Sperm microRNA Research Articles

Expression of exosomal microRNAs miR-34a and miR-210 in male infertility: relationship with morphokinetic parameters and sperm DNA fragmentation

Introduction. Infertility affects tens of millions of men and women across the globe. In approximately half of cases, male factors are the cause of infertility. In recent decades, there has been a significant decline in the quality of male ejaculate, which is characterized by reduced sperm concentration and motility. The insufficient diagnostic and prognostic value of routine semen analysis results highlights the challenge of developing effective diagnostic tools and searching for reliable biomarkers of male infertility. One of the most promising approaches may be assessing sperm microRNA expression.Objective. To study the role of sperm exosomal microRNAs miR-34a and miR-210 in the development of male infertility.Materials & methods. The retrospective study included 150 men aged 25 – 49 years; of these, 96 patients were diagnosed with idiopathic infertility. The comparison group consisted of 54 fertile men. To assess the structure and motility of sperm, the results of a standard ejaculate study (WHO, 2021) and computer data analysis using MMC Sperm (MMCSoft, St. Petersburg, Russia) software were used. The degree of DNA fragmentation was assessed using the TUNEL method. To analyze the expression of miR-34a and miR-210, quantitative real-time PCR was performed using the miRCURY LNA SYBR Green PCR Kit («Qiagen» GmbH, Hilden, Germany) and the Rotor-Gene Q PCR product detection system («Qiagen» GmbH, Hilden, Germany).Results. The study of ejaculate using the Computer Assisted Sperm Analysis System (CASA) method revealed a statistically significant relationship between the level of DNA fragmentation of sperm and indicators of the speed of their movement: rectilinear (VSL) (r = -0.522726; p < 0.01), curvilinear (VCL) (r = -0.499096; p < 0.01), along the middle path (VAP) (r = -0.429533; p < 0.01), as well as with the amplitude of lateral head displacement (ALH) (r = -0.294779, p < 0.01), the linearity of their curvilinear path (LIN) (r = -0.385796; p < 0.01), the degree of straight-line movements (STR) (r = -0.268248; p < 0.05) and their progressive mobility (r = -0.411547; p < 0.01). A study of the level of microRNA expression in sperm exosomes revealed a statistically significant decrease in its miRNA-34a pool (p = 0.0116). According to the Chaddock scale, the strength of the correlation between miR-210 expression and the effectiveness of ART programs was moderate (0.437993). The inverse relationship between miR-34a expression and IVF and ICSI results was weak (0.135314).Conclusion. The analysis of exosomal microRNA-34a and microRNA-210, which are involved in the regulation of spermatogenesis, reveals a direct correlation between their variations and changes in the kinetic and morphological parameters of gametes. It also indicates a relationship with the state of DNA fragmentation. These findings suggest varying levels of gene expression among infertile patients, men with proven fertility, and those undergoing assisted reproductive technology (ART) treatments, both with successful and repeated unsuccessful outcomes.

Sperm-borne miR-34c-5p and miR-191-3p as markers for sperm motility and embryo developmental competence.

Sperm-borne microRNAs play a pivotal role in influencing essential cellular processes during fertilization, impacting the quality of embryo development. Dysregulated microRNA profiles have been associated with compromised embryonic development and increased incidences of pregnancy loss. This study aimed to investigate the potential associations between the abundance of miR-34c-5p and miR-191-3p in human spermatozoa with sperm quality, as well as with embryo quality and metabolic performance during in vitro development. Thirteen couples who underwent a total of 13 cycles participated in this study. The sperm quality was assessed using conventional methods following World Health Organization guidelines. Quantitative polymerase chain reaction was employed to measure microRNA abundance in spermatozoa. Embryos were categorized as good, lagging, or bad based on morphokinetic evaluation. Evaluation of embryo metabolic performance involved tracking changes in specific metabolites within the cultured media using nuclear magnetic resonance spectroscopy. Statistical analysis was conducted to explore the correlation between microRNA abundance in human spermatozoa and all other collected data. Our findings revealed a negative correlation between the abundance of miR-34c-5p (but not miR-191-3p) and total sperm motility, potentially mediated by the modulation of key signaling pathways. Additionally, higher levels of miR-34c-5p in spermatozoa were strongly associated with the consumption or release of key metabolites by developing embryos, particularly those linked with lipid and glucose metabolism, suggesting enhanced metabolic performance, while miR-191-3p was mostly associated with glucose consumption. Concurrently, only miR-34c-5p content in spermatozoa correlated with higher embryo quality. This study provides evidence suggesting that the abundance of miR-34c-5p in spermatozoa is correlated not only with total sperm motility but also with markers of embryo developmental competence, highlighting the potential significance of this sperm microRNA content as a biomarker in assisted reproduction.

Bisphenol A Negatively Impacts Human Sperm MicroRNA and Protein Profiles

AbstractBisphenol A (BPA), a widely prevalent endocrine disruptor, has detrimental effects on human health and male reproduction. Elevated BPA levels have been linked to reduced sperm quality and production, while paternal exposure may result in poor reproductive outcomes and offspring health. For many years, the effects of BPA exposure in human reproduction were mainly attributed to the deregulation of the hypothalamic–pituitary–gonadal (HPG) axis. In the past decades, other mechanisms of action have been proposed, and studies highlighting epigenetic alterations linked to BPA in sperm emerged. Nevertheless, the impact of BPA on human sperm miRNA and protein profiles and its implications for fertilization and post-fertilization events remain unexplored. This study aimed to investigate the association between BPA concentration in seminal plasma and seminal quality and to examine the alterations in human sperm miRNA and protein expression among men with varying BPA levels in seminal plasma. BPA levels were measured in seminal plasma from 102 Portuguese men, and it was detected in 88% of the samples. No correlation was observed between BPA levels and age or seminal parameters. The small RNA content of 15 normozoospermic samples was evaluated using small RNA sequencing, revealing that 15 miRNAs exhibited a correlation with BPA levels. Gene ontology analysis of their target genes indicated their involvement in embryonic development and response to stress. The proteome of 20 normozoospermic human sperm samples, categorized into four groups based on BPA levels, was accessed by liquid chromatography–mass spectrometry. Data are available via ProteomeXchange with the identifier PXD047280. Sixty-two differentially expressed proteins were identified across groups. The biological processes most affected in samples with higher BPA levels were “protein sumoylation” and “cytoplasmic translation”. The altered miRNAs and proteins identified in spermatozoa from men environmentally exposed to BPA represent potential biological markers of exposure to this endocrine disruptor and may help elucidate cases of idiopathic male infertility, fertilization failure, and abnormal embryo development.

Expression of sperm microRNAs related to bull fertility: A systematic review

In this study we proposed to address the following question: “Are there differentially expressed sperm microRNAs related to fertility in bulls?”. A systematic review of scientific literature until November 2022 was performed, in accordance with PRISMA guidelines. The main outcome was differentially expressed sperm microRNA from bulls with low versus high fertility profiles identified by using different methods such as field fertility evaluation and sperm laboratory analysis. Were identified 786 documents, of which 13 were selected for qualitative analysis. A total of 182 unique differentially expressed miRNAs were identified, among these, 49 miRNAs were found in common between at least two studies. It is believed that from these 49 miRNAs, it is possible that miRNAs such as miR-10a, −10b, −103, −15b, −122, −125b, −126-5p, −151-5p, −193a-5p, −196a, −27a-5p and -99b could be potential universal biomarkers to assess the reproductive potential of males.

Age-Dependent Alterations in Semen Parameters and Human Sperm MicroRNA Profile.

The trend to delay parenthood is increasing, impacting fertility and reproductive outcomes. Advanced paternal age (APA), defined as men's age above 40 years at conception, has been linked with testicular impairment, abnormal semen parameters, and poor reproductive and birth outcomes. Recently, the significance of sperm microRNA for fertilization and embryonic development has emerged. This work aimed to investigate the effects of men's age on semen parameters and sperm microRNA profiles. The ejaculates of 333 Portuguese men were collected between 2018 and 2022, analyzed according to WHO guidelines, and a density gradient sperm selection was performed. For microRNA expression analysis, 16 normozoospermic human sperm samples were selected and divided into four age groups: ≤30, 31-35, 36-40, and >40 years. microRNA target genes were retrieved from the miRDB and TargetScan databases and Gene Ontology analysis was performed using the DAVID tool. No significant correlation was found between male age and conventional semen parameters, except for volume. Fifteen differentially expressed microRNAs (DEMs) between groups were identified. Enrichment analysis suggested the involvement of DEMs in the sperm of men with advanced age in critical biological processes like embryonic development, morphogenesis, and male gonad development. Targets of DEMs were involved in signaling pathways previously associated with the ageing process, including cellular senescence, autophagy, insulin, and mTOR pathways. These results suggest that although conventional semen parameters were not affected by men's age, alterations in microRNA regulation may occur and be responsible for poor fertility and reproductive outcomes associated with APA.

Paternal immune activation by Poly I:C modulates sperm noncoding RNA profiles and causes transgenerational changes in offspring behavior

Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content.

59 Delivery of exogenous sperm microRNAs increases cleavage rates and changes gene expression in embryos, leading to an increment on blastocyst and development rates in low

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Association between Sperm Morphology and Altered Sperm microRNA Expression

Evaluation of male infertility has been based on semen analysis for years. As this method can be subjective at times, there is a scientific tendency to discover stable and quantifiable biomarkers. This study included 28 couples who underwent an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. The couples were assigned into two groups, according to sperm morphology. Couples where the males were normozoospermic were placed in the control group (15 participants), while couples where males had teratozoospermia were placed in the study group (13 participants). Thirteen candidate miRNAs were selected for qPCR analysis, based on our literature search. We determined significant under-expression of nine miRNAs (miR-10a-5p/-15b-5p/-26a-5p/-34b-3p/-122-5p/-125b-5p/-191-5p/-296-5p and let-7a-5p) in spermatozoa from patients with teratozoospermia compared to the controls, whereas expression levels of four miRNAs (miR-92a-3p/-93-3p/-99b-5p/-328-3p) did not significantly differ between the study and control groups. The expression levels of all 13 included miRNAs were significantly positively correlated with each other and significantly positively associated with spermatozoa morphology, excluding miR-99b-5p. There were no other significant associations between miRNA expression and sperm quality parameters. Only expression levels of miR-99b-5p were significantly positively correlated with good-quality day 3 embryo rate (ρ = 0.546; p = 0.003), while other variables of the IVF/ICSI cycle outcome showed no significant associations with miRNA expression profiles. This is one of the rare studies providing an insight directly into miRNA profiles in regard to sperm morphology. We identified nine miRNAs that could serve as biomarkers of spermatozoa quality in regard to teratozoospermia.

Paternal eNOS deficiency in mice affects glucose homeostasis and liver glycogen in male offspring without inheritance of eNOS deficiency itself

Aims/hypothesisIt was shown that maternal endothelial nitric oxide synthase (eNOS) deficiency causes fatty liver disease and numerically lower fasting glucose in female wild-type offspring, suggesting that parental genetic variants may influence the offspring’s phenotype via epigenetic modifications in the offspring despite the absence of a primary genetic defect. The aim of the current study was to analyse whether paternal eNOS deficiency may cause the same phenotype as seen with maternal eNOS deficiency.MethodsHeterozygous (+/−) male eNOS (Nos3) knockout mice or wild-type male mice were bred with female wild-type mice. The phenotype of wild-type offspring of heterozygous male eNOS knockout mice was compared with offspring from wild-type parents.ResultsGlobal sperm DNA methylation decreased and sperm microRNA pattern altered substantially. Fasting glucose and liver glycogen storage were increased when analysing wild-type male and female offspring of +/− eNOS fathers. Wild-type male but not female offspring of +/− eNOS fathers had increased fasting insulin and increased insulin after glucose load. Analysing candidate genes for liver fat and carbohydrate metabolism revealed that the expression of genes encoding glucocorticoid receptor (Gr; also known as Nr3c1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a; also known as Ppargc1a) was increased while DNA methylation of Gr exon 1A and Pgc1a promoter was decreased in the liver of male wild-type offspring of +/− eNOS fathers. The endocrine pancreas in wild-type offspring was not affected.Conclusions/interpretationOur study suggests that paternal genetic defects such as eNOS deficiency may alter the epigenome of the sperm without transmission of the paternal genetic defect itself. In later life wild-type male offspring of +/− eNOS fathers developed increased fasting insulin and increased insulin after glucose load. These effects are associated with increased Gr and Pgc1a gene expression due to altered methylation of these genes.Graphical abstract

The Transmission of Intergenerational Epigenetic Information by Sperm microRNAs.

Epigenetic information is transmitted from one generation to the next, modulating the phenotype of offspring non-genetically in organisms ranging from plants to mammals. For intergenerational non-genetic inheritance to occur, epigenetic information must accumulate in germ cells. The three main carriers of epigenetic information—histone post-translational modifications, DNA modifications, and RNAs—all exhibit dynamic patterns of regulation during germ cell development. For example, histone modifications and DNA methylation are extensively reprogrammed and often eliminated during germ cell maturation and after fertilization during embryogenesis. Consequently, much attention has been given to RNAs, specifically small regulatory RNAs, as carriers of inherited epigenetic information. In this review, we discuss examples in which microRNAs have been implicated as key players in transmitting paternal epigenetic information intergenerationally.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) dietalters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.

Sperm microRNAs confer depression susceptibility to offspring.

Evidence that offspring traits can be shaped by parental life experiences in an epigenetically inherited manner paves a way for understanding the etiology of depression. Here, we show that F1 offspring born to F0 males of depression-like model are susceptible to depression-like symptoms at the molecular, neuronal, and behavioral levels. Sperm small RNAs, and microRNAs (miRNAs) in particular, exhibit distinct expression profiles in F0 males of depression-like model and recapitulate paternal depressive-like phenotypes in F1 offspring. Neutralization of the abnormal miRNAs in zygotes by antisense strands rescues the acquired depressive-like phenotypes in F1 offspring born to F0 males of depression-like model. Mechanistically, sperm miRNAs reshape early embryonic transcriptional profiles in the core neuronal circuits toward depression-like phenotypes. Overall, the findings reveal a causal role of sperm miRNAs in the inheritance of depression and provide insight into the mechanism underlying susceptibility to depression.

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting

BackgroundThe use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull.ResultsIn total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21–5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss.ConclusionsA set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire’s suitability for the production of sex-sorted sperm.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Sperm microRNA pairs: new perspectives in the search for male fertility biomarkers

To identify candidates of fertility biomarkers among pairs of human sperm microRNAs. Expression data of 736 sperm microRNAs from fertile and infertile individuals characterized in previous published studies by means of TaqMan quantitative polymerase chain reaction (PCR) were reexamined. A set of microRNA pairs with the best biomarker potential were selected and validated by means of quantitative real-time (qRT) PCR in an independent cohort. University laboratory. Semen samples were obtained from fertile (n = 10) and infertile (asthenozoospermia, n = 10; teratozoospermia, n = 10; oligozoospermia, n = 10; unexplained male infertility [UMI], n = 8) individuals. The validation cohort included 9 fertile donors and 14 infertile patients with different seminal alterations. None. Spearman test was used to select microRNA pairs with a correlated expression in fertile individuals and a noncorrelated expression in each infertile group. The biomarker potential of these pairs was determined with the use of receiver operating characteristic curves. The differential relative expression of each pair in fertile and infertile populations was verified (Mann-Whitney test). Those pairs with best results were validated by qRT-PCR. Forty-eight pairs showed significant correlations in the fertile group. The pairs that were uncorrelated in the infertile populations and displayed the best biomarker potential were hsa-miR-942-5p/hsa-miR-1208 (asthenozoospermia), hsa-miR-296-5p/hsa-miR-328-3p (teratozoospermia), hsa-miR-139-5p/hsa-miR-1260a (oligozoospermia), and hsa-miR-34b-3p/hsa-miR-93-3p (UMI). The hsa-miR-942-5p/hsa-miR-1208 pair showed the greatest potential for detecting seminal alterations in the validation cohort (85.71% true positives). The pairs hsa-miR-942-5p/hsa-miR-1208 and hsa-miR-34b-3p/hsa-miR-93-3p have the potential to become new molecular biomarkers that could help to diagnose male infertility, especially in cases of UMI or when seminal parameters are close to the threshold values.

Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen–thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Disrupted sperm mirna expression profiles revealed a fingerprint of impaired spermatogenesis in oligozoospermia males

MicroRNAs (miRNAs) are short, single stranded noncoding RNA molecules that function as post-transcriptional regulators inhibiting mRNA translation. Sperm microRNAs have been recognized as playing a key role in testicular function. However, there are limited studies investigating the importance of miRNAs in male factor infertility. The aim of this study was to correlate specific sperm miRNA expression in association with male factor infertility diagnoses to further the understanding of miRNAs in impaired spermatogenesis. Research study. Sperm samples were donated with patient consent: oligozoospermia (<10 million/ml; n=7; mean paternal age = 37.7 years), 0% normal morphology (Kruger strict criteria; n=6; mean paternal age = 41.7 years), and normozoospermic controls (n=7; mean paternal age = 38.1 years). miRNA was isolated from individual sperm samples (n=19) to determine the expression profiles of 10 specific miRNAs using qPCR with Taqman® miRNA assays (Thermo Scientific). Statistical analysis was performed with REST© software (Qiagen), significance at P<0.05. There were no significant differences observed between the groups for paternal age. None of the 10 specific miRNAs displayed significant differences in expression levels for the 0% normal morphology group compared to normozoospermic controls or oligozoospermia males. Conversely, oligozoospermia males displayed altered sperm miRNA expression profiles compared to normozoospermic controls, specifically, 3 miRNAs (122, 449 and 15b) exhibited decreased expression (P<0.05), while 2 miRNAs (141 and 200a) showed increased expression (P<0.05). Target gene analysis of these altered miRNAs identified several important spermatogenesis genes including, among others BCL2 (a regulator of spermatogonial apoptosis) and NOTCH1 (differentiation and survival of germ cells). Interestingly, both upregulated miRNAs, miR-141 (6.02 fold; P<0.05) and 200a (3.05 fold; P<0.05), are known suppressors of sperm associated antigen 9 (SPAG9), which is a critical gene located in the acrosomal compartment that has been implicated in capacitation and the sperm-oocyte interaction. Significant suppression of SPAG9 expression could represent one molecular mechanism contributing to compromised spermatogenesis in oligozoospermic males. MiRNAs function post-transcriptionally to regulate downstream target gene expression typically by inhibiting or repressing mRNA translation. Oligozoospermia displayed significant disruptions to specific sperm miRNA expression profiles revealing a fingerprint of impaired spermatogenesis resulting in low sperm concentration. In contrast, it would appear that for these specific miRNAs there was no association with abnormal sperm morphology. Determining the underlying mechanisms associated with male factor infertility will assist in improving clinical diagnosis and patient management during infertility treatment.

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations

BackgroundSmall RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed.To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized.ResultsSperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT).ConclusionsSmall RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.

Normalization matters: tracking the best strategy for sperm miRNA quantification.

What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). Not applicable. This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Autònoma de Barcelona). The authors report no conflict of interest.