Oocyte Freezing Research Articles

Oocyte Freezing: Insurance or False Security?

Oocyte Freezing: Insurance or False Security?

Vitrification for embryo and oocyte freezing

Vitrification for embryo and oocyte freezing

Cryopreservation in Assisted Reproductive Technology: New Trends

During the last few years, cryopreservation has become a relevant addition to therapeutic concepts in reproductive medicine. New data and publications have made it difficult to maintain an overview of all of the new developments and their results. The focus of interest more recently, especially with the cryopreservation of human oocytes and human ovarian tissue, has been vitrification as an interesting alternative to slow freezing methods. Even though studies investigating the slow freezing of human mature oocytes have resulted in very different survival rates, it could be an option for donor oocyte programs, in the case of threatened ovarian loss or when there is an objection to embryo freezing. An optimal freezing protocol and later use of thawed human ovarian tissue is still a point of discussion. There are encouraging results regarding different kinds of autotransplantation, and recently the first birth after orthotopic autotransplantation of cryopreserved/thawed human ovarian tissue was described in the literature. Independent of any objections to cryopreservation in general, vitrification is a potential and effective alternative to conventional slow cryopreservation, especially for oocytes and embryos. Vitrification might be also be an option for human ovarian tissue; however this is only in its infancy and requires much additional investigation. Our article discusses new trends and results of actual studies regarding these issues.

102 DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES VITRIFIED AT DIFFERENT STAGES OF MATURATION IN VITRO

Cryopreservation of unfertilized oocytes at very low temperature (-196�C) is carried out to ensure their continuous availability during different assisted reproductive techniques. However, various problems associated with the freezing of oocytes influence their developmental competence, resulting in suboptimal embryo production. The present study was planned to assess the developmental competence of buffalo oocytes vitrified at different meiotic stages of maturation. Expression profile of developmentally important genes, viz, heat shock protein 70 (HSP 70) and glucose transporter 1 (Glut1), was verified in these vitrified warmed oocytes. Buffalo cumulus-oocyte complexes were collected from slaughterhouse ovaries and divided into six groups: control (no vitrification); 0 h group (vitrified before maturation), and 6-, 12-, 18-, and 24-h groups [vitrified respectively at 6, 12, 18, and 24 h post-in vitro maturation (IVM)]. Vitrification solution consisted of propylene glycol (40% w/v), and trehalose (0.25 mol/L) in PBS + BSA (4% w/v) and vitrification was carried out by directly plunging 0.25-mL French mini-straws into liquid nitrogen. After a minimum storage period of 7 days, the straws were thawed at 37�C for 30 sec. In all groups, the oocytes completed 24-h of maturation. After 24 h maturation, a few oocytes from each of the six groups were stained with ethidium bromide to reveal their nuclear status. The remaining oocytes were co-incubated with frozen thawed buffalo semen in fertilization TALP with 6 mg/mL fatty acid free BSA and 10 �g/mL heparin sodium salt for 18 h. Presumptive zygotes were cultured in mSOF for 8 days. Vitrified warmed oocytes were subjected to total RNA isolation and RT-PCR for detection of mRNA transcripts of HSP 70 and Glut1 genes. Data were analyzed by one-way ANOVA and F-test analysis. Differences of P < 0.05 were considered significant. The percentage of oocytes recovered from all five vitrification groups varied from 89 to 92 out of which 84-91% of oocytes were morphologically normal. A higher proportion of nonvitrified control oocytes (72.8%; 40/55) reached the metaphase II stage than for the oocytes vitrified at 24 (60%; 36/60), 18 (54.4%; 31/57), 12 (42.3%; 22/52), 6 (33.3%; 20/60), and 0 (31.7%; 19/60) h of IVM. The cleavage rate of nonvitrified control oocytes was higher (36.8%) than that of oocytes vitrified at 0 (1.6%), 6 (2.0%), 12 (3.2%), 18 (5.3%), and 24 (5.2%) h of IVM. With regard to subsequent development, 0- and 6-h oocytes were blocked at 8 cells, whereas in other groups development reached the late morula (4.8%) and blastocyst (3.5%) stages, confirming that the stage of maturation at which oocytes are vitrified influenced the nuclear maturation and developmental competence. Total RNA content was 2.24 � 0.40 ng/oocyte in the control group and 2.11 � 0.22 ng/oocyte in the group vitrified after 24 h of IVM. The expression pattern of HSP 70 and Glut1 was identical in control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these genes.

Effect of slush LN2 and sodium-depleted medium on the survival of the mouse and human oocytes after vitrification

Oocyte freezing has remained one of the most elusive tasks in the field of human assisted reproductive technology (ART). Although several pregnancies from oocyte cryopreservation have been reported, the adverse effects of freezing including vitrification on the integrity and developmental capacity of the oocytes have been observed. The purpose of this study was to improve the integrity of oocytes after vitrification by introducing slush LN2 for higher cooling rate and sodium-depleted medium as a basic solution for cryopreservation.

Guidelines for oocyte donation

Guidelines for oocyte donation

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

Optimization of oocyte freezing system contributes to improving the efficacy of various infertility treatment program, such as oocyte banking and ovum donation. Selecting of proper oocyte cryopreservation method is a critical factor in development of effective banking system. In our previous study, human oocytes matured in vitro after slow freezing at the germinal vesicle stage displayed a significant increase in incidence of chromosomal and spindle abnormalities. When vitrification method using ethylene glycol (EG)-based cryoprotectants and electron microscopy (EM) grid were used on human oocytes, higher rate of post-thawed egg survival and subsequent embryo development were observed.

Freezing of human immature oocytes using cryoloops with Taxol in the vitrification solution.

In human frozen immature oocytes, there has been little successful delivery. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. We set four experimental groups that immature oocytes has cumulus cells or not, or including Taxol or not in the vitrification solution. Frozen-thawed oocytes have been performed IVM, ICSI, and IVC. There were no significant differences in survival, maturation, and fertilization rate, respectively. However, in the group enveloped by cumulus cells and including Taxol in the vitrification solution, one embryo was developed to blastocyst. Our results showed that using vitrification solution with Taxol proved so effective.

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures.

The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.

Oocytes and assisted reproduction technology

AbstractA number of methods can now be used to store the female germ plasm from farm species. New assisted reproductive techniques such as ultrasound guided oocyte pickup, followed by the in vitro maturation of oocytes together with cryopreservation enable the collection and storage of germinal vesicle or metaphase II secondary oocytes and more practically embryos after in vitro fertilisation. Following freezing using equilibration or vitrification protocols, oocytes and embryos can be stored at liquid nitrogen temperatures for as long as required. Despite much research interest, the efficiency of secondary oocyte freezing is low and the developmental potential of stored oocytes is directly affected by the local cellular and hormonal environment during maturation, fertilisation and extended culture in vitro. An alternative strategy which avoids many of the technical difficulties associated with mature oocyte freezing may be to cryopreserve primordial oocytes in situ within ovarian cortex. This approach has the added advantage that it may also provide a means of conserving the oocytes of rare species and it can be used to bank cells obtained from postmortem tissue samples or species for which IVF protocols may not have been fully optimised. Although the methodology is still in its infancy, when ovarian cryopreservation is combined with autografting, xenografting, or follicle culture, ovarian tissue freezing has the potential to restore or extend the fertility of domestic animals so maximising their genetic potential.

Healthy girl born after cryopreservation of gametes and ICSI in a patient with seminoma

This study reports birth in a case of intracytoplasmic sperm injection with cryopreserved oocytes and spermatozoa banked after radiotherapy and prior to chemotherapy due the occurrence of two non-synchronous seminomas. A 30-year-old male with a diagnosis of seminoma cryopreserved six vials of spermatozoa. After oncological treatment was completed, his partner, a 24-year-old woman, underwent ovarian stimulation. Seventeen oocytes were retrieved; one was at the germinal vesicle stage and two were injected, resulting in two embryos. Fourteen metaphase II oocytes were frozen. The woman presented moderated ovarian hyperstimulation syndrome, and embryo cryopreservation was indicated. After endometrial preparation, two embryos were transferred and a pregnancy was achieved. The woman suffered eclampsia during week 28 of gestation. Caesarean section was performed and a preterm girl weighing 1000 g was born, but died 2 weeks after delivery. A year later, a second procedure was begun. Frozen oocytes and one vial of semen were thawed. Eight of the 14 oocytes survived and were microinjected; two became fertilized and one good quality cleaved embryo was transferred. Pregnancy was achieved and a healthy girl was born with a birth weight of 2800 g. Oocyte cryopreservation associated with sperm banking in cancer patients is a useful tool for couples seeking deferred parenthood.

Conventional technologies for cryopreservation of human oocytes and embryos.

AbstractIn the last few years, there has been a significant resurgence of interest in the potential benefits of human oocyte freezing. Essentially, these benefits are formation of donor “egg banks” to facilitate and lessen the cost of oocyte donation for women unable to produce their own oocytes; provision of egg cryostorage for women who wish to delay their reproductive choices; and convenient cryopreservation of ovarian tissue from women about to undergo therapy deleterious to such tissue, which may threaten their reproductive health.KeywordsZona PellucidaGerminal VesicleHuman OocyteOocyte DonationBlastocyst TransferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Oocyte freezing: here to stay?

Oocyte freezing is an established technology but, in contrast to embryo freezing, it has very limited application in clinical IVF programmes. Is there a chance that oocyte freezing will become an integrated routine in assisted reproductive technology? The delicate cytological architecture of the oocyte with a cold-sensitive spindle and a hardening zona have made the frozen oocyte 'unwanted' in assisted reproductive technology. Nevertheless, empirical improvements in freezing protocols and the use of ICSI for fertilization have led to an increasing number of live births. This mitigates against a simple ban on oocyte freezing. While efficiency of oocyte freezing can certainly be further improved by basic research, it is clear that there are humanitarian reasons for considering oocyte freezing as a future fully utilized assisted reproductive technology. The storage of the female genome as a particulate entity can provide an alternative in case of moral, ethical, legal or religious concerns about embryo freezing. Oocyte freezing can also offer hope for oocyte donation and preservation of fertility for women facing ovarian loss. The message is one of cautious optimism when looking for a place for oocyte freezing in routine assisted reproductive technology.

Freezing of human immature oocytes using cryoloops with Taxol in the vitrification solution

Objective: Oocytes freezing is very useful for Assisted Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including our own have been reported after the freezing of human mature oocytes. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer.Design: A prospective study.Materials and Methods: The immature oocytes allocated to this study were recovered from patients undergoing ICSI. Cumulus cells were removed by the hyaluronidase treatment and those having a visible germinal vesicle oocytes were used for the study (C(-)). Some oocytes that had a remaining two or three layers of cumulus cells, and had visible germinal vesicle oocytes were defined as C(+) immature oocytes. Vitrification using cryoloop was performed using ethylene glycol and dimethyl sulfoxide (DMSO) with or without Taxol (T(+) or T(-)). We divided four freezing group; C(+)T(+), C(+)T(-), C(-)T(+) and C(-)T(-). After thawing, immature oocytes were treated in a maturation culture, and metaphase a oocytes were treated using ICSI and cultured.Results: There were no significant differences in survival rate, maturation rate and fertilization rate. In the group of C(+)T(+), there was a significant difference in cleavage rate with C(+)T(-) and C(-)T(+) (87.5%, 42.9% and 44.4%, respectively). In the group of C(+)T(+), one embryo was developed to blastocyst at day 5 after ICSI.Conclusions: Our results showed that vitrification of immature oocytes is a good method of developing mature oocytes. Moreover, using vitrification solution with Taxol proved so useful that it developed one embryo to blastocyst stage. But most embryos were arrested until 8cell stages, and the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about immature oocytes on unstimulated cycles are needed. Objective: Oocytes freezing is very useful for Assisted Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including our own have been reported after the freezing of human mature oocytes. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. Design: A prospective study. Materials and Methods: The immature oocytes allocated to this study were recovered from patients undergoing ICSI. Cumulus cells were removed by the hyaluronidase treatment and those having a visible germinal vesicle oocytes were used for the study (C(-)). Some oocytes that had a remaining two or three layers of cumulus cells, and had visible germinal vesicle oocytes were defined as C(+) immature oocytes. Vitrification using cryoloop was performed using ethylene glycol and dimethyl sulfoxide (DMSO) with or without Taxol (T(+) or T(-)). We divided four freezing group; C(+)T(+), C(+)T(-), C(-)T(+) and C(-)T(-). After thawing, immature oocytes were treated in a maturation culture, and metaphase a oocytes were treated using ICSI and cultured. Results: There were no significant differences in survival rate, maturation rate and fertilization rate. In the group of C(+)T(+), there was a significant difference in cleavage rate with C(+)T(-) and C(-)T(+) (87.5%, 42.9% and 44.4%, respectively). In the group of C(+)T(+), one embryo was developed to blastocyst at day 5 after ICSI. Conclusions: Our results showed that vitrification of immature oocytes is a good method of developing mature oocytes. Moreover, using vitrification solution with Taxol proved so useful that it developed one embryo to blastocyst stage. But most embryos were arrested until 8cell stages, and the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about immature oocytes on unstimulated cycles are needed.

Comparison of ionophore induced Ca2+ release in fresh and cryopreserved oocytes

Comparison of ionophore induced Ca2+ release in fresh and cryopreserved oocytes

Comments on oocyte cryopreservation

Recently, Robert Winston stated publicly that freezing human oocytes could cause irreparable harm to future generations. In this paper, this affirmation is refuted with arguments and facts. With regard to the low efficacy of the oocyte freezing technique, data are provided on the number of frozen oocytes required to achieve one pregnancy: 100, 17 and 25 in three different papers, compared with 50 fresh oocytes in another. Two recent articles are cited. One shows similar results (pregnancy/cycle) when using frozen oocytes and frozen embryos (17.2 and 18.7% respectively). The other reported birth rates with fresh oocytes (57.9%), frozen embryos (52.9%) and frozen oocytes (45.8%). The legal situation with regard to freezing oocytes in Spain is described, as is the legal uncertainty experienced by the CEFER Reproduction Institute when it began freezing human oocytes for use in assisted reproduction.

More successful oocyte freezing.

More successful oocyte freezing.

Embryo technologies in the horse

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 °C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 μm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipient’s oviduct results in a 70–80% pregnancy rate compared with a 30–40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55–82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced.

Comparison of two freezing methods for human immature oocytes with cumulus or cumulus free: Slow freezing versus vitrification (including the effect of Taxol)

Objective: Oocyte freezing is very useful for Assisited Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including ours were reported after the freezing of human mature oocytes in both slow-freezing (SF) and vitrification (V). We examined the feasibility using either enclosed cumulus cells or cumulus free cells using both SF and V methods (including the effect of Taxol) to find the best results of IVF-IVC. Design: A prospective study. Materials/Methods: At first, mature oocytes were collected from more than 10 mm diameter follicles of the patients stimulated by HMG-HCG with GnRH agonist. After that, cumulus-enclosed oocytes were collected from less than 10mm diameter follicles for this study. SF using straw was performed as cryoprotectants 1,2-propanediol(PROH) and sucrose(S). V using cryoloop was performed using ethylene glycol (EG) + dimethyl sulfoxide (DMSO)+S. We divided human immature oocytes into 5 groups: (Group A;SF, with cumulus, Group B;SF, cumulus free, Group C; V, with cumulus, Group D; V, cumulus free, Group E; V, with cumulus, Taxol 1μM). Results: Survival rate, maturation rate, fertilization rate, cleavage rate (Group A, B, C, D,E) were 100%(2/2), 100%(2/2), 100%(2/2), 100%(2/2): (GroupA), 84.0%(21/25),71.4%(15/21), 60.0%(9/15) 66.7%(6/9), (GroupB) 69.2%(9/13), 88.9%(8/9), 62.5%(5/8), 40%(2/5): (GroupC), 79.3%(23/29), 82.6%(19/23) 57.9%(11/19), 63.6%(7/11): (GroupD), 88.2%(15/17), 93.3%(14/15), 71.4%(10/14), 90.0%(9/10) (GroupE) respectively. There was a statistically significant difference between GroupC and GroupE in cleavage rate (p <0.05). Almost embryos arrested until 8 cell stages. Only one embryo of Group E developed to morula stage. Conclusions: Both slow freezing and vitrification methods using either enclosed cumulus or cumulus free immature oocytes were similar good results. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of human immature oocytes.But it is said that the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about human immature oocytes on unstimulated cycles are needed. Supported by: None.

Guidelines for oocyte donation

Guidelines for oocyte donation