Environmental Factors Environmental Factors and Embryo Cultureand Embryo Culture

Environmental Factors and Environmental Factors and Embryo CultureEmbryo Culture

Tim Schimmel, John Garrisi, Jacques CohenTim Schimmel, John Garrisi, Jacques CohenInstitute for Reproductive Medicine and ScienceInstitute for Reproductive Medicine and ScienceSaint Barnabas Medical Center, Livingston, NJ, USASaint Barnabas Medical Center, Livingston, NJ, USA

Antonia GilliganAntonia Gilligan Alpha Environmental Inc., NJ, USAAlpha Environmental Inc., NJ, USA

Michael CecchiMichael Cecchi GenXGenX International Inc., CN, USAInternational Inc., CN, USA

•• Gonad & TractGonad & Tract•• Culture mediaCulture media•• Solid materialsSolid materials•• Physical conditionsPhysical conditions•• Compounds in air Compounds in air

(Volatile Organics and (Volatile Organics and AldehydesAldehydes))

EnvironmentEnvironment

•• Sick Building Syndrome (TVOC)Sick Building Syndrome (TVOC)•• Anecdotal reportingAnecdotal reporting•• OSHA thresholds?OSHA thresholds?•• Incubator air is poorIncubator air is poor•• HEPA?HEPA?•• Detection in air Detection in air

from from mg/mmg/m33 to to µg/mµg/m33

•• 200200--300 300 µg/mµg/m3 3 may cause effectmay cause effect

Cohen J, Gilligan A,Esposito W, Schimmel T and Dale B: Cohen J, Gilligan A,Esposito W, Schimmel T and Dale B: Ambient air and its potential effects on conception Ambient air and its potential effects on conception inin--vitro. vitro. Hum. Hum. ReprodReprod.. 12, 174212, 1742--1749 (1997).1749 (1997).

Hall J, Gilligan A, Schimmel T, Cecchi M and Cohen J: Hall J, Gilligan A, Schimmel T, Cecchi M and Cohen J: The origin, effects and control of air pollution in The origin, effects and control of air pollution in laboratories used for human embryo culture.laboratories used for human embryo culture. Hum. Hum. ReprodReprod ((supplsuppl): 1998.): 1998.

Mayer J et al. Mayer J et al. Prospective Randomized Analysis of the Prospective Randomized Analysis of the Impact of an IVF Incubator Air Filtration System on Impact of an IVF Incubator Air Filtration System on Clinical Pregnancy Rates.Clinical Pregnancy Rates. 11Th World Congress on In 11Th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Vitro Fertilization and Human Reproductive Genetics, 1999 Sydney Australia1999 Sydney Australia

Boone W. Boone W. Control of air quality in an assisted reproductive Control of air quality in an assisted reproductive technology laboratory.technology laboratory. FertFert. & . & SterilSteril. Vol. 71, No. 1, 1999. Vol. 71, No. 1, 1999

•• CapturingCapturing•• CryoCryo--concentrationconcentration•• Gas Chromatography (GC)Gas Chromatography (GC)•• Mass Spectroscopy (MS)Mass Spectroscopy (MS)•• Adsorbent tubes (Adsorbent tubes (aldehydesaldehydes))•• High performance liquid High performance liquid chromatography (HPLC)chromatography (HPLC)

Measuring techniquesMeasuring techniques

VOC Sources• Car exhaust• Adhesives and paints• Packaging and Insulation• Some plastics• Equipment off-gassing• Humans• Neighbors • Alcohol and anesthetic gasses

•• ExteriorExterior•• InteriorInterior•• Compressed gasCompressed gas•• Disposable offDisposable off--gassinggassing•• Equipment offEquipment off--gassinggassing•• HumansHumans

LaboratoryLaboratory Air PollutionAir Pollution

Exterior pollution

Resurfacing of the Resurfacing of the Parking Lot with a Parking Lot with a

SealantSealant

Aldehydes in incubators

0

20

40

60

80

1 3 4 5 7

AcroleinAcetealdehydeNonanalTotal

µg/m3

Laboratory Location

Effects of Effects of AcroleinAcrolein on Mouse on Mouse EmbryosEmbryos

0

20

40

60

80

100

0 0.01 0.025 0.038 0.05

% blastocyst

mMmM acroleinacrolein

Exterior pollutionInterior PollutionInterior Pollution

Isopropyl Alcohol Exposure

0

500

1000

1500

2000

2500

3000

Out

side

air

Ret

urn

air

Hal

lway

s

Proc

edur

ero

om

Lam

inai

rflo

w u

nits

Incu

bato

rs

Select Volatile Compounds Exposure

0

5

10

15

20

25O

utsi

deai

r

Ret

urn

air

Hal

lway

s

Proc

edur

ero

om

Lam

inai

rflo

w u

nits

Incu

bato

rs

µg/m3BenzeneTolueneXylenesStyrene

Exterior pollutionCompressed GasCompressed Gas

VOC levels in compressed COVOC levels in compressed CO22

0

20

40

60

80

100B

enze

ne

Freo

n

IPA

n-pe

ntan

e

acet

alde

hyde

microgram/m3

Exterior pollutionOffOff--Gassing Of Gassing Of

DisposablesDisposables

Nunclon DishMaterial ng/sample• Styrene 920• Toluene 180• Acetone 150• 2-Butanone 130• Acetaldehyde 100• n-Butane 100• Benzaldehyde 100• 19 Other materials 595Total VOC’s 2275

Styrene in mouse culture media

-10

10

30

50

70

90

oil-free oil overlay

blastocysts

(n=21)

(n=80)

Percent development

Styrene• Styrene: An oil soluble pollutant• A common air pollutant from

plastics ranges form 0-20 µg/m3

• In embryo culture with mineral oil the styrene is selectively adsorbed.

• Log P= Log (Butanol) / (Water)• Styrene Partition Coefficient = 3.05

Pollutants and Partition CoefficientMethanol -0.74Ethanol -0.30Acetone -0.24Acrolien -0.01Isopropanol 0.05Formaldehyde 0.35Acetaldehyde 0.45Propanol 0.59Benzene 2.13Styrene 3.05Xylene 3.2

Exterior pollutionEquipment Equipment

OffOff--GassingGassing

Total VOC levels in incubators

0

20

40

60

80

100

120

140

Incu

bato

r 1

Incu

bato

r 2

New

Incu

bato

r3

mg/m3

'

Total VOC levels in incubators using prototype filter

200

400

600

800

1000

1200

1400

BEFORE AFTER

microgram/m3

Exterior pollutionVariability Between Variability Between

ClinicsClinics

Variability in VOC content

0

2000

4000

6000

8000

10000

12000

Outside Inside Incubator

MinimumMaximum

Counter measuresCounter measures(I) (Re)assessment(II) Reconstruction (III) Relocation(IV) Alternative culture(V) Solid media filtration

1. Carbon adsorption2. Oxidation

Solid Media FiltrationSolid Media Filtration•HVAC system•Inside laboratory•In-line•Inside incubator

Incubator Filters Incubator Filters -- Prospectively Randomized Prospectively Randomized StudyStudy

(The Jones Institute (The Jones Institute -- Norfolk VA: Jake Mayer et al, 1999)Norfolk VA: Jake Mayer et al, 1999)

30

52

10

30

50

70

Normal CODA

ClinicalPregnancy

129 retrievals129 retrievalsPP<0.02<0.02

• Variability among clinics• Complex dynamics between

physical systems: oil and water sinks• Filters may be quality control tool• Gimmick or good clinical practice?• There is a need for bio-assays• There is a need for fast and simple testing• Counter measures are experimental• Prolonged culture?

Conclusions

29

30

31

32

33

34

35

36

37

30 120 240 360 600

Hot Plate only - 35mmNuncHeated Objective setat 36.0Heated Objective setat 37.4

Effect of Time of Observation on Temperature

Seconds after incubator opening