Placenta
Volume 29, Supplement 2 , Pages 143-146, October 2008

Oocyte Cryopreservation

  • E. Albani

      Affiliations

    • UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy
    • Corresponding Author InformationCorresponding author. Tel.: +39 0282244517; fax: +39 0282244699.
    • ,
  • J. Barbieri

      Affiliations

    • UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy
    • ,
  • P.V. Novara

      Affiliations

    • UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy
    • ,
  • A. Smeraldi

      Affiliations

    • UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy
    • ,
  • G. Scaravelli

      Affiliations

    • Italian National Institute of Health, Rome, Italy
    • ,
  • P.E. Levi Setti

      Affiliations

    • UO di Medicina della Riproduzione, IRCCS Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy

Accepted 7 August 2008.

Article Outline

Abstract 

Over the last few years, there has been renewed interest and scientific debate concerning human oocyte cryopreservation. The aim of this study was to analyse the clinical data coming from our long experience of slow-freezing oocytes. Between 2001 and 2007, 1280 thawing cycles were carried out using oocytes previously frozen by means of a slow 1,2 propaniedol+sucrose protocol. A total of 7585 oocytes were thawed, of which 4409 survived and 3622 were microinjected; 144 clinical pregnancies were obtained. The number of thawing cycles increased from 19 in 2001 to 268 in 2007, and the number of thawed oocytes from 197 to 1652. Although the survival rate was significantly lower in the period 2002–2005 than in the period 2006–2007, pregnancy and implantation rates steadily improved from respectively 6.7% and 2.4% in 2001 to 15% and 8.2% in 2007. Our data demonstrate a clinically important improvement in oocyte crypreservation over the years in a Centres with proved experience, and can be offered as a standard of care not only before cancer treatment but also for couples refusing embryo crypreservation or in countries with very restrictive limitations on embryo or zygote freezing.

Keywords: Oocytes, Cryopreservation

 

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1. Introduction 

In October 2007, the American Society for Reproductive Medicine (ASRM) Practice Report [1] confirmed its previous position that oocyte cryopreservation is still considered an experimental procedure. It also advised fertility clinics to limit its use to single women with cancer or another illness requiring immediate treatment with no other viable option. These conclusions reflected their experience in oocyte cryopreservation based on the preservation of the fertility of patients about to undergo cancer chemotherapy (which is highly likely to be harmed) or healthy women that pay to cryopreserve their eggs when that are in their 20s or 30s before the decline in fertility experienced at the age of 36 years, and the results of a meta-analysis [2] comparing 4564 frozen and 4000 thawed oocytes in 397 thawing cycles, which led to 95 clinical pregnancies in the slow-freezing (SF) group. The control group consisted of 62 embryo transfers (ETs), with 141 transferred embryos after the thawing of 181 frozen embryos leading to 39 clinical pregnancies. The authors concluded that even after the inclusion of the most recent reports concerning SF (after June 2005), the data do not support the assumption that SF success rates are improving.

However, it is difficult to understand how it is possible to compare data coming from centres with different experiences and pregnancy rates in fresh and cryopreserved cycles with the results of a single Centre with very high pregnancy rates in fresh and cryopreserved cycles, and where extensive pre-freezing and post-thawing selections are allowed [3]. It would probably be more appropriate to compare the 2004 results concerning fresh and embryo or zygote thawing presented by the ESHRE European Register [4] with oocyte cryopreservation.

On the basis of many recent data coming from Italian studies [5], [6], [7], [8], it is possible to say that using the cryopreservation procedure under different clinical conditions, such as national restrictions or a patient's desire not to cryopreserve an embryo for personal or ethical reasons, can lead to acceptable results and clinical consequences. In support of this assertion, we summarise some clinical studies, including our own based on our long experience of cryobiology.

It has been shown that spindle integrity is maintained in frozen-thawed eggs [9] and that a correct karyotype follows frozen-thawed oocyte fertilisation [10], and more recent analyses have reported enhanced post-thaw survival rates and multiple births following oocyte cryopreservation [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Furthermore, Boldt et al. have shown that oocytes can be successfully and reproducibly cryopreserved using a sodium(Na)-depleted culture medium: about 70% of the oocytes survived freezing and thawing, approximately 60% were fertilised by means of ICSI (intracytoplasmic sperm injection), and the per transfer pregnancy rate was 36.4% [23]. Similar results were obtained by Quintans et al. [24].

We have previously published data, based on 303 cycles of oocyte freezing (286 patients) and 159 thawings carried out between October 1999 and December 2003, indicating that the process of cryopreservation does not seem to affect fertilisation and cleavage rates, which were respectively 66.6% and 97.6% with fresh cycles, and 67.5% and 89% with thawing cycles [25]. In this preliminary experience, before new legal restrictions came into force in Italy [26], there were no significant differences in fertilisation and cleavage rates between fresh and frozen oocytes using a 0.3mol/l sucrose protocol and injecting a mean of 8.3 fresh oocytes or 4.3 frozen oocytes. In 75 thawing cycles, the patients had both embryos and oocytes cryopreserved. The pregnancy rate with the fresh cycles was 47.9% per transfer, with an implantation rate of 23.2%; the corresponding figures with the oocyte thawing cycles were respectively 12.4% and 5.7%.

Borini et al. have recently published data concerning the clinical significance of oocyte freezing in 510 patients undergoing thawing cycles between 2004 and the end of 2006, which indicate high survival, fertilisation and cleavage rates (68.1%, 76.1% and 91.8%) even after the introduction of restrictions imposing the injection of only three fresh or thawed eggs. Using a 15mol/l PROH+0.3mol/l sucrose protocol, the pregnancy rate was 14.9% [6].

We have been involved in oocyte cryopreservation since 1998. In the present study, we describe our clinical experience up to December 2007, starting with the first thawing cycles in January 2001, and analyse the results year by year. Fewer thawing cycles were carried out in the first three years but, as from 2004 (when an Italian law was introduced that prohibited the insemination of more than three oocytes at a time), there was an increase in the number of “supernumerary” oocytes per stimulation cycle and in the number of frozen-thawed oocytes.

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2. Materials and methods 

2.1. Study population 

This retrospective study analysed 1280 oocyte thawing cycles carried out between January 2001 and December 2007, and survival, fertilisation, pregnancy and implantation rates year by year.

All of the patients signed a cryopreservation informed consent form that described the experimental nature of the procedure. Many of the cycles formed part of a multicentre trial promoted by the National Institute of Health in order to investigate different sucrose concentrations.

2.2. Oocyte cryopreservation and thawing 

After retrieval, the oocytes were prepared for freezing by enzymatically and mechanically removing the cumulus and corona cells by means of hyaluronidase (80IU/ml; Sigma, St Louis, MO) and fine-bore glass pipettes. All the oocytes were examined and the ones showing a polar body (in the metaphase II stage) were frozen 3–4h after retrieval.

We used a slow-freezing 1,2 propaniedol+0.3mol/l sucrose protocol from 1998 to 2002, when we adopted a slow-freezing 1,2 propaniedol+0.2mol/l sucrose protocol.

The oocytes were transferred into an equilibration solution (1.5mol/l PROH; Sigma Aldrich Srl, Milan, Italy) with 0.2mol/l sucrose (Sigma Aldrich, Srl) and a serum protein supplement (Pacific Andrology, CGA/Diasint, Florence, Italy). The eggs were then loaded into plastic straws (Paillettes Cristal, 133mm; Cryo Bio System, Paris, France) and transferred to an automated Kryo 10 series III biological vertical freezer (Planer Kryo 10/1.7GB; PLANER plc, Middlesex, UK) in which the temperature was slowly reduced from 23°C to −8°C at a rate of 2°C/min. Manual seeding was performed at −8°C, and then the straws were slowly cooled to −30°C at a rate of 0.3°C/min. The temperature was further decreased to −150°C at a rate of 50°C/min before the straws were finally plunged into liquid nitrogen and stored.

Thawing was performed by removing the straws from the liquid nitrogen and leaving them to warming in air for 30s, after which they were plunged into warm water at 30°C for 40s before the eggs were expelled into a multiwell dish and the cryoprotectants were diluted in a stepwise manner.

2.3. Statistical analysis 

The data were statistically analysed by means of one-way ANOVA followed by Dunn's post hoc test using SigmaStat 3.0 software. A p value of p<0.05 was considered statistically significant.

The patient ages are given as mean age per year±SD.

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3. Results 

This study included thawing cycles involving 833 patients, and the data were analysed by year (Table 1).

Table 1. Results of thawing cycles from January 2001 to December 2007
ALL2001200220032004200520062007
Number of thawing cycles12801931109265303285268
Patients833162676179192176168
Age (mean±SD)36±3.934±5.334.6±3.935.2±3.635.7±3.835.9±3.736.3±3.936.6±4.1
Cycles with no survived oocytes %411303366
Cycles with failed fertilization %91107711127
Survival rate % (mean±SD)61.3±27.169.3±19.780.2±22.271.1±25.165.1±26.563.5±25.755.4±27.454.6±27.3
Fertilization rate % (mean±SD)67.3±33.658.8±3273.8±23.166.6±27.869.1±30.767.4±34.263.7±3769.3±35.2
Number of transfer cycles11131530101239261234233
Pregnancies144141227343135
Pregnancy per thawing %11.35.312.911.010.211.210.913.1
Pregnancy per transfer %12.96.713.311.911.313.013.215.0
Implantation rate %6.62.45.76.35.46.66.78.2

In 2001, there were 19 thawing cycles involving 16 patients (two cycles performed at the end of 2000 were also included). A total of 197 eggs were thawed with a survival rate of 69.3%, and 95 oocytes were microinjected for a fertilisation rate of 58.8%. The pregnancy rate per transfer was 6.7%, with an implantation rate of 2.4%; one pregnancy was achieved. The mean age of the patients was 34±5.3 years (range 25.7–44.2).

In 2002, there were 31 thawing cycles involving 26 patients; 160 oocytes were injected, of which 120 were fertilised (73.8%); the survival rate was 80.2%. Only four pregnancies were achieved (a pregnancy rate per transfer of 13.3%, with an implantation rate of 5.7%). The patients' mean age was 34.6±3.9 years (range 25.8–41.4).

The annual number of thawing cycles significantly increased from 2003. In 2003, there were 109 cycles (76 patients, mean age 35.2±3.6 years; range 26.1–42.5) and 680 frozen oocytes were thawed; the survival rate was 71.1% (467 eggs). A total of 449 oocytes were microinjected for a fertilisation rate of 66.6%, giving rise to 252 cleaved embryos. The pregnancy rate per transfer was 11.9% and the implantation rate was 6.3%.

In 2004, there were 265 thawing cycles involving 179 patients whose mean age was 35.7±3.8 years (range 23.7–44.3). A total of 1412 frozen oocytes were thawed and the survival rate was 65.1% (845 eggs); the fertilisation rate was 69.1% (531 oocytes); the pregnancy rate per transfer was 11.3% (corresponding to 517 cleaved embryos); and the implantation rate 5.4%.

There were 303 thawing cycles in 2005 involving 192 patients with a mean age of 35.9±3.7 years (range 23.6–43.9). During this year, 1685 eggs were thawed and 801 microinjected (the survival rate after thawing was 63.5%) and the fertilisation rate was 67.4%; 549 embryo transfers were made with pregnancy and implantation rates of respectively 13% and 6.6%.

In 2006, there were 285 cycles in which 1728 oocytes from 176 patients were thawed; the survival rate was 55.4% (905 oocytes survived). The microinjection of 707 oocytes led to a fertilisation rate of 63.7%. The pregnancy rate per transfer was 13.2%, and the implantation rate 6.7%. The patients' mean age was 36.3±3.9 (range 25.7–46.2).

In 2007, there were 268 thawing cycles involving 168 patients (mean age 36.6±4.1; range 22.6–45.2). A total of 660 of the surviving oocytes were microinjected (875 eggs survived and the survival rate was 54.6%), 488 were fertilised (69.3%), and 486 were cleaved. There were 233 embryo transfers for a pregnancy rate per transfer of 15% and an implantation rate of 8.2% (Fig. 1).

  • View full-size image.
  • Fig. 1 

    Clinical data: the number of thawing cycles per year, and annual survival, fertilisation and implantation rates. p<0.001; ‘a’ versus ‘c,d,e,f’ and p<0.001 ‘b’ versus ‘e,f’.

During the studied period there were significant differences in survival rates (p<0.001) between 2002 and 2004, 2002 and 2005, 2002 and 2006, 2002 and 2007. There were significant differences also comparing 2003–2006 and 2003–2007. The differences in fertilisation rates (p=0.279) and pregnancy rates per embryo transfer (p=0.423) were not statistically significant, and there were no significant differences in implantation rates (p=0.812).

During the whole period from January 2001 to December 2007, there were 1280 thawing cycles: the rates of fertilisation, pregnancy per transfer, and implantation were, respectively, 67.3%, 12.9% and 6.6%. During the same period, 7143 fresh cycles were carried out (838 of which were suspended, mainly because of a poor response) and 1722 pregnancies were obtained for an implantation rate of 21.1%. The fertilisation rate was 72%, and the pregnancy rate per transfer 29.1%.

A mean of 1.5±0.9 thawing cycles with less than 50% of thawed oocytes were performed for 833 patients, leading to a cumulative pregnancy rate of 17.3%.

The patients' mean age increased significantly during the studied period from 34±5.3 years in 2001 to 36.6±4.1 years in 2007 (p<0.001) (Table 1).

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4. Discussion 

Our results reveal significant differences in survival rates during the study period, which are probably related to differences in the freezing/thawing protocol as has been shown by other studies investigating changes in the sucrose concentrations of freezing or thawing solutions, seeding temperatures, etc. [27]. However, this difference in survival did not seem to affect fertilisation rates and, although not statistically significant, there is a trend towards improved pregnancy and implantation rates.

Analysing our data on a yearly basis does not clarify the effect of the freezing/thawing protocol on survival, pregnancy and implantation rates because we used different protocols over the years. Nevertheless, despite using different protocols, treating significantly older patients, and having to cope with very restrictive limitations on the number of oocytes used and no embryo selection, our experience led to a clinically significant improvement although, as often is the case in reproduction studies, it would require an impossibly large sample size to detect a statistically significant mean 5% difference in implantation and pregnancy rates.

Although considerable efforts have been made to optimise cryopreservation techniques throughout the world over the last few years, the results from different centres can be compared in terms of pregnancy rates only when looking at a few centres with extensive experience and specific research interests in this field. This demonstrates that there is still a need for even greater standardisation among Centres.

Vitrification seems to be an alternative to slow freezing, and recent studies have suggested that the results are better [28] and that it may be safer and less traumatic than slow freezing in relation to some critical physiological parameters such as meiotic spindles [29], [30]. However, in our opinion, vitrification still requires further investigation because most of the good results come from donor programmes. The results in infertile patients are less consistent (as in the case of slow freezing) and it is still too early to say whether it is possible to overcome differences among operators, which may be even more important than in the case of slow freezing.

We believe that oocyte cryopreservation can be considered a valid alternative to the elimination of “supernumerary” oocytes, particularly in countries with restrictive laws or when the couple refuses embryo cryopreservation. When comparing embryo and zygote cryopreservations, it must be remembered that the latest European Register data come from cycles carried out in 2004, and show an overall pregnancy rate per transfer of 19.1%, with national rates of as low as 13.3% [4].

Consideration of pregnancy outcomes goes beyond the scope of this study, but is clearly a very important question. Although ours is one of the most extensive experiences described in the literature, the sample of achieved pregnancies is still too small to make comparisons with natural conception or other ART techniques. However, a multicentre study of children born after oocyte freezing is currently being conducted.

Here we describe what is probably one of the most extensive experiences of oocyte cryopreservation in the world and, unlike a few years ago, the cryopreservation procedure is now considered a common laboratory technique in our Centre. Our results show that the careful selection of oocytes on the basis of morphological criteria at the time of cryopreservation and the thawing stage can improve implantation rates.

Our pregnancy rate/embryo transfer in thawed cycles is not high (12.9%), but needs to be considered in the light of current Italian law, which allows the fertilisation of only three fresh or frozen eggs. Patients benefit from a pregnancy rate of 17.3% after a mean of a 1.5 thawing cycles, and the cumulative pregnancy rate after a single retrieval ranges from 29.1% after fresh cycles to 46.4% if thawed cycles are included.

Although they can clearly be improved, our results are not so different from those obtained in Europe in 2004, or in countries with established embryo/zygote freezing programmes [4].

Finally, we agree with the ASRM statement that patients looking for the cryopreservation of their oocyte (before cancer therapy, or because they have ethical objections against embryo freezing or donor sperm) need to be advised to contact the few centres in the world with proven experience and consolidated results in this particular aspect of reproductive medicine and fertility preservation.

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5. Conflict of interest 

The authors do not have any conflicts of interest.

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Acknowledgement 

We would like to thank the medical and biological staffs of the Unit of Reproductive Medicine, IRCCS Istituto Clinico Humanitas, who contributed to the results of this study. The study was partially supported by Grant No. 3174, from the Italian National Health Institute, Rome.

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PII: S0143-4004(08)00239-7

doi:10.1016/j.placenta.2008.08.002

Placenta
Volume 29, Supplement 2 , Pages 143-146, October 2008

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