Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media

1992 ◽  
Vol 31 (3) ◽  
pp. 189-194 ◽  
Author(s):  
J. A. Lawitts ◽  
John D. Biggers
Keyword(s):  
Sodium Chloride ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Joint Effects ◽  
Mouse Preimplantation Embryo

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p < 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p < 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

The composition of human preimplantation embryo culture media and their stability during storage and culture

2019 ◽  
Vol 34 (8) ◽  
pp. 1450-1461 ◽  
Author(s):  
M Tarahomi ◽  
F M Vaz ◽  
J P van Straalen ◽  
F A P Schrauwen ◽  
M van Wely ◽  
...  
Keyword(s):  
Amino Acids ◽  
Embryo Culture ◽  
Human Liver ◽  
Linear Models ◽  
Liver Enzymes ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Secondary Analysis ◽  
Success Rates ◽  
Expiry Date

Abstract STUDY QUESTION What is the composition and stability during storage and culture of fifteen commercially available human preimplantation embryo culture media? SUMMARY ANSWER No two culture media had the same composition, and both storage and culture had an effect on the concentrations of multiple components. WHAT IS KNOWN ALREADY The choice of embryo culture medium not only affects the success rate of an IVF treatment, but also affects the health of the future child. Exact formulations of embryo culture media are often not disclosed by manufacturers. It is unknown whether the composition of these media changes during storage or culture in the IVF laboratory. Without details on the exact concentrations, it is not possible to determine which components might be responsible for the differences in IVF success rates and health of the resulting children. STUDY DESIGN, SIZE, DURATION Between October 2014 and October 2015, all complete human preimplantation embryo culture media, i.e. ready to use for IVF, that were commercially available at that time, were included (n = 15). Osmolality and the concentration of thirty seven components including basic elements, metabolites, immunoglobulins, albumin, proteins and 21 amino acids were tested immediately upon arrival into the IVF laboratory, after three days of culture without embryos (sham culture) starting from the day of arrival, just before the expiry date, and after three days of sham culture just before the expiry date. PARTICIPANTS/MATERIALS, SETTING, METHODS Ions, glucose, immunoglobulins, albumin and the total amount of proteins were quantified using a combination of ion selective electrodes and photometric analysis modules, and lactate, pyruvate and 21 amino acids were analysed by ultra performance liquid chromatography mass spectrometry. Osmolality was analysed by an advanced micro-osmometer. Statistical analysis was done using multivariate general linear models. MAIN RESULTS AND THE ROLE OF CHANCE The composition varied between media, no two media had the same concentration of components. Storage led to significant changes in 17 of the 37 analyzed components (magnesium, chloride, phosphate, albumin, total amount of proteins, tyrosine, tryptophan, alanine, methionine, glycine, leucine, glutamine, asparagine, arginine, serine, proline, and threonine). Storage affected the osmolality in 3 of the 15 media, but for all media combined this effect was not significant (p = 0.08). Sham culture of the analyzed media had a significant effect on the concentrations of 13 of the 37 analyzed components (calcium, phosphate, albumin, total amount of proteins, tyrosine, alanine, methionine, glycine, leucine, asparagine, arginine, proline, and histidine). Sham culture significantly affected the osmolality of the analysed culture media. Two media contained 50% D-lactate, which a toxic dead-end metabolite. In a secondary analysis we detected human liver enzymes in more than half of the complete culture media. LIMITATIONS, REASONS FOR CAUTION The analyzed culture media could contain components that are not among the 37 components that were analyzed in this study. The clinical relevance of the varying concentrations is yet to be determined. WIDER IMPLICATIONS OF THE FINDINGS The presence of D-lactate could be avoided and the finding of human liver enzymes was surprising. The wide variation between culture media shows that the optimal composition is still unknown. This warrants further research as the importance of embryo culture media on the efficacy and safety in IVF is evident. Companies are urged to fully disclose the composition of their culture media, and provide clinical evidence supporting the composition or future changes thereof. STUDY FUNDING/COMPETING INTEREST(S) None.

pH stability of human preimplantation embryo culture media: effects of culture and batches

2018 ◽  
Vol 37 (4) ◽  
pp. 409-414 ◽  
Author(s):  
Majid Tarahomi ◽  
Annemieke A de Melker ◽  
Madelon van Wely ◽  
Geert Hamer ◽  
Sjoerd Repping ◽  
...  
Keyword(s):  
Media Effects ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Ph Stability

The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro

2002 ◽  
Vol 14 (7) ◽  
pp. 443 ◽  
Author(s):  
F. Figueiredo ◽  
G. M. Jones ◽  
G. A. Thouas ◽  
A. O. Trounson
Keyword(s):  
Culture Medium ◽  
Preimplantation Embryo ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Sulfate Proteoglycan ◽  
Preimplantation Embryo Development ◽  
Cell Numbers ◽  
Mouse Preimplantation Embryo

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte–macrophage colony-stimulating factor.

The proteome of human Fallopian tube lavages during the phase of embryo transit reveals candidate proteins for the optimization of preimplantation embryo culture

2020 ◽  
Author(s):  
D T Fujii ◽  
E Yohannes ◽  
E D Por ◽  
L Gillette ◽  
R D Beesley ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Fallopian Tube ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Development Rate ◽  
Protein Abundance ◽  
Menstrual Phase ◽  
Blastocyst Development ◽  
Human Fallopian Tube

Abstract STUDY QUESTION Are there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media? SUMMARY ANSWER The human tubal lavage proteome during the ES phase relative to the menstrual phase reveals substantial differential protein abundance in pathways such as glycolysis, redox homeostasis and activation of 14-3-3 zeta-mediated signaling. WHAT IS KNOWN ALREADY The Fallopian tube is uniquely suited to the development of the preimplantation embryo as it transits the tube during the ES phase of the menstrual cycle. Euploid cleavage-stage embryo arrest may reflect incomplete recapitulation of in-vivo conditions by current media formulations. STUDY DESIGN, SIZE, DURATION Proteome-wide analysis of distal tubal lavage specimens collected from 26 healthy women undergoing open microtubal anastomosis surgery from January 2013 to January 2018 was performed. Specimens were grouped by menstrual cycle phase in order to analyze phase-specific differences in protein abundance. For the murine embryo assay, single-cell embryos (N = 482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5 days for the assessment of blastocyst development. PARTICIPANTS/MATERIALS, SETTING, METHODS Human tubal lavage specimens were processed for label-free mass spectrometry. Reported menstrual cycle day was confirmed by measuring serum hormones. Key protein targets in the ES phase were validated via immunoblot. The ES phase-specific increase in 14-3-3 zeta protein was confirmed via ELISA of conditioned media obtained from primary human Fallopian tube epithelial cell culture. A murine embryo assay was performed to investigate the impact of graduated concentrations of 14-3-3 zeta on the blastocyst development rate. MAIN RESULTS AND THE ROLE OF CHANCE Comparison of the ES and menstrual phase human tubal lavage proteomes revealed 74 differentially expressed proteins with enrichment of pathways and biological processes involved in the regulation of carbohydrate metabolism, oxidative stress and cell survival. The adapter-regulator protein 14-3-3 zeta was among the most significantly increased in the ES phase. Supplementation of embryo culture media with 14-3-3 zeta at concentrations tested did not significantly improve the murine blastocyst development. LIMITATIONS, REASONS FOR CAUTION Although select associations were recapitulated in the conditioned media from sex steroid exposed primary human tubal epithelial cells, cell culture represents an in-vitro approximation. Changes to embryo culture media, such as protein supplementation, must undergo rigorous preclinical safety testing prior to adoption for human use. WIDER IMPLICATIONS OF THE FINDINGS This study represents the first description of the human Fallopian tube lavage proteome across the menstrual cycle, revealing a unique proteomic signature during the ES phase. Although supplementation of culture media with 14-3-3 zeta at appropriate concentrations showed no significant impact on the murine blastocyst development rate, other biologically plausible candidate proteins for individual or high throughput testing strategies are identified. STUDY FUNDING/COMPETING INTEREST(S) This work was funded in part by an Army Medical Department Advanced Medical Technology Initiative grant from the United States Army Medical Research and Materiel Command’s Telemedicine and Advanced Technology Research Center. There are no competing interests. TRIAL REGISTRATION NUMBER N/A

Sign in / Sign up

Export Citation Format

Share Document