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. Temperature Hyaluronidase Hyaluronidase Probe Wash Probe WashC D B
Hyaluronidase
Probe
Wash
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. pH
Mean pHi
Human 7.12 ± 0.01 (Phillips et al., 2000)
Mouse ± 0.01 (Edwards et al., 1998)
Hamster (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)

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 mOs/L (Borland et al., 1977; Van Winkle et al., 1990; Dawson et al., 1998)
Human mOs/L (Collins and Baltz, 1999; Li et al., 2007)
Bovine 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 Osmolarity Total cell count (Mean±SD) Control (260) mOsM
82.25±4.0311
280 mOsM
59.75±4.113
300 mOsM
43.5±4.1231
310 mOsM
26.5±3.7

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