Individually Cultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in an Extremely Confined Environment.

The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips.

Introduction: We report the development and preliminary evaluation of a novel dynamic bioreactor to culture ovarian cortical tissue strips that leverages tissue response to enhanced oxygen transport and adequate mechanical stimulation. In vitro multistep ovarian tissue static culture followed by mature oocyte generation, fertilization, and embryo transfer promises to use the reserve of dormant follicles. Unfortunately, static in vitro culture of ovarian tissue does not promote development of primordial to secondary follicles or sustain follicle viability and thereby limits the number of obtainable mature oocytes. Enhancing oxygen transport to and exerting mechanical stimulation on ovarian tissue in a dynamic bioreactor may more closely mimic the physiological microenvironment and thus promote follicle activation, development, and viability. Materials and Methods: The most transport-effective dynamic bioreactor design was modified using 3D models of medium and oxygen transport to maximize strip perifusion and apply tissue fluid dynamic shear stresses and direct compressive strains to elicit tissue response. Prototypes of the final bioreactor design were manufactured with materials of varying cytocompatibility and assessed by testing the effect of leachables on sperm motility. Effectiveness of the bioreactor culture was characterized against static controls by culturing fresh bovine ovarian tissue strips for 7 days at 4.8 × 10-5 m/s medium filtration flux in air at -15% maximal total compressive strain and by assessing follicle development, health, and viability. Results and Conclusions: Culture in dynamic bioreactors promoted effective oxygen transport to tissues and stimulated tissues with strains and fluid dynamic shear stresses that, although non-uniform, significantly influenced tissue metabolism. Tissue strip culture in bioreactors made of cytocompatible polypropylene preserved follicle viability and promoted follicle development better than static culture, less so in bioreactors made of cytotoxic ABS-like resin.

Dynamic in vitro culture of bovine and human ovarian tissue enhances follicle progression and health.

In vitro ovarian cortical tissue culture, followed by culture of isolated secondary follicles, is a promising future option for production of mature oocytes. Although efforts have been made to improve the culture outcome by changing the medium composition, so far, most studies used static culture systems. Here we describe the outcome of 7 days cultures of bovine and human ovarian cortical tissue in a dynamic system using a novel perifusion bioreactor in comparison to static culture in conventional and/or gas permeable dishes. Findings show that dynamic culture significantly improves follicle quality and viability, percentage and health of secondary follicles, overall tissue health, and steroid secretion in both species. Model predictions suggest that such amelioration can be mediated by an enhanced oxygen availability and/or by fluid-mechanical shear stresses and solid compressive strains exerted on the tissue.

Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system.

PURPOSE: Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). METHODS: The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. RESULTS: We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. CONCLUSION: SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.

Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development.

Reactive oxygen species (ROS) generated at low levels during mitochondrial respiration have key roles in several signaling pathways. Oxidative stress (OS) arises when the generation of ROS exceeds the cell's antioxidant scavenging ability and leads to cell damage. Physiological ROS production in spermatozoa regulates essential functional characteristics such as motility, capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion. OS can have detrimental effects on sperm function through lipid peroxidation, protein damage, and DNA strand breakage, which can eventually affect the fertility of an individual. Substantial evidence in the literature indicates that spermatozoa experiencing OS during in vitro manipulation procedures in human- and animal-assisted reproduction are increasingly associated with iatrogenic ROS production and eventual impairment of sperm function. Although a direct association between sperm OS and human assisted reproductive techniques (ART) outcomes after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) is still a matter of debate, studies in animal models provide enough evidence on the adverse effects of sperm OS in vitro and defective fertilization and embryo development. This review summarized the literature on sperm OS in vitro, its effects on functional ability and embryo development, and the approaches that have been proposed to reduce iatrogenic sperm damage and altered embryonic development.

Mitochondrial Dysfunction and Oxidative Stress Caused by Cryopreservation in Reproductive Cells.

Mitochondria, fundamental organelles in cell metabolism, and ATP synthesis are responsible for generating reactive oxygen species (ROS), calcium homeostasis, and cell death. Mitochondria produce most ROS, and when levels exceed the antioxidant defenses, oxidative stress (OS) is generated. These changes may eventually impair the electron transport chain, resulting in decreased ATP synthesis, increased ROS production, altered mitochondrial membrane permeability, and disruption of calcium homeostasis. Mitochondria play a key role in the gamete competence to facilitate normal embryo development. However, iatrogenic factors in assisted reproductive technologies (ART) may affect their functional competence, leading to an abnormal reproductive outcome. Cryopreservation, a fundamental technology in ART, may compromise mitochondrial function leading to elevated intracellular OS that decreases sperm and oocytes' competence and the dynamics of fertilization and embryo development. This article aims to review the role played by mitochondria and ROS in sperm and oocyte function and the close, biunivocal relationships between mitochondrial damage and ROS generation during cryopreservation of gametes and gonadal tissues in different species. Based on current literature, we propose tentative hypothesis of mechanisms involved in cryopreservation-associated mitochondrial dysfunction in gametes, and discuss the role played by antioxidants and other agents to retain the competence of cryopreserved reproductive cells and tissues.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development.

Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 µg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.

Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture.

OBJECTIVE: To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended in vitro culture. DESIGN: Randomized experimental study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9 days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. RESULT(S): The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term in vitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. CONCLUSION(S): Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Effect of superoxide dismutase, catalase, and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs.

This study investigated the correlation between oxidative stress status and key canine sperm parameters and the effect of addition of a superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) combination in egg yolk tris-citrate glucose (EYT-G) extender on semen during 10 days of storage at 4℃. Ten Boxer dogs were divided into two groups, fertile (F) and hypofertile (H), depending on pregnancy and live birth rate status in the previous year. Semen evaluation was performed on the day of collection (D0) and after 5 (D5) and 10 (D10) days of cooled storage. Sperm motility, kinetic parameters, and DNA integrity were assessed. A correlation between oxidative status and key semen parameters in both F and H groups was observed. Total and progressive motilities were significantly higher in the treated (SOD, CAT, and GPx addition) versus control groups at D10 in both F and H groups, and at D5 in the H group. DNA integrity was significantly higher in both treated groups (H and F) at D5 and D10. In conclusion, the addition of SOD, CAT, and GPx in the extender allows preservation of semen quality for up to 10 days of storage at 4℃ in both fertile and hypofertile dogs.

Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles' health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

Supplementation of sperm media with zinc, D-aspartate and co-enzyme Q10 protects bull sperm against exogenous oxidative stress and improves their ability to support embryo development.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine-xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

Successful slush nitrogen vitrification of human ovarian tissue.

OBJECTIVE: To study whether slush nitrogen vitrification improves the preservation of human ovarian tissue. DESIGN: Control vs. treatment study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected from nine women (aged 14-35 years) during laparoscopic surgery for benign gynecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian cortical strips of 2 × 5 × 1 mm were vitrified with liquid or slush nitrogen. Fresh and vitrified cortical strips were analyzed for cryodamage and viability under light, confocal, and transmission electron microscopy. RESULT(S): Compared with liquid nitrogen, vitrification with slush nitrogen preserves [1] follicle quality (grade 1 follicles: fresh control, 50%; liquid nitrogen, 27%; slush nitrogen, 48%); [2] granulosa cell ultrastructure (intact cells: fresh control, 92%; liquid nitrogen, 45%; slush nitrogen, 73%), stromal cell ultrastructure (intact cells: fresh control, 59.8%; liquid nitrogen, 24%; slush nitrogen, 48.7%), and DNA integrity (TUNEL-positive cells: fresh control, 0.5%; liquid nitrogen, 2.3%; slush nitrogen, 0.4%); and [3] oocyte, granulosa, and stromal cell viability (oocyte: fresh control, 90%; liquid nitrogen, 63%; slush nitrogen, 87%; granulosa cells: fresh control, 93%; liquid nitrogen, 53%; slush nitrogen, 81%; stromal cells: fresh control, 63%; liquid nitrogen, 30%; slush nitrogen, 52%). CONCLUSION(S): The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification with slush nitrogen compared with liquid nitrogen.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation.

BACKGROUND: Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation. METHODS: Semen samples, obtained from forty-four patients (23-30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation. RESULTS: Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation. CONCLUSIONS: Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.

Replacement of sodium with choline in slow-cooling media improves human ovarian tissue cryopreservation.

Ovarian tissue cryopreservation is a promising technique for fertility preservation in young female cancer patients and efforts have been made to improve its effectiveness. During cooling and thawing, sodium ions significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, should not contribute to the intracellular solute load. The present study assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles (fresh control n=266; cryopreserved n=363), collected from ovarian biopsies of 11 women (22-40years) during laparoscopic surgery, were studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. The results demonstrate that choline substitution leads to: (i) an improved preservation of oocytes and follicular cells; (ii) the recovery of a higher percentage of grade-1 follicles negative for p53, p21 and Apaf-1 apoptotic markers; (iii) a reduced mitochondrial damage as observed at an ultrastructural level; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media may represent a valuable tool to improve human ovarian tissue cryopreservation. Ovarian tissue cryopreservation is a promising fertility preservation approach for cancer patients before undergoing treatments that irreversibly reduce the ovarian reserve. Autotransplantation of ovarian cortical strips has resulted in viable offspring in animal models and human. Worldwide, 20 live births have been reported thus far following autotransplantation of frozen-thawed ovarian tissue. However, currently the success rate of this technology is far from being satisfactory. This could be due to inappropriate cryopreservation procedures that might impair the physiology of ovarian follicles. Sodium ions contained in freezing media significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, would not be expected to contribute to the intracellular solute load. In the present study we assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles, collected from ovarian biopsies of 11 women (aged 22-40years) during laparoscopic surgery, have been studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. Results demonstrated that choline substitution allowed: (i) a better preservation of oocytes and follicular cells; (ii) the recovery of an higher percentage of healthy follicles negative for apoptotic markers; (iii) a lower mitochondria ultrastructural damage; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media could represent a valuable tool to cryopreserve human ovarian tissue for fertility preservation.