P–203 Applying artificial intelligence for ploidy prediction: The concentration of IL–6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Aparici. Ruiz ◽  
L Bori ◽  
E Paya ◽  
M A Valera ◽  
A Quiñonero ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
General Description ◽  
Ann Model ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Testing Dataset ◽  
Standard Deviations ◽  
The Impact

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL–6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL–6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL–6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL–6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL–6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL–6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL–6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL–6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL–6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL–6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings: The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number Not applicable

P-203 Applying artificial intelligence for ploidy prediction: The concentration of IL-6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Aparicio Ruiz ◽  
L Bori ◽  
E Paya ◽  
M A Valera ◽  
A Quiñonero ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
General Description ◽  
Ann Model ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Testing Dataset ◽  
Standard Deviations ◽  
The Impact

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL-6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL-6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL-6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL-6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL-6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL-6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL-6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL-6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL-6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL-6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number not applicable

scholarly journals Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?

2019 ◽  
Vol 26 (1) ◽  
pp. 16-42 ◽  
Author(s):  
Megan Leaver ◽  
Dagan Wells
Keyword(s):  
Genetic Testing ◽  
Clinical Diagnosis ◽  
Embryo Culture ◽  
Culture Media ◽  
Genetic Material ◽  
Preimplantation Embryos ◽  
Original Research ◽  
Preimplantation Genetic Testing ◽  
Non Invasive ◽  
The Impact

Abstract BACKGROUND Preimplantation genetic testing (PGT) encompasses methods that allow embryos to be tested for severe inherited conditions or for chromosome abnormalities, relevant to embryo health and viability. In order to obtain embryonic genetic material for analysis, a biopsy is required, involving the removal of one or more cells. This invasive procedure greatly increases the costs of PGT and there have been concerns that embryo viability could be compromised in some cases. The recent discovery of DNA within the blastocoele fluid (BF) of blastocysts and in spent embryo culture media (SCM) has led to interest in the development of non-invasive methods of PGT (niPGT). OBJECTIVE AND RATIONALE This review evaluates the current scientific evidence regarding non-invasive genetic assessment of preimplantation embryos. The success of different PGT methodologies in collecting and analysing extra-embryonic DNA is evaluated, and consideration is given to the potential biological and technical hindrances to obtaining a reliable clinical diagnosis. SEARCH METHODS Original research and review papers concerning niPGT were sourced by searching PubMed and Google Scholar databases until July 2019. Searches comprised the keywords: ‘non-invasive’; ‘cell-free DNA’; ‘blastocentesis’; ‘blastocoel fluid’; ‘spent culture media’; ‘embryo culture medium’; ‘preimplantation genetic testing’; ‘preimplantation genetic diagnosis’; ‘preimplantation genetic screening’; and ‘aneuploidy’. OUTCOMES Embryonic DNA is frequently detectable in BF and SCM of embryos produced during IVF treatment. Initial studies have achieved some success when performing cytogenetic and molecular genetic analysis. However, in many cases, the efficiency has been restricted by technical complications associated with the low quantity and quality of the DNA. Reported levels of ploidy agreement between SCM/BF samples and biopsied embryonic cells vary widely. In some cases, a discrepancy with respect to cytogenetic data obtained after trophectoderm biopsy may be attributable to embryonic mosaicism or DNA contamination (usually of maternal origin). Some research indicates that aneuploid cells are preferentially eliminated from the embryo, suggesting that their DNA might be over-represented in SCM and BF samples; this hypothesis requires further investigation. WIDER IMPLICATIONS Available data suggest that BF and SCM samples frequently provide DNA templates suitable for genetic analyses, offering a potential means of PGT that is less expensive than traditional methods, requires less micromanipulation skill and poses a lower risk to embryos. Critically, DNA isolation and amplification protocols must be optimised to reproducibly obtain an accurate clinical diagnosis, whilst minimising the impact of confounding factors such as contamination. Further investigations are required to understand the mechanisms underlying the release of embryonic DNA and to determine the extent to which this material reflects the true genetic status of the corresponding embryo. Currently, the clinic al potential of niPGT remains unknown.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Biricik ◽  
V Bianchi ◽  
F Lecciso ◽  
M Surdo ◽  
M Manno ◽  
...  
Keyword(s):  
Data Analysis ◽  
Embryo Culture ◽  
Culture Media ◽  
Embryo Biopsy ◽  
Non Invasive ◽  
Culture Time ◽  
Ngs Data Analysis ◽  
High Level ◽  
Ngs Data

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (>84%) of concordance rate for ploidy and sex, sensitivity (>88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (>5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

scholarly journals Modern Methods for Assessment of the Implantation Potential of Embryos in Assisted Reproductive Programs

Doctor Ru ◽  
2021 ◽  
Vol 20 (8) ◽  
pp. 12-18
Author(s):  
G.V. Savostina ◽  
◽  
S.G. Perminova ◽  
A.V. Timofeeva ◽  
M.A. Veyukova ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
High Sensitivity ◽  
Visual Assessment ◽  
Non Invasive ◽  
Non Coding Rna ◽  
Study Results ◽  
Modern Methods ◽  
Invasive Method ◽  
Aneuploidy Testing

Objective of the Review: To analyse the modern methods for assessment of the implantation potential of embryos in assisted reproductive programs. Key Points. We present the study results for selection of a most optimal embryo for transfer, using visual assessment of embryo quality, preimplantation genetic aneuploidy testing, analysis of metabolomic, proteomic, transcriptomic profiles of culture media and embryo blastocele. We have paid special attention to assessment of small non-coding RNA (sncRNA) in embryo culture medium. Conclusion. Due to the high sensitivity, objectivity and biomarker resistance to degradation, the most promising non-invasive method to assess the implantation potential of an embryo is analysis of the sncRNA profile in embryo culture media. Keywords: aneuploidy, pre-implantation genetic testing, small non-coding RNAs, proteomic analysis, metabolomic analysis.

The Impact of Two Embryo Culture Media, SOF and Commercial BO, on Pre- and Post-Implantation Development of Cloned Sannen Goat Embryos

2020 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohsen Rahimi ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Specific Gene ◽  
Limited Information ◽  
Major Drawback ◽  
Animal Cloning ◽  
The Impact ◽  
Post Implantation

Abstract Background: The ingredients of embryo culture media developed by different companies are disclosed. Thus, it is impossible to determine which ingredients might be responsible for differences in pre-and post-implantation embryo development. To address this gap, we performed an experiment to compare two embryo culture media, namely, SOF and commercial BO, on pre- and post-implantation development of cloned Sannen goat embryos. Cumulus oocyte complexes derived from slaughterhouse ovaries were used for in vitro embryo production . In vitro development of IVF, parthenogenetic and SCNT embryos were assessed in both BO and SOF media. The expression of 16 genes, including AKT , OCT4 , SOX2 , BMPR1 , FGFR4 , CDC25 , CDX2 , GCN5 , PCAF , FOXD3 , SMAD5 , FZD , LIFR1 , CTNNB , ERK1 , and IFNT , belonging to 7 important pathways, i.e. pluripotency, FGF, TGFβ, cell cycle and proliferation, histone transferase, trophectoderm, and WNT, were examined in the goat SCNT and IVF blastocysts from both BO and SOF media. Results: The blastocyst rate in BO medium was significantly higher than that of the SOF medium in SCNT embryos ( P < 0.05). All of the genes examined showed increased expression levels in SCNT embryos compared to IVF embryos. In the IVF group, OCT4 , BMPR1 , and GCN5 showed significantly higher expression in the SOF medium compared to the BO medium. In this group, AKT , FGFR4 , SOX2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, FGFR4 , GCN5 , FZD , CTNNB , BMPR1 , and FGFR4 showed significantly higher expression in SOF medium compared to BO medium. In vivo development did not differ significantly between the two groups. Conclusions: Based on these results, we concluded that the limited information available on the allocations of ICM and TE cells in SCNT embryos and embryo-specific gene expression may be the major drawback IVC medium and an impediment to successful animal cloning.

scholarly journals The impact of selected embryo culture conditions on ART treatment cycle outcomes: a UK national study

2020 ◽  
Vol 2020 (1) ◽  
Author(s):  
Catherine M Castillo ◽  
Joyce Harper ◽  
Stephen A Roberts ◽  
Helen C O’Neill ◽  
Edward D Johnstone ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Wald Test ◽  
Culture Conditions ◽  
Oxygen Level ◽  
Success Rates ◽  
Blastocyst Culture ◽  
Clinic Site ◽  
Medium Type

Abstract STUDY QUESTION Are selected embryo culture conditions namely media, oxygen level, and incubator type, associated with IVF live birth rate (LBR) and the health of singleton offspring at birth? SUMMARY ANSWER There were statistically significant differences in LBR between the eight culture media systems analysed; however, none of the embryo culture factors showed statistically significant associations with birth weight (BW) in multivariable regression analyses. WHAT IS KNOWN ALREADY In clinical ART culture media is the initial environment provided for the growth of human embryos. Pre-implantation development is a critical period of developmental plasticity, which could have long-lasting effects on offspring growth and health. Although some studies have shown an impact of culture medium type on BW, the interaction between culture medium type and associated culture conditions on both treatment success rates (LBR) and offspring BW is largely unexplored. This study aimed to examine these factors in a large multicentre national survey capturing the range of clinical practice. STUDY DESIGN, SIZE, DURATION In this cross-sectional study, data from a survey circulated to all UK IVF clinics requesting information regarding culture medium type, incubator type, and oxygen level used in ART between January 2011 and December 2013 were merged with routinely recorded treatment and outcome data held in the Human Fertilisation and Embryology Authority Register up to the end of 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS Forty-six (62%) UK clinics responded to the survey. A total of 75 287 fresh IVF/ICSI cycles were captured, including 18 693 singleton live births. IVF success (live birth, singleton or multiple; LB), singleton gestation and singleton gestation-adjusted BW were analysed using logistic and linear regression models adjusting for patient/treatment characteristics and clinic-specific effects. MAIN RESULTS AND THE ROLE OF CHANCE Culture medium type was shown to have some impact on LBR (multivariable logistic regression, (MRL); post-regression Wald test, P &lt; 0.001), but not on BW (MLR; post-regression Wald test, P = 0.215). However, blastocyst culture had the largest observed effect on odds of LBR (odds ratio (OR) = 1.35, CI: 1.29–1.42), increased the risk of pre-term birth even when controlling for oxygen tension (MLR; OR = 1.42, CI: 1.23–1.63), and gestation-adjusted BW (MLR, β = 38.97 g, CI: 19.42–58.53 g) when compared to cleavage-stage embryo culture. We noted a very strong effect of clinic site on both LBR and BW, thus confounding between treatment practices and clinic site may have masked the effect of culture conditions. LIMITATIONS, REASONS FOR CAUTION Larger datasets with more inter-centre variation are also needed, with key embryo culture variables comprehensively recorded in national treatment registries. WIDER IMPLICATIONS OF THE FINDINGS This study is the largest investigation of laboratory environmental effects in IVF on both LBR and singleton BW. Our findings largely agree with the literature, which has failed to show a consistent advantage of one culture media type over another. However, we noted some association of LBR with medium type, and the duration of embryo exposure to laboratory conditions (blastocyst culture) was associated with both LBR and singleton health at birth. Because of the strong effect of clinic site noted, further randomized controlled trials are needed in order to reliably determine the effect of embryo culture on IVF success rates and the growth and health of subsequent offspring. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the EU FP7 project grant EpiHealthNet (FP7-PEOPLE-2012-ITN -317 146). The authors have no competing interests to declare.

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

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.

80 DEVELOPMENT OF A SYNTHETIC MEDIUM FOR THE IN VITRO CULTURE OF BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 154 ◽  
Author(s):  
D. Moreno ◽  
A. Neira ◽  
L. Dubreil ◽  
L. Liegeois ◽  
S. Destrumelle ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Culture ◽  
Disease Transmission ◽  
Culture Medium ◽  
Culture Media ◽  
Synthetic Medium ◽  
Wilcoxon Test ◽  
Blastocyst Development ◽  
Negative Effect

In the majority of media for embryo culture, 2 of typical components used are FCS or BSA; however, the presence of FCS in the culture medium has been shown to have a negative effect on embryo quality and the use of animal-derived proteins in culture media increases the risks of disease transmission through in vitro embryo production. The aim of this study was to develop an in vitro embryo culture medium free from FCS and BSA, but with the addition of various growth factors and cytokines (GF-CYK: IGF-I, IGF-II, bFGF, LIF, GM-CSF) 50 ng mL–1 and (TGF-β1) 100 ng mL–1 supplemented with hyaluronan (HA) and recombinant albumin (RA). Bovine oocytes (n = 1043, 6 replicates) from abattoir ovaries were matured in TCM-199 medium with 60 μg mL–1 penicillin, 60 μg mL–1 streptomycin, and 10 ng mL–1 EGF for 24 h at 39°C and 5% CO2 in humidified air. Afterward, the oocytes were fertilized in IVF-TALP medium with 6 mg mL–1 fatty acid-free BSA and 1.7 IU mL–1 heparin for 18 h under the same conditions. After fertilization, presumptive zygotes were divided into two groups and cultured in 30 μL droplets of SOF supplemented with (1) 0.4% BSA + 5 μg mL–1 insulin, 5 μg mL–1 transferrin, and 5 ng mL–1 selenium (ITS) as a control; or (2) GF-CYK + 0.5 mg mL–1 HA + 0.15% RA (M1). Droplets were preserved under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Blastocyst development and blastocyst diameter was observed at 7 and 8 days post-fertilization (dpf). Developmental and diameter data were analysed using the Wilcoxon test by using R software. The blastocyst rates were not significantly different between the control and M1 medium: at 7 dpf (22.9% ± 4.8 and 30.2% ± 3.0), and at 8 dpf (29.6% ± 5.1 and 37.4% ± 2.0 respectively; P > 0.05). The blastocyst diameter obtained with the M1 medium was significantly greater (P < 0.05) than that of the control at 7 dpf (173.3 μm ± 4.9 and 157.2 μm ± 4.1, respectively); however, no significant differences were observed at 8 dpf (190.3 μm ± 5.2 and 179.7 μm ± 5.3, respectively). In conclusion, the FCS- and BSA-free medium with GF-CYK, HA, and RA (M1) showed a comparable development rate to the control medium at 7 and 8 dpf. These growth factors and cytokines in association with hyaluronan and recombinant albumin have a synergistic action by promoting an increase in the blastocyst diameter at 7 dpf. This is fully synthetic method of embryo culture; it presents a valuable tool to reduce the risks of disease transmission via embryo transfer.

136 ADDITION OF LOW DENSITY LIPOPROTEIN TO CHEMICALLY DEFINED PORCINE EMBRYO CULTURE MEDIUM CAN PARTIALLY REPLACE BOVINE SERUM ALBUMIN

2008 ◽  
Vol 20 (1) ◽  
pp. 148
Author(s):  
L. D. Spate ◽  
K. A. Walker ◽  
C. E. McHughes ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Blastocyst Stage ◽  
Differential Staining ◽  
Low Density ◽  
Cell Stage ◽  
Defined Culture

Embryo culture media typically contain undefined biologicals such as BSA. Our goal is to develop chemically defined culture media that are based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos at various stages of development and determined that the message for the low density lipoprotein receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of low density lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous–oocyte complexes (COC) identified, and the COC were matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in modified Tris buffered medium and cocultured with 0.25 � 106/mL frozen thawed porcine semen for 5 h. The presumptive zygotes were then transferred to either porcine zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 28 h, cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3 + 20 µg mL–1 LDL, 3. PZM4, 4. PZM4 + 10 µg mL–1 LDL, 5. PZM4 + 20 µg mL–1 LDL, 6. PZM4 + 50 µg mL–1 LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a–cP ≤ 0.05). The percentage blastocyst was 51.3 � 0.09a, 51.6 � 0.09a, 33.1 � 0.99c, 35.8 � 0.09c, 36.9 � 0.09c, and 41.3 � 0.06b, for treatments 1–6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 µg mL–1 of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts, and preliminary results suggest that the ICM to trophectoderm ratio in the high LDL treatment group is closer to the ratio found in in vivo produced embryos. This project was supported by USDA CSREES NRI (2006-35203-17282) and Food for the 21st Century.

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