SP22 sperm protein as a potential biomarker of fertility in humans: A preliminary study.

Infertility affects approximately 15% of couples of reproductive age, and 50% of the cases are directly related to men. The evaluation of male fertility is based on analyses of routine seminal parameters and the use of more advanced techniques can help identify fertility biomarkers. SP22 sperm protein is considered a biomarker in murine species since its concentration is highly correlated with sperm fertility. As the role of this protein as a biomarker is already well-established in other species, we hypothesized that this same correlation could apply to human. Thus, the present study aimed to investigate possible correlations between SP22 concentration and sperm parameters in fertile and infertile men. For this, a study was carried out on 21 volunteers' seminal samples who were grouped according to fertility as fertile (n = 10) or infertile (n = 11). Conventional and functional sperm analyses, membrane protein extraction, quantification and immunolocalization of SP22 were performed. The infertile volunteers showed an increase in the percentage of sperm with abnormalities in head morphology and a decrease in the percentage of sperm with intact plasma membrane and damaged acrosomal membrane. Serum concentration of the hormone SHBG was also decreased in infertile volunteers. The damage to the plasma membrane was positively correlated with the superoxide anion production. Although none of the functional parameters were correlated with SP22 concentration, type D sperm motility was negatively correlated and type A+B sperm motility was positively correlated. This preliminary study opens new paths in the characterization of SP22 as a non-invasive biomarker for predicting fertility/infertility.

lncRNA 1700101O22Rik and NONMMUG030480.1 Are Not Essential for Spermatogenesis in Mice.

Many testis-specific lncRNAs are highly expressed in late spermatogenesis, especially in spermiogenesis. However, their functions and the underlying mechanisms in male fertility are largely unknown. Here, we screened two highly expressed lncRNAs, 1700101O22Rik (O22Rik) and NONMMUG030480.1 (NM480) in testes, to investigate the roles in spermatogenesis using lncRNA knockout (KO) mouse generated by CRISPER/Cas9 technology. Both testis-specific lncRNAs were mainly expressed from secondary spermatocytes to round spermatids, suggesting that they might be involved in spermiogenesis. Phenotypic analysis showed that the deletion of O22Rik or NM480 did not affect the development of testis and epididymis or spermatogenesis. These results were confirmed in both young and middle-aged male mice. In addition, there was no significant difference in sperm morphology and other parameters including concentration and motility between wild type (WT) and KO mice. Fertility tests showed that litter size was significantly lower in O22Rik KO mice compared with WT controls. Although O22Rik did not exert dramatic roles in spermatogenesis, on molecular levels, its surrounding gene expression was disturbed significantly. Gm32773 was decreased; however, Gm32828 was increased in KO mice. In conclusion, lncRNA O22Rik and NM480 are not individually essential for spermatogenesis in mice.

Interleukin 22 Expression During the Implantation Window in the Endometrium of Women with Unexplained Recurrent Pregnancy Loss and Unexplained Infertility Compared to Healthy Parturient Individuals.

We evaluated the expression of interleukin-22 (IL-22) in the endometrium of women with unexplained recurrent pregnancy loss (uRPL) and unexplained infertility (UI) compared to the women with normal pregnancies. Endometrial tissues were collected from 20 women with UI, 20 women with uRPL, and 24 healthy women as a control group. Immunohistochemical expression and gene expression of IL-22 were analyzed by immunohistochemistry (IHC) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methods. The controls showed lower IL-22 expression than the uRPL group (P > 0.05) using PCR. It was also found that patients with UI had lower levels of IL-22 expression compared to the uRPL group (P > 0.05). Although IL-22 expression in the endometrium of patients with UI was higher than the control group, this difference was not statistically significant (P < 0.05). IL-22 immunoreactivity was observed in the endometrial glands and stromal tissues using IHC. We found the lowest IL-22 expression in the control group and the highest in uRPL samples (P < 0.05). Our findings suggest that a significant increase in IL-22 expression in uRPL patients may affect fertility and pregnancy outcomes or even have a considerable impact on immune function deficits. Further studies on the critical function of IL-22 during pregnancy are suggested.

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.

SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility.

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.

Spread of multidrug resistance among Ureaplasma serovars, Tunisia.

Background: Ureaplasma spp. have been implicated in a variety of clinical conditions and certain serovars are likely to be disease-associated. Hence, the ascending trend of Ureaplasma spp. resistance to antimicrobials should deserve more attention. Here we assessed the extent of antimicrobial resistance of Ureaplasma serovars in Tunisia, and investigated the underlying molecular basis. Methods: This study included 101 molecularly typed Ureaplasma spp. clinical strains isolated over a 12-year time period (2005-2017). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Neighbor-joining tree was constructed to establish the phylogenetic relationships among isolates. Results: We found that all ureaplasma isolates were resistant to ciprofloxacin and erythromycin, intermediately resistant to azithromycin, and susceptible to doxycycline, moxifloxacin and josamycin. Ofloxacin and levofloxacin resistance was found in 73.27 and 17.82%, respectively, while 37.62% of isolates proved resistant to tetracycline. Consequently, we detected an elevated multidrug resistance rate among ureaplasma isolates (37.62%), particularly among serovars 2, 5, 8, and 9 (77.77% overall), as well as serovars 4, 10, 12, and 13 (52.63% overall). In most cases, drug resistance was found to be associated with known molecular mechanisms, yet we have identified two novel mutations in the L22 protein, which might be associated with macrolide-resistance. Conclusion: To our knowledge, this is the first study that reports the widespread expansion of multidrug resistance among Ureaplasma serovars, a finding of importance in terms of both surveillance and antimicrobial usage.

Spermatogenesis associated 22 is required for DNA repair and synapsis of homologous chromosomes in mouse germ cells.

Analysis of the N-ethyl-N-nitrosourea (ENU)-induced repro42 mutation previously identified spermatogenesis associated 22 (Spata22) as a gene required for meiotic progression and fertility in both male and female mice, but its specific contribution to the process was unclear. Here, we report on a novel, null allele of Spata22 (Spata22Gt ) and confirm its requirement for germ cell development. Similar to repro42 mutant mice, histological and mating analyses indicate that gametogenesis is profoundly affected in Spata22Gt/Gt males and females, resulting in infertility. Cytological examination confirms that germ cells do not progress beyond zygonema and meiotic arrest is linked to impairment of both synapsis and DNA repair. Analysis of SPATA22 distribution reveals that it localizes to foci associated with meiotic chromosomes during prophase I and that the number of foci peaks at zygonema; there are also more SPATA22 foci in oocytes than in spermatocytes. Furthermore, SPATA22 co-localizes with a number of proteins involved in meiotic recombination, including RAD51, DMC1, and MLH1, and is present until mid-pachynema, suggesting a role in resolution of recombination intermediates. In fact, SPATA22 co-localizes with MLH1 in more than 20% of foci at pachynema. Analysis of Spata22Gt/Gt meiocytes confirms that SPATA22 is required for localization of MEIOB but not RPA (two proteins known to interact with SPATA22), and immunoblotting corroborates that production of MEIOB is indeed decreased in the absence of SPATA22. Together, these data suggest that SPATA22 is required for both meiotic recombination and synapsis during meiosis in mice.

Organic cation/carnitine transporter OCTN2 (SLC22A5) -207C>G (rs2631367) polymorphism is not associated with male infertility.

Carnitine is an important factor in the initiation of progressive sperm motility and end stage of sperm maturation. The compound is transported by an organic cation/carnitine transporter (OCTN2), which is expressed in the male reproductive system. OCTN2 is encoded by SLC22A5 gene with proven -207C>G functional polymorphism. The aim of the case-control study was to investigate a potential association between the -207C>G SLC22A5 polymorphism and male infertility. The -207C>G SLC22A5 polymorphism was determined by means of TaqMan assay in 206 infertile Caucasian males and 256 ethnically matched controls. Besides genotyping study, sperm mitochondrial function was assessed using NADH-dependent NBT assay. The distribution of SLC22A5 genotypes in infertile men was as follows: CC - 29.6%, CG - 53.9%, GG - 16.5% and in fertile men: CC - 32.0%, CG - 50.0%, GG - 18.0%, and was comparable in both evaluated groups. Likewise, the studied polymorphism did not affect sperm mitochondrial function. The results of the current study demonstrated that -207C>G polymorphism of the SLC22A5 gene is not associated with male infertility.