Microbial Contamination in Assisted Reproductive Technology: Source, Prevalence, and Cost
Borges ED1,2 and Vireque AA2*
1Department of Obstetrics and Gynecology, University of São Paulo, Brazil
2Invitra - Assisted Reproductive Technologies LTD, Supera Innovation and Technology Park, Brazil
Submission: February 12, 2019; Published: March 28, 2019
*Corresponding author: Alessandra A Vireque, Invitra, Reproductive Technologies LTD, Supera Innovation and Technology Park; 1805 Dra. Nadir Aguiar Avenue, Ribeirão Preto, São Paulo, SP, Brazil
How to cite this article: Borges ED, Vireque A. Microbial Contamination in Assisted Reproductive Technology: Source, Prevalence, and Cost. Adv Biotech & Micro. 2019; 13(4): 555869. DOI: 10.19080/AIBM.2019.13.555869
Abstract
In vitro fertilization techniques are susceptible to multi-stage contamination. Although improper sterile technique at any stage of the process can result in contamination, the most common source of contamination is follicular fluid and semen, two potential sources of pathogenic microorganisms. Microbiological contamination may result in gametes DNA fragmentation, poor quality embryos, and early pregnancy loss or preterm birth. Antibiotics are commonly added to culture media as an attempt to prevent the growth of pathogenic microorganisms, but several microorganisms such as mycoplasmas and anaerobic bacteria (the majority of bacterial species identified within human follicular fluids) escape from antibiotic therapy imposed on embryo culture. Contaminated cycles often result in the demise of the patient’s embryos and additional costs to both the patient and the clinics managing these cycles. Cases of contamination in fertilization clinics and laboratories should, therefore, be identified, registered, and reported in order to guide legislation that regulates protocols executed during assisted reproduction procedures to ensure the long-term safety of IVF and the health of offspring.
Keywords: contamination, assisted reproduction, IVF, embryo, gametes, bacteria, pregnancy, epigenetics
Abbrevations: FF: Follicular Fluid; IVF: In vitro Fertilization; ART: Assisted Reproductive Technology
Introduction
Human gametes and their accompanying fluids are known to be able to host and carry a number of infections. As such, biological fluids like semen, ovarian Follicular Fluid (FF), Fallopian tube washings, peritoneal fluid, and endometrial aspirates are possible gateways to microbiological infection into the in vitro Fertilization (IVF) system, with consequent contamination of embryos and its carrier [1]. When transferred, contaminated embryos can carry microorganisms to microenvironment of the uterus compromising their implantation and survival during pregnancy, and potentially inducing epigenetic modifications on embryonic cells. In Assisted Reproductive Technology (ART) procedures, special attention is given to viral pathogens such as HIV, HBV, HCV, with many guidelines and protocols directly aimed to minimize the risks of transmission of these viruses, known to cause incurable, often fatal, infections [2]. However, sperm, oocytes, and embryos are also affected by contaminants of bacterial and fungal origins with worrisome results.
In a series of studies, Pelzer, et al. [3-5] demonstrated that the human FF is colonized by a number of microbial species that are not necessarily related to an active infection. They observed that microorganisms isolated from FF could persist for more than 28 weeks without any exogenous nutrient. Most of bacterial species identified within fluid samples were anaerobes and generally not targeted by penicillin, streptomycin or gentamicin, the antimicrobials traditionally included in IVF culture media. Ultimately, the presence of certain bacterial species was linked to adverse IVF outcomes. Nonetheless, biological fluids are far from being the only possible source of contamination in ART laboratories. Reagents, devices, equipment, personnel, and even the air pose a risk [6,7]. That is why each procedure and manipulation step should be performed by applying strict aseptic techniques. As affirmed in the latest consensus on IVF laboratory environment [8], exposure time of gametes to the outside, not controlled, environment should be kept at minimum. The air in incubators must be purified and incoming gasses as well as culture media should be filtered to minimize bacteria and fungi contamination.
However, even the strictest laboratories are prone to have at least a few cases of contamination. Even though the guidelines demand that all IVF clinics and laboratories keep records of all procedures, their annual report rarely includes information about the prevalence of contaminations. In the past 15 years, a single study, by Kastrop et al. [9], tackles the subject. They report data from an 8-year observation on their own laboratory showing an incidence of 0.86% of contamination in all IVF procedures (0.68% when ICSI procedures, that did not present any case of contamination, are included). The incidence ranged from 0.40% to as high as 1.30% and is in accordance to older studies that reported incidences of 0.35% and 0.69% [10,11]. The number of studies, however, is not enough to have a precise estimate of the real number of occurrences. Of note, in 2004, a study in Brazil reported a prevalence of 4.8% [12], of bacteria and fungi contamination despite following sanitary steps and supplementing culture media with recommended antibiotics.
Although the numbers seem to be low, when the total number of yearly procedures are weighed in, the absolute number of contamination cases raises an alarm, especially considering that it may cause serious damage to cultured oocytes or embryos, resulting in cancelation or delaying of a fresh embryo transfer. This also represents a big waste of money and resources that ultimately affects the accessibility of ART procedures.
Discussion
Impact of microbial contamination on IVF outcomes
As previously mentioned, the presence of certain microorganisms in the FF was associated to negative effects on IVF outcome. Pelzer et al. [5] showed that Propionibacterium spp., Streptococcus spp., Actinomyces spp., Staphylococcus spp., and Bifidobacterium spp. colonies in either ovaries negatively affected embryo transfer and pregnancy rates of women seeking ART. The observed infection might be either pre-existing or acquired during IFV procedures, highlighting the importance of a more in-depth study about this correlation. Similarly, seminal fluid might also host infections, even after washing techniques. Elevated bacteriospermia in semen has been correlated to DNA fragmentation and poorer seminal parameters overall, which in turn are connected to some types of male infertility [13]. More recently, bacterial colonization (mainly by Escherichia coli, Staphylococcus spp., and Streptococcus spp.) of the embryo transfer catheter was also associated with a reduction in the clinical pregnancy rate [14]. Additionally, yeast-colonized embryo dishes and detrimental effects in IVF have been evidenced in different reports [11,15,16]. Embryo transfer and pregnancy are the usual parameters considered when IVF outcome is discussed, however, only a longitudinal study following IVF-born individuals would be able to assess all possible effects of bacterial and fungi contamination during embryo production.
Epigenetics studies gives us some hints of possible consequences. According to Bierne, et al. [17], bacterial pathogens can be considered as potential epimutagens able to reshape the epigenome with long-lasting effects on host cells. It is known that epigenetics effects on spermatozoa might lead to oligozoospermia, one of the most common causes of male infertility [18]. The consequences, should the target of epigenetic changes caused by contamination be the embryo, could be even more critical [19].
Estimated costs of microbial contamination
The damage caused by microbial contamination in ART procedures can also be translated in costs to the laboratory and clinic that might end up affecting people seeking the service. The cost of microbial contamination can be estimated from the prevalence of these contaminations (about 0.7%), the number of IVF cycles per year, and the cost of the IFV procedure. Approximately 231,936 IVF cycles were carried out in the United States in 2015 [20]. Therefore, nearly 1,623 cycles resulted in microbiological contamination without embryo transfer in most cases. Noteworthy, the estimated cost for these contaminations, per patient, would be about $10,000 with in an overall cost of about $16.2 million. In addition, these estimates do not include the costs to woman that did not get pregnant due to potentially non-detectable contamination of embryo cultures which might have had reduced the pregnancy rates. Taking these into account, the overall effect is impressive
Conclusion
Following the guidelines to keep a clean, controlled environment is essential to minimize the risks of contaminations and all staff should be involved. Microbiological monitoring is mandatory and must be a part of the quality control routine in order to assess the source and amount of microorganisms present and guide preventive and corrective measures even though current monitoring methods are not able to identify all microbial contaminants [21].
The screening of patient samples collected during IVF treatment cycles has been indicated as an approach to reducing the impact of microbial contamination on the IVF culture. Culture plates are the best collection site to check for microbiological contamination and its impact on IFV outcomes, since the potential biological contaminants converge to them, and may interfere with pregnancy and birth rates. Some techniques, like the transfer of zona-free frozen blastocysts that were previously contaminated during IVF culture has been suggested as a successful approach to prevent effects of contamination on pregnancy rates [22]. However, there is no conclusive data on the safety of this procedure.
In general, preventive methods and new approaches to modify ART laboratories routine and the screening of microbial contamination should be discussed and developed with focus on improving the safety of ART procedures by reducing the subtle and overt-contamination impacts. Continued efforts should be made towards microorganisms’ research in the ART field in order to identify possible threats, learn their mechanisms of action, and find a way to stop them without interfering with embryo quality and subsequent development. And finally, longitudinal studies should be considered to identify possible adverse effects on adult life of embryos conceived in vitro that were subject to microorganism infections.
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