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.     

Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI

BACKGROUND: The value of oocyte cryopreservation remains controversial. Two major problems exist: poor survival and injury to the oocyte meiotic spindle after freezing and thawing. METHODS: For slow oocyte cryopreservation, we used 1.5 mol/l 1,2-propanediol and 0.3 mol/l sucrose. We waited 3 h after thawing for possible recovery of the meiotic spindles before performing ICSI. RESULTS: Forty-three women undergoing IVF or ICSI cycles cryopreserved some or all of their harvested oocytes; of these, 20 thawed their cryopreserved oocytes for personal use and one for donation. The survival rate of oocytes after thawing was 75%, with 67% of oocytes fertilizing normally after ICSI. All 21 cycles (100%) resulted in fertilization and embryo transfers. Seven pregnancies (33%) resulted. Four women delivered five babies with normal karyotypes. Three conceptions are ongoing. Compared to 38 cycles of frozen-thawed embryos at the pronuclear stage in the same period, the percentages of survival, pregnancy and implantation were similar. Additionally, four unmarried women with white blood cell diseases underwent oocyte freezing before preconditioning treatment for haematopoietic stem cell transplantation. CONCLUSIONS: This protocol achieved reproducible success of survival, fertilization and pregnancy for freezing and thawing of human oocytes. The 3 h post-thaw incubation could permit restoration of the meiotic spindles, thus facilitating normal fertilization.     

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.     

Dynamics of human follicular growth and in-vitro oocyte maturation

The physiological trigger for meiotic resumption in the human oocyte is the surge of luteinizing hormone, but it can also occur spontaneously if oocytes are released from antral follicles and cultured in vitro. The development of novel techniques for the culture of murine oocytes has raised the possibility of growing human oocytes to maturity in vitro. Such a system could open the door to a number of techniques with revolutionary consequences. It would clearly be of benefit in basic physiological studies of follicular development, as well as being used to test the effect of toxicological substances on oocyte maturation. More significantly, such a system could provide a source of human oocytes for in-vitro fertilization (IVF) where immature or germinal vesicle oocytes are cultured to maturity before being fertilized. If this can be achieved, it might facilitate oocyte cryopreservation, where surplus oocytes are stored, thus avoiding the need for repeated superovulation. A combination of immature oocyte cryopreservation for later maturation and IVF will provide the opportunity to establish oocyte banks and help overcome some of the practical and ethical dilemmas that are currently shadowing the field of reproductive medicine.     

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.     

Intracytoplasmic sperm injection into zona-free human oocytes results in normal fertilization and blastocyst development

Zona-free human oocytes are frequently encountered in in-vitro fertilization (IVF) laboratories. The oocytes escape out of the zona pellucida, following zona fracture, which can occur during oocyte retrieval or manipulation, but occasionally may be the result of increased zona fragility. Some of the zona-free oocytes are mature and morphologically healthy; nevertheless, all are typically discarded. In this report, we demonstrate that zona-free oocytes can be fertilized normally using intracytoplasmic sperm injection (ICSI) and can subsequently develop without zona to the blastocyst stage in vitro. We therefore suggest that those mature and morphologically normal zona-free oocytes may be rescued, fertilized with ICSI and then cultured to the blastocyst stage for subsequent transfer or cryopreservation.     

Fertilization and early embryology: Parthenogenetic activation of human oocytes following cryopreservation using 1, 2-propanediol

Fresh and aged human oocytes were cryopreserved using 1, 2-propanediol(PROH). After thawing, the oocytes were cultured for 20 h andexamined for parthenogenetic activation using light microscopyand an ultraviolet DNA stain. Control fresh or aged oocytesand oocytes exposed to PROH without cryopreservation were alsoexamined for activation. No control oocytes were observed toactivate spontaneously (n = 43) and parthenogenetic activationwas not induced by exposure to PROH alone (n = 26). In bothfresh and aged cryopreserved oocytes, 27 and 29% of the oocytesrespectively were activated, and these proportions were significantlyelevated compared with the controls (P < 0.01). Althougha similar rate of activation was observed for the cryopreservedfresh and aged oocytes, the form of parthenogenetic activationvaried between these two types of oocyte. A single pronucleuswas observed in 18% of the fresh and 5% of the aged cryopreservedoocytes. In contrast, the presence of two or more pronucleiwas observed in 0% of the fresh and 19% of the aged cryopreservedoocytes.     

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.     

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.     

Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: Case report

This case report describes an ongoing pregnancy after cryopreservation of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome (PCOS). Oocyte retrieval was performed on day 18. The patient was administered 10 000 IU of hCG s.c. 36 h prior to oocyte collection. A total of 61 immature oocytes was obtained. Following incubation for 24-72 h in the YS maturation medium supplemented with 30% follicular fluid (hFF), 1 IU/ml FSH, 10 IU/ml hCG and 10 ng/ml rhEGF, 65.6% (40/61) of the oocytes were at the metaphase II stage. Thirty-eight oocytes (38/40, 95.0%) were fertilized after ICSI with the patient's husband's sperm and the 2PN oocytes were co-cultured with cumulus cells in YS medium supplemented with 10% hFF. Four embryos were transferred into the uterus on day 4 following oocyte retrieval but this failed to result in pregnancy. Eight embryos were developed to expanded blastocyst stage. The blastocysts were vitrified on electron microscope grids. Two years after cryopreservation, four blastocysts were thawed, three re-expanded and these frozen-thawed blastocysts were transferred to the uterus. A viable twin pregnancy was confirmed by ultrasound scan.     

Triploid pregnancy after ICSI of frozen testicular spermatozoa into cryopreserved human oocytes: case report

Although freezing oocytes is ethically more acceptable than cryopreservation of embryos, variable oocyte survival, fertilization rate and possible risk of increased ploidy after cryopreservation have precluded the widespread clinical application of oocyte cryopreservation in assisted reproduction techniques. We report a triploid pregnancy from intracytoplasmic sperm injection of recombinant FSH-stimulated frozen/thawed testicular spermatozoa into cryopreserved oocytes in a hormone replacement cycle. To our knowledge, this is the first report of a pregnancy where both gametes have been frozen. It illustrates the need for further research when applying new techniques in assisted reproduction.     

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.     

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.     

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.     

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.     

Fertilization and early embryolgoy: Human oocyte activation following intracytoplasmic injection: the role of the sperm cell

The aim of this study was to investigate whether the human spermatozoonparticipates in the activation of human oocytes following intracytoplasmicsperm injection (ICSI) and if so, by what mechanism. In thefirst series of experiments, we randomized human oocytes whichhad remained unfertilized after in-vitro fertilization (IVF)or ICSI, for intracytoplasmic injection with live spermatozoa,spermatozoa presumed to be dead and no spermatozoa. Secondly,unfertilized human oocytes and freshly ovulated mouse oocyteswere randomized for intracytoplasmic and sub-zonal injectionwith human sperm cytosolic fraction (CF) before and after heattreatment. We found that oocyte injection with initially motilespermatozoa induces human oocyte activation at a significantlyhigher rate than injection with dead spermatozoa (61 versus0%; P < 0.001) or injection without a spermatozoon (61 versus14%; P < 0.001). Intracytoplasmic injection of CF activatedboth human and mouse oocytes at the same rate as sperm injectionof human oocytes (activation rates of 70 and 65% respectively).This effect was greatly reduced by heat treatment of the CF.From these experiments we conclude firstly that the human spermatozooninjected intracytoplasmically contributes to human oocyte activationand secondly that the spermatozoon releases into the oocytea heat-sensitive, intracellularly active factor, which is notspecies-specific.     

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.     

Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome

This case report describes the birth of a healthy infant after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from an anovulatory woman with polycystic ovarian syndrome (PCOS). To initiate the treatment cycle, the patient received intravaginal progesterone at night for 10 days to induce a withdrawal bleed. Oocyte retrieval was performed on day 11 following a withdrawal bleed. The patient was administered 10,000 IU of HCG subcutaneously 36 h prior to oocyte collection. A total of 63 immature oocytes were obtained; 10 were morphologically abnormal. Following incubation for 24--48 h in the maturation medium, TC-199 supplemented with 20% patient's own serum, 75 mIU/ml FSH and LH, 77.4% (41/53) of the oocytes were at the metaphase-II stage. Thirty-one (31/41, 75.6%) were fertilized using ICSI with her husband's spermatozoa, 15 fertilized oocytes were cultured for embryo transfer and 16 were frozen at the pronuclear stage. Pregnancy ensued following fresh embryo transfer. Unfortunately, the pregnancy was miscarried eight weeks later. However, the second frozen-thawed embryo transfer attempt resulted in a full-term pregnancy with delivery of a healthy male infant.     

Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination.

BACKGROUND: Birefringent spindles imaged with the Polscope can predict fertilization rates after intracytoplasmic sperm injection (ICSI). The present study examined the development of human oocytes with or without birefringent spindles, imaged with the Polscope before ICSI. METHODS: Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. Spindles were imaged with the Polscope combined with a computerized image analysis system. After imaging and ICSI, oocytes with or without spindles were cultured separately for examination of fertilization and embryo development. A total of 1544 oocytes from 136 cycles were examined with the Polscope and inseminated by ICSI. RESULTS: Spindles were imaged in 82% of oocytes. After ICSI, more oocytes (P < 0.05) with spindles (69.4%) fertilized normally, forming 2 pronuclei, than oocytes without spindles (62.9%). At day 3, more oocytes (P < 0.01) with spindles (66.3%) developed to 4-11 cell stages than oocytes without spindles (55.4%). Significantly more (P < 0.001) oocytes with spindles developed to morula and blastocyst by day 5 (51.1 versus 30.3%) and day 6 (53.2 versus 29.3%) compared with oocytes without spindles. CONCLUSIONS: The results indicate that the presence of a birefringent spindle in human oocytes can predict not only higher fertilization rate, but also higher embryo developmental competence.     

Ovary and ovulation: DNA fragmentation of oocytes in aged mice

To investigate whether female fertility decreases with age dueto poor oocyte quality, we examined the presence of DNA fragmentationin ovulated oocytes from young, mature and aged mice. Oocytesfrom three age groups of female mice (7–8, 20–24and 40–48 weeks) were retrieved from the ovlducts 15 hafter human chorionic gonadotrophin (HCG) injection. Oocytesfrom each mouse were incubated in a CO₂ Incubator for 0–60h in human tubal fluid (HTF). After incubation, each oocytewas stained with the terminal deoxynucleotidyl transferase-mediateddUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation(interpreted as apoptotic changes) was significantly higherfor oocytes from aged mice, and the fertilization rate was significantlylower, compared with oocytes from young and mature mice. Ourresults suggest that DNA fragmentation of oocytes might be oneof the reasons for poor oocyte quality and lower fertility inthe aged group.