1 Assisted Fertility Services, Community Health Network, Indianapolis, IN 46256, 2 Reproductive Endocrinology Associates, 2020 W 86th Street, Indianapolis, IN 46260 and 3 Reproductive Surgery and Medicine, 8040 Clearvista Parkway, Indianapolis, IN 46256, USA
4 To whom correspondence should be addressed. e-mail: jboldt{at}ecommunity.com
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Abstract |
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Key words: cryopreservation/IVF/oocyte/pregnancy/sodium
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Introduction |
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Isolated pregnancies with oocyte cryopreservation have been reported for a number of years, using either mature or immature oocytes (Chen, 1988; Van Uem et al., 1987
; Tucker et al., 1998
). However, overall success rates with respect to survival of oocytes post-thaw and pregnancy rates were very low, discouraging routine application of oocyte cryopreservation (Gook and Edgar, 1999
; Paynter, 2000
; Coticchio et al., 2001
). In addition to low survival rates, studies showed that oocyte cooling and/or freezing could cause significant disruption of the oocytes meiotic spindle and other subcellular structures as well as have adverse effects on the zona pellucida, possibly due to premature cortical granule release (Al-Hasani et al., 1987
; Sathananthan et al., 1987
; 1988; Pickering et al., 1990
; Van Blerkom and Davis, 1994
).
More recent results provide optimism for the use of oocyte freezing. In a series of reports, Gook and colleagues showed, using a slow freezerapid thaw protocol, that spindle and cortical granule integrity was maintained in frozenthawed human oocytes (Gook et al., 1993), that normal karyotypes with absence of stray chromosomes occurred after fertilization of frozenthawed human oocytes (Gook et al., 1994
), and that frozenthawed human oocytes could be fertilized and cultured to hatching blastocyst stage (Gook et al., 1995
). Several reports, including more traditional slow freezing approaches (Porcu et al., 1997
; 2000; 2002; Yang et al., 1998
; 1999; 2002; Winslow et al., 2001
), as well as vitrification methods (Hong et al., 1999
; Kuleshova et al., 1999
; Yoon et al., 2000
; Chung et al., 2000
; Wu et al., 2001
) have shown enhanced oocyte survival rates post-thaw as well as multiple births following oocyte cryopreservation. Success rates for oocyte cryopreservation as well as subsequent developmental rates remain variable, however, suggesting that more work is required to define specific freezing and thawing approaches that will maximize recovery and developmental rates.
In this paper we share our initial experience with oocyte cryopreservation as an adjunct to IVF therapy in couples who did not wish to undergo embryo cryopreservation for various ethical and religious reasons. Our work focuses on the use of sodium (Na)-depleted culture media for oocyte freezing, which has been shown to be successful for mouse oocyte cryopreservation (Stachecki et al., 1998a;b; 2002). Quintans et al. (2002
) have used a similar approach and have achieved pregnancies with human oocyte freezing. We report that good survival, pregnancy and delivery rates can be obtained routinely using Na-depleted media for human oocyte cryopreservation.
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Materials and methods |
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For IVF, patients were stimulated using several protocols including luteal phase leuprolide acetate suppression, flare protocols, or GnRH antagonist protocols. Patients were generally given 10 000 IU hCG when there were at least two follicles of >18 mm diameter. Oocyte retrievals were done by transvaginal ultrasound 36 h after hCG. Aspirates were collected into tubes containing 1 ml of phosphate-buffered saline (PBS) from Irvine Scientific (USA), and immediately scanned for the presence of an oocyte. After retrieval, oocytes were either stretched on the bottom of a Falcon 3003 dish briefly and scanned for the presence of a first polar body, or had the bulk of the cumulus cell mass removed with 22 G needles followed by exposure to hyaluronidase (80 IU/ml Type III; Sigma, USA) for 30 s and aspiration through narrow bore micropipettes to complete cumulus and corona cell removal. The former technique was used if we planned to use a standard insemination approach, and the latter was used in ICSI cycles. Oocytes with a confirmed first polar body were then selected for either insemination or cryopreservation. Three to four oocytes were generally used for insemination, with all additional mature oocytes cryopreserved. Immature (metaphase I or germinal vesicle) stage oocytes were not frozen, but were cultured overnight and observed; if they had matured and only one or two oocytes had fertilized from initial insemination then the in-vitro matured oocytes were inseminated on the day post-retrieval in an effort to increase the number of embryos available for transfer. Only mature oocytes recovered on the day of retrieval were frozen. Freezing was routinely initiated within 13 h post-retrieval.
Two freezing protocols were used in the patients reported in this study. The first was a standard 1,2-propanediol (PrOH)sucrose freezing method (Gook et al., 1993), and was used in seven of the freezethaw cycles. Oocytes had their cumulus and corona cells removed as described above, were incubated for 10 min at room temperature in PBS20% synthetic serum substitute (SSS; Irvine Scientific) containing 1.5 mol/l PrOH (Sigma), and then were transferred to 1.5 mol/l PrOH containing 0.1 mol/l sucrose (cell culture grade; Sigma). Oocytes were frozen in Nunc vials containing 1.0 ml of PrOHsucrose using the following freezing ramps: room temperature to 7.0°C at 1.0°C per min, hold at 7.0°C for 5 min, seed, hold for an additional 10 min at 7.0°C, cool at 0.3°C to 35°C, then plunge into liquid nitrogen. All freezing runs were performed with a Planar Kryo 10 controlled rate freezer (TS Scientific, USA).
The second freezing protocol employed the use of Na-depleted PBS as the base medium for the cryopreservation solution, and was used for 15 of the cycles reported. The following recipe was used to prepare Na-depleted (Na) medium (all chemicals were cell culture grade from Sigma): choline chloride 137 mmol/l, KCl 2.6 mmol/l, NaH2PO4 8.0 mmol/l, KHPO4 1.4 mmol/l. In this recipe, choline chloride was substituted for NaCl on an equimolar basis; the medium is best referred to as Na-depleted because of the NaH2PO4 salt. All salts were dissolved in sterile cell culture grade water from Sigma, and the medium was kept at 4°C for up to 3 months before use. The pH was 7.4. The cryopreservation solution consisted of Na-depleted medium with 20% SSS, and supplemented with 1.5 mol/l PrOH and 0.2 mol/l sucrose. Before freezing, cumulus and corona cells were removed, and the oocytes were then transferred into 12 ml of cryopreservative solution and kept at room temperature (2224°C) for 20 min. The oocytes shrank rapidly upon exposure to the freezing solution and appeared slightly shrunken at the end of the 20 min equilibration period. After the 20 min equilibration, oocytes were transferred into Nunc vials containing 0.5 ml of the same cryopreservative solution, and placed into the freezing chamber. The freezing ramps were as follows: room temperature to 6.0°C at 2.0°C/min, hold for 5 min, seed then hold an additional 10 min at 6.0°C, cool at 0.3°C to 33°C, plunge into liquid nitrogen.
For thawing cycles, endometrial preparation involved the use of estradiol pills such as with donor oocyte cycles. Once the endometrium reached a thickness of 9 mm, i.m. progesterone supplementation was started, and oocytes were thawed on the first day of progesterone administration. For either freezing protocol, vials were thawed by immersion in a 32°C water bath until all ice crystals had disappeared. The contents of the freezing vial were pipetted onto a 3003 dish, and oocytes identified. For freezing protocol 1, oocytes were transferred to a dish containing 12 ml of PBS containing 1.5 mol/l PrOH0.2 mol/l sucrose, and were stepped down through a graded series of PrOH and/or sucrose before transfer to culture medium. For protocol 2, oocytes were transferred into a dish containing 12 ml of 0.5 mol/l sucrose. After incubation at room temperature for 10 min, oocytes were transferred to 12 ml of 0.2 mol/l sucrose for 10 min before transfer into culture medium. Several different culture media, including P1 (Irvine Scientific), IVC-1 (InVitro Care, USA), or Quinns fertilization medium (SAGE Biopharma, USA) were used in this population of patients. Oocytes were observed after 3060 min to confirm viability, and ICSI was performed
24 h after thawing. Oocytes were examined 1620 h post-ICSI, and the presence of two pronuclei was taken as evidence of normal fertilization. Embryo transfers were done on day 3 after thawing under ultrasound guidance using either a Wallace (Cooper Surgical, USA) or Echotip (Cook, USA) catheter under ultrasound guidance. Embryos were examined daily and all embryos showing progression of cleavage were transferred regardless of quality grade. In several instances embryos arrested prior to the day of transfer (defined as failure of cell division over a 24 h period) and were not then considered for transfer. Embryos were graded for cell number and quality between 06000800 hours on the day of transfer. The grading scale was as follows: grade 4: even sized/shape blastomeres with no fragmentation; grade 3: <10% fragmentation and/or slightly irregular-shaped blastomeres; grade 2: 1050% fragmentation and/or irregular size/shape blastomeres; grade 1:
50% fragmentation with irregular size/shape blastomeres. Assisted hatching with acid Tyrodes was performed on all transferred embryos. Patients were discharged
30 min after completion of the transfer, and pregnancy tests carried out 12 days after embryo transfer. Patients with positive serum hCG levels were followed for 12 weeks before referral for obstetric care.
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Results |
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Our results from the second approach to oocyte freezingthawing are shown in Table II. There were 15 patients with 16 thaw cycles performed in this series. In every cycle we had at least one oocyte survive thawing with subsequent insemination. The overall survival rate of oocytes post-thaw was 74.4%, with 59% of oocytes fertilized after ICSI. This fertilization rate was lower than the 75% rate we have obtained with fresh oocytes (data not shown), and was due in part to the rather high damage rate (18.2%) observed in this series of patients. There were two cycles in which fertilization was not achieved, and one of these involved a cycle in which the sperm sample used for insemination had no motile sperm available for ICSI. The 16 thaw cycles resulted in 11 embryo transfer procedures. Of the remaining five, two had no transfer because of fertilization failure, two cycles yielded fertilized oocytes that arrested at pronuclear or early cleavage stages (with one and two fertilized oocytes respectively), and one involved a cycle in which 2-pronuclear stage embryos did not reach blastocyst stage for a planned day 5 transfer. Overall there was an 84.6% (33/39) cleavage rate of fertilized oocytes in this series. In the 11 embryo transfer cycles, there was an average of 2.7 embryos transferred/cycle (range 15), with a mean cell number per embryo of 5.6 and a mean embryo score of 2.7. There were four clinical pregnancies established after transfer of two embryos in two cycles, four embryos in one cycle, and five embryos in one cycle. In the latter two cases, each patient wanted to have all oocytes thawed and utilized as they had indicated they did not want to continue with treatment if the procedure was unsuccessful. Three of the pregnancies resulted in the delivery of healthy female offspring, and the fourth pregnancy resulted in the birth of healthy twin girls. The percentage pregnancy rate per thaw cycle initiated was 25% (4/16) and 36.4% (4/11) per embryo transfer cycle.
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Discussion |
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Several other studies also have demonstrated reproducible success with oocyte cryopreservation. Porcu et al. (2000) reported on two series of patients. In the first series oocyte cryopreservation was carried out in 23 women, with a 59.5% survival rate, 64.4% fertilization rate, and three pregnancies and one delivery achieved. In the second series 96 patients undergoing 112 retrieval cycles had 1769 oocytes thawed, with a 54.1% survival rate and 57.7% fertilization rate achieved. Sixteen pregnancies and nine deliveries occurred in this series, but the number of patients having a thaw procedure was unclear. Winslow et al. (2001
) reported 16 births from oocyte freezethaw cycles performed in 33 patients. Taken together, these results suggest that oocyte freezing can be performed on a routine basis and provide a realistic chance of achieving successful pregnancy.
For oocyte freezing to be considered as an alternative to embryo freezing, equivalent survival and pregnancy rates between the two methods would need to be achieved. While our results are from a small series, our initial data compare favourably with both our own as well as national data on embryo cryopreservation. Since the inception of our programme in 1998, our laboratory has a 33.3% pregnancy rate and a 14% implantation rate per thawed embryo transfer, which is similar to the 36.4% pregnancy rate and 15.6% implantation rate per transfer we obtained with frozen oocytes. Data from the 1999 Centers for Disease Control and Prevention report for programmes in the USA and Canada indicate an 18.5% delivery rate with frozen embryo transfer. Porcu et al. (2002) recently have shown similar pregnancy rates post-thaw when comparing use of frozen oocytes versus frozen embryos. Identical pregnancy rates with frozen oocytes versus embryos have been reported by Yang et al. (1999
), with a mix of donor and non-donor oocyte cycles, and Yang et al. (2002
) have shown identical implantation rates when comparing frozen oocytes versus embryos obtained from donor oocyte cycles. Taken together, the results of our investigation and others indicate that oocyte cryopreservation can provide clinical results comparable with embryo freezing. Thus, patients that have ethical or religious reasons against embryo cryopreservation would not appear to compromise their chances at pregnancy by opting for oocyte freezing.
We chose to use Na-depleted media based on studies with mouse oocytes demonstrating good survival and developmental rates when using Na-depleted conditions for freezing (Stachecki et al., 1998a;b; 2002). Goud et al. (2000
) have also shown a benefit of diminished Na in the freezing solution for preservation of human germinal vesicle and in vitro matured human oocytes. Stachecki et al. (1998a
;b) have discussed two potential reasons for enhanced oocyte survival under low Na conditions. One would involve a solute effect, involving the transport of large amounts of Na across the cell membrane through the plasma membrane-associated NaK pump. In regular (i.e. relatively high Na) media excess Na may be pumped into the cytoplasm, but be unable to be transported out during the freezethaw process, leading to excess accumulation of intracellular Na and ultimately cell death following thawing. Another alternative is that the choline used to substitute for Na in our freezing medium may have a stabilizing effect on the cell membrane directly, protecting against freezing damage. Further work, perhaps involving use of other Na replacements and/or other potentially membrane-stabilizing agents, would be needed to address this question.
Two other modifications in our cryopreservation methods that appeared to improve results were seeding at a relatively higher temperature (6 versus 7°C) and the use of high sucrose concentrations in the thawing solution. Trad et al. (1998) working with either GV stage, failed fertilization or polyspermic human oocytes showed that human oocytes benefit from seeding at a higher temperature in order to allow for dehydration of the cell prior to induction of intracellular ice formation. Significantly higher survival rates were found by Trad et al. (1998
) when oocytes (frozen in straws) were seeded at 4.5 versus 6.0°C or 8.0°C. Using vials with 0.5 ml solution, we found that seeding was difficult to achieve when the temperature was
5.5°C, thus we have used 6°C as the seeding temperature in our work.
Our change to using high sucrose concentrations for thawing was based on our observations with our first approach to freezing, in which oocytes appeared initially intact but rapidly swelled and degenerated. Oocytes appear to be quite osmotically sensitive post-thaw. This would agree with studies using bovine oocytes (Agca et al., 2000) and would be expected given the large size of the cell. By using a high (0.5 mol/l) sucrose concentration initially during thawing, oocytes shrank rapidly, and appeared to re-equilibrate more easily by passage through 0.2 mol/l sucrose prior to transfer to culture media. After thawing, we chose to use ICSI to inseminate all oocytes because of concern that the freezing process may cause modifications in the oocytes zona pellucida that would hinder normal fertilization (Kazem et al., 1995
). While we obtained an almost 60% fertilization rate, the oocyte lysis rate post-ICSI was somewhat high at 18.2% of injected oocytes. Early results (data not shown) suggest that extension of the time interval between thawing and sperm injection to a minimum of 3 h may help lower the damage rate.
Several other studies have used slow freeze methods for oocyte freezing. The techniques reported to date vary somewhat in terms of cryoprotectants used, exposure times, and thawing protocols. Porcu et al. (1997; 2000) used 1.5 mol/l PrOH0.2 mol/l sucrose for freezing, and obtained a 59.5% % survival rate post-thaw. Fabbri et al. (2001
) have shown that the time of exposure to cryoprotectant as well as concentration of extracellular cryoprotectant (e.g. sucrose) influences success rates. They showed that a 15 min exposure time to cryo protectant as well as increasing sucrose concentration in the cryopreservative solution yielded higher survival rates post-thaw. In particular, preincubation at room temperature in 1.5 mol/l PrOH0.2 mol/l sucrose for 15 min yielded a 54% survival rate. This approach is quite similar to the one used in our study, and our slightly higher survival rate might be due to the use of Na-depleted media or differences in the thawing procedure. Fabbri et al. (2001
) also showed that increasing the amount of sucrose from 0.2 to 0.3 mol/l in the freezing solution may enhance survival rates. Yang et al. (1998
; 1999; 2002) have also enhanced survival rates by extending the exposure time to cryoprotectant as well as using higher sucrose concentrations (0.2 mol/l) in their freezing solution. Yang et al. (1998
; 1999; 2002) did their pre-incubation at 37°C, as opposed to room temperature; we elected not to pursue this approach to avoid potential for enhanced toxicity of cryoprotectants at elevated temperature. Another difference between our work and that of Yang et al. (1998
; 1999; 2002) is that donor oocyte cycles were included in the latter, whereas we had no donor oocyte cycles in our group of patients. It might be expected that donor oocytes would have higher rates of survivability due to enhanced oocyte quality.
Concern has been expressed that oocyte freezing may induce damage to the meiotic spindle as well as cause other subcellular alterations that would lead to chromosomal or other cellular anomalies (Al-Hasani et al., 1987; Van Blerkom and Davis, 1994
; Sathananthan et al., 1997
; 1998) or that the prolonged exposure to cryoprotectants in slow freeze protocols may be more toxic to the oocyte during freezing (Kuleshova and Lopata, 2002
). Several studies are more reassuring in this regard. Using a slow PrOH/sucrose method, Gook et al. (1993
) showed that 60% of meiotic spindles remained intact post-thaw, as well as normal chromosome constitutions both in the thawed oocyte as well as in the embryo post-fertilization (Gook et al., 1994
; 1995). In addition, Cobo et al. (2001
) showed no increase in numerical chromosomal abnormalities in embryos derived from oocytes slow-frozen in PrOH compared with non-frozen controls. Our results as well as others showing very good survival rates post-thaw, as well as the births of a number of healthy infants, also argue against any significant toxicity effect.
In summary, our study provides a reproducible method for successful freezing and thawing of human oocytes. Other methods for oocyte cryopreservation have also been shown to be successful, including variations on slow freezing methods (Porcu et al., 1997; 2000; Yang et al., 1998
; 1999; 2002) as well as fast freezing using vitrification (Kuleshova et al., 1999
; Yoon et al., 2000
). While further study will be needed to determine which, if any, particular method is the most effective for oocyte cryopreservation, it seems clear that oocyte freezing can be accomplished on a routine basis. This offers real hope for individuals at risk from a loss or diminution of fertility, and provides a viable alternative for couples with religious or ethical concerns about embryo freezing.
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Note added at proof |
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References |
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Submitted on January 7, 2003; accepted on February 19, 2003.