Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures

BACKGROUND: Despite the recent increase in pregnancies from cryopreserved human oocytes, success in terms of births per thawed oocyte is still poor. Modifications to cryopreservation protocols have not been based on measurement of the osmotic response of oocytes, and methodologies are often poorly described or protocols not strictly adhered to, inevitably resulting in variability. METHODS: Volume change of mature human oocytes was measured on exposure to cryoprotectant. Oocytes were exposed to either 0.75 mol/l propane-1,2-diol (PrOH) for 10 min; 1.5 mol/l PrOH for 10 min, having been exposed to 0.75 mol/l PrOH for 7.5 min; or 1.5 mol/l PrOH plus 0.2 or 0.3 mol/l sucrose for 10 min, having been exposed to 1.5 mol/l PrOH for 10 min. RESULTS: On exposure to PrOH alone, oocytes shrank and then re-expanded, having reached 75 and 84% of their starting volume in 0.75 and 1.5 mol/l, respectively. Oocytes shrank continuously in PrOH plus sucrose, reaching 67 or 55% of their initial volume in 0.2 or 0.3 mol/l sucrose, respectively. CONCLUSIONS: To improve consistency following cryopreservation, protocols must be strictly adhered to; small changes in duration of exposure to cryoprotectant can result in drastic changes in cellular hydration and thus the fate of the cell during freezing/thawing.     

Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates

Human oocytes were exposed to the cryoprotectant dimethyl-sulphoxide (DMSO) at either 4 or 37 degrees C. Subsequent fertilization of these oocytes showed that exposure to DMSO at 37 degrees C was associated with a greatly reduced fertilization rate when compared to untreated control oocytes, whereas no such reduction was seen in oocytes exposed to DMSO at 4 degrees C. The significance of these results for the potential cryopreservation of human oocytes is discussed.     

Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte

At ovulation, the mouse oocyte is arrested at metaphase of thesecond meiotic division. Since microtubules are thermo-and chemosensitivestructures, the effects of 1.5 M dlmethylsulphoxide and 1.5M 1,2-propanediol were studied at room temperature on the morphologyof the meiotic spindle. Oocytes incubated at 37°C or atroom temperature served to estimate the effect of temperaturein the experiment. The meiotic spindle was visualized by immunogold-silverstaining of microtubules. In the control group at 37°C,88% of oocytes had normal spindles. After incubation at roomtemperature for the same time, 89% of oocytes showed abnormalspindles. In the oocytes exposed to dimethyl sulphoxide or 1,2-propanediolat room temperature a protective effect on spindle morphologycould be recognized. Subsequent incubation at 37°C resultedin partial restoration of the observed abnormalities after coolingto room temperature and after exposure to dhnethytsulphoxide.incubation at 37°C after exposure to 1,2-propanediol atroom temperature induced spindle absence in the majority ofoocytes. Although this latter condition allowed fertilizationwithout increased incidence of ploidy abnormalities, a rolefor 1 as an activating agent is hypothesized.     

Assessment of the developmental potential of frozen-thawed mouse oocytes

Mouse oocytes were cryopreserved by a protocol shown previouslytominimize damage tot the zona peelucida and cytoskeletal system.After thawing, the incidence of fertilization didnot differfrom that in control groups of oocytes, and after fertilizaiton,the ability of the fertilized frozen–thawed oocytes todevelop tothe balstocyst stage in vitro was only slightly less(77%) than that of the controls (87 and 89%). Transfer of frozen–thawedand fertilized oocytes after their culture to the balstocyststage in vitro resulted in a lower implantation rate (46%) thanfor the controls (68–73%), but of the implanting embryosthe same proportions in experimental and control groups survivedtoyield vaiable fetuses. In contrast, transfer after culturein vitro to the 2-to 4-cell stage rsulted in similar implantationrates for control and frozen–thawed fertilized oocytes(70–84%), but the spontaneous abortion rate was higherfor the embryos derived from frozen–thawed oocytes. Overallthe cumulative survival rate for frozen oocytes transferrd atthe 2-cell stage (36%) was better than after transfer at theblastocyst stage (30%), but both were less than for the transferat any stage of the control oocytes (47–55%). The cumulativesurvival of cryopreserved oocytes to viable fetuses was 30–40%less than that of the control oocytes. These results are comparedwith those form previous studies and the main remaining obstaclesto completely successful cryopreservation are identified     

Current status of the cryopreservation of human unfertilized oocytes

Cryopreservation facilitates the long-term storage of oocytes from patients in danger of losing ovarian function and allows greater flexibility in fertility services for other patients. If some of the oocytes collected following ovulation stimulation are stored prior to fertilization, this alleviates many of the ethical concerns associated with embryo preservation. Concerns that cryopreservation could lead to disruption of the spindle and chromosomes, thus leading to genetic abnormalities of the offspring produced, mean that this procedure is not permitted in some countries. The recent spate of human live births from thawed oocytes has prompted the granting of the first licence allowing the use of thawed oocytes in the UK. However, the success rate of this procedure is still low and further research is required to refine these techniques and to develop new ones.     

Birth following vitrification of a small number of human oocytes: case report

We report the birth of a healthy baby girl at 37 weeks gestation to a 47 year old recipient, after vitrification of mature oocytes from four in-vitro fertilization (IVF) patients. A total of 17 oocytes was vitrified in 1-2 microl of ethylene glycol (40%) and 0.6 mol/l sucrose (20.54%) in open pulled straws. Eleven oocytes survived after vitrification and five pronuclear zygotes were obtained after intracytoplasmic sperm injection (ICSI). Three embryos were transferred to three patients, two of whom were the original oocyte donors and pregnancy was not established. The third embryo was donated to a 47 year old infertile woman after preimplantation diagnosis had confirmed euploidy for chromosomes X, 13, 14, 15, 16, 18, 21 and 22. The successfully completed pregnancy is encouraging for further research to explore the potential benefits of vitrification for the cryopreservation of human oocytes, given the relatively low success of conventional freezing of human oocytes by slow cooling methods.     

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.     

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.     

Fertilization and early empryology: Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition

The addition of 20% fetal bovine serum (FBS) to media used formouse oocyte cryopreservation prevents hardening of the zonapellucida that otherwise can occur due to premature releaseof cortical granule contents (George et al., Hum. Reprod., 7,401–412, 1992). Protection of human oocytes would ideallybe achieved by using a human macromolecular source or a moredefined bovine source than total FBS. Here we investigate whetherFBS can be replaced by human serum, human cord serum, humanserum albumin or fetuin, the major protein component of FBS.Only fetuin was found to be effective.     

Use of fetal bovine serum to protect against zona hardening during preparation of mouse oocytes for cryopreservation.

Addition of 20% fetal bovine serum (FBS) to media used for cryopreservation does not reduce the premature release of cortical granules but does prevent their action on the zona pellucida and thereby prevents zona hardening. In this paper, it is shown that the washing period required for removal of FBS is less than 12 min for cumulus-free oocytes and between 150 and 170 min for cumulus-intact oocytes. When these washing periods are observed after exposure of oocytes to the cryoprotectant dimethylsulphoxide (DMSO; 1.5 M) in the presence of 20% FBS, a subsequent exposure to calcium ionophore A23187 or a second exposure to 1.5 M DMSO both lead to zona hardening. This result suggests that sufficient cortical granules remain to elicit a block to polyspermy at fertilization. Oocytes, which had been exposed to DMSO and FBS and then washed free of both, were fertilized in vitro; the incidence of polyspermy was not found to be elevated over the level found in controls.     

Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants

Pieces of ovarian cortical tissue (03–2 mm in diameter)were obtained during gynaecological operations by biopsy oras a result of oophorectomy from 19 women aged 19–44 years.The tissue was frozen in a programmable freezer using one oftwo different cryoprotectants, either 1.5 M dimethylsulphoxide(DMSO), or a combination of 1, 2-propanediol (1.5 M) and sucrose(0.1 M). After cryopreservation lasting from 24 h to 5 weeks,the ovarian pieces were thawed and studied histologically. Specimenstaken before and after cryopreservation with either pro-tectantshowed no signs of tissue necrosis. Follicles at similar developmentalstages were found before and after freezing. The proportionsof follicles showing signs of atresia, 27% in the non-frozentissue and 19% in the frozen-thawed tissue, were not significantlydifferent. Oocytes, too, had the same appearance after freezingand thawing with both cryoprotectants as was seen in the specimenstaken before freezing. These results suggest that cryopreservationof human ovarian tissue is feasible. However, the normalityof the oocytes taken from tissue which has been frozen stillneeds to be established. Cryopreservation of ovarian tissuewould be potentially an excellent method for storage of humanoocytes once methods for their maturation in vitro have beendeveloped.     

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)     

Permeability characteristics and osmotic sensitivity of rhesus monkey (Macaca mulatta) oocytes

BACKGROUND: Permeability characteristics and sensitivity to osmotic shock are principal parameters that are important to derive procedures for the successful cryopreservation of mammalian oocytes. METHODS AND RESULTS: The osmotically inactive volume of rhesus monkey oocytes was determined by measuring their volumes in the presence of hypertonic solutions of sucrose from 0.2 to 1.5 mol/l, compared with their volume in isotonic TALP-HEPES solution. Boyle-van't Hoff plots at infinite osmolality indicated that the non-osmotic volumes of immature and mature oocytes were 20 and 17% respectively. Osmotic responses of oocytes exposed to 1.0 mol/l solutions of glycerol, dimethylsulphoxide (DMSO) and ethylene glycol (EG) were determined. Rhesus monkey oocytes appeared to be less permeable to glycerol than to DMSO or to EG. Sensitivity of oocytes to osmotic shock was determined by exposing them to various solutions of EG (0.1 to 5.0 mol/l) and then abruptly diluting them into isotonic medium. Morphological survival, as measured by membrane integrity, of oocytes diluted out of EG depended significantly on the concentration of EG (P < 0.01). CONCLUSION: Determination of permeability characteristics and sensitivity to osmotic shock of rhesus oocytes will aid in the derivation of procedures for their cryopreservation.     

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.     

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing

BACKGROUND: Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. METHODS: Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. RESULTS: Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. CONCLUSIONS: Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.     

A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin

BACKGROUND: The objective of this study was to determine whether folliclesgrown within human ovarian cortical strip culture for 6 daysin serum-free medium could be isolated at the secondary stageof pre-antral development and grown in vitro to the late pre-antral/earlyantral stage during a 4 day culture period. METHODS: Ovarian cortical biopsies were obtained from six women aged26–40 years, with informed consent, during elective Caesareansection. Small tissue slices of ovarian cortex, with underlyingstromal tissue removed, were cultured in serum-free medium for6 days and at the end of this period pre-antral (secondary)follicles were dissected from the strips. Seventy-four intactpre-antral follicles ranging in size (66–132 µm)(mean size 100 µm ± 3.4) were selected for furtherculture. Follicles were placed individually within V-shapedmicrowell culture plates in serum-free medium in the presence(n = 38) or absence (n = 36) of 100 ng/ml of human recombinantactivin A. RESULTS: Pre-antral follicles grown for 4 days in the presence of activinA grew to a larger size (mean diameter 143 µm ±7.4) than those grown in control medium (mean diameter 111 µm± 8) (P < 0.005). Ninety percent of follicles culturedin the presence of activin A increased in size during the first2 days of culture compared with only 36% of follicles in controlmedium (P > 0.005). Of the follicles surviving the entireculture period, 30% of those cultured in the presence of activinA showed normal morphology with intact oocytes and antral formation.None of the follicles grown in control medium developed antralcavities and >90% of those follicles collected at the endof the culture period showed signs of oocyte degeneration. CONCLUSIONS: The results reported here demonstrate that under certain conditions,it is possible to achieve accelerated oocyte/follicle developmentfrom human primordial/primary follicles. This provides the firstencouraging step towards achieving full in vitro growth of humanoocytes.     

Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture.

This study aimed to: (i) determine whether oocytes are present in cultures of human fetal ovary; (ii) identify whether meiotic anomalies are evident; and (iii) assess whether preparation or culture conditions influence oocyte survival and meiotic progression. Ovaries were collected from fetuses after termination at 13-16 weeks. Oocyte assessment utilized antibodies specific for synaptonemal complex proteins (associated with chromosomes only during meiosis), and antibodies to centromeric proteins. Fragments of tissue were cultured in minimal essential medium + 10% serum +/- follicle stimulating hormone (100 mIU/ml). The sera were fetal calf serum (FCS), FCS for embryonic stem cells (ES-FCS) and human female serum. The numbers and stages of oocytes were assessed after 7-40 days, and particular arrangements of chromosome synapsis identified. Results in fresh tissue included oocytes at leptotene, zygotene, pachytene and diplotene in three of five samples. Four specimens remained viable in vitro, and three had detectable oocytes after culture. The numbers of oocytes and the proportions of zygotene and pachytene cells increased with time in culture. The proportion of degenerate cells in culture was initially higher than in fresh samples, but declined subsequently. More oocytes were detected in ES-FCS and human serum than in FCS. We conclude that human oocytes survive in culture and that progression through prophase I continues.     

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation

BACKGROUND: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human. METHODS: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I. RESULTS: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control. CONCLUSIONS: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.     

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.     

Maturation in vitro of immature human oocytes for clinical use

Human oocyte maturation is considered as the reinitiation andcompletion of the first meiotic division from the germinal vesiclestage (prophase I) to metaphase II, and the accompanying cytoplasmicmaturation for fertilization and early embryonic development.Immature human oocytes obtained from patients undergoing gynaecologicalsurgery, or ovulation induction or having polycystic ovary syndrome(PCOS) can be matured and fertilized in vitro. To date, 80%of immature oocytes matured to metaphase II when cultured inmaturation medium supplemented with gonadotrophins and 85% ofmatured oocytes fertilized and cleaved in vitro. Following transferof these embryos, pregnancies and live births have been achieved.However, the capacity for oocyte maturation was different whenthe immature oocytes were retrieved from PCOS patients and whenthe oocytes were cryopreserved at germinal vesicle stage.