Mouse embryos generated from frozen–thawed oocytes can successfully survive a second cryopreservation

Ariel Revel1, Naama Moshe, Aharon Helman, Anat Safran, Alex Simon and Moriah Koler

In Vitro Fertilization Unit, Department of Obstetrics and Gynecology, Hadassah University Hospital, Ein Kerem, Jerusalem, Israel

1 To whom correspondence should be addressed at: In Vitro Fertilization Unit, Department of Obstetrics and Gynecology, Hadassah University Hospital, Ein Kerem, P.O.Box 12000, Jerusalem 91120, Israel. e-mail: Revel{at}md.huji.ac.il


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: To determine whether mouse embryos generated from frozen–thawed oocytes can successfully survive a second cryopreservation. METHODS: Immature C57BL6*BALB/c female mice underwent superovulation and the collected oocytes were divided into three groups. Group A oocytes (n = 107) underwent IVF. Group B oocytes (n = 167) underwent IVF and embryos generated were then cryopreserved. Group C oocytes (n = 94) were cryopreserved, thawed and underwent IVF. Two–four-cell stage embryos were re-cryopreserved and thawed. Embryos from all groups were then cultured to the blastocyst stage. RESULTS: Cleavage rates to the 2–4-cell stage were 78, 71 and 46% for groups A, B and C respectively. Blastulation rates from 2–4 cell-stage embryos were 37/83 (45%), 27/118 (23%) and 8/35 (23%) for groups A, B and C respectively. Development to blastocysts was observed in 37/107 oocytes (35%), 27/167 oocytes (16%) and only 8/94 oocytes (9%) for groups A, B and C respectively. CONCLUSION: Oocyte cryopreservation results in reduced fertilization rates. Embryo cryopreservation reduces blastulation rates by half regardless of whether the oocytes were fertilized fresh or frozen–thawed. Nevertheless, embryos generated from cryopreserved oocytes can survive cryopreservation and develop to the blastocyst stage at rates comparable with embryos obtained from fresh oocytes.

Key words: blastocyst/cryopreservation/IVF/mouse/oocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gamete and embryo cryopreservation expand the possibilities of modern assisted reproductive techniques. It is well recognized that the ability to cryopreserve unfertilized human oocytes would make a significant contribution to infertility treatments. Oocyte cryopreservation is a possible solution for the ethical problems related to embryo storage, and the only available technique for preserving fertility in young women who have to undergo chemotherapy or radiotherapy. Pregnancies from cryopreserved oocytes have been reported after thawing, insemination and transfer of the subsequent embryos (Porcu et al., 2000Go). The recently reported pregnancies obtained by human oocyte cryopreservation are encouraging (Wininger and Kort, 2002Go).

Oocyte cryopreservation has been applied with varying success to a number of different species including the human (Gook et al., 1993Go; Bouquet et al., 1995Go). Several studies typically reported different rates of survival (20–80%), fertilization (30–60%) and cleavage (32–100%) in human oocytes (Fabbri et al., 2000Go). This variability of results raises doubts regarding the usefulness of oocyte cryopreservation in IVF treatment cycles. The main problems with oocyte cryopreservation concern the survival and fertilization rates although the introduction of ICSI led to an increase in these rates. Further investigation of various biophysical changes during oocyte cryopreservation could improve success rates.

Recently, fundamental studies on the effects of cooling, membrane permeability, cryoprotectant addition and ice formation have been performed on human oocytes (Wininger and Kort, 2002Go). It is likely that successful human oocyte cryopreservation will only follow once these factors are fully understood, but the existing base of knowledge should provide a platform for further improvements in the techniques currently employed. The accurate determination of the freezing conditions that promote intracellular ice formation is crucial for designing cryopreservation protocols for oocytes (Trad et al., 1999Go).

If better oocyte survival after cryopreservation is obtained, an increased fertilization rate would be anticipated with an increased number of embryos being found suitable for transfer. All spare embryos generated from the frozen–thawed oocytes could then be subjected to cryopreservation followed by thawing when needed. Thus, embryos generated from these frozen oocytes would undergo freezing–thawing more than once. The aim of this study was to assess the efficacy of this process in a mouse model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Superovulation and study groups
Approval from the animal facility ethics committee at the Hebrew University Medical School was obtained. CB6F1 (C57BL6*BALB/c) female mice aged 4–5 weeks were injected i.p. with 5 IU of pregnant mare’s serum gonadotrophins (PMSG) (Sigma Chemical Co., USA) followed by 5 IU of hCG (Chorigon; Teva, Israel) given 46–48 h later. Cumulus–oocyte complexes (COC) were collected from the Fallopian tubes 12–14 h after hCG injection and transferred into M-2 medium (Sigma, USA) supplemented with 4 mg/ml bovine serum albumin (BSA) (Sigma, USA). The oocytes were randomly divided into the following three groups. Group A (control oocyte group): oocytes (n = 107) were inseminated and cultured to the blastocyst stage in Whittingham’s medium according to Hogan et al. (1994)Go. Group B (embryo cryopreservation group): fresh oocytes (n = 167) underwent IVF and the resulting embryos were cryopreserved at the 2–4-cell stage, then thawed and cultured in Whittingham’s medium to the blastocyst stage (Whittingham, 1971Go). Group C (double-freezing group): oocytes (n = 94) were cryopreserved and thawed. Surviving oocytes underwent IVF and the developed embryos were frozen at the 2–4-cell stage. Subsequently, these embryos were thawed and assessed for their blastulation potential in Whittingham’s medium.

Oocyte cryopreservation
Oocytes in study group C were cryopreserved using a slow freezing method (Fabbri et al., 2001Go). Briefly, oocytes were denuded from corona and cumulus cells in M-2 solution (Sigma, USA) containing 4 mg/ml BSA and 300 µg/ml hyaluronidase. Following denudation, oocytes were rinsed with PBS solution supplemented with 12 mg/ml human serum albumin (HSA) (Sigma, USA). Oocytes were then transferred to equilibration solution [PBS with 1.5 mol/l 1,2-propandiol (PROH) and 12 mg/ml HSA] for 10 min at room temperature, followed by 15 min in loading solution (PBS with 1.5 mol/l 1,2-PROH, 0.3 mol/l sucrose and 12 mg/ml HSA) at room temperature. The oocytes were loaded into cryo-tubes containing 0.25 ml loading solution. Cryopreservation was performed using a slow-freezing protocol in a programmed biological freezer (Planer Kryo 10; Planer products Ltd, UK) using the following cryopreservation protocol: starting temperature was 23°C, and the temperature was reduced at a rate of 2°C/min down to –7°C. At that point, manual seeding was performed. After seeding, the temperature was reduced by 0.3°C/min to –30°C followed by a decline rate of 50°C/min to –150°C. The tubes were then lowered to liquid nitrogen containers.

Thawing was performed by holding tubes in the air for 60 s and then in a 35°C water-bath for a further 90 s. Oocytes were moved into PBS with 1 mol/l PROH, 0.3 mol/l sucrose and 12 mg/ml HSA for 5 min at room temperature. The oocytes were transferred to PBS containing 0.5 mol/l PROH, 0.3 mol/l sucrose and 12 mg/ml HSA, for another 5 min at room temperature, followed by 10 min in PBS containing 0.3 mol/l sucrose and 12 mg/ml HSA. The oocytes were then transferred into PBS solution containing 12 mg/ml HSA for 10 min at room temperature and another 10 min in 37°C. Finally, the oocytes were transferred into Whittingham’s medium for incubation at 37°C at 5% CO2 in air.

Oocyte survival was determined by the presence of an intact zona pellucida and a healthy-looking cytoplasm. Survival was also determined by re-expansion of the oocyte leaving a small normal-sized perivitelline space.

IVF
Epididyma were collected from 10–12 week old CB6F1 males, and sperm were recovered into Whittingham’s medium by pressing each cauda epididymis with a pair of forceps. The sperm were incubated (37°C, 5% CO2 in air) in Whittingham’s medium under paraffin oil for 1.5 h to allow capacitation.

Control as well as frozen–thawed oocytes were transferred to plates containing 200 µl Whittingham’s medium (10–12 oocytes in each drop), covered by light paraffin oil, and incubated until insemination. Sperm were added to the insemination plates at a final concentration of 1–2x106 cells/ml. For groups A and B, insemination was performed 13.5–15 h after hCG injection. In the double-freezing group (group C), only oocytes which survived the cryopreservation were inseminated.

Fertilization and embryo growth
Four hours after insemination, oocytes were washed three times and transferred to 60 µl drops (10–12 oocytes in each drop) of Whittingham’s medium covered with paraffin oil. On the following day, the number of oocytes that had reached the 2-cell stage was recorded. These 2–4-cell embryos were further cultured for 72 h, and the rate of blastocyst formation was determined. Assessment of cleavage and embryo development was performed employing inverted contrast microscopy.

Embryo cryopreservation
Embryos from groups B and C were cryopreserved using a slow-freezing technique. The embryos were placed in HTF–HEPES medium containing 12 mg/ml HSA (enriched HTF-Hepes) with 1.5 mol/l PROH for 10 min at room temperature. They were then transferred into cryo-tubes containing 0.25ml of enriched HTF–HEPES medium containing 1.5 mol/l PROH and 0.1 mol/l sucrose, and placed in the programmable freezer. The freezing protocol was the same as for oocytes. Thawing was performed for 60 s in air, and then in a water-bath at 35°C for 90 s. The embryos were transferred to a series of solutions (5–10 min each) containing 0.2 mol/l sucrose and reducing concentrations of PROH (1.0, 0.5, 0 mol/l). The embryos were washed in M-2 medium with 4 mg/ml BSA at room temperature, followed by 10 min incubation in this medium at 37°C. The embryos were finally cultured in Whittingham’s medium.

Blastocyst culture
Fresh (group A) as well as frozen–thawed (groups B and C) embryos were cultured in Whittingham’s medium, at 37°C, 5% CO2 in air, and were allowed to develop to the blastocyst stage for an additional 72 h. The embryos were examined for morphology and cleavage rate. Examination was performed by the same investigator using Normarski’s inverted optics. Good morphology blastocysts were classified as those with a distinct inner cell mass (ICM), a well-differentiated trophectoderm, and a single large blastocoelic cavity on day 3 of development.

Statistical analysis
Statistical analysis was performed using Pearson’s {chi}2-test. Blastulation rates were compared also with the statistical linear-by-linear test. P < 0.05 was considered as statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cleavage rates for groups A, B and C were 83/107 (78%), 118/167 (71%) and 35/77 (46%) respectively. Group C originally included 94 oocytes of which 77 (82%) survived the slow-freezing procedure and were used for the study. Cleavage rate of cryopreserved oocytes (46%) was significantly (P < 0.01) lower when compared to that obtained in fresh oocytes of groups A or B. The cleavage rates in groups A and B were comparable at 78 and 71% respectively (Table I).


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Table I. Cleavage rates and development to the blastocyst stage in mouse embryos obtained from three different groups; embryos developed from fresh oocytes (group A), embryos frozen at the 2–4-cell stage (group B) and embryos obtained from frozen–thawed oocytes, which then underwent a second freezing (group C)
 
Development rates of oocytes to the blastocyst stage were 37/107 in group A (35%), 27/167 in group B (16%) and only 8/94 (9%) in group C. These rates differ significantly between group A and group B or C (P < 0.01) (Table I). These results show linear association (P < 0.001), demonstrating that each cryopreservation step added to the procedure significantly lowered blastulation rate.

Blastulation rate in group A was seen in 37/83 2–4-cell embryos (45%). This was significantly different (P < 0.01) from groups B and C where development to the blastocyst stage from 2–4-cell stage embryos was 27/118 (23%) in group B and similarly 8/35 (23%) in group C (Table I). Thus frozen–thawed embryos attained similar blastulation rates regardless of whether they originated from fresh (group B) or frozen–thawed (group C) oocytes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Various attempts to cryopreserve human oocytes have been performed with conflicting results. Thus, although human oocyte cryopreservation has been attempted for more than two decades (Chen, 1986Go), this technique is still considered an experimental one.

In the present study we demonstrate that cryopreservation of mouse oocytes results in a 32% decrease in cleavage rate (from 78 to 46%). This finding has already been reported (Mandelbaum, 1991Go; Wood et al., 1992Go; Gook et al., 1993Go; Baka et al., 1995Go; Bouquet et al., 1995Go; Eppig and O’Brien, 1996Go) and reflects damage to the oocyte’s intracellular organelles, plasma membrane and the zona pellucida.

Nevertheless, it appears that tolerance for freezing and thawing differs significantly between human and mouse oocytes. Opposing results, in favour of human oocytes, were found when compared with mouse oocytes cryopreserved by the same method (Gook et al., 1993Go). Human oocytes are thus also expected to withstand double cryopreservation better.

The meiotic spindle of mature oocytes has been reported to suffer significant damage during cryopreservation (Pickering and Johnson, 1987Go; Baka et al., 1995Go). However, fluorescence microscopy has demonstrated that most oocytes that survived cryopreservation had a normal spindle (Gook et al., 1993Go) and normal chromosomal status (Gook et al., 1994Go). Other targets of oocyte cooling damage include damage to cortical granule vesicles (Vincent and Johnson, 1992Go), aneuploidy (Bouquet et al., 1995Go), zona hardening (George and Johnson, 1993Go) and chilling damage which is associated with Ca2+ oscillation (Ben-Yosef et al., 1995Go).

Nevertheless, recent advances (Fabbri et al., 2001Go; Wininger and Kort, 2002Go) indicate that the clinical applications of oocyte cryopreservation could broaden in the future. This will result in a larger number of embryos obtained from frozen–thawed oocytes. Since the worldwide tendency is to reduce the number of embryos transferred to the uterus to minimize multiple pregnancies, the demand for embryo cryopreservation is on the rise (Mandelbaum et al., 1998Go). The need to cryopreserve embryos obtained from frozen–thawed oocytes prompts the debate on the effects of double freezing. Little is known about these effects. Cryopreservation of mouse embryos was shown to increase the rates of fertilization failure (Carroll et al., 1989Go) and polyploidy (Glenister et al., 1987Go; Bouquet et al., 1995Go).

No study has previously prospectively examined the outcome of double oocyte freezing. A few human clinical pregnancies following two embryo freeze–thaw cycles have been reported (Check et al., 2001Go; Farhat et al., 2001Go; Yokota et al., 2001Go; Estes et al., 2003Go). Normal in vitro development from 8–16-cell mouse embryos to the blastocyst stage was reported even after three successive embryo freeze–thaw cycles (Vitale et al., 1997Go).

Our study is the first to compare double freezing of oocytes with embryo cryopreservation results. The endpoint chosen was development to the blastocyst stage and thus reflects potential late effects on the oocytes. Moreover, development to the blastocyst stage is known to show a high correlation with the chance of attaining pregnancy. Future experiments will be important to test the pregnancy rates after embryo transfer using such embryos.

The question we tried to answer was whether the oocyte damage caused by the cryopreservation process would present beyond the fertilization. We hypothesized that if the oocyte suffered severe damage in the freezing process, it would have a significantly smaller chance of reaching the blastocyst stage compared with non-frozen oocytes. However, our results have clearly shown that although cryopreserved oocytes had only a 9% chance of attaining the blastocyst stage, this was due to a decrease in fertilization. Indeed the oocytes that successfully fertilized and were re-frozen reached blastulation rates similar to those of fresh oocytes which were frozen–thawed at the 2-cell stage. Taken together, these results clearly show that damage to the oocyte organelles and membranes only prevents fertilization. If fertilization takes place, frozen–thawed oocytes behave like fresh ones.


    Acknowledgements
 
We thank Mrs Tali Bdolach for help with statistical analysis and Mrs June Scher for proofing the English. This work was supported by a grant from HWZOA absorption fund to Ariel Revel.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Baka SG, Toth TL, Veeck LL, Jones HW Jr, Muasher SJ and Lanzendorf SE (1995) Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod 10,1816–1820.[Abstract]

Ben-Yosef D, Oron Y and Shalgi R (1995) Low temperature and fertilization-induced Ca2+ changes in rat eggs. Mol Reprod Dev 42,122–129.[ISI][Medline]

Bouquet M, Selva J and Auroux M (1995) Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development, and chromosomal abnormalities. Mol Reprod Dev 40,110–115.[ISI][Medline]

Carroll J, Warnes GM and Matthews CD (1989) Increase in digyny explains polyploidy after in-vitro fertilization of frozen–thawed mouse oocytes. J Reprod Fertil 85,489–494.[Abstract]

Check JH, Brittingham D, Swenson K, Wilson C and Lurie D (2001) Transfer of refrozen twice-thawed embryos do not decrease the implantation rate. Clin Exp Obstet Gynecol 28,14–16.[Medline]

Chen C (1986) Pregnancy after human oocyte cryopreservation. Lancet 1,884–886.[CrossRef][Medline]

Eppig JJ and O’Brien MJ (1996) Development in vitro of mouse oocytes from primordial follicles. Biol Reprod 54,197–207.[Abstract]

Estes SJ, Laky DC, Hoover LM, Smith SE, Schinfeld JS and Somkuti SG (2003) Successful pregnancy resulting from cryopreserved pronuclear and cleaved embryos thawed and cultured to blastocysts, refrozen and transferred. A case report. J Reprod Med 48,46–48.[ISI][Medline]

Fabbri R, Porcu E, Marsella T, Primavera MR, Rocchetta G, Ciotti PM, Magrini O, Seracchioli R, Venturoli S and Flamigni C (2000) Technical aspects of oocyte cryopreservation. Mol Cell Endocrinol 169,39–42.[CrossRef][ISI][Medline]

Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16,411–416.[Abstract/Free Full Text]

Farhat M, Zentner B, Lossos F, Bdolah Y, Holtzer H and Hurwitz A (2001) Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage: case report. Hum Reprod 16,337–339.[Abstract/Free Full Text]

George MA and Johnson MH (1993) Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition. Hum Reprod 8,1898–1900.[Abstract]

Glenister PH, Wood MJ, Kirby C and Whittingham DG (1987) Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen–thawed oocytes fertilized in vitro. Gamete Res 16,205–216.[ISI][Medline]

Gook DA, Osborn SM and Johnston WI (1993) Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 8,1101–1109.[Abstract]

Gook DA, Osborn SM, Bourne H and Johnston WI (1994) Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 9,684–691.[Abstract]

Hogan B, Beddington R, Costantini F and Lacy E (1994) In vitro culture of eggs, embryos, primordial germ cells and teratocarcinoma cells. In: Manipulating the Mouse Embryo, a laboratory manual. Cold Spring Harbor Laboratory Press Publication, New York 385–414.

Mandelbaum J (1991) Cryopreservation of oocytes and embryos. Curr Opin Obstet Gynecol 3,662–667.[ISI][Medline]

Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, Alnot MO and Salat-Baroux J (1998) Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 13(Suppl 3),161–174; discussion 175–167.[Abstract]

Pickering SJ and Johnson MH (1987) The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 2,207–216.[Abstract]

Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S and Flamigni C (2000) Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 169,33–37.[CrossRef][ISI][Medline]

Trad FS, Toner M and Biggers JD (1999) Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes. Hum Reprod 14,1569–1577.[Abstract/Free Full Text]

Vincent, C and Johnson, MH (1992) Cooling, cryoprotectants, and the cytoskeleton of the mammalian oocyte. Oxf Rev Reprod Biol 14,73–100.[Medline]

Vitale NJ, MyersMW, Denniston RS, Leibo SP and Godke RA (1997) In-vitro development of refrozen mouse embryos. Hum Reprod 12,310–316.[Abstract]

Whittingham DG (1971) Culture of mouse ova. J Reprod Fertil (Suppl 1) 14,7–21.[Medline]

Wininger JD and Kort HI (2002) Cryopreservation of immature and mature human oocytes. Semin Reprod Med 20,45–49.[CrossRef][ISI][Medline]

Wood MJ, Whittingham DG and Lee SH (1992) Fertilization failure of frozen mouse oocytes is not due to premature cortical granule release. Biol Reprod 46,1187–1195.[Abstract]

Yokota Y, Yokota H, Yokota M, Sato S and Araki Y (2001) Birth of healthy twins from in vitro development of human refrozen embryos. Fertil Steril 76,1063–1065.[CrossRef][ISI][Medline]

Submitted on July 28, 2003; resubmitted on October 17, 2003; accepted on November 24, 2003.





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