The effects of radiotherapy and chemotherapy on female reproduction.

High dose chemotherapy and radiotherapy have radically increased long-term survival of young cancer patients, but major side effects of these treatments are ovarian failure and infertility. Knowledge of the risks and probabilities of ovarian failure caused by treatment is crucial for patients and physicians in order to make informed choices that will best serve patients' interests. This review presents data on ovarian damage and failure following exposure to radiotherapy, chemotherapy and ablative therapy. The risk is evaluated from the published literature according to patient's age, treatment protocol and also according to the diagnosis of some common malignancies. Many of these patients will not be sterilized immediately following treatment, but will suffer from premature menopause. In order to prevent sterilization, ovarian transposition before pelvic irradiation is mandatory. Besides cryopreservation of ovarian tissue and embryos before administration of chemotherapy, the possible protective effect of pituitary-ovarian down-regulation is discussed. The mechanism of primordial follicles damage induced by radio/chemotherapy is presented as well as the role of apoptosis signalling pathways underlying destruction. Increased knowledge of these mechanisms could help to identify potential effective inhibitors that can block the path of primordial follicles destruction and reduce ovarian failure rate.     

The use of LH activity to drive folliculogenesis: exploring uncharted territories in ovulation induction

LH plays critical roles in the control of folliculogenesis and ovarian function in humans. LH activity administration during gonadotrophin ovulation induction can enhance ovarian response and optimise treatment. More specifically, LH activity (both LH and low-dose hCG) can support the growth and stimulate the maturation of larger ovarian follicles as a result of specific granulosa cell receptors that develop after a few days of FSH priming. This action of LH is independent of FSH, and it has been shown recently that the last stages of follicular development can be supported by sole administration of LH activity in the form of low-dose hCG, without causing premature luteinization. Reproductively competent oocytes and pregnancy can be obtained with this regimen. Furthermore, LH activity is capable of reducing the development of small ovarian follicles (<10 mm) that may predispose patients to developing complications such as the ovarian hyperstimulation syndrome. Thus, better understanding of the dynamics and mechanisms that control human folliculogenesis and a more rational and selective use of LH activity administration may allow a reduction in cost and increased safety, while maintaining a high efficacy of the ovulation induction regimens used in assisted reproduction.     

Regulation of follicle development and novel approaches to ovarian stimulation for IVF

Current ovarian stimulation regimens for IVF are complex and not without risk. Increasing our knowledge of the physiology of follicle development and dominant follicle selection may enable the design of less complex, safer and cheaper ovarian stimulation regimens for IVF. Decremental serum FSH concentrations during the follicular phase of the menstrual cycle are required for single dominant follicle selection. Only the most mature follicle will continue its development due to increased sensitivity for stimulation by FSH. FSH stimulation becomes insufficient for less mature follicles and remaining cohort follicles will therefore go into atresia. The number of days during which FSH is above the threshold for stimulation of follicle development is limited, resulting in a narrow FSH window. More medium sized and large pre-ovulatory follicles and increased oestradiol output can be induced by the administration of small doses of exogenous FSH during the mid- to late follicular phase, preventing the physiological decrease in FSH stimulation. Intervention with decremental serum FSH concentrations in combination with gonadotrophin-releasing hormone (GnRH) antagonists to prevent a premature rise in serum LH may induce ongoing growth of multiple follicles sufficient for IVF. The benefits and risks of these minimal hyperstimulation protocols require further evaluation.     

Cryopreservation of oocytes from pre-antral follicles

Cryobiology is a very important tool in reproductive biology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Another goal is to establish a genetic resource bank for endangered or commercially important animal species. Cryopreservation of oocytes from pre-antral follicles has been studied during the past decade. Procedures can be divided between the cryopreservation of either ovarian tissue or isolated follicles. Most studies describe a slow freezing/rapid thawing protocol to cryopreserve ovarian fragments. Histology shows that the follicles maintain their morphological integrity, and transplantation of ovarian tissue demonstrates that the follicles can restart their growth and eventually ovulate. Some research groups have obtained offspring using this procedure in mice and sheep. With regard to the cryopreservation of isolated follicles, the few studies reported in this area used the same freezing protocol, and some of them described follicular growth using in-vitro culture. The best result was obtained in mice, with animal birth after follicular cryopreservation and culture. However, additional studies are necessary for a better understanding of the events during follicular cryopreservation and to establish a standard protocol for ovarian transplantation or follicle culture.     

Regulation of primordial follicle assembly and development

The assembly of the primordial follicles early in ovarian development and the subsequent development and transition of the primordial follicle to the primary follicle are critical processes in ovarian biology. These processes directly affect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause occur. The inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure (POF) and infertility. Primordial follicle assembly and development are coordinated by locally produced paracrine and autocrine growth factors. Endocrine factors such as progesterone have also been identified that influence follicular assembly. Locally produced factors that promote the primordial to primary follicle transition include growth factors such as kit ligand (KL), leukaemia inhibitory factor (LIF), bone morphogenic proteins (BMP's), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). Factors mediating both precursor theca-granulosa cell interactions and granulosa-oocyte interactions have been identified. A factor produced by preantral and antral follicles, Müllerian inhibitory substance, can act to inhibit the primordial to primary follicle transition. Observations suggest that a complex network of cell-cell interactions is required to control the primordial to primary follicle transition. Elucidation of the molecular and cellular control of primordial follicle assembly and the primordial to primary follicle transition provides therapeutic targets to regulate ovarian function and treat ovarian disease.     

Endocrine signaling in ovarian surface epithelium and cancer

Ovarian cancer is the sixth most common cancer and the fifth leading cause of cancer-related death among women in developed countries. Greater than 85% of human ovarian cancer arises within the ovarian surface epithelium (OSE), with the remainder derived from granulosa cells or, rarely, stroma or germ cells. The pathophysiology of ovarian cancer is the least understood among all major human malignancies because of a poor understanding of the aetiological factors and mechanisms of ovarian cancer progression. There is increasing evidence suggesting that several key reproductive hormones, such as GnRH, gonadotrophins and sex steroids, regulate the growth of normal OSE and ovarian cancer cells. The objective of this review was to highlight the effects of these endocrine factors on ovarian cancer cell growth and to summarize the signalling mechanisms involved in normal human OSE and its neoplastic counterparts.     

Current perspective on primordial follicle cryopreservation and culture for reproductive medicine

Mature oocytes are rare and precious cells. A technology which generates larger numbers would be very welcome in clinical practice, animal production technology and research. Since de-novo formation of female germ cells has ceased by the time of birth, the most attractive strategy, in theory, is to harvest and culture primordial follicles, the most abundant stage in the ovary at all ages. So far, there has been more success with cryopreservation of primordial follicles than with culture, and frozen-thawed ovarian tissue grafts have restored fertility to a number of species after oophorectomy. However, in-vitro development of isolated follicles is not sustained beyond the primary follicle stage. To meet their requirements for growth, metabolism and differentiation, a multistage protocol will probably be required for the prolonged period of development to maturity. The mouse is the only model, to date, in which a live offspring has ever been produced after growing follicles completely in vitro. A triple-stage process was required, involving culture of ovarian explants followed by isolation of granulosa-oocyte complexes and, finally, suitable conditions for completing meiotic maturation. Achievement of this goal for the larger and more slowly developing follicles from human and farm animal ovaries is still a remote possibility.     

Primate and bovine immature oocytes and follicles as sources of fertilizable oocytes

This review discusses the mechanisms underlying ovarian follicle development and the potential of immature follicles and oocytes from non-rodent mammalian species particularly human and bovine to serve as sources of oocytes for the in-vitro production of embryos. Factors that regulate growth and differentiation of unilaminar (primordial and primary) and multilaminar (secondary) follicles and the maturation of oocytes are highlighted. We conclude that many obstacles must still be overcome before fertilizable oocytes can be obtained from human and bovine ovaries, and more research on the quality of and culture conditions for immature oocytes and follicles is required before these can be considered as a source for in-vitro production.     

The menopause transition: an update

The menopausal transition is an area of active clinical investigation and epidemiological research. Recent results from several worldwide cohorts of women are beginning to provide a paradigm by which to explain the epidemiological and clinical manifestations of the ovarian process of follicle attrition. Genetic research has also provided some insight into differential ovarian ageing and disease/risk susceptibility in populations of women. Atresia of follicles occurs throughout reproductive life in women. The ever-shrinking follicle pool does not appear to result in any overt disruption of cycles for most women until they are in their forties. Subtle hormonal changes precede overt cycle disruption in mid-life. Obvious failures of ovulation or folliculogenesis lead to menstrual disturbances, and these provide the first clinical indication that the menopause transition is underway. A variety of hormonal patterns have been identified and an overall physiological progression appears to exist. The likelihood that reproductive hormone patterns influence symptomatology is high, although the analysis of the nature of such relationships is under way. For most women, effective lifestyle changes or hormonal therapy is available for symptoms associated with the menopause transition. This review will cover the existing body of knowledge, particularly regarding the clinical management of women who present with symptomatology during their menopausal transitions.     

How to make a good oocyte: an update on in-vitro models to study follicle regulation

The ability to develop the technology to mature oocytes from immature oocytes in vitro is the ambition of many IVF clinics. If this can be successfully achieved then these techniques would be available to women with fertility problems. This would aid women at risk of premature ovarian failure, and possibly result in women no longer requiring an expensive drug regime and monitoring programme, which they currently have to undergo. The idea of harvesting immature oocytes for growth in vitro is not a new one, but progress has been slow in developing and optimizing techniques for use on humans and domestic species. At present, there are many technical reasons for the lack of progress in these species, such as length of culture and difficulty of follicle isolation. However, the major problem is our lack of knowledge of how the oocyte acquires developmental competence during its growth within the follicle. To date, culture systems have been developed that can support the growth and development of immature oocytes. These systems are beneficial in improving our knowledge of how autocrine/paracrine factors are involved in regulating and controlling oocyte development. However, only when we have a more in-depth understanding of what is required during development to make a viable oocyte, will we perhaps be able to develop in-vitro culture systems for clinical application. This review will focus on how analysis of early follicular growth and development, using in-vitro culture systems, has advanced our knowledge of the factors and process involved in the regulation of oocyte and somatic cell development.     

Why does ovarian surgery in PCOS help? Insight into the endocrine implications of ovarian surgery for ovulation induction in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a complex disorder with heterogeneity of clinical and endocrine features. Ovarian surgery for ovulation induction has been used in the management of clomiphene citrate-resistant anovulatory women with PCOS. Various types of ovarian surgery have been employed (wedge resection, electrocautery, laser vaporization, multiple ovarian biopsies and others) and all procedures result in an altered endocrine profile after surgery. The mechanism behind the reversal of endocrinological dysfunction in PCOS after ovarian surgery remains incompletely understood. This review scans the literature systematically to identify the endocrine changes after ovarian surgery in PCOS, in order to glean some knowledge of the mechanism involved. After ovarian surgery in PCOS, a rapid reduction in serum levels of all ovarian hormones is seen, in combination with increased serum levels of pituitary hormones. Folliculogenesis is then initiated and ovarian hormone production increases, synchronically with a reduction of pituitary hormones. Continuation of follicle growth in subsequent cycles after ovarian surgery occurs in an environment with less androgens and lower LH and FSH levels compared with pretreatment levels. The endocrine changes found after ovarian surgery in PCOS women seem to be governed by the ovaries themselves. Rapid reduced secretion of all ovarian hormones restores feedback to the hypothalamus and pituitary, resulting in appropriate gonadotrophin secretion. Initiation of follicular development seems to be induced by increasing FSH levels following a reduction of the follicle excess and (intra-ovarian) androgen levels. Additionally, anti-Müllerian hormone and gonadotrophin surge attenuating factor probably have a role in the endocrine changes.     

Applications of ovarian tissue transplantation in experimental biology and medicine

Nowadays, high-dose chemotherapy and radiotherapy treatments for cancer are more effective but can severely affect the ovarian follicular store, compromising the fertility of surviving young patients. A promising alternative to prevent fertility loss in these patients is the cryopreservation and transplantation of ovarian tissue. Slices of animal and human ovarian tissue have been shown to survive the cryopreservation process. After transplantation, follicular development and restoration of hormone secretion have been observed in animal and human studies. This review addresses recent developments on ovarian tissue transplantation in animals and humans. We also illustrate the indications and technical difficulties of the procedure and the ethical issues that should be considered.     

Predictors of ovarian response: progress towards individualized treatment in ovulation induction and ovarian stimulation

Ovarian stimulation is applied in the clinic to restore mono-ovulatory cycles in anovulatory women (ovulation induction) or to induce the development of multiple dominant follicles for assisted reproduction. Ovarian response is the endocrine and follicular reaction of the ovaries to stimulation. Achieving an appropriate ovarian response to anti-estrogens or exogenous gonadotrophins is central to ovulation induction and ovarian stimulation protocols. However, achieving an adequate response, without cycle cancellation or adverse events related to under- or over-stimulation, is complicated by high intra- and inter-individual variability. To predict each patient's ovarian response to medication for ovarian stimulation and to individualize the starting dose of exogenous gonadotrophin or the need for exogenous luteinizing hormone, various clinical, endocrine, ovarian ultrasonographic and genetic characteristics have been explored. Some of these features have been incorporated into prediction models. In this review, the methodology behind predictive factors and prediction models and their potential clinical applicability across ovulation induction and ovarian stimulation are explored.     

Endometrial receptivity markers, the journey to successful embryo implantation

Human embryo implantation is a three-stage process (apposition, adhesion and invasion) involving synchronized crosstalk between a receptive endometrium and a functional blastocyst. This ovarian steroid-dependent phenomenon can only take place during the window of implantation, a self-limited period of endometrial receptivity spanning between days 20 and 24 of the menstrual cycle. Implantation involves a complex sequence of signalling events, consisting in the acquisition of adhesion ligands together with the loss of inhibitory components, which are crucial to the establishment of pregnancy. Histological evaluation, now considered to add little clinically significant information, should be replaced by functional assessment of endometrial receptivity. A large number of molecular mediators have been identified to date, including adhesion molecules, cytokines, growth factors, lipids and others. Thus, endometrial biopsy samples can be used to identify molecules associated with uterine receptivity to obtain a better insight into human implantation. In addition, development of functional in vitro systems to study embryo-uterine interactions will lead to better definition of the interactions existing between the molecules involved in this process. The purpose of this review was not only to describe the different players of the implantation process but also to try to portray the relationship between these factors and their timing in the process of uterine receptivity.     

Ovarian follicle development and transgenic mouse models

Ovarian follicle development is a complex process that beginswith the establishment of what is thought to be a finite poolof primordial follicles and culminates in either the atreticdegradation of the follicle or the release of a mature oocytefor fertilization. This review highlights the many advancesmade in understanding these events using transgenic mouse models.Specifically, this review describes the ovarian phenotypes ofmice with genetic mutations that affect ovarian differentiation,primordial follicle formation, follicular growth, atresia, ovulationand corpus luteum (CL) formation. In addition, this review describesthe phenotypes of mice with mutations in a variety of genes,which affect the hormones that regulate folliculogenesis. Becausestudies using transgenic animals have revealed a variety ofreproductive abnormalities that resemble many reproductive disordersin women, it is likely that studies using transgenic mouse modelswill impact our understanding of ovarian function and fertilityin women.     

Clinical management of low ovarian response to stimulation for IVF: a systematic review

Poor response is not a rare occurrence in ovarian stimulation. Although not fully accepted, the most dominant criteria for poor ovarian response are small numbers of follicles developed or oocytes retrieved, and low estradiol (E2) levels after the use of a standard stimulation protocol. There is no ideal predictive test as the poor responder is revealed only during ovulation induction; however, increased levels of day 3 FSH and E2 as well as decreased levels of inhibin B can be used to assess ovarian reserve. Several protocols have been proposed for clinical management of low ovarian response in IVF. Although high doses of gonadotrophins have been used by the vast majority of authors, results have been controversial and prospective randomized studies have shown little or no benefit. The few available relevant studies do not indicate that recombinant FSH improves outcome. Flare-up GnRH agonist protocols (including all dosage varieties) produce better results than standard long luteal protocols. Luteal initiation GnRH agonist 'stop' protocols were shown to improve ovarian response according to prospective studies with historical controls, but this was not confirmed by well-designed prospective, randomized, controlled studies. The few available data obtained with GnRH antagonists have not shown any benefits. Adjuvant therapy with growth hormone (GH) or GH-releasing factors results in no significant improvement. The use of corticosteroids reduces the incidence of poor ovarian response in women undergoing IVF treatment. The limited data obtained with nitric oxide donors are encouraging. Pretreatment with combined oral contraceptives prior to stimulation may help ovarian response. No benefit was observed with standard use of ICSI or assisted hatching of zona pellucida. Finally, natural cycle IVF has produced results which are comparable with those obtained with stimulated cycles in true poor responders. Well-designed, large-scale, randomized, controlled trials are needed to assess the efficacy of these different management strategies.     

Oocyte--granulosa cell interactions

In the past, different protocols of ovulation induction, aimed to overcome problems of anovulatory infertility in humans, have been developed during IVF programmes. However, administration of exogenous hormones may cause severe health problems, e.g. ovarian hyperstimulation syndrome. To overcome this problem an attractive alternative is to develop in-vitro systems that allow follicle and oocyte growth and maturation. This paper reviews the current status of research on oocyte-granulosa cell interactions and on the autocrine and paracrine factors involved in follicle development. The ovarian follicle is a morphological and functional unit in which the somatic and germ cell components are intimately associated and interdependent. The co-ordinate development of follicle and oocyte leads to a number of modifications in the growing oocyte necessary for the acquisition of competence to mature correctly and to undergo fertilization and embryo development. The search for the optimal culture conditions and the correct balance of hormones necessary to obtain a fertilizable oocyte in vitro is extremely important for clinical and agricultural applications.     

Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients.

Cancer treatment modalities are increasingly more effective in achieving complete remission and cure. Aggressive chemotherapy and radiotherapy, as well as bone marrow transplantation, results in >90% cure in many cancers of children and young women. As a result of this success however, a new problem has arisen. Many young women survive to live the rest of their lives in menopause, and have no chance of conceiving on their own. Oocyte cryopreservation has resulted in a handful of pregnancies, but the technique may not be applicable to young women and children. Ovarian tissue cryopreservation and transplantation has emerged against this background, first in successful rodent studies, and then in sheep and human ovarian xenograft studies. Because of the encouraging results with animals and xenografts, a human ovarian transplantation trial was launched. Pelvic auto-transplantation of frozen-banked ovarian tissue resulted in ovulation in one patient. Several other patients received fresh grafts subcutaneously, and preliminary results indicated antral follicle development at least in one patient. With the addition of promising data from humans, ovarian tissue cryopreservation from selected patients before cancer treatment, and in those requiring oophorectomy for benign causes, is now advocated.