Introduction

The first birth of a human baby in 1983 in Australia from frozen embryo startled the world. Unexpectedly human oocyte cryopreservation was introduced clinically in 1986 following inadequate experimentation on the basis of mice eggs because embryo cryopreservation was not ethically acceptable. The cryopreservation of zygotes and embryos significantly increased clinical profits as well as the potential rate of conception for a couple after a single cycle of ovarian stimulation and in vitro fertilization (IVF). Additional apparent benefits comprise the opportunity of evading fresh embryo transfer in stimulated cycles with possibility of ovarian hyperstimulation syndrome.

Vitrification is an alternative approch to cryopreservation, and it refers to the physical procedure by which an aqueous solution forms an unstructured glassy solid, instead of crystallizing. Moreover, ultra-rapid freezing is the procedure of freezing using high concentration of cryoprotrctant to freeze the cell in a glass condition without ice formation. The mechanical programmable freezers that are usually used for slow freezing procedures are not vital for vitrification and the authentic time required for freezing the specimens is considerably reduced.

Very few microorganisms in nature are capable of living at temperatures of around −196ºC. However, there is a need to guarantee the complete sterility of liquid Nitrogen (LN₂) in view of its significant purposes in cryobiology. Generally, liquid nitrogen has a very low microbial count during manufacturing. Contamination, however, may arise through storage and distribution. There has been a clear demonstration of Hepatitis B transmission in a liquid nitrogen storage tank between frozen bone marrow samples¹. This raises the possibility of pathogen transmission between samples in ART laboratories.

Reducing the rates of contamination is vital for groups using assisted reproduction^{2, 3}. There now exists significant evidence that liquid nitrogen which is used for food storage can be a source of contamination⁴, in addition to other reported contaminants that may gain entry to the system via infected donors, serum, culture medium, co-culture cells, cryopreserved zonae, and incubators.

The objective of this study is to investigate the effect of microbial contamination induced during the preservation of ova via Vitrification (Open System) using mice ova.

Materials and Methods

Bacterial strains for contamination

The bacterial strains to contaminate the mice ova were obtained from King Khaled University Hospital. The bacterial strains were Streptococcus mutans, Pseudomonas spp., Candida albicans, Bacillus subtilis, and Escherichia coli B.

Cell Line – Mice Oocyte

Inbred female mice (Balb/c)(20 young adult females 8-12 wk. age), from the animal house at the Zoology Department at King Saud University. For the ova collection ovarian stimulation were done by inter peretonial (ip) injection of the females with 10 IU of Follicle Stimulating Hormone (FSH). Then after 48 hr. for the super ovulation the females were injected (ip) with 10 IU of human chorionic gonadotropin (HCG). After 16-20hrpost (HCG) injection females were dissected and ova were collected from the oviduct of the females in culture media and divided into several groups according to the protocol blew.

Superovulation

This was induced in 4- to 6-wk-old female mice by intraperitoneal injection of  10.0 IU (for inbred) pregnant mare serum gonadotropin (Calbiochem, La Jolla, CA) followed by intraperitoneal injection of 10.0 IU HCG (Sigma, St Louis, MO) 48hrs later.

Removing Cumulus for Oocyte Counting

The technique requires euthanizing the female mouse, removing the oviducts, and dissecting cumulus oocyte masses from the ampullae. Cumulus oocyte masses were collected into a culture dish containing PBS with 4 mg/mL. Cumulus cells were then dissociated from the oocytes with hyaluronidase (0.3 mg/mL) and gentle pipetting at ambient temperature. The numbers of live, dead, and fragmented oocytes collected from the left and right oviduct of each mouse were counted and recorded. The percentage of live oocytes was then calculated as: no. live oocytes / total no. oocytes × 100%.

Oocyte contamination

Nutrient broth was used to make bacterial suspensions for all the type of bacteria in this study. The second group of oocytes were divided into 5 subgroups each one has 15 oocytes. Each subgroup was contaminated with one microbe by adding 1 ml from its suspension to the oocyte culture medium and incubated for 4hrs before applying the vitrification technique that will be described later (Individual open cryoleaf has been used for each group labeled by microbe name). Each subgroup was loaded into three cryoleaves. Last, all the samples were stored in one LN₂ container to allow microbial cross-contamination throw the LN₂.

Vitrification Method

The equilibration medium was warmed at room temperature for at least 30 minutes. A reservoir with enough liquid nitrogen to allow complete submersion of a goblet on a cryocane was prepared. A goblet was attached to the bottom of the cryocane and submerged in the liquid nitrogen and then placed near the microscope.The content of the equilibration medium and the vitrification medium were mixed via vials by a few gentle inversions. 1ml of equilibration Medium and the vitrification medium were placed in separate wells or dishes. Using a suitable pipette, 2-3 oocytes were transferred into the Equilibration Medium. The cells initially shrank before re-expanding to their original size. Equilibration was considered complete once the oocytes have re-expanded. The equilibration step took around 5 – 15 minutes. Following this, the oocytes in minimum volume were transferred into the Vitrification Medium (the cells shrink again). The time from transfer of the oocytes into the vitrification medium until vitrificationdid not exceed 1 minute. The oocytes were quickly loaded onto the vitrification carrier and vitrified according to Instructions for Use of the vitrification carrier. Following vitrification, the cryocane and goblet containing the vitrification device and vitrified cells were quickly transferred to the storage tank. It was ensured that the vitrified cells were submerged under liquid nitrogen at all times.

Thawing

The warming medium was pre-warmed to 37°C and the Dilution Medium 1 and Dilution Medium 2 and Washing medium to room temperature for at least 30 minutes. A reservoir with enough liquid nitrogen to allow complete submersion of a goblet on a cryocane was prepared. The cryocane and goblet containing the carrier device with vitrified oocytes from the storage container were collected and quickly transferred to the liquid nitrogen reservoir. It was ensured that carrier device remained submerged under liquid nitrogen. The content of the individual vials were mixed by a few gentle inversions prior to use.In separate dishes, 2ml of Dilution Medium 1, Dilution Medium 2 and two times 2ml Washing Medium were placed, respectively. 2ml of the 37°C Warming Medium were placed in a pre-warmed dish just before opening the carrier device according to the manufacturer’s instruction for use. The oocytes were then quickly transferred into Warming Medium, and left for a maximum of 3 minutes (at this point, the cells were still shrunken).Using a suitable pipette and minimum volume, the oocytes were transferred into Dilution Medium 1 at room temperature and left for 3 minutes (at this point, the cells will start to re-expand).In minimum volume, the oocytes were transferred into the Dilution Medium 2 and leave for 3 minutes (at this point the cells continued to expand). Then, the oocytes were transferred into Washing Medium and leave for 3 minutes. The washing step was repeated by transferring the oocytes to another dish with washing Medium (at this point the cells had fully re-expanded). Finally, the oocytes were transferred into the preferred culture medium equilibrated according to the manufacturer’s instruction for use and allowed to rest in the incubator for a minimum of two hours before visual inspection.

Microbial Isolation methods

Samples were screened by electro-microscope for the post-freeze eggs directly. The post-freeze eggs (without passing it through the thawing media) were then added to the embryonic culture medium and cultured it for one week to see if any microbial growth has been occurred.All intentionally contaminated eggs were thawed and followed the same processes to compare with the target eggs (control). King Saud University, The Department of Botany and Microbiology approved all procedures used in this study, and all mice were maintained at animal house in accordance with institutional protocols and the Guide for the Care and Use of Laboratory Animals.

Specimen preparation for Scanning Electron Microscopy

The specimens were prepared from the samples of LN₂ infected with the selected bacterial pathogens in this study.

Fixation

I cm square from each sample were fixed in 2% (v/v) glutraldehyde in 0.1M Cacogylate buffer at pH 7.2 for a period of 4 hours. It was then kept overnight in the same buffer at a temperature of 4⁰C. The buffer was replaced by a cold 2% (w/v) osmium tetra-oxide in 0.1M Cacodylate buffer at pH7.2 and kept for 3 hours at room temperature. The leaf tissue was washed 3 times for a period of 15 minutes each in the same buffer and then washed twice for a period of 15 minutes each in distilled water.

Dehydration

The specimen was dehydrated using ethanol by changing the serial concentration at 30% (v/v) for 15 minutes at room temperature, then at 50% (v/v) for 15 minutes, at 70% (v/v) for 15 minutes or at  70% (v/v) containing 2% uranyl acetate for a period of 12 hours, at 90% (v/v) for 15 minutes and then finally at a concentration of 100% (v/v) for a period of 30 minutes. Each sample was kept in 0.5 ml of 100% ethanol in a closed tube.

The specimens were then examined and photographed using a scanning electron microscope (JEOL – JSM6060LV) with an accelerating voltage of 14kV at the Electron Microscopy Unit, Central Laboratory, Faculty of Science, King Saud University.

Results

The overall study result shows that the samples were negative except the intentional contaminated eggs which are cultured in microbial medium directly without passing it through the thawing media (Table 1).

Table 1: Shows the overall mice ova micro-organism contamination during cryopreservation.

The Detection and Analysis by Light Microscopy and Electron Microscopy

The results obtained from the Light and Electron Microscopy Micrographs demonstrated that although the LN₂ environment surrounding the oocytes were contaminated with pathogenic bacteria, the oocytes remained contamination free. Although, the oocytes were not contaminated, they were deformed in shape (Fig.1-5). Shape deformation may have been due to the presence of damage or fractures in the ZonaPellucida (ZP) (Fig.1 (b)), the presence of some chromosomal damage or damage in the DNA; hence the deformed shape or the viability of the oocytes is most likely to be due to cytological factors rather than due to the presence of bacterial contamination in the LN_2. The results indicate further that the presence of bacterial contamination may not always mean that such bacteria are able to grow at such low temperatures (-196 ^oC) of the LN2.

Discussion

The results of this study demonstrate that contamination and the cross-contamination between tubes in a liquid nitrogen tank rarely take place. The very low temperature coupled with the toxicity of the component of vitrification media may affect both the survival and growth of microbial cells. Cryopreserved oocytes/embryos can become contaminated with viral agents [e.g. HIV-1, hepatitis C virus (HCV), Epstain–Barr virus, foot-and-mouth virus (FMDV), BTV, BVDV, BHV-1] as well as pathogenic bacteria such as P. aeruginosa, Staphylococcus spp., Streptococcus spp., Corynebactrium spp., N. gonorrhoeae, E. coli, Candida species andUreaplasmaurealiticum, in spite of the presence of antibiotics^5,6,7,8,9,10.

With regards to cryopreservation, oocytes are very different from sperm or embryos. The mammalian oocyte has a volume that three or four times larger than that of the spermatozoa, hence it has lower surface-to-volume ratio and as a result it is very sensitive to chilling and highly susceptible to intracellular ice formation^11,12,13,14. At the metaphase II (MII) stage of maturation, the plasma membrane of oocytes has a low permeability coefficient which slows down the movement of permeating cryoprotectants (CPs) and water¹². Oocytes are surrounded by zonapellucida (ZP) acting as an extra barrier to the movement of water and CPs in and out of the oocyte. The freeze-thaw process results in premature cortical granule exocytosis occurring whereby the ZP hardens and thus making sperm penetration and fertilization impossible^{15, 16}. Chilling sensitivity also increases as oocytes have high level of cytoplasmic lipid content¹². Oocyte membrane is also fragile due to having less submembranous actin microtubules. Cytoskeleton disorganisation and chromosome and DNA abnormalities can occur as a result of cryopreservation¹⁷. Additionally, cryopreservation may compromise the meiotic spindle formed during the MII stage which is very sensitive to chilling¹⁸. After thawing or warming and IVC, the meiotic spindle may recover to a certain degree; however, the rate of recovery is faster after vitrification than after slow freezing¹⁸. Reactive oxygen species can cause a damaging effect on oocytes¹⁹.

Oocyte (ovulated, mature or immature) cryopreservation is still not regarded as an established procedure and remains to be labelled as experimental technique notwithstanding the many advances in the field of cryopreservation²⁰. With regards to human medicine, less than 200 births were reported from cryopreserved oocytes²¹. This number rose to only 500 by 2009²².

Vitrification results in a higher oocyte survival rate than slow freezing, however the pregnancy rate per thawed/warmed oocyte remains low. Chilling (at the nuclear level), freezing, and thawing procedures seem to have a lesser damaging effect on immature oocytes. Controlled-rate freezing²³or vitrification²⁴van is used to cryopreserve such oocytes. The difficulties facing maturation of early-stage oocytes in vitro arise out of the need to develop the complex endocrine system that supports the development at different stages as well as other culture conditions that will ensure survival (oxygen pressure for example).

To prevent oocyte cell contamination and infection, the ZP should be maintained intact during cooling, warming and postwarming manipulation. This will also ensure the survival and viability of the oocytes. The OIE Animal Health Code Appendices have stressed the importance of multiple washing of embryos of domestic animals prior to cryopreservation. Additionally, they have called for a sharp inspection of the ZP for continuity using the IETS recommended methodology²⁵. The abnormal morphological characteristics of oocytes observed in this study may have been affected by faulty chromosomes or DNA.

Irreversible damage or lack of ZP can be induced by the application of commonly applied techniques in human embryology. Contamination is most probable in oocyte/embryonic cells with damaged or no ZP in comparison to those with an intact ZP. Prior to cryopreservation and storage in LN₂, it is imperative to screen oocyte donors for blood-borne diseases, including HIV, hepatitis B and C and herpes viruses. Despite this screening, there must be efficient and adequate washing of oocytes and embryos in the IVF laboratory before cryopreservation. To reduce infectivity and prevent contamination from many viral and bacterial pathogens, rigorous washing procedures of embryos and oocytes can be effective and efficient²⁶.

Following thawing or warming, the further washing of oocytes would potentially reduce the risk of infectivity and also that of cross-contamination between samples during cryobanking. Finally, to eliminate the risk of disease transmission as far as possible to recipients, germplasm could be stored in a quarantine cryotank until the donors have been tested for seroconversion and/or the samples have been tested for the presence of infectious agent(s).

To eliminate cross-contamination occurring out of leaking seals or broken straws, it would be safe and appropriate to store the oocytes from a patient suspected or known to be infected in a separate or distinct LN₂ tank. Gametes obtained from possibly infected patients with the same pathogenic agent could be quarantined together. At all times, the storage of germplasm from a ‘clean’ patient and that from a potentially infected patient should not be in the same LN₂ tank. The processing of gametes obtained from infected patients should be undertaken at designated facilities²⁷. In the absence of such facilities, such gametes should be processed during allocated times and under rigorous laboratory cleaning and disinfection. Alternatively, the handling of embryos of a clean patient and those of an infected patient could be done on separate days, with that of the clean patient taking priority.

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