BackgroundMultiple morphological anomalies of the sperm flagella (MMAF) is a term used to describe abnormalities in sperm morphology, which lead to primary infertility in males. Intracytoplasmic sperm injection (ICSI) is an effective treatment for MMAF. However, ICSI failure rates remain high in MMAF patients. Our purpose was to investigate novel gene mutations in a cohort of nineteen patients with MMAF and assess the impact of these mutations on assisted reproductive therapy.MethodsWe recruited nineteen infertile patients with MMAF and twenty healthy men with proven fertility at the Affiliated Yantai Yuhuangding Hospital of Qingdao University. The morphology of the spermatozoa was observed using Papanicolaou staining and the ultrastructure of the spermatozoa was inspected using transmission electron microscopy. Gene mutations were evaluated using whole-exome sequencing and novel mutations were further validated in patients and their parents using Sanger sequencing. The effect of these novel mutation sites on the expression of DNAH1 was analysed using immunofluorescence, and the effect of these novel mutations on pregnancy outcome was analysed using intracytoplasmic sperm injection (ICSI). ResultsSpermatozoa from 19 patients presented with a typical MMAF phenotype including severe ultrastructural defects. We identified ten novel mutation sites in the DNAH1 locus from six of these patients, none of which were identified in DNAH1 or the other MMAF-related genes from the twenty men with proven fertility. In the sperm from these patients, DNAH1 was absent. Three patients with DNAH1 mutation who underwent intracytoplasmic sperm injection (ICSI) had a good outcome. ConclusionHere, we describe several novel compound heterozygous mutations and a novel homozygous mutation in DNAH1 from six independent MMAF patients. This study adds to the body of knowledge surrounding the genetic landscape of MMAF and DNAH1 improving our ability to diagnose and treat MMAF efficiently.

Abstract Background The intraflagellar transport protein 140 homolog (IFT140) is involved in the process of intraflagellar transport (IFT), a process that is essential for the formation and maintenance of most eukaryotic cilia and flagella. Variants IFT140 have been reported to account for ciliopathy but association with male fertility has never been described in humans. Here we report the identification of two novel variants of IFT140 which caused spermatogenic dysfunction and male infertility. Methods Whole‐exome sequencing was performed in a 27‐year‐old infertile man presented with severe oligozoospermia, asthenozoospermia, and teratozoospermia (OAT) without other physical abnormality. Sanger sequencing was used to verify gene variants in the patient, his healthy brother, and their parents. Morphology and protein expression in the patient's sperm were examined by transmission electron microscopy (TEM) and immunofluorescence staining. Function of gene variants was predicted by online databases. Results Compound heterozygous variants of IFT140: exon16: c.1837G > A: p.Asp613Asn and exon31: c.4247G > A: p.Ser1416Asn were identified in the patient, both of which showed autosomal recessive inheritance in his family, and had extremely low allele frequency in the population. Morphological abnormalities of the head, nucleus, and tails and the absence of IFT140 from the neck and mid‐piece of the patient's spermatozoa were observed. Mutation Taster database predicted a high probability of damage‐causing by both variations. Conclusion This study for the first time reported IFT140 variants that cause infertility in humans.

Oligoasthenoteratozoospermia (OAT) is characterized as low sperm count, decreased sperm motility and structural abnormalities of the sperm head in the same patient. However, very few studies reported the genetic alterations associated with OAT. Here we report a 38-year-old patient with OAT from a consanguineous family, with 2-6 million/mL sperm density, 2.1-3.8% normal sperm morphology and immotile sperm. Whole-exome sequencing (WES) identified homozygous variant c.1259A > G:p.Y420C in the TDRD6 gene. TDRD6 is a testis specific expressed protein that was localized to the chromatoid bodies in germ cells and played an important role in the nonsense-mediated decay pathway. This rare variant co-segregated with the OAT phenotype in this family. Bioinformatic analysis also suggested the variant a pathogenic mutation. Two intracytoplasmic sperm injection (ICSI) cycles were carried out in the patient's wife, but she did not become pregnant after embryo transfer. So the mutations in TDRD6 may be associated with human male infertility and early embryonic lethality.

Dear Editor, Male infertility, which affects approximately 20 million people worldwide, is commonly caused by spermatogenic dysfunctions, including severe oligozoospermia, cryptozoospermia, and nonobstructive azoospermia, which are largely genetic in origin.123 Here, we report a case of cryptozoospermia in a 33-year-old patient, who sought treatment for primary sterility that had been ongoing for 5 years. The patient had intercourse with his spouse without contraception two to three times per week, but they had not achieved pregnancy. The patient was born of consanguineous parents who were maternal cousins (Figure 1). He had no history of adverse sexual contact or inappropriate hobbies. He was 161 cm tall and weighed 80 kg, and his external genital organs were normally developed, with both testes around 6 ml in size, and no palpable abnormality in his bilateral spermatic veins.Figure 1: Pedigree chart of the patients. The arrow indicates the proband.The patient underwent three semen examinations in our hospital and other institutions. In our hospital, the semen analysis was carried out according to the guidelines in the WHO Laboratory Manual for the Examination and Processing of Human Semen.4 The patient's semen volume was 3.0–3.5 ml, pH 7.2–7.5, and spermatozoa were absent from his semen smear. Semen centrifugal sediment smear showed a sperm count of 0-2 cells per high-power field (HPF), and very few active spermatozoa were observed. Staining with Diff-Quik indicated normal morphology in 2.5%-4.0% of spermatozoa. Sex-hormone levels were follicle-stimulating hormone (FSH) 20.8 mIU ml−1 (reference value 1.5–12.4 mIU ml−1), luteinizing hormone (LH) 10.4 mIU ml−1 (reference value 1.7–8.6 mIU ml−1), testosterone (T) 2.9 ng ml−1 (reference value 2.5–8.4 ng ml−1), estradiol (E2) 40.5 pg ml−1 (reference value 7.6–42.6 pg ml−1), and prolactin (PRL) 5.1 ng ml−1 (reference value 2.6–13.1 ng ml−1). No abnormalities were revealed by peripheral-blood chromosomal-karyotype analysis. Y-chromosome-microdeletion screening was carried out according to the European Academy of Andrology (EAA) guidelines by real-time fluorescent PCR using the Y Chromosomal Microdeletion Test Kit (Shanghai Tellgen Corporation, Shanghai, China), which detected the sY84 and sY86 sequence-tagged sites (STSs) of azoospermia factor a (AZFa), the sY127 and sY134 STSs of AZFb, and the sY254 and sY255 STSs of AZFc. All six STSs were present, indicating that the patient did not have a Y-chromosome microdeletion. The initial clinical diagnosis was primary infertility with cryptozoospermia, bilateral testicular dysplasia, and high-gonadotropin gonadal-function decline. With the approval of the Ethics Committee of Yantai Yuhuangding Hospital and with the patient's informed consent, peripheral blood was extracted for exome sequencing. Because the patient's parents were consanguineous, bioinformatics analysis was performed to identify inheritance of recessive characteristics. All the homozygous mutations were screened to identify potentially pathogenic gene alterations related to spermatogenesis, through phenotype and genotype correlation analysis. A novel nonsense mutation was identified at exon 1:c.6934G>A (p.R2312X) of the testis-expressed 15 (TEX15) gene, which resulted in a truncated TEX15 protein. This mutation was confirmed by Sanger sequencing. Both parents of the proband were carriers of this mutation (Figure 2). We speculated that this mutation was at least in part the cause of the spermatogenic dysfunction. Because of the extremely limited availability of the patient's spermatozoa, detection of TEX15 mRNA or TEX15 protein in spermatozoa was not possible.Figure 2: Sanger-sequencing results showing the homozygous c.6934G>A mutation in the proband and the heterozygous c.6934G>A mutation in the parents of proband, as well as the normal control.Spermatogenesis is a highly complex process of cell differentiation, which is necessary for the formation of haploid spermatozoa. The core of this process is meiosis in spermatocytes, during which synapsis and recombination of homologous chromosomes occur. TEX15 was first identified as a protein that is required for chromosomal synapsis and meiotic recombination in 2008.5 In TEX15-deficient male mice, DNA double-strand breaks (DSBs) are formed and not repaired, suggesting that TEX15 functions in the repair of DSBs via regulation of the loading of DNA repair proteins (RAD51 and DMC1) onto sites of DSBs. Homozygous deletion of TEX15 in mice also leads to significantly reduced testis volume.5 In an analysis of single-nucleotide polymorphisms (SNPs) in TEX15, rs323346 and rs323347 were identified as genetic risk factors for spermatogenic failure in the Chinese Han population.6 In 2015, Okutman and colleagues reported that a nonsense mutation (c.2130T>G, p.Y710X) in TEX15 caused infertility in three of seven brothers in a Turkish family with consanguineous parents.7 The sperm concentrations of patients with TEX15 mutations declined over time, and early-stage sperm cryopreservation was recommended.7 In summary, our study identified a novel homozygous nonsense mutation in TEX15 in a patient with cryptozoospermia. This mutation could be the cause of cryptozoospermia, with TEX15 deficiency resulting in the failure of spermatogenesis. The mutation was inherited from the patient's parents, who were both heterozygous carriers, indicating a recessive pattern of inheritance. With the development of precision medicine, TEX15 could become a clinical marker for detection of nonobstructive azoospermia or cryptozoospermia. AUTHOR CONTRIBUTIONS YWS and ZLG designed the study; XW analyzed data and wrote the manuscript; HRJ analyzed high-throughput sequencing data and screened for candidate genes; YQC extracted DNA and performed Sanger sequencing; and JC collect clinical data. All authors read and approved the final manuscript. COMPETING INTERESTS All authors declared no competing interests. ACKNOWLEDGMENTS This study was supported by the Science and Technology Planning Project (Grant No. 3502Z20154033), the Major/Important Disease Research Project (Grant No. 3502Z20159022), the Young/Middle-aged Talent Cultivation Project (Grant No. 2015-ZQN-JC-44), and the Science and Technology Guided Project of Fujian Province (Project No. 2016Y0101).

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease that causes primary infertility. However, the genetic causes for approximately half of MMAF cases are unknown. Whole exome sequencing analysis of the 27 patients with MMAF identified several CFAP44 mutations (3 homozygous: c.2935_2944del: p.D979*, c.T1769A: p.L590Q, c.2005_2006del: p.M669Vfs*13; and putative compound heterozygous: c.G3262A: p.G1088S and c.C1718A: p.P573H.) and CFAP43 acceptor splice-site deletion (c.3661-2A>-) mutations in 5 and 1 patients, respectively. Real-time quantitative polymerase chain reaction assays also demonstrated that CFAP44 expression was very weak in patient (P)1 and P3, and CFAP43 expression was lower in P6 than in the control. Immunofluorescence analysis of CFAP43 showed lower CFAP43 protein expression levels in P6 than in the normal control. This study demonstrated that biallelic mutations in CFAP44 and CFAP43 cause MMAF. These results provide researchers with a new insight to understand the genetic etiology of MMAF and to identify new loci for genetic counselling of MMAF.

在成年后就诊的男性21-羟化酶缺陷症患者临床少见,本文报道了1例合并无精症的单纯男性化型21-羟化酶缺陷症患者的临床资料.