Cryopreservation of immature and mature human oocytes

Cryopreservation of oocytes facilitates the long-term storage of oocytes for patients in danger of losing ovarian function. It also alleviates many of the ethical concerns associated with embryo cryopreservation. Problems associated with metaphase II oocyte cryopreservation include zona pellucida hardening and spindle damage. The cryopreservation of germinal vesicle-stage oocytes has been undertaken as a means of circumventing the problem of spindle damage in mature oocytes. One of the main disadvantages of immature oocyte cryopreservation is the fact that in vitro maturation is required post-thaw. The majority of live births from oocyte cryopreservation have involved the use of 1,2-propanediol and slow freezing protocols. Various methods have been used in an attempt to improve survival rates. These include vitrification and use of novel cryopreservatives. Future areas of concentration should include in vitro maturation, vitrification, and alternate cryopreservatives.     

Successful ultrarapid freezing of unfertilized oocytes

Successful application of ultrarapid freezing techniques to unfertilized murine oocytes has not been reported. In an effort to improve results, preovulatory murine oocytes were exposed to three ultrarapid freezing protocols involving varying sucrose concentrations (0.25, 0.5, and 1.0M) and 3.5M dimethyl sulfoxide (DMSO) as cryoprotectants prior to direct immersion in liquid nitrogen. Postthaw morphology and rates of in vitro fertilization and embryo development were compared with those obtained after freezing oocytes employing two established programmed cooling techniques. The rates of fertilization and development to the blastocyst stage in vitro of oocytes undergoing ultrarapid freezing after exposure to 3.5M DMSO and 0.5M sucrose were similar or superior to those obtained with programmed cooling techniques. Of oocytes which appeared morphologically normal postthaw, only those which underwent ultrarapid freezing with 0.25 or 0.5M sucrose and 3.5M DMSO reached the blastocyst stage at rates similar to those of controls. Ultrarapid freezing may represent a viable option for successful murine oocyte cryopreservation.Presented in part at 44th Annual Meeting of the American Fertility Society, October 10–13, 1988, Atlanta, GA.     

Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol

Background Although cryopreservation of human preembryos has been carried out with success, the cryostorage of oocytes, which pose fewer controversial moral, ethical, and legal problems has been much less successful. Various attempts to cryopreserve human oocytes have been mostly unsuccessful and the search for an optimal protocol for oocyte cryopreservation remains elusive. We therefore undertook this study to determine the effect of oocyte cryostorage in 1,2-propanediol.Method Mature human oocytes with or without their cumuli were cryopreserved in precooled 1,2-propanediol, then thawed and inseminated with sperms for in vitro fertilization. The outcome of insemination and subsequent embryonic development were also recorded and compared.Results Postthaw cryosurvival rate was significantly better when cryostorage was carried out with the oocyte cumulus intact as compared to those oocytes denuded of their cumuli (54 versus 27%, respectively; P <0.05). Eight (44%) of 18 surviving postthaw oocytes with intact cumuli were fertilized normally, with cleavage in six, as compared to two (25%) and one, respectively, of those denuded of their cumulus prior to cryostorage. Development to the blastocyst stage was achieved in three embryos derived from oocytes with an intact cumulus at cryostorage. Conclusion We conclude that 1,2-propanediol can be used with success in oocyte cryopreservation, although the issue of parthenogenecity is still to be resolved. Oocyte's with intact cumulus survive cryostorage better than those without it.     

Embryonic behavior of two-cell mouse embryos frozen by the one- and two-step ultrarapid techniques

Purpose A modified two-step ultrarapid freezing technique was compared to the one-step ultrarapid freezing technique. Two-cell mouse embryos were frozen-thawed using the two freezing protocols, and postthaw cryoprotectant removal was carried out in either a single- or a multiple-step procedure.Results Statistically similar cryosurvival (96.95–100%) and blastocyst formation rates (87.95–91.47%) were obtained with both freezing groups. In addition, the method of cryoprotectant removal did not have any significant effect on the survival and development of the frozenthawed embryos in both groups. Blastocysts formed following single-step cryoprotectant removal had significantly lower inner cell mass counts in the one-step than in the two-step group (26.14 and 27.59, respectively; P <0.05). Embryo transfer studies showed that the implantation and fetal formation rates of embryos frozen by the two-step technique (61.67 and 60.0%, respectively) were similar to those of embryos frozen by the one-step technique (74.12 and 71.76%, respectively). Conclusion These results demonstrate that the ultrarapid two-step technique is as effective in cryopreserving two-cell mouse embryos as the ultrarapid one-step technique.     

Cryopreservation of human ovarian tissue

Approaches to the cryopreservation of human ovarian tissue are typically characterised by the use of slow freezing/rapid thawing methods using dimethyl sulfoxide or 1,2-propanediol (PROH) as cryoprotectants. This paper reviews current experience with these procedures.     

Zona pellucida surface of immature and in vitro matured mouse oocytes: Analysis by scanning electron microscopy

Purpose The aim of this work was to determine the morphology of the zona pellucida surface of immature and in vitro matured mouse oocytes by scanning electron microscopy. For this purpose two groups of immature oocytes (germinal vesicle group and metaphase I group) were studied either before or after in vitro maturation.Results Before in vitro maturation, the germinal vesicle immature group showed mainly an unstructured zona pellucida surface with smooth cumulus cells. The metaphase I immature group showed a more structured zona pellucida with smooth or blebbing cumulus cells. After in vitro maturation, development of the zona pellucida toward a mature surface, related to the initial degree of oocyte maturity, was observed in both groups.Conclusions These observations show a correlation between the morphology of the zona pellucida surface and the degree of oocyte maturity; the in vitro maturation process can give rise to a proper development of this endowment when immature oocytes are used.     

Correlation between human follicular diameter and oocyte outcomes in an ICSI program

Purpose: To determine the correlation between the follicular sizes and oocyte recovery, metaphase II oocyte recovery, fertilization rate and good embryo quality from mature and immature oocytes in an intracytoplasmic sperm injection (ICSI) program. Methods: 991 follicles obtained from 72 ICSI cycles were classified into three groups according to their diameters as measured by transvaginal ultrasound including group A (<10 mm), group B (10–14 mm), and group C (>14 mm). All obtained oocytes were classified according to their nuclear maturation: germinal vesicle (GV), metaphase I (MI) and metaphase II (MII). Mature oocytes underwent ICSI while immature oocytes were further cultured until maturity before ICSI was performed. The rates of fertilization and good quality embryos at day 3 were evaluated. Results: A progressive and significant increase in the rates of oocyte recovery and MII oocyte recovery were observed from group A follicles compared to the other groups (p < 0.001). The fertilization rate of mature and in vitro matured oocytes, as well as the rate of good quality embryos showed a tendency to increase from group A to group C follicles, but not significantly. The corresponding fertilization rates were 78 and 55.3% (p < 0.001) for mature and in vitro matured oocytes, respectively. Conclusion: Collection of oocytes from small follicles, especially with a mean diameter less than 10 mm, and in vitro maturation of immature oocytes before fertilization may allow the total number of good quality and transferable embryos to be increased.     

丁内酯-Ⅰ对小鼠卵母细胞体外成熟及发育能力的影响

目的:观察丁内酯-Ⅰ(BL-Ⅰ)对小鼠未成熟卵的体外成熟及发育能力影响。方法:用不同浓度的BL-Ⅰ培养液培养小鼠未成熟卵3h或6h,观察卵母细胞生发泡破裂(GVBD)的发生率。将6.25μmol/L和100μmol/L的BL-Ⅰ培养液分别培养未成熟卵3h和6h,以抑制其核的成熟,然后移入成熟培养液继续培养成熟,行体外受精,观察受精率和胚胎发育至囊胚的能力,并与对照组进行比较。结果:BL-Ⅰ培养液培养3h时,6.25μmol/LBL-Ⅰ就可抑制未成熟卵发生GVBD;培养6h时,100μmol/L才能抑制未成熟卵的GVBD。两种情况下,各组间的成熟率无差异。6.25μmol/L组卵母细胞的受精率(83.1%)和囊胚形成率(25.4%)虽低于体内成熟组(93.3%和46.4%),但显著地高于体外成熟组(59.9%和4.9%)。结论:BL-Ⅰ能可逆性地抑制小鼠未成熟卵的核成熟且不影响其成熟能力,适量的BL-Ⅰ对核成熟的短暂性抑制可显著提高小鼠未成熟卵的发育能力。     

Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.     

Cryopreservation of embryos and ova

Development of techniques for cryopreservation of embryos of several species, principally the mouse, laid the foundation for cryopreservation of human embryos. As IVF has become more widely available and the need for the cryopreservation of human embryos has become apparent, pressure for technical development has increased. The ideal method would be simple, inexpensive, and effective. The most effective method for cryopreservation of early human embryos, such as those at the 1-cell pronuclear stage and up to the 4-cell stage, now appears to be stepwise cooling in 1,2-propanediol with sucrose in plastic ministraws. The preferred method for intermediate stage embryos uses DMSO with cooling and thawing at slow rates in a programmed biologic freezer. For the human blastocyst, slow cooling in glycerol and rapid thawing is the only method reported with survival rates comparable to those achieved for intermediate stage embryos using DMSO. The rates of survival from freezing and thawing blastocysts are not sufficiently high, however, to justify the losses associated with prolonged in vitro incubation. Even at the current level of technical achievement, cryopreservation of human embryos provides the clearest opportunity to improve the clinical results obtained with IVF. Research now underway in the modification of methods for vitrification and ultrarapid freezing holds promise for both simplification of technology and improvement of outcome. In view of legal and ethical considerations involved in embryo preservation, the desirability of ova preservation is widely accepted. Although a small number of human unfertilized mature ova have been cryopreserved using various methods, success rates are still low. Methods for the cryopreservation of eggs should be developed, but these methods probably should be proved by animal experiments to be safe, especially with regard to genetic damage, before a policy of transfer of embryos derived from frozen-thawed human ova is applied on a large scale.     

Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes

The aim of this study was to evaluate the impact of different cryopreservation protocols on the repolymerization of metaphase (M)II spindles in human oocytes. Fresh aspirated donor oocytes were cryopreserved 3-4 h after retrieval using four different protocols: slow freezing using 1.5 mol/l 1,2-propanediol (PROH) + 0.2 mol/l sucrose (n = 36); 1.5 mol/l PROH + 0.3 mol/l sucrose (n = 34); 1.5 mol/l PROH + 0.3 mol/l sucrose with Na(+) depleted-choline replaced media (n = 27), and vitrification by the Cryotip method (n = 23). The control group comprised 34 fresh oocytes. Three hours after thawing, surviving and control oocytes were fixed for meiotic spindle/chromatin assessment. Survival rates were 63.8, 73.5, 74.1 and 86.9% respectively for the four protocols described above. Survival for vitrified oocytes was higher than that observed for slow freezing with 0.2 and 0.3 mol/l sucrose (P < 0.05). The proportion of oocytes showing normal spindle configuration was similar for the four protocols (81, 73.9, 88.9 and 81.3% respectively) and 88.5% for controls, showing that the MII spindle returns to its normal configuration after 3 h of post-thawing incubation under standard conditions, irrespective of the cryopreservation technique used.     

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage

Objective: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes.Design: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours.Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea.Patient(s): Oocytes were obtained from patients undergoing gynecologic surgery.Main Outcome Measure(s): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin.Result(s): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3.Conclusion(s): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.     

The current challenges to efficient immature oocyte cryopreservation

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.     

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

OBJECTIVE: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. DESIGN: Prospective, randomized, controlled trial. SETTING: University hospital. PATIENT(S): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. INTERVENTION(S): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. MAIN OUTCOME MEASURE(S): Oocyte maturation and fertilization rates, embryonic developmental quality. RESULT(S): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). CONCLUSION(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not an appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles.     

Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation

The authors describe a rapid freezing method (ultrarapid freezing) that has been developed for cryopreservation of early cleavage stage embryos. In the present experiments, 2-cell mouse embryos were frozen under a wide range of conditions in an attempt to optimize their survival and viability in vitro and in vivo. The experiments show that embryos exposed briefly (2 to 2.5 minutes) to relatively high concentrations of dimethyl sulfoxide (3 to 4 M) and 0.25 M sucrose survive and develop when plunged directly into liquid nitrogen and thawed in a 37 degrees C waterbath when sealed in 0.25-ml plastic pailettes. Survival and viability rates of ultrarapidly frozen embryos after thawing were comparable to those obtained with conventional slow-freezing techniques. The authors believe that this freezing technique can be further improved and that the speed, ease, and low cost of the method make it a very attractive alternative to more conventional methods for freezing early cleavage stage embryos.     

Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles

The present experiments compared the maturational and developmental competence of immature oocytes derived from stimulated cycles, following culture in a newly designed in-vitro maturation medium (IVM-medium) or in standard tissue culture medium (TCM-199; control). The results indicated that maturation and fertilization rates were comparable when the cumulus-free M-I stage oocytes were matured in the IVM-medium (78.6%) or the control medium (70.8%). However, there was a significant difference in blastocyst development (P < 0.05) when M-I oocytes were matured in these two media (19.6 versus 7.7%). Both maturation and early embryonic development rates of GV-stage oocytes were significantly higher (P < 0.01) in the IVM-medium (maturation: 75.7%; blastocyst: 12.9%) compared with control (maturation 55.7%; blastocyst: 0.0%). Moreover, embryos developed to the blastocyst stage at a higher rate in both media if GV-stage oocytes had matured within 24 h compared with 48 h of culture. These results demonstrate that immature human oocytes derived from stimulated ovaries can achieve maturation and early embryonic development in vitro, especially in the new IVM-medium, which may allow additional embryos to be produced for clinical use at embryo transfer.     

Cryopreservation of immature and in-vitro matured human oocytes by vitrification

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.     

Generierung und Konservierung von Keimzellen

In vitro maturation (IVM) is a technique which allows the maturation of oocytes from the germinal vesicle stage up to the stage of the fertilization-competent metaphase-II oocytes. Immature oocytes are primarily retrieved from small antral follicles. Their successful maturation is usually documented by formation of the first polar body as an indicator of completion of the first meiotic division. The quality of in vitro matured oocytes can now be judged by polarisation microscopy. This technique allows better quality assessment and is hence a unique instrument for optimising existing IVM protocols. In combination with the now realistic option of successful cryopreservation of mature and immature oocytes by the technique of vitrification, IVM will soon enter new fields of application.     

Follicle stimulating hormone effects on immature human oocytes: In vitro maturation and hormone production

Purpose: Our purpose was (1) to determine if in vitro maturation of unstimulated oocytes could be improved with the addition of urofollitropin; (2) to evaluate the output of estradiol, testosterone, progesterone, and androstenedione by the cultured oocyte-cumulus complex; and (3) to ascertain if steroid hormone production of the oocyte-cumulus complex correlates with final oocyte maturation stage. Methods: Fifty-eight immature oocytes were obtained from 11 regularly cycling women undergoing oophorectomy. The oocyte-cumulus complexes were randomly assigned to control medium (Ham’s F-10 supplemented with 7.5% fetal bovine serum) or test medium (control medium supplemented with 75 mIU/ml of urofollitropin). Results: (1) The addition of urofollitropin to oocyte culture medium does not significantly increase the ability of the oocyte to achieve the metaphase II stage; (2) the addition of urofollitropin significantly increases the production of progesterone, testosterone, and androstenedione by the oocyte-cumulus complex; and (3) there is no difference in the production of estradiol, progesterone, testosterone, and androstenedione by the oocyte-cumulus complex at the germinal vesicle, metaphase I or metaphase II stage of oocyte maturation. Conclusions: This information is of importance in the use of oophorectomy specimens for patients who must undergo an oophorectomy but desire to attempt pregnancy using their oocytes, in the use of oophorectomy specimens for donor oocytes, or for patients undergoing in vitro fertilization using immature oocyte collection.