Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium

Carlos J. Quintans1, Mónica J. Donaldson, M. Victoria Bertolino and R. Sergio Pasqualini

Halitus Instituto Médico, Affiliated to the University of Buenos Aires, Marcelo T. de Alvear 2084 (C1122AAF), Buenos Aires, Argentina


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21–41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.

Key words: birth/choline chloride/cryopreservation media/oocyte cryopreservation/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Development of a technique for routine metaphase II (MII) oocyte cryopreservation still remains a technical challenge in assisted reproductive technology (ART). Undoubtedly, a successful oocyte-freezing programme would overcome many practical, legal and ethical problems and thus enhance efficiency in oocyte use. Oocyte cryopreservation techniques have been applied with some success in mice (Whittingham, 1977Go), but extrapolation of methods to human oocytes renders insufficient results to be applied routinely in ART (Bernard and Fuller, 1996Go). Several groups used the propanediol (PROH)-sucrose technique, reported to be successful for human pronucleate embryo freezing (Testart et al., 1986Go), as an approach to freeze MII oocytes; but only a small number of successful pregnancies have been achieved worldwide so far (Porcu, 2001Go). Recently, the use of a choline-based medium has been described in mice to avoid the deleterious effects of high solute concentrations occurring during the freezing process (`solution effects') (Stachecki et al., 1998Go). Sodium salts are the major components of all cell-handling media and are thought to be one of the major threats for cell damage related to cryopreservation. Additionally, the excess of sodium ions is removed by energy-dependent sodium pumps, which may be impaired by the freezing–thawing procedure. Substitution of sodium by choline, which seems not to diffuse into the cell, could overcome intracellular cation load and subsequent cell injury (Stachecki et al., 1998Go). The aim of the present report was to describe our preliminary results, obtained after cryopreserving human oocytes in a medium in which most of the sodium was replaced by choline.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design
Retrospective analysis was undertaken of data belonging to 12 women in 12 cycles of ART, who had some (10 cases) or all (two cases) of their oocytes cryopreserved in a choline-based medium. All patients underwent treatment in Halitus, a Fertility Clinic affiliated to the University of Buenos Aires. Institutional Review Board approval was granted, and both partners signed an informed consent.

Patients
Median female age was 33 years (range 21–41, 95% CI: 29.7–35.0). Infertility aetiology was severe male factor in all the treated couples. Pituitary desensitization was achieved in all cycles with leuprolide acetate (Lupron®; Abbott, Buenos Aires, Argentina) following a long down-regulation protocol. Ovarian stimulation was performed with recombinant FSH (Gonal F®; Serono, Buenos Aires, Argentina) in all patients. Preovulatory oocytes were retrieved transvaginally by ultrasound guidance, 34 to 36 h after 10 000 IU of hCG (Profasi HP®; Serono) injection. All oocytes were placed in human tubal fluid (hTF) medium (prepared in-house) (Quinn et al., 1985Go) plus 10% serum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA, USA), in Nunc 4-well dishes (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere with 5% CO2 in air. Afterwards, the oocytes were separated into those for subsequent fertilization and those for cryopreservation.

Freezing, thawing, ICSI and embryo transfer
Cumulus and corona cells were removed by a brief exposure to 0.1% hyaluronidase type IV-S (Sigma Chemical Co., St Louis, MO, USA). Oocytes were then washed several times in HEPES buffered HTF with 10% SSS and finally incubated for about 30 min in HTF 10% SSS at 37°C in 5% CO2 before freezing. Only morphologically normal MII oocytes were selected for cryopreservation.

The basal medium used for freezing and thawing oocytes was a modified phosphate buffered saline (mPBS) in which sodium chloride was replaced by 137 mmol/l choline chloride as published (Stachecki et al., 1998Go). SSS, sucrose (SU), and PROH were added to mPBS as needed. Equilibration with freezing medium was performed at room temperature (22–24°C). Briefly, oocytes were first equilibrated for 5 min in mPBS with 20% SSS and 0.5 mol PROH, then were changed to mPBS with 20% SSS, 1.0 mol PROH for another 5 min, and then mPBS, 20% SSS, 1.5 mol PROH for 10 min and finally to mPBS 20% SSS, 1.5 mol PROH, 0.1 mol SU for 5 min. At this last step, oocytes were loaded into 0.25 ml French-type mini straws (Minitüb, Tiefenbach, Germany) and heat-sealed. A slow freezing protocol was used in a Bio-cool II biological freezer (FTS Systems Inc., Stone Ridge, NY, USA). From 22°C to –6°C the cooling rate was 1.5°C/min. At –6°C seeding was performed with cold tweezers, and after a 10 min holding period, cooling to –36°C was started at a 0.3°C/min rate. At –36°C straws were plunged into liquid nitrogen (–196°C).

If pregnancy was not attained in the fresh transfer cycle, thawing and fertilization of the cryopreserved oocytes was carried out. Rapid thawing was achieved by holding the straws in the air at room temperature for 40 s and afterwards stirring the straws in a 30°C water bath until thawing. The content of the straws was thoroughly mixed with mPBS 20% SSS, 1.0 mol PROH and 0.2 mol SU in Nunc 4-well dishes at room temperature. Oocytes were recovered and washed stepwise for 5 min periods as follows: firstly in mPBS, 20% SSS, 1.0 mol PROH, 0.2 mol SU; secondly in mPBS with 20% SSS, 0.5 mol PROH, 0.2 mol SU; thirdly in mPBS with 20% SSS, 0.2 mol SU; and finally transferred to HTF with 10% SSS equilibrated with 5% CO2 at 37°C. ICSI was performed 4–6 h after thawing, and fertilization was assessed 12–16 h later. Intrauterine embryo transfer was carried out 48–72 h after ICSI at the 2- to 8-cell stage using a Frydman catheter (Laboratoire C.C.D., Paris, France). Endometrial stimulation was performed with 4 mg/day of oral 17ß-estradiol (Ronfase; Rontag, Buenos Aires, Argentina) from days 2–4 of the menstrual cycle and 6 mg from day 5 onwards. On day 15, 100 mg/day of i.m. progesterone (Proluton; Schering, Buenos Aires, Argentina) was added.

In those patients that achieved intrauterine pregnancy, hormonal support with estrogen and progesterone was maintained until week 15 of gestation.


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 Materials and methods
 Results
 Discussion
 References
 
Table IGo shows details of patient's age, number of oocytes retrieved, cryopreserved, thawed, and number of oocytes surviving freezing and thawing. Table IIGo shows numbers of injected oocytes, damaged oocytes, normally fertilized oocytes, number of cleaving embryos, transferred embryos, observed pregnancies and pregnancy outcomes.


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Table I. General data regarding oocyte source, freezing and thawing
 

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Table II. Results after the injection of thawed oocytes
 
Six clinical pregnancies were achieved, the pregnancy rate per transfer was 50%. Implantation rate per embryo transferred was 25%. Two of these pregnancies went to term. Amongst the other four cases, one pregnancy was ectopic and three aborted spontaneously in the first trimester. Genetic analysis of the aborted material was performed in two cases, revealing no pathology (one case) and a 45,XO karyotype (one case); in the third case there was no material available for the study.

The proportion of implanted embryos that were lost was 71%, (5/7), or 67% (4/6) if only the intrauterine pregnancies are taken into account, which is very high. Live birth rate per transfer was 18% (2/11).

Term pregnancies
Details of the pregnancies that went to term are described as follows. In one case, two embryos at the 2-cell stage, one graded A and the other graded B, were transferred into the uterus 48 h after thawing the oocytes. On day 15 post-transfer, the ßhCG subunit was 1980 IU/ml. Ultrasound at 4 weeks gestation revealed two gestational sacs with cardiac activity. Four weeks later only one sac remained alive. Pregnancy developed without complications and on January 24, 2000, at week 34 of gestation, a baby boy with an Apgar score 8/9, weighing 2050 g and 44 cm in height was delivered vaginally. He had to remain in the neonatal intensive care unit for 23 days due to respiratory difficulties, hyperbilirrubinaemia, and hypoactive suckling reflex.

In the other case, four embryos were transferred 48 h after oocyte thawing, one at the 2-cell stage, graded B, and three at the 4-cell stage, one graded B and two graded C. On day 15 post-transfer, the ßhCG subunit was 205 IU/ml. Ultrasound at 4 weeks post-transfer revealed one gestational sac with a vital embryo. The pregnancy developed without complications and on January 1, 2001, at week 39 of gestation, a 2785 g baby girl, 47 cm height, Apgar 9/10 was delivered by Caesarean section due to unfavourable cervical conditions. Both children thrived well, and showed normal development up to the most recent paediatric and psychological evaluation, that took place on May 16, 2002.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human MII oocyte seems to be especially susceptible to damage caused by the freezing and thawing procedure. This is related to some of its particular characteristics, such as its surface to volume ratio, temperature sensitivity of the metaphase spindle, cytoskeletal architecture elements, and zona pellucida. Several factors may affect oocyte survival after a freezing and thawing cycle; however, a detailed analysis is well beyond the scope of this report.

Published data on post-thawing survival rates of cryopreserved human oocytes in media with standard sodium content show a high variability, ranging from 25–95%, depending on each particular study (Porcu, 2001Go). The low number of thawed oocytes, and the inclusion of sub-optimal grade oocytes may explain this variability (Porcu, 2001Go). In our study MII oocytes cryopreserved in a choline chloride based medium gave a median survival rate of 63%. In a small group of patients whose oocytes were frozen in standard sodium freezing medium, we have obtained a lower oocyte survival rate than in choline-based medium (unpublished results). These data do not allow statistical comparison of choline versus sodium media on oocyte survival to freezing and thawing. As previously stated, sodium salts, the major components of all cell-handling media, are thought to be one of the main threats in cell damage related to cryopreservation. Substitution by choline (Stachecki et al., 1998Go) could overcome intracellular cation load and subsequent cell injury.

The use of choline at physiological concentrations is known to be highly beneficial for embryo development. Choline is a dietary compound that has been classified recently as an essential nutrient for humans (Guo-Ross et al., 2002Go), is required for methylation, acetylcholine and phospholipid biosynthesis and cell signalling. Perturbation in choline uptake and metabolism causes developmental abnormalities in neurulating mouse embryos (Fisher et al., 2001Go). Hatching of peri-implantation hamster embryos is diminished in the absence of choline chloride (McKiernan et al., 2000). Choline is also known to act as an osmolyte. In contrast with inorganic ions, that at high concentrations destabilize protein structure, osmolytes exert a stabilizing influence on intracellular proteins (Kirk, 1997Go). On the other hand, the possibility that choline chloride may have some toxicity at the concentrations used here cannot be completely ruled out due to the lack of experimental evidence.

It has been reported that zona hardening as a consequence of freezing may affect fertilization in conventional IVF (Bernard and Fuller, 1996Go). This was not a problem here, because all post-thaw oocytes underwent ICSI. Reported fertilization rates of thawed oocytes are quite variable and range from 13–71% (Porcu, 2001Go). In our study, the median normal [2 pronuclei (PN)] fertilization rate was 59%, which is lower than the mean normal fertilization rate for non-frozen oocytes in our ICSI programme (72%). Contrary to Goud (Goud et al, 2000Go) we did not observe 3PN fertilization. Oocyte damage caused by microinjection fell within the accepted ranges for fresh oocytes (Stoddart and Fleming, 2000Go).

Implantation and pregnancy rates seem to be encouraging, even though they lack statistical significance due to the small sample size. As already mentioned, amongst the six pregnancies, only two resulted in the delivery of two babies. Of the other four, one was ectopic and three aborted spontaneously. This high pregnancy loss seems to be a common problem in pregnancies arising from cryopreserved human oocytes (Tucker et al., 1996Go). Considering our data, no solid assumptions could be drawn on the reasons underlying fetal loss. One may be tempted to attribute it to chromosomal anomalies, caused by meiotic spindle impairment during the freezing–thawing procedure (Pickering et al., 1990Go).

Recent experiments in mice seem to indicate that parameters related to the environment, where embryo development occurs, might be playing an important role in post-implantation viability of embryos arising from cryopreserved oocytes (Stachecki et al., 2002Go).

In summary, IVF outcome after oocyte thawing seemed to be improved in a medium where most of the sodium content was replaced by choline. However, the small size of the sample prevents us from drawing any categorical conclusion.

As far as we know the present study reports the first pregnancies and term births attained after oocyte cryopreservation in a choline-based medium. Considering our results we believe it may be worth undertaking further research on human oocyte cryopreservation in low sodium medium.


    Notes
 
1 To whom correspondence should be addressed at: Marcelo T. de Alvear 2084 (C1122AAF), Buenos Aires, Argentina.E-mail: cquintans{at}halitus.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 21, 2002; resubmitted on July 3, 2002; accepted on August 8, 2002.