Dror Meirow

Toxicity of Chemotherapy and Radiation on Female Reproduction

One of the most devastating consequences of cancer treatment in the young female population is ovarian damage, resulting in diminished fertility potential. The extent of damage is related to age, chemotherapeutic regimen, and dose of pelvic radiation received. It is crucial that physicians know the impact each of these factors has on future fertility to advice patients on fertility preservation options. Anticancer drugs injure the female reproductive system through ovarian follicular and stromal damage. Although the exact mechanisms of damage remain unclear, it is essential to better understand these mechanisms to develop methods to diminish ovarian injury.

Cyclophosphamide Triggers Follicle Activation and “Burnout”; AS101 Prevents Follicle Loss and Preserves Fertility

Cyclophosphamide activates dormant follicle growth, leading to depletion of ovarian reserve, and AS101 prevents this activation, rescuing fertility in mice. Of Mice and Women: Protecting Cancer Patients from Treatment-Induced Infertility Cancer, especially in advanced stages, is a multisystem disease that can affect a patient in a myriad ways. Unfortunately, cancer treatments such as chemotherapy can also wreak havoc on the body, and some of their side effects can be felt for the rest of the patient’s lifetime. One notable consequence of chemotherapy is infertility, which is particularly problematic in young cancer patients who receive their treatments before they’ve had an opportunity to have children. Alkylating agents such as cyclophosphamide (Cy) carry a high risk of ovarian toxicity and are among the worst offenders with regard to risk of future infertility. Now, Kalich-Philosoph et al. present findings that show how Cy exerts its toxic effects on ovarian cells, as well as a potential method of protecting the ovaries and preserving fertility. In a mouse model of Cy treatment, the authors demonstrated that this chemotherapy drug attacks the ovaries by a twofold mechanism. It is toxic to dividing cells and kills actively growing ovarian follicles. At the same time, it also activates the quiescent follicles, inducing them to grow and proliferate, which makes them susceptible to the effects of the drug as well. In this way, Cy treatment depletes the ovarian reserve, leading to early ovarian failure and infertility. The authors also showed that an experimental drug called AS101 may provide protection against this adverse effect of cancer treatment. Mice treated with AS101 in conjunction with Cy fared much better than their counterparts receiving chemotherapy alone. Their primordial ovarian follicles remained dormant, did not start proliferating prematurely, and survived through the entire treatment. Subsequently, the mice that received AS101 along with Cy had normal fertility, whereas the ones treated with Cy alone had a lower rate of pregnancy and fewer total offspring. Future experiments will be needed to translate this work from mice into human patients and confirm the effectiveness of AS101 for preserving fertility in the clinical setting. Promisingly, AS101 is already in phase 2 clinical trials and appears to be safe for human use. Moreover, AS101 itself appears to have anticancer effects and may be able to reinforce for the therapeutic action of Cy while counteracting its reproductive toxicity. Premature ovarian failure and infertility are major side effects of chemotherapy treatments in young cancer patients. A more thorough understanding of the mechanism behind chemotherapy-induced follicle loss is necessary to develop new methods to preserve fertility in these patients. We show that the alkylating agent cyclophosphamide (Cy) activates the growth of the quiescent primordial follicle population in mice, resulting in loss of ovarian reserve. Despite the initial massive apoptosis observed in growing, though not in resting, follicles of Cy-treated mice, differential follicle counts demonstrated both a decrease in primordial follicles and an increase in early growing follicles. Immunohistochemistry showed that granulosa cells were undergoing proliferation. Analysis of the phosphatidylinositol 3-kinase signaling pathway demonstrated that Cy increased phosphorylation of proteins that stimulate follicle activation in the oocytes and granulosa cells. Coadministration of an immunomodulator, AS101, reduced follicle activation, thereby increasing follicle reserve and rescuing fertility after Cy, and also increased the efficacy of Cy against breast cancer cell lines. These findings suggest that the mechanism in Cy-induced loss of ovarian reserve is accelerated primordial follicle activation, which results in a “burnout” effect and follicle depletion. By preventing this activation, AS101 shows potential as an ovarian-protective agent, which may be able to preserve fertility in female cancer patients.

Occasional involvement of the ovary in Ewing sarcoma

BACKGROUNDnEwing sarcoma (EWS) is a highly metastatic malignancy in young patients. Ovarian cryopreservation is often an option for fertility preservation in cancer patients of reproductive age, specifically in minors. Thus, the possibility of ovarian involvement in EWS needs to be elucidated.nnnMETHODSnEight patients aged 13-20 years with EWS participated in the study. Ovarian samples were fixed and prepared for light microscopy, and frozen in liquid nitrogen for RNA extraction followed by RT-PCR. Histological studies, including immunostaining for the adhesion receptor CD99, were used to detect histopathological features. Sensitive molecular methods were used to detect translocations causing the formation of tumor-specific EWS-Friend leukemia virus integration site 1 fusion gene (EWS-FLI1).nnnRESULTSnIn seven patients, there was no evidence of EWS in the ovaries from pathological/molecular studies. However, in one patient, the RT-PCR showed the EWS translocation, although there was no pathological evidence.nnnCONCLUSIONSnOvarian involvement is possible in EWS. Therefore, in patients with EWS ovarian tissue should be examined for traces of malignancy at both the pathological and molecular levels prior to the grafting of cryopreserved tissue in order to minimize the risk of reseeding the cancer.

Prolonged conservative treatment of endometrial cancer patients: more than 1 pregnancy can be achieved.

Background: Preserving reproductive function in young patients with early endometrial cancer is an accepted concept today. The safety and feasibility of long-term conservative treatment, allowing more than 1 pregnancy, remain to be ascertained. Methods: This study was a retrospective chart review of a 27 women with endometrioid adenocarcinoma of the endometrium, who were treated conservatively at 2 tertiary-care institutions. Treatment comprised oral high-dose progestins with or without a levonorgestrel-releasing intrauterine device. Endometrial biopsy was repeated every 2 to 3 months. Results: Over 7.8 to 412 months (median, 57.4 months), tumors regressed completely in 24 (89%) of 27 patients and partially in 2 patients, with 79% responding within 1 to 17 months. Of the complete responders, 15 (62%) of 24 had a recurrence; 4 underwent hysterectomy, and 11 underwent subsequent progestational treatment. All 11 responded, and 3 subsequently conceived. After 2 to 4 years, 5 patients again had a recurrence, of whom 3 underwent hysterectomy. Overall, 2 patients developed ovarian adenocarcinoma. All patients are currently disease-free. Conception occurred in 14 (51.8%) of 27 patients, in 5 more than once. There were 17 live births, and 2 patients are pregnant. Conclusions: According to our data, prolonged progestational therapy for early-stage endometrial adenocarcinoma, allowing women to conceive, is feasible and apparently does not alter clinical outcome. Patients should be advised of the high recurrence rate and possible concomitant ovarian malignancy.

Prevention of chemotherapy-induced ovarian damage: possible roles for hormonal and non-hormonal attenuating agents

BACKGROUNDnCurrent options for female fertility preservation in the face of cytotoxic treatments include embryo, oocyte and ovarian tissue cryopreservation. However these methods are limited by the patient age, status or available timeframe before treatment and they necessitate invasive procedures. Agents which can prevent or attenuate the ovotoxic effects of treatment would provide significant advantages over the existing fertility preservation techniques, and would allow patients to retain their natural fertility without the necessity for costly, invasive and risky procedures. Recent studies have contributed to our understanding of the mechanisms involved in cytotoxicity-induced ovarian follicle loss and highlight a number of agents that may be able to prevent or reduce this loss.nnnMETHODSnThis paper reviews the relevant literature (research articles published in English up to December 2013) on the mechanisms of cytotoxic-induced ovarian damage and the implications for fertility preservation. We present a comprehensive discussion of the potential agents that have been shown to preserve the ovarian follicle reserve in the face of cytotoxic treatments, including an analysis of their respective advantages and risks, and mechanisms of action.nnnRESULTSnMultiple molecular pathways are involved in the cellular response to cytotoxic treatments, and specific cellular reactions depend on variables including the drug class and dose, cell type, and cell stage. A number of agents acting on different elements of these pathways have demonstrated potential for preventing or reducing ovarian follicle loss, although in most cases, the studies are still very preliminary.nnnCONCLUSIONSnAdvances in our understanding of the mechanisms and pathways involved in both cytotoxic ovarian damage and follicle growth and development have opened up new directions for fertility preservation. In order to bring these agents from the lab to the clinic, it will be vital to accurately evaluate the efficacy of each agent and additionally to demonstrate that co-treatment with these agents will not interfere with the anti-cancer activity of the chemotherapy drugs, or produce genetically comprised embryos.

Ovarian Tissue Cryopreservation and Transplantation: A Realistic, Effective Technology for Fertility Preservation

It is clear that ovarian tissue cryopreservation can serve a very important role in providing fertility preservation. To date, more than 30 live human births have resulted from the transplantation of cryopreserved ovarian tissue, proving the successful implementation of this technique. The ideal conditions for ovarian tissue cryopreservation have yet to be determined, indicating the crucial need for more research in this field. Nonetheless, it is recommended that ovarian tissue cryopreservation be offered as an option to women before undergoing chemotherapy treatment in instances where there is no time to delay for hormonal stimulation and oocyte retrieval.

FOXL2 C402G mutation detection using MALDI-TOF-MS in DNA extracted from Israeli granulosa cell tumors

OBJECTIVEnTo develop a rapid, sensitive and reliable method to detect FOXL2 C402G mutation in granulosa cell tumor (GCT) and to investigate the prevalence of FOXL2 mutation in granulose cell tumors among Israeli patients.nnnMETHODSnWe designed and optimized a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) genotyping assay to detect FOXL2 C402G mutation in DNA isolated from formalin-fixed paraffin-embedded tissue samples. We examined 20 tumor samples obtained from Israeli patients diagnosed with granulose cell tumor.nnnRESULTSnEighteen out of 20 samples were found to harbor FOXL2 C402G mutation. Pathological review of the two tumors harboring wild type FOXL2 (C402) concluded that they were adenocarcinomas and has been misclassified at initial diagnosis. We found that the prevalence of FOXL2 mutations among Israeli patients with GCT (100%) is similar to previous reports.nnnCONCLUSIONSnOur results indicate that the FOXL2 mutations can be reliably detected by MALDI-TOF-MS genotyping. MALDI-TOF-MS genotyping is a simple, robust and highly sensitive method to detect FOXL2 C402G mutation. Our results confirm previous studies reporting over 95% prevalence of FOXL2 mutation in GCT. Furthermore, we suggest that testing for the presence of the FOXL2 C402G mutation may improve diagnostic accuracy.

Follicle activation and ‘burn-out’ contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness

STUDY QUESTIONnWhat are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation?nnnSUMMARY ANSWERnTransplantation of reduced-thickness ovarian grafts led to intense activation and burn-out a short time after transplantation resulting in significant folllicle loss.nnnWHAT IS KNOWN ALREADYnTransplantation of fresh and frozen-thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing-thawing process and to post-transplantation hypoxia.nnnSTUDY DESIGN, SIZE, DURATIONnBovine ovarian tissue strips (n = 55) were prepared in two groups of conventional-thickness strips (1-2 mm) or thin strips (0.5-0.9 mm). Fresh or frozen-thawed samples were xenotransplanted into sterilized immune-deficient mice (n = 49). Non-transplanted conventional size fresh samples were used as controls (n = 6). Grafts from all study groups were recovered after 7 days for analysis.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnMorphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm(2). P-values <0.05 were considered statistically significant.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P < 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-GFs in the fresh, and more so in the frozen-thawed, samples (P < 0.05). Extensive stromal fibrosis and high blood-vessel density were observed in all grafts with no advantage in the thin prepared grafts.nnnLIMITATIONS, REASONS FOR CAUTIONnThis study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied.nnnWIDER IMPLICATIONS OF THE FINDINGSnFollicle activation and burn-out appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent burn-out will potentially improve follicle pool survival.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No. 12-3081). The authors have no competing interest to declare.

IVF for fertility preservation in breast cancer patients--efficacy and safety issues.

BackgroundPotential risks on future fertility have become a dominant issue in consultation and management of newly diagnosed young cancer patients. Several fertility preservation strategies are currently available. Of those, ovarian stimulation followed by IVF and embryo cryopreservation is the most established one and is especially applicable in reproductive aged breast cancer patients.AimThe aim of this study is to provide a comprehensive review on ovarian stimulation and IVF for fertility preservation in newly diagnosed breast cancer patients.MethodsReview of relevant literature is available through PubMed and Google scholar.ResultsThe use of IVF for fertility preservation in breast cancer patients raises dilemmas regarding efficacy and safety of controlled ovarian stimulation. Among these are the suggested role of malignancy and BRCA mutation in reducing ovarian response to stimulation, strategies designated to protect against hyper-estrogenic state associated with stimulation (co-treatment with tamoxifen or letrozole), and possible adjustments to accommodate oncologic-related time constraints.ConclusionOvarian stimulation followed by IVF forms an important fertility preservation strategy for newly diagnosed young breast cancer patients, though live born rates following thawed embryo transfer in these patients are still lacking. Recent advances in controlled ovarian stimulation protocols provide practical options for some of the challenges that breast cancer patients present.

BRCA mutation carriers show normal ovarian response in in vitro fertilization cycles

OBJECTIVEnTo evaluate the association between carriage of BRCA1/2 mutations and ovarian performance, as demonstrated by in vitro fertilization (IVF) outcomes.nnnDESIGNnRetrospective cohort study.nnnSETTINGnTwo tertiary IVF centers.nnnPATIENT(S)nBRCA mutation carriers undergoing IVF for preimplantation genetic diagnosis (PGD) or fertility preservation were compared with non-BRCA PGD or fertility preservation patients, matched by age, IVF protocol, IVF center, and cancer disease status.nnnINTERVENTION(S)nIn vitro fertilization cycles for PGD and fertility preservation.nnnMAIN OUTCOME MEASURE(S)nOutcome of IVF: oocyte yield, poor response rate, number of zygotes, pregnancy rates.nnnRESULT(S)nA total of 62 BRCA mutation carriers and 62 matched noncarriers were included; 42 were fertility preservation breast cancer patients, and 82 were PGD non-cancer patients. Mean (± SD) age of patients was 32 ± 3.58 years. Number of stimulation days and total stimulation dose were comparable between carriers and noncarriers. Their cycles resulted in comparable oocyte yield (13.75 vs. 14.75) and low response rates (8.06% vs. 6.45%). Number of zygotes, fertilization rates, and conception rates were also comparable.nnnCONCLUSION(S)nBoth healthy and cancer-affected BRCA mutation carriers demonstrated normal ovarian response in IVF cycles.