Brief and long co-incubation of sperm and oocytes for in vitro fertilization: a meta-analysis of randomized controlled trials.

BACKGROUND: There is still no consensus on the optimal time of oocyte-sperm co-incubation during in vitro fertilization and embryo transfer (IVF-ET). The aim of this meta-analysis was to compare the effects of brief (1-6 h) and long (16-24 h) gametes co-incubation time on IVF outcomes. METHODS: The study protocol was registered online through PROSPERO (CRD42022337503) and PRISMA guidelines were followed in the present study. The following databases were searched from inception to May 2022 for randomized controlled trials (RCTs): PubMed, Embase, Cochrane library, Web of Science, using search terms related to IVF, gametes, time of co-incubation and reproductive outcome measure. Studies comparing outcomes of brief co-incubation to that of long co-incubation during IVF, and reporting primary outcome (live birth rate), secondary outcomes (clinical pregnancy rate; ongoing pregnancy rate; miscarriage rate; normal fertilization rate; polyspermy rate; top-quality embryo rate; implantation rate) were searched. A total of 11 studies were included in the meta-analysis. Combined odds ratio (OR) and 95% confidence interval (CI) were calculated for the data. Statistical heterogeneity analysis between studies was assessed by Cochran Q and I2 statistic with a significant threshold of P < 0.05. Methodologic quality assessment of RCTs was made for potential risk of bias with Cochrane Risk of Bias Tool. RESULTS: Compared to long-term co-incubation, brief co-incubation had an advantage in increasing implantation rate (OR: 1.97, 95% CI: 1.52-2.57), ongoing pregnancy rate (OR: 2.18, 95% CI: 1.44-3.29) and top-quality embryo rate (OR: 1.17, 95% CI: 1.02-1.35). However, brief co-incubation of gametes had no advantages in the live-birth rate (OR: 1.09, 95% CI: 0.72-1.65), miscarriage rate (OR: 1.32, 95% CI: 0.55-3.18), clinical pregnancy rate (OR: 1.36, 95% CI: 0.99-1.87) and polyspermy rate (OR: 0.80, 95% CI: 0.48-1.33) than long-term co-incubation. Additionally, the brief co-incubation was associated with lower normal fertilization rate (OR: 0.89, 95% CI: 0.80-0.99), compared with long co-incubation. CONCLUSIONS: Brief co-incubation of gametes had the advantages in increasing implantation rate, ongoing pregnancy rate and top-quality embryo rate than long-term co-incubation. However, the live-birth rate displayed no difference between the two in vitro fertilization methods. Gametes co-incubation time should be individualized according to each patient's IVF history, infertility causes and the semen parameters.

Differential effects of short co-incubation of gametes and early removal of cumulus cells in patients with different fertilizing capabilities.

The objective of this retrospective study was to evaluate the embryological and clinical outcomes of short co-incubation of gametes and early removal of cumulus cells (SCGERCC) in patients with good IVF capability and those with complete fertilization failure treated by rescue intracytoplasmic sperm injection (ICSI). The study included 257 couples with >60% fertilization rate (SCGERCC group) in the SCGERCC cycle, 72 couples with complete fertilization failure in the SCGERCC cycle given early rescue ICSI treatment (early rescue group), and 219 couples who underwent IVF cycles with overnight co-incubation of gametes with >60% fertilization rate (traditional IVF group). The results showed that the SCGERCC group had a higher multi-pronuclei rate (13.34%, P < 0.001) than the early rescue ICSI (5.15%) and traditional IVF (6.15%) groups. The good quality embryo rate was higher in the SCGERCC group, but implantation, clinical pregnancy and live-birth rates (37.21%, 36.92% and 38.20% for SCGERCC, early rescue and traditional IVF groups, respectively) were comparable in all three groups. The study indicated that SCGERCC followed by rescue ICSI helped couples with initial complete fertilization failure attain clinical outcomes comparable with the other two groups, but it significantly increased the multi-pronuclei rate in couples with good fertilizing capabilities.

Does reducing gamete co-incubation time improve clinical outcomes: a retrospective study.

PURPOSE: The objective of this retrospective study was to determine whether patients undergoing in vitro fertilization (IVF) benefit from reducing the gamete co-incubation time. METHODS: Patients (n = 570) were enrolled, including 281 patients in the reduced incubation time group (2-h incubation) and 289 patients in the standard IVF group (18-h incubation). RESULTS: The observed outcomes, including the clinical pregnancy rate (CPR), implantation rate (IR), live birth rate (LBR), and miscarriage rate (MR), were similar between the two groups. When the data were divided into two subgroups based on the maternal age (≤30 and >30 years), the rates of top-quality embryos (30.83 vs. 25.89 %; p = 0.028), CPR (66.67 vs. 42.11 %; p = 0.013), and IR (41.90 vs. 31.25 %, p = 0.019) of the 2-h incubation group were significantly higher in the younger subgroup. However, for older patients, only a lower MR (7.59 vs. 20.83 %; p = 0.019) was achieved. Reducing the time of incubation still improved the CPR (OR = 1.993, 95 % CI 1.141-3.480) and MR (OR = 3.173, 95 % CI 1.013-9.936) in the younger and older subgroups, respectively, after it was adjusted for potential confounders. CONCLUSIONS: Reducing incubation time improves the clinical results of IVF, although the LBR is not statistically different between the 2- and 18-h incubation time groups. And the specific clinical outcomes of reducing incubation time varied between the >30-year-old and the ≤30-year-old.

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos.

Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

The Impact of Streptomyces griseus Protease Reserved for Protein Evaluation of Ruminant Feed on Carbohydrase Activity during Co-Incubation.

For protein evaluation of feedstuffs for ruminants, the Streptomyces griseus protease test provides a solely enzymatic method for estimating ruminal protein degradation. Since plant proteins are often structured in carbohydrate complexes, the use of carbohydrase during the test might improve its accuracy. It is advisable to co-incubate protease and carbohydrase, risking that the carbohydrase activity is reduced under the influence of the protease. The present study was conducted to investigate this impact by using α-amylase or the multi-enzyme complex Viscozym® L as carbohydrase. The detection of active protease was determined fluorescence photometrically using internally quenched fluorogenic substrates (IQFS). Cellulose, pectin, and starch degradation were determined spectrophotometrically using 3,5-dinitro salicylic acid as a colorimetric agent. The Streptomyces griseus protease mixture proved to be active for the selected IQFS immediately after the start of measurements (p < 0.05). Starch hydrolysis catalyzed by α-amylase or Viscozym® L, respectively, was decreased by co-incubation with protease mixture by maximal 3% or 37%, respectively, at 5 h incubation time (p > 0.05). Pectin and cellulose hydrolysis catalyzed by Viscozym® L, respectively, was not significantly influenced by co-incubation with a protease mixture (p > 0.05). Although a decrease in carbohydrase activity during co-incubation with Streptomyces griseus protease occurred, it was only numerical and might be counteracted by an adapted carbohydrase activity.

Exploiting the Opportunity to Use Plant-Derived Nanoparticles as Delivery Vehicles.

The scientific community has become increasingly interested in plant-derived nanoparticles (PDNPs) over the past ten years. Given that they possess all the benefits of a drug carrier, including non-toxicity, low immunogenicity, and a lipid bilayer that protects its content, PDNPs are a viable model for the design of innovative delivery systems. In this review, a summary of the prerequisites for mammalian extracellular vesicles to serve as delivery vehicles will be given. After that, we will concentrate on providing a thorough overview of the studies investigating the interactions of plant-derived nanoparticles with mammalian systems as well as the loading strategies for encapsulating therapeutic molecules. Finally, the existing challenges in establishing PDNPs as reliable biological delivery systems will be emphasized.

Characterization of Campylobacter jejuni proteome profiles in co-incubation scenarios.

In dynamic microbial ecosystems, bacterial communication is a relevant mechanism for interactions between different microbial species. When C. jejuni resides in the intestine of either avian or human hosts, it is exposed to diverse bacteria from the microbiome. This study aimed to reveal the influence of co-incubation with Enterococcus faecalis, Enterococcus faecium, or Staphylococcus aureus on the proteome of C. jejuni 81-176 using data-independent-acquisition mass spectrometry (DIA-MS). We compared the proteome profiles during co-incubation with the proteome profile in response to the bile acid deoxycholate (DCA) and investigated the impact of DCA on proteomic changes during co-incubation, as C. jejuni is exposed to both factors during colonization. We identified 1,375 proteins by DIA-MS, which is notably high, approaching the theoretical maximum of 1,645 proteins. S. aureus had the highest impact on the proteome of C. jejuni with 215 up-regulated and 230 down-regulated proteins. However, these numbers are still markedly lower than the 526 up-regulated and 516 down-regulated proteins during DCA exposure. We identified a subset of 54 significantly differentially expressed proteins that are shared after co-incubation with all three microbial species. These proteins were indicative of a common co-incubation response of C. jejuni. This common proteomic response partly overlapped with the DCA response; however, several proteins were specific to the co-incubation response. In the co-incubation experiment, we identified three membrane-interactive proteins among the top 20 up-regulated proteins. This finding suggests that the presence of other bacteria may contribute to increased adherence, e.g., to other bacteria but eventually also epithelial cells or abiotic surfaces. Furthermore, a conjugative transfer regulon protein was typically up-expressed during co-incubation. Exposure to both, co-incubation and DCA, demonstrated that the two stressors influenced each other, resulting in a unique synergistic proteomic response that differed from the response to each stimulus alone. Data are available via ProteomeXchange with identifier PXD046477.

Interactions between Blastocystis subtype ST4 and gut microbiota in vitro.

BACKGROUND: Blastocystis ST4 is a common protistan parasite of the gastrointestinal tract of humans and a wide range of animals. While it has been suggested that colonization with ST4 is associated with healthy gut microbiota, how ST4 influences the gut microbiota remains poorly studied. This study aimed to examine the interactions between ST4 and several intestinal bacteria using in vitro co-culture systems, and to further investigate the mechanism of interaction and its effect on the epithelial barrier integrity of HT-29 cells. METHODS: Seven intestinal bacteria Bacteroides fragilis, Bifidobacterium longum, Bacillus subtilis, Bacteroides vulgatus, Escherichia coli, Enterococcus faecalis, and Lactobacillus brevis were co-cultured with Blastocystis ST4 in vitro. Flow cytometry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to determine the role of reactive oxygen species (ROS) and bacteria oxidoreductase genes, respectively, in response to Blastocystis co-incubation. Transepithelial electrical resistance (TEER) and flux assays were performed to assess the effect of microbiota representatives on the integrity of the intestinal epithelial barrier. RESULTS: Co-incubation with Blastocystis ST4 showed a beneficial influence on most intestinal bacteria, while ST4 significantly inhibited the growth of B. vulgatus, a common pathogen in the genus Bacteroides. The decrease in B. vulgatus when co-incubated with Blastocystis ST4 was associated with high levels of ROS and the upregulation of oxidative stress-related genes. Furthermore, co-incubation with Blastocystis ST4 was able to protect the intestinal epithelial barrier from damage by B. vulgatus. CONCLUSIONS: This study demonstrated, for the first time, that Blastocystis ST4 has beneficial effects on intestinal commensal bacteria in vitro, and can inhibit the growth of pathogenic B. vulgatus. Combined with previous microbiome research on ST4, our data suggest that ST4 may be a beneficial commensal.

Bifidobacterium adolescentis (DSM 20083) and Lactobacillus casei (Lafti L26-DSL): Probiotics Able to Block the In Vitro Adherence of Rotavirus in MA104 Cells.

Rotavirus is the leading worldwide cause of gastroenteritis in children under five years of age. Even though there are some available vaccines to prevent the disease, there are limited strategies for challenging diarrhea induced by rotavirus infection. For this reason, researchers are constantly searching for other approaches to control diarrhea by means of probiotics. In order to demonstrate the ability of some probiotic bacteria to interfere with the in vitro rotavirus infection in MA104 cells, strains of Lactobacillus sp. and Bifidobacterium sp. were tested in MA104 cells before the viral infection. As a preliminary assay, a blocking effect treatment was performed with viable bacteria. In this screening assay, four of initial ten bacteria showed a slight reduction of the viral infection (measured by percentage of infection). L. casei (Lafti L26-DSL), L. fermentum(ATCC 9338), B. adolescentis (DSM 20083), and B. bifidum (ATCC 11863) were used in further experiments. Three different treatments were tested in order to evaluate protein-based metabolites obtained from mentioned bacteria: (i) cell exposure to the protein-based metabolites before viral infection, (ii) exposure to protein-based metabolites after viral infection, and (iii) co-incubation of the virus and protein-based metabolites before viral infection to the cell culture. The best effect performed by protein-based metabolites was observed during the co-incubation assay of the virus and protein-based metabolites before adding them into the cell culture. The results showed 25 and 37% of infection in the presence of L. casei and B. adolescentis respectively. These results suggest that the antiviral effect may be occurring directly with the viral particle instead of making a blocking effect of the cellular receptors that are needed for the viral entrance.

Shortening gametes co-incubation time improves live birth rate for couples with a history of fragmented embryos.

Short gamete co-incubation (SGCO) consists in decreasing the duration of contact between oocytes and sperm from the standard overnight insemination (SOI) toward 2 hours. However, the effectiveness of this technique to improve in vitro fertilization and embryo transfer (IVF-ET) outcomes remains controversial. Our study was designed to evaluate the efficiency of SGCO in a poor prognosis population with a history of fragmented embryos defined by the presence of at least 50% of the embryos with more than 25% of cytoplasmic fragments. From January 2010 to January 2014, 97 couples were included in a SGCO protocol. We separated women into 2 subgroups: younger and older than 35 years. Compared to SOI, after SGCO, 2-cell stage embryos were higher in all women (p<0.001) and less fragmented in women over 35 years (p<0.05). On day 2, top quality embryos obtained and transferred were higher with SCGO than with SOI, independently of the age of the women (p<0.001). Moreover, the number of embryos with less than 25% of fragmentation was higher after SGCO than SOI (p<0.001) whereas the number of multinucleated embryos was lower (p<0.001). We observed that after fresh ET, independently of the age of the women, the clinical pregnancy rate was 3 times higher after SGCO than after SOI. However, the live-birth rate was 4 times higher with SGCO than with SOI in women above 35 years but 3 times higher with SGCO than with SOI in women younger than 35 years. The present results indicate that for a particular indication, reducing the time of oocytes and sperm co-incubation may improve IVF-ET outcomes in terms of live-birth rate. ABBREVIATIONS: AMH: anti mullerian hormone; COC: cumulus-oocytes complex; E2: estradiol; ET: embryo transfer; FET: frozen embryo transfer; FSH: follicle stimulating hormone; GnRH: gonadotrophin releasing hormone; hCG: human chorionic gonadotropin hormone; hMG: human menopausal gonadotropin hormone; IRB: institutional review board; IVF: in vitro fertilization; IVF-ET: in vitro fertilization and embryo transfer; MNB: multinucleated blastomere; mRNA: messanger ribonucleic acid; OC: oocyte retrieval; O2: oxygen; ROS: reactive oxygen species; SGCO: short gamete co-incubation; SOI: standard overnight insemination.

Can dendritic cells improve whole cancer cell vaccines based on immunogenically killed cancer cells?

Immunogenic cell death (ICD) offers interesting opportunities in cancer cell (CC) vaccine manufacture, as it increases the immunogenicity of the dead CC. Furthermore, fusion of CCs with dendritic cells (DCs) is considered a superior method for generating whole CC vaccines. Therefore, in this work, we determined in naive mice whether immunogenically killed CCs per se (CC vaccine) elicit an antitumoral immune response different from the response observed when immunogenically killed CCs are associated with DCs through fusion (fusion vaccine) or through co-incubation (co-incubation vaccine). After tumor inoculation, the type of immune response in the prophylactically vaccinated mice differed between the groups. In more detail, fusion vaccines elicited a humoral anticancer response, whereas the co-incubation and CC vaccine mainly induced a cellular response. Despite these differences, all three approaches offered a prophylactic protection against tumor development in the murine mammary carcinoma model. In summary, it can be concluded that whole CC vaccines based on immunogenically killed CCs may not necessarily require association with DCs to elicit a protective anticancer immune response. If this finding can be endorsed in other cancer models, the manufacture of CC vaccines would greatly benefit from this new insight, as production of DC-based vaccines is laborious, time-consuming and expensive.

Recovery of redox homeostasis altered by CuNPs in H4IIE liver cells does not reduce the cytotoxic effects of these NPs: an investigation using aryl hydrocarbon receptor (AhR) dependent antioxidant activity.

The generation of reactive oxygen species (ROS) and consequent oxidative stress is regarded as a relevant mechanism for nanoparticle toxicity. In cells, the activation of the aryl hydrocarbon receptor (AhR) triggers a cascade of defence responses against oxidative stress. By increasing AhR dependent cellular anti-oxidant activity, we tested the extent to which the cytotoxic effect of copper nanoparticles (CuNPs) is governed by oxidative stress. H4IIE rat hepatoma cells were challenged with high ROS levels after exposure to CuNPs, while the AhR-induced cellular anti-oxidant defence was simultaneously activated by the AhR ligand beta-Naphthoflavone (ßNF). Activation of phase II detoxification enzymes (as glutathione-S-transferases, GSTs) and anti-oxidants (glutathione, GSH) led to a complete abrogation of CuNP-induced ROS production. However, a concurrent reduction in cytotoxicity was not detected, thereby indicating that CuNPs exert non-oxidative stress mediated cytotoxic effects. Transmission electron microscopy analysis pointed to a direct physical perturbation of cellular structures by CuNPs, thus contributing to their cytotoxicity. Our observations highlight that distinct mechanisms underlie the toxicity of ions and NPs and indicate that while ROS elicitation is CuNP-specific, the cytotoxic action of these particles may not be directly related to their pro-oxidative activity. These findings have important implications with respect to the oxidative stress paradigm used to explain NP toxicity.

The inhibitory effect of a Lactobacillus acidophilus derived biosurfactant on biofilm producer Serratia marcescens.

OBJECTIVES: Serratia marcescens is one of the nosocomial pathogen with the ability to form biofilm which is an important feature in the pathogenesis of S. marcescens. The aim of this study was to determine the anti-adhesive properties of a biosurfactant isolated from Lactobacillus acidophilus ATCC 4356, on S. marcescens strains. MATERIALS AND METHODS: Lactobacillus acidophilus ATCC 4356 was selected as a probiotic strain for biosurfactant production. Anti-adhesive activities was determined by pre-coating and co- incubating methods in 96-well culture plates. RESULTS: The FTIR analysis of derived biosurfactant revealed the composition as protein component. Due to the release of such biosurfactants, L. acidophilus was able to interfere with the adhesion and biofilm formation of the S. marcescens strains. In co-incubation method, this biosurfactant in 2.5 mg/ml concentration showed anti-adhesive activity against all tested strains of S. marcescens (P<0.05). CONCLUSION: Our results show that the anti-adhesive properties of L. acidophilus biosurfactant has the potential to be used against microorganisms responsible for infections in the urinary, vaginal and gastrointestinal tracts, as well as skin, making it a suitable alternative to conventional antibiotics.

Time of insemination culture and outcomes of in vitro fertilization: a systematic review and meta-analysis.

BACKGROUND Conflicting results have been reported regarding the technique of brief insemination used in IVF. The aim of this meta-analysis was to determine if better clinical outcomes of IVF are associated with a brief co-incubation of gametes than with a standard overnight co-incubation. METHODS A computerized search was conducted of the published literature of four databases, using search terms related to gamete, time of co-incubation and outcome measure. Eligible studies compared outcomes of IVF with a brief co-incubation of gametes to that of a control group of standard insemination and reported rates of live birth (primary outcome), normal fertilization, polyspermy, good quality embryos, implantation, clinical pregnancy or ongoing pregnancy (secondary outcomes). A total of 11 studies were included in the meta-analysis. Pooled risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for the data. Statistical heterogeneity was tested using Cochran Q and I² values. RESULTS Brief co-incubation of gametes was associated with significantly higher rates of clinical pregnancy (RR: 1.84, 95% CI: 1.24-2.73) and ongoing pregnancy (RR: 1.73, 95% CI: 1.27-2.33) than standard insemination. Brief co-incubation of gametes was associated also with a significantly higher rate of implantation (RR: 1.80, 95% CI: 1.43-2.26) than standard insemination. However, the rates of normal fertilization (RR: 0.98, 95% CI: 0.93-1.02), good quality embryos (RR: 1.24, 95% CI: 1.0-1.53) and polyspermy (RR: 0.84, 95% CI: 0.7-1.01) were not significantly different with brief co-incubation of gametes compared with standard insemination. CONCLUSIONS Reduced gamete exposure time may be associated with beneficial outcomes. Drawbacks inherent to the quality of several studies limit the quality of the available evidence. Adequately powered randomized controlled studies need to be performed to evaluate the efficacy of brief insemination.