Oocytes cryopreservation: State of art

Reproductive Toxicology 22 (2006) 250–262

Review

Oocytes cryopreservation: State of art G. Ambrosini a , A. Andrisani a,∗ , E. Porcu d , E. Rebellato a , A. Revelli c , D. Caserta b , E. Cosmi a , R. Marci b , M. Moscarini b a

Department of Gynecological Science and Reproductive Medicine, University of Padua School of Medicine, Padua, Italy b Department of Gynecology and Obstetrics Unit, S. Andrea Hospital, University of Rome “La Sapienza”, Rome, Italy c Deptarment of Obstetrical and Gynecological Sciences Reproductive Medicine and IVF Unit, University of Torino, S. Anna Hospital, Torino, Italy d Department of Obstetrics and Gynecology, University of Bologna, Bologna, Italy Received 1 March 2006; received in revised form 26 April 2006; accepted 27 April 2006 Available online 19 June 2006

Abstract In the present review article we sought to analyze, on the basis of a systematic review, the indications, rationale of oocytes cryopreservation, as well as the techniques that improved the aforementioned procedure in order to higher the pregnancy rate in women undergoing that procedure. Moreover, we pointed out the importance of oocytes cryopreservation in the research field as oocyte banking may be of utmost importance to increase the availability of oocytes for research applications such as genetic engineering or embryo cloning. Oocyte freezing has 25 year of history alterning successes and setbacks. Human oocytes have a delicate architecture but are freezable. Clinical efficiency remains low, but healthy children have been born, indicating that chromosomally normal embryos can originate from frozen oocytes. Freezing protocols are not yet optimal and it is now desirable to combine empirical and theoretical knowledge. © 2006 Elsevier Inc. All rights reserved. Keywords: Oocyte cryopreservation; Freezing and thawing; Vitrification; Cryoprotectant agents; Meiotic spindle; Mammalian species; Survival and fertilization rate; Development rate

Contents 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of oocyte cryopreservation in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of the mammalian oocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rationale for oocyte cryopreservation in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Slow freezing method for mammalian oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryopreservation-related injury to the mammalian oocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryodamage in human oocytes: cytoskeleton and meiotic spindle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Cooling to room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Cooling to 0 ◦ C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zona pellucida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro enviroment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current status of oocyte cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Children from cryopreserved oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +39 0347 9001591; fax: +39 049 8213409. E-mail address: [email protected] (A. Andrisani).

0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2006.04.024

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1. Introduction The recovery of oocytes from low temperature has proven to be more effective compared to preservation of embryos and spermatozoa. In fact the oocyte has a short life span and unless it fuses with a single spermatozoon in order to be fertilized, it will degenerate. The oocyte needs to maintain the integrity of several unique structural features, in order to undergo fertilization and development, many of which are affected by low temperatures, e.g. the zona pellucida, the cortical granules, the microtubular spindel and condensed chromosomes. The first success with oocyte freezing was obtained in the mouse in 1977. However, the interest to that approach has been lost as the first IVF baby obtained in 1978 and in 1983 the first human pregnancies and live births from frozen embryos were announced [1,2]. Because embryo freezing was not generally morally acceptable, human oocyte freezing was suddenly introduced clinically in 1986 based on the limited experience in mouse oocytes [3]. 2. History of oocyte cryopreservation in mammals The history of oocyte cryopreservation in mammals begins in 1958, when the possibility of survival of unfertilized mouse oocytes after freezing and thawing was first demonstrated [4]. Approximately 20 years later, the fundamental cryobiology of the mouse oocyte began to be systematically studied [5,6], and in 1977 the first successful IVF and live offspring from cryopreserved mouse oocytes frozen and stored under liquid nitrogen was reported [7]. Subsequently, relatively successful cryopreservation of oocytes has been achieved for both the mouse (by far the most studied species) and several other species including the hamster, rabbit, pig, cat and cow [8–16]. Overall, however, the procedures developed for oocyte cryopreservation are far less effective than those used for embryos. Although several studies have been accomplished in order to cryopreserve immature (GV) or mature (MII) oocytes from several mammalian species, the final result is still unsatisfactory and the main reason for this is the specific biological features of the oocyte and the severity of problems that are encountered when this cell undergoes freezing–thawing procedures. 3. Structure of the mammalian oocyte Mammalian oocytes are very large cells, ranging from 80 to 120 mcm in diameter depending upon the species. Immature oocytes at the germinal vesicle (GV) stage are characterized by a large diploid nucleus (prophase I), a dense band of filamentous actin subjacent the oolemma, and several other organelles scattered in the ooplasm, such as mitochondria, endoplasmic reticulum, and Golgi apparatus. The Golgi of the oocyte produces lysosome-like vesicles, the cortical granules, that are randomly distributed throughout the cytoplasm. The nuclearly mature oocyte (metaphase of the second meiotic division, MII) is characterized by a large, peripheral spindle apparatus with microtubules extending from pole to pole and

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from each pole to the kinetochores of the chromosomes aligned along the metaphase plate. The meiotic spindle of mammalian oocytes lacks centrioles but contains some pericentriolar material which serves as the organizing center for tubulin polymerization at each pole during spindle formation [17]. Abundant actincontaining microfilaments are distributed in the perinuclear and cortical ooplasm, where they direct organellar distribution and polar body extrusion during meiosis. In the MII oocyte, cortical granules migrate to the periphery of the ooplasm just beneath the actin band, where they are ready to undergo exocytosis at the time of fertilization [18]. 4. Rationale for oocyte cryopreservation in mammals Oocyte cryopreservation in mammals has a series of potential applications aimed at the maintenance of biodiversity. From a clinical point of view, in animal farming there is a need for cryostoring oocytes from domestic livestock animals (e.g. cattle, pigs and sheep) with high economic or genetic value in order to enhance the development of improved breeding programs. The oocytes from some laboratory animals can be cryostored in order to preserve specific strains for experimental purposes, without the need and the high cost of continuous breeding. Moreover, oocyte cryopreservation and banking may be important to preserve endangered species. In the research field, oocyte banking may be of utmost importance to increase the availability of oocytes for research applications such as genetic engineering or embryo cloning. In fact, strains of transgenic mice have been produced from frozen–thawed oocytes after microinjection of DNA, a valuable method to maintain genetically valuable strains [19]. Moreover, frozen bovine oocytes have been used as recipients for nuclear transfer procedures, even if embryo development following fertilization of oocytes reconstructed by nuclear transfer has been found to be poorer when using frozen instead of fresh oocytes, probably because of the cryodamage of the cell’s cytoplasm [20,21]. The feasibility of nuclear transfer with frozen–thawed oocytes has also been shown in the mouse, where frozen immature oocytes have been used both as germinal vesicle (GV) donors and recipients in GV-transfer procedures [22]. Another interesting option to cryopreserve genetically precious strains is freezing an oocyte after parthenogenetical activation, subsequently using the female pronucleus of the haploid embryo to build a reconstructed zygote together with a male haploid hemizygote obtained by female pronuclear removal from a fertilized oocyte (hemi-cloning). This methodology has been demonstrated successful as viable offsprings have been obtained from rabbit and mouse parthenogenotetically activated oocytes [23,24]. 5. Slow freezing method for mammalian oocytes The standard method used for mammalian oocyte cryopreservation is conventionally called “slow freezing” or “equilibrium cooling” [25], as the cooling rate is slow and it implies the use of solutions containing cryoprotectant agents (CPAs).

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In slow freezing, oocytes are immersed in hypertonic CPA solutions, and an outward flux of water is generated by osmosis because the cell membranes are more permeable to water than to the CPA. When the equilibrium point is reached, the cell volume is restored, but cooling the cell to subzero temperatures causes a second volume variation due to the fact that at first water freezes extracellularly and the salt concentration rises outside the oocyte; water therefore leaves the cell again by osmosis [26]. To avoid the highly unstable state of supercooling of cell water, that will lead to sudden ice nucleation and have lethal consequences on the cell, “seeding” is usually induced around 4–7 ◦ C. In order to minimize cellular stress due to volume and salt concentration changes, an optimal rate of cooling is critical. Below a temperature of −150 ◦ C, metabolic activity ceases, cells are stable and may be stored in liquid nitrogen. The risk of intracellular ice formation is higher when temperature is changing, whether by cooling or rewarming. Hence, during thawing the temperature is usually raised very rapidly through the critical range [27]. Structural integrity inside the oocyte can be affected not only by ice crystal formation, but also by the abrupt increase in salt concentration that occurs during cellular dehydration (“solution effect”). CPAs are able to lower the freezing point and protect the cell during dehydration. After CPA addition, the intracellular medium becomes viscous and form a glassy solid rather than crystallizing. Among CPAs, dimethylsulphoxide (DMSO), 1,2-propanediol (PROH) and ethylene glycol (EG) are the most widely used for oocyte cryopreservation [28], whereas glycerol, that is frequently employed for sperm, was observed to be poorly effective for oocytes [29]. 6. Cryopreservation-related injury to the mammalian oocyte Mammalian oocytes have a complex subcellular structure an most of the components are particularly sensitive to temperature and osmotic pressure. The developmental stage of the oocyte (from GV to MII stage) affects its cryobiological properties as the plasma membrane, the mitochondria, some cytoskeletal elements and other organelles relevantly change during maturation. Also the granulosa cells of the cumulus and the zona pellucida play an important role in the development of immature oocytes; a stress produced to cumulus cells during the freezing procedure might be reflected in an adverse effect on the oocyte. A direct contact between cumulus cells and oocyte during follicle growth improves oocyte quality, and a loss of association between oocyte and cumulus cells was observed to be one of the consequences of cryopreservation [30,31]. During cryopreservation, the oocyte undergoes dramatic volume changes due to different osmotic pressures between the intracellular and the extracellular solutions, which cause rapid water transport across the plasma membrane [32]. These changes in cell volume affect the integrity of the oolemma as well as that of the subcellular structures. The ideal osmotic response has been estimated for MII mouse oocytes [33] as well as for immature and mature bovine oocytes [34], but oocyte osmotic tolerance has to be determined for each species and oocyte devel-

opmental stage in order to avoid excessive shrinkage/swelling during freezing/thawing procedures. Cryopreservation has been shown to induce premature extrusion of the cortical granules, in turn leading to an abrupt hardening of the ZP; this phenomenon explains the decreased rates of fertilization in frozen–thawed mouse oocytes after IVF, and the almost absolute need for microinjection procedures (ICSI) to achieve fertilization [35–39]. Cooling mouse oocytes to 4 ◦ C for more than 5 min dramatically alters the ZP, resulting in an irreversible decrease in chimotrypsin sensitivity [40]. Changes in the ZP are associated with a numerical decrease in cortical granules and do not occur in isolated ZP, strongly suggesting that the underlying cause is a premature cortical granule exocytosis. At low temperatures some CPAs, such as dimethylsulphoxide (DMSO), counteract cortical granule exocytosis, stored at room temperature the effect is opposite, as it promotes the fusion between oolemma and cortical granules [41]. Cryoinjury to the oocyte appears to be cumulative with each procedural step (addition of CPAs, cooling, freezing, thawing). Hypothermia during initial cooling is one of the most important causes of the reduced developmental competence of cryopreserved oocytes. Moreover, chilling per se does not seem to alter the morphology of oocytes from most species [42], but an injury may be manifested morphologically at later developmental stages [43]. Chilling does not appear to compromise the structural integrity of the oolemma, yet hydraulic conductivity (membrane permeability to water and CPAs) appears to be decreased in oocytes cooled to 0 ◦ C [44,45]. As a general roule, oocytes from domestic animals are extremely sensitive to chilling in the temperature range between 0 ◦ C and seeding down to −7 ◦ C. Porcine oocytes undergo particularly relevant damages by chilling [42]. Fertilization rates of in vitro matured bovine oocytes appears to be significantly reduced after 2 min at 0 ◦ C, and even the cleavage rate significantly decreases when oocytes are cooled to 0 ◦ C or up to −7 ◦ C [46]. With the slow cooling rates associated with slow freezing cryopreservation procedures, chilling sensitivity alone can account for a relevant part of the low development potential of frozen–thawed mammalian oocytes. The most important effect of cooling MII oocytes below body temperature is the disassembly of the meiotic spindle apparatus [47]. The meiotic spindle is composed of microtubules consisting of alpha and beta tubulin dimers, that continuously change their state in equilibrium between the free and the polymerized state. Sensitivity of the spindle to cooling and rewarming is affected by temperature, duration of exposure and species-specificity. For example, the spindles of mouse and bovine oocytes are partially disassembled after cooling at 25 ◦ C and are completely disaggregated at 4 ◦ C [48–50]. After rewarming to 37 ◦ C for 60 min, the chilling effect on the spindle is completely reversed in mouse oocytes [51], whereas in bovine oocytes spindle morphology remains abnormal even after rewarming, due to a deficiency in the pericentriolar structures that are required for tubulin polymerization [41,50]. Complete recovery of spindles in slowly freezed mouse oocytes occurs 1 h after-thawing: oocytes inseminated immediately after thawing exhibit anormal spindles with an increased rate of chromosomal abnormalities, whereas those inseminated

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after 1 h incubation at 37 ◦ C frequently display normal fertilization [52]. Disassembly of the meiotic spindle is associated with dispersion of the metaphase chromosomes and with subsequent abnormalities such as aneuploidy or polyploidy [53,54]. If the spindle is completely disrupted and extrusion of the second polar body is prevented, polyploidy is the consequence. An increased polyploidy rate in mouse embryos from frozen–thawed mouse oocytes has been repeatedly shown [53,55–57]. A spindle injury to a lesser degree may lead to aneuploidy linked to unequal distribution of chromosomes and chromatids [58]. The presence of two female pronuclei and one male pronucleus (digynic triploidy) may also be observed, and is a sign of the important damage of the spindle. The effect of CPAs on the meiotic spindle is deeply influenced by their concentration and by temperature. In fact, at low doses (up to 1.0 M) propanediol (PROH) causes disorganization of the meiotic spindles of mouse oocytes, whereas its effect becomes stabilizing at higher concentrations (1.5–2.0 M) [59], although a temporary loss of the spindle upon rewarming to 37 ◦ C still persists [60]. The main problem when PROH is used as a CPA is of different nature, as it may induce parthenogenetic activation in mouse oocytes, rendering them non-fertilizable [61,62]. On the other hand, DMSO (1.5 M) at 37 ◦ C causes the formation of multiple tubulin asters; this phenomenon proceeds at the expense of the spindle, finally resulting in its disassembly. At 4 ◦ C the tubulin polymerizing activity of DMSO ceases because of the decreased membrane permeability to the CPA, and the final effect is that DMSO counteracts cold-induced spindle disassembly [63]. Thus, the temperature of exposition to DMSO is critical, and digynic polyploidy and maternal aneuploidy are largely procedure-dependent for cryopreservation protocols with DMSO [64]. Microfilaments which constitute much of the cortical cytoskeleton of the oocyte do not seem to be directly modified by cooling. An exception is represented by mouse oocytes, in which a thick network of actin filaments is located in the cortex overlying the metaphase chromosomes [65]. In mouse oocytes cooled to 4 ◦ C and stained with phallacidin, an irregular expansion of this microfilament network has been observed [38]; this finding is a reflection of the spindle disassembly and the resulting chromosome dispersion, which causes withdrawal of the actin associated with the chromosomes [17]. Once again, the effects of CPAs on microfilaments in mammalian oocytes are dependent upon temperature. DMSO 1.5 M at 37 ◦ C reduces the density of microfilaments in the area over the meiotic spindle of mouse oocytes, but this effect is almost absent when the CPA is added at 4 ◦ C [41]. Since the major concerns about spindle damage in oocyte freezing are all linked to the use of mature oocytes (MII), several attempts were made to freeze immature oocytes (GV), in which the meiotic spindle is not yet formed [66–69]. Unfortunately not the nucleus, but the cytoplasm appears to be the main problem in immature oocytes, as alterations of the cytoplasm are probably at the basis of the frequently impaired blastocyst development observed after in vitro maturation of GV oocytes, fertilization and embryo culture [12,62,69–71].

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7. Cryodamage in human oocytes: cytoskeleton and meiotic spindle It has been shown that the spindle is sensitive to cooling although repair is possible if the cell are returned to 37 ◦ C for sufficient time. However, in the human oocyte, some work suggested that the spindle may undergo irreversible disruption following exposure to room temperature for 10–30 min. A study on the kinetics of spindle disruption has recently been undertaken using freshly collected immature human oocytes which were matured in vitro to the metaphase II stage. After 1 min at 0 ◦ C spindle damage was negligible, but after 2–3 min the spindle was shortened. At 4–9 min, disruption was increasingly severe and by 10 min the spindles had completely disappeared. Human oocytes have also been cooled to various temperatures from room temperature to 0 ◦ C in order to assess spindle changes [72,73]. 7.1. Cooling to room temperature Human oocytes are rapidly affected by brief exposure to room temperature. Almeida and Bolton showed that abnormalities were identical when oocytes were exposed to 32 ◦ C or 25–27 ◦ C. In cooled oocytes, 77–89% of spindels displayed anomalies and 50% had chromosomal dispersal versus 69 and 13%, respectively, in control oocytes. Microtubule depolymerisation begins even with a fall in temperature of only 5 ◦ C in humans and seem irreversible when oocytes are exposed to 27 ◦ C. Spindle disassembly is dependent on the extent of temperature decrease and its duration [74]. 7.2. Cooling to 0 ◦ C Exposure to 0 ◦ C is highly detrimental to the meiotic spindle. Chromosomes remained close together, a usual feature suggesting that chromosome separation triggered by spindle dynamics does not occur after the rapid disassembly of the spindle [74]. Cooling to 0 ◦ C for 20–60 min has been shown to result in disorganised spindles in human oocytes in the presence or absence of Me2 SO [75] although widespread scattering of the chromosomes was not observed. Human oocytes exposed to PROH, either with or without freezing, displayed no stray chromosomes despite absence or abnormality of the spindle [76]. However, the use of several protocols which minimise these potentially dangerous alterations has been shown to be effective. One must also be aware of the numerous biases that may lead to overestimation of the true rate of damage caused by cooling and/or cryoprotectants to the oocyte spindle. The age of the patients included in the studies is an important parameter, as it has been shown that spindle and chromosomal anomalies are significantly and positively correleted to incresing age. More non-disjunction and predivision of chromatids are found in oocytes of patients older than 35 years [77].

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8. Zona pellucida The zona pellucida, the glycoprotein coat surrounding the oocyte, plays an important role in protection and regulation of fertilisation. Premature release of cortical granules from the oocytes during cryopreservation techniques may result in “zona hardening” that prevents the penetration of spematozoa into the oocyte. Conversely, the zona pellucida may be damaged or the cortical granules caused to migrate to the centre of the oocyte, resulting in multiple sperm entry and abnormal fertilisation. Whether or not zona hardening occurs following cryopreservation has less significance now that ICSI can be used to bypass the zona pellucida and inject a single spermatozoa. Studies comparing conventional IVF with ICSI fertilised oocytes that have been cryopreserved, showed comparable or better fertilisation and clevage with ICSI [78,79]. However, zona hardening may have other consequences on the ability of the embryo to hatch the zona pellucida prior to implantaion in the uterus. A number of live births have been reported using fertilisation by ICSI, with percentage of live births from thawed oocytes varying from 1 to 10. The first live births using this technique was described by Porcu et al. in 1997. In this case report, the survival rate of cryopreserved oocytes was poor (33.3%). In addition the fertilization rate with ICSI was not optimal (50%). Other investigators have reported high fertilization rate using ICSI with cryopreserved oocytes, suggesting the superiority of this method in optimizing fertilization and embryo cleavage [79]. 9. In vitro enviroment The quality of the oocytes can be modified not only by the process of cryopreservation but also by the in vitro enviroment in ART cycles. After the oocyte retrival an embryologist examines the aspirates under a stereo dissecting miscroscope with a transmitted illumination base and heated stage [80]; then the aspirates are poured in two Petri dishes and are carefully scanned searching oocytes. The oocytes appear within varying quantities of cumulus cells and, if very mature are pale, immature are dark. Granulosa cells are clearer often in iridescent clumps, blood clots, should be dissected to check for presence of cumulus cells. The presence of a single polar body indicates that it has reached the stage of methaphase II [80]. Tissue culture dishes or plates are equilibrated in the CO2 incubator overnight. Before each egg treatment the embryologist must be ensure that the heating blocks, stages and trays are warmed at 37 ◦ C. Even the collection test tubes and Petri dishes for scanning aspirates are usually overnight warmed. The clinical use of matured oocytes is a well known procedure in IVF treatment, and culture media are determined in every IVF laboratory to improve fertilization and pregnancy rate. Numerous events affect oocyte maturation and the acquisition of developmental competency, including: interactions between somatic cells of the follicle, in particular cumulus cells and the oocyte. Culture conditions do not reflect the follicular environment and metabolites included in media such as glucose,

pyruvate, oxygen and amino acids have been shown to have differential influences on oocyte maturation and competency. The role of ascorbic acid in the neutralisation of free radical species has been studied and the antioxidant properties of ascorbic acid also affect the process of follicular cell death [81]. Reactive oxygen species (ROS) have a role in the modulation of gamete quality and gamete interaction; ROS in culture media may impact post-fertilization development, i.e. cleavage rate, blastocyst yield and quality; so antioxidant strategies should be able to intercept both extracellular and intracellular ROS [82]. The body possess several defense mechanisms against ROS including antioxidant enzymes such as superoxidedismutase, catalase, glutathione peroxidase, pyruvate and also small molecules like Vitamins A, C and E. An oxidative stress is one of the main sources of embryo loss in IVF treatment: for this reason it is important to protect either gametes and embryos during assisted reproductive techniques [83]. Apoptosis has been reported in oocytes and this apoptotic fate could be modulated by sub-optimal culture conditions. Action of antioxidant agents is well known in animal in vitro fertilization [84], Retinoids, carotenoids and alpha-tochopherol may improve oocyte maturation and viability [85]. Moreover, the high percentage of oocytes demonstrating DNA fragmentation in the unfertilised oocytes could be correlated with fertilization failure. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. 10. Current status of oocyte cryopreservation A better oocyte survival (up to 64%) was reported following cryopreservation using a slow-cooling, rapid-thawing method in the presence of PROH and sucrose [76]—a method that has been successfully applied to the cryopreservation of human embryos [86]. The karyotypes of four human oocytes cryopreserved using the same technique were found to be normal following fertilization, although a later study showed 27% of thawed oocytes to be parthenogenetically activeted, while neither the control untreated oocytes, nor oocytes exposed to CPA without freezing, were activated [79]. Contrasting results are reported in the literature regarding the human oocyte survival rate after cryopreservation with or without the cumulus oophorus and using a solution consisting of 1.5 mol/L PROH to which 0.1 mol/l sucrose was added. It was observed [87] that the presence of the cumulus mass or the partial or the total removal of cumulus cells did not significantly modify the oocyte survival rate. By contrast, it was also described that the oocytes surrounded by a total cumulus and corona mass, as retrieved at ovum recovery, had a significantly reduced survival rate compared with those oocytes which had the mass removed prior to freezing, suggesting that the presence of cumulus cells and the cumulus matrix causes a different rate and extent of dehydration during cryopreservation [76]. The cumulus–corona complex may also form a more rigid structure limiting the distortion of oocyte shape which occurs during ice formation in the cytoplasm [88]. The results of the study of Fabbri et al. agree with Mandelbaum’s findings since the presence or the absence of the cumulus

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oophorus does not influence the survival rate of the oocytes cryopreserved in the presence of 0.1 mol/l sucrose. The main biophysical factor affecting the human oocyte survival is the intracellular ice formation that generally pierces the membrane causing cell lysis. Intracellular ice formation can be affected by the presence of the cryoprotectants in the freezing solution, and by the freezing and thawing rate [89]. The cryoprotectants generally used in oocyte freezing protocols are 1,2-propanediol (PROH, membrane-permeating cryoprotectant) and sucrose (membrane-non-permeating cryoprotectant). Their protective action is very complex and attributable to several properties, the most important because of 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 [90]: the increase of the extracellular solute concentration generates an osmotic gradient across the cell membrane, which draws water out of the cell, causing the cell to dehydrate before the freezing procedure. For oocytes cryopreservation procedures, cryoprotectant concentration are usually 1.5 mol/l, many times higher than any other component in the medium. Thus the cryoprotectants enter the cell by osmosis. While the cryoprotectants readily cross the cell membranes, water usually crosses even more readily. The results presented by Fabbri et al., showed that a double and a tripled sucrose concentration significantly increased the oocyte survival rate. Probably a 0.1 mol/l sucrose concentration was not sufficient to allow suitable oocyte dehydration before lowering the temperature. On the other hand, a 0.3 mol/l sucrose concentration probably causes a more adequate loss of intracellular water without excessive oocyte shrinkage which could lead to the collapse of the cellular membranes. A recent study presented by Ching-Ching Tyer describes the use of slow freezing method with 1.5 M PROH and 0.3 M sucrose in conjunction with a loading time of 15 min for enhancing the dehydration of oocytes before cryopreservation. Because the optimal timing of insemination of frozen–thawed human oocytes remains to be determined, they had chosen the insemination time of the frozen oocytes at 4 h after thawing. The implementation of a longer incubation time is based on their speculation that full restoration of the temperature-sensitive meiotic spindles of human oocytes may require more than 3 h after thawing. In this study, the oocyte survival rate was 71.4%, the fertilization and clevage rates were 80% and 100%, respectively. The report helps to provide a further clinical confirmation of the benefits and safety relating to the use of PROH with a higher sucrose concentration as cryoprotectant and a longer exposure time of oocytes in these media before freezing. Improvements in cryopreservation protocols continue to be made as our understanding of the underlying mechanisms deepens. Replacement of sodium chloride with choline in the cryoprotectant vehicle solution has been found beneficial for mature murine oocytes [91] and for immature and in vitro matured human oocytes [92] after slow cooling in PROH. This replacement of sodium chloride with choline to obviate salt-induced damage during slow cooling is now being tested in a clinical setting [93], but remains to be fully evaluated.

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Another area in which an improvment in survival has been seen is the use of a higher subzero temperature at which ice formation is induced (“ice seeding”) prior to cooling. An increase from −8 to −6 ◦ C and −4.5 ◦ C improved survival of immature and failed to be fertilised human oocytes from 32 to 56% to 955, respectively [94]. The use of sodium-depleted medium and an “ice-seeding” temperature of −6 ◦ C have been combined to produce five live births and four pregnancies in 11 women receiving embryo transfer after ICSI of cryopreserved oocytes [95]. The intraoocyte injection of a sugar (trehalose), which can stabilise biomembranes during freezing, has been shown to improve survival and is compatible with normal fertilisation and live births in a murine model [96]. 11. Vitrification As previously stated, slow freezing methods imply the precipitation of water as ice. During this process two types of cell damage may occur: (a) formation of intracellular ice crystals, that occurs when the sample is cooled too quickly, cell dehydration is inadequate and water is entrapped inside the cell, (b) damage due to the high solute concentration generated when water precipitates as ice [97–99] (Table 1). Vitrification is the transition of acqueous solutions from the liquid state to the glassy state (solid), bypassing the crystalline state. It is a sort of extreme increase of viscosity and requires both ultrarapid cooling rates and a very high CPA concentration; both factors synergyze in preventing ice crystal formation and increasing viscosity at low temperatures. During vitrification, the solution composition remains unchanged during cooling and ice crystals do not form [100]. Although vitrification eliminates ice crystal formation, it implies an increased probability of other forms of injury. Different approaches are used to minimize toxic, osmotic and other damages: application of less toxic chemicals, combination of two or three CPAs, stepwise addiction and/or exposure of cells to precooled concentrated solutions [101–110]. The high cooling rate used during vitrification significantly decreases chilling injury to the oocyte, passing rapidly through the dangerous temperature zone [111–114]. However, the cooling rates used to vitrify require approximately 5–7 M CPA concentrations, which is much higher (approximately 4–7 times) Table 1 Vitrification: studies and results Author

Oocytes

Results

Kola (1988) (58)

Mouse oocytes

Hunter (1995) (37)

Human oocytes

Kuleshova (1999) (191) Hong (1999) (190)

17 human oocytes 106 human oocytes

Chen (2000) (208) Yoon (2003) (211)

84 human oocytes 474 human oocytes

Aneuployd zygotes & malformed fetus 65% survival rate, no further development 1 birth 100% survival rate; 38–71% fertilization 65–96% survival rate 69% survival rate; 7 births

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than needed for traditional slow freezing. A considerably high CPA concentration is required to maintain the solutes at a concentration tolerated by the cell. There is a critical CPA concentration needed for vitrification, but on the other side for some CPA this minimum concentration may result in either osmotic or chemical toxicity. Therefore during vitrification a balance is needed, which is aimed to maximize the cooling rate and contemporaneously minimize the CPA concentration. Since both a small volume of solution and an increase of the hydrostatic pressure may facilitate vitrification, an approach to minimize CPA concentration may be to increase the hydrostatic pressure on the solution during the vitrification process, returning to atmospheric pressure during subsequent storage [101]. Another option is to add high molecular weight polymers (such as polyvinylpirrolidone) to the solution, in order to vitrify the extracellular milieu (that requires more CPA) with the same CPA concentration that is needed to vitrify intracellularly [101]. Nearly all the vitrification techniques are based on direct contact between the cryoprotectant solution and the liquid nitrogen. The easiest way to obtain this contact is the direct plunging into liquid nitrogen [115,116], but the size required for the oocytecontaining droplet in this case is relatively large (5–6 mcm at least), and its cooling rate may be lower than optimal because of the flotation on the surface on the liquid nitrogen before submerging. To avoid these problems, oocytes may be placed on the surface of electron microscope grids before submerging into liquid nitrogen [112,117]. Even the cryoloop technique, in which a thin nylon loop is used to suspend a CPA film containing the oocytes before direct plunging, has yelded good results in terms of oocyte survival and embryo formation [118,119]. Alternatively, the “open pulled straw” (OPS) technology have been used: in this case, the oocyte carrier is a narrow plastic tube and a volume of 1 mcl CPA solution is loaded into the open end [120]. These methods have allowed to obtain a high-speed vitrification (cooling rate around 20000 ◦ C/min) and to use less concentrated and less toxic CPA solutions [102]. It must be considered that liquid nitrogen may be a source of contamination by viruses or viral particles [121]. This danger may be eliminated using sterile liquid nitrogen for freezing and then wrapping the frozen oocytes in a hermetic container (e.g. narrow and thin-walled, heat-sealed straws) before plunging them for long-term storage [122–124]. A cell that has been vitrified and stored in liquid nitrogen must be warmed to ambient temperature before used, and during warming it is susceptible to devitrification and ice crystallization. Warming protocols based simply on thermal conduction are not feasible for vitrified oocytes because heat diffusion via conduction mode does not adequately prevent ice crystallization and cell damage [125]. In order to achieve a fast warming rate, electromagnetic or microwave heating at proper frequency may be preferable. Most publications comparing traditional oocyte slow freezing and vitrification report either similar survival rates or slightly improved results after vitrification [126–133]. The vitrifying solution VS1 (20.5%, w/v DMSO, 15.5%, w/v acetamide, 10%, w/v propylene glycol and 6%, w/v polyethylene glycol in mod-

ifyed phosphate buffer) and its later derivatives (VS2, VS3 and VS3a) have been widely used in these comparative studies [103]. Using VS1 to vitrify mouse oocytes, a high frequency of oocytes survived (80%) and fertilized in vitro (85%) was observed, but relatively few (50%) were able to further develop [134,135]. Shortening time exposure of mouse oocytes to VS1 prior to cooling was found to be associated with higher cryosurvival rates [136]. A major progress with vitrification was achieved in cryopreservation of bovine oocytes. The blastocyst rates after fertilization of vitrified oocytes and in vitro culture were found to be similar to those of non-cryopreserved control oocytes [112,122,116,119,137] and several live offsprings were reported [122]. Overall after oocyte vitrification a relatively high initial survival and fertilization rate was reported, but it was frequently followed by a relatively low in vivo development rate of the embryos. The reason for this has not been fully clarified, but bisbenzimide labelling of the blastocysts derived from vitrified oocytes revealed that the inner cell masses were formed by a number of cells significantly lower than normal [138]. Similarly to what happens with slow cooling, problems of genetic nature or fetal defective development raise some concern even in vitrification [54,75,137,139]. The meiotic spindles of mouse oocytes were found to disappeared when exposed to a vitrification solution of 5.5 M ethylene glycol and 1.0 M sucrose [140]. Recovery of the spindle structure in vitrified mouse oocytes was observed to be more extensive after 2 or 3 h than after 1 h post-thawing [141,142]. In fact, blastocyst formation of vitrified mouse oocytes inseminated 1 h after warming was significantly lower than the one observed for oocytes inseminated after 2 or 3 h of incubation after thawing [143]. Oocytes vitrified in a very small amount of vitrification solution (like in OPS technique) is claimed to better preserve the meiotic spindles when compared to conventional straws [140,142]. Some technical modifications seem to be particularly important for very cold-sensitive oocytes such as bovine oocytes [144,145]. From a practical point of view, vitrification is appreciated because it does not require expensive automatic coolers or special skill and can be performed quite quickly. The vitrification procedure is simpler that the slow freezing, although but the most tricky part of the latter is done automatically. Vitrification technique takes just a few minutes; however, it must be considered that each straw must be cooled individually, and therefore the overall time required per patient might not be very short. 12. Children from cryopreserved oocytes Since the first pregnancies and births, children conceived from frozen–thawed oocytes seemed to be apparently normal and healthy all around the world (Table 2). The concern about possible abnormalities arose from the thesis of intrinsic vulnerability of the mammalian eggs when exposed to low temperatures together with cryoprotectants, complex manipulation and so on. The first human pregnancies and live births were announced in 1986–1988 [146–148]. Apparently, those children were nor-

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Table 2 Pregnancies and births from frozen human oocytes Authors

Year

Cryoprotectants

Freezing Protocol

Oocyte stage

Chen Al-Hasani et al. Diedrich et al. Van Uem (32) Siebzehnrubl et al. Porcu et al. Tucker et al. Polak de Fried et al. Young et al. (203) Nawroth and Kissing Yang et al. Antinori et al. Borini et al. Tucker et al. Porcu Porcu et al. Porcu et al. Kuleshova et al. Yoon et al. Porcu et al.

1986, 1987 1986, 1987 1987 1987 1989 1997 1998 1998 1998 1998 1998 1998 1998 1998 1999 1999a 1999b 1999 2000 2000

DM PR/DM DM DM PR/DM PR PR PR PR PR PR PR PR PR PR PR PR EG + S EG + S PR

Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Vitri Vitri Slow

MII MII MII MII MII MII MII MII MII MII MII MII MII GV MII MII MII MII MII MII

DM: dimetilsulfoxide; PR: 1,2-propanediol; EG: ethinilclicol; S: sucrose; Slow: slow freezing protocol; MII Metaphase II; GV germinal vesicle; Vitri: vitrification.

mal and healthy. Chen [146] reported this result obtained with a technique involving the reduction in size of the oocyte/cumulus–oophorus complex, the addition of dimethylsulphoxide (DMSO) as a one-step procedure, slow cooling between −7 and −36 ◦ C after seeding, and rapid freezing to −196 ◦ C before storage in liquid nitrogen. Thawing was rapidly achieved by warming in a 37 ◦ C water-bath, followed by dilution of the cryoprotectant as a single step. The oocytes were examined for morphological evidence of survival. Further development of the gametes required the transfer to the regular culture medium, and at appropriate time insemination was carried out. Van Uem et al. [148] obtained the second birth reported in the literature after oocyte cryopreservation with a freezing technique different from the one described by Chen. In attempt to overcome cell damage due to super cooling, he developed a computer-controlled “open-vessel” freezing device (CTE 8100). This device permits seeding to take place automatically in the ideal temperature range around the freezing point of the medium (“self-seeding”). Further, van Uem adopted the technique of slow-freezing and slow-thawing. As Chen did, van Uem also reduced the cumulus by needle dissection, but he used a freezing medium of phosphate-buffered saline containing 10% heatinactivated fetal cord serum and 1.5 mol/l DMSO not chilled before the addition to the oocyte. Therefore, from those early experiences, the cryoprotectant DMSO, the slow freezing and both the slow and rapid thawing procedures appeared to be safe and effective. However, those early successes could be hardly reproduced in the subsequent years. Researchers were very careful and tried to find basic experimental proofs of the safety of egg cryopreservation. Unfortunately, a number of studies used bad quality disgharged eggs and the results were often misleading. On the other hand, reassuring basic studies were conducted by Gook team [76,79].

Sustained by those studies, it was only after several years that another birth of a healthy female coiceived with cryopreserved human oocytes was reported [149]. This was the first child born after intracytoplasmic sperm injection (ICSI) of oocytes cryopreserved with the propandiol slow freezing technique. Compared to the early deliveries, this birth derived from the combination of two new variables: (1) the use of propanediol, believed to be potentially less toxic than DMSO; (2) the introduction of intracytoplasmic sperm injection which appeared to be a winning choise for the insemination of cryopreserved eggs. The combination of two novel techniques were potentially harmful for the health of conceptuses but in actual facts the several additional pregnancies obtained subsequently by the same team revealed normal fetuses and children [150–158]. The perinatal parameters and the follow up displayed regular growth and development of the children [154]. Shortly after other groups adopted the same technique with good results and healthy newborns [159–164]. In the same period and with the same technique, Tucker et al. [165] obtained the first birth of a healthy child from a cryopreserved germinal vesicle oocyte. Cryopreservation of immature oocytes at prophase I (GV stage) was proposed as an alternative to standard oocyte cryopreservation, as it was thought these oocytes were less sensitive to cryoinjury, due to the missing spindle and different membrane permeability [166–168]. Actually, no advantage seem to derive from immature oocyte cyopreservation in terms of survival rate, fertilization rate, developmental ability; moreover, this method requires oocyte in vitro maturation after thawing and has also been related to an increased incidence of chromosomal abnormalities. A study performed in 1997 [169] comparing 128 frozen immature oocytes with 91 control oocytes, showed an increased frequency of chromosomal anomalies (77.8% versus 31.8%) and spindle anomalies in (70% versus 22.2%); these percentages were similar (70% versus 22%

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of spidle anomalies) in another more recent study [170]. On the contrary, other authors did not find differences in spindle anomalies between cryopreserved MII or GV stage oocytes [171,172]. However, as far as now, only one child has been born from early stage cryopreserved oocytes matured in vitro and fertilized by ICSI [165]. The following year, Kuleshova [145] announced the birth of the first child from oocytes stored by vitrification. Also in this case, the newborn was normal and healthy. Subsequently, vitrification was successfully adopted by other Authors with the publication of ten additional pregnancies with normal children [173,174]. Children follow up has been done by two groups so far [155]. Both teams reported apparently normal development and growth of the children born from frozen eggs. It is crucial to extend the follow up to all these children. The international register of frozen egg babies has recently be started to collect data. 13. Conclusions Oocyte freezing has 25 year of history alterning successes and setbacks. Human oocytes have a delicate architecture but are freezable. In practice, the PROH-based protocol is used as the current standard. Clinical efficiency remains low, but healthy children have been born, indicating that chromosomally normal embryos can originate from frozen oocytes. Freezing protocols are not yet optimal and it is now desirable to combine empirical and theoretical knowledge. About the vitrification we have not enough knowledge and human results also if most publications comparing slow freezing protocol and vitrification report similar survival rates. There is clearly no urgent global need for implementation of oocyte freezing in current assisted reproductive technology, excepted for the country where the law does not permit the embryo-freezing (for example in Italy). On our opinion oocyte freezing should not be used as a substitute for embryo cryopreservation but rather as a method with its own specific application. This technique during the years is constantly improving in better but pregnancy rates remain higher using cryopreserved embryos. Furthermore, there are specific areas where oocyte freezing can be valid, such as in donor oocyte programmes, in the case of threatening ovarian loss and women that have to undergo chemioterapy or radioterapy or, like in Italy, in the case of objection against embryo freezing. In other words, oocyte freezing should be considered as routine assisted reproductive technology when used appropriately. References [1] Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983;305:707–9. [2] Zeilmaker GH, Alberda ATh, et al. Two pregnancies following transfer of intact frozen–thawed embryos. Fertil Steril 1984;42:293–6. [3] Van der Elst J. Oocyte freezing: here to stay? Hum Reprod Update 2003;9:463–70. [4] Sherman JK, Lin TP. Survival of unfertilized mouse eggs during freezing and thawing. Proc Soc Exp Biol Med 1958;98:902–5.

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