotting and immunofluorescence staining were used to confirm the untoward impact of the variant on the expression of LRRC6, as well as on the dynein arm proteins in the patient's spermatozoa. RESULTS: We identified a homozygous nonsense variant c.749G>A (p.W250*) of LRRC6 in the PCD patient. This variant severely impaired LRRC6 expression and further led to negative effects on dynein arm protein expression in the spermatozoa of the affected individual, which eventually caused defects in sperm ultrastructure and motility. Moreover, we are the first to report a positive prognosis using intracytoplasmic sperm injection (ICSI) for LRRC6-associated male infertility. CONCLUSIONS: Our findings strongly implicated the homozygous mutation of c.749G>A (p.W250*) in LRRC6 as a new genetic cause of PCD, uncovering its involvement in defective sperm flagella and poor sperm motility. Furthermore, we posit that patients with LRRC6 mutations may have good outcomes with ICSI treatment. These findings add to the literature on the genetic diagnoses and treatment of male infertility associated with PCD.

[Homozygous CFAP65 mutation induces multiple morphological abnormalities of sperm flagella: A preliminary genetic study].

OBJECTIVE: To search for the possible pathogenic genes for multiple morphological anomalies of sperm flagella (MMAF). METHODS: We performed whole exome sequencing (WES) of a typical case of MMAF and analyzed its possible pathogenic genes. We examined the semen sample from the patient and identified the ultrastructural characteristics of the sperm flagella under the scanning electron and transmission electron microscopes, and analyzed the expression pattern of cilia and flagela-associated protein 65 (CFAP65) in spermatogenesis by immunofluorescence assay. RESULTS: The MMAF patient was found with a homozygous pathogenic mutation of the CFAP65 gene c.2675G>A(p.Trp892*). Scanning electron microscopy showed that the sperm of the patient had typical characteristics of MMAF, that is, without tails or with folded tails, curly tails, short tails or irregular tails. Transmission electron microscopy revealed the loss and disorder of the "9+2" structure in the sperm flagellum, with abnormal assembly of the fibrous sheath, accompanied by loss of central microtubules and dynamin arms. Cellular immunofluorescence assay suggested that the CFAP65 gene was expressed at all levels of mouse germ cells. CONCLUSIONS: The CFAP65 gene is involved in the assembly of the sperm flagellum structure, and its mutation can cause the phenotype of MMAF, leading to male infertility.

Loss-of-function mutation in DNAH8 induces asthenoteratospermia associated with multiple morphological abnormalities of the sperm flagella.

Asthenozoospermia is a common cause of male infertility associated with the reduced motility and/or abnormal morphology of spermatozoa, although its etiology remains incompletely understood. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the main causes of asthenozoospermia. However, the MMAF-associated genes identified to date cannot explain all the human MMAF cases. Herein, a loss-of-function mutation of DNAH8 was identified in an asthenozoospermia patient with MMAF. Moreover, the negative effect of this mutation on DNAH8 expression was confirmed by immunofluorescence staining and western blotting. Remarkably, it is the first time that DNAH8 is suggested to be associated with human MMAF. Our findings provide strong evidence that a loss-of-function mutation in DNAH8 can cause male infertility with MMAF and that DNAH8 is essential for sperm flagellar formation.

Evaluation of the role of B7-H3 haplotype in association with impaired B7-H3 expression and protection against type 1 diabetes in Chinese Han population.

BACKGROUND: Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disorder caused by the destruction of insulin-secreting cells. B7-H3 (CD276) plays a vital role in T cell response. However, B7-H3 expression and its clinical significance in T1D remain unclear. The aim of this study was to investigate the correlations between the expression of B7-H3 and clinical parameters in T1D patients. The possible role of B7-H3 gene variants with T1D was also discussed. METHODS: Four B7-H3 single nucleotide polymorphisms (SNPs) were genotyped in 121 T1D patients and 120 healthy controls by polymerase chain reaction (PCR) direct sequencing. Expression of membrane B7-H3 (mB7-H3) in peripheral blood lymphocytes was determined by flow cytometry. Levels of soluble B7-H3 (sB7-H3) in serum were analyzed by enzyme linked immunosorbent assay (ELISA). RESULTS: The B7-H3 haplotype T-A-C-T was less frequently observed in T1D patients compared to the controls (OR: 0.31, 95% CI: 0.16-0.61). B7-H3 expression on monocytes showed significant upregulation in T1D patients and was positively correlated with several clinical features including ALT, fast C-peptide 120 min, HbAlc, IFN-γ, IL-6 and TNF-α (P < 0.05). The concentration of sB7-H3 in serum increased in T1D patients (P < 0.0001). We also observed that B7-H3-T-A-C-T was associated with the decreased release of sB7-H3 but not the membrane form. CONCLUSIONS: B7-H3 may act as a potential biomarker related to the pathogenesis of T1D. The B7-H3-T-A-C-T polymorphism variant is associated with the low risk of T1D as well as less release of sB7-H3.

The breeding and the performance of 'Liaoning 4' walnut cultivar.

Walnut is an important cultivated tree with high economic value and wide distribution across China. The objective was to breed a new walnut cultivar with high yield and desirable nut and tree qualities that could meet the demands of the walnut industry in China. 'Liaoning 4' walnut cultivar is lateral bearing which originated from a controlled cross between the Persian walnut (J. regia) selections 'Liaoning Chaoyang big and rough walnut' and '11001'. During long term assessment and research, 'Liaoning 4' walnut performed well and it was released as a new cultivar in China on 15 Oct. 2018. It has a high yield and excellent nut traits and has thus been widely planted in North China.

Novel biallelic loss-of-function variants in ZP1 identified in an infertile female with empty follicle syndrome.

PURPOSE: Empty follicle syndrome (EFS) refers to the inability to obtain mature oocytes after appropriate ovarian stimulation during the process of in vitro fertilization (IVF). However, the specific cause and mechanism of action underlying EFS remain to be further explored. Herein we aimed to investigate the clinical and genetic characteristics of EFS. METHODS: After data were collected in an infertile family, we performed whole-exome sequencing (WES) on the patient and confirmed the pathogenic mutations through Sanger sequencing. Western immunoblotting, immunofluorescence staining, and minigene assay were further used to investigate the negative effects of these mutations. RESULTS: Absence of oocytes was observed over 2 cycles of IVF in the patient, and we evaluated the novel compound heterozygous mutations c.2T>A (p. M1K) and c.1112+1G>T of the zona pellucida glycoprotein 1 gene (ZP1, MIM# 195000) by WES. For the family under study, EFS was classified as an autosomal recessive inheritance pattern. The results of western blotting and immunofluorescence staining analyses confirmed that the missense mutation of c.2T>A [p. M1K] resulted in almost missing protein production. Additionally, using a minigene assay, we demonstrated the deleterious effect on the ZP1 gene of the splice site mutation c.1112+1G>T, which caused truncation of ZP1 protein. CONCLUSIONS: The compound heterozygous mutations of ZP1 gene identified in this study by genetic and functional experiments constituted a novel genetic cause of EFS, and further study will expand its use in the clinical and molecular diagnoses of EFS.