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
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Abstract |
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Key words: blastocyst/cryopreservation/IVF/mouse/oocyte
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Introduction |
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Oocyte cryopreservation has been applied with varying success to a number of different species including the human (Gook et al., 1993; Bouquet et al., 1995
). Several studies typically reported different rates of survival (2080%), fertilization (3060%) and cleavage (32100%) in human oocytes (Fabbri et al., 2000
). 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, 2002). 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., 1999
).
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 frozenthawed oocytes could then be subjected to cryopreservation followed by thawing when needed. Thus, embryos generated from these frozen oocytes would undergo freezingthawing more than once. The aim of this study was to assess the efficacy of this process in a mouse model.
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Materials and methods |
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Oocyte cryopreservation
Oocytes in study group C were cryopreserved using a slow freezing method (Fabbri et al., 2001). 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 Whittinghams 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 1012 week old CB6F1 males, and sperm were recovered into Whittinghams medium by pressing each cauda epididymis with a pair of forceps. The sperm were incubated (37°C, 5% CO2 in air) in Whittinghams medium under paraffin oil for 1.5 h to allow capacitation.
Control as well as frozenthawed oocytes were transferred to plates containing 200 µl Whittinghams medium (1012 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 12x106 cells/ml. For groups A and B, insemination was performed 13.515 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 (1012 oocytes in each drop) of Whittinghams medium covered with paraffin oil. On the following day, the number of oocytes that had reached the 2-cell stage was recorded. These 24-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 HTFHEPES 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 HTFHEPES 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 (510 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 Whittinghams medium.
Blastocyst culture
Fresh (group A) as well as frozenthawed (groups B and C) embryos were cultured in Whittinghams 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 Normarskis 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 Pearsons 2-test. Blastulation rates were compared also with the statistical linear-by-linear test. P < 0.05 was considered as statistically significant.
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Results |
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Blastulation rate in group A was seen in 37/83 24-cell embryos (45%). This was significantly different (P < 0.01) from groups B and C where development to the blastocyst stage from 24-cell stage embryos was 27/118 (23%) in group B and similarly 8/35 (23%) in group C (Table I). Thus frozenthawed embryos attained similar blastulation rates regardless of whether they originated from fresh (group B) or frozenthawed (group C) oocytes.
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Discussion |
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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, 1991; Wood et al., 1992
; Gook et al., 1993
; Baka et al., 1995
; Bouquet et al., 1995
; Eppig and OBrien, 1996
) and reflects damage to the oocytes 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., 1993). 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, 1987; Baka et al., 1995
). However, fluorescence microscopy has demonstrated that most oocytes that survived cryopreservation had a normal spindle (Gook et al., 1993
) and normal chromosomal status (Gook et al., 1994
). Other targets of oocyte cooling damage include damage to cortical granule vesicles (Vincent and Johnson, 1992
), aneuploidy (Bouquet et al., 1995
), zona hardening (George and Johnson, 1993
) and chilling damage which is associated with Ca2+ oscillation (Ben-Yosef et al., 1995
).
Nevertheless, recent advances (Fabbri et al., 2001; Wininger and Kort, 2002
) indicate that the clinical applications of oocyte cryopreservation could broaden in the future. This will result in a larger number of embryos obtained from frozenthawed 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., 1998
). The need to cryopreserve embryos obtained from frozenthawed 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., 1989
) and polyploidy (Glenister et al., 1987
; Bouquet et al., 1995
).
No study has previously prospectively examined the outcome of double oocyte freezing. A few human clinical pregnancies following two embryo freezethaw cycles have been reported (Check et al., 2001; Farhat et al., 2001
; Yokota et al., 2001
; Estes et al., 2003
). Normal in vitro development from 816-cell mouse embryos to the blastocyst stage was reported even after three successive embryo freezethaw cycles (Vitale et al., 1997
).
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 frozenthawed 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, frozenthawed oocytes behave like fresh ones.
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Acknowledgements |
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References |
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Submitted on July 28, 2003; resubmitted on October 17, 2003; accepted on November 24, 2003.
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