Control of Variables; The key to success in the IVF laboratory A/Prof Cecilia Sjoblom Westmead...

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

A/Prof Cecilia SjoblomA/Prof Cecilia Sjoblom

Westmead Fertility Centre, University of SydneyWestmead Fertility Centre, University of Sydney

BackgroundBackground

Success of assisted reproductive technology (ART) affected by:

Patient factorsQuality of the Laboratory Culture protocolsSupplies Methods

Equipment

(Alper et al., 2002; Higdon et al., 2007; Fujiwara et al., 2007)

BackgroundBackground

• Embryos resembles primitive cells

• Pre-Compaction they can not regulate changes to– pH– Temperature– Osmolarity

Control of Physical ParametersControl of Physical Parameters

• Temperature

• pH

• Osmolarity

• Gas Composition

• Full Control

TemperatureTemperature

• 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


• 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)


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)


HyaluronidHyaluronidasease

ProbeProbe

WashWash

A

CD

B

HyaluronidaseHyaluronidase

ProbeProbe

WashWash

• Temperature measurements were taken every 20 seconds• Digital thermometer with ± 0.1°C accuracy


36.6

29.75 32.02

0

5

10

15

20

25

30

35

40

45

UNDER OIL 4 WELL DISH 4 WELL DISH IN WARMING BLOCK

Tem

pera

ture

(°C

)

Denudation protocol

a

bc

37°C

32 °C29.8°C

29.8 °C for 3 min

29.8 °C

32.3 °C


• Temperature

• pH

• Osmolarity

• Gas Composition

• Full Control

pHpH

7.6-7.9

7.2-7.3

• 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)

pHpH

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)

Micro pH Probe in 50ul Drop under OilMicro pH Probe in 50ul Drop under Oil

7.2

7.4

7.6

7.8

8

8.2

8.4

8.6

8.8

0 10 20 30 40 50 60 70

time (min)

pH

pH in a Drop under OilpH in a Drop under Oil

7,3

7,4

7,5

7,6

7,7

7,8

7,9

0 5 10 15 20 25 30 35

time (min)

pH

3 minutes 5 minutes 10 minutes

pH in Drop under OilpH in Drop under Oil

Mouse EmbryosMouse Embryos

The effect of exposure time on cell number in blastocysts cultured in vitro

(11)

(12)(18)

(10) (10)

0

10

20

30

40

50

60

70

In vivo Control 3 min 5 min 8min

Time spend outside the incubator (min)

Nu

mb

er o

f ce

lls

pHpH

• 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


• Temperature

• pH

• Osmolarity

• Gas Composition

• Full Control

OsmolarityOsmolarity

• 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


• 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

Osmolarity

240

250

260

270

280

290

300

0 24 hrs 48 hrs 72 hrs

4 Well Dish60 mm dishCentre well dish


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


• 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


• Temperature

• pH

• Osmolarity

• Gas Composition

• Full Control

Gas CompositionGas 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


• 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


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)


• Temperature

• pH

• Osmolarity

• Gas Composition

• Full Control

Full ControlFull 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

High IVF Live Birth Rates is achieved High IVF Live Birth Rates is achieved through Full Controlthrough Full Control

Control of Variables; The key to success in the IVF laboratory A/Prof Cecilia Sjoblom Westmead...

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