Hyaluronan Binding Research Articles

Extracellular Superoxide Dismutase Content Modulates Hypoxia-induced Platelet Activation, Interstitial Macrophage Accumulation/Reprogramming and Hyaluronan Binding

Extracellular Superoxide Dismutase Content Modulates Hypoxia-induced Platelet Activation, Interstitial Macrophage Accumulation/Reprogramming and Hyaluronan Binding

Distinct chemical structures inhibit the CEMIP hyaluronidase and promote oligodendrocyte progenitor cell maturation

Growing evidence supports pathogenic roles for chronically elevated hyaluronidase activity in numerous conditions. Elevated expression of one such hyaluronidase, the Cell Migration Inducing and hyaluronan binding Protein (CEMIP), has been implicated in the pathogenesis and progression of several cancers as well as demyelinating diseases in the central nervous system (CNS). Developing effective and selective CEMIP inhibitors could therefore have efficacy in treating a variety of conditions where CEMIP is chronically elevated. Using two distinct screens for novel hyaluronidase inhibitors, we identified two synthetic thiocarbamates and one plant-derived flavonoid, sulfuretin, that effectively blocked CEMIP activity in live cells, including a tumorigenic cell line and primary cultures of oligodendrocyte progenitor cells (OPCs). None of these agents influenced cell proliferation, but they had differential dose-dependent and cell type specific effects on cell survival. Furthermore, we found that each of these agents could promote oligodendrocyte maturation by OPCs in the presence of high molecular weight (>2 Mda) hyaluronan, the accumulation of which is linked to the inhibition of OPC maturation and remyelination failure in demyelinating diseases. These findings indicate that CEMIP can be inhibited through distinct chemical interactions, and that CEMIP inhibitors have potential efficacy for treating demyelinating diseases or other conditions where CEMIP is elevated.

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

Biomimetic Hyaluronan Binding Biomaterials to Capture the Complex Regulation of Hyaluronan in Tissue Development and Function.

Within native ECM, Hyaluronan (HA) undergoes remarkable structural remodeling through its binding receptors and proteins called hyaladherins. Hyaladherins contain a group of tandem repeat sequences, such as LINK domains, BxB7 homologous sequences, or 20-50 amino acid long short peptide sequences that have high affinity towards side chains of HA. The HA binding sequences are critical players in HA distribution and regulation within tissues and potentially attractive therapeutic targets to regulate HA synthesis and organization. While HA is a versatile and successful biopolymer, most HA-based therapeutics have major differences from a native HA molecule, such as molecular weight discrepancies, crosslinking state, and remodeling with other HA binding proteins. Recent studies showed the promise of HA binding domains being used as therapeutic biomaterials for osteoarthritic, ocular, or cardiovascular therapeutic products. However, we propose that there is a significant potential for HA binding materials to reveal the physiological functions of HA in a more realistic setting. This review is focused on giving a comprehensive overview of the connections between HA's role in the body and the potential of HA binding material applications in therapeutics and regenerative medicine. We begin with an introduction to HA then discuss HA binding molecules and the process of HA binding. Finally, we discuss HA binding materials anf the future prospects of potential HA binding biomaterials systems in the field of biomaterials and tissue engineering.

Adaptor protein CEMIP reduces the chemosensitivity of small cell lung cancer via activation of an SRC-YAP oncogenic module.

Small cell lung cancer (SCLC) is a recalcitrant malignancy with dismal prognosis due to rapid relapse after an initial treatment response. More effective treatments for SCLC are desperately needed. Our previous studies showed that cell migration-inducing hyaluronan binding protein (CEMIP) functionally promotes SCLC cell proliferation and metastasis. In this study, we investigated whether and how CEMIP regulates the chemosensitivity of SCLC. Through the GDSC database, we found that CEMIP expression levels were positively correlated with the IC50 values of several commonly used chemotherapeutic drugs in SCLC cells (cisplatin, gemcitabine, 5-fluorouracil and cyclophosphamide). We demonstrated that overexpression or knockdown of CEMIP in SCLC cells resulted in a notable increase or reduction in the IC50 value of cisplatin or etoposide, respectively. We further revealed that CEMIP functions as an adaptor protein in SCLC cells to interact with SRC and YAP through the 1-177 aa domain and 820-1361 aa domain, respectively, allowing the autophosphorylation of Y416 and activation of SRC, thus facilitating the interaction between YAP and activated SRC, and resulting in increased phosphorylation of Y357, protein stability, nuclear accumulation and transcriptional activation of YAP. Overexpressing SRC or YAP counteracted the CEMIP knockdown-mediated increase in the sensitivity of SCLC cells to cisplatin and etoposide. The combination of the SRC inhibitor dasatinib or the YAP inhibitor verteporfin and cisplatin/etoposide (EP regimen) displayed excellent synergistic antitumor effects on SCLC both in vitro and in vivo. This study demonstrated that targeted therapy against the CEMIP/SRC/YAP complex is a potential strategy for SCLC and provides a rationale for the development of future clinical trials with translational prospects.

The spatial separation of basic amino acids is similar in RHAMM and hyaluronan binding peptide P15-1 despite different sequences and conformations.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.

<span>Identification of the potential compounds for inhibition CD44 target of human breast cancer stem cells by docking method</span>

In a recent cell-based assay, it has been demonstrated that the F-19848A compound inhibits the binding of hyaluronan to the CD44 receptor, which is a cell-surface glycoprotein and a receptor for hyaluronan, a major component of the tumor extracellular matrix. The interaction between CD44 and hyaluronan has been shown to promote breast cancer metastasis according to evidence. In this study, the PubChem database contains more than 112 million compounds. This data is inputted for virtual screening find out top hits by combining Lipinski’s rule and docking method. With 20 configurations obtained by docking method, the lowest binding affinity ΔEbind achieved in the best docking mode was chosen as a scoring function for picking out top ligands. For inhibition the CD44 target, the top-leads compounds with binding energy less than -9.0 kcal.mol-1 and F-19848A have selected. By docking method, the binding site and other quantities were determined such as the number of hydrogen bonds (HB), non-bond contacts (NBC) of top ligands with CD44 target. Besides, the results also showed that the non-bonded contacts dominate over hydrogen bonds in the interaction between top ligands with CD44 target.

The non-mitotic role of HMMR in regulating the localization of TPX2 and the dynamics of microtubules in neurons.

A functional nervous system is built upon the proper morphogenesis of neurons to establish the intricate connection between them. The microtubule cytoskeleton is known to play various essential roles in this morphogenetic process. While many microtubule-associated proteins (MAPs) have been demonstrated to participate in neuronal morphogenesis, the function of many more remains to be determined. This study focuses on a MAP called HMMR in mice, which was originally identified as a hyaluronan binding protein and later found to possess microtubule and centrosome binding capacity. HMMR exhibits high abundance on neuronal microtubules and altering the level of HMMR significantly affects the morphology of neurons. Instead of confining to the centrosome(s) like cells in mitosis, HMMR localizes to microtubules along axons and dendrites. Furthermore, transiently expressing HMMR enhances the stability of neuronal microtubules and increases the formation frequency of growing microtubules along the neurites. HMMR regulates the microtubule localization of a non-centrosomal microtubule nucleator TPX2 along the neurite, offering an explanation for how HMMR contributes to the promotion of growing microtubules. This study sheds light on how cells utilize proteins involved in mitosis for non-mitotic functions.

"Modeling Diffusive Search by Non-Adaptive Sperm: Empirical and Computational Insights".

During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ~106-1011 cells to 101-102 cells (depending on the species). Classical sperm competition theory addresses the positive or 'stabilizing' selection that acts on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because 'low fitness' sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm 'fitness' during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm 'fitness' as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

The Role of Hyaluronan/Receptor for Hyaluronan-Mediated Motility Interactions in the Modulation of Macrophage Polarization and Cartilage Repair

Hyaluronan (HA), a negatively charged linear glycosaminoglycan, is a key macromolecular component of the articular cartilage extracellular matrix. The differential effects of HA are determined by a spatially/temporally regulated display of HA receptors, such as CD44 and Receptor for Hyaluronan Mediated Motility (RHAMM). HA signaling through CD44 with RHAMM has been shown to stimulate inflammation and fibrotic processes. This study shows an increased expression of RHAMM in pro-inflammatory macrophages. Interfering with HA/RHAMM interactions using a 15mer RHAMM-mimetic, HA binding peptide (P15-1) together with high molecular weight (HMW)HA reduced the expression and release of inflammatory markers and increased the expression of anti-inflammatory markers in pro-inflammatory macrophages. Interfering with HA/RHAMM interactions in vivo during the regeneration of a full thickness cartilage defect after microfracture surgery in rabbits using three intra-articular injections of P15-1 together with HMWHA reduced the number of pro-inflammatory macrophages and increased the number of anti-inflammatory macrophages in the injured knee joint and greatly improved the repair of the cartilage defect compared to intra-articular injections of HMWHA alone. These findings suggest that HA/RHAMM interactions play a key role in cartilage repair/regeneration via stimulating inflammatory and fibrotic events, including increasing the ratio of pro-inflammatory to anti-inflammatory macrophages. Interfering with these interactions reduced inflammation and greatly improved cartilage repair.

Lespedeza maximowiczii flower absolute promotes skin epithelization, barrier properties, and moisturization-related beneficial responses in human keratinocytes

Lespedeza maximowiczii (LM), a member of the legume family, has tyrosinase inhibitory and estrogenic activities. However, its effects on skin-related biological activities remain unclear. Therefore, the present study aimed to explore the effects of LM flower absolute (LMFAb) on skin-related biological events, especially skin re-epithelization, barrier and moisturizing-related keratinocyte (HaCaT cell) responses. In this study, LMFAb was isolated from LM flowers via solvent extraction and its chemical composition analysis was performed using gas chromatography/mass spectrometry. 5-bromo-2′-deoxyuridine incorporation, Boyden chamber, sprout outgrowth, enzyme-linked immunosorbent, and Western blot assay were used to analyze the biological effects of LMFAb on HaCaT cells (a human epidermal keratinocyte cell line). Twelve components were identified in LMFAb. LMFAb promoted cell proliferation, migration, and sprout outgrowth in HaCaT cells. The absolute enhanced the activations of MAPKs (ERK1/2, JNK, and p38), PI3K and AKT proteins in HaCaT cells and elevated collagen type I and IV levels in HaCaT cell conditioned medium. In addition, LMFAb induced an increase in the expression levels of epidermal barrier proteins (filaggrin and involucrin) in HaCaT cells. Furthermore, LMFAb increased hyaluronan (HA) production and expression of HA synthases (HAS-1, HAS-2, and HAS-3) but decreased HYBID (HA binding protein involved in HA depolymerization) level in HaCaT cells. These findings demonstrate that LMFAb might promote skin re-epithelization, barrier and moisturizing-related beneficial responses in keratinocytes. This study suggests that LMFAb should be considered a potential starting material for the development of cosmetic or pharmaceutical agents that restore the functions of damaged skin.

Abstract 16891: High Molecular Weight Hyaluronan Inhibits Macrophage Phagocytosis

Background: After a myocardial infarction (MI), macrophages are recruited to the site of injury to begin the process of wound healing. Macrophages are important because they clear cellular debris via phagocytosis. This period of acute inflammation in the heart coincides with extracellular matrix (ECM) remodeling. Hyaluronan (HA), one of the most abundant ECM components, accumulates in a high molecular weight form (HA HMW ). The relationship between HA HMW accumulation and macrophage function in is not fully understood. Goal: To determine the extent to which hyaluronan impacts phagocytosis in polarized macrophages. Methods and Results: Tissue sections were collected from mice one-week post-MI and stained for hyaluronan binding protein (HABP) to assess HA accumulation. A significant increase in HA was seen in both male (n=4, p=0.0031) and female (n=4, p=0.0210) MI hearts compared to sham hearts. Separately, bone marrow was isolated from naïve mice and cells were treated in culture with macrophage colony stimulating factor (M-CSF) for 7-10 days. The resultant macrophages were then polarized into a pro-inflammatory (M1) phenotype using LPS and INF-γ, or pro-reparative phenotype (M2) using IL-4 and IL-13. Macrophage polarization was confirmed via qRT-PCR. In additional experiments, macrophages were polarized for 24 hours and then incubated for 30 min with FITC-labeled, IgG-coated latex beads in the presence or absence of 1 μM HA HMW . Flow cytometry was used to assess phagocytosis of the beads. HA HMW treatment resulted in a significant reduction in the percent of FITC positive cells in M1 female (n=6, p=0.005), M2 female (n=5, p=0.0406), and M2 male (n=5, p=0.0314) macrophage subsets indicating a decrease in phagocytosis. No significant changes were seen in the M0 and M1 male macrophage subsets. Additionally, the mean fluorescence intensity (MFI) measured in the FITC channel was significantly decreased in all the female subsets as well as the M2 male macrophages (n=5-6, p<0.05). No significant changes were seen in the M0 and M1 male subsets. Conclusion: HA HMW impairs opsonin-induced phagocytosis in macrophages. Given that HA HMW accumulates post-MI, these findings may partially explain the incomplete immune response in the failing heart.

Age-Related COVID-19 Influence on Male Fertility.

The impact of coronavirus on the reproductive health of men attracts the special attention of many researchers. While studies suggest changes in sperm parameters and the possibility of testicular inflammation, further studies are needed to elucidate any potential age-related changes in these findings, which is the purpose of the present study. The semen quality parameters, cytokine concentration, and markers of the pro- and antioxidant system were assessed in 60 men five to seven months after the coronavirus infection and in 77 controls (without a history of coronavirus infection). Additionally, participants were divided into two age groups: less than 35 years and 35 years or older. Notably increased round cell count in ejaculate and reduced sperm hyaluronan binding ability were observed among post-infection patients younger than 35 years. In the same group, a decline in seminal plasma zinc levels and nitrotyrosine in the cell fraction was found. In men over 35 years of age, Coronavirus Disease 2019 (COVID-19) led to increased sperm DNA fragmentation, a decrease in the total antioxidant capacity, and an elevation in the levels of interleukin-1β and interleukin-10. The concentration of interleukin-1β decreased over time following recovery in all affected patients. The data obtained suggest the potential adverse impact of the coronavirus infection on male reproductive health; however, these effects appear to be age-dependent.

Urinary Protein-Biomarkers Reliably Indicate Very Early Kidney Damage in Children With Alport Syndrome Independently of Albuminuria and Inflammation

Alport syndrome (AS) is a hereditary type IV collagen disease. It starts shortly after birth, without clinical symptoms, and progresses to end-stage kidney disease early in life. The earlier therapy starts, the more effectively end-stage kidney disease can be delayed. Clearly then, to ensure preemptive therapy, early diagnosis is an essential prerequisite. To provide early diagnosis, we searched for protein biomarkers (BMs) by mass spectrometry in dogs with AS stage 0. At this very early stage, we identified 74 candidate BMs. Of these, using commercial enzyme-linked immunosorbent assays (ELISAs), we evaluated 27 in dogs and 28 in children, 50 with AS and 104 healthy controls. Most BMs from blood appeared as fractions of multiple variants of the same protein, as shown by their chromatographic distribution before mass spectrometry. Blood samples showed only minor differences because ELISAs rarely detect disease-specific variants. However, in urine , several proteins, individually or in combination, were promising indicators of very early and preclinical kidney injury. The BMs with the highest sensitivity and specificity were collagen type XIII, hyaluronan binding protein 2 (HABP2), and complement C4 binding protein (C4BP). We generated very strong candidate BMs by our approach of first examining preclinical AS in dogs and then validating these BMs in children at early stages of disease. These BMs might serve for screening purposes for AS before the onset of kidney damage and therefore allow preemptive therapy.

The inflammatory oxidant peroxynitrous acid modulates the structure and function of the recombinant human V3 isoform of the extracellular matrix proteoglycan versican

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO−/ONOOH. Both reagent ONOO−/ONOOH and SIN-1 (a thermal source of ONOO−/ONOOH) modified Tyr, Trp and Met residues. ONOO−/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO−/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO−/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

P-048 Vitrification of human semen impairs the mobility of spermatozoa when compared to the conventional slow-freezing

Abstract Study question Does vitrification provide a better method for human sperm cryopreservation than slow freezing in terms of survival, mobility, maturity, and DNA fragmentation? Summary answer Vitrification of human semen yields similar outcome as slow-freezing considering survival, mobility, DNA fragmentation, and maturity but the mobility after thawing is lower. What is known already Slow freezing is the most common technique for cryopreservation of human sperm. Unfortunately, many sperm cells die during the process or lose their mobility and are thereafter unable of fertilization. Meanwhile, vitrification of animal sperm showed good outcomes in terms of sperm survival and motility while being easier to perform than slow freezing. So far, many studies focused on survival and mobility of human sperm after vitrification compared to slow freezing, with results still not unified. They also neglected the importance of sperm maturity and DNA fragmentation caused by the process. DNA damage can affect embryo development or cause abortion. Study design, size, duration The study was performed from March January to July 2022. It included 69 male participants who underwent a diagnostic spermiogram at the Division of gynaecology and obstetrics of University Medical Centre Ljubljana. All participants gave written consent of using the remaining of sample, after performing the spermiogram, for our study. Sperm samples were divided in three aliquots, one saved as native sample, one intended for vitrification, and one intended for slow freezing. Participants/materials, setting, methods Participants in the study had normal sperm quality parameters according to 5th edition of WHO manual. Slow freezing was performed using Sperm Freezing medium (Origio). For vitrification sperm was diluted in solution consisting of Sperm preparation medium (Origio), sucrose, and HSA in 1:1 (v/v) ratio, incubated for five minutes and then vitrified by adding 30 microliter droplets directly into liquid nitrogen. After thawing sperm survival, mobility, maturity, and DNA fragmentation were assessed. Main results and the role of chance The data are presented as mean values (± standard deviation) when normally distributed and as medians (IQR) when not normally distributed. The patient’s age was 35.0±5.1 years. In native sperm sample, sperm concentration was 69.6±59.0x106 cells per millilitre and total motility was assessed to be 60.0% (50.0%-70.0%). After thawing we noticed significantly lower mobility (p < 0.001), while also the mobility was significantly lower after vitrification when compared to slow-freezing (17.6% (12.4%-25.3%) and 14.5% (9.0%-22.6%), p = 0.007). Interestingly, there was no difference in survival after thawing the samples (20.5±10.7 % after slow freezing and 23.7±13.7 % after vitrification). Also there was no difference in maturity of sperm when checked with protamine staining (native vs. slow-freezing vs. vitrification; 53.6±15.2 % vs. 53.9±16.0 % vs. 52.4±15.2 %, p = 0.905) and also when checked with hyaluronan binding assay (78.0% (67.5%-82.4%) vs. 79.4% (71.7%-82.95%) vs. 75.3% (67.5%-81.1%), p = 0.607) . While sperm maturity was not affected with cryopreservation, DNA fragmentation was significantly higher after cryopreservation (native vs. slow-freezing vs. vitrification; 19.7±9.5%, vs. 24.7±11.6 vs. 27.1±15. %, p = 0.049). Limitations, reasons for caution We believe our study would be better by obtaining higher number of participants and by being performed over longer time period. We would also like to further optimise vitrification protocol. More knowledge is also needed in poor sperm samples, since we only included sperm samples of good quality. Wider implications of the findings We believe our finding are of great importance for the field of andrology. Our results show vitrification of human sperm can be comparable to slow freezing in most of parameters analysed, but lower mobility after vitrification is reason for caution. Trial registration number 20210024

Binding of different hyaluronan to CD44 mediates distinct cell adhesion dynamics under shear flow.

As a known receptor-ligand pair for mediating cell-cell or cell-extracellular matrix adhesions, cluster of differentiation 44 (CD44)-hyaluronan (HA) interactions are not only determined by molecular weight (MW) diversity of HA, but also are regulated by external physical or mechanical factors. However, the coupling effects of HA MW and shear flow are still unclear. Here, we compared the differences between high molecular weight HA (HHA) and low molecular weight HA (LHA) binding to CD44 under varied shear stresses. The results demonstrated that HHA dominated the binding phase but LHA was in favour of the shear resistance phase, respectively, under shear stress range ≤ 1.0 dyne·cm-2 . This difference was attributed to the high binding strength of the CD44-HHA interaction, as well as the optimal distribution matching between both CD44 and HA sides. Activation of the intracellular signal pathway was sensitive to both HA MW and shear flow. Our findings also indicate that only CD44-HHA interaction under shear stress of 0.2 dyne·cm-2 could significantly enhance the clustering of CD44, as well as induce the increase in both CD44 and CD18 expression. The present study offers the basis for further quantification of the features of CD44-HA interactions and their biological functions.

Hyaluronan regulates sperm-induced inflammatory response by enhancing sperm attachment to bovine endometrial epithelial cells via CD44: in-silico and in-vitro approaches.

Recently, we reported that sperm induce cluster of differentiation 44 (CD44) expression and Toll-like receptor 2 (TLR2)-mediated inflammatory response in bovine uterus. In the present study, we hypothesized that the interaction between CD44 of bovine endometrial epithelial cells (BEECs) and hyaluronan (HA) affects sperm attachment and thereby enhancing TLR2-mediated inflammation. To test our hypothesis, at first, in-silico approaches were employed to define the binding affinity of HA for CD44 and TLR2. Further, an in-vitro experiment using the sperm-BEECs co-culture model was applied to investigate the effect of HA on sperm attachment and inflammatory response. Here, low molecular weight (LMW) HA at different concentrations (0, 0.1, 1, or 10 µg/mL) was incubated with BEECs for 2 h followed by the co-culture without- or with non-capacitated washed sperm (106/ml) for additional 3 h was performed. The present in-silico model clarified that CD44 is a high-affinity receptor for HA. Moreover, TLR2 interactions with HA oligomer (4- and 8-mers) target a different subdomain (h-bonds) compared to TLR2-agonist (PAM3) which targets a central hydrophobic pocket. However, the interaction of LMW HA (32-mers) with TLR2 revealed no stability of HA at any pocket of TLR2. Notably, the immunofluorescence analysis revealed the HA localization in both endometrial stroma and epithelia of ex-vivo endometrial explant. Moreover, ELISA showed significant levels of HA in BEECs culture media. Importantly, BEECs pretreatment with HA prior to sperm exposure increased the number of attached sperm to BEECs, and upregulated the transcriptional levels of pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs in response to sperm. However, BEECs treated with HA only (no sperm exposure) did not show any significant effect on the transcript abundance of pro-inflammatory genes when compared to the non-treated BEECs. Altogether, our findings strongly suggest a possible cross-talk between sperm and endometrial epithelial cells via HA and HA binding receptors (CD44 and TLR2) to induce a pro-inflammatory response in bovine uterus.

Influence of a hyaluronan-binding system for sperm selection in intracytoplasmic sperm injection cycles on embryo morphokinetic parameters and in vitro fertilization cycle outcomes.

Although the impact of the paternal contribution to embryo quality and blastocyst formation is a well-known phenomenon, the current literature provides insufficient evidence that hyaluronan-binding sperm selection methods improve assisted reproductive treatment outcomes. Thus, we compared the cycle outcomes of morphologically selected intracytoplasmic sperm injection (ICSI) with hyaluronan binding physiological intracytoplasmic sperm injection (PICSI) cycles. A total of 2415 ICSI and 400 PICSI procedures of 1630 patients who underwent in vitro fertilization cycles using a time-lapse monitoring system between 2014 and 2018 were analyzed retrospectively. Fertilization rate, embryo quality, clinical pregnancy rate, biochemical pregnancy rate and miscarriage rate were evaluated, differences in morphokinetic parameters and cycle outcomes were compared. In total, 85.8 and 14.2% of the whole cohort were fertilized with standard ICSI and PICSI, respectively. The proportion of fertilized oocytes did not significantly differ between groups (74.53 ± 1.33 vs. 72.92 ± 2.64, p > 0.05). Similarly, the proportion of good-quality embryos according to the time-lapse parameters and the clinical pregnancy rate did not significantly differ between groups (71.93 ± 4.21 vs. 71.33 ± 2.64, p > 0.05 and 45.55 ± 2.91 vs. 44.96 ± 1.25, p > 0.05). No statistically significant differences were found between groups in clinical pregnancy rates (45.55 ± 2.91 vs. 44.96 ± 1.25, p > 0.05). Biochemical pregnancy rates (11.24 ± 2.12 vs. 10.85 ± 1.83, p > 0.05) and miscarriage rates (24.89 ± 3.74 vs. 27.91 ± 4.91, p > 0.05) were not significantly different between groups. The effects of the PICSI procedure on fertilization rate, biochemical pregnancy rate, miscarriage rate, embryo quality, and clinical pregnancy outcomes were not superior. The effect of the PICSI procedure on embryo morphokinetics was not apparent when all parameters were considered.