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.     

Gamete quality and preserving fertility

The main advantage of freezing oocytes is preserving the future fertility of women whose ovarian function is threatened by medical or surgical treatments and facilitating oocyte donation. The results of slow freezing of oocytes seem relatively dependent on the protocols used, most particularly on the sucrose concentration in the solutions of cryoprotective agents used. Today it seems that the use of low or medium-level concentrations of sucrose during the freezing process gives better results in terms of pregnancies and implantation than higher concentrations (0.3 mol/l), despite the sometimes less favorable results on oocyte survival after thawing. The cumulated pregnancy rates from frozen/thawed oocyte transfer are nearly equivalent to the results observed with frozen/thawed embryo transfer, which makes oocyte cryopreservation a valid alternative to embryo cryopreservation. Moreover, it seems that these results can be improved by the vitrification technique.     

Cryopreservation of human oocytes,zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification) methods

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.     

Ovarian tissue cryopreservation in young cancer patients for fertility preservation

Several options are currently available to preserve fertility and give female cancer survivors a chance to have children at a later date, including the cryopreservation of embryos, oocytes, and ovarian tissue. Selection of the most suitable strategy to preserve fertility depends on the type and timing of anticancer therapy, the cancer, the patient's age, and the presence of the patient's partner. Several studies have shown that the ovarian tissue can be successfully frozen and later grafted in the human womb. To date, approximately 30 live births have been achieved after the transplantation of frozen‐thawed ovarian tissue. At present, the standard procedure for cryopreservation of ovarian tissue is the slow‐cooling method. The slow‐cooling method uses an optimal cooling rate for the target cells, and relies on extracellular ice crystals to gradually dehydrate and equilibrate the tissue. Several groups reported that slow cooling is more efficient than vitrification for the cryopreservation of human ovarian tissue. However, vitrification can be performed under a variety of conditions, and therefore, the choice of methods is important. In addition, vitrification traps aqueous solutions in an amorphous, “vitreous” solid phase that prevents ice crystal formation in tissues. Vitrification methods that were developed using mice and monkey have recently been shown to improve the viability of vitrified ovarian tissues. In this review article, recent topics of ovarian tissue cryopreservation are described.     

Autotransplantation of cryopreserved ovarian tissue – effective method of fertility preservation in cancer patients

Abstract

Objective: To review the literature and to present the latest advances in the autotransplantation of cryopreserved ovarian tissue.

Materials and methods: A literature review was conducted for all relevant articles assessing the fertility preservation, ovarian tissue transplantation, standard freezing and vitrification of ovarian tissue.

Results: One of the promising and effective methods for fertility preservation may be the autotransplantation of cryopreserved ovarian tissue. At present, 30 babies have been born after orthotopic autotransplantation of frozen-thawed human ovarian tissue. Restoration of ovarian activity occurs between 3.5 months and 6.5 months. The longevity of autotransplanted ovarian tissue is about 5–7 years. The follicles are similarly preserved after all freezing methods; however, the ovarian stroma is significantly better preserved after vitrification than after slow freezing. An important topic for further research is preparation of the “vascular bed”, optimization of vitrification technique and the development of alternative procedures to avoid the transmission of cancer cells via ovarian tissue autotransplantation – “artificial ovary”.

Conclusions: Cryopreservation of ovarian tissue has unique advantages over other strategies. This method: (1) does not delay cancer treatment; (2) is safer for hormone dependent malignancy; (3) can be done independent of menstrual cycles; (4) is the only option for prepubertal girls; (5) can restore not only fertility but endocrine function.     

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。     

Reprint of: Theoretical and experimental basis of slow freezing

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement.In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins of improvement.     

Vitrification as an alternative means of cryopreserving ovarian tissue

Because of the simplicity of vitrification, many authors have investigated it as an alternative to slow freezing for cryopreserving ovarian tissue. In the last decade, numerous studies have evaluated vitrification of ovarian tissue from both humans and animals.Different vitrification solutions and protocols, mostly adapted from embryo and oocyte vitrification, have been applied. The results have been discrepant from species to species and even within the same species, but lately they appear to indicate that vitrification can achieve similar or even superior results to conventional freezing. Despite the encouraging results obtained with vitrification of ovarian tissue from humans and different animal species, it is necessary to understand how vitrification solutions and protocols can affect ovarian tissue, notably preantral follicles. In addition, it is important to bear in mind that the utilization of different approaches to assess tissue functionality and oocyte quality is essential in order to validate the promising results already obtained with vitrification procedures. This review summarizes the principles of vitrification, discusses the advantages of vitrification protocols for ovarian tissue cryopreservation and describes different studies conducted on the vitrification of ovarian tissue in humans and animal species.     

Methods for cryopreservation of human ovarian tissue

Human ovarian tissue can be successfully cryopreserved, with good survival and function after thawing. Experimental animal studies regarding ovarian tissue cryopreservation resulting in live-born offspring preceded the present freezing systems in humans. On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow programmed freezing, using human serum albumin-containing medium, and propanediol, dimethylsulphoxide (DMSO) or ethylene glycol as a cryoprotectant, combined with sucrose. Vitrification is still at the experimental stage. Whole organ cryopreservation is an interesting experimental option. Transplantation of the frozen-thawed tissue is a feasible method to utilize the tissue in infertility treatment. Ovarian function has been restored in humans. Because one healthy child has already been born from cryopreserved tissue, tissue cryopreservation should perhaps be offered to all young girls and women who can be predicted to undergo premature ovarian failure due to cancer treatment or genetic causes. Maturation of follicles in vitro from frozen-thawed tissue is another option that is still under development.     

Cancer and fertility: strategies to preserve fertility

Fertility preservation is a key component of cancer management in young people. The Fourth Evian Annual Reproduction Workshop Meeting was held in April 2009 to discuss cancer and fertility in young adults. Specialists in oncology, assisted reproduction, embryology and clinical genetics presented published data and ongoing research on cancer and fertility, with particular focus on strategies to preserve fertility. This report is based on the expert presentations and group discussions, supplemented with publications from literature searches and the authors' knowledge. Fertility preservation should be considered for all young people undergoing potentially gonadotoxic cancer treatment. A variety of options are required to facilitate safe and effective fertility preservation for individual patients. Sperm banking is a simple and low-cost intervention. Embryo cryopreservation is the only established method of female fertility preservation. Oocyte cryopreservation offers a useful option for women without a male partner. Emergency ovarian stimulation and cryopreservation of ovarian tissue (followed by tissue transplantation or in-vitro maturation of oocytes) are experimental techniques for women who require urgent cancer treatment. Further prospective studies are required to validate cryopreservation of oocytes and ovarian tissue, in-vitro maturation of oocytes and new vitrification techniques and to identify any long-term sequelae of slow freezing of embryos.     

Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique

This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n=49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5h prior to freezing had previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility preservation; however, before applying the method clinically, each laboratory should perform thorough validation of their technique.     

兔卵巢组织玻璃化冷冻的实验研究

目的:探讨玻璃化冷冻法保存兔卵巢组织的效果。方法:随机将25只新西兰雌兔分为对照组(5只)、慢速冷冻组(10只)和玻璃化冷冻组(10只),比较各组冻融前后卵巢组织学、超微结构、卵泡凋亡(原位末端标记法,TUNEL)和子宫系膜内移植后卵巢功能的恢复情况。结果:新鲜组织、慢速冷冻复苏组织和玻璃化冷冻复苏组织中正常形态卵泡比例分别为87.36%、81.96%和82.72%,两冷冻组正常卵泡比例均低于对照组,差异有统计学意义?P(0.05),但玻璃化冷冻组与慢速冷冻组差异无统计学意义(P>0.05)。3组间卵泡凋亡比率分别为21.4%、13.5%和17.1%,差异无统计学意义(P>0.05);3组移植后兔动情周期出现率均为100%,动情周期出现天数差异无统计学意义?P>0.05);移植存活的卵巢组织内可见各级形态正常的卵泡发育。结论:玻璃化冷冻可有效保存卵巢组织的结构和功能,是一种简单、可行的兔卵巢组织冷冻保存法。     

Theoretical and experimental basis of slow freezing

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement.     

Human oocyte cryopreservation: comparison between slow and ultrarapid methods

The success of reproductive technologies is facilitated by the cryopreservation of embryos and gametes. In Italy, where legislation prohibits zygote and embryo cryopreservation, clinics have extensively introduced oocyte cryopreservation. Two different strategies of oocyte cryopreservation are available: slow freezing or ultrarapid cooling (vitrification). Although the results are very encouraging with both methods, there is still controversy regarding both the procedure itself and the most suitable method to use. This study reports the routine application of the two different oocyte cryopreservation methods in programmes running in two consecutive periods. The study centre carried out 286 thawing cycles for a total of 1348 thawed oocytes cryopreserved by the slow-freezing method and 59 warming cycles for a total of 285 warmed oocytes cryopreserved by vitrification. Comparison of the outcomes obtained with the slow-freezing method versus vitrification in women who underwent IVF for infertility showed survival, fertilization, pregnancy and implantation rates of 57.9% versus 78.9% (P < 0.0001), 64.6% versus 72.8% (P = 0.027), 7.6% versus 18.2% (P = 0.021) and 4.3% versus 9.3% (P = 0.043) respectively. These results suggest that oocyte vitrification is associated with a better outcome than the slow-freezing method.     

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.     

Is vitrification standard method of cryopreservation

Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins. Vitrification is a cryopreservation technique that leads to a glass-like solidification. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Moreover, the use of vitrification technology for ovarian tissue cryopreservation to freeze eggs offers such an elderly women who sometime find more difficulty in conceiving or in maintaining pregnancy till full term because of old age compared to relatively younger women who might get better chances to get a healthy pregnancy. Furthermore, vitrification helps cancer patients who are looking to preserve their fertility later on after completing their treatment.     

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.     

Live birth from vitrified–warmed human oocytes fertilized with frozen–thawed testicular spermatozoa

A couple with male infertility due to non-obstructive azoospermia were referred to the fertility centre for treatment. Testicular biopsy was performed on the male partner and testicular samples were frozen. The female partner underwent ovarian stimulation and 31 mature oocytes were recovered by ultrasound-guided vaginal aspiration. Twelve oocytes were cryopreserved by the Cryotop vitrification method and 19 oocytes were inseminated by intracytoplasmic sperm injection (ICSI) using frozen–thawed testicular spermatozoa. Nine out of 19 oocytes were fertilized and the resulting embryos were cryopreserved by slow freezing. Four months later, two out of six thawed embryos were transferred, but no pregnancy resulted. One year later, the couple decided to attempt pregnancy using vitrified oocytes and frozen testicular spermatozoa. Six vitrified–warmed oocytes were injected with frozen–thawed testicular spermatozoa and four were fertilized. On the day of transfer, two cleavage stage embryos (4-cell, 2-cell) were obtained. Serum β-HCG test 14 days after embryo transfer was positive. Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation. A healthy baby boy weighing 3.09 kg was delivered by elective Caesarean section at 38 weeks of gestation. This case report demonstrates that oocyte cryopreservation by the Cryotop vitrification method does not compromise oocyte developmental competence.     

Are programmable freezers still needed in the embryo laboratory? Review on vitrification

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.