Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

Zona-opening of hamster oocytes: a comparative study using macro- and micro-manipulation methods

The present study compared a new macro-manipulation techniquefor zona-opening of hamster oocytes with existing micro-manipulationtechniques. In experiment I the zona pellucida of hamster oocyteswas partially opened with a 32 gauge steel needle (macro-manipulation)and these oocytes were then co-incubated with human spermatozoaat 36.5°C in 5% CO₂ in air for 3.5 h. Zona-free and zona-intactoocytes similarly inseminated served as controls. Of 113 oocytes,30 (26%) lysed following zona-opening with the macro technique.The sperm penetration rates of zona-opened and zona-free oocyteswere 37% (31/83) and 60% (49/81) respectively (P<0.01), with12 and 28% oocytes respectively showing polyspermia (P<0.05).No sperm penetration occurred in zona-intact oocytes. In experimentII the zona pellucida of hamster oocytes was partially openedby macro-or micro-manipulation techniques before the oocyteswere coincubated with human spermatozoa as in experiment I.Nine of 156 (6%) macro-manipulated and 11 of 133 (8%) micro-manipulatedoocytes lysed following zona-opening. Of 147 remaining macro-manipulatedand 122 remaining micro-manipulated oocytes, 80 (55%) and 79(64%) respectively were penetrated (P>0.05), with 30 and37% respectively showing polyspermia (P>0.05). These resultsdemonstrate that macro-manipulation results in similar penetrationand polyspermia rates as compared to conventional micro-manipulation.     

Abnormal chromosomal arrangements in human oocytes

Ninety-one human oocytes, lacking signs of fertilization 50h after insemination in vitro, were investigated cytogeneticallyto assess the frequency and type of chromosomal abnormalities.Chromosome spreading permitted adequate karyotyping in 55 oocytes.Non-determined numerical aberrations occurred with the followingfrequencies: hypohaploidy, 10.9% (6/55), hyperhapJoidy, 14.5%(8/55) and hyperdiploidy, 3.6% (2/55). Total aneuploidy occurredwith a frequency of 29.1% and was observed in oocytes from 30patients. No correlation was found between specific chromosomalaberrations and type of infertility, stimulation treatment orgonadotrophin levels. On the other hand, the frequency of aneuploidywas significantly higher (P < 0.05) in patients >35 yearsof age. Two chromosomal complements (3.6%) had structural rearrangements;one oocyte had both structural and numerical chromosomal abnormalitiesand the other had differently condensed regions on the longarms of three chromosomes from group C. The overall frequencyof chromosomal aberrations was 32.7%. Only two samples containedan additional set of polar body chromosomes. Thirteen oocytespresented sperm chromosomes in an arrested stage of prematurechromosome condensation of the G₁, phase and four oocytes showedasynchronous condensation of pronuclear chromosomes. Finally,it was concluded that the high proportion of chromosomal aberrationsobserved in human oocytes may contribute significantly to abnormalembryonic development in vitro.     

Fertilization and early embryology: Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection

A cytogenetic-cytological study was performed on unfertilizedhuman oocytes (first polar body visible) after intracytoplasmicsperm injection (ICSI) with respect to the rate of prematurelycondensed sperm chromosomes (G₁-PCC). Out of 163 prepared oocytesderived from 41 ICSI cycles, 133 (-82%) could be analysed successfully.A total of 60 oocytes (45.1%) showed metaphase II chromosomesin the haploid range along with an intact sperm head and 27oocytes (20.3%) were missing the sperm head, but two of themshowed an approximately diploid set of chromosomes; 38 oocytes(28.6%) exhibited the maternal metaphase II chromosomes as wellas G₁-PCC of the sperm nucleus showing a remarkable variationin the degree of condensation. Ten ICSI cycles (each followedby an embryo transfer) were characterized each by 2–3oocytes demonstrating G₁-PCC. It is concluded that the maincause of failed fertilization after ICSI is the failure of oocyteactivation. When the sperm nucleus is able to act with the chromosomecondensing factors and the oocyte does not become activated,this will lead to the induction of PCC. Absence of the spermhead might be due to injection or ejection of the spermatozoonin the perivitelline space except for two cases in which fertilizationmight have occurred. Finally, the observation of both a singlechromatin region (n = 6) or two chromatin regions (n = 2) indicatedoocyte activation which, however, was followed by developmentalarrest.     

Endocrinology and paracrinology: Activation of elements of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin E2

The current study was designed to examine the effects of prostaglandin(PG) E² on progesterone production by primate luteal cells collectedduring the late luteal phase. PGE₂ inhibited basal and humanchorionic gonadotrophin (HCG)-stimulated progesterone production(P <0.01) in late luteal phase corpora lutea. The abilityof PGE² to activate a second messenger system (phosphatidylinositolpathway) in corpora lutea of rhesus monkeys was also assessed.PGE² significantly increased the accumulation of inositol phosphates(P <0.05). This stimulation was not apparent in the earlyluteal phase but was manifested in the mid-late luteal phase.PGE² also caused a rapid, yet transient, increase (P <0.01)in intracellular free calcium ion concentrations ([Ca²⁺]_i) ina large proportion of primate luteal cells. The proportion ofluteal cells that responded to PGE² with an increase in [Ca²⁺]_iwas smaller (P <0.05) in corpora lutea collected during theearly luteal phase (12%) in comparison with those collectedduring the latter half of the luteal phase (63–66%). Changesin [Ca²⁺]_i in response to PGE₂ were similar in small and largeluteal cells. This study demonstrates that PGE₂ activates elementsof the phosphatidylinositol pathway in primate corpora lutea.This activation is augmented as the luteal phase progresses.Thus, the inhibitory effects of PGE₂ on luteal progesteroneproduction observed in the late luteal phase are associatedwith activation of elements of the phosphatidylinositol pathway. corpus luteum/phosphatidylinositol/progesterone/prostaglandin/rhesus monkey     

Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques.

Both glycerol and dimethyl sulphoxide (DMSO) equilibration prior to cryopreservation produced adequate rates of survival and development to the hatching blastocyst stage for mouse oocytes using the two chosen cooling and warming regimes. Successful fertilization of human oocytes and further development of the embryos produced, was achieved following equilibration with DMSO at 0 degrees C. Although fertilization of fresh human ova previously exposed to glycerol was recorded, all oocytes with two pronuclei failed to continue to undergo cell division by 48 h in culture. Following cryopreservation, human oocytes were successfully inseminated after equilibration with both glycerol and DMSO. However, cell division to the 2-cell stage was recorded in only one oocyte which had undergone exposure to DMSO, freezing and thawing. No cell divisions were recorded after glycerol cryopreservation, or even after simple exposure to glycerol. Therefore it appears that DMSO and slow cooling may be the best protocol, although further evaluation will be necessary.     

Manifestations of diabeters mellitus on mouse preimplantation developement: effect of elevated concentration of metabolic intemediated

The metabolic derangements of pregnancies complicated by diabetesmellitus, specifically hyperglycaemia and hyper-ketonaemia,are known to be teratogenic during the period of organogenesisin animals. We have shown previously that poorly controled diabetesmellitus impairs in-vivo and in-vitro mouse preimplantationembryo growth, and that culturing embryos in elevated glucoseconcentrations only partially recreated this developmental dealy.To extend this observation we examined the effect on mouse preimplantationembryo growth of elevated concentrations of other metabolicintermediates, which may be deranged in diabetes mellitys, namelylipids, lactate, glycerol, amino acids, and ketones. Two-cellembryos from ovulation-induced B₆C₃F₁ mice were cultured for72 h in the presence of added lipids (250 mg/dl), lactate (5mM), glycerol (160 µM) or mixed amino acids (8.5% travosol,7 mM) and showed no significant difference in growth over 72h verus their control groups. However, growth of preimplantationembryos in acetoacetate (10 mM) or in the racemic micture ofDL--hydroxybutyrate (16 and 32mM) revealed marked retardationversus controls when assessed either by distribution of developmentalstages over time (24, 48, 72 h, P <0.001) or by the differencein the average rank of sums indicating a delay in maturation(P<0.0001). We conclude that elevated ketone concentrationsadversely affect preimplantation embryo development. These findingsextend previous studies which correlate uncontrolled diabetesmellitus as well as hyperglycaemia with abnormal organogenesis,and demonstrate tht exposure to metabolic derangements may alsohinder reproductive performane at even earlier stages in gestation.     

Effect of cumulus cell mass and follicle quality on in-vitro maturation of cynomolgus monkey oocytes

Cynomolgus monkey oocytes were recovered from healthy or atreticfollicles > 1000 µm in diameter at day 8 of gonadotrophinstimulated cycles and cultured in vitro, cumulus enclosed orcumulus free, for 2 days. Germinal vesicle breakdown (GVBD)occurred in 26.7% of healthy cumulus enclosed oocytes (n= 60)compared to 52.6% of atretic cumulus enclosed oocytes (n = 38).The mechanical removal of the cumulus cell mass increased theGVBD rate of the healthy oocytes (56.5%, n = 23) and acceleratedthe passage of the atretic oocytes (n = 23) from metaphase I(4.3%) to metaphase II (47.8%). In the healthy primate follicle,the dktyate stage could be maintained by aninhibitory substancesecreted by the cumulus; foUicular atresia could either decreasethe synthesis of this substance and/or induce the metabolismof a stimulatory substance.     

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol

Rational design of a cryopreservation protocol was demonstratedby using theoretical models of the cryopreservation processto develop an optimal freezing protocol for mouse oocytes. Acoupled mechanistic model of the processes of freeze-inducedcell dehydration and intracellular ice formation was developed,and cryomicroscopical measurements of intracellular ice formationkinetics were used to determine biophysical parameters requiredby the model, and to test model predictions of the freezingbehaviour of mouse oocytes. A simple phenomenological modelfor oocyte damage resulting from exposure to concentrated electrolyteand cryoprotectant solutions during cryopreservation was obtainedby defining a cost function equal to the duration of the freezingprotocol. A two-step freezing protocol was theoretically optimizedby using a sequential simplex algorithm to minimize the costfunction, subject to the constraint that the predicted probabilityof intracellular ice formation remain below 5%, yielding a putativeoptimum at the cooling rate B = 0.59°C/min, and plunge temperatureTp = –67°C. By systematically varying B and Tp aboutthese values in experiments with mouse oocytes cryopreservedin 1.5 M dimethyl sulphoxide, the maximal recovery of intactoocytes with a normal morphology (82%) was obtained for B =0.59°C/min and Tp = –80°C. Further evaluationof the fertilizability and developmental capacity of oocytescryopreserved using the optimized protocol yielded cleavageto the 2-cell stage in 65% of oocytes inseminated, and blastocystformation in 50% of these 2-cell embryos.     

The thiol reagent, thimerosal induces intracellular calcium oscillations in mature human oocytes

The aim of this study was to investigate the effects of thimerosalon intracellular calcium in human oocytes related to the stageof nuclear maturity. A total of 20 oocytes from superovulatedwomen undergoing gamete intra-Fallopian transfer (GIFT) werestudied. The calcium-sensitive fluorescent dye Fura-2 was usedto monitor intracellular calcium. Regular oscillations in theconcentration of cytosolic calcium were observed in 13 out of20 oocytes following exposure to thimerosal; five oocytes didnot respond to thimerosal treatment, and spontaneous oscillationsof cytosolic free calcium were recorded in two oocytes. Thimerosalinduced oscillations of intracellular calcium in a significantlyhigher proportion of metaphase II oocytes (10/11) compared withmetaphase I oocytes (3/8; P < 0.2). These findings demonstratethat thimerosal is a potentially useful agent for the studyof the calcium signalling processes in human eggs and suggestthat the underlying cellular mechanisms develop at a relativelylate stage of oocyte maturation.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

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.     

Follicular fluid pH changes following intraperitoneal exposure of Graafian follicles to carbon dioxide: a comparative study with follicles exposed to ultrasound

Follicular aspiration to obtain oocytes is generally performedvia laparoscopy after creating a pneumoperitoneum with carbondioxide. Such a procedure has been shown to reduce the rateof in-vitro fertilization of human oocytes and affect the rateof cleavage of rabbit embryos. These adverse effects may becaused by a reduction in follicular fluid pH due to diffusionof carbon dioxide into the folilde. In laparoscopic oocyte retrievals,a negative correlation was observed between duration of CO₂exposure and follicular fluid pH, whereas In ultrasound-guidedretrievals, the pH remaIned unchanged. The mean pH In 78 folliclesaspirated at laparoscopy was 7.22 ± 0.03 compared with7.62 ± 0.01 in 35 follicles aspirated under ultrasoundguidance (P = 0.0003). The results also indicate that oocytesin preovulatory follicles are surrounded by fluid that is morealkaline than plasma. Hence, the acidic environment treatedby CO₂ may be deleterious to sub sequent reproductive functionof the oocyte.     

Andrology: Insulin-like growth factor-I and {alpha}2-macroglobulin in seminal plasma correlate with semen quality

Insulin-like growth factor-I (IGF-I) and ₂-macroglobulin (₂-M)are believed to be involved in the development of germ cells.IGF-I is mainly controlled by concentrations of human growthhormone (HGH), influences cell proliferation and differentiationand its action is mediated by insulin-like growth factor-bindingproteins (IGFBP), placental protein 14 (PP14) and prostate-specificantigen (PSA). ₂-M acts as a broad spectrum proteinase inhibitorand a binding protein for many cytokines and hormones, e.g.inhibin and activin. This study was designed to identify concentrationsof these molecules in seminal plasma in normal semen samplesof healthy men, correlations with semen quality, the relationshipof IGF-I and ₂-M with factors affecting male fertility, andwhether vasectomy influences the concentrations of these molecules.Concentrations of IGF-I and₂-M in human seminal plasma wererelated to semen quality, basal concentrations of HGH, testosterone,IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 inseminal plasma and to serum concentrations of luteinizing hormone(LH) and follicle stimulating hormone (FSH). Commercially availableassays were used to analyse 69 semen samples of various qualityand 11 post-vasectomy samples. IGF-I concentrations in seminalplasma were significantly correlated with the percentage ofmorphologically normal spermatozoa (r = 0.748, P = 0.00001)and sperm concentration (r = 0301, P = 0.011), but negativelycorrelated with serum FSH (r = –0.506, P = 0.00006) andPSA in seminal plasma (r = –0388, P = 0.0009). Total ₂-Mwas significantly correlated with sperm count (r = 0.423, P= 0.0005), percentage of progressively motile spermatozoa (r= 0.444, P = 0.00019), quality of motility (sperm motile efficiency,r = 802, P = 0.00001) and straight line velocity (r = 0.411,P = 0.0013). Correlation between the sperm concentration andHGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomyreduced the concentration of total ₂-M (P = 0.00008) and HGH(P = 0.0068) in the seminal plasma; IGF-I was also reduced aftervasectomy when the total ejaculate amount was considered. ThusIGF-I and ₂-M are significant for the germ cell development:IGF-I hi the maturation of spermatozoa and ₂-M in progressivemotility.     

Clinical--pharmacological studies on human menopausal gonadotrophin

Using a randomized, cross-over design, the clinical, pharmacokineticand pharmacodynamic properties of commercially available gonadotrophinpreparations were studied. Following i.v. administration of150 IU of follicle-stimulating hormone (FSH) in the form ofHumegon® Pergonal® and Metrodin®, the maximum concentrations(C_Max were 27.5, 24.1 and 26.5 IU/I, respectively which werereached after 15.4, 16.9 and 16.9 mm (T_Max respectively. Thehalf lives (t) were 1.6, 2.3 and 2.0 h, respectively (fast compartment)and 11, 10 and 7.3 h, respectively (slow compartment). The tof lutelnizing hormone (LH) could only be estiniated in a surgicallyhypophysectomized patient: the fast compartment was 1.2 and1.3 h after the administration of Humegon® and Pergonal®,respectively and the slow compartment was 3.3 h in both cases.Marked individual differences of the same type were found inplasma FSH profiles, ovarian images and peripheral oestradlollevels after the administration of both preparatIons (150 IUdaily for 8 days) and the approximate t of ESH exceeded 40 h.It is concluded that the pharmacokinetic and pharmacodynamicproperties of the preparations studied are similar, if not identical,and that there are marked individual differences in patientresponsiveness, which are unrelated to the preparation administered.     

Follicular fluid prostaglandin E2:prostaglandin F2{alpha} ratio in relation to outcome of matched oocyte

The prostaglandins PGE₂ and PGF₂ and the steroid hormones oestradiol(E₂) and progesterone (P) were measured in 345 follicular fluidsof patients undergoing ovarian hyperstimulation for in-vitrofertilization (IVF). The measured concentrations were analysedin relation to the outcome of the matched oocyte. Progesteronelevels were significantly tower in the unfertilized group (P<0.005)compared to the fertilized group but there was no differencebetween ‘pregnancy’ and ‘no pregnancy’.No differences were shown in either E₂ levels or the E₂:P ratio.No significant differences were shown among the groups in theconcentration of either PGE₂ or PGF₂ but there were highly significantdifferences shown when the PGE₂:PGF₂ ratios were compared. ThePGE₂:PGF₂ ratio fell within a much narrower range for the ‘pregnancy’group compared with any of the other groups. The ratio was significantlylower (P<0.001) when ‘pregnancy’ was comparedwith ‘no pregnancy’. The range of the prostaglandinratio found for the ‘pregnancy’ group may reflectthe moment when conditions are optimal within the follicle forthe associated oocyte to go on to establish pregnancy.     

Elevated tissue type plasminogen activator in human granulosa cells correlates with fertilizing capacity

An increased production of plasminogen activators, able to convertplasminogen into plasmin, has been found in experiments in vivoon rat ovarian granulom cells at the time of ovulation, indicatingan involvement in follicular rupture. The graoulosa cells of49 follicles from 20 patients undergoing in-vitro fertilizationwere obtained by laparclscopy and tested for the content ofurokinase-type plasminogen activator (u-PA), tissue-type plasminogenactivator (t-PA) and inhibitor of plasmhogen activator (PAI).In the respective follicular fluids the concentrations of oestradiol(E₂), progesterone (P) and testosterone (T) were determinedand the levels of these enzymes and of the follicular steroidcontent were related to the fertilizing behaviour of the respectiveoacytes. Follicles containing oocytes which could be fertilized,revealed significantly higher follicular fluid E₂ and P levelsand significantly lower T levels than follicles with unfertilizedoocytes. The respective granulosa cells of fertilized oocytesexhibited higher levels of t-PA compared to their unfertilizedcounterparts, whereas no significant difference occurred inthe levels of u-PA and PAI. These data suggest that successfulfertilization of human oocytes is associated with a high contentof t-PA in granulom cells and high E₂ and P levels in the follicularfluid.     

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.     

Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts

BACKGROUND: Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 micromol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.     

Efficacy of the sperm survival test for the prediction of oocyte fertilization in culture

The present study was carried out to investigate the predictivevalue of the sperm survival test (SST) with respect to the fertilizationof oocytes in culture. In general, our laboratory uses a totalof 50 000–150 000 motile spermatozoa to inseminate eachoocyte. The remaining material is evaluated for motility beforeand after 24 h of incubation at 37°C in a 5% CO₂ atmosphere.A total of 250 oocytes from 50 cases (mean ± SD, 5.0± 2.4 oocytes per retrieval) were inseminated and thefinal rate of cleaved embryos obtained was 52.5%. The SST (%)was considered normal when the ratio (final density of progressingspermatozoa after 24 h x 100/initial density of progressingspermatozoa) was 50% or more. Any other result was consideredabnormal. Cases presenting one or more cleaved embryos (n =40) were separated from those in which no embryo formation occurred(n = 10) and the results were compared in terms of the respectivesperm survival rates over a period of 24 h: normal SST (oneor more cleaved embryos, 37; none, five), abnormal SST (oneor more cleaved embryos, three; none, five). The specificityof the SST was 0.92 and sensitivity 0.50, the predictive valueof the abnormal test was 0.62 and the predictive value of thenormal test 0.88. The efficacy of the test was estimated at0.71, which was better than the conventional parameters of spermanalysis. A receiver — operating characteristics curvefor SST confirmed that the test can be useful for the predictionof fertilizability of oocytes in the laboratory.