IVF Medium Research Articles

Control of sperm penetration using stereumamide A derived from Trichaptum fuscoviolaceum in the in vitro fertilization of pig oocytes.

Fungal metabolites are known to have potent and diverse properties such as antiviral, antidiabetic, antitumour, antioxidant, free radical scavenging, and antibacterial effects which can be utilized to treat diseases. In this study, we investigated the functional activity of stereumamide A (StA) derived from a culture broth of Trichaptum fuscoviolaceum during the in vitro fertilization (IVF) of pig oocytes, to determine its effects on sperm penetration. Oocytes matured in vitro were fertilized in the absence or presence of varying concentrations of StA (0-50 μg/ml StA). When StA was directly added into the IVF medium, significantly lower fertilization rates were seen with the 20 or 50 μg/ml StA (2.0-17.5%) treatments compared with those of 10 μg/ml StA or the controls (60.9-62.3%), whereas StA had no influence on the survival of oocytes and spermatozoa throughout the IVF process. For evaluating the control of sperm entry, mature oocytes were pre-incubated in a medium containing 20 μg/ml StA for 1 h, and then IVF was subsequently performed. The incidence of polyspermy was significantly reduced when oocytes were pre-incubated with StA (15.0% vs. 50.4-57.5% in controls). In conclusion, sperm penetration was inhibited in the medium in the presence of StA during IVF, while StA did not affect sperm motility and fertility competence. Fertilization was controlled when mature oocytes were incubated with StA prior to IVF, suggesting the possible use of the fungal metabolite in assisted reproductive technology for humans and animals.

In vitro exposure of porcine sperm to functionalized superparamagnetic nanoparticles.

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co-incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and invitro fertilization-IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p > .05). In BTS, althoughsome kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p > .05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p > .05). In conclusion, invitro exposure of boar sperm to OVGP1-functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP.

Incubating frozen-thawed buffalo sperm with olive fruit extracts counteracts thawing-induced oxidative stress and improves semen quality

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress.Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 106/mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes.The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity.

P-109 Non-invasive Genetic Testing (niPGTA) and Laser Assisted Hatching (LAH) may improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age

Abstract Study question Does niPGTA and LAH improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age? Summary answer niPGTA increases pregnancy and clinical pregnancy rates in patients aged ≥38 years compared to those without tested embryos, while LAH may further improve pregnancy outcomes. What is known already Embryos release cell-free DNA (cf-DNA) in the IVF culture media, which can be analyzed for chromosomal constitution. The benefit that niPGTA holds over PGT-A is that it is a non-invasive test and could potentially produce comparable results. Therefore, the time to pregnancy and the risk of miscarriage could be reduced in comparison to an IVF treatment with non-tested embryos. Previous studies have shown high concordance with trophectoderm biopsies in PGT-A patients and improved outcomes with LAH in frozen-thawed cycles. However, there is a lack of studies investigating clinical pregnancy rates with niPGTA. Study design, size, duration Retrospective cohort study including 74 patients aged ≥38 years, who underwent Single Frozen Embryo Transfer (FET) from May 2021 to February 2023. Patients who had Endometrial Receptivity Assay (ERA) were excluded. Participants/materials, setting, methods 32 couples had niPGTA; Spent Blastocyst Media (SBM) for each blastocyst were collected on Day 6 and analyzed using the EMBRACE test. Embryos were vitrified. 42 couples had their blastocysts vitrified on Day 5/6 without testing. LAH was introduced to all patients in October 2021. Before that, no LAH was performed. Embryos were transferred to patients 2 hours post-warming after LAH. Pregnancy test was performed 10 days after FET and clinical pregnancy was established. Main results and the role of chance There was a statistical significant increase of pregnancy rate (p = 0.017, Pearson’s Chi-Squared test) and a trend towards higher clinical pregnancy rate for niPGTA patients versus those who had a transfer of not tested embryos. Regarding LAH, there was a trend towards higher pregnancy and clinical pregnancy rates in both niPGTA and not tested group. Results for pregnancies and clinical pregnancies are presented in Table 1. Limitations, reasons for caution More patients are required to confirm clinical significance of pregnancy and clinical pregnancy rates of niPGTA over not tested embryos and if LAH can improve the outcomes. A review of patients who had previous implantation failures and miscarriages in addition to ERA could yield clearer conclusions. Wider implications of the findings The results of this study are encouraging for niPGTA combined with LAH for patients aged ≥38 years. Further studies on niPGTA and modifications of the protocol for increased concordance rates and shorter time in embryo culture should be explored. Trial registration number Not applicable

Evaluation of epididymal and frozen sperm to produce goat embryos through in vitro fertilization

Preservation of the livestock genetic makeup could be performed by application of in vitro fertilization (IVF) using spermatozoa collected from cauda epididymis. Therefore, this study as sessed the development of goat embryos following IVF of spermatozoa collected from cauda epididy mis compared to those kept frozen. Oocytes were in vitro cultured in a maturation medium for 24 hours at a temperature of 38.5°C, 5% CO2, and 95% humidity. Following maturation, the oocytes (n = 370) were in vitro fertilized by fresh epididymal sperm (G1) and G2 frozen-thawed sperm, and the Fert-TALP medium was used as the IVF medium. In addition, in vitro developed embryos were cultured in GT-L medium. The fertilization rate, percentage of morula, and blastocytes (p&lt;0.01) were significantly higher in oocytes inseminated with epididymal than in frozen sperm. In conclusion, in vitro embryo production of goat oocytes may be successfully performed using epididymal spermatozoa for IVF.

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.

Effect of environmental factors on seminal microbiome and impact on sperm quality.

This review provides a comprehensive overview of the existing research on the seminal microbiome and its association with male infertility, while also highlighting areas that warrant further investigation. A narrative review was conducted, encompassing all relevant studies published between 1980-2023 on the male reproductive tract microbiome in humans. This review considered studies utilizing culture-based, polymerase chain reaction (PCR)-based, and next-generation sequencing (NGS)-based methodologies to analyze the microbiome. Data extraction encompassed sample types (semen or testicular tissue), study designs, participant characteristics, employed techniques, and critical findings. We included 37 studies comprising 9,310 participants. Among these, 16 studies used culture-based methods, 16 utilized NGS, and five employed a combination of methods for microorganism identification. Notably, none of the studies assessed fungi or viruses. All NGS-based studies identified the presence of bacteria in all semen samples. Two notable characteristics of the seminal microbiome were observed: substantial variability in species composition among individuals and the formation of microbial communities with a dominant species. Studies examining the testicular microbiome revealed that the testicular compartment is not sterile. Interestingly, sexually active couples shared 56% of predominant genera, and among couples with positive cultures in both partners, 61% of them shared at least one genital pathogen. In couples with infertility of known causes, there was an overlap in bacterial composition between the seminal and vaginal microbiomes, featuring an increased prevalence of Staphylococcus and Streptococcus genera. Furthermore, the seminal microbiome had discernible effects on reproductive outcomes. However, bacteria in IVF culture media did not seem to impact pregnancy rates. Existing literature underscores that various genera of bacteria colonize the male reproductive tract. These organisms do not exist independently; instead, they play a pivotal role in regulating functions and maintaining hemostasis. Future research should prioritize longitudinal and prospective studies and investigations into the influence of infertility causes and commonly prescribed medication to enhance our understanding of the seminal microbiota's role in reproductive health.

Plasminogen activation and plasmin inhibition during in vitro fertilization in bovine: implications for fertilization parameters and early embryo development

Components of the plasminogen/plasmin system, known to be present in the oocyte, play a key role in maturation and fertilization. The objective of this study was to examine the effect of plasminogen activation and plasmin inhibition by exogenous supplementation of the IVF medium with streptokinase (SK) or ɛ-aminocaproic acid (ε-ACA), respectively, on fertilization parameters and preimplantation embryo development. After in vitro maturation, bovine cumulus-oocyte complexes (COCs) were inseminated in the presence of SK or ε-ACA. The addition of SK to the IVF medium facilitated the adhesion of the spermatozoa to the zona pellucida without affecting the percentages of monospermy. Cleavage rates and blastocyst yield were similar between the SK and Control groups while they were lower with the ε-ACA treatment. Additionally, we found that the expression levels of embryo quality-related genes (SDHA and DNMT3A) could be modified in blastocysts by the addition of SK or ε-ACA during IVF. The results obtained indicate that supplementation of the IVF medium with SK did not greatly alter the embryonic developmental parameters related to embryo quality in blastocysts. Moreover, we noticed that ε-ACA treatment compromises the success of in vitro embryo development, thus highlighting the importance of the plasminogen/plasmin activity during the early stages of embryogenesis in bovine.

Assessment of developmental rate of mouse embryos yielded from in vitro fertilization of the oocyte with treatment of melatonin and vitamin C simultaneously

BackgroundIn recent decades, in vitro fertilization (IVF) has been widely used as a method of assisted reproductive technology (ART) to improve fertility in individuals. To be more successful in this laboratory method, we used the presence of two common types of antioxidants (melatonin and vitamin C) simultaneously and exclusively in IVF medium.MethodsThe cumulus-oocyte complexes (COCs) were obtained from Gonadotropin-releasing hormone (GnRH) and Human Chorionic Gonadotropin (HMG) -stimulated mice. Subsequently, metaphase II (MII) oocytes were fertilized in vitro. In the experiment, the IVF medium was randomly divided into two equal groups: The control group did not receive any antioxidants. In the treatment group, 100 µM melatonin and 5 mM vitamin C were added to the IVF medium. Finally, oocytes and putative embryos transferred into developmental medium and cultured 120 h after IVF to the blastocyst stage. After and before IVF, oocytes and putative embryos were stained with dichlorodihydrofluorescein diacetate (DCFDA) and the H2O2 level was measured with an inverted fluorescence microscope using ImageJ software. At the end of the fifth day after IVF, the expression of Bax and B cell lymphoma 2 (Bcl2) was evaluated using real-time PCR.ResultsThe levels of reactive oxygen species (ROS) in oocytes and putative embryos observed in the treatment group demonstrated a significant reduce compared to the control group (p ≤ 0.01. (.Furthermore, the number of embryos in the blastocycte stage(P < 0.05), the expression level of the Bcl2 (P < 0.05) gene, the Bax unlike gene, significantly increased compared with the control group.ConclusionWe conclude that the presence of melatonin and vitamin C antioxidants simultaneously and exclusively in the IVF medium leads to a reduction in ROS and ,as a result, improves the growth of the embryo up to the blastocyst stage.

O-007 Association of successful embryo development and pregnancy outcome with redox state as measured by human non-mercaptalbumin in embryo culture medium

Abstract Study question Is the redox state in the embryo culture medium, as measured by oxidized albumin, associated with successful embryo development and pregnancy outcome? Summary answer The level of oxidized albumin in the culture medium was the most important variable in predicting blastocyst formation andwas associated with miscarriage after transfer. What is known already Human serum albumin (HSA), the most abundant protein in the plasma, exerts important antioxidant activities against oxidative damage. HSA exists as oxidized human non-mercaptalbumin (HNA) and reduced human mercaptalbumin. HNA is attracting attention because of its novel role as a marker reflecting oxidative state, and HNA levels are known to increase in oxidative stress diseases such as kidney disease, diabetes, liver disease or Parkinson's disease. On the other hand, in human IVF, most culture medium are supplemented with HSA as a protein source, but no previous studies have examined the relationship between HNA and IVF outcomes. Study design, size, duration We followed the pregnancy outcomes for 40 cycles of single blastocyst transfer out of the retrospective study, which enrolled a total of 173 embryos cultured to the blastocyst stage from a total of 91 patients who underwent IVF/ICSI cycle between February 2018 and July 2018. Participants/materials, setting, methods Prior to using the medium for embryo culture, the redox state was assessed with HNA level. HNA level, patient age, IVF/ICSI and oocyte maturity were analyzed as factors associated with blastocyst formation on day 5 and 6 after fertilization, and a machine learning model was developed by using the Random Forest (RF) algorithm to predict blastocyst formation. We also examined whether the factors made a difference in pregnancy outcomes. Main results and the role of chance The median %HNA in the culture medium was 89.36% (range, 80.12% to 94.84%), which was much higher than the value for blood in healthy humans, (approximately 25%). Blastocyst formation was observed in 41.04% (71/173) embryos. In both univariate and multivariate analyses, successful blastocyst development was associated with a lower %HNA in the culture medium (p = 0.001), a younger patient age (p &amp;lt; 0.001), and the use of standard IVF (p = 0.007). A prediction model for successful blastocyst formation was developed using a RF algorithm with four factors (%HNA, patient age, fertilizationmethod, and oocyte maturation stage). The RF model developed using 70% of samples (training set, n = 121) was validated in the remaining testing set (n = 52) and produced an area under the curve of 0.761, where %HNA in the culture medium was the most important variable for the prediction of blastocyst formation, followed by patient age. The HNA levels were significantly higher in the group of embryos that resulted in miscarriage after blastocyst transfer (93.15% vs. 89.28%, P = 0.0498). Limitations, reasons for caution This study was conducted in G-TL medium only. Further studies are needed to elucidate the association of %HNA and embryo development, and to predict the transfer success rate by using other commonly used media. Wider implications of the findings In the IVF medium, which is supposed to mimic the body environment, the %HNA showed a high oxidized state compared to the body itself; it is suggesting it may cause unsuccessful IVF results and lower live birth rate. Controlling the oxidation level may help to create a more appropriate environment. Trial registration number not applicable

O-235 Antioxidants in culture media improve ICSI fertilisation rate: results from a randomised controlled trial

Abstract Study question Does addition of three antioxidants to G-Series media during gamete collection, insemination, and embryo culture increase the clinical pregnancy rate from fresh blastocyst transfers? Summary answer Antioxidants increased the ICSI fertilization rate, and consequently the number of blastocysts utilised, but the clinical pregnancy rate from fresh blastocyst transfers was not affected. What is known already Supplementation of IVF media with a combination of three antioxidants known as A3 (10 μmol/l acetyl-L-Carnitine, 5 μmol/l α-Lipoic acid and 10 μmol/l N-acetyl-L-cysteine) is beneficial in mouse IVF, embryo culture and cryopreservation, and improves post-transfer outcomes. Further, in a prospective randomised trial on sibling oocytes, addition of A3 to G-1/G-2 media (Vitrolife, Sweden) resulted in more good quality embryos on day 3 (Gardner et al., 2020). The study was not powered to determine the effect of A3 on pregnancy rate, but an increase in ongoing pregnancy rate was observed in women aged 35-40 years following frozen blastocyst transfer. Study design, size, duration Single-centre, prospective randomised controlled trial, superiority study comparing G-series media (G-MOPS, G-IVF and G-TL (Vitrolife)) with or without the addition of A3 from January 2019 to November 2021. 1482 patients were randomized before egg collection. Patients and their doctors were blinded to the treatment group. The primary endpoint was clinical pregnancy per randomised couple from the fresh transfer of a single blastocyst. Participants/materials, setting, methods Patients undergoing IVF/ICSI cycles and intending to undergo a fresh transfer of a single blastocyst were recruited. Exclusion criteria were previous participation in the study, freeze-all cycle, or extraction of sperm from testicular biopsy. 743 patients were randomised to the A3 media, 739 to the control. Main results and the role of chance Patients randomised to A3 media had significantly higher fertilisation rate per inseminated oocyte (64.4 ± 25.6%) compared to the control (59.2% ± 26.3%, P &amp;lt; 0.001). This was more pronounced in patients undergoing ICSI (68.1% ± 24.9% vs 57.9% ± 27.2%, P &amp;lt; 0.0001), where the number of cycles with failed fertilisation decreased from 8.0% to 3.9% with A3 media (P &amp;lt; 0.05). There was no effect of A3 on fertilisation rate following IVF. Blastocyst development rate was unaffected by A3, but the higher fertilisation rate resulted in more blastocysts available for transfer or cryopreservation per patient in the A3 group (3.06 ± 2.97 vs 2.67 ± 2.60, P &amp;lt; 0.01). Clinical pregnancy rate from fresh cycles was not different between the control (26.1%) and A3 media (23.0%; P &amp;gt; 0.05; RR 0.88, 95% CI 0.73-1.05). When patients who did not have a fresh transfer were excluded (due to freeze-all or no embryo available), there was also no difference between the control (35.8%) and A3 media (32.6%; P &amp;gt; 0.05; RR 0.91; 95% CI 0.77-1.08). Limitations, reasons for caution This was a single-centre study, so the effects of A3 supplementation in clinics with different media and protocols are unknown. Detection of fetal heart by ultrasound was the primary endpoint, birth outcomes are currently being investigated. Outcomes of frozen embryo transfers and cumulative pregnancy rates are yet to be determined. Wider implications of the findings Addition of antioxidants to media during gamete collection, incubation and ICSI can increase fertilisation rate and reduce the frequency of failed fertilization cycles. This results in more blastocysts available for transfer and cryopreservation per egg collection, potentially leading to higher cumulative pregnancy rates. Trial registration number ACTRN12618001479291

How and when to measure pH in IVF culture media: validation of a portable blood gas analyzer in two IVF culture dishes for time lapse and conventional incubators.

Maintaining a stable pH at optimal level in human embryo culture media is crucial for embryo development but poses a challenge for all IVF laboratories. We validate analytically reliable conditions for pH measurement that are as close as possible to the embryo microenvironment during IVF. This was a multicentric study. A Siemens EPOC portable blood gas analyzer was used. The analytical validation was carried out under the culture medium (Global Total HSA®) conditions of use (microdroplets, under oil overlay, in a IVF incubator with (EmbryoScope®) or without a time lapse system (K system G210+®) and using IVF dishes. The validation included repeatability ("within-run" precision), total precision (between-day precision), trueness based on inter-laboratory comparison, inaccuracy based on external quality assessment and comparison to the reference technique. We also assessed the pre-analytical medium incubation time required to obtain a target value. A measurement after an incubation period of 24 to 48 h is more representative of the pH to which the embryo will be exposed throughout the culture. The "within-run" and "between-day" precision show very low coefficients of variation (CV%): 0.17 to 0.22% and 0.13 to 0.34%, respectively, with IVF culture media. Trueness (% bias) range from - 0.07 to - 0.03%. We demonstrate good correlation between EPOC and reference pH electrode with an overestimation of 0.03 pH units of EPOC. Our method demonstrates good analytical performance for IVF laboratories wishing to implement a robust quality assurance system to monitor pH in embryo culture media. Compliance with stringent pre-analytical and analytical conditions is essential.

Lipid Peroxidation Status in Embryo Culture Media: The Impact on Fertilization and Embryo Quality during IVF Cycles

Background: The process of preimplantation embryo development in vitro represents a key phase during in vitro fertilization (IVF) cycles. It involves several regulatory signaling pathways as well as an optimized in vitro culture system. The resulting embryo quality helps to determine embryo competence before implantation and pregnancy outcomes. Reactive oxygen species (ROS) are known to play a major role in influencing the process of embryo development. Their role can be reflected in the regulation of signaling pathways as second messengers or in the irreversible cell alterations due to oxidative stress following an excess of ROS levels.&#x0D; Methods: In this study, we investigated the association between morphological embryo quality (fertilization, cleavage quality, and fragmentation levels) and lipid peroxidation levels (Malondialdehyde) in embryo culture media. After intracytoplasmic sperm injection (ICSI), a total of 103 oocytes were evaluated on day 1 and day 3 of their development, and their corresponding culture media were analyzed by estimating MDA levels using thiobarbituric acid.&#x0D; Results: The results showed no significant association between MDA levels in culture media and fertilization rate (p=0.3), sperm quality (p=0.99; p= 0.17; p=0.46; p=0.30; p=0.65; p=0.44; p=0.09; p=0.15; p=0.56), embryo fragmentation levels (p=0.79; p=0.40), AMH levels (p=0.31; p=0.36) and female age (p=0.60; p=0.34). However, we revealed a significant association between cleavage quality and MDA levels in the embryo environment (p=0.03).&#x0D; Conclusion: We conclude that oxidative stress in IVF culture media might be mainly associated with delayed embryonic development.

Embryotoxicity evaluation of Gentamicin, an aminoglycoside antibiotic added to human embryo culture medium, using the zebrafish (Danio rerio) model

Infertility gives rise to a lot of social and psychological problems. At present, assisted reproductive technology (ART) is an important way to solve infertility. However, the live birth rate of in vitro fertilization and embryo transfer (IVF-ET) is less than 50 %. Medium is essential for the culture of embryos in vitro. Therefore, we want to explore whether the composition of the culture medium affects the survival rate of embryos. Gentamicin (GM) is an aminoglycoside antibiotic that is used to treat various bacterial infections. It is widely used in IVF medium, but it is not known whether it has a toxicity effect on embryonic development. Here, we used zebrafish embryos to investigate the embryotoxicity of GM which is an ingredient in culture medium. Our results found that there was no significant effect on the zebrafish embryo development, including survival rate, malformation rate and developmental time course, while zebrafish embryos were treated with GM at the culture medium concentration (10 mg/L, 17.8 μM) compared with the control group. To research the potential embryotoxicity of GM, we treated zebrafish embryos with GM with high concentration (range from 17.8 μM to 3000 μM). The results showed that the lethal concentration of 50 % (LC50) at 48-h post-fertilization (hpf) value of zebrafish embryos for GM was 1150 μM; the survival rate and malformation rate of zebrafish embryos were significantly changed in a dose-dependent manner. Furthermore, transcriptomics, metabolomics and epigenomics (m6A-MeRIP-seq) were used to investigate the molecular mechanism of embryotoxicity, and results showed cell cycle, dorso-ventral axis formation and collecting duct acid secretion pathway were altered significantly in treated embryos. In conclusion, there are no adverse effects on embryonic development with the working concentration of GM in human culture medium, suggesting that GM is safe for embryo culture at working concentration.

The impact of various antioxidant supplementation on ram's sperm quality, fertilization, and early embryo development, in vitro

The in vitro embryo production (IVEP) is very stressful for gametes. Gametes are subjected during in vitro manipulation to many different types of stress; oxidative stress is the most prominent one, which will cause damage or alter the genetic material of the sperm and reduce the quality of the oocytes, and has a crucial impact on the possibility of developing embryos even after implantation. This study aimed to determine the influence of antioxidants on the achievement of In vitro culture (IVC) and sperm's ability to adhere to and penetrate further into In vitro maturated oocytes. For this purpose, we have incubated ram sperm using four different treatments in terms of antioxidants: melatonin, cysteamine, vitamin C, and vitamin E. They were incubated by the standard methods of maturation and capacitation of sperm. The oocytes were fertilized by spermatozoa that had been capacitated with two groups of fertilization media, the first group containing melatonin and the second group containing cysteamine. Compared with other groups, sperms treated with melatonin demonstrated hyperactivity, and the fertilization rate was significantly increased. As for the IVF medium containing melatonin, it was superior to cysteamine in embryo development rates. In conclusion, melatonin could be a promising tool for improving sperm competence for fertilizing oocytes and embryo development in sheep.

“Thick and Viscous” or “Light and Easy”: An Oil Overlay Comparison

Background: Oil overlay in IVF culture media is essential in the maintenance of pH and prevention of evaporation. The recent proliferation of “Heavy oil” raises questions of how oil density and viscosity affect CO2 diffusion into and out of culture media. Aim: To compare the effects of “Heavy oil” versus standard oil on pH stability in embryo culture dishes. Method: Density (g/ml) readings of each oil (Ovoil, Ovoil Heavy and LifeGuard) were obtained in triplicate using a volumetric flask and analytical balance in both refrigerated (4[Formula: see text]C) and warmed state (37[Formula: see text]C). Viscosity testing was performed externally by Intertek laboratory. pH was measured at regular intervals using a blood gas analyser (EPOC system) in culture dishes under standard and ‘Heavy’ oil overlays. Results: Density measurements showed Ovoil Heavy to be the least dense (0.8227g/ml) followed by Ovoil (0.8476g/ml), with LifeGuard the most dense at 0.8640g/ml. There was a significant difference (p&lt;0.0001) when comparing densities between oil types in both 4[Formula: see text]C and 37[Formula: see text]C. Viscosity testing reported LifeGuard the most viscous at 153.6mm2/s followed by Ovoil Heavy at 57.98mm2/s and Ovoil at 30.68mm2/s. Media equilibrated more slowly under denser oil, requiring 5 hours to read pH 7.4 under LifeGuard with lighter oil requiring 3 hours to reach equilibration. Equilibrated dishes exposed to reduced CO2 concentration (4%) showed quicker pH loss in Ovoil at 0.049/hour when compared to LifeGuard at 0.034/hour (p=0.006). Culture dishes placed in a humidicrib with open cuffs, emulating real world settings, showed slower pH loss under LifeGuard (0.06/hour) compared to Ovoil (0.08/hour). Conclusion: Higher density oil overlay takes longer to equilibrate to optimal pH but can slow down loss of pH when dishes are exposed to ambient conditions.

P-226 Efficiency of different antioxidants in reducing the supra-physiological Oxidation Reduction Potential Levels in human embryo culture media

Abstract Study question Does every antioxidant compounds perform similarly in reducing the excessive levels of Oxidation Reduction Potential (ORP) in IVF culture media? Summary answer Not all antioxidant compounds perform similarly in maintaining physiological ORP levels in IVF culture media. What is known already During assisted reproduction technologies (ART), several external factors are responsible for disturbing the ORP equilibrium in gametes and embryos. Therefore, antioxidant supplementation of culture media has been evaluated and reported. However, there is lack of consensus on the efficiency of antioxidant action in embryo culture media. Antioxidants are known to lower the levels of free radical concentration to physiological levels, preventing cellular damage caused by oxidative stress. Therefore, it is important to determine which antioxidant compound are the most effective in reducing ORP levels in embryo culture media. Study design, size, duration In this prospective study was performed at CITMER Reproductive Medicine, Mexico city. ORP levels were measured by MiOXSYS System (UAB Caerus Biotechnologies, Vilnius, Lithuania) in embryo-free culture medium (Global® Total®, LifeGlobal®, Connecticut, US). Embryo-free culture medium was supplemented with cumene hydroperoxide and seven different antioxidants were tested each day for 5 continuous days (n = 40). Follicular fluid ORP levels from dominant follicles of 40 oocyte donors were used as control and considered as a physiological target. Participants/materials, setting, methods Cumene hydroperoxide (CH) 1mM, was utilized to increase ORP values in culture media from 224 mV to 415 mV. The following compounds considered as antioxidants according to literature were supplemented at 2 mM to the CH culture media: Ascorbic acid (ASC), L-Carnitine (CAR), L-Cysteine (CYS), Glutathione (GLT), Curcumin (CU), Resveratrol (RSV) and b-Mercaptoethanol (BME). ORP levels were quickly evaluated each time. Main results and the role of chance In this study, CH was used as a culture media oxidant to measure the antioxidants capacity in lowering the excessive ORP values of CH culture media. ORP in CH solution was 415 mV ± 4.3. All antioxidants showed a statistically different capacity (p &amp;lt; 0.05) to lower the ORP values when added to CH solution: RSV decreased the ORP to 348.3 mV ± 3.6 p = 0.0449, CAR to 316.32 mV ± 4.7 (p &amp;lt; 0.0001), CU to 315.7 mV ± 4.4 (p &amp;lt; 0.0001) and GLT to 258.4 mV ± 15.1 (p = 0.00002). The compounds that showed higher antioxidant capacity were CYS 158.4 mV ± 4.7 (p &amp;lt; 0.00001), ASC 154.1 mV ± 3.1 (p &amp;lt; 0.00001) and BME 136.1 mV ± 9.6 (p &amp;lt; 0.00001). CYS, ASC and BME ORP values reached the physiological ORP values found in follicular fluid from donors, which are 89 mV±23.6 mV. Limitations, reasons for caution Further studies should focus on the comparison between physiological dynamics of antioxidant supplementation on culture media and molecular and biochemical effect of antioxidants on embryos and gametes. Wider implications of the findings According to our results, CYS, ASC and BME were significantly more potent antioxidants in lowering the ORP in culture medium to physiological levels. The measurement of antioxidant capacity in lowering excess of ROS in culture media may assist in developing the most effective antioxidant supplements, thus improving ART outcomes. Trial registration number none

P-759 Comparison of DNA methylation profiles of human embryo cultured in either uninterrupted or interrupted incubators

Abstract Study question Are there are differences in the DNA methylation profiles of human embryos cultured in Time-lapse imaging (TLI) and standard incubators (SI)? Summary answer The genome-wide DNA methylation landscape was globally similar between the SI and TLI groups. What is known already Early embryonic development is a special biological process, along with dynamic changes of DNA methylation. In vitro culturing ensured the successful development of human embryo, and is the most important step of ART cycle. TLI is one type of newly developed ART embryo culture systems, which allows for continuous assessment of embryonic development. Our previous study performed transcriptome analysis to compare SI and TLI and found that the global transcriptomic profiles were similar between the two groups. However, whether there are differences in the DNA methylation profiles of human embryos cultured in TLI and SI still unknown. Study design, size, duration This study was designed to explore the influence of SI and TLI incubator culturing on genome-wide DNA methylation of human eight-cell embryos. A total of 9 women who received IVF treatment, ≤ 30 years old (range: 20–30 years), without a history of genetic diseases or smoking were included in this study. Participants/materials, setting, methods Transvaginal oocyte retrieval was performed 36 h after HCG injection. Cumulus-enclosed oocytes were collected in 2.5 mL IVF medium and incubated in 5% O2, 6% CO2, and 37 °C incubators for insemination. The fertilized oocytes were transferred into pre-equilibrated Embryoslides. Then the embryoslides were cultured in either TLI or SI at 37 °C with 5% O2 and 6% CO2 until embryo transfer on Day 3. Samples were sequenced by the Illumina HiSeq 4000 with a 150-bp paired-end. Main results and the role of chance The genome-wide methylation patterns and CpG methylation levels in transposable elements and imprinting control regions of TLI-cultured embryos were similar to those of the SI-cultured embryos. However, a small number of differentially methylated regions (DMRs) were detected, and these DMRs mainly occurred in exons other than promoters. Functional annotation revealed that the genes in DMRs tended to execute functions such as cell cycle, DNA damage stimulus, histone modification, mitochondrial, glucose import, and MAPK signaling pathway. Limitations, reasons for caution Small sample size. Wider implications of the findings Evaluated the safety of TLI culture system from the perspective of DNA methylation at single-cell level, and provided an important reference for understanding the association between embryo culture condition and epigenetic regulation. Trial registration number not applicable

P-397 How do extracellular vesicles secreted by human embryos impact decidualized endometrial stromal cells?

Abstract Study question What is the transcriptomic response of decidualized endometrial stromal cells (DESCs) to the uptake of extracellular vesicles (EVs) secreted by human embryos? Summary answer Human embryo derived EVs can impact DESCs transcriptomic profile via a cargo that can induce a positive effect on the expression of some decidualization-related genes. What is known already The establishment of an appropriate embryo–endometrial communication is essential for the implantation process and a successful pregnancy. Embryo-released factors are able to elicit biological effects on the endometrium. Among these, EVs secreted by embryos carry molecules potentially able to modulate endometrial function and favour the implantation process. However, most results come from animal species as few studies were performed using EVs derived from human embryos, due to difficulty in sample collection or ethical reasons. Study design, size, duration EVs were isolated from one pool of IVF media where embryos were cultured between day 3 and 5 of development (blastocyst). Initially, DESCs(n = 4) were treated with different concentrations of EVs at different time points and RNA-sequencing was performed to define optimal conditions.Subsequently, EVs were isolated from one pool of IVF media where blastocysts diagnosed as euploid were cultured (EVe). To increase the reliability of the results, DESCs(n = 5) were treated with EVe using the optimal conditions. Participants/materials, setting, methods EVs were isolated by differential ultracentrifugation from spent media of in vitro cultured human blastocyst stage embryos and from an equal volume of control medium (not exposed to embryos). DESCs were in vitro decidualized (E2:10nM+ P4:1uM+cAMP:0.5mM) and treated with different concentrations of EVs (10µg,50µg,100µg) for 24hours and 48hours. RNA-sequencing analysis was then performed. Once established the conditions that allow to better detect differences in trascriptomic profile, RNA-sequencing analysis on DESCs treated specifically with EVe was performed. Main results and the role of chance The best response of DESCs to EVs uptake was induced by treatment with 10µg of EVs for 48hours. Using this condition, differential gene expression analysis allowed the identification of 166 upregulated and 124 downregulated genes in DESCs treated with EVe, when compared to DESCs treated with control. Based on Over-Representation analysis, DESCs treated with EVe were found to be enriched in transcripts of genes involved in signalling and cell communication. Moreover, based on Gene Set Enrichment Analysis results, EVe may regulate decidualization and the implantation process by targeting cholesterol biosynthesis and metabolism pathways, being among the most enriched pathways. The depletion of transcripts related to inflammatory and IL1R pathways in DESCs treated with EVe was also observed, suggesting a role of EVe in the fine regulation of inflammatory/immunomodulatory events occurring during implantation. At the intersection of genes whose expression was modified by EVe-treatment and those modified by the decidualization treatment, thirty-one transcripts exhibited the same trend, demonstrating that EVe exerted a positive effect on decidualization-related genes expression in DESCs. Specifically, expression of genes involved in the implantation process (PTGER2, IL15, WNT4) increased after decidualization and were further up-regulated after EVe-treatment, confirming the crucial role of EVs in the embryo-endometrium cross-talk. Limitations, reasons for caution Larger studies are needed to validate these findings. Wider implications of the findings EVs derived from human euploid embryos can evoke a specific transcriptional response in DESCs. Moreover, we speculate that these EVs may exert favourable effects on the endometrial tissue, increasing its receptivity for embryo implantation by modulating decidualization-related genes expression. Trial registration number not applicable

Extracellular vesicles from seminal plasma improved development of in vitro-fertilized mouse embryos.

In vitro fertilization (IVF) has wide application in human infertility and animal breeding. It is also used for research on reproduction, fertility and development. However, IVF embryos are still inferior to their in vivo counterparts. Some substances in seminal plasma appear to have important roles in embryo development, and during the traditional IVF procedure, the seminal plasma is washed away. In this study, extracellular vesicles (EVs) were concentrated from seminal plasma by ultracentrifugation, visualized using transmission electron microscopy, and particle size distributions and concentrations were determined with a NanoSight particle analyzer. We found particles of various sizes in the seminal plasma, the majority having diameters ranging from 100 to 200 nm and concentrations of 6.07 × 1010 ± 2.91 × 109 particles/ml. Addition of seminal plasma EVs (SP-EVs) to the IVF medium with mouse oocytes and sperm significantly increased the rate of blastocyst formation and the inner cell mass (ICM)/trophectoderm (TE) cell ratio, and reduced the apoptosis of blastocysts. Our findings provide new insights into the role of seminal plasma EVs in mediating embryo development and it suggests that SP-EVs may be used to improve the developmental competence of IVF embryos, which has important significance for assisted reproduction in animals and humans.