Control of Variables; The key to success in the IVF laboratory

1. Control of Variables;The key to success in the IVF laboratory A/Prof Cecilia Sjoblom Westmead Fertility Centre, University of Sydney

2. Background • Success of assisted reproductive technology (ART) affected by: • Patient factors • Quality of the Laboratory • Culture protocols • Supplies • Methods • Equipment • (Alper et al., 2002; Higdon et al., 2007; Fujiwara et al., 2007)

3. Background • Embryos resembles primitive cells • Pre-Compaction they can not regulate changes to • pH • Temperature • Osmolarity

4. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

5. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

6. Temperature • Temperature is a key determinant of gamete viability and embryonic growth • The micro-environment for culture should be held a temperature of 37 ± 0.2 °C • Various types of warming devices • (Yeung et al., 2004; Hansen 2007) Lane et al 2008

7. Temperature • Meiotic spindles • Oocyte quality • Chromosome alignment and separation during MI and MII • Cooling and overheating produce disassembled meiotic spindles • Human (Wang et al., 2001) • Mouse (Sun et al.,2004) • Porcine (Suzuki et al., 2007) • Cow (Pollard et al., 1996; Ju et al., 1999)

8. Temperature • Insemination of oocytes with disrupted meiotic spindles results in: • Failed fertilization • Abnormal fertilization • Aneuploidy • Low embryo developmental competence • Apoptosis/ fragmentation • Gene expression? • (Wang et al., 2001; Sun et al., 2004; Massaro et al., 2007; Zeng et al., 2007; Lane et al., 2008)

9. Hyaluronidase B A Wash D C Probe Temperature Hyaluronidase Probe Wash • Temperature measurements were taken every 20 seconds • Digital thermometer with ± 0.1°C accuracy

10. Temperature

11. 37°C 32.3 °C 32 °C 29.8°C 29.8 °C 29.8 °C for 3 min

12. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

13. Mean pHi Human 7.12 ± 0.01 (Phillips et al., 2000) Mouse 7.17-7.22 ± 0.01 (Edwards et al., 1998) Hamster 7.19-7.22 (Lane et al., 1998) During the in vitro manipulation of mammalian embryos the extra-cellular pH (pHo) should be maintained close to pHi in order to reduce stress(Edwards et al., 1998; Lane et al.,1998) Currently 7.35 ±0.05 in an environment of 5-6% CO2 (Sjöblom, 2004; Quinn, 2004) pH 7.6-7.9 7.2-7.3

14. pH • A precise control over pHi is essential for numerous cellular processes • Enzyme activity (Lane et al., 1999a;1999b) • Cell differentiation; growth and proliferation (Ozawa et al.,2006) • Cell division; membrane transport; cell-cell communication (Lane et al., 1998) • Protein and DNA synthesis (Squirrell et al., 2001) • Respiration (Lane, 2001) • Metabolism, calcium level modulation and cytoskeletal dynamics (Squirrell et al., 2001)

15. Micro pH Probe in 50ul Drop under Oil

16. pH in a Drop under Oil

17. pH in Drop under Oil

18. Mouse Embryos

19. pH • The pH becomes sub optimal after just 3 min outside the incubator • No activity should take longer than 3 min • A stop watch is an embryologists best friend

20. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

21. Osmolarity • Zygotes are more sensitive than 2-cell embryos • In response to changes in osmolarity • Embryos will act by changing their volume to regulate osmotic pressure across their membrane • Irreversible damage to cyto-skeleton • Changes to gene expression and possibly imprinted genes

22. Osmolarity • The osmolarity of the reproductive tract is high for most species • Mouse 310- 360 mOs/L (Borland et al., 1977; Van Winkle et al., 1990; Dawson et al., 1998) • Human 320-360 mOs/L (Collins and Baltz, 1999; Li et al., 2007) • Bovine 330-370 mOs/L (Baltz 2001; Hwang et al., 2008) • Rat 290 mOs/L (Baltz 2001; Hwang et al., 2008) • Attempts to culture embryos in vitro at these high osmolarities have failed • The optimal osmolarity for pre-implantation embryos is 260 mOS/L

23. Osmolarity

24. Osmolarity

25. Osmolarity • Making up dishes in advance is common practice in the IVF lab • To allow for pre-equilibration • For smooth running of the day • Making up dishes too early can effect the osmolarity of the culture medium • This can in turn have detrimental effects on embryo development • Cover micro-drops IMMEDIATELY

26. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

27. Gas Composition • CO2 is used in cell culture as a part of the CO2 - HCO3- buffer system • CO2 is NOT a metabolite for cells or embryos • Concentration of CO2 is depending on • What pH you aim for • The concentration of HCO3- • O2 is a metabolite and crucial in the embryos utilisation of Pyruvate as an energy substrate

28. Gas Composition • A total of 573 patients, 7312 oocytes • 689 consecutive IVF and ICSI cycles at NURTURE • Prospectively randomised to culture in • 7% oxygen (275 patients, 325 cycles) • Ambient conditions at approximately 20% oxygen (298 patients, 364 cycles). • No difference between the two groups in; • IVF:ICSI ratio (56:44) • Patient age (34±0.3 years) • Infertility background

29. Gas Composition • Lowering oxygen to more physiological levels is associated with; • Significant improvement in embryo quality • Significantly higher pregnancy rate per oocyte retrieval (47% versus 39% p< 0.05) • There was no difference in the pregnancy rate per ET(48% and 42% respectively, p=0.11) • Significant difference in live birth rate (45% and 39% respectively, p< 0.05)

30. Control of Physical Parameters • Temperature • pH • Osmolarity • Gas Composition • Full Control

31. Full Control • You can improve your results by having full control over variables • It is crucial that the laboratory has full control over physical parameters and that embryologists are fully aware of the implications of their actions

32. High IVF Live Birth Rates is achieved through Full Control