There is no ART(IVF) laboratory setup with 100% sterile environment, but ensuring contamination free environment to work is crucial and essential. Regardless of the care and precautionary protocols followed for sterile environment , there always happens to be a window of possible routes for microbes to travel and contamination will always occur if pathogens are carried out from unintended source to a host body where they can grow. Ivf cultures is the media used for the in-vitro development of the embryo. While the gametes are maturing in the incubators they might be home to the contamination which will further result into apoptosis , embryo demise and failure to achieve pregnancy.
Sources of Contamination.
Although, the environment we work in, is full of bacteria, fungi and viruses, but also the biggest carriers of the contaminants is the female and male body. The IVF procedure is another method to achieve pregnancy but it also is an opportunity for screening biological fluids and products such as semen
analysis , morphological study related to both eggs and sperm , ovarian follicular fluid, Fallopian ube washings, peritoneal fluid and aspirates of endometrial. Regardless ,of the care taken to provide a sterile environment, when eggs(post ovum retrieval), sperms and embryos are cultured, these cultures contain microbes. These microbes that results into contamination of embryo-culture
• – Transvaginal oocyte retrieval (TVOR) for oocyte retrieval in the follicular fluid .
• – Collection of the semen and improper sterile collection technique
• – Aspirants of the endometrium.
• – Non sterile use of disposables and needle for ovum pick-up.
• – Non sterile culture media.
• – Fluid from the fallopian tube or the peritoneal cavity.
• – Non sterile laboratory environment. media. The source of contamination in every instance is variable but the carriers of these remain definite. The sources of contamination and time of occurrence are :
Presence and Recognition of Microorganisms
Reportedly , the most common sources of contamination were semen and follicular fluids collected for oocyte retrieval . However, the commonly identified contamination causing microbes are bacterium and fungi (yeast or mould). With wide diverse range of microbes that can probably cause contamination , and considering the fact that humans(the practitioners) are the biggest carriers of contaminants. The most commonly identified species are Escherichia coli (E. coli), Aspergilllus, Candida albicans(C. Albicans) and gram negative cocci. Some of the microbial species were specifically found in the follicular fluid and the vaginal lining (during ovum pick-up) are Lactobacillus spp. , Bifidobacterium spp. and Staphylococcus spp. Particularly in males , the bacteria responsible for semen contaminations originates from the urinary tract. The most frequently isolated microorganism from semen contamination is E. coli. During the ovum pick-up , semen collection and series of procedures of embryo culture , unsterilised glass wares and disposables leads to contamination through wide range of microbes whose host is nearly impossible to be detected.
Testing the Presence of Microbes
While working on the gametes , it is essential to strictly follow the protocol with limited to least movement in the laboratory. During egg retrieval or semen collection , it is important to incubate for minimum 1 hour , so as to ensure safe maturation. Further, the IVF or ICSI cultures suspected with bacterial or fungal contamination , should be sampled and tested in microbiological lab. These samples are cultured standard protocol and procedures. The Samples are inoculated on: Blood agar plates or MacConkey agar plates or on Mout agar plates. The Plates are incubated at 37°C in aerobic conditions for almost 24 hours. The samples are tested to check the presence of bacterial , yeast or fungal infections. Another efficient procedure to detect and analyse the presence of microorganism in IVF cultures
through Polymerase Chain Reaction(PCR). The principle of this method is direct as the use of DNA polymerase as an in- vitro replication of specific DNA sequences. Results show that , if the sequence of interest is indeed present in the DNA extract, it will selectively replicate it.
Eliminating Sources of Contamination
In the IVF Laboratories, precautions to ensure that contamination does do not occur at any state before successful embryo transfer is very crucial. The laboratories are equipped with HEPA filtration which reduces the risk for the presence of airborne particles that harbour microorganisms. The laboratories are further engineered with positive air-flow (maintaining positive pressure) so as to ensure that the cleaner air flows into the laboratory and with lesser risk of pathogens travelling in the laboratory. To survive the colonisation of the bacteria get into culture media, almost all of the media now used in IVF cultures contains antibiotics(penicillin , gentamicin , etc) to inhibit bacterial growth. The reduced risk of infections and contaminations is during the ICSI procedure that is due to the removal of cumulus and corona cells, the use of hyaluronidase or the extensive denudation procedure. Another efficient treatment is through Anti Microbial Therapy(AMT). antimicrobial therapy is adequately described as the therapeutic option to overcome both unexplained and symptomatic infertility that might be caused by microbial agents. In assisted reproductive technology (ART), antimicrobial therapy, using antimicrobial agents directed as wide range of conditions, is often considered in order to manage infertility either in men and women or the couple.
Although, main objective of an ART-IVF laboratory is to create artificial in- vivo environment as that of the female reproductive system. IVF Culture media for the gamete collection, preparation and further for embryo development can be complex media at times because it can be host to many contamination causing microorganisms. One of the most important concerns culture media is not to host contamination which is significant reason for ivf failure. Since media is an excellent environment with combination of nutrients and growth elements , it also provides a platform for most of the biological and chemical reactions. It is susceptible to contamination by gametes , media and also through air contact. Therefore , determination and termination of factors effecting the media culture and eliminating factors causing contamination will only lead to successful ivf culture and positive pregnancy outcome. For such pervasive problems it is necessary that the effects of these microbes on embryo culture is understood and developing methods to eliminate the effects of these microbial contamination from human embryo culture.