1 Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan 250021, China, 2 Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and 3 Center for Human Reproduction, North Shore University Hospital, NYU School of Medicine, Manhasset, NY 11030, USA
4 To whom correspondence should be addressed at: Center for Human Reproduction, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030, USA. Email: hfeng{at}nshs.edu
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Abstract |
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Key words: cryopreservation/embryo/human fertilization in vitro/ooctyes
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Introduction |
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In addition, oocyte cryopreservation is a successful alternative for storing the excess of oocytes during the ART therapies, thus avoiding ethical, moral and religious dilemmas. However, in spite of several successes being reported (Porcu et al., 1997; Kuleshova et al., 1999
; Porcu et al., 2000
; Van der Elst, 2003
), there are still technical problems associated with oocyte freezing. Studies have shown that oocyte survival rates after cryopreservation could be affected by morphological and biophysical factors. Morphological characteristics of oocytes such as maturity and size are particularly important. Biophysical factors such as cryoprotectant compositions are also important. For oocyte cryopreservation with slow-freezing method, cryoprotectant solution consists of usually
1.5 M membrane-permeating cryoprotectant (i.e. propanediol) and 0.1 M sucrose. To limit damage occurring during the cryopreservation procedure, it is necessary to reduce the amount of intracellular water by an increasing dehydration process. It was reported that increasing the sucrose concentrations could benefit the survival of the frozen-thawed human oocytes (Fabbri et al., 2001
).
In the present study, in order to evaluate the effects of different concentrations of sucrose in the cryoprotectant solution on the developmental potential of human oocytes at different maturity stages, we have frozen different maturity oocytes with a slow-freezing rapid-thawing protocol using 1.5 M 1,2-propanediol with 0.1 M or 0.2 M sucrose.
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Materials and methods |
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Cryoprotectant solutions
All cryoprotectant solutions were prepared by using Dulbecco's phosphate-buffered solution (DPBS) (Sigma), 1,2-propanediol (PROH) (Sigma) and 5 mg/ml human serum albumin (HSA) (Sigma). We used the following freezing solutions: 1.5 M PROH+5 mg/ml HSA in the PBS solution (S1) and 1.5 M PROH+sucrose (0.1 M or 0.2 M)+5 mg/ml HSA (S2). For thawing procedure, the oocytes were thawed in solutions with decreased PROH concentration gradients of 1.0 M, 0.5 M and 0.0 M in the presence of 0.2 M sucrose (Fabbri et al., 2001).
Retrieval of oocytes
The recovered oocytes from large follicles were used for the patients' in vitro maturation (IVM)/IVF procedure. The oocytes from the small follicles (diameter <1 cm) were included in this study. The participants signed the consent forms. The cells of the cumulus and corona radiata were completely removed by a brief exposure to HEPES-buffered solution containing 80 IU/ml hyaluronidase (Irvine Scientific, USA) and by gentle aspiration in and out of a hand-drawn glass pipette (Sage BioPharma, NJ). The denudated oocytes were then evaluated by assessment of their nuclear maturation stage under the microscope. The assessment included identification of germinal vesicle (GV) stage, metaphase I (MI) stage and metaphase II (MII) stage oocytes. Different maturity oocytes were divided randomly into three experimental groups: Group A was not frozen; Group B was frozen with cryoprotectant solution containing 0.1 M sucrose and 1.5 M PROH; Group C with 0.2 M sucrose and 1.5 M PROH. Both groups (B and C) had the same concentration of 1.5 M PROH.
Cryopreservation and thawing
The oocytes from the two cryopreservation groups were incubated in human tubal fluid (HTF) plus 10% serum substitute supplement (SSS) (Irvine scientific, CA) for no more than 3 h before being transferred to culture dishes containing DPBS supplemented with 5 mg/ml human serum albumin (HSA) (Irvine scientific, CA) at room temperature. After pretreatment in S1 solution for 10 min, the oocytes were transferred to S2 solutions. Then the oocytes were loaded into 0.25 ml plastic straws (IMV-Technologies Lalgle, France) and transferred into an automated program freezer (Planer Kryo-10, England). The time of the oocytes in the cryoprotectant was <20 min in all.
The initial chamber temperature was 20°C in the freezer. The temperature was reduced slowly to 7°C at a rate of 2°C/min. Ice nucleation was induced manually by ice-forceps. After a hold time of 10 min at 7°C, the temperature was cooled slowly to 30°C at a rate of 0.3°C/min followed by rapid cooling to 120°C at a rate of 30°C/min. The straws were transferred into liquid nitrogen tanks and stored until thawing.
All the cryopreserved oocytes were stored in the liquid nitrogen for 6 months. To thaw, the oocytes were air-warmed for 40 s and then immersed in a 30°C water bath for 40 s until all traces of ice had disappeared. The cryoprotectant was removed at room temperature by stepwise dilution of PROH in the thawing solutions. The contents of the melted straws were expelled in the 1.0 M PROH and 0.2 M sucrose solution and equilibrated for 5 min and then in 0.5 M PROH and 0.2 M sucrose for another 5 min. After that the oocytes were transferred to 0.2 M sucrose for 5 min, and placed into the final solution of DPBS supplemented with 5 mg/ml HSA. Finally, the immature oocytes were transferred to the M-199 culture medium for in vitro maturation; the mature oocytes were cultured in HTF for further incubation in a 37°C, 5% CO2 incubator.
Two hours after thawing, the oocytes were checked for survival according to morphological criteria. The oocytes were classified as high-quality oocytes and low-quality oocytes. High-quality oocytes means that the zona pellucida and plasma membrane were intact, the perivitelline space was clear and normal in size and there was no evidence of cytoplasmic leakage or oocyte shinkage, which could be observed under the inverted microscope. They also had good refractive cytoplasm and smooth plasma membrane. In the survived but low-quality oocytes, the perivitelline space was enlarged and the cytoplasm was darker, though refraction was just fair under the dissecting microscope.
In vitro maturation, ICSI and embryo culture
The fresh immature oocytes and frozen-thawed immature oocytes were subjected to (IVM) in M-199 medium supplemented with 20% fetal bovine serum (Gibco, USA), penicillin G 50 IU/ml, streptomycin sulfate 50 µg/ml, pyruvate sodium 25 mM (Sigma, USA), recombinant FSH 0.075 IU/ml, HCG 0.15 IU/ml (Gonal-F; Serono Laboratories) and epidermal growth factor (EGF) 2 ng/ml (Goud et al., 1998). After in vitro culture for 2448 h, the matured high-quality oocytes were subjected to ICSI if the first polar bodies were extruded and the cytoplasm was homogeneous with good refraction.
The thawed MII oocytes were cultured in HTF supplemented with 10% SSS for 24 h, and then ICSI performed. A fertilization check was performed 1620 h later and the 2PN zygotes were followed for another 4048 h in P-1 (Irvine Scientific, USA) for cleavage. According to Puissant's standard for embryo grades, four cell embryos with <30% fragmentation were specified as high quality embryos (Puissant et al., 1987). Embryos in the non-frozen group were cryopreserved for future replacement into the patient's uterus. Some of the embryos in the frozen MII oocytes group were transferred to patients who needed embryo donation, and obtained data will be submitted for publication elsewhere.
Statistical analysis
Differences in the rates of survival, maturation, fertilization, cleavage and embryo quality were analyzed with Fisher's exact test or 2 test as appropriate. SPSS version 10.0 was used for all statistical analyses. Values were considered significant when P<0.05.
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Results |
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The results of the MII oocytes cryopreservation indicated that the 0.2 M sucrose concentration is beneficial for the survival of frozenthawed human oocytes. The survival rate of the frozenthawed MII oocytes in Group C was significantly higher than that in Group B (P<0.01). There was also a significant difference in the cleavage rate of the zygotes from Groups B and C (22.73% versus 55.56%, P<0.05). No such significant difference was found in the fertilization and high-quality embryos rates between Groups B and C. However, cleavage rate and high-quality embryo rate in Group B were much less than that in Group A (P<0.01) (Table I).
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Discussion |
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Several plausible explanations have been offered to address the difficulty of storing oocytes (MII) at low temperatures. First of all, there is evidence that the zona pellucida (ZP) hardens and results in premature cortical granule exocytosis during the cryopreservation process (Pickering and Johnson, 1987; Vincent et al., 1991
). This change halts the penetration of spermatozoa and inhibits embryonic hatching. This problem can be bypassed using micromanipulation techniques (Gook et al., 1995
). Secondly, it is of great concern that there is the possibility of raising the incidence of aneuploidy by depolymerization of the spindle apparatus of the mature oocyte during cellular cooling. The spindle is a dynamic structure composed of microtubules that are being assembled continuously at one end and removed at the other in a treadmill fashion. Although the chromosomes reassemble and align along the spindle equator at cell rewarming, there is a risk of chromosomal loss and of the occurrence of aneuploidy during the first maturation division (Pickering et al., 1998
). Thirdly, it has been show that the oocyte cytoskeleton gets damaged by the cryopreservation process, which could lead to significant changes in the organization and trafficking of molecules and organelles (Vincent and Johnson, 1992
). Therefore, the quality of frozen oocytes after thawing is a crucial factor to guarantee oocyte fertilization and development potential. The better the quality, the higher the survival rate will be. The high survival rate is particularly affected by freezethaw protocols.
An overview of the literature shows that most studies use a similar freezing and thawing procedure (slow-freezing rapid-thawing), similar seeding points and cryoprotectants (1,2-propanediol and sucrose) (Fabbri et al., 2001). The slow-freezing program, as a standard method suitable for large cells, has been successfully used in the cryopreservation of human embryos. In the present study, human oocytes were cryopreserved by using slow-freezing program. The cryoprotectants generally used in oocyte freezing protocols are 1,2-propanediol (membrane-permeating cryoprotectant) and sucrose (membrane non-permeating cryoprotectant). Their protective action is very complex and attributable to a number of properties, the most important of which is the beginning of the dehydration process. In particular, sucrose does not enter the cell, but exerts its beneficial effects by causing cellular dehydration through changes in osmotic pressure (Fabbri et al., 2001
). In this study, the effects of different sucrose concentrations on oocyte freezing were investigated. The results indicated that 0.2 M sucrose was more beneficial to the survival of the frozenthawed oocytes than the 0.1 M sucrose (80% vs 68.07%, P<0.05). A significantly higher developmental potential was found in the second group (0.2 M sucrose) with respect to the first group (0.1 M sucrose). We suggest that the increase in the sucrose concentration generates an osmotic gradient across the cell membrane, which draws water out of the cell, causing the cell to dehydrate sufficiently before and during the freezing procedure. Similar results were obtained in the previous study on the matured oocytes; 0.3 M and 0.2 M sucrose concentrations in the freezing solutions gave higher oocyte survival rates (82% and 60%, respectively) than 0.1 M sucrose (34%) (Fabbri et al., 2001
). It was also reported that using Na-depleted media along with other alterations in freezing and thawing procedures in human oocyte cryopreservation can provide excellent survival (74.4%), fertilization (59%) and live birth rates (36.4%) (Boldt et al., 2003
). Furthermore, several babies have been born by using oocyte cryopreservation according to a recent report (Fosas et al., 2003
). There were also studies suggesting that longer exposure time in cryoprotectants could increase the survival rate pertaining to the dehydration of oocytes, an observation that needs further investigation (Fabbri et al., 2001
).
Although the survival rates of the frozenthawed immature oocytes appeared to be higher than the frozenthawed mature oocytes, no significant difference in the survival rate was found between oocytes at different maturity stages (P>0.05). Our results indicated that there was a higher fertilization rate in mature oocytes as compared to immature oocytes, which is similar to previous reports (Fabbri et al., 2001). The cleavage rate was not superior to other studies (91% vs 100%; Gook et al., 1995
; Park et al., 2001
). At the same time, the GV oocytes could be matured in vitro, but the fertilization and cleavage rates were not as good as in normal mature oocytes, which could be due to freezing injury of the cellular cytoskeleton, resulting in fertilization failure and embryo development arrest (Van der Elst, 2003
).
The cryopreservation of immature GV oocytes raises a lot of controversial questions. First, no definite conclusion has been drawn about immature oocytes being more suitable for cryopreservation then the mature oocytes. Secondly, the most critical problem is the in vitro maturation of the frozenthawed immature oocytes. A good in vitro maturation system not only means benefit for nucleus maturation, but also benefit for cytopasmic maturation. Although we had a slightly better overall maturation rate of 65.17% with MI and GV oocytes than that reported by Park et al. (61%) (Park et al., 1997), the maturation rate is still lower than the rates obtained with the maturation of fresh oocytes using the same protocol in our center (83%) (Li et al., 2003
). The cryopreservation of immature oocytes has been successfully used in animals (Amorim et al., 2003
). However, only a few pregnancies and births of normal infants from embryos derived from frozenthawed immature oocytes have been reported in human (Tucker et al., 1998
; Goud et al., 2000
; Wu et al., 2001
). These reports suggest that immature human oocytes could survive the freezing and thawing and process of fertilization in vitro as used for mature oocytes, but only with a very low developmental efficiency. So it seems that the same problems are encountered in human as in mouse immature oocyte maturation with or without freezing. Namely, not the nucleus but the cytoplasm was of concern (Van der Elst, 2003
). Therefore, it is necessary in the future to optimize in vitro maturation systems and freezethawing procedures for immature oocytes.
In summary, our study indicates that an increased sucrose concentration (0.2 M) enhanced oocyte survival and quality, which is beneficial for the developmental potential of the embryos. Further studies are required to optimize freezing and thawing conditions in order to improve the oocyte survival rate and increase the potential of oocyte fertilization and embryonic cleavage rates after thawing.
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Acknowledgements |
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References |
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Submitted on January 2, 2004; resubmitted on May 24, 2004; accepted on July 6, 2004.