Extended embryo culture and it potential

Extended Embryo Culture

1. IS EXTENDED EMBRYO CULTURE THE KEY TO IMPROVING IVF SUCCESS? UNDERSTANDING ITS POTENTIAL Dr P DURAI PhD Head, Embryology Krishna Institute of Medical Sciences Hyderabad

2. WHAT WE DISCUSS TODAY Past, Present and Future What is extened embryo culture Why we need extended culture system Warnock Report Factors Influencing Extended Culture Current Research and Future DATA set “The black box” Ethical, technological in extended embryo culture

3. Global IVF projections and success rates IVF success still averages ~30–40% per cycle globally. Variability in implantation and live birth Need for advanced embryo selection: precision over probability Can extended culture enhance embryo selection, improve implantation, and reduce multiple pregnancies?

4. What is Extended Embryo Culture? Standard vs. extended culture: Day 3 vs. Day 5–7 Extended culture allows embryos to grow for an additional 2–4 days. To improve the accuracy of selecting embryos To reduce the risk of multiple pregnancies As per Indian ART and Surrogacy Act, up to how many days can we culture? Why Such restriction?

5. 2011- Vienna Consensus: Blastocyst development as a key performance indicator Historical Overvi ew of Embryo Culture 1980S 1990S Cultu re limited to Day 2-3, cleavage stage embryos Ext ension to Day 5 Technological Advances Media Development

6. Why Day 5 is still Gold Standard Hanover and Tyke Vienna Consensus ASRM Recent Meta-Analysis Hanover and Tyke Considers blastocyst development rate as a Key Performance Indicator (KPI) in IVF, focusing on embryos developed by Day 5 Supports Day 5 and Day 6 blastocyst cryopreservation in laboratory practice. Found that Day 5 blastocysts are associated with better clinical outcomes compared to Day 6 blastocysts in frozen cycles, suggesting a decreased implantation potential for embryos cultured to Day 6.

7. What Day 7 Tells Us D7 Euploid Blastocysts show significantly compromised developmental potential when compared to D5 or D6 euploid blastocysts. Live Birth Rate for D7 euploid blastocysts is observed to be half of that of D5/D6 blastocysts, despite being chromosomally normal. Nutrient depletion, stress responses, and changes in epigenetic regulation during extended culture DNA methylation or histone modification, may occur during extended culture, influencing the gene expression of embryos the data suggests that extended culture to D7 for blastocyst biopsy can still be a viable clinical option for patients with poor prognosis Couns el patients appropriately regarding the risks and benefits of extended culture

8. DAY 7 OUTCOME Day 5 Day 5 Day 5 Ueno, et al (2020). Establishment of day 7 blastocyst freezing criteria using blastocyst diameter for single vitrified-warmed blastocyst transfer from live birth outcomes: a single-center, large cohort, retrospectively matched study. Journal of Assisted Reproduction and Genetics. 37. 1-9. 10.1007/s10815-020-01882-8. Day 5 Day 6 Niu, X., Wang, C.T., Li, R. et al. Is day 7 culture necessary for in vitro fertilization of cryopreserved/warmed human oocytes?. Reprod Biol Endocrinol 18, 4 (2020). https://doi.org/10.1186/s12958-020-0565-9 Day 6 Day 6 Day 2 Day 2 Day 7 Day 7

9. EPIGENETIC IMPACT: ARE WE CROSSING THE LINE? Study plan Chromosomal Analysis 30 high-quality Day 3 embryos donated after Day 3 blastomere biopsy (single-gene defect) All embryos cultured identically from Day 3 to Day 5 Whole-Genome Amplification + array CGH Results for Euploid Embryos Finding Developed to Blastocyst: 7 (58.3%) Arrested by Day 5: 12 (66.6%) Aneuploidy was not the primary cause of arrest Most arrested embryos were chromosomally normal Culture conditions and non-chromosomal factors play a significant role in embryo arrest Orvieto, et al (2022).

10. Extended Culture for Advanced Maternal Age Blastocyst formation decreases with age, but extended culture still allows better embryo selection but reduced live birth rates due to the high risk of chromosomal abnormalities   23–29 years 30–33 years 34–36  years 37–43   years Blastocyst transfer rate 86% 82% 83% 83% Implantation rate 47% 48% 39% 30% Clinical pregnancy rate per oocyte pick-up 40% 40% 34% 30% Miscarriage rate per clinical pregnancy 14% 14% 14% 23% Live birth rate per oocyte pick-up 33.5% 33% 29% 22% Multiple pregnancy rate per live birth 8% 8% 10% 13%

11. Developmental potential of D3 embryo with different blastomeres number by extended culture Group Embryo number Blastocyst formation rate (%) High quality blastocyst rate (%) Available blastocyst rate (%) 4–5 cells 12,426 28.17 9.77 7.11 6 cells 8148 41.73 12.59 12.98 7–9 cells 15,290 53.83 29.85 30.69  > 9 cells 3012 58.27 37.89 36.35 Group Cycles Clinical pregnancy rate (%) Implantation rate (%) Abortion rate (%) 4–5 cells 45 64.44 39.78 20.69 6 cells 82 73.17 48.54 23.33 7–9 cells 514 71.98 52.91 14.59  > 9 cells 87 81.61 66.67 7.04 Pregnancy outcomes of double blastocysts transfer after extended culture of D3 embryos with different blastomeres number Parameter Blastocyst ET n = 688 Cleavage ET n = 344 Female age (years) a 28.79 ± 5.12 28.90 ± 5.19 Total number of embryos (n) a 3.26 ± 1.6 3.16 ± 1.61 Clinical pregnancy rate (%) b 202/688 ( 29.3%) 92/344 ( 26.7%) Can we predict possible blastocyst conversion on day 3? Li B, et al 2021

12. Extended Culture for Advanced Maternal Age Blastocyst Formation Decline with Age Cell Organization Unaffected by Age Increased Risk of Miscarriage Reduced Risk of Multiple Pregnancies Reduced Live Birth Rates higher risk of chromosomal abnormalities Extended embryo culture with PGT-A Allows for higher quality embryo selection Extended Culture for Advanced Maternal Age 23-29 years 34-36 years 30-33 years 23-29 years 23-29 years 30-33 years 30-33 years 34-36 years 34-36 years 37-43 years 37-43 years

13. CASE STUDY DAY 3 DAY 4 DAY 5 Day 3 transfer may not always allow accurate embryo selection. Day 5 blastocyst transfer, despite fewer embryos, provided better implantation and birth outcomes due to natural selection in culture. Extended culture helped select the embryo with the highest implantation potential.

14. 01 02 03 04 05 Mechanisms Beh ind Extended Embryo Culture Mitochondrial Stress Leads to increased reactive oxygen species (ROS) Impairing embryo development Epigenetic Drift Histone modification and DNA methylation patterns may be altered Affect Gene expression Nutrient Depletion Extended time without fresh media may lead to shortages of nutrients Reduce embryo growth Gas/pH issues Affect cell metabolism ATP production Protein folding Cellular Arrest Due to cumulative stress metabolic epigenetic environmental

15. Decision Tree when to consider Extended Embryo Culture ≥4 good Embryos Yes Yes (≥ 35yrs) < 4 Embryos Extended culture Yes Few Embryos on day 5 Freeze/Transfer Yes- Consider day 6 or day 7 Consider day 3 transfer Need for PGT? Advanced maternal Age Culture to Day 5/6 for Biopsy Extended culture to improve embryo selection 2 Day 3 preferred when embryo numbers are low or if lab conditions are suboptimal Day 5 & 6 -higher implantation and Synchrony with endometrium Day 7 culture consider cautiously Required counselling Predictive AI can support in decision-making

16. we go to extreme lengths to select the ‘best’ embryo provide a more natural embryo environment Balance between blastocyst and cleavage stage embryo transfer

17. Sub mitted by Warnock Committee in 1984 Chairman: Dame Mary Warnock Enshrined in the UK’s Human Fertilisation and Embryology (HFEA) Act 1990, Th e committee investigated the social and ethical impact of new reproductive technologies Warnock Report / Where should draw the line

18. 14-Day Rule In most countries limit embryo culture to 14 days Rule regulates the invitro culture of human embryos for research and ART The primitive streak, a visible line of cells around the 15th day of embryo development. Cells differentiate and form Endoderm Mesoderm Ectoderm, for forming tissues and organs. Despite its widespread adoption, the 14-day rule remains a subject of debate Primitive streak risks the embryo experiencing pain and suffering Extension of the limit, for understanding early development and preventing miscarriages. To maintain ethical standards

19. “The black box” 14th and 28th day of embryo development Learn about the developing nervous system without any risk of neural connections being present Ethical Debate: Gain a better understanding of the early development of organs Should the 14-day rule be extended for better scientific understanding? Concerns the potential for human cloning

20. The black box opened Zernicka-Goetz et al. (2016) successfully cultured human embryos to Day 13. University of Cambridge

21. C urrent Research AI in Embryo Selection Predicting embryo quality using real-time data Synthetic Culture Systems Nutrient profiles to optimize embryo growth 3D Embryo Culture Exploring materials like Matrigel and Hydrogel Long-Term Health Outcomes Investigating epigenetic effects of extended culture extrusion-based, laser-assisted, and inkjet-based methods used in 3D Bioprinting.

22. Challenges and Controversies Embryo Arrest & Chromosomal Abnormalities Potential c oncerns about embryo discard policies Legal limits Extended culture requires m ore resources, raising costs Culture-Induced Stress impact on embryo health and gen e expression Biological Ethical Operational Physiological

23. Conclusion

24. Aspect Day 3 Day 5 Day 6 Day 7 Embryo Stage Cleavage stage Blastocyst Blastocyst Blastocyst Embryo Development Potential Moderate High Moderate to High Lower Live Birth Rate Lower compared to later stages Highest among all stages Lower than Day 5 Lowest among all stages Chromosomal Abnormalities More common in arrested embryos Lower risk of chromosomal anomalies Similar to Day 5 Higher risk of chromosomal abnormalities Nutrient Depletion Less concern Moderate Moderate High Risk of Embryo Arrest High Moderate Higher than Day 5 Very high Impact on Implantation Lower implantation potential Highest implantation potential Moderate Lower implantation potential Clinical Outcomes Less favorable in frozen cycles Better clinical outcomes Lower success in frozen cycles Much lower success, even if euploid Epigenetic Changes Minimal Minimal Moderate Significant potential for changes Usage Standard practice Ideal for embryo transfer Suitable for certain cases Suitable for poor prognosis cases Ethical Concerns Low Low Low Higher due to extended culture K ey aspects of embryo culture on Days 3, 5, 6, and 7

25. Enhances embryo choice, Improves implantation, R educes multiple pregnancies Continued research into advanced media and AI predictions A promising area for improving IVF outcomes Reuires individualized decicions based on clinical profile. “one size not fit for all” Optimal for : patients with more embryos. >age, PGT cases Extended culture is a “selection strategy”, not a treatment Conclusion