Improved Blastocyst Development Research Articles

O-064 Can artificial removal of the zona pellucida with severe fragmentation improve culture and pregnancy outcomes?

Abstract Study question Can artificial removal of the zona pellucida (ZP-free) in patients whose embryos had severe fragmentation improve culture and pregnancy outcomes? Summary answer In patients with severe fragmentation, blastulation and live birth rate were improved by ZP-free procedure, and 14 live babieshave been obtained so far. What is known already In 2020, Yumoto et al. first reported that ZP-free procedure at the pronuclear stage decreased the rate of fragmentation and improved blastulation of 3 pronuclei embryos. Therefore, the ZP may not always be necessary for normal embryonic development after the pronuclear stage. The same group presented at the 37th ESHRE meeting in 2021, that the transfer of blastocyst derived from 2 pronuclei embryos (2PN) after ZP-free procedure resulted in normal pregnancy and live births in two patients who had had poor-quality embryos only before ZP-free procedure. We also presented culture outcomes improved by ZP-free procedure at the 2023 ESHRE meeting. Study design, size, duration This study included 571 embryos derived from 2PN on 118 cycles in 61 couples obtained between July 2021 and November 2023, whose previous oocyte retrieval cycle had shown massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI) in two institutes. Mean age of women was 40.0±3.8 years. Culture and pregnancy outcomes after blastocyst transfer were compared with 1272 embryos derived from 2PN in the past 247 cycles without ZP-free procedure (ZP-intact). Participants/materials, setting, methods All participants provided written informed consent, and IRB approval was obtained. Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking at the pronuclear stage. The ZP was partially removed in non-adhesive areas with a laser system. Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. The ZP-free embryos were cultured in TLI up to 7 days, followed by blastocyst cryopreservation for future embryo transfer cycles. Main results and the role of chance Day 3 embryos were divided into 3 groups using the Veeck’s classification: 8 cell embryos with grade 2 or higher were defined as good, embryos with grade 4 or 5 regardless of the number of cells were defined as poor, and others were defined as fair. We compared the culture outcomes of ZP-free cycle and previous ZP-intact cycle. For day 3 embryos, good, fair and poor grade were 15.6%, 55.3% and 29.1% in ZP-free embryos compared with 3.7%, 42.5%, and 53.8% in ZP-intact embryos, respectively (p < 0.001). As to the blastocysts, blastocysts development and good-quality blastocysts (G3BB or more on day 5 by the Gardner’s classification) rates were 36.9% and 19.4% in ZP-free embryos compared with 23.1% and 6.0% in ZP-intact embryos, respectively (p < 0.001). Clinical pregnancy rates for ZP-free and ZP-intact blastocyst transfer were 36.1% (22/61) and 15.0% (17/113), miscarriage rates were 18.2% (4/22) and 82.4% (14/17), and ongoing pregnancy rates were 29.5% (18/61) and 2.7% (3/113), respectively, with statistical significance after ZPF procedure (P < 0.01). Fourteen live babies have given birth by single blastocyst transfer with ZP-free procedure so far. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only two fertility centers. It is necessary to conduct sibling oocytes study or extensive randomized controlled trial to create criteria for patients who are benefit from ZP-free procedure. Moreover, safety of ZP-free culture should be evaluated. Wider implications of the findings ZP-free procedure in patients with severe fragmentated embryos at the pronuclear stage, reduced fragmentation and improved blastocyst development and clinical outcomes. We suggest that ZP-free embryos could be proposed as a new treatment option for patients who have had poor quality embryos with severe fragmentation. Trial registration number Not applicable

P-165 Benefits of ultra-low oxygen tension (2%) in patients with low quality embryos or previous arrest: the patient as its own control

Abstract Study question Is reduction in oxygen tension from 5% to 2% after D3 of extended culture beneficial for blastocyst development in patients with previous failed culture? Summary answer Ultra-low oxygen tension in extended culture improves blastocyst development (overall blastulation rate OBR), quality (top blastulation rate TPR) and increases the pregnancy rate (PR). What is known already The practice ESHRE guidelines recommends human embryo culture at low oxygen concentrations (2-8%). Traditionally atmospheric conditions (20% O2) were used but better outcomes were observed at low monophasic 5% O2. Human embryo leaves the oviduct (5-7% O2) to cross the uterus-tubal junction around D3-4 and reaches the uterin ultra-low O2 tension (2%). This decreasing gradient coincides with the shift from pre-compaction oxidative phosphorylation in the oviduct, to post-compaction glycolysis in the uterus. Few studies with ultra-low 2% O2 incubation suggested that the best O2 level may be stage-dependent and its reduction after D3 may be a more physiological approach. Study design, size, duration Prospective observational study in a single private hospital during 2023. 71 patients experienced a failed cycle of extended culture (n = 29 cIVF, n = 37 ICSI and n = 5 Sibling oocytes) with a poor embryo quality and low OBR, under standard monophasic 5% O2 incubation. In the subsequent cycle for these 71 patients (n = 24 cIVF and n = 46 ICSI and n = 1 Sibling oocytes) the O2 level was reduced to 2% from D3 onwards. Participants/materials, setting, methods After a failed extended culture, considered as the control cycle, the patients were enrolled for the study. Blastocysts were rated by Gardner’s classification. In the control cycle embryos were cultured under monophasic 5% O2 until D6. In the subsequent cycle embryos were cultured under 5% O2 until D3 and in ultra-low 2% O2 from D3 to D6. Sage 1-Step™ media was used for both cycles. The statistical analyses were done by chi-square and fisher test. Main results and the role of chance 71 patients were included in the study regardless of age, indication or technique. Each patient was its own control. The mean age was not different between the 2 groups (36.5±4 vs 37.2±4) but the attempt rank was logically statistically higher in the study cycle (2.1±1.4 vs 3.4±1, p<0.001). Oocyte maturity (75% vs 76.4%) and fertilization rate (66.8 vs 70.3%) were similar between groups. The average embryo quality at D3 was poor in both groups. OBR and TBR (40.7% vs 52.4%, p=0.004 and 15.8% vs 37.5%, p<0.001) were statistically higher after culture in ultra-low O2 conditions. Extended culture failure with absence of usable blastocyst (57.7% vs 19.7%, p<0.001) was statistically lower in ultra-low incubation group and the pregnancy rate PR per cycle (3.6% vs 25%, p=0.004) was seven fold higher after ultra-low culture. Limitations, reasons for caution The small size of the sample needs to be expanded in order to confirm the encouraging findings in a larger population. The study will continue during 2024 and will compel the live birth rate and the cumulative live birth rate. Wider implications of the findings Providing embryos with the most physiologic culture system would improve IVF cost-effectiveness and a better infertility management. Embryo energetic adaptation to suboptimal conditions results in impaired viability and lower pregnancy rates. In our study extended culture of low-quality embryos under 2% O2 after D3 yielded better culture and pregnancy outcomes. Trial registration number NA

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

In vitro culture alters cell lineage composition and cellular metabolism of bovine blastocyst†.

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.

Lipid Enriched Reduced Nutrient Culture Medium Improves Bovine Blastocyst Formation.

The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.

Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

Sibling oocytes cultured in a time-lapse versus benchtop incubator: how time-lapse incubators improve blastocyst development and euploid rate.

The aim was to study whether a limited exposure of embryos outside the incubator has an effect on embryo development, blastocyst quality and euploid outcomes. This retrospective study was performed at ART Fertility Clinics, Abu Dhabi, United Arab Emirates (UAE) between March 2018 and April 2020 and included 796 mature sibling oocytes that were split randomly between two incubators after intracytoplasmic sperm injection (ICSI): an EmbryoScope™ (ES) incubator and a benchtop incubator, G185 K-SYSTEMS (KS). The fertilization, cleavage, embryo/blastocyst qualities, useable blastocyst and euploid rates were assessed to evaluate the incubator performance. In total, 503 (63.2%) mature oocytes were cultured in the EmbryoScope and 293 (36.8%) in the K-SYSTEMS. No differences were observed in fertilization rate (79.3% vs 78.8%, P = 0.932), cleavage rate (98.5% vs 99.1%, P = 0.676) and embryo quality on Day 3 (P = 0.543) between both incubators, respectively. Embryos cultured in the EmbryoScope, had a significantly higher chance of being biopsied (64.8% vs 49.6%, P < 0.001). Moreover, a significantly higher blastocyst biopsy rate was observed on Day 5 in the EmbryoScope (67.8% vs 57.0%, P = 0.037), with a highly significant increased euploid rate (63.5% vs 37.4%, P = 0.001) and improved blastocyst quality (P = 0.008). We found that exposure of embryos outside the incubator may negatively affect the in vitro blastocyst development and euploid rate on Day 5.

Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.

MICROFLUIDIC SPERM SORTING COMPARED WITH TRADITIONAL DENSITY GRADIENT CENTRIFUGATION: A COST ANALYSIS

It has been advocated that the use of a microfluidics device may improve blastocyst development and euploidy rates during in vitro fertilization (IVF) cycles. However, the cost of this additional laboratory add-on has not been previously analyzed. We sought to analyze the cost of using the Zymot™ Multi (850 μL) Sperm Separation Device compared with traditional density gradient centrifugation for processing sperm to be used for intracytoplasmic sperm insemination (ICSI).

Artificial Oocyte Activation to Improve Blastocyst Development

Background and Aim: Artificial oocyte activation (AOA) can be used to induce the calcium oscillations required for oocyte activation. Using calcium ionophores for AOA has been shown to overcome fertilisation failure in cases attributed to failed oocyte activation. It was recently suggested that AOA may also restore calcium signalling deficiencies in cleavage stage embryos which arrest in cases of failed blastocyst development (Ebner et al. 2015). This study investigated the effects of AOA on 34 patients with previous poor or failed blastocyst development between the period July 2018 and January 2020.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

P-215 Use of hyaluronan-based solution as an alternative to polyvinylpyrrolidone to improve blastulation in ICSI

Abstract Study question This study aimed to analyze whether hyaluronan as an alternative to polyvinylpyrrolidone (PVP) for sperm immobilization, aspiration, and injection into the ooplasm improves ICSI outcomes. Summary answer The use of hyaluronan solution as an alternative to PVP improves the blastocyst formation rate and good quality blastocyst formation rate compared to PVP. What is known already The PVP solution facilitates ICSI procedures, such as sperm immobilization, aspiration, and injection into the ooplasm. However, it has been reported that injection of a small amount of PVP along with the sperm into the ooplasm negatively affects subsequent embryo development. Hyaluronan is a natural component of the extracellular matrix of the cumulus-oocyte complex. Balaban et al. (2003) reported that hyaluronan can replace PVP during ICSI procedures in the early embryo transfer cycle without adversely affecting pregnancy outcomes. This study analyzed whether using hyaluronan-based solution for the ICSI procedure would improves blastocyst development compared with ICSI using PVP. Study design, size, duration This retrospective study analyzed clinical medical reports at Takahashi Women’s Clinic, Japan. We included 4002 oocytes retrieved from 411 patients under 39 years of age who underwent ICSI using autologous oocytes between December 2019 and August 2021. Of the oocytes used for ICSI, 1909 underwent sperm aspiration and injection into the ooplasm with hyaluronan (Sperm Slow; Origio), and 2093 oocytes with 7% PVP (NakaMedical). We did not perform preimplantation genetic testing-aneuploidy for any cycles. Participants/materials, setting, methods We used PVP droplets for sperm selection under 400× magnification in both groups. In hyaluronan-ICSI, the selected sperm were transferred to a hyaluronan droplet and washed three times. The sperm was then aspirated with hyaluronan, and ICSI was performed. In PVP-ICSI, all procedures were performed using PVP. The ICSI and embryo transfer outcomes were compared between hyaluronan-ICSI and PVP-ICSI by logistic regression analysis considering patient age, BMI, and basal level of anti-mullerian hormone. Main results and the role of chance Normal fertilization rates were 74.3% (1556/2093) in PVP-ICSI and 75.5% (1442/1909) in hyaluronan-ICSI. There was no significant difference in the normal fertilization rate between PVP-ICSI and hyaluronan-ICSI groups (p = 0.437, aOR:1.06, 95% CI: 0.92–1.22). We cultured 1323 2PN embryos in PVP-ICSI and 1237 2PN embryos in hyaluronan-ICSI until the blastocyst stage. Blastocyst formation rates were 48.1% for PVP-ICSI and 52.3% for hyaluronan-ICSI, and this difference was statistically significant (aOR, 1.20; 95% CI: 1.02–1.40; p = 0.024). Moreover, the good grade (Gardner criteria ≧BB) blastocyst formation rates were significantly higher in the hyaluronan-ICSI group (36.9% and 41.0%, aOR: 1.21, 95% CI: 1.03–1.42, p = 0.022). During the study period, we performed 163 and 169 cryo-thawed blastocyst transfer cycles in PVP-ICSI and hyaluronan-ICSI, respectively. The clinical pregnancy rate (50.9% vs. 54.4%, aOR: 1.19, 95% CI: 0.77–1.83, p = 0.443) and miscarriage rate (19.3% vs. 13.0%, aOR: 0.66, 95% CI: 0.3–1.44, p = 0.295) after embryo transfer were not significantly different between PVP-ICSI and hyaluronan-ICSI. Limitations, reasons for caution The study was conducted at a single IVF center, and the oocytes included in this study were collected from patients aged &amp;lt; 39 years. Embryo transfer result is based on ongoing pregnancy, while the live birth data for all pregnancies are not yet available. Wider implications of the findings Hyaluronan facilitates ICSI procedures such as sperm immobilization, aspiration, and injection. Moreover, the hyaluronan improves blastocyst development. The present study indicates that using hyaluronan as an alternative to PVP during the ICSI procedure is recommended. Trial registration number not applicable

O-099 The use of cumulus cells (CCs) to select spermatozoa for intracytoplasmic sperm injection improves blastocyst development and quality

Abstract Study question Is there a role for sperm selection based on the capacity to pass through cumulus oophorous column in improving the laboratory outcomes in ICSI cycles? Summary answer A higher rate of blastocyst formation and quality are obtained with the use of CCs to select spermatozoa for ICSI compared to conventional density gradients. What is known already The sperm selection for ICSI is an operator-dependent procedure based on sperm morphology and motility. ICSI bypasses all natural barriers of sperm selection, which may impact embryo development. During natural fertilization, only the spermatozoa that pass-through cumulus cells will have the chance to fertilize the egg. Spermatozoa capable of traverse COCs have better morphology, higher chromatin integrity and greater acrosomal reaction. Hyaluronic acid is an important component of these cells and plays a key role in the selection of spermatozoa with intact DNA. However, there is scarce data regarding this selection method for ICSI on the development of the embryo. Study design, size, duration This is a prospective randomized sibling oocyte pilot study (February-December 2021). A total of 996 metaphase II (MII) oocytes were randomized into two groups. In the control group (Group 0, n = 489 MII), ICSI was performed with sperm selected by conventional density gradients. In the study group (Group 1, n = 507 MII) the oocytes were microinjected with sperm selected through CCs. The main outcome was blastocyst formation rate and quality. Reproductive results were analyzed as secondary outcomes. Participants/materials, setting, methods Ninety-eight couples undergoing ICSI (private center) with ≥6 fresh own MII obtained after ovarian stimulation, embryo culture until blastocyst stage and ejaculated sperm samples were included in the study. All seminal samples were processed using density gradients and those in the study group were additionally passed through CCs (the patient's own CCs were used in all cases). Fertilization and day 5 blastocyst formation rates were assessed. Embryo quality was scored according to Gardner's criteria. Main results and the role of chance Data was similar with regards to cause of infertility, maternal age (36.47 vs 36.63 y.o.), sperm count (60.53 vs 58.25 mill/ml) and progressive motility (57.45 vs 57.50%) between group 0 vs 1, respectively. Fertilization rate was similar between groups (74.0% vs 78.7%; p = 0.419. Group 0 vs 1, respectively). However, the blastocyst formation rate was significantly higher in the study group compared to control (70.9% vs 60.1%; p = 0.002). In addition, high-quality blastocyst rate was significantly higher in the CCs compared to the control group (49.6% vs 38.5%; p = 0.003). After the analysis of 145 treatments (131 frozen vs 14 fresh transfers) the cumulative clinical pregnancy rate in the study group was 47.2%, higher than in the control group (32.1%), but without reaching statistical difference (p = 0.07). Limitations, reasons for caution This is an exploratory study, aimed to evaluate the effectiveness of CCs as a more “physiologic” selection of spermatozoa for ICSI treatments in terms of laboratory outcomes (blastocyst formation rate and quality). Albeit not reaching statistical significance, the higher cumulative pregnancy rate in the study group seems clinically relevant. Wider implications of the findings Our results suggest that CCs can select more competent sperm for ICSI. This “physiologic-resembling”, operator-independent selection method has the potential of increasing pregnancy outcomes in ICSI cycles, pending of confirmation in larger studies including cumulative embryo-transfer cycles. Trial registration number NA

Novel Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) culture medium improves development and cryotolerance of in vitro produced Holstein embryos.

In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, and then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.

In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

MITOQUINONE RESCUES STRESS INDUCED MITOPHAGY IN AGED OOCYTES

It is well established that decreased oocyte quality from advanced maternal age (AMA) women are associated with dysfunctional mitochondria. Mitophagy is a critical pathway to regulate mitochondrial quality. Our previous research demonstrated reduction in key mitophagy protein Parkin (PRKN) and its activity in oocytes from AMA women. The objective of this study was to investigate if maturing aged mouse oocytes with Mitoquinone (MQ) would improve oocyte mitochondrial quality by rescuing mitophagy. First, oocytes from young (2 months) and aged (13.5 months) mice were matured with or without MQ and the levels of p-PRKN and PRKN were analyzed by Western blot (Jess, ProteinSimple). In the second experiment, young oocytes with or without MQ were matured under normal (6.5%) or high oxygen (20%) environment, and the expression of p-PRKN and PRKN, mitochondria DNA (mtDNA) copy number (analyzed by qPCR) and blastocyst development after in vitro fertilization, blastocyst hatching, and inner cell mass (ICM) ratio were examined. We observed a significant decrease in p-PRKN (0.32±0.09, p≤0.01) and PRKN (0.30±0.1, p≤0.05) in aged compared to young mouse oocytes (normalized to 1). Treatment of aged oocytes with MQ rescued p-PRKN (1.11±0.19 p≤0.01) and PRKN (0.67±0.04, p≤0.05) protein expression. Oocytes matured under high oxygen environment exhibited significantly reduced p-PRKN (0.5±0.1, p≤0.05) compared to oocytes matured under normal oxygen conditions. This coincided with an increase in mtDNA copy number (Young: 56957, High oxygen: 72758 copies). MQ treatment significantly increased p-PRKN protein expression (1.2±0.2, p≤0.05) as well as decreased mtDNA copy number (50312 copies) in oxidatively stressed oocytes. Addition of MQ significantly improved blastocyst development (High O2: 39.5%, High O2+MQ: 70%, p≤0.05), hatching (High O2: 59%, High O2+MQ: 93%, p≤0.05), and ICM ratio (High O2: 14.61%, High O2+MQ: 22.5%, p≤0.0001) of oxidatively stressed oocytes that showed decreased trends in blastocyst development potential. We have demonstrated previously that oocytes from AMA women (>38 years old) exhibited significantly less p-PRKN and PRKN than oocytes from young women (<32 years old). Similar PRKN expression pattern was obtained in young and aged mice and we also observed decreased p-PRKN in oxidatively stressed mouse oocytes, indicating that mitophagy via Parkin pathway is altered in aged and oxidatively stressed oocytes. We have shown for the first time that treating aged and oxidatively stressed oocytes with MQ rescues p-PRKN and PRKN protein expression to ameliorate mitophagy. MQ also improved blastocyst development potential of oxidatively stressed oocytes.

Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0−5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30−60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.

Optimization of donor cell cycle synchrony, maturation media and embryo culture system for somatic cell nuclear transfer in the critically endangered Vietnamese Ỉ pig

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese Ỉ pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized Ỉ pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of Ỉ pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Fatty acids present in commercial albumin preparations differentially affect development of murine embryos before and during implantation

To characterize fatty acid (FA) profile of commercially available albumin products and determine their effect on embryonic development. Research study. Private research facility. Outbred mice aged 4-8 weeks. Gas chromatography-mass spectrometry was used to quantify the FA content of 15 commercial albumins. Embryos were produced in media containing different albumin products, with or without carnitine or exogenous FA supplementation, to determine their effect on embryo development invitro. Total micrograms of FA per milligram of albumin for the 15 albumin products, blastocyst development, cell number, allocation to the trophectoderm (TE) or inner cell mass (ICM), and evaluation of morphology during implantation. The albumin products contained 0.07-16.77 μg total FA/mg albumin. Compared to media with with >1.4 μg FA/mg albumin, media with <0.5 μg FA/mg albumin supported improved blastocyst development, and addition of carnitine mitigated this difference. Addition of palmitoleic acid or oleic acid individually did not improve blastocyst development and decreased ICM:TE ratio. However, in the presence of carnitine, there was improved blastocyst development and maintenance of the ICM:TE ratio. Embryos cultured inVitrolife human serum albumin with supplementation of carnitine, palmitoleic acid, and oleic acid were more likely to develop cells positive for POU5F1 in an extended embryo culture than embryos cultured in Origio serum protein substitute. Commercial albumin products contain FAs, which vary in abundance. These FAs have different effects on embryo development and quality before and during the implantation stage. Several of these albumin preparations are routinely used for human-assisted reproductive technologies; therefore, serious consideration is warranted when selecting a product for clinical use.