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.     

Immature oocyte retrieval in the luteal phase to preserve fertility in cancer patients

As cancer treatment outcomes improve, the number of women with cancer seeking fertility preservation increases. Currently, embryo/oocyte cryopreservation appears to provide the best fertility preservation option. However, patients may not have sufficient time to undergo ovarian stimulation prior to chemotherapy and/or the hormones used in ovarian stimulation are contraindicated for certain tumours. In-vitro maturation has been suggested as an effective treatment for these patients. This report presents three women aged 21, 30 and 40 years, without male partners, seeking fertility preservation prior to chemotherapy. They were first seen during the luteal phase of their menstrual cycle and were to undergo gonadotoxic treatment imminently. They underwent immature oocyte retrieval in the luteal phase and seven, five and seven immature oocytes were recovered, respectively. After in-vitro maturation, five, three and five metaphase II (MII) oocytes were vitrified. Two patients later underwent one and two more retrievals, respectively, in the follicular phase of the next cycle(s) and additional oocytes were cryopreserved. These results suggest that immature oocytes recovered in the luteal phase can successfully be matured in vitro; therefore, if there is not sufficient time for conventional follicular-phase oocyte retrieval in a stimulated/unstimulated cycle prior to chemotherapy, a retrieval in the luteal phase could be considered.     

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.     

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.     

In-vitro maturation of human oocytes: before or after vitrification?

Purpose

This study aims to determine if in-vitro maturation (IVM) of human immature oocytes should be performed before or after vitrification.

Methods

A total of 184 immature oocytes were randomly divided into two different groups: 100 were vitrified at metaphase II (MII) stage 24 h-48 h after IVM (group 1) and 84 were immediately vitrified at germinal vesicle (GV) or metaphase I (MI) stages and in vitro matured after warming (group 2).

Results

Survival rate after warming was similar in both groups (86.9% versus 84.5%). However, oocyte maturation rate per collected oocyte was significantly higher for oocytes matured before vitrification (group 1, 46%) than for oocytes vitrified before IVM (group 2, 23.8%) (p < 0.01). Consequently, the number of MII oocytes inseminated per oocyte collected was significantly higher for group 1 (40%) than for group 2 (23.8%) (p < 0.05).

Conclusion

IVM procedure is more efficient when it is performed before oocyte vitrification.     

Nitrogen vapor shipment of vitrified oocytes: time for caution

Oocyte cryopreservation for fertility preservation, in both medical and elective situations, has significantly increased as freezing technology has improved. Slow freezing techniques demonstrated ～ 50-80% survival of mature oocytes, however vitrification with ～ 97% survival has become the preferred method for oocyte cryopreservation around the world. Our work investigated the effect of transporting cryopreserved oocytes to and from a long-term storage facility. Our findings demonstrate that extra caution should be practiced for vitrified oocytes, especially when handling and transferring between shipping and long-term cryopreservation storage containers.     

In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising “ex vivo” method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe

Purpose

We present our center’s experience with 34 consecutive cases who underwent in vitro maturation (IVM) of oocytes obtained from ovariectomy specimens and compare our data with updated literature data.

Methods

Feasibility and efficiency of oocyte collection during ovarian tissue processing was assessed by the recovery rate, maturation rate, and embryological development after IVM.

Results

On average, 14 immature oocytes were retrieved per patient during ovarian tissue processing in 33/34 patients. The overall maturation rate after IVM was 36 %. The maturation rate correlated with the age of the patient and the duration of IVM. Predominately, oocyte vitrification was performed. Eight couples preferred embryo cryopreservation. Here, a 65 % fertilization rate was obtained and at least one good-quality day 3 embryo was cryopreserved in 7/8 couples. The retrieval of oocytes ex vivo resulted in mature oocytes or embryos available for vitrification in 79 % of patients. One patient with ovarian insufficiency following therapeutic embolization of the left uterine and the right ovarian artery because of an arteriovenous malformation had an embryo transfer of one good-quality warmed embryo generated after IVM ex vivo, which resulted in an ongoing clinical pregnancy.

Conclusions

IVM of oocytes obtained ex vivo during the processing of ovarian cortex prior to cryopreservation is a procedure with emerging promise for patients at risk for fertility loss, as illustrated by the reported pregnancy. However, more data are needed in order to estimate the overall success rate and safety of this novel approach.     

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.     

State of the art in in-vitro oocyte maturation

PURPOSE OF REVIEW: The recovery of immature oocytes followed by in-vitro maturation (IVM) and in-vitro fertilization is an attractive alternative to conventional in-vitro fertilization treatment in which controlled ovarian stimulation with gonadotropins is used to increase the number of available oocytes and embryos. Significant progress has been made to improve pregnancy and implantation rates from in-vitro matured oocytes. This review summarizes current knowledge and achievements in human oocyte in-vitro maturation for clinical application, and will highlight recent advances reported in in-vitro maturation treatment. RECENT FINDINGS: It has been demonstrated that priming of ovarian immature oocytes with follicle-stimulating hormone or human chorionic gonadotropin prior to immature oocyte retrieval improves oocyte maturation rates and embryo quality as well as pregnancy rates in infertile women with polycystic ovaries or polycystic ovary syndrome. The size of follicles may be important for the subsequent embryonic development, but the developmental competence of oocytes derived from the small antral follicles is not adversely affected by the presence of a dominant follicle. However oocyte maturation in vitro is profoundly affected by culture conditions. Currently more than 300 healthy infants have been born following immature oocyte retrieval and in-vitro maturation. In general, the clinical pregnancy and implantation rates have reached 30-35% and 10-15% respectively in infertile women with polycystic ovaries or polycystic ovary syndrome. SUMMARY: In-vitro maturation treatment can now be offered as a successful option to infertile women with polycystic ovaries or polycystic ovary syndrome. It is possible to combine natural cycle in-vitro fertilization with immature oocyte retrieval followed by in-vitro maturation, and thus offer women with various causes of infertility reasonable pregnancy and implantation rates without recourse to ovarian stimulation. Further research remains to be done to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of oocytes matured in vitro.     

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.     

Retrieval of immature oocytes followed by in vitro maturation and vitrification: a case report on a new strategy of fertility preservation in women with borderline ovarian malignancy

BACKGROUND: We report a novel fertility preservation strategy in a woman with borderline ovarian tumors involving retrieval of immature oocytes, in vitro maturation (IVM) and subsequent cryopreservation. CASE: A 43-year-old woman underwent laparotomy for cystic ovarian masses on day 18 of her menstrual cycle. A diagnosis of bilateral borderline ovarian tumors was made following histological frozen section analysis. Left salpingo-oophorectomy, right ovarian cystectomy, omentectomy and lymph node sampling were performed. All visible follicles on the surface of the removed ovary were aspirated. Four immature oocytes were retrieved and underwent IVM. Three oocytes matured after 48 h and were cryopreserved. CONCLUSION: Immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, matured in vitro and cryopreserved, providing a possible strategy for fertility preservation in this group of women.     

超排卵周期未成熟卵体外培养的研究

目的:研究来源于超排卵周期中的未成熟卵在拆除卵丘细胞后进行体外成熟培养(IVM)的成熟、受精及胚胎发育能力,探讨IVM技术的临床应用。方法:选取46名体外受精/卵胞浆内单精子显微注射-胚胎移植(IVF/ICSI-ET)患者为研究对象,比较MI和GV期不成熟卵的体外成熟情况,并比较体内成熟卵和体外成熟卵进行ICSI后的正常受精、异常受精、卵裂和优质胚胎形成情况。结果:体外培养中69.8%的MI期卵和77.2%的GV期卵均在24小时内达到成熟,其24小时和48小时的成熟率、总成熟率均无明显差异(P>0.05)。体外成熟卵与体内成熟卵相比较,正常受精率、异常受精率和卵裂率均无明显差异(P>0.05),优质胚胎形成率较低,差异有显著性(P<0.05)。结论:常规超排卵周期中的未成熟卵在拆除卵丘细胞后能够继续体外发育成熟,具有与体内成熟卵相似的ICSI受精、卵裂能力。虽然优质胚胎的形成率低于体内成熟卵,但增加了可移植胚胎和冷冻胚胎数量,提高了助孕成功率。     

Fertility preservation in oncology

Survival rates of childhood and pre-pubertal female cancer patients are constantly increasing. However the lifesaving treatments carry a significant risk for infertility. Chemotherapy and radiotherapy might induce oocyte and follicular loss, infertility and premature ovarian failure. In order to preserve the fertility potential, several options are currently available, many of those should be considered as experimental. Ovarian transposition out of the radiation field may considerably reduce the radiation dose and should be considered for patients younger than 40 years of age. The benefits of GnRH analog are not clear yet and apoptosis inhibiting agents are not available. Embryo cryopreservation is a well established technique and should be offered to patients with spouses; when the patient does not have a male partner, oocyte cryopreservation or vitrification can be performed. When the cancer treatment cannot be delayed for ovarian stimulation or the tumor is hormone sensitive then collection of immature oocytes from unstimulated ovaries is particularly useful. The oocytes are matured in-vitro and either fertilized and cryopreserved as embryos or vitrified as mature oocytes. Ovarian tissue cryopreservation has the potential of preserving thousands of primordial follicles. The thawed ovarian tissue can be autotransplented orthotopically or heterotopically. Until now, only one human live birth has been reported and critical issues like the potential risk of transplanting malignant cells and the survival of the grafts have to be addressed. The strategy for preservation of fertility prior to cancer treatment should be tailored according to the patients age, presence of a partner, type of malignant disease, therapeutic agent, and time interval available. The patient should obviously be informed that some of the methods are still experimental.     

Human oocyte vitrification: in-vivo and in-vitro maturation outcomes

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.     

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.     

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.     

Cryoloop vitrification in assisted reproduction: analysis of survival rates in > 1000 human oocytes after ultra-rapid cooling with polymer augmented cryoprotectants

While human oocytes have been successfully cryopreserved using traditional slow-rate freezing protocols, inconsistent results post-thaw have limited the routine clinical application of oocyte cryopreservation. Despite interest in the potential benefits of vitrification as an alternative laboratory approach to long-term oocyte preservation in assisted reproduction, there is little agreement on how best to configure such cryopreservation protocols to optimize oocyte viability. To comparepost-thaw oocyte survival rates,we performed cryoloop vitrification of human oocytes utilizing two different cryoprotectant mixtures that included polymer macromolecules. Human oocytes (n = 1120) were obtained from consenting patients undergoing in vitro fertilization, but only failed-matured (uninseminated) or failed-fertilized (inseminated but without 2pn development) were included in this investigation. Protocol A consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.4 M sucrose and 20% synthetic serum substitute. Protocol B consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.65 M sucrose, 1 mg/ml polyethylene glycol, 10 mg/ml Ficoll and 20% synthetic serum substitute. Following cryostorage for 10-14 d at -196 degrees C, the survival rate for oocytes vitrified with protocol A was 80.9%, whereas the post-thaw viability among protocol B oocytes was 80.6% (p > 0.005). Our results indicate that an ethylene glycol + dimethyl sulphoxide mixture (with or without polymer macromolecules) can be an effective cryoprotectant strategy for human oocyte vitrification; either approach can be employed without any observed compromise in post-warming survival and/or morphology.     

Vitrification of embryos and oocytes for fertility preservation in cancer patients

As survival rates and the life expectancy of those with malignancy have increased, more women in their reproductive years are referred for fertility preservation. Chemotherapy and radiotherapy can severely affect ovarian function, and the effect is irreversible. Therefore, it is optimal to attempt fertility preservation before chemotherapy and radiotherapy are initiated. Oocyte and embryo cryopreservation is the most common option for fertility preservation in women. Several reports have proven that embryo and oocyte cryopreservation can achieve a successful pregnancy. This review discusses the impact of chemotherapy and radiotherapy on ovarian function, and the importance of oocyte and embryo cryopreservation for fertility preservation. In addition, the current status of pregnancy outcomes and potential for cryopreserved oocytes to result in live births in cancer patients was reviewed. This may provide useful information for decision‐making in cancer patients regarding oocyte and embryo cryopreservation and fertility preservation.     

人类未成熟卵母细胞玻璃化冷冻研究

目的:探讨玻璃化冷冻未成熟卵母细胞的有效性。方法:根据有无颗粒细胞将实施玻璃化冷冻的GV期卵母细胞分为含颗粒细胞(非裸卵)组和不含颗粒细胞(裸卵)组;将部分GV期卵母细胞体外培养至MⅡ期卵母细胞实施玻璃化冷冻,比较非冷冻IVM组与MⅡ卵玻璃化冷冻组间、裸卵组与非裸卵组间的存活率、成熟率、受精率、卵裂率及囊胚形成率。结果:非裸卵组的成熟率大于裸卵组(P<0.05),而存活率、受精率、2-细胞形成率、>2-细胞形成率之间均无统计学差异(P>0.05)。另外,非冷冻IVM组与GV玻化组间成熟率、受精率、卵裂率均存在显著性差异(P<0.05);非冷冻IVM组与MⅡ期卵玻化组间成熟率、受精率、卵裂率间均存在统计学差异(P<0.05);GV玻化组与MⅡ玻化组间存活率、成熟率、受精率、卵裂率间均无统计学差异(P>0.05)。结论:玻璃化冷冻未成熟卵母细胞需要保留颗粒细胞,同时初步构建了人GV期卵的玻璃化冷冻联合IVM技术的雏形。     

Closed vitrification of human oocytes and blastocysts: outcomes from a series of clinical cases

Purpose

High survival rates and clinical outcomes similar to those from fresh oocytes and blastocysts have been observed with open oocyte vitrification systems. It has been suggested that the extremely fast cooling rates that are only achieved with open systems are necessary for human oocyte and blastocyst vitrification. However, there is a potential risk of introducing contamination with open systems. The aim of this study was to assess whether similar survival and subsequent implantation rates could be achieved using a closed vitrification system for human oocytes and blastocysts.

Methods

Initially, donated immature oocytes that were matured in vitro were vitrified using the cryoprotectants ethylene glycol (EG) + dimethyl sulphoxide (DMSO) + sucrose and either a closed system (Rapid-i®) or an open system (Cryolock). The closed system was subsequently introduced clinically for mature oocyte cryopreservation cases and blastocyst vitrification.

Results

Using in vitro matured oocytes, a similar survival was achieved with the open system of 92.4 % (73/79) and with the closed system of 89.7 % (35/39). For clinical oocyte closed vitrification, high survival rate of 90.5 % (374/413) and an implantation rate of 32.7 % (18/55) from the transfer of day 2 embryos was achieved, which is similar to fresh day 2 embryo transfers. Blastocysts have also been successfully cryopreserved using the Rapid-i closed vitrification system with 94 % of blastocysts having an estimated ≥75 % of cells intact and a similar implantation rate (31.5 %) to fresh single blastocyst transfers.

Conclusion

Closed vitrification can achieve high survival and similar implantation rates to fresh for both oocytes and blastocysts.