CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

STUDY QUESTION: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN SIZE DURATION: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND THE ROLE OF CHANCE: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

Microencapsulation and nanowarming enables vitrification cryopreservation of mouse preantral follicles.

Preantral follicles are often used as models for cryopreservation and in vitro culture due to their easy availability. As a promising approach for mammalian fertility preservation, vitrification of preantral follicles requires high concentrations of highly toxic penetrating cryoprotective agents (up to 6 M). Here, we accomplish low-concentration-penetrating cryoprotective agent (1.5 M) vitrification of mouse preantral follicles encapsulated in hydrogel by nanowarming. We find that compared with conventional water bath warming, the viability of preantral follicles is increased by 33%. Moreover, the cavity formation rate of preantral follicles after in vitro culture is comparable to the control group without vitrification. Furthermore, the percentage of MII oocytes developed from the vitrified follicles, and the birth rate of offspring following in vitro fertilization and embryo transfer are also similar to the control group. Our results provide a step towards nontoxic vitrification by utilizing the synergistic cryoprotection effect of microencapsulation and nanowarming.

The vertebrate- and testis- specific transmembrane protein C11ORF94 plays a critical role in sperm-oocyte membrane binding.

Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far, spermatozoon factor IZUMO1 and the IZUMO1 counter-receptor JUNO on the oocyte membrane has been identified as a protein requiring fusion. Some sperm membrane proteins such as FIMP, SPACA6 and TEME95, have been proved not to directly regulate fusion, but their knockout will affect the fusion process of sperm and oocytes. Here, we identified a novel gene C11orf94 encoding a testicular-specific small transmembrane protein that emerges in vertebrates likely acquired via horizontal gene transfer from bacteria and plays an indispensable role in sperm-oocyte binding. We demonstrated that the deletion of C11orf94 dramatically decreased male fertility in mice. Sperm from C11orf94-deficient mice could pass through the zona pellucida, but failed to bind to the oocyte membrane, thus accumulating in the perivitelline space. In consistence, when the sperm of C11orf94-deficient mice were microinjected into the oocyte cytoplasm, fertilized oocytes were obtained and developed normally to blastocysts. Proteomics analysis revealed that C11orf94 influenced the expression of multiple gene products known to be indispensable for sperm-oocyte binding and fusion, including IZUMO1, EQTN and CRISP1. Thus, our study indicated that C11ORF94 is a vertebrate- and testis-specific small transmembrane protein that plays a critical role in sperm binding to the oolemma.

A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals.

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient's meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.

Biallelic HFM1 variants cause non-obstructive azoospermia with meiotic arrest in humans by impairing crossover formation to varying degrees.

STUDY QUESTION: Do variants in helicase for meiosis 1 (HFM1) account for male infertility in humans? SUMMARY ANSWER: Biallelic variants in HFM1 cause human male infertility owing to non-obstructive azoospermia (NOA) with impaired crossover formation and meiotic metaphase I (MMI) arrest. WHAT IS KNOWN ALREADY: HFM1 encodes an evolutionarily conserved DNA helicase that is essential for crossover formation and completion of meiosis. The null mutants of Hfm1 or its ortholog in multiple organisms displayed spermatogenic arrest at the MMI owing to deficiencies in synapsis and severe defects in crossover formation. Although HFM1 variants were found in infertile men with azoospermia or oligozoospermia, the causal relationship has not yet been established with functional evidence. STUDY DESIGN, SIZE, DURATION: A Pakistani family, having two infertile brothers born to consanguineous parents, and three unrelated Chinese men diagnosed with NOA were recruited for pathogenic variants screening. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the patients were diagnosed with idiopathic NOA and, for the Chinese patients, meiotic defects were confirmed by histological analyses and/or immunofluorescence staining on testicular sections. Exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of identified variants was assessed and studied in vivo in mice carrying the equivalent mutations. MAIN RESULTS AND THE ROLE OF CHANCE: Six variants (homozygous or compound heterozygous) in HFM1 were identified in the three Chinese patients with NOA and two brothers with NOA from the Pakistani family. Testicular histological analysis revealed that spermatogenesis is arrested at MMI in patients carrying the variants. Mice modeling the HFM1 variants identified in patients recapitulated the meiotic defects of patients, confirming the pathogenicity of the identified variants. These Hfm1 variants led to various reductions of HFM1 foci on chromosome axes and resulted in varying degrees of synapsis and crossover formation defects in the mutant male mice. In addition, Hfm1 mutant female mice displayed infertility or subfertility with oogenesis variously affected. LIMITATIONS, REASONS FOR CAUTION: A limitation of the current study is the small sample size. Owing to the unavailability of fresh testicular samples, the defects of synapsis and crossover formation could not be detected in spermatocytes of patients. Owing to the unavailability of antibodies, we could not quantify the impact of these variants on HFM1 protein levels. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide direct clinical and in vivo functional evidence that HFM1 variants cause male infertility in humans and also suggest that HFM1 may regulate meiotic crossover formation in a dose-dependent manner. Noticeably, our findings from mouse models showed that HFM1 variants could impair spermatogenesis and oogenesis with a varying degree of severity and might also be compatible with the production of a few spermatozoa in men and subfertility in women, extending the phenotypic spectrum of patients with HFM1 variants. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (31890780, 32070850, 32061143006, 32000587 and 31900398) and the Fundamental Research Funds for the Central Universities (YD2070002007 and YD2070002012). The authors declare no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

RAD51AP2 is required for efficient meiotic recombination between X and Y chromosomes.

Faithful segregation of X and Y chromosomes requires meiotic recombination to form a crossover between them in the pseudoautosomal region (PAR). Unlike autosomes that have approximately 10-fold more double-strand breaks (DSBs) than crossovers, one crossover must be formed from the one or two DSBs in PARs, implying the existence of a sex chromosome­specific recombination mechanism. Here, we found that RAD51AP2, a meiosis-specific partner of RAD51, is specifically required for the crossover formation on the XY chromosomes, but not autosomes. The decreased crossover formation between X and Y chromosomes in Rad51ap2 mutant mice results from compromised DSB repair in PARs due to destabilization of recombination intermediates rather than defects in DSB generation or synapsis. Our findings provide direct experimental evidence that XY recombination may use a PAR-specific DSB repair mechanism mediated by factors that are not essential for recombination on autosomes.

Whole-exome sequencing of consanguineous families with infertile men and women identifies homologous mutations in SPATA22 and MEIOB.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal pathogenic mutations in two consanguineous Pakistani families with infertile patients? SUMMARY ANSWER: A homozygous spermatogenesis associated 22 (SPATA22) frameshift mutation (c.203del), which disrupts the interaction with meiosis specific with OB-fold (MEIOB), and a MEIOB splicing mutation (c.683-1G>A) that led to loss of MEIOB protein cause familial infertility. WHAT IS KNOWN ALREADY: MEIOB and SPATA22, direct binding partners and functional collaborators, form a meiosis-specific heterodimer that regulates meiotic recombination. The protein stability and the axial localization of MEIOB and SPATA22 depend on each other. Meiob and Spata22 knockout mice have the same phenotypes: mutant spermatocytes can initiate meiotic recombination but are unable to complete DSB repair, leading to crossover formation failure, meiotic prophase arrest, and sterility. STUDY DESIGN, SIZE, DURATION: We performed WES for the patients and controls in two consanguineous Pakistani families to screen for mutations. The pathogenicity of the identified mutations was assessed by in vitro assay and mutant mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two consanguineous Pakistani families with four patients (three men and one woman) suffering from primary infertility were recruited. SPATA22 and MEIOB mutations were screened from the WES data, followed by functional verification in cultured cells and mice. MAIN RESULTS AND THE ROLE OF CHANCE: A homozygous SPATA22 frameshift mutation (c.203del) was identified in a patient with non-obstructive azoospermia (NOA) from a consanguineous Pakistani family and a homozygous MEIOB splicing mutation (c.683-1G>A) was identified in two patients with NOA and one infertile woman from another consanguineous Pakistani family. The SPATA22 mutation destroyed the interaction with MEIOB. The MEIOB splicing mutation induced Exon 9 skipping, which causes a 32aa deletion in the oligonucleotide-binding domain without affecting the interaction between MEIOB and SPATA22. Furthermore, analyses of the Meiob mutant mice modelling the patients' mutation revealed that the MEIOB splicing mutation leads to loss of MEIOB proteins, abolished SPATA22 recruitment on chromosome axes, and meiotic arrest due to meiotic recombination failure. Thus, our study suggests that SPATA22 and MEIOB may both be causative genes for human infertility. LIMITATIONS, REASONS FOR CAUTION: As SPATA22 and MEIOB are interdependent and essential for meiotic recombination, screening for mutations of SPATA22 and MEIOB in both infertile men and women in larger cohorts is important to further reveal the role of the SPATA22 and MEIOB heterodimer in human fertility. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide direct clinical and functional evidence that mutations in SPATA22 and MEIOB can cause meiotic recombination failure, supporting a role for these mutations in human infertility and their potential use as targets for genetic diagnosis of human infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Developmental Program of China (2018YFC1003900, 2018YFC1003700, and 2019YFA0802600), the National Natural Science Foundation of China (31890780, 31630050, 32061143006, 82071709, and 31871514), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Semen parameters in men recovered from COVID-19.

The novel coronavirus disease (COVID-19) pandemic is emerging as a global health threat and shows a higher risk for men than women. Thus far, the studies on andrological consequences of COVID-19 are limited. To ascertain the consequences of COVID-19 on sperm parameters after recovery, we recruited 41 reproductive-aged male patients who had recovered from COVID-19, and analyzed their semen parameters and serum sex hormones at a median time of 56 days after hospital discharge. For longitudinal analysis, a second sampling was obtained from 22 of the 41 patients after a median time interval of 29 days from first sampling. Compared with controls who had not suffered from COVID-19, the total sperm count, sperm concentration, and percentages of motile and progressively motile spermatozoa in the patients were significantly lower at first sampling, while sperm vitality and morphology were not affected. The total sperm count, sperm concentration, and number of motile spermatozoa per ejaculate were significantly increased and the percentage of morphologically abnormal sperm was reduced at the second sampling compared with those at first in the 22 patients examined. Though there were higher prolactin and lower progesterone levels in patients at first sampling than those in controls, no significant alterations were detected for any sex hormones examined over time following COVID-19 recovery in the 22 patients. Although it should be interpreted carefully, these findings indicate an adverse but potentially reversible consequence of COVID-19 on sperm quality.