Introduction of a single step medium from fertilization through blastocyst stage by supplementation of the culture media with L -Carnitine

The nonhuman primate (NHP) is a valuable translational model for human health studies and is widely used to investigate pre-implantation embryo development. Central to these investigations is the dependency on in vitro embryo culture (IVC). Since 2001, the single-step hamster embryo culture medium (HECM) has been the accepted standard for NHP IVC. With recent advances in formula optimization for IVC in human clinics, a re-examination of optimal NHP IVC media is warranted. Thus, two types of commercially available IVC media routinely used in human applications were compared with HECM-9: Global (single-step; LifeGlobal Group, Guilford, CT, USA), and Quinns Advantage (sequential; SAGE, Trumbull, CT, USA). Normally cycling, adult rhesus monkeys (n = 3) underwent controlled ovarian stimulations, and follicles were aspirated via laparoscope. Recovered ova were fertilized in vitro and the resultant zygotes (n = 138) were cultured for 9 days in HECM-9, Global, or Quinns with 10% protein supplement at 37.5°C in humidified tri-gas (6% CO2, 5% O2, and 89% N). Single-step media (HECM-9 and Global) were refreshed every two days while embryos were cultured for Days 1–3 in Quinns Advantage Cleavage medium without being replaced and in Quinns Advantage Blastocyst medium for Days 4–9 with medium changes every 2 days. Embryos were observed for cleavage, compaction, and blastocyst development. Proportional data with equal variance and normal distribution were analysed by one-way ANOVA, and significance was determined post-hoc by the Holm-Sidak method with P < 0.05. Developmental stage data ± s.e.M are presented in Table 1; a change in superscript indicates a significant difference within the column. There was no difference in embryonic cleavage or morula compaction between the three culture media evaluated, indicating no obvious differences in their effects on embryonic development 1 to 3 days after fertilization. However, a greater proportion of blastocysts developed in Global medium compared with HECM-9, and though it was not statistically different, embryos cultured in Global tended to reach the blastocyst stage more frequently than those in Quinns. Although not significant due to large variances in each group, blastocyst expansion also tended to occur more frequently in Global medium than in HECM-9 or Quinns. Taken together, these data indicate that single-step Global is as supportive of early embryonic development as HECM-9 but is better formulated to facilitate later stage differentiation and would be better suited for use in updated standard NHP IVC protocols. Table 1.Cleavage, compaction, blastocysts, and expansion of embryos in HECM-9, Global, and Quinns media

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Quality of embryos on day 7 after medium refreshment on day 6: a prospective trial

Human Reproduction ◽

10.1093/humrep/deab038 ◽

2021 ◽

Vol 36 (5) ◽

pp. 1253-1259

Author(s):

Iris G Insogna ◽

Andrea Lanes ◽

Elizabeth S Ginsburg ◽

Catherine Racowsky

Keyword(s):

Adverse Effect ◽

Continuous Medium ◽

Culture Media ◽

Prospective Trial ◽

Blastocyst Stage ◽

Single Step ◽

Fresh Medium ◽

Preimplantation Genetic Testing ◽

Younger Women ◽

Competing Interest

Abstract STUDY QUESTION Are embryos that fail to meet biopsy or freezing criteria on day 6 (D6) more likely to meet these criteria on day 7 (D7) if cultured in fresh medium from D6 to D7? SUMMARY ANSWER Refreshment of medium on D6 did not increase the proportion of usable embryos on D7, with an adverse effect for women ≥40 years old. WHAT IS KNOWN ALREADY Embryo development in continuous single-step medium, from fertilization to the blastocyst stage, is equivalent to that using a sequential media protocol. However, there remains a theoretical benefit of refreshing the culture environment by transitioning slowly developing D6 embryos to a fresh medium droplet of the same composition, with a renewed source of nutrients and a milieu free of metabolic toxins. STUDY DESIGN, SIZE, DURATION This was a prospective trial of culture media exposure in which embryos were randomized on D6 to remain in the same culture medium from D3 to D7 (continuous, n = 620) or be moved to fresh medium (fresh, n = 603) on D6, with re-evaluation on D7. Data were collected from IVF cycles, with or without ICSI, between 29 March 2019 and 17 February 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos from 298 women, aged 18–44 years, from cycles with or without preimplantation genetic testing (PGT) that did not meet criteria for biopsy and/or freeze on D6 were included in the study. Embryos were only included if there was a minimum of two embryos meeting the inclusion criteria in any cohort. Only the first cycle undertaken by each woman in the study period from which embryos were randomized was included. MAIN RESULTS AND THE ROLE OF CHANCE A total of 1254 embryos were randomized from 312 cycles (209 non-PGT and 103 PGT) including 200 women undergoing IVF without PGT and 98 women who underwent PGT. The proportion of usable blastocysts on D7 did not differ between groups: 10.1% (61/603) in fresh versus 9.7% (60/620) in continuous medium (relative risk (RR) 1.05, 95% CI 0.74–1.47)). Embryos from women ≥40 years old had a significantly decreased likelihood of achieving a usable blastocyst on D7 after culture in fresh versus continuous medium: 3.5% versus 12.2%; RR 0.29, 95% CI 0.08–0.98. In total, 9.9% of embryos otherwise discarded on D6 met the criteria for biopsy and/or freeze on D7. LIMITATIONS, REASONS FOR CAUTION Future work investigating implantation, clinical pregnancy and miscarriage rates with D7 embryos is still needed. WIDER IMPLICATIONS OF THE FINDINGS Refreshment of medium on D6 did not increase the proportion of usable embryos on D7 overall. Younger women were more likely to develop D7 embryos after refreshment of medium on D6, while an adverse effect was seen in women ≥40 years old. However, by extending the culture of embryos to D7, additional blastocysts become available for clinical use. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided through the Department of Obstetrics and Gynecology at Brigham and Women’s Hospital. I.G.I. works with Teladoc Health. A.L. has no disclosures. E.S.G. works as a consultant for Teladoc Health, and a writer and editor for UpToDate and BioMed Central. C.R. is a board member of the American Society for Reproductive Medicine and works with UpToDate. TRIAL REGISTRATION NUMBER N/A.

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P–168 RCT comparing the effect of Continuous ( Single Step ) embryo culture system versus a Sequential embryo culture system on the outcome of IVF/ICSI cycles

Human Reproduction ◽

10.1093/humrep/deab130.167 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

R Singh ◽

M Singh

Keyword(s):

Embryo Culture ◽

Culture System ◽

Culture Media ◽

Poor Quality ◽

Blastocyst Stage ◽

Single Step ◽

Blastocyst Formation ◽

Cleavage Stage ◽

Media Systems ◽

Single Medium

Abstract Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

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Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657308 ◽

1983 ◽

Vol 49 (01) ◽

pp. 024-027 ◽

Cited By ~ 20

Author(s):

David Vetterlein ◽

Gary J Calton

Keyword(s):

Monoclonal Antibody ◽

Light Chain ◽

Culture Media ◽

Biological Fluids ◽

Single Step ◽

High Yield ◽

Step Method ◽

Commercial Preparation ◽

E Coli ◽

Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

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P–174 Globulin-rich protein supplements improve blastulation efficiency in culture and promote implantation in vitro

Human Reproduction ◽

10.1093/humrep/deab130.173 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

S Ojosnegros ◽

A Seriola ◽

E Aroca ◽

A Godeau ◽

D Denkova ◽

...

Keyword(s):

Molecular Markers ◽

Culture Media ◽

Blastocyst Stage ◽

Reproductive Age ◽

Human Embryos ◽

Protein Supplement ◽

Age Cohorts ◽

The Matrix ◽

Basal Media ◽

Post Implantation

Abstract Study question Can globulin-rich compared to albumin (HSA) supplements improve blastulation and support embryo development towards post implantation? Summary answer Yes, globulin supplements with clinical-grade quality increase blastulation efficiency by 20% (50% in older mothers) and support the transition of embryos towards post-implantation development. What is known already During embryonic development at the morula stage there is a metabolic transition towards glycolysis as demand from outsourced energy increases. Therefore as cleavage proceeds, the demand for nutrients in the embryo increases accordingly. With few exceptions, HSA from human plasma or recombinant origin has been the main an only protein supplement used in almost all IVF-procedures. Globulin rich supplements are available but their use is not widespread and little is known about their efficiency in post-implantation development. Study design, size, duration We have cultured more than 600 mouse embryos in continuous media containing a protein supplement#1 (PS#1), from 1-cell up to blastocyst stage. At blastocyst stage embryos were replaced into fresh media containing protein supplement#2 (PS#2). The embryos were allowed to hatch naturally and then transferred into a proprietary matrix for further development and implantation for an additional 48h. Participants/materials, setting, methods: The blastulation rate, measured for HSA-supplemented embryo cohort was compared with embryos cultured in PS#1. Hatching efficiency was reported for embryos cultured in transfer media including PS#2. Once embedded in the matrix, advanced label-free imaging techniques and custom algorithms to measure matrix implantation strength were used. Key molecular markers (i.e. OCT4, CDX2) for correct post-implantation lineage patterning were documented by conventional 3D confocal immunofluorescence imaging. Main results and the role of chance Embryos supplemented with PS#1 reached blastocyst with overall 21% higher efficiency than embryos supplemented by HSA. When separated by age cohorts, embryos obtained from older females (ex-colony breeders, >14 weeks old) reached blastocyst stage with 55% higher efficiency than the same type of embryos cultured in the presence of HSA. Embryos obtained from females at optimal reproductive age reached blastocyst stage 10% more efficiently under PS#1 supplementation than with HSA. Hatching efficiency was 45% higher for embryos cultured with PS#2 than embryos supplemented with HSA. For every variable tested (e.g.% of arrested or degenerated embryos) or condition implemented (e.g. mouse basal media, human basal media from different brands, etc.) PS#1 and PS#2 outperformed, without exception, the supplementation with HSA. When embedded in the implantation matrix, the embryos cultured with PS#1 (cleavage) and transferred to PS#2 at blastocyst stage showed a remarkable implantation ability as measured by trophoblast outgrowth and matrix deformations. The embryos in PS#2 medium exerted stronger force into the matrix and also survived longer times than the embryos in HSA. PS#2 supported the transition of blastocyst towards post-implantation stages of development showing the correct lineage patterning of embryonic and extraembryonic molecular markers, including Oct4, CDx2, EOMES or GATA4. Limitations, reasons for caution This is a study based on an animal model. These observations need to be confirmed by ongoing experiments with human embryos. Wider implications of the findings: This work constitutes a proof-of-concept for the use of globulin-rich supplements as higher performance substitute of albumin in the culture of IVF embryos, both as (i) a standard protein source for culture media and (ii) as a supplement for transfer media to capacitate the embryo for implantation. Trial registration number Not applicable

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160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab160 ◽

2004 ◽

Vol 16 (2) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

W.F. Swanson ◽

A.L. Manharth ◽

J.B. Bond ◽

H.L. Bateman ◽

R.L. Krisher ◽

...

Keyword(s):

Culture Media ◽

Ionic Composition ◽

Cell Number ◽

Blastocyst Stage ◽

Domestic Cat ◽

Embryo Viability ◽

Blastocyst Development ◽

In Vitro Development ◽

Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

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P–187 Multinucleation and reverse cleavage, sings of early embryo self-repair mechanisms

Human Reproduction ◽

10.1093/humrep/deab130.186 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Munuer. Puigvert ◽

V. Montalv Pallès ◽

J Mass. Hernáez ◽

A García-Faura ◽

B Marquè. López-Teijón ◽

...

Keyword(s):

Embryo Development ◽

Culture Media ◽

Live Birth Rate ◽

Time Lapse ◽

Blastocyst Stage ◽

Early Embryo ◽

Control Group ◽

Complete Fusion ◽

Depth Analysis ◽

Self Repair

Abstract Study question Have multinucleation and reverse cleavage any effect on embryo development and clinical outcomes on IVF treatments? Summary answer Embryos capable of repairing dysmorphisms and developing up to blastocyst stage keep intact their ability to become healthy babies. What is known already Time-lapse systems allow IVF laboratories to perform in-depth analysis of embryo development using the continuous monitoring tool. Some events that are impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered a negative factor when the embryo quality assessment is performed. However, it has been described the possibility that embryos have self-repair intrinsic methods. Study design, size, duration Retrospective study including data from 3,577 cycles with 21,274 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to day 5/6 between 2014 and 2019. Participants/materials, setting, methods Three embryo groups were considered: Control group, embryos without multinucleation or reverse cleavage (CG; n = 16,897); Multinucleation group, embryos with at least one blastomere multinucleated on D + 2/3 (MNC; n = 3,879) and Reverse Cleavage group, embryos undergoing complete fusion of two blastomeres on D + 2/3 (RC; n = 498). Single embryo transfer was performed on blastocyst stage. Clinical outcome rates were compared between groups and analyzed by Chi-square test. Main results and the role of chance As published by other groups, the 2.3% of our embryos showed at least one reverse cleavage event and we observed multinucleation in the 18.2% of the embryos. Blastocyst rate of dysmorphism groups was significantly lower (p < 0.05) than Control group (MNC=20.0%; RC = 27.7%; CG = 58.0%). Once transferred, MNC and RC evolutive embryos showed significantly lower pregnancy (MNC=47.9%; RC = 46.8%; CG = 60.8%; p < 0.05) and clinical pregnancy rates (MNC=39.4%; RC = 40.4% CG = 50.6%; p < 0.05) than the Control group (p < 0.05). However, during the post-implantational development the negative effect of dysmorphisms disappears, reaching values of live birth rate comparable to the Control group (MNC=28.3%; RC = 31.9% CG = 33.8%; p = 0.17). These results prove the importance of blastocyst culture and the inherent capability of the embryos to overcome some abnormal dynamics as multinucleation and reverse cleavage. Thus, these embryos showing the poor-prognosis events can be considered for transfer or vitrify. Limitations, reasons for caution There is a wide difference on sample size between groups despite the fact that the statistical analysis considers that into account. There are some ongoing pregnancies in all groups. Wider implications of the findings: When analyzing the development of embryos undergoing reverse cleavage and multinucleation, we hypothesize that these embryos could be showing a self-correction mechanism for some type of error detected. Embryos capable of repairing and developing up to blastocyst stage keep intact their ability to become healthy babies. Trial registration number Not applicable

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P–561 Male and female blastocyst: any difference other than the sex?

Human Reproduction ◽

10.1093/humrep/deab130.560 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

B Carrasc. Canal ◽

M C Pons ◽

M Parriego ◽

M Boada ◽

S García ◽

...

Keyword(s):

Embryo Transfer ◽

Sperm Quality ◽

Time Lapse ◽

Blastocyst Stage ◽

Study Data ◽

Single Step ◽

Chromosomal Anomalies ◽

Low Oxygen ◽

Male And Female ◽

Normal Copy Number

Abstract Study question Is there any imbalance in the sex ratio (SR) and in the aneuploidy rate of male and female human blastocysts from a PGT-A programme? Summary answer Although SR in human blastocysts is significantly male-biased, more aneuploidies are observed among male blastocysts, resulting in comparable euploid male and female embryos available. What is known already More boys than girls are born worldwide, meaning that the SR at birth is biased towards males. Differences in the SR of children born after ART have been also reported. Factors such as the insemination technique or the day of embryo transfer have been shown to be related to the SR at birth, but whether the SR is shifted during the preimplantation and/or postimplantation development remains unknown. Study design, size, duration: Embryos from patients undergoing 921 PGT-A cycles from September 2017 to February 2020 were included in the study. Data from the chromosomal constitution of 2637 biopsied blastocysts was retrospectively analysed. Participants/materials, setting, methods Embryos were cultured in time-lapse incubators with low oxygen tension (5%) (Embryoscope®; Geri®) using single-step medium (Global®, LifeGlobal®; GTL™, Vitrolife). Blastocyst biopsy was performed between D5-D7 followed by immediate vitrification (Cryotop®, Kitazato). Trophectoderm samples were analysed by NGS. Embryos were categorized as euploid, aneuploid or mosaic. Embryos were called as mosaic when the deviation from the normal copy number was ≥30% and <70%. Main results and the role of chance Overall biopsies from 2637 blastocysts were analysed, 1320 on day 5 (50.1%), 1169 on day 6 (44.3%) and 148 on day 7 (5.6%). Sex distribution among the embryos analysed was skewed in favor of male sex with 1401 diagnosed as male (53.1%) and 1236 were female (46.9%), [OR (95%CI):1.13(1.05–1.22)]. As a consequence of this biased SR, more male embryos reached the blastocyst stage and were biopsied both on day 5/6 (708/1320, 53.6% on day 5 and 619/1169, 53% on day 6). Embryos biopsied on day 7 were balanced between sexes with 50% being male and 50% being female. Following biopsy and PGT-A, 1086 (41.2%) of the embryos were classified as euploid, 1349 (51.16%) as aneuploid, and 202 (7.7%) as mosaic embryos. More chromosomal anomalies were observed among male blastocysts when compared to the female ones, 738 (52.7%) vs 611 (49.4%). Similarly, mosaicism was more frequents in male as compared with female blastocysts, 123 (8.8%) vs 79 (6.4%). (P = 0.000). As more aneuploidies are observed among male blastocysts, the final number of available euploid blastocysts for embryo transfer was comparable between sexes (540 male/546 female), [OR (95%CI): 0.99 (0.87–1.11)]. Limitations, reasons for caution This is a retrospective study. Only embryos at the blastocyst stage have been analyzed. Potential confounding factors such as sperm quality or the female age have not been analyzed. No data regarding the SR at birth have been analyzed in these study. Wider implications of the findings: In our study, more male embryos develop to the blastocyst when compared to female ones. It can be hypothesized that female embryos can be more affected by an early arrest at cleavage stages. SR at birth would be expected to be similar as more aneuploidy is observed in male embryos. Trial registration number Not applicable

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MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

International Journal of Molecular Sciences ◽

10.3390/ijms21238888 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8888

Author(s):

Bárbara Melo-Baez ◽

Yat S. Wong ◽

Constanza J. Aguilera ◽

Joel Cabezas ◽

Ana C. F. Mançanares ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fatty Acid Biosynthesis ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Target Cells ◽

Bovine Embryos ◽

Developmental Potential ◽

Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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In vitro culture and non-invasive metabolic profiling of single bovine embryos

Reproduction Fertility and Development ◽

10.1071/rd17446 ◽

2019 ◽

Vol 31 (2) ◽

pp. 306

Author(s):

Monika Nõmm ◽

Rando Porosk ◽

Pille Pärn ◽

Kalle Kilk ◽

Ursel Soomets ◽

...

Keyword(s):

Molecular Weight ◽

Metabolic Profiling ◽

Culture Medium ◽

Culture Media ◽

Blastocyst Stage ◽

Growth Media ◽

Low Molecular Weight ◽

Bovine Embryos ◽

Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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