Use of sugars in cryopreserving human oocytes

RBMOnline - Vol 9. No 2. 2004 179-186 Reproductive BioMedicine Online; www.rbmonline.com/Article/1328 on web 23 June 2004

Article Use of sugars in cryopreserving human oocytes Diane Wright is Laboratory Director of the Vincent IVF Unit at Massachusetts General Hospital, Boston, Massachusetts, USA. After obtaining a BSc in Biomedical Science, she proceeded to complete a MSc in Physiology of Reproduction, both degrees being obtained from Texas A&M University. A PhD was received from a joint programme with Old Dominion University and Eastern Virginia Medical School, Norfolk, Virginia, USA. Dr Wright’s doctoral research was conducted within the Jones Institute for Reproductive Medicine. Her areas of interest reside with oocyte and embryo cryopreservation, sperm selection, and embryonic stem cells.

Dr Diane Wright Diane L Wright1,4, Ali Eroglu2, Mehmet Toner3, Thomas L Toth1 1Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, VBK 220, Boston, MA 02189, USA; 2Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, CB-2803, Augusta, GA 30912, USA; 3Centre for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, Shriners Burns Hospital, 51 Blossom Street, Boston, MA 02114, USA 4 Correspondence: Tel: +1 617 7241807; e-mail: [email protected]

Abstract In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and approximately 50 ongoing pregnancies being reported. While the ability to freeze human embryos has become a standard of practice in assisted reproductive technologies, obtaining reliable techniques for oocyte cryopreservation has been more difficult. The unique properties of the mature oocyte, such as the meiotic stage with sensitive spindle structure as well as the large cell volume, are responsible for the limited success obtained to date. There have been two approaches to cryopreserving the oocyte: (i) slow freeze–rapid thaw, and (ii) vitrification protocols with rapid cooling–rapid warming. Both methods have incorporated sugars (sucrose) as a beneficial non-permeating extracellular cryoprotectant. Studies of organisms that survive extreme conditions of freezing/dehydration have demonstrated the ability to accumulate intracellular sugars to afford protection and survival. A novel technique using microinjection of sugars into the oocyte for cryopreservation has been developed as an alternative approach to external addition of sugars. Freezing the human oocyte has been a challenging goal; however, developing research and efforts will, in the near future, provide women with an important option for their reproductive health. Keywords: birth, human, oocyte cryopreservation, slow freezing, sugar, vitrification

Introduction After almost three decades of successful cryopreservation of mouse embryos (Whittingham et al., 1972) and over two decades with human embryos (summarized by Ludwig et al., 1999), advances in the preservation of oocytes have been limited since the original report in the mouse (Parkening et al., 1976). Men have been experiencing the benefit of successful gamete preservation for several decades. In fact, spermatozoa were successfully cryopreserved half a century ago in the bull (Polge and Lovelock, 1952). However, women are still experiencing the limitations of the current state of the art for oocyte preservation. The difficulties of cryopreserving an oocyte versus an embryo appear to be inherent to the structure of the DNA and microtubular arrangement, which is unique to the mature oocyte. Premature zona pellucida hardening (Carroll et al.,

1990), disorganization of cytoskeletal structure such as microfilaments and spindle microtubules (Magistrini and Szollosi, 1980; Johnson and Pickering, 1987; Pickering and Johnson, 1987; Vincent et al., 1990; Aigner et al., 1992; for review Parks and Ruffing, 1992; for review Vincent and Johnson, 1992), and oocyte lysis are all injuries associated with cryopreservation of mammalian oocytes. Chen et al. (2003) provide an extensive review of these specific effects of cryopreservation on the mature oocyte. Depolymerization of the spindle can create significant problems during the fertilization process if recovery after cryopreservation is not complete. In the mouse, Eroglu et al. (1998b) found that oocytes inseminated immediately after thawing resulted in a decrease in normal fertilization with significant alterations in development of the second polar body, the mitotic spindle and pronuclear migration. The significance of the damage to the cytoskeletal structure of the oocyte appears related to many

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factors, including cryopreservative concentrations and low temperature exposure (Chen et al., 2003). A recent publication (Stachecki et al., 2004) found little evidence of prolonged spindle damage in several species after cryopreservation using a choline-based slow cooling–rapid thaw protocol. Baka et al. (1995) found the spindle apparatus of the mature human oocyte susceptible to significant damage during cryopreservation when compared with the immature oocyte that was frozen and subsequently in-vitro matured. A related study conducted in the mouse (Eroglu et al., 1998a) also demonstrated that cryopreserving germinal vesicle stage oocytes was a superior option to the metaphase II mature oocyte. The disruption of the metaphase plate in the mouse oocyte during cryopreservation is depicted in Figure 1. In an attempt to avoid the delicate nature of the mature human metaphase II oocyte, some researchers have explored the possibility of cryopreserving immature oocytes where the DNA is more densely packed (Toth et al., 1994). In addition to freezing immature oocytes, Toth and coworkers (1994) also demonstrated improved results when sugars were utilized. Most of the current work with human oocytes has centred on the use of the mature metaphase II oocyte for preservation.

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The first successful human oocyte cryopreservation resulting in a live birth was reported by Chen (1986). Since this initial report nearly two decades ago, approximately 40 live births have been reported in peer reviewed publications (Table 1) and more than 50 pregnancies described (Al-Hasani et al., 1987; Diedrich et al., 1988; Siebzehnruebl et al., 1989; Tucker et al., 1996; Antinori et al., 1998; Borini et al., 1998; Nawroth and Kissing 1998; Young et al., 1998; Porcu et al., 2000; Winslow, 2001; Chen et al., 2002; for review, see Van der Elst, 2003). While these results suggest promise, efforts have fallen short of offering dependable technology. A reliable method for long-term storage of human oocytes would be beneficial to many women for very diverse reasons. Those who are anticipating loss of gonadal function due to radiation, chemotherapy or extirpative therapy could greatly benefit by preservation of some gametes. Current statistics suggest that 1 in 52 females under the age of 40 are diagnosed with cancer (Jemal et al., 2002). In recent years, chemotherapy and radiation treatment of patients with cancer have become increasingly successful, and sustained remissions are more commonly achieved. The 5-year relative survival rate, over all

Figure 1. Cytoskeletal organization and chromosome alignment of metaphase II mouse oocytes during the period after thawing. Overlaid fluorescence images depict microfilaments (f-actin) (red), microtubules (green), and DNA (blue). (A) An unfrozen control oocyte fixed after 1 h of incubation at 37°C. Arrow indicates the f-actin-rich ring outside the relatively f-actin-depleted intermediate zone. (B) A cryopreserved oocyte fixed immediately upon thawing (without removal of cryoprotectants). Arrowhead shows one of several microtubular asters; arrow indicates the first polar body. (C) A cryopreserved oocyte fixed immediately after the removal of cryoprotectants. (D) A cryopreserved oocyte fixed after the removal of cryoprotectants and subsequent incubation for 1 h at 37°C. Arrowhead indicates f-actin-rich ring; arrow shows the first polar body. Bar = 20 μm. Reprinted from Eroglu et al. (1998b), with permission from The American Society of Reproductive Medicine © 1998.

Article - Cryopreserving human oocytes - DL Wright et al.

Table 1. Published oocyte cryopreservation studies resulting in live births. COC = cumulus–oocyte complex, infertile patient = infertile patient cohort, MII = only metaphase two oocytes, all = all oocyte maturational stages. n/a = not/available, only percentage described in publication. Authors

Method of freeze/thaw

Oocyte source

Sucrose freeze/initial thaw (mol/l)

Survival rate (%)

Fertilization rate (%)

Chen (1986)

Slow/rapid

0/0

32/40 (80)

25/30 (83)

2

20

van Uem et al. (1987)

Slow/slow

0/0

7/28 (25)

2/4 (50)

1

28

Porcu et al. (1997)

Slow/rapid

0.2/0.2

4/12 (33)

2/4 (50)

1

12

Tucker et al. (1998) two study groups

Slow/rapid

Infertile patient – COC Infertile patient – COC Infertile patient – MII Donor – MII

0.1/0.2

75/311 (24) 38/75 (51)

3

104

8/61 (13)

4/8 (50)

1

61

3/10 (30)

2/3 (67)

1

10

12

153

58/79 (73)

5

18

58/144 (40) 33/58 (57)

2

72

Polak de Fried et al. (1998) Porcu et al. (2000) Fosas et al. (2003) Quintans et al. (2002)

Boldt et al (2003) two study groups (traditional and Na depleted)

Infertile 0.1/0.2 patient – All Slow/rapid Donor – 0.1/0.2 COC Slow/rapid Infertile 0.2/0.2 patient – MII Slow/rapid Donor – 0.3/0.3 MII Slow/rapid Infertile 0.1/0.2 (Na depleted) patient – MII (male factor only) Slow/rapid Infertile traditional patient – 0.1/0.2 MII Na depleted 0.2/0.5

Kuleshova et al. (1999)

Vitrification

Yoon et al. (2003)

Vitrification

Infertile patient – MII Infertile patient – COC

1013a/1840 n/a (57–64) (55) 79/88 (90)

Number of infants

6/49 (12)

3/6 (50)

67/90 (74)

39/66 (59)

5

18

0.6/0.4

11/17 (65)

5/11 (45)

1

17

1.0/1.0

325/474 (69) 142/198 (72)

7

68

40

79

Total

1688/3164 (53)

353/542 (65)

0

Number of oocytes/infant

–

aActual number survived not published, estimated from percentage described.

cancers combined, is now 63% (American Cancer Society, 2004). Embryo cryopreservation is an alternative for some female patients, but preservation of the unfertilized oocyte would be preferable in many instances. Ethical, religious and legal issues abound when freezing an embryo versus a gamete. Many couples are constrained during an IVF attempt by their inability to preserve excess embryos due to religious beliefs or government-mandated regulations. The ability to reliably freeze the oocyte would address many of these concerns. In addition, cryopreservation of the human oocyte would be advantageous in donor oocyte programmes. Preservation of the gametes would allow for appropriate testing of infectious

diseases as is the current standard of care when using sperm donors. Some women might elect to freeze their oocytes when they were younger if they anticipated a delay in pregnancy.

Oocyte cryopreservation methods All cryopreservation protocols have in common the goal to cool cells to subzero temperatures that effectively stop biological activity with subsequent return to physiological temperatures without damaging viability. Traditional cryopreservation methods for cells and oocytes can be grouped as (i) slow freeze–rapid thaw protocols that use 1–2 mol/l

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conventional penetrating cryoprotectants (e.g. dimethylsulphoxide (DMSO), glycerol, 1,2-propanediol (prOH)), and (ii) vitrification protocols that use high concentrations (i.e. 4–8 mol/l) of cryoprotectant mixtures with rapid cooling–rapid warming methods. Both require a fine balance between potential toxic effects to the cells related to cooling rate and cryoprotectant concentrations. The slow freeze protocols use a slow rate of cooling and dehydration of the cells to subvert nucleation of lethal intracellular ice. A potential problem with these protocols is the toxic effects of long exposure time to increased electrolyte concentrations (Mazur, 1984). Vitrification protocols are limited by potential toxicity through the use of high concentrations of cryoprotectants. While still controversial, some researchers propose the use of the permeating cryoprotectants is important for their ability to interact with and replace some water molecules in the cell, resulting in a decrease in the actual freezing point. Another dramatic difference between the two types of protocols is the ‘open system’ required with the ultrarapid vitrification method. Increased oocyte survival outcomes were noted in domestic animal studies when vitrification protocols were modified to allow for ultrarapid cooling by the use of ‘open’ systems such as electron microscope grids (Martino et al., 1996), open pulled straws (Vajta et al., 1998), precooled solid surface (Dinnyes et al., 2000) and nylon loops (Lane and Gardner, 2001). Cells are generally exposed directly to liquid nitrogen during the vitrification process, where slow freeze methods employ an enclosed straw or vial. The guiding principle for both of the procedures is to minimize or eliminate intracellular ice formation. Conventional slow freezing methods induce extracellular ice formation (–5 to –7°C), causing the solute concentration to increase resulting in dehydration of the cell by water transport out of the cell into the extracellular medium. The same process is completed in vitrification protocols by the use of high concentrations of cryoprotectants for a shorter duration, with the same intent to dehydrate the cell prior to rapid cooling. This rapid transition also serves to limit the time available for intracellular ice formation. There is a fine balance required with vitrification protocols, to limit the toxic affects of the high concentration of cryoprotectants while sufficiently removing the intracellular water.

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Subsequent to the freezing process is the need to successfully thaw the cells. Recrystallization and osmotic shock are two potential problems that occur during this process. The transition from the storage temperature of –196°C to physiological temperatures can cause the same cell damage due to ice crystal formation as freezing. Many protocols use a rapid thaw method to prevent this formation by limiting the actual time available for ice to form. Osmotic shock can be diminished by using stepwise dilutions of decreasing cryoprotectants. Another option is to use non-permeating cryoprotectants, such as sucrose, to minimize osmotic stress of the cell. When inside the cell, sugars may serve to stabilize membranes, proteins and supramolecular structures during dehydration (Crowe et al., 1998). However, sugars are unable to cross the cell membrane naturally, limiting their beneficial effects to enhancing cellular dehydration by osmotic pressure. Addition of extracellular sucrose to most human cryopreservation protocols has become the standard.

Protocols used for live birth outcomes (see Table 1 for summary) Slow freeze without the use of sucrose The two earliest reports in the human of successful oocyte cryopreservation with resulting live births (Chen, 1986; van Uem et al., 1987) were described just a few years after the first published success with human embryos (Trounson and Mohr, 1983; Zeilmaker et al., 1984). Chen (1986) and van Uem et al. (1987) described similar protocols that incorporated 1.5 mol/l DMSO as the cryoprotectant within the base medium phosphate-buffered saline (PBS) and 10% fetal bovine serum (FBS). Van Uem and coworkers chose a slow thaw regimen versus the rapid thaw procedure described by Chen. Decreasing dilutions of cryoprotectant, from four to six dilutions, were used to minimize osmotic stress on the oocytes. Survival (80 and 25%) and fertilization (83 and 50%) rates were higher for Chen’s rapid thaw protocol. Both studies described insemination of the thawed cumulus–oocyte complexes with conventional IVF techniques. Two pregnancies resulted from these reports. Chen described the birth of twins and van Uem et al. described one singleton birth.

Slow freeze–rapid thaw with the addition of sucrose It was another 10 years before the next live birth with oocyte cryopreservation was reported by Porcu and coworkers (1997). A publication followed 3 years later chronicling the continued success in Italy with live births from oocyte cryopreservation (Porcu et al., 2000). An additional three publications (Tucker et al., 1998; Polak de Fried et al., 1998; Fosas et al., 2003) have reported live births using slightly modified versions of the protocol described by Porcu et al. (1997). All five studies used PrOH as the permeating cryoprotectant at 1.5 mol/l and sucrose as the non-permeating cryoprotectant within different defined CO2 independent salt solutions (PBS, Earle’s, and HTF + HEPES). Each protocol shared the use of a slow freeze–rapid thaw method with decreasing concentrations of cryoprotectant in 3–5 dilutions. The suggestion that hardening of the zona pellucida, due to premature cortical granule release, may impede fertilization after oocyte cryopreservation, led researchers to use intracytoplasmic sperm injection (ICSI) as the preferred method for fertilization (Vincent et al., 1990; Kazem et al., 1995). All viable oocytes were inseminated with freshly ejaculated spermatozoa by use of ICSI. The major difference between the three studies was the concentration of sucrose used (0.1, 0.2 and 0.3 mol/l). Increasing the concentration of sucrose in the freezing media was studied by Fabbri et al. (2001), showing improved survival results with oocyte cryopreservation when the concentration of sucrose was increased to 0.2 and 0.3 mol/l. This change in protocol was incorporated by Fosas et al. (2003). Tucker et al. (1998) and Fosas et al. (2003) also evaluated the use of a donor oocyte population. Given that these oocytes would come from young fertile females, it has been suggested that the outcome of cryopreservation could potentially be better. However, only Fosas et al. demonstrated an improved result using this population of oocytes. In the review by Porcu et al. (2000), four additional live births were described with one involving the use of a frozen semen sample

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and the other three from the use of epididymal and testicular spermatozoa. These four births are excluded from Table 1 due to the confounding factor of sperm source.

Slow freeze–rapid thaw with the addition of sucrose in low-sodium medium More recently, the concern regarding high solute concentrations as a source of cellular injury during cryopreservation led to the evaluation of replacing sodium, the main contributor to the solution effect, with choline as the major extracellular cation (Stachecki et al., 1998). The investigation was conducted using mouse oocytes, and found the highest survival and developmental rates in the choline-based medium when compared with the conventional sodium-based medium. These original findings were followed by a complete study through implantation and fetal development in the mouse, demonstrating normal development and viable fetuses with the use of the choline-based medium (Stachecki et al., 2002). Quintans et al. (2002) described the first human births associated with oocytes frozen in choline-based medium. This study population was a cohort of patients experiencing only male factor related infertility. Less that half the oocytes survived cryopreservation. Those oocytes that survived were fertilized by ICSI; after transfer of the resultant embryos, two infants were born. The choline-based freezing protocol was similar to the conventional method published by Tucker et al. (1998), with the modification of using PBS and 20% synthetic serum substitute (SSS) as the protein source. A second more successful attempt evaluated the difference between the conventional freezing medium and the choline-based medium, finding improved results using the latter (Boldt et al., 2003). Both protocols studied in this investigation were based on the method described by Tucker et al. (1998), using the base medium of PBS and 20% SSS. However, in the portion of the study using choline, the initial thaw solution had an elevated concentration of sucrose (0.5 mol/l) as compared with other previous reports. Boldt and coworkers described improved survival and fertilization rate (ICSI) with the choline-based medium. The four live births (five infants) reported in this publication resulted from oocytes frozen in the choline-based medium.

Vitrification Limited success in oocyte cryopreservation has also been achieved using the alternate approach of vitrification. The earliest report was in 1999 by Kuleshova and co-investigators. This original publication was followed by another live birth study from Korea (Yoon et al., 2000). Yoon and coworkers summarized their entire experience with oocyte cryopreservation in 2003. Both sets of investigators used similar protocols for vitrification. Ethylene glycol (EG) and sucrose were the cryoprotectants in the base medium PBS, supplemented with either 10 mg/ml human serum albumin (HSA) (Kuleshova et al., 1999) or 10% FBS (Yoon et al., 2003). Kuleshova et al. used three increasing concentrations of EG (10, 20, 40%), adding 0.6 mol/l sucrose in the final dilution for vitrification. Yoon limited the dilution steps to two with 1.5 mol/l EG followed by 5.5 mol/l EG and 1 mol/l sucrose for the final solution. The oocytes were vitrified on two different devices. Kuleshova et al. utilized an open pulled straw system

and Yoon found success using an electron microscope copper grid. The type of oocyte vitrified differed between the two groups, with Kuleshova et al. electing to clean the surrounding cells away and freeze only the metaphase two oocytes. Yoon and co-investigators cryopreserved the entire cumulus–oocyte complex without differentiation of mature and immature oocytes, which helps to explain the lower number of oocytes injected for ICSI when compared with the number that actually survived the vitrification process. Surviving oocytes in both studies were inseminated using ICSI. The oocytes were thawed in a similar fashion between both publications, using only sucrose in the dilutions. However, the number of dilutions and concentration varied. Kuleshova and co-authors began with 0.4 mol/l sucrose followed by 0.25 mol/l and finally 0.125 mol/l. Yoon et al. began at a much higher concentration of sucrose, 1 mol/l, followed by three additional decreasing dilutions (0.5, 0.25, 0.125 mol/l). Survival rates were similar for each group (65 and 69%) and fertilization rates were higher (72 and 45%) in the more extensive study by Yoon et al. The early publication by Kuleshova et al. described one live birth from the 17 oocytes frozen. The larger study by Yoon et al. reported six live births (seven infants) from their 5-year experience.

Use of intracellular sugars In nature, a wide variety of organisms including arctic frogs, salamanders, tardigrades, some nematodes, rotifers, insects, brine shrimp, bacteria, yeasts, fungi and their spores, some plant seeds, and cysts of certain crustaceans tolerate extreme conditions (Crowe et al., 1992; Potts, 1994). The ability to survive such extreme dehydration conditions is related to the accumulation of large amounts of intracellular sugars (as much as 20% of their dry weight). Research has demonstrated that sugars are useful in the stabilization of lipid membranes and proteins when cells are dehydrated (Crowe et al., 1998). Cells contain free water and water that is bound to atoms within various proteins and membrane phospholipids (Shaw and Jones, 2003). As cells are dehydrated during cryopreservation, a fine balance must be maintained between the removal of free water that could form ice crystals without the excessive removal of bound water, resulting in loss of structural support to the proteins and lipids. It is thought that sugars may serve as a replacement for bound water on the membranes to diminish injury from dehydration stresses. Mammalian cell membranes are impermeable to sugars, and consequently vital supramolecular structures, proteins, and lipids inside cells cannot receive the benefit from the stabilizing properties of sugars during freezing. Given the knowledge that sugar inside the cell could improve desiccation and subsequent cryopreservation results, several researchers have embarked on novel ventures to artificially introduce sugars to the intracellular domain. Beattie et al. (1997) introduced trehalose into pancreatic islet cells using thermal poration in the presence of 2 mol/l DMSO. This study resulted in previously unobtainable survival rates of human pancreatic islets with the combination of intracellular trehalose and DMSO versus DMSO alone, indicating beneficial effects of trehalose in the cryopreservation of mammalian cells. Another successful method to move trehalose through the mammalian cell membrane was described using a genetically engineered pore forming protein (Eroglu et al., 2000). The authors were able to effectively control the opening and closing

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of these pores to provide predetermined concentrations of intracellular trehalose in two cell lines. Trehalose alone, at a concentration of 0.2 mol/l intra- and extracellularly, was able to afford adequate protection for the cells during cryopreservation, with survival rates of >70%. Other investigators have introduced sugars by transfecting mammalian cells with a bacterial trehalose synthase gene (Garcia de Castro and Tunnacliffe, 2000; Guo et al., 2000; Puhlev et al., 2001). While some studies have demonstrated the beneficial nature of having sugar inside the cell when desiccating or freezing, most of the methods investigated thus far would be unacceptable for use with human gametes. A novel method was developed that would utilize current techniques for introducing spermatozoa into oocytes and bypass the membrane impermeability limitations related to sugars. Eroglu et al. (2002) were the first to describe the introduction of trehalose into the human oocyte by microinjection (Figure 2). Briefly, the technique involves the quantitative microinjection of trehalose, through the use of calibrated micropipettes, into the oocyte prior to the initiation of a slow-cooling protocol. In a process similar to ICSI, a concentrated (0.8 mol/l) solution of trehalose is injected into the oocyte to reach an internal concentration of 0.15 mol/l trehalose. The injected oocytes are then stepped through two dilutions (0.3 and 0.5 mol/l) of trehalose. Oocytes were frozen within straws and thawed by exposure to room air. Thaw dilutions consist of 0.5 mol/l galactose followed by 0.2 mol/l galactose. The study used discard human oocytes (n = 158) that failed to fertilize during normal IVF procedures. Three different groups of the oocytes were treated with the same slow cooling–rapid thawing protocol: (i) control group with no addition of trehalose; (ii) extracellular trehalose with 0.5 mol/l in the freezing medium; (iii) intracellular trehalose with 0.15

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Figure 2. Microinjection of trehalose solution into a failed-tofertilize human oocyte that also contained a fluorophore to offer visualization of the injection procedure. Reprinted from Eroglu et al. (2002), with permission from The American Society of Reproductive Medicine © 2002.

mol/l intracellular and 0.5 mol/l extracellular trehalose. Using only trehalose as the cryoprotectant, Eroglu and co-workers (Eroglu et al., 2002) were able to obtain >60% survival when cells were cooled to –60°C with 0.15 mol/l intracellular and 0.5 mol/l extracellular (Figure 3). This evaluation was followed by a more extensive study of the technique in the mouse (Eroglu et al., 2003a). A natural concern when introducing a substance to a cell is the level of toxicity. The authors found that injecting 0.15 mol/l trehalose into the onecell zygote did not alter its ability to develop to the blastocyst stage. In addition, when these injected embryos (0.1 mol/l) were transferred to foster mothers, implantation occurred and healthy pups were delivered (Table 2). No difference was noted in implantation sites, viable fetuses or mean fetal weight when compared with controls. Metabolism studies have followed (Eroglu et al., 2003b) to determine the fate of this microinjected trehalose during embryonic development. A high performance liquid chromatography and pulsed amperometric detection protocol was developed to ascertain the amount of sugar in a single cell. Trehalose was progressively eliminated from the embryo during development from the microinjected oocyte (6 ng) to the blastocyst stage embryo (<2 ng).

Conclusion Nearly 20 years ago, the first live birth was reported from human oocyte cryopreservation. Advances in technology have been slow to develop, primarily due to the unique nature of an oocyte as compared with most other cells in the body. Two general methods have been employed by researchers in an attempt to bring oocyte cryopreservation to general laboratory practice. The majority of publications reporting live births use a slow freeze–rapid thaw protocol with variations in cryoprotectant concentrations and sodium concentrations.

Figure 3. Survival of human oocytes frozen in the absence and presence of trehalose to different temperatures. The total number of oocytes (n) for each experiment is shown on the figure. The survival rates represent mean ± SEM for more than three replicates. Reprinted from Eroglu et al. (2002), with permission from The American Society of Reproductive Medicine © 2002.

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Table 2. Implantation and fetal development to day 16 of gestation after microinjection to 0.1 mol/l intracellular trehalose. Untreated control and microinjected pronuclear eggs were cultured in the hypermedium overnight and two-cell embryos were transferred into pseudopregnant recipient mice. The data summarize outcome of three experiments.

Control (non-injected) Trehalose (microinjected)

No. 2-cell embryos transferred

No. implantation sites

No. viable fetuses

Mean fetal weights (g) ± SD

13

10/13 (77)

7/13 (54)

0.29 ± 0.03

14

12/14 (86)

8/14 (57)

0.27 ± 0.04

Values in parentheses are percentages.

Other investigators have explored the use of vitrification to avoid intracellular ice formation with some success. The addition of extracellular sucrose has become a standard in oocyte cryopreservation protocols with improved survival rates. With the knowledge from nature that intracellular sugars may offer additional protection for a cell during dehydration and freezing, a novel technique has been developed to insert a sugar, trehalose, into the oocyte with demonstrated success in the mouse model and preliminary studies in human oocytes. Continued research and development in this essential field of study are nearing the goal of offering reliable cryopreservation of the human oocyte.

Acknowledgements This study was supported in part by a research grant from the Lance Armstrong Foundation.

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Paper based on contribution presented at the ‘PCOS Symposium: Current Concepts, Treatment and Ovulation Induction’ in Antalya, Turkey, September 2003. Received 8 March 2004; refereed 5 April 2004; accepted 16 June 2004. Declaration of interest: Thomas Toth and Mehmet Toner are consultants for Viacell.