Cryopreservation Of Immature Oocytes Research Articles

The dawning of a new ice age for human oocyte cryopreservation

The dawning of a new ice age for human oocyte cryopreservation

Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing

The cryopreservation of immature oocytes permits oocyte banking for patients at risk of losing their fertility. However, the optimum protocol for such fertility preservation remains uncertain. The present study investigated the survival, maturation, cytoskeletal and chromosome organization of sibling immature oocytes leftover from controlled ovarian stimulation cycles, that were either slow-frozen (with choline-substitution) or vitrified. A comparison group included oocytes that were never cryopreserved. Among the three groups, comparable rates were observed for both survival (67-70%) and polar body extrusion (59-79%). Significantly more oocytes underwent spontaneous activation after IVM following slow-freezing compared with either vitrification or no cryopreservation. Likewise, the incidence of spindle abnormalities was greatest in the slow-frozen group, with no differences in spindle morphometrics or chromosome organization. While the overall incidence of mature oocytes with normal bipolar spindles from warmed immature oocytes was low, the yield using Cryoleaf vitrification was slightly superior to choline-based slow-freezing.

Fertility Preservation in Women With Breast Cancer

Fertility preservation is an important issue for young women diagnosed with breast cancer. The most well-established options for fertility preservation in cancer patients, embryo and oocyte cryopreservation, have not been traditionally offered to breast cancer patients as estradiol rise during standard stimulation protocols may not be safe for those patients. Potentially safer stimulation protocols using tamoxifen and aromatase inhibitors induce lower levels of estradiol whereas similar results in terms of number of oocyte and embryo obtained to standard protocols. Cryopreservation of immature oocytes and ovarian cortical tissue, both still experimental methods, are also fertility preservation options for breast cancer patients.

The future potential of cryopreservation for assisted reproduction

Cryopreservation of human gonadal tissue and oocytes has brought about new and exciting research in reproductive medicine, as well as new cryopreservation techniques that are dramatically improving post-thaw viability and freezing damage. The work done on gonadal tissues is aimed at improving the quality of life for infertile patients and for prepubertal patients undergoing gonadotoxic chemotherapy, patients for whom hormonal stimulation/IVF is not an option, and women without partners. Cryopreservation of mature oocytes is the best model for timing IVF. Vitrification is likely to benefit the field, and since 2005, implantation and pregnancy rates from vitrified oocytes have matched or eclipsed results from conventional methods, due to new cell-specific methods and formulas. Cryopreservation of immature oocytes leads to a new direction of egg banking in future. Preserving ovarian tissue for autografting is still promising and has resulted in folliculogenesis, resumed hormone production and live births in limited cases. The use of small cortical blocks, or mechanical/chemical digestion of ovarian tissue for isolation of follicles is a new direction for ovary preservation for reasons of cryoprotectant permeation and graft revascularization. Maturation of follicles in vitro has become more feasible with the use of alginate microencapsulation. Testicular tissue preservation has taken a sharp turn towards preservation of gonadal stem cells. Research into the mechanism for spermatogenesis points to the ability for male germ cells to resume spermatogenesis. The cryopreservation of minced testicular tissue for isolation of germ cells via chemical digestion has produced viable cells, however, structural damage that may be avoided by vitrification has been noted to the surrounding cell junctions and supporting cells.

Congélation lente et vitrification des ovocytes humains matures et immatures

Performance and security questions in human oocyte cryopreservation have been taking researchers for about two decades. Oocytes are usually frozen at metaphase II. Immature oocytes cryopreservation is still a research alternative. Two techniques are currently available for oocyte cryopreservation: slow freezing and vitrification. Experimental data suggest that vitrification has less impact on oocyte physiology than classical slow freezing. After slow freezing of mature oocytes, survival and fertilization rates reach 70 to 80% whereas cleavage rates are around 90%, leading to five implantations and 1.2 births per 100 thawed oocytes. After vitrification of mature oocytes, survival and cleavage rates reach 90% leading to 11 implantations and 1.8 births per 100 thawed oocytes. The obstetrical and neonatal prognosis of these pregnancies is reassuring. No increased risk of congenital anomalies has been observed. However, further evaluation is needed to guarantee the safety of cryopreservation procedures. Immature oocyte cryopreservation is not currently perfected but some indications appear of great interest.

Vitrification of immature mouse oocyte using stepwise equilibration before or after in vitro maturation

Immature mouse oocytes before or after in vitro maturation (IVM) were vitrified by the use of either conventional one-step or three-step equilibration. Although vitrification after IVM using the stepwise method does not affect the cleavage rate, the early embryonic development was profoundly impaired in both protocols.

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

82 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP™: EFFECT ON MEIOTIC MATURATION

Oocyte cryopreservation will become an important tool for the creation of genetic resources bank in domestic animals. Many problems have been described following cryopreservation of MII oocytes, including spindle disorganization (Ledda et al. 2007 Reprod. Fertil. Dev. 19, 13–23) and loss or clumping of microtubules resulting in some scattered chromosomes (Sathananthan et al. 1988 Hum. Reprod. 3, 968–977). Freezing of immature oocytes at the geminal vesicle stage might circumvent these problems because at this stage the genetic material is contained within the contours of a nuclear envelope (Siqeira-Pyles and Landim-Alvarenga 2005 Anim. Reprod. Sci. 193, 176–182). However, the number of reports relating to cryopreservation of immature oocytes remains low and it has been suggested that immature oocytes are more susceptible to cryoinjuries (Ledda et al. 2007). The aim of the present work was to study the effect of CryoLoop™ (Hampton Research, Aliso Viejo, CA, USA) vitrification on survival and subsequent meiotic maturation of immature ovine oocytes. Cumulus–oocyte complexes (COCs) were isolated from ovaries of slaughtered sheep. COCs were washed three times in basal medium [BM (HEPES–TCM-199 supplemented with 10% fetal bovine serum)], and then exposed to equilibration solution (10% ethylene glycol (EG) and 0.25 m trehalose (T) in BM for 3 min. After that, COCs were exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the CryoLoop within 1 min, and immersed in liquid nitrogen. Following cryopreservation, COCs were warmed by exposure of oocytes to (1) 10% EG and 1 mT in BM, (2) 0.5 mT in BM, and (3) BM for 3 min in each solution at 39�C. Then oocytes were matured in vitro for 24 h in maturation medium, as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). The nuclear maturation of oocytes was determined using aceto-orcein staining. Oocytes were classified into four stages: germinal vesicle (GV), metaphase-I (MI), anaphase-I + telophase-I (AI/TI), and metaphase-II (MII). Oocytes at MII stage were considered matured. Data were analyzed using chi-square test. The survival rate of oocytes after vitrification and warming based on oocyte morphology was 72.6% (61/84). Numbers of oocytes remaining at the GV stage were significantly higher in the vitrified group than in the control group (43.4 v. 12.6%, respectively; P < 0.01). Maturation to MII stage was higher (63.2%, 55/87) in the control group than in the vitrified group (43.4%, 23/53); however, the difference was not significant. Also, there were no significant differences between vitrified and control groups in terms of MI and AI/TI (7.5 and 5.7 v. 10.3 and 13.8%, respectively). In conclusion, immature ovine oocytes can be vitrified using CryoLoop with high survival and maturation rates.

Natural history of the mammalian oocyte

Combining cryopreservation of immature oocytes with in-vitro growth/maturation techniques is the ambition of many IVF clinics. Whilst these techniques have been demonstrated in rodents their application to humans and domestic species has been slow. There are many technical reasons for the lack of progress in these species, but the major problem is that we have very little knowledge of how the oocyte acquires developmental competence during its growth within the follicle. The life history of the mammalian oocyte involves a complex series of co-ordinated developmental processes that in the human take place over several months. This review will consider: (i) growth and development of the oocyte; (ii) the newly regenerated debate on the existence of germ-line stem cells in the mammalian ovary; and (iii) strategies for producing oocytes in vitro.

Effects of cryopreservation on the meiotic spindle of human oocytes.

The microtubular meiotic spindle of most mammals, including humans, is very sensitive to cooling [Hum. Reprod. 16 (2001) 2374; Fertil. Steril. 54 (1990) 102; Fertil. Steril. 75 (2001) 769; Zygote 3 (1995) 357] and is rapidly depolymerised even after a slight reduction in temperature to 33 degrees C. Spindle disassembly is dependent on the extent of temperature decrease and its duration. After rewarming, the recovery is far from complete. Cryoprotectants themselves may alter the spindle structure, depending on the duration and temperature of exposure, the duration of recovery at 37 degrees C and the species [Hum. Reprod. Update 2 (1996) 193]. Damage to the meiotic spindle is considered to be the cause of aneuploid embryos, by inducing chromatid non-disjunction and chromosome scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Nevertheless, a consensus arose from all the studies: appropriate exposure to cryoprotectants and appropriate rates of cooling and thawing allow the cryopreservation of mature oocytes without any significant changes in their second meiotic spindle organisation and without any increase in the rate of aneuploid embryos [Mol. Hum. Reprod. 2 (1996) 445; Hum. Reprod. 8 (1993) 1101; Hum. Reprod. 9 (1994) 684; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 75 (2001) 354]. These fundamental studies in humans, showing good preservation of cell structures after freeze-thaw procedures opened the way to new successful clinical trials with embryos derived from cryopreserved mature oocytes [Fertil. Steril. 68 (1997) 724]. Considering immature oocyte freezing at prophase I (germinal vesicle (GV) stage), a stage which was thought to be less sensitive to cryoinjury, pooled data from the literature showed no advantage in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, conflicting results are reported on the effects of freezing on the spindle-chromosome configuration of immature oocytes or in vitro matured oocytes, highlighting the need for large scale studies [Hum. Reprod. 10 (1995) 1816; Hum. Reprod. 13 (Suppl. 3) (1998) 161; Hum. Reprod. 17 (2002) 1885; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 68 (1997) 920]. One child has been born after the use of cryopreserved immature oocytes at GV stage, matured in vitro and fertilised by ICSI [Hum. Reprod. 13 (1998) 3156], demonstrating at least the feasibility of this technique. Improvements are required so as to make mature and immature oocyte cryopreservation an established and safe technique for ART.

MAP kinase phosphorylation in relation to cytoskeletal behavior in mouse oocytes cultured in vitro after vitrification at the germinal vesicle stage

Objective: Successful cryopreservation of immature oocytes may be an alternative approach in the ovum bank because cryopreservation will be performed before spindle formation. However, relatively few studies have examined the attempts of freezing germinal vesicle (GV) stage oocytes. There was a report showing that an incidence of chromosomal abnormalities in human GV stage oocytes after freezing and thawing was increased (Park et al., 1997). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior. In previous study, we have found that MAP kinase phosphorylation was correlated with cytoskeletal behavior in mouse oocytes after vitrification at the mature stage and that vitrification induced changes in microtubule organization and MAP kinase phosphorylation level in mature oocytes. The main objective of this study was to show a possible correlation between cytoskeletal change and MAP kinase phosphorylation level in mouse oocytes matured in vitro after vitrification at the germinal vesicle stage.

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.

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.

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.

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation

Objective: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. Design: Case report. Setting: Private infertility clinic. Patient(s): A 28-year-old woman with tubal infertility undergoing IVF therapy. Intervention(s): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. Main Outcome Measure(s): Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. Result(s): None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partner’s sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. Conclusion(s): This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation.

Die prämature Menopause

In western countries menopause is expected to occur at 50-52 years of age. Cessation of ovarian function before the age of 40 is defined as premature ovarian failure (POF). POF can be associated with chromosomal aberrations as well as with autoimmune diseases or general endocrine or metabolic disorders. Prevalence of POF is significantly increased in patients undergoing chemotherapy or radiation for treatment of malignant diseases. In some patients however etiology of POF remains unknown, a situation which is defined as idiopathic premature ovarian failure. If POF is definite therapeutic options for restoration of fertility are limited. In a few cases spontaneous recurrence of fertility has been reported. During chemotherapy, GnRH-analogues have been applied for gonadal protection with conflicting results. There are several ways of preserving patients' fertility by cryopreservation techniques. Principally either immature oocytes, fertilized ova or ovarian tissue can be cryopreserved. Whereas cryopreservation of pronucleus stages is an established procedure, outcome of cryopreservation of immature oocytes and ovarian tissue is poor. The disadvantage of pronucleus stage-cryopreservation is that in Germany only married couples can benefit from this approach. Immature oocytes and ovarian tissue can be preserved irrespective of marital status. However before these procedures can be applied as routine strategy several other problems concerning in vitro maturation of oocytes and the risk of transmitting malignant cells by autografting cryopreserved ovarian biopsies remain to be solved.

Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes

This study examined the effects of cryopreservation on cellular organization, chromosomal complement, and developmental potential of immature and mature mouse and human oocytes. Chromosomal analyses were performed by DNA fluorescence microscopy and karyotyping on the same metaphase II-stage oocytes before and after freezing. Cellular analyses involved electron microscopy, time-lapse video recording, and fluorescent-probe microscopy of cortical granules. The findings demonstrate that while profound cytoplasmic, nuclear, and nucleolar alterations occur in the immature oocyte during cryopreservation, an apparently normal nucleus and cytoplasm is re-established progressively after thawing and culture. The resulting oocytes mature at high frequency and for the mouse, are fertilizable and capable of normal preimplantation of embryogenesis. Cryopreservation of mature mouse and human oocytes is not accompanied by a significant increase in the frequency of aneuploidy. However, cryopreserved human oocytes, while fertilizable, arrest development during the early cleavage stages and display aberrant patterns of cytokinesis. The possible etiologies of developmental failure in the human embryo that may be related to oocyte cryopreservation, as well as the potential benefits of cryopreservation of the immature oocyte, are discussed with respect to clinical and commercial applications.