Ovarian damage from chemotherapy and current approaches to its protection.

BACKGROUND: Anti-cancer therapy is often a cause of premature ovarian insufficiency and infertility since the ovarian follicle reserve is extremely sensitive to the effects of chemotherapy and radiotherapy. While oocyte, embryo and ovarian cortex cryopreservation can help some women with cancer-induced infertility achieve pregnancy, the development of effective methods to protect ovarian function during chemotherapy would be a significant advantage. OBJECTIVE AND RATIONALE: This paper critically discusses the different damaging effects of the most common chemotherapeutic compounds on the ovary, in particular, the ovarian follicles and the molecular pathways that lead to that damage. The mechanisms through which fertility-protective agents might prevent chemotherapy drug-induced follicle loss are then reviewed. SEARCH METHODS: Articles published in English were searched on PubMed up to March 2019 using the following terms: ovary, fertility preservation, chemotherapy, follicle death, adjuvant therapy, cyclophosphamide, cisplatin, doxorubicin. Inclusion and exclusion criteria were applied to the analysis of the protective agents. OUTCOMES: Recent studies reveal how chemotherapeutic drugs can affect the different cellular components of the ovary, causing rapid depletion of the ovarian follicular reserve. The three most commonly used drugs, cyclophosphamide, cisplatin and doxorubicin, cause premature ovarian insufficiency by inducing death and/or accelerated activation of primordial follicles and increased atresia of growing follicles. They also cause an increase in damage to blood vessels and the stromal compartment and increment inflammation. In the past 20 years, many compounds have been investigated as potential protective agents to counteract these adverse effects. The interactions of recently described fertility-protective agents with these damage pathways are discussed. WIDER IMPLICATIONS: Understanding the mechanisms underlying the action of chemotherapy compounds on the various components of the ovary is essential for the development of efficient and targeted pharmacological therapies that could protect and prolong female fertility. While there are increasing preclinical investigations of potential fertility preserving adjuvants, there remains a lack of approaches that are being developed and tested clinically.

How do chemotherapeutic agents damage the ovary?

BACKGROUND: Chemotherapy treatment in premenopausal women is associated with an increased risk of premature ovarian failure (POF) but the exact mechanism through which this occurs is uncertain. In this review we examine the current evidence for the direct action of chemotherapeutic agents on the ovary and discuss possible molecular pathways through which follicle loss may occur. METHODS: A systemic search of the databases, PubMed and Google Scholar, was made for all English language articles through to 2011 in each subject area discussed. RESULTS: POF results from the loss of primordial follicles but this is not necessarily a direct effect of the chemotherapeutic agents. Instead, the disappearance of primordial follicles could be due to an increased rate of growth initiation to replace damaged developing follicles. Likewise, the loss of oocytes need not necessarily be a direct result of damage: evidence suggests that chemotherapy drugs can also induce oocyte death indirectly via damage to somatic cells. Specific molecular mechanisms and likely ovarian targets are discussed for some of the anti-cancer drugs most commonly used to treat premenopausal women. Finally, we consider current and prospective methods of preserving fertility. CONCLUSIONS: It is likely that different chemotherapeutic drugs act through a range of mechanisms and on different target cells. More research into the cellular mechanisms underpinning chemotherapy-induced follicle loss could lead to the generation of treatments specifically designed to prevent POF.

Genomic imprinting and reproduction.

Genomic imprinting is the parent-of-origin specific gene expression which is a vital mechanism through both development and adult life. One of the key elements of the imprinting mechanism is DNA methylation, controlled by DNA methyltransferase enzymes. Germ cells undergo reprogramming to ensure that sex-specific genomic imprinting is initiated, thus allowing normal embryo development to progress after fertilisation. In some cases, errors in genomic imprinting are embryo lethal while in others they lead to developmental disorders and disease. Recent studies have suggested a link between the use of assisted reproductive techniques and an increase in normally rare imprinting disorders. A greater understanding of the mechanisms of genomic imprinting and the factors that influence them are important in assessing the safety of these techniques.

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture.

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.

Combined effect of follicle-follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro.

Follicle selection occurs throughout an adult female's reproductive life, with selected, dominant follicle(s) developing to the preovulatory stage whereas the remaining, subordinate follicles within the growing cohort instead undergo atresia and die. To date, most research into follicle dominance has concentrated on its endocrine regulation, although it seems likely that intraovarian mechanisms are also involved in its regulation. We demonstrate here that the response of singly cultured murine follicles to declining concentrations of FSH depends on their developmental stage, with follicles at an earlier stage of development being much more susceptible than mature follicles to a lowering of FSH levels. We then extrapolate this information to follicle cocultures, in which a large dominant follicle was grown with a small subordinate follicle in a manner that maintained a dominant/subordinate relationship, with follicle health assessed by a terminal transferase-mediated 2'-deoxyuracil 5'-triphosphate nick end-labeled reaction on whole-follicle mounts. Our investigations show a combined negative effect of coculture and FSH withdrawal on small subordinate follicles, such that subordinate follicles cocultured with dominant follicles and subjected to a lowering of FSH levels during the culture period exhibit a greatly increased incidence of apoptosis in the granulosa cells (750% increase) compared with that exhibited by the dominant follicles (97% increase). We suggest that a similar interaction between endocrine and intraovarian factors regulates follicular dominance in vivo, such that dominant follicles, in addition to bringing about a fall in FSH levels via the hypothalamic-pituitary axis, exert local, direct effects on subordinate follicles, with both of these influences combining to induce atresia in subordinate follicles.

Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro.

Ascorbic acid has three known functions: it is necessary for collagen synthesis, promotes steroidogenesis and acts as an antioxidant. Within the ovary, most studies have concentrated on the role of ascorbic acid in luteal formation and regression and little is known about the function of this vitamin in follicular growth and development. Follicular growth and development were investigated in this study using an individual follicle culture system that allows the growth of follicles from the late preantral stage to Graafian morphology. Follicles were isolated from prepubertal mice and cultured for 6 days. Control media contained serum and human recombinant FSH. Further groups of follicles were cultured in the same media but with the addition of ascorbic acid at concentrations of either 28 or 280 micromol l(-1). Addition of ascorbic acid at the higher concentration significantly increased the percentage of follicles that maintained basement membrane integrity throughout culture (P < 0.001). Ascorbic acid had no effect on the growth of the follicles or on oestradiol production. Metalloproteinase 2 activity tended to increase at the higher concentration of ascorbic acid and there was a significant concomitant increase in the activity of tissue inhibitor of metalloproteinase 1 (P < 0.01). Follicles cultured without the addition of serum but with FSH and selenium in the culture media underwent apoptosis. Addition of ascorbic acid to follicles cultured under serum-free conditions significantly reduced apoptosis (P < 0.05). From these data it is concluded that ascorbic acid is necessary for remodelling the basement membrane during follicular growth and that the ability of follicles to uptake ascorbic acid confers an advantage in terms of granulosa cell survival.

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles.

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.

Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-alpha knockout mice.

Estrogen receptor-alpha (ERalpha) knockout (ERalphaKO) female mice are infertile. Initially, they exhibit normal follicular development, but by 4-5 wk of age, they begin to develop hemorrhagic ovarian cysts. Follicles in adult ERalphaKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERalpha is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERalphaKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERalphaKO and WT mice. Gonadotropin-treated ERalphaKO females that ovulated produced 7.09 +/- 0.77 oocytes per mouse, whereas gonadotropin-treated WT female mice had 16.17 +/- 0.84 oocytes. Surprisingly, ruptured ERalphaKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERalphaKO mice had 3-fold higher concentrations of serum progesterone than did control ERalphaKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERalphaKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERalpha, although to a lesser extent than in WT mice.

Failure of founder transgenic male mice to transmit an attenuated HSV thymidine kinase transgene results from mosaicism and sperm competition.

Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.

Follicular development in vitro.

There has been tremendous interest in recent years in the culture of oocytes and follicles. Although much of the research using follicle culture aims to increase understanding of the regulation of follicle development, an important goal has been to develop a method that will eventually allow maturation of human oocytes from the primordial follicle to the mature Graafian stage. We are still some way from this at present, although it has now been achieved in the mouse. In this article, we consider various methods of follicle culture for primordial, preantral, and antral follicles. In vitro development of primordial follicles has used primarily whole ovaries or ovarian fragments as a source of follicles. Culture of later stages of follicle development uses mainly isolated follicular units, either whole (with an intact basement membrane and, in some cases, attached thecal cells) or nonintact (oocyte-somatic cell complexes, which may or may not have remnants of basement membranes and/or thecal cells attached).

The role of intra-ovarian interactions in the regulation of follicle dominance.

The processes that precisely control the selection of ovulatory follicles from a growing cohort are poorly understood. This reduction in follicle number occurs through several phases of selection, consequently we limit the use of the term 'selection' to the first major reduction of growing follicles, at the pre- to early antral stage. The final process of selection, achieving the appropriate ovulatory number, is referred to as 'dominance'. We discuss possible mechanisms that could bring about these reductions and highlight intra-ovarian involvement, particularly via follicle-follicle interactions. Analogies are drawn between local ovarian events and processes commonly reported in the determination of cell fate in developmental biology. Two facets of intra-follicular interactions are proposed: initially that follicle-follicle interactions mediate early selection processes at the preantral stage, and later that during antral development dominant follicles directly affect the fate of the subordinate cohort members.

Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro.

A whole follicle culture system has been used to investigate the actions of gonadotrophic hormones, oestrogen and progesterone in the regulation of follicular development and steroidogenesis. Recombinant human FSH was required for the growth of preantral follicles and for Graafian morphogenesis, whereas recombinant LH was ineffective. While pure FSH was sufficient for growth and morphogenesis, production of oestrogen was greater when androstenedione or LH was present in combination with FSH, confirming that there is a two-cell mechanism for oestradiol production in the mouse follicle. When an antiserum to oestrogen or to progesterone or an oestrogen receptor antagonist were added to the culture medium, there was no significant effect on either follicular growth or oestradiol production. Thus, physiological concentrations of oestradiol are not needed for follicle development, although a role cannot be completely ruled out. In conclusion, the obligatory role of FSH was demonstrated. It appears to be sufficient for follicle development even in the absence of LH, and the paracrine or autocrine effects of oestradiol and progesterone, if any, appear to be minor in the mouse ovary.

Effect of androgens on the development of mouse follicles growing in vitro.

The effects of androgens on ovarian follicular development have been investigated using a whole follicle culture system. Follicles obtained from mouse ovaries and cultured in the presence of anti-androgen serum grew more slowly than control follicles. This effect was reversed by the addition of androstenedione to the medium. A similar effect was obtained when receptor-mediated effects of androgens were blocked using an androgen receptor antagonist. When follicles were grown in concentrations of FSH that are marginal for follicle development, they developed faster in the presence of a non-aromatizable androgen, dihydroxytestosterone. The results indicate that androgens exert a direct, stimulatory role on the growth and development of mouse antral follicles, in vitro.

Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3',5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone.

Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studying the regulation of preantral follicle survival and growth. We have evaluated preantral follicle survival in vivo and in vitro, and have developed a serum-free rat follicle culture system that can be used to characterize the regulation of preantral follicle growth and differentiation. Analysis of ovarian cell DNA fragmentation during the first wave of follicle growth in the infantile rat indicated negligible apoptosis up to day 16 of age. However, a major increase in apoptosis was found by day 18, a time point associated with the appearance of large antral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral follicles mechanically dissected from ovaries of 12- or 14-day-old rats in serum-free conditions led to major increases in follicle cell apoptosis, similar to that seen in cultures of antral and preovulatory follicles. In contrast to antral and preovulatory follicles, treatment of preantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppressed apoptosis by 75% in cultured preantral follicles. In situ analysis identified granulosa cells as the cell type susceptible to apoptosis regulation. Taking advantage of the ability of the cGMP analog to suppress apoptosis, we evaluated the potential of FSH as a growth factor. In the absence of serum, FSH treatment for 48 h did not affect follicle size compared to controls; however, treatment with the cGMP analog together with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot analysis further indicated that the inhibin-alpha content of the cultured follicles was increased by treatment with the combination of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGMP pathway promotes the survival of cultured preantral follicles and that in the presence of alpha cGMP analog, FSH is a growth and differentiation factor for preantral follicles. The present serum-free follicle culture model system will be useful in further evaluation of the regulation of growth and differentiation of preantral follicles.

Programmed cell death in the reproductive system.

The reproductive system presents some of the best examples of programmed cell death, which is to be expected considering the dramatic cycles of tissue growth and regression in females. Hormones from the pituitary gland, gonads and uterus are responsible for coordinating cycles in which the preservation of cell survival and inhibition of apoptosis are important. In the ovary, atresia regulates the size of the follicle cohort for ovulation and is an archetype of apoptosis as induced by hormone withdrawal. The fate of an antral follicle--growth or atresia--is determined by circulating levels of gonadotrophins, and follicle-stimulating hormone (FSH) in particular. At the end of a menstrual cycle or pregnancy or lactation, hormone withdrawal triggers cell death and tissue remodelling and initiates a fresh cycle. In the endometrium, breast and prostate gland, steroid hormones are the principal survival factors and castration triggers regression of responsive tissues, which is sometimes decisive in the fight against disease. But while the primary trigger of cell death varies between tissues, underlying cellular mechanisms are more conservative and cell death/survival genes, such as bcl-2, bax and others that are expressed in other tissues, play important roles in the reproductive system too.

Clozapine treatment in polydipsia and intermittent hyponatremia.

BACKGROUND: Recent case reports indicate that clozapine treatment diminishes excessive diurnal weight gain and alleviates hyponatremia observed in some chronically psychotic patients. We examined the influence of clozapine on sodium metabolism and water regulation across a group of patients with the syndrome of polydipsia and intermittent hyponatremia. METHOD: Eleven patients with treatment-resistant DSM-III-R schizophrenia or schizoaffective disorder were studied. Each had a history of repeated diurnal weight gains of greater than 10% with at least one documented bout of hyponatremia in the 6 months before clozapine treatment. We utilized a target weight protocol and serial laboratory measures to compare changes in sodium metabolism and water regulation during 26 weeks of standard antipsychotic medication and 26 weeks of clozapine treatment. RESULTS: Across patients, we found significant improvement in routinely monitored 6 a.m. and 4 p.m. serum sodium, reflecting normalization of sodium metabolism. We also found that the frequency (as reflected by diurnal weight gain), severity (lowest serum sodium), and estimated quantity (calculated urine volume) of polydipsia improved across patients. Improvement in polydipsia and hyponatremia was associated with decreased necessity for monitoring and restrictive interventions, and tended to be associated with psychiatric improvement. CONCLUSION: We found a corrective and stabilizing effect of clozapine on polydipsia and intermittent hyponatremia. Future studies need to examine the relationship of psychiatric improvement and alterations in the regulation of sodium and water physiology to our findings.

The establishment of follicular dominance in co-cultured mouse ovarian follicles.

An in vitro model of dominant and subordinate ovarian follicles was developed to allow a closer investigation of the phenomenon of follicular dominance. Preantral mouse ovarian follicles were cultured either alone or in pairs. Pairs of follicles were either in direct contact or in shared medium, but without physical contact. The experiments showed that where contact was allowed to develop between follicles one follicle invariably became dominant, while the other would grow and develop little during the culture period. In contrast, there was no effect of co-culture on follicle development in the absence of contact between the follicles. There was, therefore, no evidence of secretion of a diffusible factor by a dominant follicle that could affect the development of neighbouring follicles. After 6 days of co-culture with contact, histological examination of the subordinate follicle showed that it was healthy, in spite of remarkably little growth during culture. In a further experiment, the subordinate follicle was separated from the dominant one after 2 days of co-culture (when a significant difference in size had already developed), and cultured alone. These 'released' follicles exhibited a spurt of growth during the remaining culture period, attaining a size and appearance indistinguishable from those of controls by the end of culture. This confirms that the dominant follicle, while depressing the growth of its neighbour, is not inducing irreversible atresia in the subordinate follicle in this model. The in vitro model will allow a more detailed study of direct influences of dominant-subordinate follicle interactions, and should increase our knowledge of a poorly understood phenomenon.