Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility.

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility.

Fat mass and obesity-associated factor (FTO)-mediated N6-methyladenosine regulates spermatogenesis in an age-dependent manner.

N6-methyladenosine (m6A) is the most prevalent reversible RNA modification in the mammalian transcriptome. It has recently been demonstrated that m6A is crucial for male germline development. Fat mass and obesity-associated factor (FTO), a known m6A demethylase, is widely expressed in human and mouse tissues and is involved in manifold biological processes and human diseases. However, the function of FTO in spermatogenesis and male fertility remains poorly understood. Here, we generated an Fto knockout mouse model using CRISPR/Cas9-mediated genome editing techniques to address this knowledge gap. Remarkably, we found that loss of Fto in mice caused spermatogenesis defects in an age-dependent manner, resulting from the attenuated proliferation ability of undifferentiated spermatogonia and increased male germ cell apoptosis. Further research showed that FTO plays a vital role in the modulation of spermatogenesis and Leydig cell maturation by regulating the translation of the androgen receptor in an m6A-dependent manner. In addition, we identified two functional mutations of FTO in male infertility patients, resulting in truncated FTO protein and increased m6A modification in vitro. Our results highlight the crucial effects of FTO on spermatogonia and Leydig cells for the long-term maintenance of spermatogenesis and expand our understanding of the function of m6A in male fertility.

CFAP61 is required for sperm flagellum formation and male fertility in human and mouse.

Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G>A, causes exon skipping/intron retention in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and head. During early stages of Cfap61-/- spermatid development, the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted. This study reveals an organ-specific mechanism of axoneme stabilization that is related to male infertility.

Delayed femoral artery injury caused by heterotopic ossification: a rare case report and review of the literature.

BACKGROUND: Arterial injury caused by heterotopic ossification (HO) following fractures is rarely reported, yet it can have catastrophic consequences. This case report presents a unique instance of femoral artery injury and hematoma organization, occurring a decade after intramedullary nail fixation for a femoral shaft fracture complicated by HO. CASE PRESENTATION: A 56-year-old male presented with right femoral artery injury and organized hematoma, a decade after suffering bilateral femoral shaft fractures with mild head injury in a traffic accident. He had received intramedullary nailing for the right femoral shaft fracture and plate fixation for the left side in a local hospital. Physical examination revealed two firm, palpable masses with clear boundaries, limited mobility, and no tenderness. Peripheral arterial pulses were intact. Radiography demonstrated satisfactory fracture healing, while a continuous high-density shadow was evident along the inner and posterior aspect of the right thigh. Computed tomography angiography identified a large mixed-density mass (16.8 × 14.8 × 20.7 cm) on the right thigh's medial side, featuring central calcification and multiple internal calcifications. The right deep femoral artery coursed within this mass, with a smaller lesion noted on the posterior thigh. Surgical consultation with a vascular surgeon led to planned intervention. The smaller mass was completely excised, but the larger one partially, as it encased the femoral artery. The inability to remove all HO was due to excessive bleeding. Postoperatively, the patient experienced no complications, and one-year follow-up revealed a favorable recovery with restoration of full right lower limb mobility. CONCLUSION: This case underscores the potential gravity of vascular injury associated with heterotopic ossification. Surgeons should remain vigilant regarding the risk of vascular injury during HO excision.

Wheat gluten deamidation: structure, allergenicity and its application in hypoallergenic noodles.

BACKGROUND: Wheat gluten (WG) containing gliadin and glutenin are considered the main allergens in wheat allergy as a result of their glutamine-rich peptides. Deamidation is a viable and efficient approach for protein modifications converting glutamine into glutamic acid, which may have the potential for allergenicity reduction of WG. RESULTS: Deamidation by citric acid was performed to investigate the effects on structure, allergenicity and noodle textural properties of wheat gluten (WG). WG was heated at 100 °C in 1 m citric acid to yield deamidated WG with degrees of deamidation (DD) ranging from DWG-25 (25% DD) to DWG-70 (70% DD). Fourier-transform infrared and intrinsic fluorescence spectroscopy results suggested the unfolding of WG structure during deamidation, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed molecular weight shifts at the 35-63 kDa region, suggesting that the deamidation mainly occurred on low molecular weight glutenin subunits and γ- gliadin of the WG. An enzyme-linked immunosorbent assay of deamidated WG revealed a decrease in absorbance and immunoblotting indicated that the intensities of protein bands at 35-63 kDa decreased, which suggested that deamidation of WG might have caused a greater loss of epitopes than the generation of new epitopes caused by unfolding of WG, and thereby reduction of the immunodominant immunoglobulin E binding capacity, ultimately leading to the decrease in allergenicity. DWG-25 was used in the preparation of recombinant hypoallergenic noodles, and the hardness, elasticity, chewiness and gumminess were improved significantly by the addition of azodicarbonamide. CONCLUSION: The present shows the potential for deamidation of the WG products used in novel hypoallergenic food development. © 2023 Society of Chemical Industry.

Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse.

Motile cilia and flagellar defects can result in primary ciliary dyskinesia, which is a multisystemic genetic disorder that affects roughly 1:10 000 individuals. The nexin-dynein regulatory complex (N-DRC) links neighboring doublet microtubules within flagella, serving as a central regulatory hub for motility in Chlamydomonas. Herein, we identified two homozygous DRC1 variants in human patients that were associated with multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility. Drc1-/-, Drc1R554X/R554X and Drc1W244X/W244X mice on the C57BL/6 background suffered from pre-pubertal mortality. However, when the ICR background was introduced, some of these mice were able to survive and recapitulate the MMAF phenotypes detected in human patients. By analyzing these animals, we determined that DRC1 is an essential regulator of N-DRC assembly in cilia and flagella. When DRC1 is absent, this results in the shortening of cilia and consequent impairment of their motility. Damage associated with DRC1 deficiency in sperm flagella was more pronounced than in cilia, as manifested by complete axoneme structural disorder in addition to the loss of the DRC structure. Altogether, these findings suggest that DRC1 is required for the structural stability of flagella but not cilia, emphasizing the key role of this protein in mammalian species.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.

Cinematic Rendering of Complex Coronary Artery Left Atrial Fistula.

Supplemental material is available for this article.

Identification of novel first-trimester serum biomarkers for early prediction of preeclampsia.

BACKGROUND: Preeclampsia (PE) is a leading cause of maternal and perinatal mortality and morbidity worldwide, but effective early prediction remains a challenge due to the lack of reliable biomarkers. METHODS: Based on the extensive human biobank of our large-scale assisted reproductive cohort platform, the first-trimester serum levels of 48 cytokines, total immunoglobulins (Igs), anti-phosphatidylserine (aPS) antibodies, and several previously reported PE biomarkers [including placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and activin A] were measured in 34 women diagnosed with PE and 34 matched normotensive controls. RESULTS: The PE group has significantly higher first-trimester serum levels of interleukin (IL)-2Rα, IL-9, tumor necrosis factor-ß (TNF-ß), RANTES, hepatocyte growth factor (HGF), total IgM, and total IgG, and aPS IgG optical density (OD) value, as well as lower first-trimester serum levels of PlGF and total IgA and aPS-IgG immune complexes (IC) OD value than the control group. Combining top five first-trimester serum biomarkers (total IgM, total IgG, PlGF, aPS IgG, and total IgA) achieved superior predictive value [area under the curve (AUC) and 95% confidence interval (CI) 0.983 (0.952-1.000), with a sensitivity of 100% and a specificity of 94.1%] for PE development compared to PlGF and PlGF/sFlt-1 independently [AUC and 95% CI 0.825 (0.726-0.924) and 0.670 (0.539-0.800), respectively]. CONCLUSION: We identified novel first-trimester serum biomarkers and developed an effective first-trimester prediction model using immune-related factors and PlGF for PE, which could facilitate the development of early diagnostic strategies and provide immunological insight into the further mechanistic exploration of PE.

Functional screening of congenital heart disease risk loci identifies 5 genes essential for heart development in zebrafish.

Congenital heart disease (CHD) is the most common birth defect worldwide and a main cause of perinatal and infant mortality. Our previous genome-wide association study identified 53 SNPs that associated with CHD in the Han Chinese population. Here, we performed functional screening of 27 orthologous genes in zebrafish using injection of antisense morpholino oligos. From this screen, 5 genes were identified as essential for heart development, including iqgap2, ptprt, ptpn22, tbck and maml3. Presumptive roles of the novel CHD-related genes include heart chamber formation (iqgap2 and ptprt) and atrioventricular canal formation (ptpn22 and tbck). While deficiency of maml3 led to defective cardiac trabeculation and consequent heart failure in zebrafish embryos. Furthermore, we found that maml3 mutants showed decreased cardiomyocyte proliferation which caused a reduction in cardiac trabeculae due to inhibition of Notch signaling. Together, our study identifies 5 novel CHD-related genes that are essential for heart development in zebrafish and first demonstrates that maml3 is required for Notch signaling in vivo.

Clinical outcomes of subtypes of mosaic single aneuploid embryos after preimplantation genetic testing for aneuploidy.

OBJECTIVE: Mosaic embryos are often characterized by different numbers (single or double or ≥ 3 aneuploidies) or types of chromosomal abnormalities (monosomy or trisomy and involving whole chromosome or chromosome segments). However, due to limitations in the number of samples, the relationship between these abnormalities and clinical outcomes is often not evaluated. METHODS: This study analyzed chromosomal abnormalities and clinical outcomes in 591 aneuploid mosaic and 3071 euploid embryos from multiple retrospective cohorts as well as from the current authors' unpublished retrospective cohort. RESULTS: Through meta-analysis, it was found that single aneuploid mosaicism reduced implantation and clinical pregnancy rates. In addition, no significant differences were noted between mosaic trisomies and mosaic monosomies in terms of their effects on implantation and clinical pregnancy rates. All subtypes of single aneuploid mosaicism were found to reduce implantation and clinical pregnancy rates for women of over 35 years old. Furthermore, it was observed that all subtypes of single aneuploid in higher-level mosaicism reduced implantation and clinical pregnancy rates. Regarding the lower-level group, only segmental mosaicism with segmental chromosome gain reduced both of the above rates. Unexpectedly, the type of chromosome abnormality was more likely to influence miscarriage rates compared with the level of mosaicism. Indeed, monosomy aneuploid mosaic embryos increased miscarriage rates in both lower- and higher-levels mosaic ratio groups, but not other subtypes. CONCLUSIONS: Although the mechanism for the above phenomenon remains unknown, it is recommended that attention should still be paid to the increased miscarriage rates caused by monosomy in aneuploid mosaic embryos.

The extraction and identification of active components of the sex pheromones of Asian citrus psyllid, Diaphorina citri.

In this study, we extracted and identified the active components of the Asian citrus psyllid, Diaphorina citri sex pheromones to provide a basis for further development of sex attractants. Under laboratory conditions, mating activity in D. citri started 3 d after emergence, which peaked at 6-7 d, and mating activity had no obvious peak during the observed period 7:00-21:00 h. Additionally, D. citri males were attracted to the emanations from conspecific females, especially to the n-hexane extracts of the pheromone. A total of 17 compounds were identified from the n-hexane extracts of female and male D. citri by gas chromatography-mass spectrometer (GC-MS). Among them, 13 compounds were identified from the female D. citri n-hexane extracts, of which 7 (dichloromethane, acetic acid, toluene, butyl acetate, ethyl carbamoylacetate, α-pinene, and 1-nonanal) were not found in the male D. citri n-hexane extracts. In addition, a total of 33 compounds were identified from the solid phase microextraction (SPME) volatiles of the male and female D. citri adults. Among these, 17 compounds were identified from the female D. citri volatiles, of which 6 (cycloheptatriene, 5-methyl-2-phenylindole, 1-dodecanol, cis-11-hexadecena, dodecyl aldehyde, and nerylacetone) were not identified in the volatiles of the D. citri males. It was found that males were significantly attracted to 0.1-10 µL/mL acetic acid and 1-nonanal with the selection rates ranging from 62.04%-70.56% and 62.22%-67.22%, respectively. Therefore, the results of this study suggest that acetic acid and 1-nonanal might be the active compounds of the female D. citri sex pheromones.

Echocardiographic Characteristics of Pulmonary Artery Intimal Sarcoma: Comparison With CTPA.

OBJECTIVES: This study examined the echocardiographic characteristics of patients with pulmonary artery intimal sarcoma (PAIS) and compared the results with those from computed tomographic pulmonary angiography (CTPA). METHOD: Twenty-six (26) patients were diagnosed with PAIS at the current institution during the study period, and 23 were eligible for analysis. Echocardiography and CTPA examinations were performed in all enrolled patients. RESULTS: The echocardiography results showed that most lesions had expansive growth in the left pulmonary artery (PA); the right PA; or a combination of the left PA, right PA, and main PA, with extension to the pulmonary valve and/or right ventricular outflow tract. These lesions also had distinctive sieve-like echogenic signals. Echocardiography also showed that some lesions had lobulated shapes, were nearly round and echolucent or with calcifications, and moved during imaging. The lesion distribution was similar in CTPA and echocardiography (p=0.361), but CTPA was more sensitive in detection of the complete shape (p=0.023). CONCLUSIONS: The unique echocardiographic characteristics of PAIS, especially the "sieve sign", could help in the diagnosis of this cancer. Transthoracic echocardiography is a non-invasive technique that appears effective in detecting PAIS.

Homozygous missense mutation in CCDC155 disrupts the transmembrane distribution of CCDC155 and SUN1, resulting in non-obstructive azoospermia and premature ovarian insufficiency in humans.

Non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) represent the most serious forms of human infertility caused by gametogenic failure. Although whole-exome sequencing (WES) has uncovered multiple monogenic causes of human infertility, our knowledge of the genetic basis of human gametogenesis defects remains at a rudimentary stage. Coiled-coil-domain-containing protein 155 (CCDC155) encodes a core component of the linker of the nucleoskeleton and cytoskeleton complex that is essential for modulating telomere-led chromosome movements during the meiotic prophase of mice. Additionally, Ccdc155 deficiency in mice causes infertility in both sexes with meiotic arrest. In this study, we applied WES to identify the pathogenic genes for 15 NOA and POI patients whose parents were consanguineous and identified a novel homozygous missense mutation in CCDC155 [c.590T>C (p.Leu197Pro)] in a pair of familial NOA and POI patients whose parents were first cousins. The affected spermatocytes were unable to complete meiotic division coupled with unresolved repair of the DNA double-strand break. This rare missense mutation with lesions in the conserved CC domain of CCDC155 blocked nuclear envelope (NE) distribution and subsequently prevented NE-specific enrichment of Sad1- and UNC84-domain-containing 1 either ex vivo or in vitro, eventually leading to disruptive NE anchoring of chromosome-induced meiotic arrest in both sexes. This study presents the first evidence of the necessity of the SUN1-CCDC155 complex during human meiosis and provides insight into the CCDC155 CC domain, thereby expanding the genetic spectrum of human NOA and POI and promoting adequate genetic counselling and appropriate fertility guidance for these patients.

ZDHHC19 localizes to the cell membrane of spermatids and is involved in spermatogenesis†.

Sperm is the ultimate executor of male reproductive function. Normal morphology, quantity, and motility of sperm ensure the normal reproductive process. Palmitoylation is a posttranslational modification mediated by palmitoyltransferases whereby palmitoyl is added to proteins. Seven palmitoyltransferases have been identified in Saccharomyces cerevisiae and 23 in humans (including ZDHHC1-9 and ZDHHC11-24), with corresponding homologs in mice. We identified two testis-specific palmitoyltransferases ZDHHC11 and ZDHHC19 in mice. The Zdhhc11 and Zdhhc19-knockout mouse models were constructed, and it was found that the Zdhhc11 knockout males were fertile, while Zdhhc19 knockout males were sterile. ZDHHC19 is located in the cell membrane of step 4-9 spermatids in the mouse testis, and phenotypic analysis showed that the testicular weight ratio in the Zdhhc19-/- mice decreased along with the number and motility of the sperm decreased, while sperm abnormalities increased, mainly due to the "folded" abnormal sperm caused by sperm membrane fusion, suggesting the involvement of ZDHHC19 in maintaining membrane stability in the male reproductive system. In addition, Zdhhc19-/- mice showed abnormal sperm morphologies and apoptosis during spermatogenesis, suggesting that spermatogenesis in the Zdhhc19-/- mice was abnormal. These results indicate that ZDHHC19 promotes membrane stability in male germ cells.

PABPN1L assemble into "ring-like" aggregates in the cytoplasm of MII oocytes and is associated with female infertility†.

Infertility affects 10-15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A recent study showed that poly(A)binding protein nuclear 1-like (PABPN1L) recruited BTG anti-proliferation factor 4 (BTG4) to mRNA 3'-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated a PABPN1L-antibody and found "ring-like" PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed "ring-like" aggregates in vitro. This phenomenon is similar with CCR4-NOT catalytic subunit, CCR4-NOT transcription complex subunit 7 (CNOT7), when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l-/- and Pabpn1l  tm1a/tm1a) simulating the intron 1-exon 2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l-/- MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and CCR4-NOT transcription complex subunit 6 like (Cnot6l) genes are necessary for the deadenylation process and Pabpn1l-/- resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4-NOT-mediated maternal mRNA degradation and PABPN1L variants on intron 1-exon 2 could be a genetic marker of female infertility.

A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

The 18S rRNA m6 A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation.

RNA modifications represent a novel layer of regulation of gene expression. Functional experiments revealed that N6 -methyladenosine (m6 A) on messenger RNA (mRNA) plays critical roles in cell fate determination and development. m6 A mark also resides in the decoding center of 18S ribosomal RNA (rRNA); however, the biological function of m6 A on 18S rRNA is still poorly understood. Here, we report that methyltransferase-like 5 (METTL5) methylates 18S rRNA both in vivo and in vitro, which is consistent with previous reports. Deletion of Mettl5 causes a dramatic differentiation defect in mouse embryonic stem cells (mESCs). Mechanistically, the m6 A deposited by METTL5 is involved in regulating the efficient translation of F-box and WD repeat domain-containing 7 (FBXW7), a key regulator of cell differentiation. Deficiency of METTL5 reduces FBXW7 levels and leads to the accumulation of its substrate c-MYC, thereby delaying the onset of mESC differentiation. Our study uncovers an important role of METTL5-mediated 18S m6 A in mESC differentiation through translation regulation and provides new insight into the functional significance of rRNA m6 A.

Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis.

BACKGROUND: Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood. METHODS: We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system. RESULTS: Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure. CONCLUSION: Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

Clinical value of resting cardiac dual-energy CT in patients suspected of coronary artery disease.

BACKGROUND: Rest/stress myocardial CT perfusion (CTP) has high diagnostic value for coronary artery disease (CAD), but the additional value of resting CTP especially dual-energy CTP (DE-CTP) beyond coronary CT angiography (CCTA) in chest pain triage remains unclear. We aimed to evaluate the diagnostic accuracy of resting myocardial DE-CTP, and additional value in detecting CAD beyond CCTA (obstructive stenosis: ≥ 50%) in patients suspected of CAD. METHODS: In this prespecified subanalysis of 54 patients, we included patients suspected of CAD referred to invasive coronary angiography (ICA). Diagnostic accuracy of resting myocardial DE-CTP in detecting myocardial perfusion defects was assessed using resting 13N-ammonia positron emission tomography (PET) as the gold standard. Diagnostic accuracy of cardiac dual-energy CT in detecting flow-limiting stenoses (justifying revascularization) by CCTA combined with resting myocardial DE-CTP, using ICA plus resting 13N-ammonia PET as the gold standard. The CCTA and DE-CTP datasets derived from a single-phase scan performed with dual-energy mode. RESULTS: For detecting myocardial perfusion defects, DE-CTP demonstrated high diagnostic accuracy with a sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) of 95.52%, 85.93%, and 0.907 on a per-segment basis. For detecting flow-limiting stenoses by CCTA alone, sensitivity, specificity, and AUC were 100%, 56.47%, and 0.777 respectively on a per-vessel basis. For detecting flow-limiting stenoses by CCTA combined with resting myocardial DE-CTP, sensitivity, specificity, and AUC were 96.10%, 95.29% and 0.956 respectively on a per-vessel basis. Additionally, CCTA combined with resting myocardial DE-CTP detected five patients (9%) with no obstructive stenosis but with myocardial perfusion defects confirmed by ICA plus 13N-ammonia PET. CONCLUSIONS: Resting cardiac DE-CTP demonstrates a high diagnostic accuracy in detecting myocardial perfusion defects and provides an additional clinical value by reducing rates of false-positive and false-negative patients beyond CCTA in patients suspected of CAD.