Noa Hasky

Interleukin-1 deficiency prolongs ovarian lifespan in mice.

Significance Females are subjected to a biological clock that dictates the end of the reproductive lifespan, on average, at 50 y of age, whereas fecundity sharply decreases after 30 y of age. Over the past decade, a current trend of postponing childbearing into advanced age has led to a corresponding upward trend in the number of in vitro fertilization (IVF) treatments. Inflammation was reported to affect both IVF outcomes and the ovarian reserve adversely. Identifying a possible culprit, such as IL-1, may offer new insight into the mechanisms responsible for oocyte loss as well as practical interventions, such as IL-1 blockade, which aims to slow down the rate at which oocytes are eliminated and improve IVF outcomes. Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1β–KO mice. IL-1α–KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α–KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α–KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1β–KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α–KO mice. The protein and mRNA of both IL-1α and IL-1β mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2–associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1β, IL-6, and TNF-α in ovaries of IL-1α–KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways.

Chemotherapy-Induced Ovarian Failure as a Prototype for Acute Vascular Toxicity

BACKGROUND Chemotherapy-related amenorrhea is a frequent side effect observed in young breast cancer patients. Studies in mice revealed that chemotherapy-induced gonadal toxicity may result from vascular damage. We prospectively evaluated ovarian blood flow and function in young breast cancer patients following chemotherapy. METHODS Young female patients with localized breast cancer undergoing adjuvant or neoadjuvant anthracycline- or taxane-based chemotherapy were evaluated using transvaginal ultrasound prior to initiation of and immediately after cessation of chemotherapy. Doppler-flow velocity indices of the ovarian vasculature-resistance index (RI), pulsatility index (PI)-and size measurements were visualized. Hormonal profiles, anti-Müllerian hormone (AMH) levels, and menopausal symptoms were assessed at the same time points. RESULTS Twenty breast cancer patients were enrolled in the study. The median age was 34 ± 5.24 years. Ovarian blood flow was significantly reduced shortly following chemotherapy: RI decreased by 52.5% and PI decreased by 24.2%. The mean ovarian size declined by 19.08%. Patients who were treated with sequential chemotherapy experienced further reductions in ovarian blood flow and ovarian size after the second sequence. AMH levels dropped dramatically in all patients following treatment. Hormonal profiles after treatment depicted a postmenopausal profile for most patients, accompanied by related symptoms. CONCLUSIONS Our results may imply a mechanism of chemotherapy-induced ovarian toxicity manifested by decreased ovarian blood flow accompanied by a reduction in ovarian size and diminished post-treatment AMH levels. Based upon our former preclinical studies, we assume that this may derive from an acute insult to the ovarian vasculature and may represent an initial event triggering a generalized phenomenon of end-organ toxicity.

Gonadotrophin-releasing hormone agonists for fertility preservation: unraveling the enigma?

STUDY QUESTION Can gonadotrophin-releasing hormone agonists (GnRH-a) preserve long-term fertility when administered prior to and concomitantly with chemotherapy? SUMMARY ANSWER GnRH-a display a differential protective effect on fertility, depending upon the specific chemotherapy-induced mechanism of ovarian injury. WHAT IS KNOWN ALREADY The role of GnRH-a in fertility preservation has been constantly debated and their use is considered experimental due to conflicting clinical evidence and paucity of data regarding their mechanism for ovarian protection. STUDY DESIGN, SIZE, DURATION In vivo model: 7-8 weeks old imprinting control region (ICR) mice were injected with GnRH-a (Leuprolide-acetate) or saline prior to and concomitantly with cyclophosphamide, doxorubicin or saline and sacrificed at various time-points on a longitudinal follow-up; 24 h (n = 36), 1 week (n = 40), 1 month (n = 36) and 9 months (n = 66) post chemotherapy treatment. Blood samples were drawn on Day 0 and on a monthly basis after chemotherapy treatment. On the day of sacrifice, blood samples were drawn and ovaries excised and processed for either immunohistochemistry (IHC), protein or RNA extraction. In vitro model: 21-23 days old Wistar-derived rats were sacrificed, their ovaries excised and primary granulosa cells (PGC) were either isolated for in vitro culture, or processed for immunofluorescence (IF) as well as for protein or RNA extraction. MATERIALS, SETTING, METHODS Ovarian reserve was estimated by serial measurements of serum anti-mullerian hormone (AMH), quantified by the AMH Gen II ELISA assay. Ovarian AMH and phosphorylated Akt (pAkt) were detected by immunoblotting. Vascular endothelial growth factor (VEGF) was measured by quantitative PCR. Ovarian GnRH receptor (GnRHR), AMH and CD34 were visualized by IHC, and apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL). MAIN RESULTS AND THE ROLE OF CHANCE Cyclophosphamide-induced ovarian injury caused a prompt decrease in AMH level (P < 0.01) and a further long-term decline in serum AMH (P = 0.017), indicating damage to the ovarian reserve. Pretreatment with GnRH-a diminished AMH-decrease (P < 0.05) and maintained serum AMH level in the long run (P < 0.05). Doxorubicin-exerted ovarian-vascular-injury is also displayed by an acute increase in ovarian VEGF level (P < 0.05) and a sustained decrease in serum AMH level (P < 0.001). This was followed by ovarian recovery manifested by increased neovascularization. GnRH-a delayed the recovery in AMH level and decreased the level of VEGF (P < 0.001), thus interfering with the vascular recovery subsequent to doxorubicin-induced vascular damage. LIMITATIONS, REASONS FOR CAUTION To portray the differential mechanism of each chemotherapy, cyclophosphamide and doxorubicin were given separately, whereas most of the clinical protocols include several types of chemotherapies. Thus, future study should explore a prospective evaluation of various chemotherapies, as well as combined chemotherapeutic protocols. WIDER IMPLICATIONS OF THE FINDINGS Our study demonstrates that different chemotherapy agents affect the ovary via diverse mechanisms and thus the administration of GnRH-a concomitantly, could be beneficial to a subpopulation of patients treated with cyclophosphamide-based protocols. STUDY FUNDING/COMPETING INTERESTS This work was partially supported by a grant from the Israel Science Foundation (ISF) to I.B.-A. The authors have no conflict of interest to disclose.

Anti-Müllerian Hormone Is a Marker for Chemotherapy-Induced Testicular Toxicity

Due to increased numbers of young cancer patients and improved survival, the impact of anticancer treatments on fertility has become a major health concern. Despite mounting research on ovarian toxicity, there is paucity of data regarding reliable biomarkers of testicular toxicity. Our aim was to evaluate anti-Müllerian hormone (AMH) as a marker for chemotherapy-induced testicular toxicity. Serum AMH and a panel of gonadal hormones were measured in male cancer patients at baseline and after chemotherapy. In the preclinical setting, mice were injected with diverse chemotherapies and were killed 1 week or 1, 3, or 6 months later. We evaluated spermatogenesis by AMH as well as qualitative and quantitative sperm parameters. Nineteen patients were enrolled, the median age was 38 years (21-44 y). Serum AMH was correlated with increased FSH and T and decreased inhibin-B in gonadotoxic protocols (cisplatin or busulfan) and remained unchanged in nongonadotoxic protocols (capecitabine). AMH expression had the same pattern in mice serum and testes; it was negatively correlated with testicular/epididymal weight and sperm motility. The increase in testicular AMH expression was also correlated with elevated apoptosis (terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling) and reduced proliferation (Ki67, proliferating cell nuclear antigen; all seminiferous tubules cells were analyzed). Severely damaged mice testes demonstrated a marked costaining of AMH and GATA-4, a Sertoli cell marker; staining that resembled the pattern of the Sertoli cell-only condition. Our study indicates that the pattern of serum AMH expression, in combination with other hormones, can delineate testicular damage, as determined in both experimental settings. Future large-scale clinical studies are warranted to further define the role of AMH as a biomarker for testicular toxicity.

Hormonal regulation of pigment epithelium-derived factor (PEDF) expression in the endometrium

Pigment epithelium-derived factor (PEDF) is highly expressed in the female reproductive system and is subjected to regulation by steroid hormones in the ovary. As the uterine endometrium exhibits morphological and functional changes in response to estrogen (E2) and progesterone (P4), we aimed at characterizing the expression of PEDF in this component of the female reproductive tract and further at exploring the hormonal regulation of its expression. We found that PEDF is expressed in human and mouse endometrium. We further showed that this expression is subjected to regulation by steroid hormones, both in vivo and in vitro, as follows: E2 decreased PEDF expression and P4 increased its levels. In human endometrial samples, PEDF levels were dynamically altered along the menstrual cycle; they were low at the proliferative and early secretory phases and significantly higher at the late secretory phase. The expression levels of PEDF were inversely correlated to that of vascular endothelial growth factor (VEGF). We also showed that PEDF receptor was expressed in the endometrium and that its stimulation reduced VEGF expression. Illustrating the pattern of PEDF expression during the menstrual cycle may contribute to our understanding of the endometrial complexity.

Long-Term Follow-Up of Chemotherapy-Induced Ovarian Failure in Young Breast Cancer Patients: The Role of Vascular Toxicity

BACKGROUND We previously reported that chemotherapy-induced ovarian toxicity may result from acute vascular insult, demonstrated by decreased ovarian blood flow and diminished post-treatment anti-Müllerian hormone (AMH) levels. In the present study, we report the continuous prospective evaluation of ovarian function in that cohort. METHODS Patients (aged <43 years) with localized breast cancer were evaluated by transvaginal ultrasound prior to initiation of chemotherapy, immediately at treatment completion, and at 6 and 12 months after treatment cessation. Doppler flow velocity indices of the ovarian vasculature (resistance index [RI], pulsatility index [PI]) were visualized. Hormone markers of ovarian reserve were assessed at the same time points. RESULTS Twenty patients were enrolled in the study. Median age was 34 ± 5.24 years. Ovarian blood flow was significantly reduced immediately following chemotherapy (both RI and PI; p = .01). These parameters were partially recovered at later points of assessment (6 and 12 months after treatment); patients aged <35 years significantly regained ovarian blood flow compared with patients aged >35 years (p < .05). AMH dropped dramatically in all patients following treatment (p < .001) and recovered in only 10 patients. Hormone markers of ovarian reserve shortly after chemotherapy depicted a postmenopausal profile for most patients, accompanied by related symptoms. Follicle-stimulating hormone (FSH) levels recovered in 14 of 20 patients and significantly returned to the premenopausal range in patients aged <35 years (p = .04); 10 of 20 resumed menses at 12 months. The pattern of vascular impairment was lessened in patients treated with a trastuzumab-based protocol, although results did not reach statistical significance (p = .068). CONCLUSION Continuous prospective evaluation of ovarian vasculature and function in a cohort of young patients during and after chemotherapy indicated that ovarian toxicity may derive from acute vascular insult. Age may affect whether patients regain ovarian function, whereas recovery of blood flow and premenopausal FSH levels at later assessment was notable in patients aged <35 years. IMPLICATIONS FOR PRACTICE This study explored the role of vascular toxicity in mediating ovarian impairment and recovery following chemotherapy. Continuous prospective evaluation of ovarian vasculature and function in a cohort of young patients during and after chemotherapy indicated that ovarian toxicity may derive from acute vascular insult. Future studies are warranted to further characterize patterns of vascular toxicity of various chemotherapies in clinical practice and to assess the role of chemotherapy-induced vascular toxicity for specific end organs such as the ovary with systemic vascular effect. Elucidating the cause of impairment may facilitate development of measures to minimize vascular toxicity and consequences of acute vascular insult.

Pigment Epithelium–Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia

Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor–positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium–derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies. Mol Cancer Ther; 14(12); 2840–9. ©2015 AACR.

Premature ovarian aging in BRCA carriers: a prototype of systemic precocious aging?

Purpose Though former evidence implies a correlation of breast cancer susceptibility gene (BRCA) mutation with reduced ovarian reserve, the data is yet inconsistent. Our aim was to investigate biomarkers of ovarian aging in a cohort of young healthy carriers of the BRCA mutation. We hypothesized that the role played by BRCA genes in aging pathways is not exclusive to the ovary. Experimental Design Healthy female BRCA carriers, 40 years or younger and healthy male BRCA carriers, 50 years or younger, were enrolled in the study. Serum anti-mullerian Hormone (AMH), fibroblast growth factor-23 (FGF-23), Klotho and IL-1 were measured by enzyme-linked immunosorbent assay (ELISA). Ovarian AMH and protein kinase B (AKT) mRNA from BRCA carriers who underwent prophylactic oophorectomy and from age-matched, healthy, non-carriers who underwent partial oophorectomy due to benign conditions were analyzed by qPCR. Results Thirty-three female (median age 35y) and 20 male (44y) BRCA carriers were enrolled into the study and matched to control non-carriers (34y and 43y, respectively). Serum AMH level was significantly lower in BRCA female carriers than in both non-carrier controls and age-matched nomograms. The levels of ovarian AMH and AKT mRNA were significantly lower in carriers than in controls. The systemic aging cytokines FGF-23, klotho and IL-1 displayed a differential expression in carriers of both genders. FGF-23 level was higher in carriers (P=0.06). Conclusions Our results suggest a link between BRCA mutation, accelerated ovarian aging and systemic aging-related pathophysiology.