Lipidomic profiling of cervical mucus reveals the potential role of pro-inflammatory derived metabolites on sperm transport across the ovine cervix.

Internationally, cervical artificial insemination (AI) in sheep yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway where vaginal AI of frozen-thawed semen to a natural oestrus yields non-return rates in excess of 60%, which has been attributed to the ewe breed used in Norway. This study used both metabolomics and an RNA-sequencing approach to assess the lipid production and composition from cervical mucus and tissue of four European ewe breeds (n = 28-30 ewes per breed) with previously reported differences in pregnancy rates following cervical AI with frozen-thawed semen. These breeds included Suffolk (exhibiting low fertility), Belclare (medium fertility) as well as Norwegian White Sheep and Fur (both with high fertility and pregnancy rates > 60%) at both a synchronised and natural oestrous cycle. The aim was to explore the differences between ewe breeds in the lipidomic profile and to identify candidate biomarkers associated with an optimal environment for cervical sperm transport. The results revealed the identification of 255 lipids, of which 170, 102 and 83 were different between ewe breeds, types of cycle and affected by their interaction, respectively (P < 0.05). Reduced levels of lipids involved in the resolution of inflammation (i.e. 14-HDoHE,17-HDoHE, 15-HETE) were identified in the low-fertility Suffolk breed compared to high-fertility ewe breeds. However, there was an up-regulation of the COX pathway accompanied by increased levels of prostaglandins in the Suffolk breed. These findings indicated a sub-optimal and pro-inflammatory environment that could have a negative effect on cervical sperm transport.

Expression Analysis of Genes Corresponding to Mucins and Their Glycans from Cervical Tissue Using RNA Sequencing.

In the female reproductive tract, mucin proteins are the main component of mucus secreted by cervical goblet cells and play an essential role in many biological functions. They act as a medium for lubrication and mainting a cervical mucosal barrier against ascending pathogens from the vagina while also allowing sperm migration. The expression of mucin genes as well as the levels of O-glycosylation changes across the oestrous cycle. Detection and characterization of mucins and their glycans is important to understand the interface between the external and the internal environment, as the cervical epithelium represents the first line of defense against infections of the upper reproductive tract. Advances in the field of molecular biology have made possible to study differences in mucin and glycan gene expression which can help to understand impeded sperm transport as well as variation in the susceptibility to infection. This chapter discusses procedures relevant for both animals and humans on how to recover cervical tissue and perform a gene expression analysis of genes corresponding to mucins and their glycans using RNA sequencing.

Metabolic signature of cervical mucus in ewe breeds with divergent cervical sperm transport: a focus on metabolites involved in amino acid metabolism.

INTRODUCTION: Cervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used. OBJECTIVES AND METHODS: This study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility). RESULTS: A total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset (P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively (P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS (P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds (P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle. CONCLUSION: The suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls.

In vitro assessment of bull semen quality is routinely used in bull semen processing centres in order to ensure that semen destined to be used in the field has passed minimum standards. Despite these stringent quality control checks, individual bulls that pass the quality control checks can still vary in field fertility by up to 25%. A genome-wide association study was undertaken to determine genetic markers associated with prefreeze and post-thaw bull sperm quality traits as well as field fertility. Genome-wide association analysis was performed using a single nucleotide polymorphism (SNP) regression mixed linear model in WOMBAT. Genes within a 250 Kb span of a suggestive (P ≤ 1 × 10-5) SNP were considered as candidate genes. One SNP was associated with adjusted pregnancy rate, and 21 SNPs were associated across the seven semen quality traits (P ≤ 1 × 10-5). Functional candidate genes include SIPA1L2 which was associated with adjusted pregnancy rate. This encodes a Rap GTPase-activating protein involved in Rap1 signalling pathway and was previously found to play a role in the process of sperm differentiation. Gene ontology (GO) analysis also identified significantly enriched biological processes involved protein tyrosine kinase activity including genes such as DYRK1A, TEC and TXK that were associated with sperm motility prior to freezing. Another candidate gene associated with post-thaw sperm motility was FHDC1 which coordinates actin filament and microtubule dynamics. The induced 11 GO terms in the ejaculates rejected after freezing trait were related to ATPase, phosphatase and hydrolase activity. These results reveal novel specific genomic regions and candidate genes associated with economically important phenotypes such as field fertility and semen quality traits.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls.

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport†.

Worldwide, cervical artificial insemination using frozen-thawed semen yields low pregnancy rates. The only exception to this is in Norway, where vaginal insemination with frozen-thawed semen yields pregnancy rates in excess of 60% and which has been attributed to the specific ewe breed used. Our previous work demonstrated differences in cervical gene expression at the follicular phase of the estrous cycle in ewe breeds with known differences in pregnancy rates. In this study, we characterized the cervical transcriptome of the same ewe breeds [Suffolk, Belclare, Fur, and Norwegian White Sheep (NWS)] during the luteal phase, as an optimal environment at the luteal phase could better prepare the cervix for sperm migration through the cervix at the subsequent follicular phase. High-quality RNA extracted from postmortem cervical tissue was analyzed by RNA sequencing. After stringent filtering, 1051, 1924, and 611 differentially expressed genes (DEGs) were detected in the low-fertility Suffolk breed compared with Belclare, Fur, and NWS, respectively. Gene ontology analysis identified increased humoral adaptive immune response pathways in Suffolk. Increased expression of multiple immune genes supports the presence of an active immune response in the cervix of Suffolk ewes, which differentiates them significantly from the other three ewe breeds. Inflammatory pathways were upregulated in the Suffolk, resulting in higher expression of the potent pro-inflammatory cytokines. Therefore, higher levels of pro-inflammatory cytokines indicate unresolved inflammation in the cervix of the low-fertility Suffolk breed that could contribute to reduced cervical sperm transport in the next follicular phase.

Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle.

BACKGROUND: Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds). RESULTS: RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P <  0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Biochemical and molecular characterization of sialylated cervical mucins in sheep†.

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Identification and characterization of O-linked glycans in cervical mucus as biomarkers of sperm transport: A novel sheep model.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen-thawed semen, which are due to reported differences in cervical sperm transport. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway (n = 28-30 ewes/breed). We identified 124 O-glycans, from which 51 were the major glycans with core 2 and fucosylated glycans as the most common structures. The use of exogenous hormones for synchronization did not affect the O-glycan composition in both high-fertility ewe breeds, but it did in the other four ewe breeds. There was a higher abundance of the sulfated glycan (Galß1-3[SO3-GlcNAcß1-6]GalNAc), fucosylated glycan (GlcNAcß1-3(Fucα1-2Galß1-3)GalNAc) and core 4 glycan (GlcNAcß1-3[GlcNAcß1-6]GalNAc) in the low-fertility Suffolk breed compared with NWS (high fertility). In addition, core 4 glycans were negatively correlated with mucus viscosity. This novel study has identified O-glycans that are important for cervical sperm transport and could have applications across a range of species including human.

Conserved and breed-specific differences in the cervical transcriptome of sheep with divergent fertility at the follicular phase of a natural oestrus cycle.

BACKGROUND: The outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle (n = 8 to 10 ewes per breed). RESULTS: High-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering (P <  0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: These breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

H3K4me2 accompanies chromatin immaturity in human spermatozoa: an epigenetic marker for sperm quality assessment.

Chromatin remodeling, including histone post-translational modifications, during spermatogenesis can affect sperm quality and fertility, and epigenetic marks may therefore be useful for clinical evaluations of sperm. Together with histone hyperacetylation, the dimethylation of histone H3 on lysine K4 (H3K4me2) is also required during protamination. Accordingly, we evaluated the utilization of this epigenetic mark for the identification of sperm with decrease quality and immature chromatin. In this study, 99 semen samples, including 22 normozoospermic (N), 63 asthenozoospermic (A), and 14 oligoasthenozoospermic (OA) samples, were comprehensively analyzed with respect to H3K4me2 levels, DNA damage (DNA fragmentation index, DFI), and sperm immaturity (high DNA stainability, %HDS), as determined by a sperm chromatin structure assay using flow cytometry. We detected a significant relationship between H3K4me2 and %HDS (r = 0.47; p < 0.001). Furthermore, we observed negative correlations between H3K4me2 and sperm concentration, motility, and mitochondrial activity (p < 0.05). The increase in immaturity as semen quality decreased (N > A > OA) indicates the importance of chromatin immaturity and histone code deviations in sperm evaluations. Using various approaches, our study elucidated H3K4me2 as a molecular marker of sperm quality with potential use in reproductive medicine.Abbreviations: A: asthenozoospermic; AO: acridine orange; ART: assisted reproductive therapy; BWW: Biggers-Whitten Whittingham; DAPI: 4',6' -diamidino-2-phenylindole; DFI: DNA fragmentation index; H3K4me2: dimethylation of lysine K4 on histones H3; HDS: high DNA stainability; HRP: horseradish peroxidase; MACS: magnetic-activated cell sorting; N: normospermic; NGS: normal goat serum; OA: oligoasthenozoospermic; PTM: post-translational modification; SCSA: sperm chromatin structure assay; SUTI: sperm ubiquitin tag assay; TBS-T: TBS with 0.5% Tween-20.

Non-Invasive Approaches to Epigenetic-Based Sperm Selection.

Since sperm size and form do not necessarily provide information on internal sperm structures, novel sperm markers need to be found in order to conduct assisted reproductive therapies (ART) successfully. Currently, the priority of andrologists is not only to select those sperm able to fertilize the oocyte, but also a high quality of sperm that will guarantee a healthy embryo. Evidence of this shows us the importance of studying sperm intensively on genetic and epigenetic levels, because these could probably be the cause of a percentage of infertility diagnosed as idiopathic. Thus, more attention is being paid to posttranslational modifications as the key for better understanding of the fertilization process and its impact on embryo and offspring. Advances in the discovery of new sperm markers should go hand in hand with finding appropriate techniques for selecting the healthiest sperm, guaranteeing its non-invasiveness. To date, most sperm selection techniques can be harmful to sperm due to centrifugation or staining procedures. Some methods, such as microfluidic techniques, sperm nanopurifications, and Raman spectroscopy, have the potential to make selection gentle to sperm, tracking small abnormalities undetected by methods currently used. The fact that live cells could be analyzed without harmful effects creates the expectation of using them routinely in ART. In this review, we focus on the combination of sperm epigenetic status (modifications) as quality markers, with non-invasive sperm selection methods as novel approaches to improve ART outcomes.