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Effectiveness and biocompatability of common IVF

laboratory disinfectants on embryonic development

William Lee | 23296712

Supervised by Dr Jim Catt, Dr Sally Catt, Dr Mulyoto Pangestu

Monash Medical Centre Clayton, Graduate Diploma in Reproductive Sciences

Embryos are sensitive to the environment and there have been various factors reported to affect

their embryonic development including the disinfectants to keep environment free of contamination.

At present ethanol is the most commonly used disinfectant in IVF laboratories as it has a broad

effect against bacteria and it is cost-effective, but it is also a known volatile organic compound (VOC).

A recent disinfecting product named Oosafe®(SparMed) was suggested by a commercial study to be

harmless to sperm. Previous studies also suggested that hydrogen peroxide acted as a good

disinfectant both medically and at home. However questions on the efficacy and safety of these

agents remain unknown. We hypothesized hydrogen peroxide could be an alternative disinfectant

because it loses water molecules unlike VOCs. We showed that all three disinfectants yielded

equivalent outcomes on eliminating microorganisms using differential bacteria count with DifcoTM

HycheckTM hygiene contact slides. Hydrogen peroxide was shown with better suppression on

microbial growth at a concentration of 6%. Interestingly we also observed that wiping with both a

wet cloth and a dry cloth had similar effects to disinfectants. The toxicity test demonstrated that all

three disinfectants were lethal to mouse embryos at 6-24 hours after direct contact with the media

or dishes. A further toxicity study growing 2 cell mouse embryos in MINC incubators recently

sterilized with the disinfectants showed that Oosafe® had no negative influence on embryonic

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growth while the other two terminated the growth. This is consistent with data suggesting that

volatile organic compounds such as alcohols evaporate and may permeate through plasma

membrane which disturbs the embryonic growth. However it also overturns our hypothesis on

hydrogen peroxide being embryo-safe, suggesting Oosafe® is a more ideal surface disinfectant at

least for IVF incubators and perhaps cleaning surfaces with water can minimize bacterial growth.

Introduction

The laboratory is the fundamental unit of the IVF clinics. Contamination by microorganisms may lead

to subtle negative outcomes over time or even lethal to growth of the embryos in vitro. For this

reason, appropriate disinfection procedures and good personal hygiene are important factors in IVF

laboratories. Though disinfection is not as effective as sterilization, it is difficult and inefficient to

sterilise the entire working environment. On the other hand, disinfecting agents may be favourable

but most of them are also embryotoxic. Thus a disinfectant that can minimise the microbial

infections and reduce the biobnurdens of embryos is preferred.

Among several disinfectants, alcohols nowadays are still favourable in most IVF laboratories.

Ethanol and iso-propanol are the most common alcohols used as disinfectants and antiseptics. At a

concen-tration of 70 percent, alcohol performs most effectively because pure alcohols coagulates

the protein inside cell wall and prevents further penetration of the bacterial cell wall, while alcohols

at 70 percent concentration denatures the protein at a slower rate, allowing alcohols to penetrate

into the nucleus and in the end kill the microbial cell(Morton, 1950, Van Engelenburg et al., 2002).

Alcohols are known as volatile organic compounds (VOCs) and are favoured as a disinfectant for

their characteristic to evaporate rapidly leaving no further damage to the surface. However it is also

highly flammable and acts less effectively against bacterial endospores and fungal spores(Piggot and

Hilbert, 2004).

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Oosafe® is a disinfectant recently introduced by SparMED (Stenløse, Denmark) for cleaning surfaces

of the CO2 incubators and the Laminar flow hood. It is composed of quaternary ammonium

compounds(SparMED, 2011). These compounds disrupt the cell membrane and are effective to a

wide spectrum of microorganisms. The product is promoted to be safe and non-toxic to IVF cultures.

However there have been few studies on this product and its effect except a study showed that it

had no significant detrimental effect on sperm motility(Brzyski and Schenken, 2010).

Hydrogen peroxide is the simplest form of peroxide which is a highly reactive oxygen species with a

strong oxidizing capacity. Hydrogen peroxide decomposes to free oxygen radicals and water

molecules. In contrast with alcohols, the water molecules of hydrogen peroxide are lost instead,

leaving more concentrated solution. The mechanisms of disinfection is that free oxygen radicals

cause damage and oxidization of DNA and protein in order to eliminate microorganisms(Tofant et al.,

2006). A 3~6% solution of Hydrogen Peroxide has been used widely as both domestic and medical

disinfectants. Use of vaporized hydrogen peroxide has been increasing(Andersen et al., 2006).

Studies showed that hydrogen peroxide has a high efficacy of decontamination against viruses and a

wide spectrum of bacteria including Staphylococcus aureus, Bacillus subtilis, Escherichia coli and

Micrococci(Bartels et al., 2008, Kahnert et al., 2005, Rogers et al., 2005, Sickbert-Bennett et al., 2005).

Hydrogen peroxide however has not yet been introduced to IVF laboratories due to lack of

knowledge on its effect on embryonic growth. Alcohols, on the other hand, are still most commonly

used in spite of its known toxicity.

The aim of this project is to investigate the efficacy of disinfection process and toxicity with 70%

ethanol, Oosafe® surface disinfectant and hydrogen peroxide, by differential bacteria counts in an

incubator sterilized by the listed disinfectants followed by the comparison of the toxic effects on

developing embryos. We postulated that all disinfectants show equivalent strength on disinfecting

while hydrogen peroxide shows no toxic influences on the growing embryos.

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Methods and Materials

All the chemicals and reagents used for this study were purchased from Sigma Chemical Company

(Sigma, St Louis, MO, USA) unless otherwise stated.

Differential Bacterial Count

DifcoTM HycheckTM hygiene contact slides (Becton, Dickinson and Company, Sparks, MD 21152 USA)

were used for the differential counts. The slides were double-sided, hinged plastic paddles with

Tryptic Soy Agar on side 1 and Rose Bengal Chloramphenicol Agar on side 2. Top surfaces of a shelf

and an incubator were divided into six areas for control, wiping with a dry cloth and clothes rinsed

with 70% ethanol, Oosafe® and 3% and 6% hydrogen peroxide. The agar slides were in contact with

separated divisions and were sealed in sterile containers for incubation under 5% Carbon Dioxide in

air at 37°C for 24 to 48 hours. An initial bacterial count was performed prior to the disinfection test

as a control. Two sets of samples were collected from the top of a shelf and one set from the top of

an incubator inside the laboratory. Another wiping procedure for the incubator was performed for

additional interest.

Animals

Ethics approval for this research project was obtained from the Monash Medical Centre Animal

Ethics Committee (AEC Approval No.MMCA08/65). CBAxB6 mice were superovulated by injecting 5

IU of ip PMSG (Folligon, Intevet, Bendigo, Australia) followed with 5 IU of ip hCG (Chorolon, Intevet,

Bendigo, Australia) 48h after. The mice were mated with the same strains.

Embryo collection

Female mice were killed by cervical dislocation 16-18h post hCG injection and the oviducts were

obtained and placed into 1ml of KSOM handling medium(Summers et al., 1995). Embryos were

collected from the dissected oviducts using KSOM HM. Only embryos at the 1-cell stage with an

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intact zona pellucida and being morphologically graded as good were selected for experiments. After

an initial wash in handling medium, the embryos were transferred to pre-prepared KSOM culture

medium for continuous culture in the incubator at 37°C within a humidified atmosphere of 5% CO2 in

air. Embryos at the 2-cell stage were used for the toxicity tests.

Residue toxicity testing

Three dishes were prepared by separately pre-washing with 70% ethanol, Oosafe® disinfectant, 6%

hydrogen peroxide. Another dish was washed with Oosafe® followed with rinsing with water. Four

dishes were air dried. Three drops of 30μl cleavage media were placed in each dish and covered with

mineral oil. The media were equilibrated at 37°C with 5% CO2 in air for 24h prior transfer of 2-cell

embryos (n=2 per treatment group). The test was repeated

Direct Contact toxicity testing

A dish with five drops of 30μl cleavage media covering with mineral oil was prepared and

equilibrated at 37°C with 5% CO2 for 24h. 2-cell embryos were transferred into each drops following

with a drop of 3μl 70% ethanol, Oosafe® and 6% hydrogen peroxide (n=2 per treatment group

leaving one drop as control and one for wash, no control group due to shortage of embryos).

Toxicity testing on disinfectants present as fumes

Three MINC mini incubator sections were previously sprayed with 70% ethanol, Oosafe® disinfectant

and 6% hydrogen peroxide and wiped with wet paper towels followed by a 1 minute outgassing. A

MINC incubator section without sterilizing was prepared for the control group. Four dishes of

mineral oil and cleavage media were equilibrated inside separately in each incubator to allow any

possible gaseous toxic particles to permeate for 24h. Three drops of 30μl of each medium were

prepared on new dishes covering with the corresponding oil. 2-cell embryos were transferred into

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each treatment group (n=10 embryos per treatment group, n=8 for ethanol, n=11 for Oosafe®).

Media were replaced with the same media prepared but covered with fresh mineral oil.

Embryonic growth was observed at 24h, 48h through 2-cell stage to blastocyst. The second replicate

of residue testing and the fume testing were also observed at 3h and 6h for additional findings.

Results

All disinfectants showed equivalent effects on microbial elimination

Differential counts showed no microbial growth on all treated samples at 24h and similarly at 48h,

except one bacterial colony was present on the 3% hydrogen peroxide treated cultures and ongoing

growth was observed in the next 48h (Table 1-3). One bacterial colony appeared on the slide for the

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first ethanol treated sample at 24h and a yeast colony was obtained from the first dry wipe sample

from 24h. However no further growth was observed for either sample. Interestingly the wiping

procedure with dry clothes showed almost the same efficacy as the disinfectants. Furthermore we

found the wet wiping procedure showed to be as effective as the dry wipe (Table 4).

Table 4 Bacterial counts on the top surface of

incubator after cleaned with a wet cloth over 24

to 72 hours (Side 1/Side 2)

Wet Wipe

24hr 0/0

48hr 0/0

72hr 0/0

All disinfectants had toxic residues which affected the embryonic growth

The residue testing showed that residues of the three disinfectants remained on the dishes after

being air dried. These residues were toxic to the embryos. In the first trial, no embryos were found

at 24h post culture in either of the treatment group. In trial 2, all embryos remained at 2-cell stage at

24h post culture (Figure 1). Embryos in the hydrogen peroxide treated group showed darkened

cellular contents at 3h (Figure 1B). Embryos in Oosafe® treatment and Oosafe® treatment following

with rinsing showed membrane degradation at 3h and cellular deformation at 24h (Figure 1C, 1D, 1H,

1I). However the 2-cell embryo in the control group showed no further growth after reaching 4-cell

stage (Figure 1E).

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Direct contact of all disinfectants caused death of embryos

Embryos in direct contact with a drop of 3μl of each disinfectant showed no growth in any treatment

group with dark cellular contents, indicating that embryos at this stage were dead.

Oosafe® showed no negative influence on embryonic growth after use to sterilize the incubators

All 2-cell embryos in the control group grew into 4-cell stage at 24h and 6 out of the 9 embryos

further grew into blastocysts at 48h (Table 2A, 2E). 6 embryos remained at 2-cell stage and 2

embryos entered 3-cell stage in the ethanol sterilized group at 24h however no further division was

observed (Figure 2B, 2F). The incomplete cleavage indicates that ethanol shows an adverse influence

Figure 1 Morphology of embryos at 3h and at 24h post treatment (A) No effect seen at 3h after transferring in ethanol; (B) Cellular content darkened at 3h in H2O2; (C) Membrane lost and cellular deformation at 3h in Oosafe®/Rinsing dish; (D) Membrane lost from embryos at 3h post culture in Oosafe® treated dish; (E) a 4-cell embryo found in control at 24h; (F) No growth of 2-cell embryo in ethanol at 24h; (G) No growth of 2-cell embryos in H2O2 while cellular contents remained dark at 24h; (H) Membrane lost and deformation of embryos in Oosafe® treatment group at 24h; (I) Membrane lost and deformation of embryos in Oosafe®/Rinse treatment group at 24h

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on embryos. Hydrogen peroxide also showed a negative impact on embryonic growth. Only one

embryo entered 3-cell stage at 24h post culture (Figure 2C, 2G). All 2-cell embryos in the Oosafe®

sterilized incubators entered 4-cell stage at 24h and 10 were observed as blastocysts while one as

morula (Figure 2D, 2H).

Discussion

Disinfection is an important part in IVF clinical research for reducing microbial infections on embryos.

Yet disinfectants are also one potential bioburden on embryos. This study used several toxicity

testings on three different disinfectants. Here the results from residue testing demonstrated that

ethanol though evaporated, can still be present in the dish in either gaseous or the residue form or

possibly causing a reaction on the dish. Furthermore, data shows that ethanol causes toxic effects on

embryos with either direct contact or by spray followed with outgassing. This consistency with data

Figure 2 Morphology of embryos and blastocysts at 24h and at 48h post treatment. (A) Embryos

at 4-cell stage in control group at 24h; (B) Embryonic growth terminated at 2-cell stage in

ethanol sterilized incubator at 24h; (C) One embryo entered 3-cell stage but no further growth

while the others remained as 2-cell in H2O2 sterilised incubator at 24h; (D) Embryos at 4-cell

stage in Oosafe® sterilized incubator at 24h; (E) Embryos in control grew into blastocysts and

morulae at 48h (the 1-cell embryo stopped growing before transferred); (F) Embryonic growth

remained at 2-cell stage in ethanol sterilised incubator at 48h ; (G) embryos at 48h remained the

same as at 24h in H2O2 sterilised incubator but the cellular contents were darker indicating

death; (H) Almost all embryos grew into blastocysts in Oosafe® sterilized incubator at 48h

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suggests that evaporated form of ethanol may still permeate through embryonic membranes.

Overall the results support our hypothesis on ethanol as a VOC being potentially embryolethal. This

is also supported by other previous studies(Lau et al., 1991).

Hydrogen peroxide shows negative effects to embryos because of the residue of free oxygen radicals

on the surface. Hydrogen peroxide though presented an almost equivalent efficacy on disinfection, it

was also shown to be harmful to embryos in the fume test inside the sterilised incubator. This rejects

our hypothesis on its possibility as a replacement of ethanol. Free oxygen radicals may still be

present in the air rather than remaining as a high concentration of peroxide residue.

Oosafe® disinfectant, on the other hand, shows a great efficacy on elimination of microbes as the

other two. The results presented here also demonstrated that embryos that grew in environment

sterilized with Oosafe® disinfectant had no adverse influences on embryonic growth. There was no

difference in the embryo survival rate nor in morphology when compared to the control group.

The large CO2 incubator sucks in air from the surrounding atmosphere, gaseous ethanol can possibly

be drawn into the machine during routine laboratory cleaning which can consequently be hazardous.

This suggests that alcohol based sanitisers for workers and equipment may increase risks in

embryonic survival rate. The MINC incubators however do not constantly draw in air from the

atmosphere, instead they have two independent sections being supplied with bottle gas. In our

study, we deliberately allowed only one minute of outgassing for the MINC incubators, which

possibly resulted in highly concentrated ethanol or oxygen radicals, therefore a thorough outgassing

time should be recommended.

Feedback from using hydrogen peroxide or Oosafe® disinfectant as an IVF disinfectant has not been

reported previously. Repeats of the fume tests are needed to be performed with longer outgas

duration. More embryos are needed to obtain better results on the residue tests. In the first trial of

residue test, the control group had only one embryo entered 4-cell stage but did not divide any

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further. This may be because of the smaller numbers of embryos grown. Embryos produce factors

that help embryonic growth. Studies on rats, cats and bovine suggest that embryonic development

appear to be improved when conspecific embryos of good quality are cultured in groups(Spindler et

al., 2006, Hoelker et al., 2009). Moreover a dose response test can possibly determine the threshold

concentrations of ethanol and hydrogen peroxide in the air that are potentially harmful to embryos.

Hallare’s study has previously showed embryos of zebrafish directly exposed to 1.5% to 2.0% ethanol

showed a significant reduction in survival rate(Hallare et al., 2006). Retardation of embryonic

development has also been observed in chicks after exposed to 15% ethanol(Chaudhuri, 2004).

Studies on the threshold concentrations of ethanol and hydrogen peroxide and the consequences

would help to determine their applications inside the laminar flow hood and under normal lab

condition. Sperm motility assay is another possible test which can be investigated. Although Brzyski

and Schenken’s study suggested that Oosafe® chemical disinfectant is not detrimental to sperm

motility and progression, the methodology of the study was unclear and the amount of chemicals

exposed to sperm was not stated in the article(Brzyski and Schenken, 2010).

In conclusion, this is the first study on comparison between ethanol, Oosafe® disinfectant and

hydrogen peroxide. Further studies are required to evaluate the possible use of these products in IVF

laboratories. Though there have not been many reports on Oosafe® disinfectant, it does show to be

a strong and safe agent in contrast to the other two. Furthermore this draws us back to re-evaluate

the guidelines and procedures for the IVF laboratory. Incubator cleaning and outgas procedures are

recommended to be done outside the working environment in order to prevent adverse effects on

cultures.

Reference

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ANDERSEN, B. M., RASCH, M., HOCHLIN, K., JENSEN, F. H., WISMAR, P. & FREDRIKSEN, J. E. (2006) Decontamination of rooms, medical equipment and ambulances using an aerosol of hydrogen peroxide disinfectant. Journal of Hospital Infection, 62, 149-155.

BARTELS, M. D., KRISTOFFERSEN, K., SLOTSBJERG, T., ROHDE, S. M., LUNDGREN, B. & WESTH, H. (2008) Environmental meticillin-resistant Staphylococcus aureus (MRSA) disinfection using dry-mist-generated hydrogen peroxide. Journal of Hospital Infection, 70, 35-41.

BRZYSKI, R. G. & SCHENKEN, R. S. (2010) Human sperm survival bioassay to examine toxicity of a new clinical laboratory equipment disinfectant. assessment, 2, 8.

CHAUDHURI, J. D. (2004) Effect of a single dose of ethanol on developing skeletal muscle of chick embryos. Alcohol, 34, 279-283.

HALLARE, A., NAGEL, K., K HLER, H. R. & TRIEBSKORN, R. (2006) Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos. Ecotoxicology and environmental safety, 63, 378-388.

HOELKER, M., RINGS, F., LUND, Q., GHANEM, N., PHATSARA, C., GRIESE, J., SCHELLANDER, K. & TESFAYE, D. (2009) Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro. Reproduction, 137, 415.

KAHNERT, A., SEILER, P., STEIN, M., AZE, B., MCDONNELL, G. & KAUFMANN, S. H. E. (2005) Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis. Letters in applied microbiology, 40, 448-452.

LAU, C. F., VOGEL, R., OBE, G. & SPIELMANN, H. (1991) Embryologic and cytogenetic effects of ethanol on preimplantation mouse embryos in vitro. Reproductive Toxicology, 5, 405-410.

MORTON, H. E. (1950) The relationship of concentration and germicidal efficiency of ethyl alcohol. Annals of the New York Academy of Sciences, 53, 191-196.

PIGGOT, P. J. & HILBERT, D. W. (2004) Sporulation of Bacillus subtilis. Current opinion in microbiology, 7, 579-586.

ROGERS, J. V., SABOURIN, C. L. K., CHOI, Y. W., RICHTER, W. R., RUDNICKI, D. C., RIGGS, K. B., TAYLOR, M. L. & CHANG, J. (2005) Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator. Journal of applied microbiology, 99, 739-748.

SICKBERT-BENNETT, E. E., WEBER, D. J., GERGEN-TEAGUE, M. F., SOBSEY, M. D., SAMSA, G. P. & RUTALA, W. A. (2005) Comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses. American Journal of infection control, 33, 67-77.

SPARMED (2011). Stenløse, Denmark. SPINDLER, R. E., CRICHTON, E. G., AGCA, Y., LOSKUTOFF, N., CRITSER, J., GARDNER, D. K. & WILDT, D.

E. (2006) Improved felid embryo development by group culture is maintained with heterospecific companions. Theriogenology, 66, 82-92.

SUMMERS, M. C., BHATNAGAR, P. R., LAWITTS, J. A. & BIGGERS, J. D. (1995) Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biology of reproduction, 53, 431.

TOFANT, A., VUCEMILO, M., PAVICIC, Z. & MILIC, D. (2006) The hydrogen peroxide, as a potentially useful slurry disinfectant. Livestock Science, 102, 243-247.

VAN ENGELENBURG, F. A. C., TERPSTRA, F. G., SCHUITEMAKER, H. & MOORER, W. R. (2002) The virucidal spectrum of a high concentration alcohol mixture. Journal of Hospital Infection, 51, 121-125.