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.     

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.     

In-vitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation

This study describes the possibility of combining two options in order to preserve female fertility: cryopreservation of human ovarian tissue and in-vitro matured germinal vesicle (GV) oocytes retrieved during tissue dissection. In contrast to ovarian tissue cryopreservation, the cryostorage of in-vitro matured unfertilized metaphaseI/II oocytes could be a more realistic option. This concept of preserving fertility before chemotherapy and/or radiotherapy without a long time delay could be an additional reason for favouring ovarian tissue cryopreservation. This concept is discussed in regard to two cases.     

In-vitro maturation: its role in fertility treatment

PURPOSE OF REVIEW: In-vitro maturation of oocytes was primarily developed to make in-vitro fertilization safer and simpler for women with polycystic ovaries and high risk of ovarian hyperstimulation syndrome. In-vitro maturation has potentially many advantages over conventional in-vitro fertilization. A simple protocol with decreased or no hormonal stimulation before oocyte retrieval and thus lower cost of the treatment cycle are clear benefits. More importantly, the risk of ovarian hyperstimulation syndrome is entirely avoided. RECENT FINDINGS: The clinical outcome has substantially improved in recent years with pregnancy rates between 20 and 54%. The birth of approximately 400 children has been reported in the literature, but about three times as many are known to have been born. The postnatal follow-up studies of the children have been reassuring. SUMMARY: Although good results have been reported by some clinics, in-vitro maturation has not yet become a mainstream fertility treatment. The most important reason for this is the lower chance of a live birth per treatment compared with conventional in-vitro fertilization. Several aspects of the clinical in-vitro maturation need to be improved to give in-vitro maturation a place among assisted reproduction techniques it deserves.     

Cryopreservation and culture of human ovarian cortical tissue containing early follicles

Maturation of oocytes from primordial follicles after cryopreservation of ovarian tissue with subsequent in vitro fertilisation would be an important method of infertility treatment for young women who are to undergo premature ovarian failure due to chemotherapy or genetic causes. Cryopreservation of human ovarian tissue is already feasible. Human primordial follicles can be cultured regularly to secondary and occasionally to early antral stages, yet cultures have to be further optimised before meiotically competent oocytes can be achieved for final maturation in vitro. Maturation in vitro (IVM) of oocytes obtained by aspiration from small antral follicles is already a feasible technique resulting in pregnancies, with many healthy infants born.     

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 and ovarian tissue cryopreservation and its application

PURPOSE: To review the recent progress in human oocyte and ovarian tissue cryopreservation, and in the application of these two technologies for preserving female fertility of patients who are undergoing cancer treatment. DESIGN: The literature on human oocyte and ovarian tissue freezing was searched with PubMed. The scientific background, current developments and potential future applications of these two methods were reviewed. RESULTS: Chemotherapy and/or radiotherapy can induce premature ovarian failure in most of female cancer patients. Consequently, there has been a greater need for options to preserve the reproductive potential of these individuals. However, options are somewhat limited currently, particularly following aggressive chemotherapy and/or radiotherapy treatment protocols. In recent years, there have been considerable advances in the cryopreservation of human oocytes and ovarian tissue. For women facing upcoming cancer therapies, cryopreservation of ovarian tissue and oocytes is a technology that holds promise for banking reproductive potential for the future. Recent laboratory modifications have resulted in improved oocyte survival, oocyte fertilization, and pregnancy rates from frozen-thawed oocytes in IVF. This suggests potential for clinical application. CONCLUSIONS: In the case of patients who are facing infertility due to cancer therapy, oocyte cryopreservation may be one of the few options available. Ovarian tissue cryopreservation can only be recommended as an experimental protocol in carefully selected patients. In ovarian tissue transplantation, more research is needed in order to enhance the revascularization process with the goal of reducing the follicular loss that takes place after tissue grafting. These technologies are still investigational, although tremendous progress has been made. The availability of such treatment will potentially lead to its demand not only from patients with cancer but also from healthy women who chose to postpone childbearing until later in life and therefore wish to retain their fertility.     

State of the art on in vitro folliculogenesis in mouse

Follicle culture systems have been developed so as to achieve in vitro fertilization of oocytes coming from immature follicles. The in vitro folliculogenesis methods would be especially useful in reproductive medicine to restore fertility in women having undergone ovarian cryopreservation. Several culture systems allowing in vitro growth of small follicles have been developed in mouse. These have proven to be successful by the birth of healthy offsprings. Some elements determine the outcome of culture: follicle isolations at a defined stage of development, follicular morphology preservation, and supplementation of growth factors or hormones. Development of follicle culture in the mouse model led to a better understanding of ovarian physiology, in particular the relation between endocrine and paracrine factors on follicle development. The in vitro techniques in mouse became a valuable tool for improving reproductive technics improvement, and for toxicology studies.     

Recent advances in oocyte and ovarian tissue cryopreservation and transplantation

Options for preserving fertility in women include well-established methods such as fertility-sparing surgery, shielding to reduce radiation damage to reproductive organs, and emergency in-vitro fertilisation after controlled ovarian stimulation, with the aim of freezing embryos. The practice of transfering frozen or thawed embryos has been in place for over 25 years, and today is a routine clinical treatment in fertility clinics. Oocytes may also be frozen unfertilised for later thawing and fertilisation by intracytoplasmic sperm injection in?vitro. In recent years, oocyte cryopreservation methods have further developed, reaching promising standards. More than 1000 children are born worldwide after fertilisation of frozen and thawed oocytes. Nevertheless, this technique is still considered experimental. In this chapter, we focus on options for fertility preservation still in development that can be offered to women. These include freezing of oocytes and ovarian cortex and the transplantation of ovarian tissue.     

<Emphasis Type="Italic">In vitro</Emphasis> growth and maturation of mouse oocyte-granulosa cell complex from cryopreserved ovaries and achievement of pup birth

Ovarian tissue banking is a feasible strategy for fertility preservation for young women after cancer treatments. Ovarian tissue, after thawing, is used for several options; orthotopic grafting (normal site), autologous heterotopic grafting and collection of ovarian follicles for culture. Recent reports of live birth encouraged clinicians and researchers to apply this technology to premature ovarian failure (POF) resulting from strong cancer therapy. Grafting, however, carries a risk of malignant cell recurrence. For safety, development of a culture method is necessary but optimum culturing conditions for less-developed follicles abundant in the ovary are not well known. In the present article, the current status of ovarian tissue cryopreservation, and in vitro oocyte growth and maturation from the preserved ovaries are reviewed.     

Fertility Preservation in Women with Turner Syndrome: A Comprehensive Review and Practical Guidelines

In this article we review the existing fertility preservation options for women diagnosed with Turner syndrome and provide practical guidelines for the practitioner. Turner syndrome is the most common sex chromosome abnormality in women, occurring in approximately 1 in 2500 live births. Women with Turner syndrome are at extremely high risk for primary ovarian insufficiency and infertility. Although approximately 70%-80% have no spontaneous pubertal development and 90% experience primary amenorrhea, the remainder might possess a small residual of ovarian follicles at birth or early childhood. The present challenge is to identify these women as early in life as is possible, to allow them to benefit from a variety of existing fertility preservation options. To maximize the benefits of fertility preservation, all women with Turner syndrome should be evaluated by an expert as soon as possible in childhood because the vast majority will have their ovarian reserve depleted before adulthood. Cryopreservation of mature oocytes and embryos is a proven fertility preservation approach, and cryopreservation of ovarian tissue is a promising technique with a growing number of live births, but remains investigational. Oocyte cryopreservation has been performed in children with Turner syndrome as young as 13 years of age and ovarian tissue cryopreservation in affected prepubertal children. However, current efficacy of these approaches is unknown in this cohort. For those who have already lost their ovarian reserve, oocyte or embryo donation and adoption are strategies that allow fulfillment of the desire for parenting. For those with Turner syndrome-related cardiac contraindications to pregnancy, use of gestational surrogacy allows the possibility of biological parenting using their own oocytes. Alternatively, gestational surrogacy can serve to carry pregnancy resulting from the use of donor oocytes or embryos, if needed.     

Healthy twins delivered after oocyte cryopreservation and bilateral ovariectomy for ovarian cancer

Anti-neoplastic treatments have significantly increased the survival of cancer patients, but female patients risk premature menopause. Oocyte cryopreservation has been proposed as a fertility-saving option. This report describes the first live birth achieved with autologous cryopreserved oocytes in an ovariectomized borderline cancer patient. A patient with a borderline ovarian tumour asked for oocyte cryopreservation after a right adnexectomy. Ovulation induction resulted in the retrieval and cryopreservation of seven mature oocytes. Thirty-nine months after a left ovariectomy, the patient asked for oocyte thawing and embryo transfer. Endometrial growth was induced using hormone replacement treatment. Three of the seven cryopreserved oocytes were thawed; they survived and, after insemination, normal fertilization took place. Three embryos were transferred into the patient's uterus. A twin pregnancy was achieved with the birth of two healthy females. Oocyte cryopreservation may be a reliable option for preserving fertility in young cancer patients who risk premature menopause due to surgery, chemotherapy or radiotherapy.     

Fertility preservation: nonsurgical and surgical options

Improved surveillance and treatment regimens have resulted in decreased mortality rates among cancer patients, allowing these women to focus on survival and quality of life, including the ability to preserve their fertility. The treatments that have improved survival among both adults and children diagnosed with cancer are often gonadotoxic, especially those that employ high doses of alkylating agents and radiation therapy directed near or toward the pelvis. The impact on the ovarian reserve is related to the accelerated depletion of the primordial germ cell pool resulting from these therapies. Nonsurgical approaches to fertility preservation, including embryo cryopreservation from in vitro fertilization, oocyte cryopreservation from controlled ovarian hyperstimulation, and in vitro maturation of oocytes, are discussed. Surgical approaches such as conservative gynecologic surgery, ovarian transposition, and ovarian tissue cryopreservation are reviewed. Guidelines from the American Society for Reproductive Medicine and the American Society of Clinical Oncology classify these treatments into established and experimental procedures, and they provide the practitioner with an optimal approach to preserve the fertility of these patients before the initiation of their cancer therapies.     

Preliminary experience of ovarian tissue cryopreservation procedure: alternatives, perspectives and feasibility

Chemotherapy and radiotherapy induce premature ovarian failure in many patients treated for oncological or benign diseases. The present paper reviews the risk of developing premature ovarian failure according to the type of treatment and the different options to preserve fertility, focusing on the cryopreservation of ovarian tissue. This technique constitutes a promising approach to preserve the fertility of young patients and offers the advantage of storing a large number of follicles that could be subsequently transplanted or cultured in vitro to obtain mature oocytes. Based on 34 requests, from which 19 were performed, the feasibility of the ovarian cryopreservation procedure is evaluated. The medical and ethical approaches of this protocol are also discussed. Cryopreservation of ovarian tissue constitutes new hope for many patients, but must still be kept for selected cases, with a significant risk of premature ovarian failure after treatments such as bone marrow transplantation.     

Gynaecologic concerns for young women exposed to gonadotoxic chemotherapy

PURPOSE OF REVIEW: Due to the improved long-term survival of adolescents and young women with systemic malignancies such as lymphomas and leukaemia undergoing gonadotoxic chemotherapy, preservation of future fertility has been the focus of recent ubiquitous interest. This review summarizes, in brief, the recent progress in the various attempts to prevent premature ovarian failure in these young women with unconsumed fertility potential. RECENT FINDINGS: The investigational endeavours of ovarian cryopreservation await the clinical experience of auto- or xenotransplantation, or in-vitro maturation of thawed primordial follicles, and in-vitro fertilization. Although promising, this procedure is not available yet. Moreover, the risk of possible reimplantation of malignant stem cells with the thawed cryopreserved ovary has been highlighted following experimental animal observations. The gonadotropin-releasing hormone agonist has been efficient in primates in a prospective study, and in young women in several nonrandomized series. The disruption of the acid sphingomyelinase gene, or sphingosine-1-phosphate, in rodents can prevent follicle destruction by ionizing radiation, possibly indicating in-vivo protection in cancer patients at risk of iatrogenic sterilization. SUMMARY: The recent enormous scientific advance lends hope that the future may hold answers to the questions regarding safety and efficiency of oocyte, follicle, or ovarian tissue cryopreservation, and the most efficient means of using the thawed tissue - auto-, hetero-, or xenotransplantation versus IVM on the one hand, and in-vivo pharmacological attempts to minimize follicle depletion by gonadotropin-releasing hormone agonist or other modalities on the other hand. Until then, a combination of all the clinically available modalities should be offered to these young women with unconsumed fertility potential who face gonadotoxic therapy.     

Preservation of fertility in young cancer patients: contribution of transmission electron microscopy

During the last decade, new technologies in reproductive medicine have emerged to preserve the fertility of women whose gonadal function is threatened by premature menopause or gonadotoxic treatments. To offer an individualized approach to these patients, different experimental procedures are under investigation, including oocyte cryopreservation and cryopreservation and transplantation of ovarian tissue in the form of cortical fragments, whole ovary or isolated follicles. This review shows that transmission electron microscopy (TEM), combined with other in-vivo and in-vitro analysis techniques, is a valuable tool in the establishment of new experimental protocols to preserve female fertility. Ultrastructural studies allow in-depth evaluation of the oocyte's unique morpho-functional characteristics, which explain its low cryotolerance, and provide essential information on follicular, stromal and endothelial cell integrity, as well as cellular interactions crucial for normal folliculogenesis. In order to be able to offer appropriate and efficient options in every clinical situation, oocyte in-vitro maturation and ovarian tissue transplantation need to be optimized. Further development of new approaches, such as follicular isolation and whole ovary transplantation, should be encouraged. Fine ultrastructural details highlighted by TEM studies will be useful for the further optimization of these emerging technologies.     

Blastocyst Development After Cryopreservation and Subcutaneous Transplantation of Mouse Ovarian Tissue

Purpose To investigate follicle survival and developmental potential with IVF of cryopreserved, subcutaneously transplanted mouse ovarian tissue.Methods Fresh and frozen mouse ovarian tissue was autologously transplanted into subcutaneous tissue. Two weeks after the transplantation, the morphology and histology of the fresh and frozen grafts were compared. Superovulation and IVF was performed to evaluate the fertility potential of the frozen ovarian graft.Results Both fresh and frozen grafts of ovarian tissue survived in 14 of 16 mice (88%). Morphologically, both types of grafts resembled fresh ovarian tissue and contained follicles at all stages of folliculogenesis. A total of 73% of follicles in fresh grafts and 62% in frozen grafts survived after transplantation compared with fresh ovarian tissue. Sixteen ICR mice underwent superovulation. A total of 56 oocytes from antral follicles were recovered from the subcutaneously transplanted cryopreserved ovarian tissue. Fourteen (25%) oocytes were in metaphase II stage, 6 were fertilized by IVF, and 2 progressed to the blastocyst stage.Conclusions Cryopreservation and subcutaneous transplantation of ovarian tissue provides a possible means of fertility preservation. The main loss of follicles occurred during grafting rather than during freezing and thawing.     

In vitro maturation of oocytes

In vitro maturation of oocytes is a safe and effective treatment offered in some fertility centers for assisted reproduction, where immature oocytes are retrieved from unstimulated ovaries. Therefore, the procedure avoids ovarian stimulation with expensive gonadotropins, side effects of the medications, and risks such as ovarian hyperstimulation syndrome. Added advantages are reduced frequency of monitoring scans and shorter treatment regimen compared with in vitro fertilization. The candidates initially considered were women with polycystic ovaries having multiple antral follicles, but the indications are widening to include women with primarily poor quality embryos in repeated cycles and poor responders to stimulation. The two new applications for in vitro maturation we are now successfully implementing at McGill Reproductive Center are for oocyte donors and for fertility preservation, especially in women with cancer who are undergoing gonadotoxic therapy. In young women without partners needing this treatment for fertility preservation, it is combined with vitrification of the oocytes. We have achieved a 38% clinical pregnancy rate per cycle in women having IVM for infertility treatment up to the age of 35 years, and 50% clinical pregnancy rate per cycle in recipients of IVM egg donation.     

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.     

冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取

目的:探求冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取。方法:取手术切除的5例患者卵巢皮质,放入1.5 mol/L的二甲基亚砜+10％血清中,应用可控程序冷冻仪逐渐降温,移入液氮中冻存。三个月后,将融解的卵巢皮质移植入89只裸鼠皮下,注射hFSH 12周,刺激卵泡生长。注射hCG 36 h后,用18号针头穿刺提取卵细胞。结果:卵泡直径为3～9 mm,穿刺获19个卵细胞,放入TC-199培养基,加20％胎牛血清,25 mmol/L丙酮酸,75 mIU/mL FSH和LH培养,获15个分裂中期的次级卵母细胞。结论:人卵巢组织冻融后可异体移植生长,且其卵细胞可在体外发育成熟。