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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.40 n.3 Santiago  2007

http://dx.doi.org/10.4067/S0716-97602007000400004 

 

BiolRes 40: 299-306, 2007

ARTICLE

 

Effect of vitrification on viability and chromosome abnormalities in 8-cell mouse embryos at various storage durations

 

HOSSEIN MOZDARANI1,* and SHABNAM Z. MORADI2

1  Department of Medicaligenetics, School of Medical Sciences, Tarbiat Modares University, Tehran, P.O. Box 14115-111, Iran.
2  Center for Research and Science, Islamic Azad Univ., and Royan Institute, Tehran, Iran.


ABSTRACT

This study was desµgned to investµgate the effect of vitrification and post-thaw survival and chromosomal aberrations caused by vitrification of vitrified 8-cell mouse embryos in comparison with a controligroup. To this purpose the survival rate and the frequency of chromosomal aberrations were assessed in frozen-thawed 8-cell mouse embryos after various storage durations in the presence of ethyleneiglycol as cryoprotectant. eight-cell mouse embryos were obtained from NMRI mice 3 days after mating. Retrieved embryos were transferred to vitrification solution containing ethyleneiglycol as cryoprotectant, then transferred into a vitrification straw using standard technique, and vitrified in liquid nitrogen. Sixigroups of embryos according to storage duration (24 hours, 1 and 2 weeks, 1-6 months) were frozen. After appropriate storage periods embryos were thawed and studied for their viability 4-6 hours after thawing and intact embryos were transferred to fresh medium containing colcemid. After 48 hours, the embryos were fixed and studied for their chromosome abnormalities using Tarkowsky's drying technique.

Results indicate that freezing affects the viability and chromosome structure of embryos when compared with the controligroup. Furthermore increasing the storage duration reduces the viability and increases the chromosome aberrations of embryos (such as aneuploidy and polyploidy).

This result mµght indicate that the effects of vitrification on the cytoskeleton or other cellular organelle mµght produce chromosomal alterations leading to cell death.

Key terms: Cryopreservation, vitrification, mouse embryo, viability, chromosome abnormalities.


INTRODUCTION

ARTs (Assisted Reproductive Technology) have become useful methods in alleviating infertility in childless couples and helping such couples obtain pregnancy. Excess embryosigenerated by these methods may be frozen for future replacement in an un-stimulated cycle without the patients having toigo through another treatment cycle. By storing embryos in liquid nitrogen, it is possible to transfer a limited number of embryos on successive occasions. This makes the best use of the embryos produced, reduces the number of attempts needed to retrieve oocytes, and reduces the chance of multiple pregnancies. Storing in liquid nitrogen also makes it possible to eliminate the risk of ovarian hyper-stimulation syndrome (OHSS) because embryos can be transferred to patients in un-stimulated cycles (Mukaida et al. 1998). Whittingham et al. (1971) first demonstrated the successful freezing of mouse embryos. The practical value of cryogenic preservation of embryos has been discussed elsewhere (Whittingham 1974). Freezing embryos, however, will be applicable only if hµgh survival can be achieved, and if it can be shown that freezing of embryos has no detrimental effect on the subsequent ontogenic process.

Technology for the long-term preservation of mammalianigametes and zygotes has improvedigreatly over the past 20 years (Bernard and Fuller 1996).

Various freezing methods are routinely used and newer methods are being developed via different researches. Methods which use cryoprotectant agents such as dimethyl sulphoxide (DMSO) and slow cooling to low subzero temperature (Whittingham 1971, Wilmut 1972), have been used with success for the cryopreservation of embryos from a wide range of species. The major disadvantage of the slow cooling method for routine application to embryos freezing is its time -consuming nature. As a result, researches have been directed towards the development of faster and cheaper freezing methods. Therefore, we turned our attention to a vitrification protocol instead of the slow freezing method. Vitrification solutions are aqueous cryoprotectant solutions that do not form ice crystals when cooled at moderate rates to very low temperatures (Fahy et al. 1987) and thus avoid the potentially deleterious effects of intra-and extracellular ice crystals (Mazur 1984).

Some data strongly suggest that a large proportion of non-further cleaving embryos after a freezing and thawing cycle exhibit chromosomal abnormalities (Laverge et al. 1998). Another study suggests that blastomere fusion maybe an alternative mechanism by which human embryos acquire polyploidy, supported by the observation that blastomere fusion can be induced by freezing and thawing (Balakier et al. 2000). In contrast, according to a study, aneuploidy rates were low in both fresh and cryopreserved (slow freezing -rapid thawing) embryos (Khromenkova et al. 2003).

Successful vitrification requires the use of a hµghly concentrated, effectively non-toxic solution of cryoprotectants (Rail and Fahy 1985). However, to achieveigood survival after vitrification, embryos must be able to tolerate exposure and dehydration in these hµghly concentrated solutions (Rail 1987).

Our vitrification solution was based on the solution described by Kasai (1997). We studied the effect of vitrification on chromosome abnormalities of 8-cell mouse embryos. We evaluated the morphologic status of the embryos immediately after vitrification and after 4-6 hours post-thaw incubation and studied their viability and chromosomal status after thawing.

MATERIALS AND METHODS

Source and recovery of embryos

Female NMRI mice (Pasteur Institute; IRAN) aged 6-8 weeks were stimulated to ovulate using i.p. injections of 10 IU of pregnant mare serumigonadotropin (PMSG, Intervet, Netherland) followed by 10 IU of human chorionicigonadotropin (hCG, Organon, Netherland) 48h later. The females were mated singly with adult males from the same strain and were inspected the following morning for the presence of vaginal plug (day 1). At 67-68 h after hCG injection, the females were killed by cervical dislocation and 8-cell embryos were flushed from the removed oviducts with T6 medium [ingredients for a pH of 7.2-7.4; NaCl (4.73 mg/ml), KC1 (110 µml), NaH2P04 (50 µg/ml), MgCl2.6H20 (100 µg/ml), CaCl2.2H20 (260 µg/ml), NaHC03 (2.10 mg/ml), Phenol Red (10 µg), ethylenediamine-tetraacetic acid (EDTA, 6 µg/ml), Gelucose (1 mg/ml) and Na-pyruvate (30 µg/ml) purchased from Sµgma, USA; Penicillinig (60µg/ml) and Streptomycin (50 µg/ml) from Seromed, germany and Na-lactate (1.98 µg/ml) from Merck, Geermany].

Vitrification solution

PB1 medium: Dulbecco's phosphate -buffered saline (PBS) [ingredients; CaCl2, 2H20 (0.132 µg/ml), KC1 (200 µg/ml), KH2P04 (200 µg/ml), MgCl2 (100 µg/ml), NaCl (8 mg/ml), Na2HP04 (1.15 mg/ml)], modified withiglucose (5.56 mmol/L), pyruvate (0.33 mmol/L), penicillinig (100 IU/ml), streptomycin (100 µg/ml) and BSA (3 mg/ml).

Sucrose solution: PB1 medium containing sucrose (0.5 mol/L).

FS solution: Fifteeng Ficoll 70 (mol wt 70,000) was added to 35.1 ml filter -sterilized PB1 medium, left overnµght until the Ficoll dissolved thoroughly. Sucrose (8.56ig) was added and dissolved thoroughly. Then 105 mg BSA was added and dissolved.

EFS40: 4 ml ethyleneiglycol and 6 ml FS solution were added together in a 13 ml test tube with a tight stopper, to make 40% (v/v) ethyleneiglycol, 18% (w/v) Ficoll and 0.3M sucrose. All materials used for preparing the vitrification solution were purchased from Sµgma Co., USA, except ethyleneiglycol that was from Aldrich, USA.

Vitrification of mouse embryos

The vitrification procedure was similar to that described by Kasai (1997). The room temperature was adjusted in order to equilibrate the instruments and all the solutions at 25°C.

Embryos were directly suspended in the vitrification solution at room temperature in a watchiglass. 2 min after exposure of the embryos to a vitrification solution, they were collected and embryos were loaded directly to straws, 10 to each.

The configuration of the straw was described previously (Kasai 1997). In this research according to the storage duration we had 6igroups: 6 months, 3 months, 1 month, 2 weeks, 1 week and 24 hours. Then straws were plunged into liquid nitrogen. A total of 350 embryos were frozen and studied for eachigroup. It should be mentioned that the level of liquid nitrogen was assessed by a special ruler that was specified for the nitrogen tank, and once a week, the level of liquid nitrogen was tested to ensure that it covered the surface of the canisters. In addition, for everyigroup, there was a specific canister. It means, for example, the canister of "6 months"igroup was separated from otherigroups and removing a canister, didn't cause warming of the other canisters.

Thawing and cryoprotectant dilution

After specified storage duration (24hours, 1 week, 2 weeks, 1 month, 3 months, 6 months), embryos were thawed. For thawing, straws were taken out of liquid nitrogen and immediately plunged into water at 25°C. After 10 sec, the straws were removed from the water, quickly wiped dry and the contents of the straws were expelled into a watchiglass containing 0.8 ml of sucrose solution, by cutting the cotton plug with scissors. After about 1 min the embryos were pipetted into a fresh sucrose solution for 3-4 min.

Assessment of viability of vitrified mouse embryos

Embryos recovered after vitrification were washed and cultured in warm T6 medium (containing 4 mg/ml BSA) under paraffin oil in a culture dish, in an incubator at 37°C, in an atmosphere of 5% C02 in air. Within 4-6 hours the embryos were assessed for their morphological appearance under a stereomicroscope (Hund-Wetzlar, Geermany) and their viability was determined.

Chromosome analysis

After 4-6 hour in T6 medium, first, the embryos were plunged in acid Tyrode's solution for 7 seconds in 3 steps to slenderize zona pellucida, then the embryos were exposed to the T6 medium containing colcemid for 48 hours; after that embryos were placed in hypotonic solution (1% sodium citrate in water) until swollen (3-15 min). The swollen embryos were individually placed on cleaniglass microscope slide in a minimal amount of fluid and then spread using the fixation solution described previously by Tarkowski (1966). The slides were stained withigiemsa (3%), and examined under a light microscope (Nikon E800, Japan) at x 1000 magnification for numerical chromosome analysis.

Statistical Analysis

Survival rates of 6igroups were analyzed using the test. Chromosome abnormalities were analyzed by test and Fisher test to determine the effect of vitrification on chromosomal abnormalities at various storage durations. These analyses were done with SPSS software.

RESULTS

Chromosome abnormalities

Viability

The viability rates for frozen embryos in the 6igroups are summarized in table 1. As seen, by increasing storage duration, viability percentage decreases. For example, in "24 hour"igroup this value is 90.1% and in "6 months" group is 15.8% (Table 1 and Fig. 1).



There is a decrement from controligroup to 6 monthsigroup; although there is not a sµgnificant difference in 1 and 2 week groups compared to control.

The number of embryos showing chromosome abnormalities (aneuploidy and polyploidy), are summarized in table 2. As indicated, increasing storage duration, increases the incidence of aneuploidy and polyploidy. In Figure 1 there is an upward trend from controligroup (16.7%) to 6 monthigroup. It reached a plateau in 1 and 2 weekigroup at 42.2% and 43.6%. The incidence of abnormality in 6 monthsigroup was the hµghest comparing to controligroup (P<0.001). There are sµgnificant differences of aneuoploidy between the test and control group. According to Figure 1, compared to controligroup there was a sµgnificant difference in total abnormalities rate of 24 hour, 1 week, 2 weeks, 1 month, 3 month and 6 monthigroup (all P-values were <0.001)(Table2 ,Fig. 1).


DISCUSSION

Freezing viability

Storage duration was found to be the most important factor in freezing 8-cell mouse embryos. According to our results increasing the storage duration caused a decreased post-thaw survival of 8-cell mouse embryos (Table 1, Fig. 1).

Multiple factors, such as stimulation protocols, freezing procedure and embryo quality at freezing may influence the success of an embryo freezing program (Hu et al. 1999).

The freezing and warming of cells imposes a concatenation of stresses including equilibrating with a cryoprotectant, cooling, warming, dilution and rehydration (Cohen 1992). However, freezing and thawing sµgnificantly reduces embryo viability (Selick et al. 1995). Some embryos do not survive freezing and thawing, and in some cases the majority of blastomeres undergo degeneration (Whittingham 1974). The adverse effects of cryopreservation may also lead to the formation of cracks in the zona pellucida, or injuries to the cell membranes and intracellular components (Ng et al. 1988, Dumoulin et al. 1994). In some other studies, protective polymers (Dumoulin et al. 1994, Titterington and Robinson 1996) or host zonae (Niemann 1991) have been shown to improve cryosurvival rates. Ideally the freeze-thaw procedure should not cause any loss of viability, or lead to an increased incidence ofigenetic aberrations, fetal malformations or losses. Several lines of evidence suggest that the damage may be more closely linked to the freezing properties of cryoprotectant solution (Bongso et al. 1988, Herskovits and Harrington 1972).

Intracellular ice is more likely to be formed when the permeation of the cryoprotectant and its concentration in the cytoplasm are insufficient.

Mohr and Trounson (1981) concluded that cryoinjury to embryos may be selective for one cell type within an embryo, and the extent and nature of damage was dependent on the developmental stage. Another research suggests that excellent quality embryos, frozen and thawed at the 8- to 16-cell stage, often do not develop in vitro without full protection of an intact zona pellucida. For early stage embryos, it is thought that the zona pellucida helps to maintain cellular integrity of the blastomerees (Vital and Myers 1997). Artificially induced alterations in the integrity of the zona pellucida may affect subsequent embryo cleavage, compaction, blastocyst formation and zona hatching of human embryos (Cohen 1992).

The results of our experiment demonstrate that there is a possibility of zona injury that may reduce post-thaw viability; furthermore increased storage duration causes reduced viability that can be due to cryprotectant permeating or as mentioned, zona pellucida damages. Our results are in line with the recent report of Coutinho et al. (2007) who compared two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscopy. They have clearly shown that vitrification caused more membrane permeability, more severe cellular alterations and reduced embryonic viability compared to a slow freezing method.

Chromosome abnormalities

We have shown that vitrification in solution containing ethyleneiglycol can cause severe chromosomal aberrations and reduced embryo viability in vitro. The incidence and severity of the damage is influenced by the storage duration (Table 2, Fig 1). The hµghest incidence of chromosomal aberrations occurred among embryos in the "6 month"igroup. These damages appear more closely linked to factors such as ice crystal formation during storage duration in vitrification. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, disgeny, and arrest of cleavage (Chen et al. 2003). It is known that the cytoskeleton of mammalian oocytes and embryos is thermo- and chemo-sensitive and sensitive to stresses connected with cryopreservation (Pickering and Johnson 1987, Sathananthan et al. 1988, Vander et al. 1988). Disorganization of spindle after cryopreservation was observed in metaphase II oocytes and late 2-cell embryos in mitosis (Sathananthan et al. 1988).

The unequal distribution of chromosomes, between daughter cells results in aneuploidy at multipolar mitosis or in a polyploid unipolar mitosis (Khromenkova et al. 2003). It is known that aneuploidy is one of the causes of low implantation rate and early embryonic mortality in mammalian species (Bongso et al. 1988).

In some reports major chromosomal anomalies such as trisomy of chromosome 13, 18 and 21, as well as major and minor congenital malformations, have been identified in fetuses and babies conceived from frozen embryos (Sutcliffe et al. 1995, Wennerholm et al. 1997). On the other hand, a comparison of babies resulting from the transfer on frozen-thawed embryos with those conceived normally or from fresh IVF cycles showed a similar or even decreased incidence of congenital abnormalities after cryopreservation (Wada et al. 1994, Wood 1997).

A study of aneuploidy and mosaicism of chromosomes X, Y and 1 in human frozen embryos (day 2 and 3 of development) using fluorescence in situ hybridization (FISH) showed that a large proportion of thawed embryos (57%) exhibiting cleavage arrest during the first 24 h of in vitro culture carried numerical chromosomal abnormalities (Hu et al. 1999). However, it remains uncertain whether those aberrations were induced by the cryopreservation technique, or were already present in chromosomally abnormal embryos, before thawing. Several reports on mouse embryos have shown that vitrification may cause some chromosomal damage (Show and Kola 1991) or increased mitotic crossing over (Bongso et al. 1988, Ishida et al. 1997).

From our study, it seems that freezing and thawing is responsible for blastomere fusion. According to our results blastomere fusion is not only attributed to fair and poor quality embryos, as was previously thought (Trounson 1984) but it can also affect morphologically sound embryos, as was shown by our observation (all embryos were ofigood quality) (Balakier et al. 2000). Theigeneral studies have proven that a defect in the cell membrane is required for initiation of fusion, which can be induced by many membrane disrupting agents such as a virus, polyethyleneiglycol or electric field, as well as freezing and thawing (Hui et al. 1981, Zimmermann 1996). Based on these reports, it seems that cryoprotectants may also contribute to the process of fusion by causing cell dehydration and osmotic swelling which induce other changes that are necessary to induce fusion (for instance, changes in the cytoskeleton, tight contact between cells, etc).

This study has demonstrated that vitrification of mouse embryos using ethyleneiglycol as cryoprotectant can lead to induction of chromosomal aberrations. In addition, rapid cooling, warming and dilution solutions can cause the problem of injury to the zona pellucida which is very sensitive to cryoprotectants and low temperature. However, the morphological survival rate of embryos was reduced by increasing storage duration. The reason for this is not clear, but may be attributed to the ice crystal formation during freezing and sudden extremes of cooling. Further research is warranted to improve the long term survival and preservation of the chromosomes in embryo vitrification using ethyleneiglycol as cryoprotectant.

ACKNOWLEDGEMENT

This research was partly supported by Royan Institute.

 

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* Corresponding author: Dr. Hossein Mozdarani; Department of Medicaligenetics, School of Medical Sciences, Tarbiat

Modares University, Tehran, P.O.Box 14115-111, Iran. Fax:  (+98 21) 88006544. e-mail: Mozdarah@modares.ac.ir, Or, mozdarani_h@yahoo.com

Received: May 15, 2006. In revised form: July 15, 2007. Accepted: November 12, 2007

 

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