Biotransport Phenomena in Freezing Mammalian Oocytes

Water transport across the cell plasma membrane and intracellular ice formation (IIF)—the two biophysical events that may cause cell injury during cryopreservation—were studied by cryomicroscopy and modeling using mammalian (Peromyscus) oocytes. Unusually high activation energy for water transport across the cell plasma membrane was identified indicating that the water transport process is unusually sensitive to temperature (and cooling rate). Although literally all studies on IIF were conducted using protocols with ice-seeding (seeding extracellular ice usually at ≥−7 °C), it is not used for cell cryopreservation by vitrification that is becoming increasingly popular today. In this article, we show that ice-seeding has a significant impact on IIF. With ice-seeding and cooling at 60 °C/min, IIF was observed to occur over a wide range from approximately −8 to −48 °C with a clear change of the ice nucleation mechanism (from surface- to volume-catalyzed nucleation) at approximately −43 °C. On the contrary, without ice-seeding, IIF occurred over a much narrower range from approximately −19 to −27 °C without a noticeable change of the nucleation mechanism. Moreover, the kinetics of IIF without ice-seeding was found to be strongly temperature (and cooling rate) dependent. These findings indicate the importance of quantifying the IIF kinetics in the absence of ice-seeding during cooling for development of optimal vitrification protocols of cell cryopreservation.     

In-vitro growth of murine pre-antral follicles after isolation from cryopreserved ovarian tissue

The low temperature storage of ovarian tissue allows patients at risk of premature menopause to preserve their fecundity. One strategy for harvesting mature oocytes may be to isolate small follicles from the stroma for in-vitro culture. The aim of this study was to assess the survival and growth of murine follicles during in-vitro culture after isolation from tissue frozen-thawed in various cryoprotective agents. The effect of different seeding and thawing temperatures was also investigated. Pre-antral follicles 100--135 microm in diameter were isolated from fresh and frozen-thawed tissue and cultured in vitro for 8 days. In the fresh control group 79 +/- 3% of follicles survived in-vitro culture and grew to antral stages. Fewer follicles survived after isolation from tissue cryopreserved in dimethyl sulphoxide (43 +/- 5%) or propylene glycol (24 +/- 2%) and none survived freeze-thawing in glycerol. Lowering the seeding temperature from -5 degrees to -7 degrees or -9 degrees C reduced follicle survival rates from 49 +/- 4% to 26 +/- 1% and 28 +/- 3% respectively. If thawing was carried out at 27 degrees C follicle survival rate was higher (38 +/- 7%) than at 37 degrees C (26 +/- 2%) or 47 degrees C (20 +/- 6%). Follicles surviving 8 days of in-vitro culture were stimulated with human chorionic gonadotrophin. The number of mature oocytes released did not differ between any experimental group. The results indicate that follicles isolated from frozen-thawed tissue can be grown to antral sizes and produce mature oocytes. The in-vitro culture system also proved a sensitive method for testing variations in the freeze-thaw protocol.     

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.     

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.     

Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity

The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.     

Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles

BACKGROUND: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. METHODS: Two cryopreservation procedures were used. Method 1 involved use of 1.5 mol/l propanediol (PrOH)-0.1 mol/l sucrose with medium containing sodium (Na) as cryoprotectant medium, seeding at -7 degrees C, and stepwise dilution of cryoprotectant post-thaw. Method 2 used Na-depleted media with 1.5 mol/l PrOH-0.2 mol/l sucrose for freezing, seeding at -6 degrees C, and use of high sucrose (0.5 and 0.2 mol/l) for cryoprotectant removal. RESULTS: The first method was used in seven patients, and gave poor (12.3%) survival results and no pregnancies. The second method was used in 15 patients (16 cycles), and yielded good survival and fertilization rates (74.4 and 59% respectively), with four pregnancies and five healthy infants born to 11 women receiving an embryo transfer. CONCLUSIONS: Using Na-depleted media along with other alterations in freezing and thawing procedures, human oocyte cryopreservation can provide excellent survival and pregnancy rates.     

Cryobiology of non-human primate oocytes

The responses to various stresses involved with cryopreserv-ationprotocols were investigated using non-human primate oocytes.Fluorescence microscopy was used to assess the status of theF-actin microfilament system of rhesus monkey oocytes afterexposure to different concentrations of glycerol. The F-actinorganization around the cortex and hi the transzonal processeswas modified by exposure to 1.0 or 2.0 M glycerol at ambienttemperature. These effects were reduced significantly when exposureto glycerol was combined with cooling to 0C. Cynomolgus monkeyoocytes were also subjected to hyperosmotic stress and observedfor morphological changes. An irregular shrinkage phenomenonwas observed with germinal vesicle or metaphase I but not metaphaseII (MIl) oocytes. The irregular shrinkage became uniform andspherical when the oocytes were pretreated with ethyleneglycol-bis-(3-aminoethylether)N, N, N', N' tetraacetic acid (EGTA) before exposure tohypertonic solution. Also, in-vitro-matured MIl oocytes fromcynomolgus monkeys were used to determine crucial biophysicalparameters for freezing primate oocytes. The permeability ofoocyte plasma membrane to water, Lpg, and its activation energy,E_LP, were determined between 0 and -12C in the absence of cryoprotectiveadditives. The Lpg was found to be 3.8 10–14 m3N/s andthe ELp was 141.5 kJ/mol. The pre-exponential kinetic and exponentialthermodynamic parameters of intracellular ice formation weredetermined to be 8108 m2/s and 2.2 109 K5 respectively. Bycombining models of water transport and intracellular ice formation,the cumulative fraction of oocytes with intracellular ice asa function of the cooling rate was also predicted, and it wasshown to correlate reasonably with experimental observations.     

Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes

BACKGROUND: Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the oocyte oolemma permeability to water and diverse cryoprotectants. METHODS: We prospectively investigated volume changes over time at different temperatures (30 degrees C, 22 degrees C and 8 degrees C) of human metaphase II (MII) oocytes (obtained in stimulated ICSI cycles and matured in vitro from the germinal vesicle stage) when exposed to changes in external osmolality. We also investigated human in vitro matured (IVM) oocytes membrane permeability characteristics at 22 degrees C to 1,2-propanediol (PG) and dimethylsulphoxide (DMSO) and at 30 degrees C, 22 degrees C and 8 degrees C to ethylene glycol (EG), and calculated corresponding oocyte oolemma permeability coefficients (Lp and Pcpa). Furthermore, we investigated the osmotic tolerance limits of IVM oocytes exposed to changes in external osmolality as assessed by their developmental competence during the course of 72 h after ICSI. RESULTS: The results of our studies describe human oocyte membrane permeability coefficients for EG at 30 degrees C (2.85+/-0.15x10(-3) cm/min), 22 degrees C (1.17+/-0.60x10(-3) cm/min) and 8 degrees C (0.37+/-0.15x10(-3) cm/min). Furthermore, at 22 degrees C the EG oolemma permeability coefficient was lower than that of PG and DMSO (1.17+/-0.60x10(-3) cm/min versus 2.15+/-0.70x10(-3) and 1.56+/-0.38x10(-3) cm/min, respectively). Our results also indicate, that human IVM MII oocytes tolerated exposure to solutions in the range of 39-2264 mOsmol/kg H2O as assessed by the oocytes' developmental competence after exposure. CONCLUSIONS: The results of the present study may contribute to a better understanding of the biology and cryobiology of human oocytes, and to the design of better and more robust cryopreservation (freezing or vitrification) protocols.     

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20,000 degrees C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200 degrees C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000 degrees C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.     

The effect of chilling on membrane lipid phase transition in human oocytes and zygotes

BACKGROUND: Chilling injury occurs when the cell membrane undergoes a transition from the liquid state to the gel state. Human oocytes and single-cell zygotes are of similar shape and size but the post-thawing survival rate of oocytes is poorer. We set out to investigate the possible difference in membrane lipid phase transition (LPT) temperature between the two cell types. METHODS: The LPT temperature was measured with a Fourier Transform Infrared analyser, which detects the change in the vibration frequency of the CH2 bond stretches of the membrane lipid molecules during temperature change. The LPT temperatures of unfertilized human oocytes, in vitro-matured oocytes, and immature germinal vesicle (GV) stage oocytes were compared with that of abnormally fertilized human zygotes. RESULTS: The LPT temperatures of zygotes and of mature and immature GV oocytes differ significantly from each other (10.0 +/- 1.2, 16.9 +/- 0.9 and 24.4 +/- 1.6 degrees C respectively; P < 0.05). CONCLUSIONS: Zygotes show a higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation protocols in order to minimize cryoinjury.     

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.     

Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy.

BACKGROUND: Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. METHODS: Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. RESULTS: Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. CONCLUSIONS: These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.     

Rapidly cooled human sperm: no evidence of intracellular ice formation

BACKGROUND: The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. METHODS: In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000 degrees C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling. RESULTS: No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000 degrees C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage. CONCLUSIONS: Cell damage to human spermatozoa, at cooling rates of up to 3000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.     

Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI

BACKGROUND: Meiotic spindles in living human oocytes can be visualized by the Polscope. This study investigated the relationship between the presence/location of the spindle in metaphase II (MII) oocytes and developmental competence of embryos in vitro. METHODS: The spindles in 626 MII oocytes were examined by the Polscope and divided into six groups (A-F) based on the presence or absence of the spindles and the angle between the spindle and the first polar body. After ICSI, the fertilization and embryo development were evaluated. RESULTS: Meiotic spindles were imaged in 523 oocytes (83.5%), while 103 (16.5%) did not have a visible spindle (group F). The majority of oocytes (68.8%) had the spindle directly beneath or adjacent to the first polar body (groups A and B: 48.2 and 20.6%). Oocytes in group C (11.2%) had the spindle located between 60 and 120 degrees angle away from the first polar body, those in group D (2.4%) had the spindle located between 120 and 180 degrees angle and those in group E (1.1%) had the spindle located at 180 degrees angle to the first polar body. The fertilization and embryonic development were similar in the oocytes with spindles regardless of spindle position. However, the rate of high quality embryos was significantly higher in the oocytes (64.2%) with visible spindles than in the oocytes (35.9%) without spindle and multipronuclear proportion showed a slight tendency to increase in oocytes without spindles. (10.7 versus 5.9%, P = 0.12; NS). CONCLUSIONS: the presence of a bi-refringent meiotic spindle in human oocytes by using the Polscope can predict a higher embryonic developmental competence. However, the relative position of the spindle within the oocyte doesn't appear to influence the developmental potential of embryos.     

In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle.

Cryopreservation of germinal vesicle (GV)-stage mouse oocytes results in a developmental block. As an approach to explain the failure in development, we have investigated the morphology of the second meiotic spindle after in-vitro maturation. Fully grown GV-stage mouse oocytes were collected from the ovaries of primed mice and kept in meiotic arrest with dibutyryl cyclic AMP. These oocytes were submitted to different variables of cryopreservation: (i) cooling to 22 degrees C or 0 degrees C; (ii) exposure to 1.5 M 1,2-propanediol at 22 degrees C or 0 degrees C; (iii) exposure to 1.5 M dimethylsulphoxide (DMSO) at 22 degrees C or 0 degrees C; (iv) ultrarapid freezing with 3.5 M DMSO/0.5 M sucrose; (v) exposure to a sucrose dehydration series according to the ultrarapid freezing protocol. The morphology of the second meiotic spindle was evaluated 16 h after release from meiotic arrest. We were able to demonstrate that following cooling, exposure to cryoprotectants or ultrarapid freezing of GV-stage mouse oocytes, a normal barrel-shaped spindle with the chromosomes in midplane position is found in 79-94% of oocytes except for two conditions with great exposure to dehydration stress. Exposure to DMSO at 0 degrees C or exposure to a sucrose dehydration series resulted in significantly lower percentages of barrel-shaped spindles, respectively 64% and 62%. The effect on spindle morphology has to be put into perspective, however, since the observed abnormalities were changes of spindle shape, such as elongation or reduction, which are assumed to be restorable, and since no polar organizational defects were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different stages of maturity

BACKGROUND: Success of human oocyte cryopreservation depends on multiple cryobiological factors that could influence the developmental potential of the oocytes. The objective of this study was to examine the effects of different sucrose concentrations on the developmental potential of human frozen-thawed oocytes at different maturity stages. METHODS: A total of 355 oocytes collected from small follicles were randomly divided into three groups and two groups (B and C) were cryopreserved using slow-freezing method. Group A included 131 oocytes at different maturity stages without freezing. Another 119 oocytes in Group B were cryopreserved with 0.1 M sucrose and 105 oocytes in Group C with 0.2 M sucrose concentration. RESULTS: The post-thaw survival rate of the oocytes and the cleavage rate in Group C were significantly higher than that of Group B (P<0.05). For immature metaphase I (MI) stage oocytes, a significant difference was found in the maturation rate between Group C and Group B (P<0.05). The maturation rate for the GV oocytes in Groups A and C was significantly higher than Group B (P<0.01). CONCLUSIONS: The results suggested that sucrose concentration of 0.2 M in the cryoprotectant solution is more suitable for human oocyte cryopreservation.     

Fertilization and early embryology: Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development

Investigations were made into the low temperature preservationof pre-ovulatory human oocytes by vitrification using a methodof brief exposure of the oocytes to the vitrification solutionat room temperature. Assessments of morphological survival,fertilization and embryonic development were recorded. All thoseoocytes exposed to the vitrification solution alone were morphologicallynormal and 86% of them were fertilized after incubation withspermatozoa. All the fertilized ova (86%) underwent cell division.Following cooling to –196°C, morphological survival(65%) and fertilization (45%) rates remained high. However,in all vitrified oocytes, embryonic cell division and furtherdevelopment were inhibited. From our study it appears that freshhuman oocytes can be vitrified using only brief exposure tocryoprotective agents and survive to undergo fertilization.However, progress remains to be made in achieving further embryonicdevelopment.