Deficiency of primate-specific SSX1 induced asthenoteratozoospermia in infertile men and cynomolgus monkey and tree shrew models.

Primate-specific genes (PSGs) tend to be expressed in the brain and testis. This phenomenon is consistent with brain evolution in primates but is seemingly contradictory to the similarity of spermatogenesis among mammals. Here, using whole-exome sequencing, we identified deleterious variants of X-linked SSX1 in six unrelated men with asthenoteratozoospermia. SSX1 is a PSG expressed predominantly in the testis, and the SSX family evolutionarily expanded independently in rodents and primates. As the mouse model could not be used for studying SSX1, we used a non-human primate model and tree shrews, which are phylogenetically similar to primates, to knock down (KD) Ssx1 expression in the testes. Consistent with the phenotype observed in humans, both Ssx1-KD models exhibited a reduced sperm motility and abnormal sperm morphology. Further, RNA sequencing indicated that Ssx1 deficiency influenced multiple biological processes during spermatogenesis. Collectively, our experimental observations in humans and cynomolgus monkey and tree shrew models highlight the crucial role of SSX1 in spermatogenesis. Notably, three of the five couples who underwent intra-cytoplasmic sperm injection treatment achieved a successful pregnancy. This study provides important guidance for genetic counseling and clinical diagnosis and, significantly, describes the approaches for elucidating the functions of testis-enriched PSGs in spermatogenesis.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Cooperation of long noncoding RNA LOC100909675 and transcriptional regulator CTCF modulates Cdk1 transcript to control astrocyte proliferation.

Astrocyte activation and proliferation contribute to glial scar formation during spinal cord injury (SCI), which limits nerve regeneration. The long noncoding RNAs (lncRNAs) are involved in astrocyte proliferation and act as novel epigenetic regulators. Here, we found that lncRNA-LOC100909675 (LOC9675) expression promptly increased after SCI and that reducing its expression decreased the proliferation and migration of the cultured spinal astrocytes. Depletion of LOC9675 reduced astrocyte proliferation and facilitated axonal regrowth after SCI. LOC9675 mainly localized in astrocytic nuclei. We used RNA-seq to analyze gene expression profile alterations in LOC9675-depleted astrocytes and identified the cyclin-dependent kinase 1 (Cdk1) gene as a hub candidate. Our RNA pull-down and RNA immunoprecipitation assays showed that LOC9675 directly interacted with the transcriptional regulator CCCTC-binding factor (CTCF). Dual-luciferase reporter and chromatin immunoprecipitation assays, together with downregulated/upregulated expression investigation, revealed that CTCF is a novel regulator of the Cdk1 gene. Interestingly, we found that with the simultaneous overexpression of CTCF and LOC9675 in astrocytes, the Cdk1 transcript was restored to the normal level. We then designed the deletion construct of LOC9675 by removing its interacting region with CTCF and found this effect disappeared. A transcription inhibition assay using actinomycin D revealed that LOC9675 could stabilize Cdk1 mRNA, while LOC9675 depletion or binding with CTCF reduced Cdk1 mRNA stability. These data suggest that the cooperation between CTCF and LOC9675 regulates Cdk1 transcription at a steady level, thereby strictly controlling astrocyte proliferation. This study provides a novel perspective on the regulation of the Cdk1 gene transcript by lncRNA LOC9675.

Histone acetyltransferase KAT2A modulates neural stem cell differentiation and proliferation by inducing degradation of the transcription factor PAX6.

Neural stem cells (NSCs) proliferation and differentiation rely on proper expression and posttranslational modification of transcription factors involved in the determination of cell fate. Further characterization is needed to connect modifying enzymes with their transcription factor substrates in the regulation of these processes. Here, we demonstrated that the inhibition of KAT2A, a histone acetyltransferase, leads to a phenotype of small eyes in the developing embryo of zebrafish, which is associated with enhanced proliferation and apoptosis of NSCs in zebrafish eyes. We confirmed that this phenotype is mediated by the elevated level of PAX6 protein. We further verified that KAT2A negatively regulates PAX6 at the protein level in cultured neural stem cells of rat cerebral cortex. We revealed that PAX6 is a novel acetylation substrate of KAT2A and the acetylation of PAX6 promotes its ubiquitination mediated by the E3 ligase RNF8 that facilitated PAX6 degradation. Our study proposes that KAT2A inhibition results in accelerated proliferation, delayed differentiation, or apoptosis, depending on the context of PAX6 dosage. Thus, the KAT2A/PAX6 axis plays an essential role to keep a balance between the self-renewal and differentiation of NSCs.

Adenine base editor-mediated splicing remodeling activates noncanonical splice sites.

Adenine base editors (ABEs) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. The discovery of split genes revealed that all introns contain two highly conserved dinucleotides, canonical "AG" (acceptor) and "GT" (donor) splice sites. ABE can directly edit splice acceptor sites of the adenine (A) base, leading to aberrant gene splicing, which may be further adopted to remodel splicing. However, spliced isoforms triggered with ABE have not been well explored. To address it, we initially generated a cell line harboring C-terminal enhanced GFP (eGFP)-tagged ß-actin (ACTB), in which the eGFP signal can track endogenous ß-actin expression. Expectedly, after the editing of splice acceptor sites, we observed a dramatical decrease in the percentage of eGFP-positive cells and generation of splicing products with the noncanonical splice site. Furthermore, we manipulated Peroxidasin in mouse embryos with ABE, in which a noncanonical acceptor was activated to remodel splicing, successfully generating a mouse disease model of anophthalmia and severely malformed microphthalmia. Collectively, we demonstrate that ABE-mediated splicing remodeling can activate a noncanonical acceptor to manipulate human and mouse genomes, which will facilitate the investigation of basic and translational medicine studies.

Homozygous pathogenic variants in ACTL9 cause fertilization failure and male infertility in humans and mice.

Total fertilization failure (TFF) can occur during in vitro fertilization (IVF) treatments, even following intracytoplasmic sperm injection (ICSI). Various male or female factors could contribute to TFF. Increasing evidence suggested that genetic variations in PLCZ1, which encodes 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta-1 (PLCζ), is involved in oocyte activation and is a key male factor in TFF. In the present study, we explored the genetic variants in male individuals that led to TFF. A total of 54 couples with TFF or poor fertilization (fertilization rate < 20%) were screened, and 21 couples were determined to have a male infertility factor by the mouse oocyte activation test. Whole-exome sequencing of these 21 male individuals identified three homozygous pathogenic variants in ACTL9 (actin like 9) in three individuals. ACTL9 variations led to abnormal ultrastructure of the perinuclear theca (PT), and PLCζ was absent in the head and present in the neck of the mutant sperm, which contributed to failed normal calcium oscillations in oocytes and subsequent TFF. The key roles of ACTL9 in the PT structure and TFF after ICSI were further confirmed in an Actl9-mutated mouse model. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in a couple with an ACTL9 variant. These findings identified the role of ACTL9 in the PT structure and the correct localization of PLCζ. The results also provide a genetic marker and a therapeutic option for individuals who have undergone ICSI without successful fertilization.

Bi-allelic mutations of DNAH10 cause primary male infertility with asthenoteratozoospermia in humans and mice.

Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated genes, highlighting the condition's genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia.

Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

SARS-CoV-2 infection is detrimental to pregnancy outcomes after embryo transfer in IVF/ICSI: a prospective cohort study.

BACKGROUND: To explore whether SARS-CoV-2 infection affects the pregnancy outcomes of assisted reproductive techniques (ART). METHODS: A prospective cohort study recruited patients for embryo transfer from December 01, 2022, to December 31, 2022. All patients were closely followed up for SARS-CoV-2 infection after embryo transfer. The SARS-CoV-2 "diagnosed group" was defined as RNA or antigen-positive. The SARS-CoV-2 "suspected infection group" was defined as having apparent SARS-CoV-2 symptoms without an RNA or antigen test, while the "uninfected group" was defined as having a negative SARS-CoV-2 RNA or antigen test and no SARS-CoV-2 symptoms. RESULTS: A total of 1330 patients participated in the study, 687 of whom were in the SARS-CoV-2 diagnosed group, 219 in the suspected infection group, and 424 in the uninfected group. There was no significant difference in basic characteristics among the three groups. The clinical pregnancy rate was 68% in the SARS-CoV-2 diagnosed group, 63% in the uninfected group, and 51% in the suspected infection group (P < 0.001). The ongoing pregnancy rate was 58% in the SARS-CoV-2 diagnosed group, 53% in the uninfected group, and 45% in the suspected infection group (P < 0.001). Upon analyzing the factors influencing clinical pregnancy, it was found that suspected infection (odds ratio [OR] 0.618, 95% CI 0.444-0.862, P = 0.005) and the short time (≤ 22 days) between embryo transfer and SARS-CoV-2 infection (OR 3.76, 95% CI 1.92-8.24, P < 0.001) were not conducive to clinical pregnancy. In addition, the concurrent presence of fever and dizziness/headache SARS-CoV-2 symptoms (OR 0.715, 95% CI 0.526-0.972, P = 0.032) decreased the clinical pregnancy rate. However, vaccination administered 2-3 times (OR 1.804, 95% CI 1.332-2.444, P < 0.001) was associated with an improvement in clinical pregnancy rate. CONCLUSIONS: This prospective cohort study shows that SARS-CoV-2 infection in a short period of time after embryo transfer is not conducive to clinical pregnancy. Reproductive physicians should advise patients to avoid SARS-CoV-2 infection shortly after embryo transfer. Meanwhile, women should be encouraged to vaccinate at least 2-3 times before embryo transfer or pregnancy.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

Human umbilical cord-derived mesenchymal stem cell transplantation improves the long COVID.

No effective treatments can ameliorate symptoms of long COVID patients. Our study assessed the safety and efficacy of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in the treatment of long COVID patients. Ten long COVID patients were enrolled and received intravenous infusions of UC-MSCs on Days 0, 7, and 14. Adverse events and clinical symptoms were recorded, and chest-high-resolution CT (HRCT) images and laboratory parameters were analyzed. During UC-MSCs treatment and follow-up, we did not observe serious adverse events, the symptoms of long COVID patients were significantly relieved in a short time, especially sleep difficulty, depression or anxiety, memory issues, and so forth, and the lung lesions were also repaired. The routine laboratory parameters did not exhibit any significant abnormalities following UC-MSCs transplantation (UMSCT). The proportion of regulatory T cells gradually increased, but it was not statistically significant until 12 months. The proportion of naive B cells was elevated, while memory B cells, class-switched B-cells, and nonswitched B-cells decreased at 1 month after infusion. Additionally, we observed a transient elevation in circulating interleukin (IL)-6 after UMSCT, while tumor necrosis factor (TNF)-α, IL-17A, and IL-10 showed no significant changes. The levels of circulating immunoglobulin (Ig) M increased significantly at month 2, while IgA increased significantly at month 6. Furthermore, the SARS-CoV-2 IgG levels remained consistently high in all patients at Month 6, and there was no significant decrease during the subsequent 12-month follow-up. UMSCT was safe and tolerable in long COVID patients. It showed potential in alleviating long COVID symptoms and improving interstitial lung lesions.

A hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

Testing the generalizability and effectiveness of deep learning models among clinics: sperm detection as a pilot study.

BACKGROUND: Deep learning has been increasingly investigated for assisting clinical in vitro fertilization (IVF). The first technical step in many tasks is to visually detect and locate sperm, oocytes, and embryos in images. For clinical deployment of such deep learning models, different clinics use different image acquisition hardware and different sample preprocessing protocols, raising the concern over whether the reported accuracy of a deep learning model by one clinic could be reproduced in another clinic. Here we aim to investigate the effect of each imaging factor on the generalizability of object detection models, using sperm analysis as a pilot example. METHODS: Ablation studies were performed using state-of-the-art models for detecting human sperm to quantitatively assess how model precision (false-positive detection) and recall (missed detection) were affected by imaging magnification, imaging mode, and sample preprocessing protocols. The results led to the hypothesis that the richness of image acquisition conditions in a training dataset deterministically affects model generalizability. The hypothesis was tested by first enriching the training dataset with a wide range of imaging conditions, then validated through internal blind tests on new samples and external multi-center clinical validations. RESULTS: Ablation experiments revealed that removing subsets of data from the training dataset significantly reduced model precision. Removing raw sample images from the training dataset caused the largest drop in model precision, whereas removing 20x images caused the largest drop in model recall. by incorporating different imaging and sample preprocessing conditions into a rich training dataset, the model achieved an intraclass correlation coefficient (ICC) of 0.97 (95% CI: 0.94-0.99) for precision, and an ICC of 0.97 (95% CI: 0.93-0.99) for recall. Multi-center clinical validation showed no significant differences in model precision or recall across different clinics and applications. CONCLUSIONS: The results validated the hypothesis that the richness of data in the training dataset is a key factor impacting model generalizability. These findings highlight the importance of diversity in a training dataset for model evaluation and suggest that future deep learning models in andrology and reproductive medicine should incorporate comprehensive feature sets for enhanced generalizability across clinics.

Fenton-like reaction triggered chemical redox-cycling signal amplification for ultrasensitive fluorometric detection of H2O2 and glucose.

An ultrasensitive fluorescent biosensor is reported for glucose detection based on a Fenton-like reaction triggered chemical redox-cycling signal amplification strategy. In this amplified strategy, Cu2+ oxidizes chemically o-phenylenediamine (OPD) to generate photosensitive 2,3-diaminophenazine (DAP) and Cu+/Cu0. On the one hand, the generated Cu0 catalyzes the oxidation of OPD. On the other hand, H2O2 reacts with Cu+ to produce hydroxyl radicals (˙OH) and Cu2+ through a Cu+-mediated Fenton-like reaction. The generated ˙OH and recycled Cu2+ ions take turns oxidizing OPD to produce more photoactive DAP, triggering a self-sustaining chemical redox-cycling reaction and a remarkable fluorescent enhancement. It is worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Benefiting from H2O2-triggered chemical redox-cycling signal amplification, the strategy was exploited for the development of an ultrasensitive fluorescent biosensor for glucose determination. Glucose content monitoring was realized with a linear range from 1 nM to 1 µM and a limit of detection of 0.3 nM. This study validates the practicability of the chemical redox-cycling signal amplification on the fluorescent bioanalysis of glucose in human serum samples. It is expected that the method offers new opportunities to develop ultrasensitive fluorescent analysis strategy.

Biallelic CFAP61 variants cause male infertility in humans and mice with severe oligoasthenoteratozoospermia.

BACKGROUND: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. OBJECTIVE: To identify the genetic cause of male infertility characterised by OAT. METHODS: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. RESULTS: We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.

Biallelic mutations in CFAP54 cause male infertility with severe MMAF and NOA.

BACKGROUND: Spermatogenic impairments can lead to male infertility by different pathological conditions, such as multiple morphological abnormalities of the sperm flagella (MMAF) and non-obstructive azoospermia (NOA). Genetic factors are involved in impaired spermatogenesis. METHODS AND RESULTS: Here, we performed genetic analyses through whole-exome sequencing in a cohort of 334 Han Chinese probands with severe MMAF or NOA. Biallelic variants of CFAP54 were identified in three unrelated men, including one homozygous frameshift variant (c.3317del, p.Phe1106Serfs*19) and two compound heterozygous variants (c.878G>A, p.Arg293His; c.955C>T, p.Arg319Cys and c.4885C>T, p.Arg1629Cys; c.937G>A, p.Gly313Arg). All of the identified variants were absent or extremely rare in the public human genome databases and predicted to be damaging by bioinformatic tools. The men harbouring CFAP54 mutations exhibited abnormal sperm morphology, reduced sperm concentration and motility in ejaculated semen. Significant axoneme disorganisation and other ultrastructure abnormities were also detected inside the sperm cells from men harbouring CFAP54 mutations. Furthermore, immunofluorescence assays showed remarkably reduced staining of four flagellar assembly-associated proteins (IFT20, IFT52, IFT122 and SPEF2) in the spermatozoa of CFAP54-deficient men. Notably, favourable clinical pregnancy outcomes were achieved with sperm from men carrying CFAP54 mutations after intracytoplasmic sperm injection treatment. CONCLUSION: Our genetic analyses and experimental observations revealed that biallelic deleterious mutations of CFAP54 can induce severe MMAF and NOA in humans.

Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Evaluation of genetic risk of apparently balanced chromosomal rearrangement carriers by breakpoint characterization.

PURPOSE: To report genetic characteristics and associated risk of chromosomal breaks due to chromosomal rearrangements in large samples. METHODS: MicroSeq, a technique that combines chromosome microdissection and next-generation sequencing, was used to identify chromosomal breakpoints. Long-range PCR and Sanger sequencing were used to precisely characterize 100 breakpoints in 50 ABCR carriers. RESULTS: In addition to the recurrent regions of balanced rearrangement breaks in 8q24.13, 11q11.23, and 22q11.21 that had been documented, we have discovered a 10-Mb region of 12q24.13-q24.3 that could potentially be a sparse region of balanced rearrangement breaks. We found that 898 breakpoints caused gene disruption and a total of 188 breakpoints interrupted genes recorded in OMIM. The percentage of breakpoints that disrupted autosomal dominant genes recorded in OMIM was 25.53% (48/188). Fifty-four of the precisely characterized breakpoints had 1-8-bp microhomologous sequences. CONCLUSION: Our findings provide a reference for the evaluation of the pathogenicity of mutations in related genes that cause protein truncation in clinical practice. According to the characteristics of breakpoints, non-homologous end joining and microhomology-mediated break-induced replication may be the main mechanism for ABCRs formation.