Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

Distinct brain state dynamics of native and second language processing during narrative listening in late bilinguals.

The process of complex cognition, which includes language processing, is dynamic in nature and involves various network modes or cognitive modes. This dynamic process can be manifested by a set of brain states and transitions between them. Previous neuroimaging studies have shed light on how bilingual brains support native language (L1) and second language (L2) through a shared network. However, the mechanism through which this shared brain network enables L1 and L2 processing remains unknown. This study examined this issue by testing the hypothesis that L1 and L2 processing is associated with distinct brain state dynamics in terms of brain state integration and transition flexibility. A group of late Chinese-English bilinguals was scanned using functional magnetic resonance imaging (fMRI) while listening to eight short narratives in Chinese (L1) and English (L2). Brain state dynamics were modeled using the leading eigenvector dynamic analysis framework. The results show that L1 processing involves more integrated states and frequent transitions between integrated and segregated states, while L2 processing involves more segregated states and fewer transitions. Our work provides insight into the dynamic process of narrative listening comprehension in late bilinguals and sheds new light on the neural representation of language processing and related disorders.

A novel variant in CFAP69 causes asthenoteratozoospermia with treatable ART outcomes and a literature review.

PURPOSE: Multiple morphological abnormalities of the sperm flagella (MMAF) are a severe form of sperm defect causing male infertility. Previous studies identified the variants in the CFAP69 gene as a MMAF-associated factor, but few cases have been reported. This study was performed to identify additional variants in CFAP69 and describe the semen characteristics and outcomes of assisted reproductive technology (ART) in CFAP69-affected couples. METHODS: Genetic testing with next-generation sequencing (NGS) panel of 22 MMAF-associated genes and Sanger sequencing was performed in a cohort of 35 infertile males with MMAF to identify pathogenic variants. Morphological, ultrastructural, and immunostaining analyses were performed to investigate the characteristics of probands' spermatozoa. ART with intracytoplasmic sperm injection (ICSI) was carried out for the affected couples to get their own progenies. RESULTS: We identified a novel frameshift variant in CFAP69 (c.2061dup, p. Pro688Thrfs*5) from a MMAF-affected infertile male with low sperm motility and malformed morphology of sperm. Furthermore, transmission electron microscopy and immunofluorescence staining revealed that the variant induced the aberrant ultrastructure and reduction of CFAP69 expression in the proband's spermatozoa. Moreover, the partner of the proband birthed a healthy girl through ICSI. CONCLUSIONS: This study expanded the variant spectrum of CFAP69 and described the good outcome of ART treatment with ICSI, which is beneficial to the molecular diagnosis, genetic counseling, and treatment of infertile males with MMAF in the future.

FMRP binds Per1 mRNA and downregulates its protein expression in mice.

FMRP, an RNA-binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue-dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function.

Patients with MMAF induced by novel biallelic CFAP43 mutations have good fertility outcomes after intracytoplasmic sperm injection.

As a specific type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by composite abnormalities, including absent, short, coiled, angulation, and irregular-caliber flagella. Mutations in cilia- and flagella-associated protein 43 ( CFAP43 ) are one of the main causative factors of MMAF established to date. To identify whether there are other CFAP43 mutations related to MMAF and to determine the clinical outcomes of assisted reproductive technology for patients with MMAF harboring different mutations, we recruited and screened 30 MMAF-affected Chinese men using a 22-gene next-generation sequencing panel. After systematic analysis, seven mutations in CFAP43 , including five novel mutations and two previously reported mutations, were identified from four families and related to MMAF in an autosomal recessive pattern. Papanicolaou staining, immunofluorescence, and electronic microscopy further clarified the semen characteristics and abnormal sperm morphologies, including disorganized axonemal and peri-axonemal structures, of the CFAP43 -deficient men. The female partners of two patients were pregnant after undergoing assisted reproductive technology through intracytoplasmic sperm injection, and one of them successfully gave birth to a healthy boy. This study significantly expands the mutant spectrum of CFAP43 , and together with the available information regarding male infertility and MMAF, provides new information for the genetic diagnosis and counseling of MMAF in the future.

Brain fingerprints along the language hierarchy.

Recent studies have shown that the brain functional connectome constitutes a unique fingerprint that allows the identification of individuals from a group. However, what information encoded in the brain that makes us unique remains elusive. Here, we addressed this issue by examining how individual identifiability changed along the language hierarchy. Subjects underwent fMRI scanning during rest and when listening to short stories played backward, scrambled at the sentence level, and played forward. Identification for individuals was performed between two scan sessions for each task as well as between the rest and task sessions. We found that individual identifiability tends to increase along the language hierarchy: the more complex the task is, the better subjects can be distinguished from each other based on their whole-brain functional connectivity profiles. A similar principle is found at the functional network level: compared to the low-order network (the auditory network), the high-order network is more individualized (the frontoparietal network). Moreover, in both cases, the increase in individual identifiability is accompanied by the increase in inter-subject variability of functional connectivities. These findings advance the understanding of the source of brain individualization and have potential implications for developing robust connectivity-based biomarkers.

Circadian Rhythm Sleep Disorders: Genetics, Mechanisms, and Adverse Effects on Health.

Nearly all living organisms, from cyanobacteria to humans, have an internal circadian oscillation with a periodicity of approximately 24 h. In mammals, circadian rhythms regulate diverse physiological processes including the body temperature, energy metabolism, immunity, hormone secretion, and daily sleep-wake cycle. Sleep is tightly regulated by circadian rhythms, whereas a misalignment between the circadian rhythms and external environment may lead to circadian rhythm sleep disorders (CRSD). CRSD includes four main kinds of disorders: the advanced sleep-wake phase disorder (ASPD), the delayed sleep-wake phase disorder (DSPD), the irregular sleep-wake rhythm disorder and the non-24-h sleep-wake rhythm disorder. Recent studies have begun to shed light on the genetic basis of CRSD. Deciphering the genetic codes for ASPD and DSPD has so far been more successful than the other CRSDs, which allow for the development of animal models and understanding of the pathological mechanisms for these disorders. And studies from humans or animal models implicate CRSDs are associated with adverse health consequences, such as cancer and mental disorders. In this review, we will summarize the recent advances in the genetics, underlying mechanisms and the adverse effects on health of ASPD and DSPD.

Concurrent Newborn Hearing and Genetic Screening in a Multi-Ethnic Population in South China.

Hearing loss is a common sensory deficit in humans with intricate genomic landscape and mutational signature. Approximately 1-3 out of 1,000 newborns have hearing loss and up to 60% of these cases have a genetic etiology. In this study, we conducted the concurrent newborn hearing and genetic screening in 20 mutations (18 pathogenic variants in GJB2, SLC26A4, and MT-RNR1 and 2 uncertain clinical significance variants in GJB3) for 9,506 normal newborns (4,977 [52.4%] males) from 22 ethnic population in South China. A total of 1,079 (11.4%) newborns failed to pass the initial hearing screening; 160 (1.7%) infants failed to pass the re-screening, and 135 (1.4%) infants presented the diagnostic hearing loss. For the genetic screening, 220 (2.3%) newborns who presented at least one of the screened mutations were more likely to fail the hearing screening and have diagnostic hearing loss than mutation-negative newborns. In comparison to the differences of distribution of mutations, we did not identify any significant difference in the prevalence of screened mutations between Han group (n = 5,265) and Zhuang group (n = 3,464), despite the lack of number of minority ethnic groups. Studies including larger number of minority ethnic populations are needed in the future.

Comparison of Two-Step Transient Evoked Otoacoustic Emissions and One-Step Automated Auditory Brainstem Response for Universal Newborn Hearing Screening Programs in Remote Areas of China.

Objective: To compare the hearing screening results of two-step transient evoked otoacoustic emissions (TEOAE) and one-step automatic auditory brainstem response (AABR) in non-risk newborns, and to explore a more suitable hearing screening protocol for infants discharged within 48 h after birth in remote areas of China. Methods: To analyze the age effect on pass rate for hearing screening, 2005 newborns were divided into three groups according to screening time after birth: <24, 24-48, and 48-72 h. All subjects received TEOAE + AABR test as first hearing screen, and those who failed in any test were rescreened with TEOAE + AABR at 6 weeks after birth. The first screening results of AABR and TEOAE were compared among the three groups. The results of two-step TEOAE screening and one-step AABR screening were compared for newborns who were discharged within 48 h. The time spent on screening was recorded for TEOAE and AABR. Results: The pass rate of TEOAE and AABR increased significantly with the increase of first screening time (P < 0.05), and the false positive rate decreased significantly with the increase of first screening time (P < 0.05). The failure rate of first screening of AABR within 48 h was 7.31%, which was significantly lower than that of TEOAE (9.93%) (P < 0.05). The average time spent on AABR was 12.51 ± 6.36 min, which was significantly higher than that of TEOAE (4.05 ± 1.56 min, P < 0.05). The failure rate of TEOAE two-step screening was 1.59%, which was significantly lower than one-step AABR. Conclusions: Compared with TEOAE, AABR screening within 48 h after birth can reduce the failure rate and false positive rate of first screening. However, compared with TEOAE two-step screening, one-step AABR screening has higher referral rate for audiological diagnosis. In remote areas of China, especially in hospitals with high delivery rate, one-step AABR screening is not feasible, and two-step TEOAE screening protocol is still applicable to UNHS screening as more and more infants discharged within 48 h after birth.

Current status of universal newborn hearing screening program at 26 institutions in China.

OBJECTIVES: The present study aimed to determine the status of a universal newborn hearing screening (UNHS) program being conducted in parts of China, by comparing differences in the program findings between 2016 and 2017, as well as across regions in China. METHODS: This study investigated a nationally representative sample of newborns from 26 provinces, autonomous regions, and municipalities in mainland China. A ''Newborn Hearing Screening Survey'' questionnaire was sent to 43 hearing screening institutions throughout China and the data were analyzed, with appropriate quality control throughout the study process. RESULTS: Twenty-six questionnaires, covering 55.88% (19/34) of the provincial administrative regions in China were appropriately completed. The overall sampling frame comprised 238,795 (year 2016) and 229,185 (year 2017) newborns, respectively. We found differences between two years, the initial screening coverage in 2017 (96.10%) was higher than that in 2016 (94.96%); the referral rate at initial screening in 2017 (9.21%) was lower than that in 2016 (10.26%); and the rescreening rate in 2017 (73.50%) was higher than that in 2016 (68.44%). We found differences across three regions, the rescreening rate were highest in West China, the referral rate at rescreening and the referral rate to diagnostic audiological assessment diagnosis were both highest, while the hearing-loss rate was lowest, in the East China in two years. Overall, 61.54% (n = 16) reported using otoacoustic emissions (OAEs), while 38.46% (n = 10) reported using OAEs in combination with automated auditory brainstem response (AABR) tests, for the initial screening. For rescreening, most sites (n = 19, 73.08%) reported using OAEs in combination with AABR, followed by OAEs only (n = 4, 15.38%) and AABR only (n = 3, 11.54%). Of the twenty-six institutions, 57.69% (n = 15) were equipped with a digital information management system for UNHS program, East China had the highest rate of it (81.82%, 9/11). CONCLUSIONS: This study indicated that implementation of a UNHS program had essentially been achieved in many regions of China under the guidance of technical specifications for newborn hearing screening. Compared with 2016, the overall quality of the UNHS program had improved in 2017 and that in East China was better than in the Midland and West China. However, national quality control of the UNHS program is still required to enhance the quality of the program and public education needs to be emphasized to improve the rescreening and reception rate.

Genetic analysis of a Chinese family with members affected with Usher syndrome type II and Waardenburg syndrome type IV.

AIMS: The purpose of this study was to identify the genetic causes of a family presenting with multiple symptoms overlapping Usher syndrome type II (USH2) and Waardenburg syndrome type IV (WS4). METHODS: Targeted next-generation sequencing including the exon and flanking intron sequences of 79 deafness genes was performed on the proband. Co-segregation of the disease phenotype and the detected variants were confirmed in all family members by PCR amplification and Sanger sequencing. RESULTS: The affected members of this family had two different recessive disorders, USH2 and WS4. By targeted next-generation sequencing, we identified that USH2 was caused by a novel missense mutation, p.V4907D in GPR98; whereas WS4 due to p.V185M in EDNRB. This is the first report of homozygous p.V185M mutation in EDNRB in patient with WS4. CONCLUSION: This study reported a Chinese family with multiple independent and overlapping phenotypes. In condition, molecular level analysis was efficient to identify the causative variant p.V4907D in GPR98 and p.V185M in EDNRB, also was helpful to confirm the clinical diagnosis of USH2 and WS4.

Etiology and diagnostic value of nasopharyngeal endoscopy in children with upper airway cough syndrome / 中国内镜杂志

Objective To explore the etiological analysis and diagnostic value of nasopharyngeal endoscopy in children with upper airway cough syndrome. Method The results of nasopharyngeal endoscopy in 120 children diagnosed with upper airway cough syndrome from January 2014 to December 2016 were analyzed. Results Of the 132 cases, 125 cases (94.70％) with upper airway lesions were found, and among the 125 cases, 43 cases (32.58％) had sinusitis, 32 cases (24.24％) had chronic rhinitis (including allergic rhinitis), 13 cases (9.85％) had adenoid hypertrophy, 11 cases (8.33％) had chronic pharyngitis, chronic tonsillitis (tonsil hypertrophy) 9 cases (6.82％). The distribution of upper airway lesions was different in different age groups (P < 0.05). Conclusion Nasopharyngeal endoscopy in the upper airway cough syndrome etiology analysis and diagnosis can be accurate and timely detection of local airway lesions, clinical diagnosis and treatment to provide good help. It is a safe and effective examination method, worthy of clinically promotion.

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles.

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments.

[Comparison of sensitivity of audiological tests to identify otitis media with effusion in newborn infants].

OBJECTIVE: To investigate the sensitive factors which were used in routine audiological tests to find out otitis media with effusion (OME) in newborn infants. METHODS: Subjects of this study were 48 infants, including 31 males and 17 females, who failed in the universal newborn hearing screening. The age ranged from 1.5 to 12 months with the average age of 4.3 months. All subjects accepted temporal bone CT and routine audiological assessments, including air-conduction and bone-conduction auditory brainstem response (ABR), 40 Hz-auditory event related potential (40 Hz-AERP), distortion-product otoacoustic emission (DPOAE), acoustic reflex, tympanometries using 226 Hz and 1000 Hz probe tone. Nine factors were statistically analyzed using Kappa test, Univariate chi(2) test and multivariate condition Logistic stepwise regression analysis, which included the results of acoustic immittance, the air-conduction and bone-conduction ABR thresholds, the difference between air-conduction and bone-conduction ABR thresholds, the latency of ABR wave I, duration between ABR wave I and V, 40 Hz-AERP thresholds, amplitudes and thresholds of DPOAE, and acoustic reflex thresholds (ART). RESULTS: Seventy-seven ears were diagnosed with OME, and 19 ears were normal. CT scan of temporal bone was set as a comparative standard. Kappa test indicated that the results of tympanometry with 1000 Hz probe tone (Kappa = 0.745, P < 0.001), the air-conduction ABR threshold (Kappa = 0.453, P < 0.001), the latency of ABR wave I (Kappa = 0.430, P < 0.001), the threshold of 40 Hz-AERP (Kappa = 0.582, P < 0.001), and DPOAE (Kappa = 0.495, P < 0.001) had agreement with the results of temporal bone CT on evaluating the function of middle ear. Univariate analysis indicated that sensitive factors of middle ear function in newborn infants were tympanometry with 1000 Hz probe tone (P < 0.001), ART (P < 0.001), the air-conduction ABR threshold (P < 0.001), the difference between air-conduction and bone-conduction ABR thresholds (P < 0.001), the latency of ABR wave I (P < 0.001), the threshold of 40 Hz-AERP (P < 0.001) and DPOAE (P < 0.001). And multivariate conditional Logistic stepwise regression model showed that tympanometry with 1000 Hz probe tone (P < 0.001) and 40 Hz-AERP threshold (P = 0.004) can be substituted into Logistic stepwise regression equation. CONCLUSIONS: Tympanometry with 1000 Hz probe tone and are sensitive factors to find out OME in newborn infants. The air conduction ABR threshold, ABR wave I latency, 40 Hz-AERP threshold and DPOAE could reflect the middle ear function of newborn infants effectively.

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China.

AIM: To determine the genotype distribution of hepatitis B virus (HBV) with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS: An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS: HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% (n = 33), 83.2% (n = 333), and 8.5% (n = 34), respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION: The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.