all the embryology laboratory should have
automatic emergency generator backup. Minimum of two incubators is recommended.
Gas cylinders should be placed in a separate room with an automatic backup
system. Frequent cleaning and sterilization is necessary for incubators whereas
nitrogen tanks should be cleaned and sanitized annually.
Safety aspects include both microbial and Non-Microbial
aspects that play a role in ART.
Microbial aspects: microbial contamination may be due to
bacterial, viral or fungal which might lead to contamination of the culture
medium, semen, follicular fluid or the laboratory environment. Donor semen or
donor embryos must be screened for microbial infections during cryopreservation
to avoid contamination of liquid nitrogen. Co-cultures of human embryos with
animal or human cells also require safety attention.
Microbiological safety includes the following:
1. Screening patient for pathogenic
micro-organisms: European Society
for Human Reproduction and Embryology (ESHRE) recommends that the women who are
undergoing IVF treatment should have undergone screening test for HIV, HBsAg,
Antibodies to Rubella, Toxoplasma. Their
partners should also undergo the screening test for these infections. In Netherlands, screening is not indicated
unless case history indicates the possibility for any infection. Screening is
not indicated if the IVF treatment does not include cryopreservation of the
embryos. If the patients serum is to be used in the medium and the embryos are
to be cryopreserved then serum should comply with quality standards such as
those used for blood banks (HIV- negative, HBsAg- negative, HSV-negative &
Syphilis -negative). Screening of patients for sexually transmitted diseases
such as gonorrhoea & chlamydia are generally carried on indications like
tubal pathologies, Cervicitis, Leukospermia etc., if the results are positive
treatment for the infection has to be given in first place before starting the
2. Contamination of the culture medium includes
a. Safety preparation of the culture medium:
Erlenmeyer flask should be cleaned according to the cell and tissue culture
procedures. Heat sterilization is preferred. Water that is used to prepare
culture medium should be virtually sterile and the prepared medium should be
sterilized using membrane filtration before storage. To secure complete safety
medium should be checked for the growth of bacterium after preparation and filtration
by incubating at 37oc for 48 hours. Cloudiness of the medium
indicates bacterial contamination. Routine examination for the endotoxin is not
necessary. Paraffin oil that is used to overlay the medium need not be
sterilised since the high standard company products are used.
b. Contamination of medium from the serum component:
If patients own serum is used for preparing the culture medium, screening
test for diseases transmissible via blood is necessary for the cryopreservation
of the embryos. During cryopreservation it is very important to avoid
contamination of liquid nitrogen. Negative results are valid for one year after
which the donor is tested again for further use of the embryos. Use of pooled
sera must be avoided since the test that is used for the detection of diseases
has its own sensitivity.
c. Contamination of the medium from follicular
fluid: Follicular fluid can cause contamination during vaginal puncture. It
is not advisable to disinfect vagina as its highly embryotoxic especially iodine.
d. Contamination of the semen: Most
e. Contamination of the medium from the
laboratory environment: It is important to maintain air quality in IVF
laboratory since it might affect the clinical outcome. Laminar Air Flow (LAF)
is used to avoid contamination of the culture medium from laboratory
environment. Laminar air flow is of two types vertical and horizontal. Vertical
air flow is safer than horizontal air flow since it provides protection to the
user as well as the culture.. Air is drawn into the HEPA filter which flows
gentle and smooth towards the operator in horizontal air flow and towards the
table in vertical air flow. There are three types of laminar hood. Class I –air
flow characterise to chemical fume hood. Provides significant protection for
the user but does not protect the culture. Class II-provides aseptic
environment .hence indicated for IVF laboratory. Class III-gas tight cabinets
and provides high protection for the user. HEPA filters can filter the
particulate size of upto 0.3 microns whereas ULPA filters filter upto 0.1 micro
particle size. Floor and the instruments can be disinfected with phenolic
compounds or 0.5%hypochlorite.UV light should be avoided since it can cause
ozone formation. Use of disposable materials of tissue culture quality is recommended.
To work with tissue cultures glass wares should be sterilised in accordance
with the procedure.it is recommended to clean the incubators with open water
system for every four weeks with 70% ethanol. To prevent the development of
fungi in the incubator copper sulphate or amphotericin can be used.
of the culture medium expected: E.coli, yeasts and fungi are the more
common contaminants. Bacterial investigation has to be carried out to discover
pathogens. In case of infection refined semen sample used for insemination has
to be subjected to microbial investigation. If semen is the contaminant, gram
negative rod shaped bacilli will be found in the culture. Yeast will be seen if
contamination is due to follicular fluid and fungal growth will be observed in
the culture if the contamination is from the incubator. Oocyte degenerates if
the culture medium is contaminated by bacteria. In case of yeast and fungal
contamination vital embryos survive but the transfers of the contaminated
embryos were not indicated due to lack of documentation.
g. Donor semen: Fresh semen sample is not
advisable for donor insemination. Minimum of six months quarantine period is
necessary for donor sample.
h. Co-cultures: Co-culture of human
embryos with animal cell or the other human cell which is followed by transfer
is not advisable, since it might result in viral contamination.
of enzyme treatment: alpha amylase or hyaluronidase should be free of
viruses or prions.
Contamination of the liquid nitrogen should be avoided to prevent the other
embryos from infection as decontamination of liquid nitrogen is not advisable.