Norah Spears

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.

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice.

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.

On Regenerating the Ovary and Generating Controversy

For more than a half a century, biologists have upheld the theory that in most mammalian species, oocytes are formed before or shortly after birth, but never in adulthood. This foundation of reproductive science has survived the rapid growth of new technology and knowledge and has remained virtually unchallenged until two recent papers were published by the group headed by Jonathan Tilly. The first paper claims that mouse germline stem cells (GSCs) replace ovarian follicles that have been rapidly lost through follicle death (Johnson et al., 2004).

Müllerian Inhibitory Substance Induces Growth of Rat Preantral Ovarian Follicles

Abstract 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-α 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)-β 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-β was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-α 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.

The role of neurotrophin receptors in female germ-cell survival in mouse and human

During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB–/– and TrkC–/– mice with a mixed (129Sv × C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB–/– mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC–/– mice. Congenic TrkB–/– mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells.

Cancer treatment and gonadal function: experimental and established strategies for fertility preservation in children and young adults

Preservation of gonadal function is an important priority for the long-term health of cancer survivors of both sexes and all ages at treatment. Loss of opportunity for fertility is a prime concern in both male and female cancer survivors, but endocrine effects of gonadal damage are likewise central to long-term health and wellbeing. Some fertility preservation techniques, such as semen and embryo cryopreservation, are established and successful in adults, and development of oocyte vitrification has greatly improved the potential to cryopreserve unfertilised oocytes. Despite being recommended for all pubertal male patients, sperm banking is not universally practised in paediatric oncology centres, and very few adolescent-friendly facilities exist. All approaches to fertility preservation have specific challenges in children and teenagers, including ethical, practical, and scientific issues. For young women, cryopreservation of ovarian cortical tissue with later replacement has resulted in at least 40 livebirths, but is still regarded as experimental in most countries. For prepubertal boys, testicular biopsy cryopreservation is offered in some centres, but how that tissue might be used in the future is unclear, and so far no evidence suggests that fertility can be restored. For both sexes, these approaches involve an invasive procedure and have an uncertain risk of tissue contamination in haematological and other malignancies. Decision making for all these approaches needs assessment of the individuals risk of fertility loss, and is made at a time of emotional distress. Development of this specialty needs better provision of information for patients and their medical teams, and improvements in service provision, to match technical and scientific advances.

Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin

Purpose Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined. Experimental design Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect. Results Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment. Conclusion Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to.

Gonadotropin Induction of Ovulation and Corpus Luteum Formation in Young Estrogen Receptor-α Knockout Mice

Abstract Estrogen receptor-α (ERα) knockout (ERαKO) 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 ERαKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERα is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERαKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERαKO and WT mice. Gonadotropin-treated ERαKO 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 ERαKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERαKO mice had 3-fold higher concentrations of serum progesterone than did control ERαKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERαKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERα, although to a lesser extent than in WT mice.

Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids

Although there have been extensive studies on the effects of gonadotrophins and steroids on follicular development, less is known as to the effects these hormones have on the acquisition of oocyte developmental competence. This study investigates the effect of altering the gonadotrophin or steroidal environment on follicular development and on oocyte viability and DNA methylation. Oocytes were obtained from pre-ovulatory follicles after individual follicle culture from the pre-antral stage; gonadotrophin or steroid levels were manipulated during the culture period. Oocytes obtained from follicles grown in gonadotrophin free conditions were able to fertilize and develop to the blastocyst stage despite their impaired follicle development. There was no effect of luteinizing hormone or steroids on follicular growth. Altering the steroidal environment did, however, affect oocyte development. The oocytes of follicles exposed to high estrogen levels had lower fertilization rates, regardless of the presence or absence of high androgen levels. The combined presence of high levels of both steroids altered the level of global methylation. This study demonstrates that gonadotrophins and steroids influence the acquisition of developmental competence of the oocyte and suggests that optimal steroid exposure during follicle development is required for the oocyte to mature correctly.

Docetaxel induces moderate ovarian toxicity in mice, primarily affecting granulosa cells of early growing follicles

Abstract Advances in cancer therapy have focused attention on the quality of life of cancer survivors. Since infertility is a major concern following chemotherapy, it is important to characterize the drug-specific damage to the reproductive system to help find appropriate protective strategies. This study investigates the damage on neonatal mouse ovary maintained in vitro for 6 days, and exposed for 24 h (on Day 2) to clinically relevant doses of Docetaxel (DOC; low: 0.1 µM, mid: 1 µM, high: 10 µM). Furthermore, the study explores the putative protective action exerted by Tri-iodothyronine (T3; 10−7 M). At the end of culture, morphological analyses and follicle counts showed that DOC negatively impacts on early growing follicles, decreasing primary follicle number and severely affecting health at the transitional and primary stages. Poor follicle health was mainly due to effects on granulosa cells, indicating that the effects of DOC on oocytes were likely to be secondary to granulosa cell damage. DOC damages growing follicles specifically, with no direct effect on the primordial follicle reserve. Immunostaining and western blotting showed that DOC induces activation of intrinsic, type II apoptosis in ovarian somatic cells; increasing the levels of cleaved caspase 3, cleaved caspase 8, Bax and cleaved poly(ADP-ribose) polymerase, while also inducing movement of cytochrome C from mitochondria into the cytosol. T3 did not prevent the damage induced by the low dose of DOC. These results demonstrated that DOC induces a gonadotoxic effect on the mouse ovary through induction of somatic cell apoptosis, with no evidence of direct effects on the oocyte, and that the damaging effect is not mitigated by T3.