Refreezing of murine intact and biopsied embryos by rapid-freezing procedure

M.A. Nowshari1,3 and G. Brem2

1 Interuniversitäres Forschungsinstitut für Agrarbiotechnologie, Tulln and 2 Ludwig Boltzmann Institute für immuno-, zyto- und molekulargenetische Forschung, Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vivo development of murine morula stage embryos frozen-thawed once or twice and embryos biopsied after one freezing cycle and refrozen was studied. Embryos (n = 860) were cryopreserved using a rapid-freezing procedure. At least 24 h after freezing, embryos were thawed and cultured in vitro for 3 h. In experiment I, morphologically intact embryos were either transferred (n = 180) into recipients or refrozen (n = 160). Unfrozen embryos (control group, n = 180) and refrozen embryos stored for at least 24 h and then thawed, were transferred into recipients. In experiment II, embryos frozen once were thawed and biopsied or sham-biopsied (n = 230 and 180 respectively) and refrozen (n = 226 and 179 respectively). They were thawed and transferred (n = 192 and 160 respectively) into recipients. Recipient mice were either killed on day 15 after embryo transfer and number of implantation sites and live fetuses recorded or pregnant recipients (n = 6, experiment II) were allowed to carry the fetuses to term. There was no difference in the survival rate of embryos at thawing between those frozen once or twice (91 versus 93%). The implantation rate and number of live fetuses in the pregnant recipients at necropsy among those transferred with unfrozen embryos (57% and 51%; 8/9), embryos frozen once (55% and 45%; 8/9) or twice (51% and 48%; 6/8) was not different. There was no difference in the survival rate of refrozen embryos biopsied or sham-biopsied after one freezing cycle (89 versus 87%). The implantation rate and number of live fetuses in pregnant animals transferred with biopsied or sham-biopsied embryos was not different (64 and 41% versus 57 and 37% respectively). All six pregnant animals allowed to carry the fetuses to term delivered normal live fetuses (n = 39). On mating 12 females with six males of the progeny born out of biopsied embryos, all became pregnant and delivered live fetuses. It may be concluded that murine biopsied and intact embryos can be successfully refrozen by rapid-freezing procedure.

Key words: biopsy/cryopreservation/mice/micromanipulation/refreezing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryo biopsy and single-cell genetic analysis now make it possible to screen human and animal embryos at preimplantation stages of development. In the human, preimplantation genetic diagnosis (PGD) techniques are being used to screen the embryos for diagnosing genetic defects causing a number of inherited diseases and other genetic conditions (Handyside and Delhanty, 1997Go). Biopsy of cells from cleaving embryos (Wilton and Trounson, 1989Go; Krzyminska et al., 1990Go; Roudebush et al., 1990Go; Krzyminska and O'Neill, 1991; Takeuchi et al., 1992Go) or from the trophectoderm of blastocysts (Monk et al., 1988Go) is a successful method of providing material for genetic analysis without impairment of subsequent development of the biopsied embryos in vitro and in vivo. Preimplantation embryo biopsy found its first clinical application in human prenatal diagnosis and sex determination of preimplantation embryos derived from IVF (Handyside et al., 1990Go). Single gene defects can also be detected in biopsies (Holding and Monk, 1989Go; Gomez et al., 1990Go). Because of the small quantity of cellular material required, polymerase chain reaction (PCR) or other methods of single gene detection (Penketh et al., 1989Go) may be widely used, although cytogenetic analysis of biopsied material (Roberts et al., 1990Go) may be useful in cases of high risk chromosomal translocations. Fluorescence in-situ hybridization technique (FISH) has also been successfully used for PGD (Harper et al., 1995Go). The same human blastomeres have been used for repeated FISH for PGD (Liu et al., 1998Go). PGD using a single cell biopsy is preferable to the more conventional amniocentesis or chorionic villus sampling at a later stage of pregnancy because only healthy embryos are transferred, thus avoiding later decisions on termination. Infertile patients with a poor prognosis (who have undergone three or more IVF failures) have been shown by preimplantation screening (FISH) of the embryos to have a high percentage of chromosomally abnormal embryos (Gianaroli et al., 1997Go). It has also been shown that there is a progressive loss of chromosomally abnormal embryos during preimplantation development and there is association between chromosomal abnormality and embryo development (Almeida and Bolton, 1996Go). In domestic animals, embryo biopsy is generally used for the determination of sex. Transferring embryos of specific gender is economically beneficial and has herd management advantages. With the success of nuclear transfer in domestic animals, PGD will gain much importance for screening of embryos for genetic defects or confirming the transgenic status of the embryos.

Depending on which type of genetic analysis is to be used and the time required to perform it, biopsied embryos may require cryopreservation. Human frozen–thawed embryos with a reduced number of cells develop and implant normally (Trounson and Mohr, 1983Go). Cryopreservation of mouse biopsied embryos has been shown to have no adverse effect on their survival and development (Krzyminska and O'Neil, 1991Go). However, the conventional freezing protocols have not been found to be optimal for freezing of human (Magli et al., 1999Go) and mouse biopsied embryos (Wilton and Trounson, 1989Go). The demonstration that embryos can be efficiently refrozen without killing them offers the opportunity to refreeze those carrying genetic defects with the possibility of repairing the defects at some unspecified time in the future. Retrospective analysis of cryopreserved embryos is increasingly attractive as new molecular probes become available to assay specific inherited defects not previously measurable. Although many types of genetic analysis can now be performed within a few hours, the type, sensitivity and reliability of such analyses continue to improve at a rapid pace. Furthermore, many academic institutions and animal breeding organizations have amassed large numbers of cryopreserved embryos in recent years. The capability to analyse such embryos retrospectively, and refreeze them in order to have time to make the most productive use of such embryos, would also increase their value. In-vitro development of refrozen human (Baker et al., 1996Go), mouse (Fields et al., 1991Go; Vitale et al., 1997Go) and bovine embryos (Vitale et al., 1994Go; Nowshari and Brem, 1999Go) and in-vivo development of mouse intact (Leibo et al., 1991Go) and biopsied (Snabes et al., 1993Go) embryos has been reported. However, in most of these studies conventional freezing procedures, which are time consuming, require freezing protocols which may not be optimal for freezing of biopsied embryos. Therefore, in this experiment in-vivo development of intact and biopsied embryos using a rapid-freezing procedure, previously found to be optimal for freezing different stages of mouse embryos (Nowshari et al., 1995Go; Nowshari and Brem, 1998aGo) and oocytes (Nowshari et al., 1994Go), was studied.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryo collection
Female virgin mice (C57BL x CBA-F1, 25–30 days old) were superovulated with 7.5 IU i.p. eCG (Folligon®; Intervet, Vienna, Austria) and HCG (Ekluton®; Intervet) administered ~48 h apart. The injections were given between 1200 and 1230 h, and lights were on between 0800 and 1900 h. At the time of HCG injection, female mice were individually caged with males and checked for copulation plugs the next morning. Mice were killed by cervical dislocation 68–72 h after HCG injection and embryos flushed as described earlier (Nowshari and Brem, 1998aGo).

For collection of genitalia, flushing and washing of embryos, medium M2 (Whittingham, 1971Go) with 10% fetal calf serum (FCS; Gibco BRL, Life Technologies, Vienna, Austria) was used. For each repetition, embryos from 4–6 mice were pooled and washed three times in the drops of flushing media and examined microscopically at x40. Morphologically intact morula-stage embryos were selected and transferred to fresh drops of flushing media. For both experiments, embryos were collected in 12 replicates. Each experiment was repeated 6–8 times.

Freezing and thawing
The freezing solutions of 1.5 mol/l ethylene glycol (Sigma, Vienna, Austria) with 0.25 mol/l sucrose (Merck, Darmstadt, Germany), 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and thawing solution of 0.5 mol/l sucrose were made with medium M2. For freezing and refreezing of embryos, freezing solutions were supplemented with 10% heat inactivated FCS. All manipulations were performed in 35 mm plastic Petri dishes (Nunc, Roskilde, Denmark) at room temperature (22–23°C).

For freezing and refreezing of embryos, a simple rapid-freezing procedure was adopted (Nowshari and Brem, 1998bGo). In short, embryos were pre-equilibrated in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose at room temperature (22–23°C) for 5 min. Embryos were then loaded directly with a mouth controlled glass pipette into the middle of a 0.25 ml freezing straw (Mini-tüb, Landshut, Germany) containing 50 µl of ethylene glycol (7.0 mol/l with 0.5 mol/l sucrose). The two ends of the straw were filled with 60 µl of 0.5 mol/l sucrose, which was separated from the medium containing the cryoprotectant by air bubbles. The straws were heat-sealed and immediately (45–60 s) dipped, slowly (to avoid cracking) and vertically, into the liquid nitrogen for periods not less than 24 h.

Straws with frozen embryos were thawed in a water bath at 20°C (Nowshari and Brem, 2000bGo). The cryoprotectants were diluted in a single step by emptying the straws into 0.5 mol/l sucrose solution. Embryos were left in this medium for 5 min and then washed three times in M2.

Embryos (n = 10–15) were transferred to 50 µl of M16 supplemented with 4 mg/ml BSA (Crystalline, ICN, Meckenheim, Germany). The droplets of M16 were maintained in an incubator with 5% CO2 in air for 3 h. Embryos were examined for their morphology and either transferred to recipients or refrozen (experiment I) or biopsied/sham-biopsied and refrozen (experiment II). Refrozen embryos were thawed as described above, examined for their morphology and transferred to recipients. Non-cryoprotectant exposed embryos (control) collected in M2 with FCS were left in droplets of M16 supplemented with BSA until their transfer to recipients.

Micromanipulation
The embryos (early morula stage) were biopsied under an inverted microscope (ID 35; Leica, Heidelberg, Germany) equipped with Leitz micromanipulators (Leitz, Wetzler, Germany), attached to a Narishige IM-6 micro-injector. The holding and aspiration pipettes were made from glass capillaries (Hilgenberg, Malsfeld, Germany) on model P-97 Sutter micropipette puller (Sutter, Novato, CA, USA). The holding pipettes had an outer diameter of 60–80 µm and an inner diameter of 30–40 µm and were polished on the microforge (Bachhofer, Reutlingen, Germany). The pulled aspiration pipettes were cut with the microforge at the place where the outer diameter was 20–25 µm. A bevel angle of 30° was made with a microgrinder (Sauer, Reutlingen, Germany). The inner diameter of the aspiration pipette was about 15–20 µm. The aspiration pipette was connected to 800 µl Narishige micrometer syringes (Narishige, Tokyo, Japan) by plastic tubing filled with lightweight paraffin oil (BDH; Promochem, Wesel, Germany).

Embryos (n = 10–15 at a time) were placed into a drop of medium (M2 without Ca2+ and Mg2+) covered with paraffin oil, positioned on a manipulation chamber mounted on the microscope stage. Embryos were fixed with a holding pipette and the aspiration pipette was forced through the zona pellucida and one blastomere was drawn into the pipette with a gentle suction which was then removed from the embryo. During sham-biopsy, aspiration pipette was forced through the zona pellucida but was withdrawn without removing a blastomere from the embryo.

After the biopsy procedure, the manipulated embryos were transferred to fresh M16 medium and cultured at 37°C in an incubator with an atmosphere of 5% CO2 in air and humidity of 95%. Survival after biopsy was assessed 3 h later under an inverted microscope at a magnification of x100. An embryo was considered to have survived the biopsy procedure if, by inspection under light microscope, all the remaining blastomeres were intact and zona pellucida was not lost.

Biopsied embryos were refrozen immediately after in-vitro culture for 3 h by the same freezing procedure as described above. Refrozen embryos were stored for at least for 24 h before thawing and subsequent transfer into recipients.

Embryo transfer to recipients
Embryos at morula stage were transferred to pseudopregnant recipients. Recipient mice received only (i) unfrozen (control), or (ii) intact frozen–thawed, or (iii) biopsied embryos. Embryos were transferred to the uterine horn (8–10 embryos per oviduct) of C57 BL x CBA-F1 recipients on day 3 of pseudopregnancy, as previously described (Hogan et al., 1986Go). Pseudopregnancy was induced by mating with proved vasectomized Him-OF1 males. The recipients were either necropsied on day 15 of pregnancy, when the numbers of implantation sites and live fetuses were counted, or were allowed to carry the fetuses to term. Early fetal resorption was confirmed by dipping the uterus in a solution of ammonium sulphate (10%) to make implantation sites visible. Each fetus was examined for size and any visible abnormality.

Statistical analysis
Differences in survival rate, implantation rate and proportion of live fetuses at necropsy between treatment groups were tested for significance with the {chi}2 test. The level of significance was set at 5%.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experiment I
There was no difference in the survival rate of embryos frozen–thawed once or twice (382/420, 91% versus 167/180, 93%) on thawing. The transfer of embryos frozen once or twice resulted in comparable pregnancy rates of 89 and 75% respectively. The implantation rates (55 versus 51%) and development of embryos frozen–thawed once or twice (45 versus 48%) to live fetuses in pregnant animals on day 15 of pregnancy was not different between the two groups (Table IGo). The rate of implantation and development to live fetuses of embryos frozen–thawed once or twice was comparable to unfrozen embryos. All the fetuses recovered were of normal size and no macro-morphological abnormalities were observed in any of the fetuses.


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Table I. Implantation rate and development of mouse morula-stage embryos frozen once or twice and unfrozen embryos (experiment I) and embryos biopsied or sham-biopsied after one freezing cycle, refrozen–thawed and transferred to recipients (experiment II)
 
Experiment II
Of the 440 embryos that underwent one cycle of cryopreservation, 410 (93%) were intact and suitable for biopsy on thawing. Embryos (n = 230 and 180 respectively) were biopsied or sham-biopsied and were refrozen (n = 226 and 179 respectively) within 3 h of biopsy. During this period embryos were left in medium M16 + 4 mg/ml BSA in an incubator gassed with 5% CO2 at 37°C. On thawing of biopsied/sham-biopsied embryos, the rate of survival of biopsied and sham-biopsied embryos was not different (89 versus 87%). The transfer of biopsied and sham-biopsied embryos to recipients resulted in comparable pregnancy rates between the two groups (10/12 animals, 83% and 8/10 animals, 80% respectively).

Six of the recipients which became pregnant after transfer of biopsied embryos were allowed to carry the fetuses to term, while the rest were necropsied on day 15 after transfer. The transfer of biopsied and sham-biopsied embryos to recipients resulted in comparable implantation rates and survival rates of live fetuses (Table IGo). The six mice allowed to carry the fetuses to term gave birth to 39 normal live fetuses. None of the fetuses born had any macromorphological abnormalities and the sex ratio was normal (1:1 male to female). After mating at 8 weeks of age of six males with 12 females born from biopsied embryos, all the females became pregnant and delivered 84 (43 males and 41 females) live fetuses. All living young of the second generation were also morphologically normal.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, it has been demonstrated that mouse intact and biopsied embryos can be refrozen with a simple rapid-freezing procedure. It has already been shown that this freezing procedure is very effective for freezing of mouse oocytes and embryos at different stages (Nowsahri et al., 1994, Nowsahri et al., 1995; Nowshari and Brem, 2000aGo) and bovine blastocysts (Nowshari and Brem, 1998bGo). Using this freezing procedure, no difference was recorded in the morphological survival of embryos frozen once or twice. Even biopsy of embryos after one freezing cycle did not affect the morphological survival of embryos when they were again frozen and thawed.

A comparable morphological survival of mouse embryos frozen twice was reported earlier (Fields et al., 1991Go; Leibo et al., 1991Go; Vitale et al., 1997Go); however, in all of these studies conventional freezing procedures were used. A higher proportion of zona damage of embryos frozen twice or three times with conventional freezing procedure, using a small number of embryos, was reported (Vitale et al., 1997Go). Intact embryos frozen once or twice with a rapid-freezing procedure in this experiment and transferred to recipients developed to live fetuses, indicating that embryos can be refrozen using this freezing procedure and an acceptable in-vivo survival of embryos can be expected upon thawing and transfer to recipients. Previously the in-vivo development of mouse embryos refrozen with a conventional freezing procedure was reported in only one study (Liebo et al., 1991). The in-vivo survival of twice frozen embryos in that study was comparable to embryos frozen twice with rapid-freezing procedure in this study. Embryo refreezing may find a practical application in embryo transfer programmes in human and domestic animals. Embryos which have been thawed accidentally or which could not be transferred to recipients because of their unsuitability, ascertainable only after thawing, need not be discarded, but rather refrozen and if suitable transferred next time.

After biopsy of once frozen–thawed embryos, 89% of biopsied embryos remained intact. This survival rate appeared to be the same between sham-biopsied embryos in experiment II and intact embryos frozen once or twice in experiment I. This indicates that embryos which do not have intact zonae pellucidae can also survive freezing and thawing procedures and can further develop in vitro and in vivo in a similar way to frozen–thawed zona-intact embryos. Complete absence of the zona pellucida has been previously shown to affect the survival and development of intact as well as demi-embryos in mouse (Nowshari and Holtz, 1998Go) and goats (Nowshari and Holtz, 1993Go). Poor results have been reported after freezing of mouse embryos with punctured zonae pellucidae using conventional freezing procedure (Wilton et al., 1989). These authors, however, improved the survival and development rate of biopsied embryos with a rapid-freezing procedure using 4.5 mol/l dimethylsulphoxide (DMSO) as cryoprotectant. A comparable survival rate of biopsied and non-biopsied embryos but a reduced developmental rate of mouse embryos which were frozen after biopsy has been reported (Thompson et al., 1995Go). That in-vitro developmental potential of biopsied embryos is not influenced by freezing embryos has also been reported by many authors (Roudebush et al., 1990Go; Krzyminska and O'Neill, 1991; Liu et al., 1993Go). However, these authors just attempted freezing of freshly biopsied embryos. The successful cryopreservation and refreezing of embryos with a punctured zona pellucida could have applications in experimental embryology other than embryo biopsy. It also should be possible to cryopreserve or refreeze mouse embryos after manipulations such as gene injection, embryo splitting, and nuclear transplantation.

The optimal stage of embryonic development at which embryo biopsy can be performed without compromising the developmental capacity is unclear. In this experiment early morula stage embryos were used and intact and biopsied embryos at this stage can be refrozen and biopsied without affecting the in-vivo development of embryos compared with freshly transferred embryos. Removal of a single blastomere at 4-cell (Wilton et al., 1989) and one or two blastomeres at 8-cell stage (Kryzminski and O'Neill, 1991; Liu et al., 1993Go) does not influence the developmental capacity of embryos in vitro and in vivo. However, impairment of the developmental capacity of embryos at blastocyst stage after removal of trophectoderm cells has been reported (Monk et al., 1988Go). Kryzminsky et al. (1990) reported that 8-cell stage mouse embryos are more suitable for embryo biopsy than 4-cell and morula stage embryos. The developmental capacity of frozen–thawed biopsied embryos seems to be mainly dependent on the number of blastomeres remaining in the embryos (Krzyminska and O'Neill, 1991; Liu et al., 1993Go). Removal of more than four blastomeres from an 8-cell stage embryo resulted in low survival of embryos in vitro and in vivo (Liu et al., 1993Go).

Results of this experiment also show that the offspring from biopsied embryos are normal. None of the offspring had any macromorphological abnormality and they developed and reproduced normally, indicating that biopsy did not affect the reproductive performance of the progeny.

In conclusion, the survival and development in vivo of embryos frozen–thawed using a simple rapid-freezing procedure is not influenced by freezing them once or twice. An acceptable survival in vivo of embryos subjected to biopsy after one freezing cycle, refrozen and transferred to recipients after thawing, can be achieved. The results suggest that under certain conditions, it may be possible to utilize cryopreservation and re-cryopreservation of embryos in strategies involving screening of embryos for PGD in human and domestic animals and for other logistic reasons.


    Acknowledgments
 
We thank E.Hörmann and Sandra Schönkhammer for animal maintenance and husbandry.


    Notes
 
3 To whom correspondence should be addressed at: Biotechnology in der Tierproduktion, Interuniversitäres Forschungsinstitut für Agrarbiotechnologie, Konrad Lorenz Str. 20, 3430-Tulln, Austria. E-mail: Nowshari{at}ifa-tulln.ac.at Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 11, 2000; accepted on September 7, 2000.