Cumulus matrix selection leads to isolation of spermatozoa with better motility, morphology, and lower DNA fragmentation.

The objective of this study was to compare the motility, morphology, and levels of DNA fragmentation of spermatozoa subjected to conventional swim-up or cumulus matrix (CM) sperm selection. Semen samples were collected from 60 normozoospermic men at a private hospital between December 2021 and March 2022. After liquefaction, semen samples were separated into two portions - one part was subjected to conventional swim-up preparation and the remaining spermatozoa were subjected to CM selection. The CM was obtained by mechanical isolation from healthy donor oocytes. Semen analysis and evaluation of sperm were performed according to WHO 6th Edition Laboratory Manual and Kruger's strict criteria, respectively. Sperm DNA fragmentation (SDF, %) of the two preparations was evaluated using Halosperm G2 detection kit (Halotech, Madrid, Spain). Wilcoxon rank-sum test was used to compare the characteristics of spermatozoa obtained by the two preparations. Spermatozoa selected by CM showed significantly better rapidly progressive motility (43.5% vs. 30.6%, respectively, p<0.001), higher percentage of morphologically normal forms (14.0% vs. 9.0%, respectively, p<0.05), and lower levels of SDF (26.0% vs. 45.0%, p<0.05) in comparison to those prepared by conventional swim-up. Moreover, the incidence of multiple sperm defects was considerably lower in the samples that underwent CM selection compared to those that did not (30.0% vs. 49.0%, respectively, p<0.05).The selection by CM significantly increases sperm motility and reduces morphologically abnormal spermatozoa and DNA fragmentation rates in comparison to the conventional swim-up preparation. The application of this selection technique may increase chances of successful IVF outcomes.

Characteristics of Pre-Lens Tear Film Behavior in Eyes Wearing Delefilcon A Silicone Hydrogel Water Gradient Contact Lenses.

The pre-lens tear film (PLTF) over (i) delefilcon A silicone hydrogel water gradient (WG; 33-80% from core to surface) contact lenses (CLs) (SHWG-CLs) and (ii) subjects' own non-WG soft CLs (SCLs) (SO-SCLs) was studied in 30 eyes of 30 subjects to assess the hypothesized PLTF stabilization over SHWG-CLs. In both eyes, delefilcon A SHWG-CLs (DAILIES TOTAL1®; Alcon, Fort Worth, TX, USA) or SO-SCLs were worn. After 15 min of wearing each lens, the tear meniscus radius (TMR, mm), lipid-layer interference grade (IG) and spread grade (SG), and non-invasive breakup time (NIBUT, seconds) were evaluated and compared between the SHWG-CLs and the SO-SCLs. The comparison between the SHWG-CL and SO-SCL groups (SHWG-CL and SO-SCL, mean ± SD) revealed that TMRs temporarily decreased and reached a plateau value after 15 min (0.21 ± 0.06; 0.21 ± 0.06) compared to the value prior to CL insertion (0.24 ± 0.08; 0.25 ± 0.08), with no significant difference between the two groups. The NIBUT, IG, and SG values after 15 min of wearing the CLs were (9.7 ± 3.7; 4.7 ± 4.2), (1.0 ± 0.2; 1.8 ± 1.0), and (1.1 ± 0.4; 1.9 ± 1.5), respectively, and all values were significantly better in the SHWG-CL group (p < 0.0001, p = 0.0039, and p < 0.0001, respectively). We found that compared to the SO-SCLs, the maintenance of the PLTF on the SHWG-CLs was supported by the thicker and more stable PLTF.

Dynamic Aspects of Pre-Soft Contact Lens Tear Film and Their Relation to Dry Eye: Basic Science and Clinical Relevance.

Soft contact lens (SCL) perturbs the intimate connection between the pre-lens tear film (PLTF) and the ocular surface in various ways, i.e., (i) decrease in tear meniscus radius and aqueous tear thickness, (ii) attenuation of tear film lipid layer spread, (iii) limited wettability of SCL surface, (iv) increased friction with eyelid wiper, etc. This often results in SCL-related dry eye (SCLRDE) manifested as PLTF instability and contact lens discomfort (CLD). In this review, the individual contributions of factors (i-iv) to PLTF breakup patterns (BUP) and CLD are considered via the tear film-oriented diagnosis framework adopted by the Asia Dry Eye Society from a clinical and basic science perspective. It is shown that SCLRDE (due to aqueous deficiency, increased evaporation, or decreased wettability) and BUP of PLTF classify within the same types as the ones observed for the precorneal tear film. The analysis of PLTF dynamics reveals that the inclusion of SCL enhances the manifestation of BUP associated with (i) decreased thickness of PLTF aqueous layer and (ii) limited SCL wettability as shown by the rapid expansion of BUP area. PLTF thinness and instability result in increased blink-related friction and lid wiper epitheliopathy as major contributor to CLD.

Surface Chemistry Study of the Interactions of Sesame Oil with Meibomian Films.

A possible approach for the treatment of meibomian gland disease (MGD) can be the supplementation of meibomian gland secretion (MGS) with nonpolar lipids (NPL) rich plant oils. Sesame oil (SO), approximately equal in monounsaturated fat (oleic acid, 40% of total) and polyunsaturated fat (linoleic acid, 42% of total), has shown multiple health benefits due to its anti-inflammatory and antioxidant effects. Thus, the interactions between SO and MGS in surface layers deserve further study. Therefore, pseudobinary films were formed with controlled MGS/SO molar ratios (0%, 10%, 30%, 50%, and 100% SO) at the air/water surface of the Langmuir trough over phosphate buffered saline (pH 7.4) subphase. Surface pressure (π)-area (A) isotherms and Brewster angle microscopy observations showed nonideal interactions where SO aggregates with MGS and complements the NPL stratum of the meibomian layers. The analysis of stress relaxation transients with Kohlrausch-Williams-Watts equation revealed that the supplementation of fixed amount of MGS with excess lipids via SO altered the dilatational elasticity of the films as reflected by the increase of the exponent ß. Thus, SO with its unique combination of high oxidative stability and abundance of long polyunsaturated acyl chains might be a useful supplement to MGS layers.

Correlations between bulk and surface properties of meibomian lipids with alteration of wax-to-sterol esters content.

In a recent study (Ewurum et al., 2021), wax (WE) and sterol esters (CE) from human meibum secretions (MGS) were separated and reconstituted with controlled WE/CE ratios (0%, 20%, 30%, 40%, 50%, 75% and 100% CE weight fractions). It was found that the alterations in the CE content of WE/CE mixtures modified the hydrocarbon chain conformation and packing of the mixture. A major question that emerges is whether the spectroscopic packing parameters determined for bulk meibum translate to a change in the performance of meibomian layers at the air/water interface, as it is the surface film functionality that is crucial for the performance of MGS at the ocular surface. The study of human meibum films with Langmuir surface balance was performed to access the surface properties at blink-like deformations of the film area. Surface pressure (π)-area (A) isocycles and stress relaxations were used to assess the layer's reorganization during area cycling and dilatational elasticity, respectively. The morphology of the films was monitored by Brewster angle microscopy. It was found that the increased order and chain melting temperature of the bulk samples correlated with a raise in the maximum surface pressure attained at minimal surface area and in the transient dilatational modulus of the meibomian layers. Such correlations may allow for development of an improved understanding between the bulk and surface properties of human meibum and of other natural and synthetic tear lipid films.

Interactions of Meibum and Tears with Mucomimetic Polymers: A Hint towards the Interplay between the Layers of the Tear Film.

Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the "healthy" tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.

Relationships between the material properties of silicone hydrogels: Desiccation, wettability and lubricity.

Silicone hydrogels (SiHy), represent composite matrices composed of hydrophobic gas permeable silicone (Si) rich core and a surface enriched with hydrophilic polymer moieties. Their utilization in contact lens design requires number of SiHy properties (hydration, wettability, lubricity) to be optimized for the challenging conditions at the ocular surface. Typical limitations in literature are that (i) these properties are studied in isolation, monitoring only one parameter but not the rest of them, and (ii) measurements are performed with hydrated samples immediately after removal from storage solutions. Here we study the simultaneous evolution of critical material properties (evaporative loss of water, water contact angle, coefficient of friction) of different SiHy subjected to continuous blink-like desiccation/rehydration cycling. SiHy with wetting agents incorporated in their core (narafilcon A, senofilcon A) were particularly susceptible to extended desiccation. Stenfilcon A, a material with only 3% bulk Si content maintained its performance for 4 h of cycling, and delefilcon A (80% surface water content) resisted extended 8 h of desiccation/rehydration runs. Strong correlation exists between the evolution of SiHy wettability and lubricity at ≥4 h of blink-like cycling. Understanding the interplay between SiHy properties bears insights for knowledge based design of novel ophthalmic materials.

Improving Stability of Tear Film Lipid Layer via Concerted Action of Two Drug Molecules: A Biophysical View.

The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.

Contribution of Mucins towards the Physical Properties of the Tear Film: A Modern Update.

Instability of the tear film (TF) protecting the ocular surface results in dry eye syndrome (DES), the most prevalent public health ophthalmic disease affecting the quality of life of 10 to 30% of the human population worldwide. Although the impact of the tear film lipid layer (TFLL) and of the aqueous tears (AT) to the TF stability is extensively studied, in contrast the contribution of the secretory mucins (SM) and of the membrane-associated mucins (MAM), i.e., one of the most abundant molecular classes in AT and in the corneal epithelium respectively, remains poorly defined. However, it is well known that in DES both types of mucins are quantitatively or qualitatively deficient. Numerous studies since the 1990s until now have proposed direct involvement of SM and MAM in the material properties (viscoelasticity, hydration, and protection of the ocular surface; synergistic cooperation with the rest of the TF layers; etc.) and stability of TF. These theories will be reviewed here in the context of the classical and modern in vitro and in vivo results that allow their reappraisal and in view of the novel mucin secretion enhancing pharmaceuticals, which have opened innovative routes for the therapy of DES.

Lipid Saturation and the Rheology of Human Tear Lipids.

Elevated levels of acyl chain saturation of meibomian lipids are associated with enhanced tear film (TF) stability in infants to shortened TF breakup time with meibomian gland dysfunction. Thus, the effect of saturation on the surface properties of human TF lipids (TFLs) using a Langmuir surface balance and Brewster angle microscopy was studied. Lipid phase transitions were measured using infrared spectroscopy. The raise in the % of saturation resulted in thicker, and more elastic films at π = 12 mN/m, with the effects being proportional to the saturation level. At the same time, at lower (≤10 mN/m) π, the raise in saturation resulted in an altered spreading and modified structure of TFL layers. The strong impact of saturation on TFL surface properties correlated with a saturation induced increase of the TFL acyl chain order, phase transition temperature, and lipid-lipid interactions. The native TFL order and πmax were significantly greater, compared with native meibum collected from the same individual. Aggregation of lipids on the tear surface due to saturation was not as significant as it was for meibum. Although the surface pressure/area isotherms for TFL were similar for meibum, differences in rheology and phase transition parameters warrant the study of both.

Impact of Air Exposure Time on the Water Contact Angles of Daily Disposable Silicone Hydrogels.

The wettability of silicone hydrogel (SiHy) contact lens (CLs) is crucial for the pre-lens tear film stability throughout the day. Therefore, sessile drop and captive bubble setups were used to study the advancing and receding water contact angles (CA) of four SiHy materials: narafilcon A (TE), senofilcon A (AOD), stenfilcon A (MD), and delefilcon A (DT). TE and AOD have 48% and 38% water content, respectively, and no surface coating. MD (54% water) implements "smart chemistry" with just 4.4% bulk silicone content, while DT has >80% water at its surface. These SiHy were subjected to continuous blink-like air exposure (10 s)/rehydration (1s) cycles for 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, and 16 h. The advancing CA, which measures the rehydration propensity of the CL surface, proved to be the most sensitive parameter to discriminate between the samples. The order of performance for the entire time scale was DT > MD >> AOD ≥ TE. The extended desiccation/rehydration cycling increased the differences between the CA of DT and MD compared to AOD and TE. This suggests that the low Si surface content and the high surface hydration are major determinants of SiHy wettability.

Effects of Lipid Saturation on the Surface Properties of Human Meibum Films.

Elevated levels of acyl chain saturation of meibomian lipids are associated with vastly different effects: from enhanced tear film (TF) stability in infants to shortened TF breakup time in meibomian gland disease patients. Thus it is important to study the effect of saturation on the surface properties of human meibum (MGS). Therefore, MGS films (1, 2, 3, 4, 5, 10, 25, 50, 67, and 100% saturation) were spread at the air/water interface of a Langmuir surface balance. The layers' capability to reorganize during dynamic area changes was accessed via the surface pressure (π)-area (A) compression isotherms and step/relaxation dilatational rheology studies. Film structure was monitored with Brewster angle microscopy. The raise in the % (at ≥10%) of saturation resulted in the formation of stiffer, thicker, and more elastic films at π ≥ 12 mN/m with the effects being proportional to the saturation level. At the same time, at low (≤10 mN/m) π the raise in saturation resulted in altered spreading and heterogeneous structure of MGS layers. The strong impact of saturation on MGS surface properties correlates with our recent spectroscopy study, which demonstrated that saturation induced increase of MGS acyl chain order, phase transition temperature, and cooperativity.

Structure-function relationship of tear film lipid layer: A contemporary perspective.

Tear film lipid layer (TFLL) stabilizes the air/tear surface of the human eye. Meibomian gland dysfunction (MGD) resulting in quantitative and qualitative modifications of TFLL major (>93%) component, the oily secretion of meibomian lipids (MGS), is the world leading cause of dry eye syndrome (DES) with up to 86% of all DES patients showing signs of MGD. Caused by intrinsic factors (aging, ocular and general diseases) and by extrinsic everyday influences like contact lens wear and extended periods in front of a computer screen, DES (resulting in TF instability, visual disturbances and chronic ocular discomfort) is the major ophthalmic public health disease of the present time affecting the quality of life of 10-30% of the human population worldwide. Therefore there is a pressing need to summarize the present knowledge, contradictions and open questions to be resolved in the field of TFLL composition/structure/functions relationship. The following major aspects are covered by the review: (i) Do we have a reliable mimic for TFLL: MGS vs contact lens lipid extracts (CLLE) vs lipid extracts from whole tears. Does TFLL truly consist of lipids only or it is important to keep in mind the TF proteins as well?; (ii) Structural properties of TFLL and of its mimics in health and disease in vitro and in vivo. How the TFLL uniformity and thickness ensures the functionality of the lipid layer (barrier to evaporation, surface properties, TF stability etc.); (iii) What are the main functions of the TFLL? In this aspect an effort is done to emphasize that there is no single main function of TFLL but instead it simultaneously fulfills plethora of functions: suppresses the evaporation (alone or probably in cooperation with other TF constituents) of the aqueous tears; stabilizes (due to its surface properties) the air/tear surface at eye opening and during the interblink interval; and even acts as a first line of defense against bacterial invasion due to its detergency action on the bacterial membranes. An effort is done to highlight how the concept on the importance of TFLL and TF viscoelasticity transpires from old and new studies and what are its clinical implications. An attempt is made to outline the future hot directions of research into the field ranging from quest for molecules that can significantly alter TFLL properties to addressing open questions on the contribution of TFLL to the overall performance of the TF.

Surface properties and exponential stress relaxations of mammalian meibum films.

The surface properties of meibomian secretion (MGS), the major constituent of the tear film (TF) lipid layer, are of key importance for TF stability. The interfacial properties of canine, cMGS, and feline, fMGS, meibum films were studied using a Langmuir surface balance. These species were selected because they have blinking frequency and TF stability similar to those of humans. The sample's performance during dynamic area changes was evaluated by surface pressure (π)-area (A) isocycles and the layer structure was monitored with Brewster angle microscopy. The films' dilatational rheology was probed via the stress-relaxation technique. The animal MGS showed similar behavior both between each other and with human MGS (studied previously). They form reversible, non-collapsible, multilayer thick films. The relaxations of canine, feline, and human MGS films were well described by double exponential decay reflecting the presence of two processes: (1) fast elastic process, with characteristic time τ < 10 s and (2) slow viscous process, with τ > 100 s-emphasizing the meibum layers viscoelasticity. The temperature decrease from 35 to 25 °C resulted in decreased thickness and lateral expansion of all MGS layers accompanied with increase of the π/A hysteresis and of the elastic process contribution to π relaxation transients. Thus, MGS films of mammals with similar blinking frequency and TF stability have similar surface properties and stress relaxations unaltered by the interspecies MGS compositional variations. Such knowledge may impact the selection of animal mimics of human MGS and on a better understanding of lipid classes' impact on meibum functionality.

Effect of hydrophilic polymers on the wettability, static and dynamic, of solid substrate covered by confluent monolayer of air-damaged SIRC cells.

The aim of this study was to evaluate the possible implementation of hydrophilic polymers as recovery agents in air-damaged corneal cells. The sessile bubble technique was implemented to measure the wetting properties of four selected polymers: hydroxyethyl cellulose (HEC), sodium chondroitin sulphate (SCS), hydroxypropyl-methylcellulose (HPMC) and poloxamer F127 (PO12), at equilibrium conditions and in the case of advancing and receding contact angle. For testing the wetting properties of the polymers, glass slides covered with a confluent monolayer of Statens Seruminstitut rabbit cornea (SIRC) cells were used. HEC showed best properties for a broad concentration range, as the polymer showed capability to maintain low values of the static (equilibrium) contact angle (average static contact angle - 36.07˚, compared to average static compact angles of HPMC - 38.44˚, PO12 - 38.92˚ and SCS - 37.85˚), i.e. better wettability. Sessile bubble technique provides quick, relatively simple and reliable approach for testing surface properties of the listed polymers. The nature of the surface damage produced by the exposition of SIRC cells was used as a plausible model of evaporative dry eye syndrome, and thus the results may have clinical implementation.