Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise.

The mechanism of chemotherapy-induced acceleration of ovarian aging is not fully understood. We used doxorubicin, a widely used cancer chemotherapeutic, in a variety of in vivo xenograft, and in vitro models to investigate the impact of chemotherapy-induced aging on the human ovary. Doxorubicin caused massive double-strand-DNA-breaks in primordial follicles, oocytes, and granulosa cells in a dose dependent fashion as revealed by accumulating γH2AX foci. This damage was associated with apoptotic oocyte death and resulted in the activation of ATM. It appeared that the repair response enabled a minor proportion of oocytes (34.7%) and granulosa cells (12.1%) to survive while the majority succumbed to apoptotic death. Paradoxically, inhibition of ATM by KU-55933 resulted in improved survival, probably via prevention of downstream activation of TAp63α. Furthermore, doxorubicin caused vascular and stromal damage in the human ovary, which might impair ovarian function both pre- and post-menopausally. Chemotherapy-induced premature ovarian aging appears to result from a complex process involving both the germ- and non-germ cell components of the ovary. These effects may have clinical implications in aging both for premenopausal and postmenopausal cancer survivors.

Anti-Mullerian hormone and antral follicle count as predictors for embryo/oocyte cryopreservation cycle outcomes in breast cancer patients stimulated with letrozole and follicle stimulating hormone.

PURPOSE: To predict embryo/oocyte cryopreservation cycle (ECC) outcomes in breast cancer patients stimulated with letrozole and follicle stimulating hormone for fertility preservation based on observed anti-mullerian hormone (AMH) levels and antral follicle counts (AFC). METHODS: The correlation between AMH and AFC and ECC outcomes were analyzed retrospectively on forty one women with breast cancer before adjuvant treatment. RESULTS: AMH and AFC had a stronger correlation with the total number of oocytes and the number of mature oocytes than age, FSH, and inhibin B. Subjects were evaluated by the number of mature oocytes retrieved to create cutoff points of AMH level, which identified 1.2 ng/mL as a potential value. Seven of 18 patients with AMH levels ≤1.2 ng/mL had low response versus none of 23 with >1.2 ng/mL, (p = 0.001). CONCLUSIONS: AMH is the most reliable serum marker of ECC outcomes, together with AFC as a biophysical marker, in breast cancer patients. Low response is highly likely when the AMH level is ≤1.2 ng/mL.

Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants.

Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether sphingosine-1-phosphate or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with sphingosine-1-phosphate, its analogs, or vehicle for 1-10 days. We found that sphingosine-1-phosphate treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW2871 had the opposite effect. In addition, sphingosine-1-phosphate accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, sphingosine-1-phosphate treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in sphingosine-1-phosphate-treated transplants. We conclude that while sphingosine-1-phosphate promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, sphingosine-1-phosphate receptor agonists appear to functionally antagonize this process. Sphingosine-1-phosphate holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants.

Determinants of access to fertility preservation in women with breast cancer.

OBJECTIVE: To evaluate socioeconomic, demographic, and medical factors that influence the referral pattern-either before cancer treatment for fertility preservation (FP, early referral) or post-chemotherapy for assisted reproductive technology (PCART, delayed referral)-in women with breast cancer. DESIGN: Secondary analysis. SETTING: Academic medical centers. PATIENT(S): Three hundred fourteen patients with breast cancer who were counseled for FP (n=218) or PCART (n=96) from June 1999 to July 2009. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Factors favoring early referrals. RESULT(S): Mean age at diagnosis was higher in FP vs. PCART (35.3±4.5 years vs. 33.9±4.7 years). Ninety percent presented with cancer stage 1 or 2. From 2000 to 2009 the proportion of referrals for FP increased continually. In 2009, nearly all (95.5%) were for FP. The majority (63.8%) was referred from an academic center. Patients with a family history of breast cancer were more likely to consult for FP (75.2% vs. 64.3% without). There was no association with occupation, income, race, ethnicity, obstetric history, and prior infertility treatment. Only 22.9% of those counseled in PCART, compared with 45.0% in the FP group, proceeded with a procedure. CONCLUSION(S): There has been an increasing trend within the last 10 years for early referral of breast cancer patients to FP. Factors favoring early referrals are older age, early-stage cancer, family history of breast cancer, and academic center involvement. Those seen before cancer treatment are more likely to receive an intervention.

Reduced fertilization after ICSI and abnormal phospholipase C zeta presence in spermatozoa from the wobbler mouse.

Failed fertilization after intracytoplasmic sperm injection (ICSI) can be due to a reduced oocyte-activation capacity caused by reduced concentrations and abnormal localization of the oocyte-activation factor phospholipase C (PLC) zeta. Patients with this condition can be helped to conceive by artificial activation of oocytes after ICSI with calcium ionophore (assisted oocyte activation; AOA). However some concern still exists about this approach. Mouse models could help to identify potential oocyte-activation strategies and evaluate their safety. In this study, the fertilizing capacity of wobbler sperm cells was tested and the efficiency of AOA with two exposures to ionomycin to restore fertilization and embryo development was studied. The quality of the obtained blastocysts was assessed and embryo transfer was performed to evaluate post-implantation development. The presence of PLCzeta in the spermatozoa and testis of the wobbler mouse was evaluated by PLCzeta immunostaining and quantitative reverse-transcription polymerase chain reaction. Sperm cells from wobbler mice had reduced fertilizing capacity and abnormalities in PLCzeta localization, but not in its expression. Artificially activating the oocytes restored fertilization and embryo development. Therefore, the wobbler mouse can be a model for failed fertilization after ICSI to study PLCzeta dynamics and aid in optimization of the AOA method.

Value of early referral to fertility preservation in young women with breast cancer.

PURPOSE: To determine whether early referral to reproductive specialists improves fertility preservation (FP) outcomes and reduces delay in adjuvant treatment in young women with breast cancer. PATIENTS AND METHODS: A secondary analysis of a prospective database of patients with breast cancer undergoing ovarian stimulation (OS) for FP by oocyte or embryo cryopreservation was performed. RESULTS: Of the 154 patients, 93 met the inclusion criteria (mean age, 35.2 ± 4.4 years). Thirty-five of the 93 patients were referred before breast surgery (PreS), and 58 patients were referred after surgery (PostS). The time periods from initial diagnosis (ID) to initiation of OS (42.6 ± 27.7 days for PreS v 71.9 ± 30.7 days for PostS; P < .001) and from ID to initiation of chemotherapy (83.9 ± 24.3 days for PreS v 107.8 ± 42.9 days for PostS; P = .045) were significantly shorter for the PreS group versus the PostS group. Nine (25.7%) of 35 patients in the PreS group versus one (1.7%) of 58 patients in the PostS group were able to undergo two FP cycles (P < .001), resulting in an increased yield of oocytes in the PreS group (18.2% [93 of 511 oocytes] v 0.6% [five of 800 oocytes], respectively; P < .001) and embryos (17.2% [40 of 233 embryos] v 0.6% [two of 357 embryos], respectively; P < .001). Patients who had an oocyte retrieval within 5 weeks of the surgery were able to complete a second cycle within 9 weeks of the surgery. CONCLUSION: FP referral before breast surgery enables earlier initiation of cryopreservation cycles and chemotherapy and, when appropriate, multiple FP cycles. Women who can undergo multiple cycles may be at advantage for FP because of a larger number of oocytes or embryos cryopreserved. This is the first study demonstrating the benefit of early FP referral in patients with cancer.

Oscillatory Ca2+ dynamics and cell cycle resumption at fertilization in mammals: a modelling approach.

Fertilization in mammals is accompanied by Ca(2+) oscillations in the egg cytoplasm, leading to exit from meiosis and entry into the first embryonic cell cycle. The signal transduction pathway linking these Ca(2+) changes to cell-cycle related kinases has not yet been fully elucidated, but involves activation of calmodulin-dependent kinase II (CaMKII). Here, we develop a computational model to investigate the mechanism by which cell cycle resumption can be sensitive to the temporal pattern of Ca(2+) increases. Using a model for CaMKII activation that reproduces the frequency sensitivity of this kinase, simulations confirm that Ca(2+) spikes are accompanied by in phase variations in the level of CaMKII activity and suggest that in most mammalian species, Ca(2+) spikes are well suited to maximize CaMKII activation. The full model assumes that CaMKII brings about a decrease in the level of cyclinB-cdk1 by two pathways, only one of which is CSF-dependent. Parameters are selected to account for the experimental observations where mouse eggs were artificially activated by different Ca(2+) stimulatory protocols. The model is then used in the context of 'assisted oocyte activation (AOA)' to investigate why the best rates of successful activation are obtained when eggs are submitted to two applications of Ca(2+) ionophores.

Effect of ionomycin on oocyte activation and embryo development in mouse.

Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 micromol/l. An improved effect of a second exposure to 5 micromol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.

Effect of N-acetylcysteine on neutrophil activation markers in healthy volunteers: in vivo and in vitro study.

BACKGROUND AND AIMS: Neutrophil activation is implicated in the pathogenesis of inflammatory processes such as chronic obstructive pulmonary disease (COPD). We wished to evaluate both in vivo and in vitro whether N-acetylcysteine (NAC) has an effect on the response of activated neutrophils. METHODS: Ten healthy volunteers took NAC (600 mg daily) for 14 days. The effects on basal and fMLP-induced respiratory burst and chemotaxis were assessed at baseline, 2 h and 14 days after NAC intake. Neutrophils from healthy volunteers (NAC naïve) were pre-incubated with NAC for 30 min and the effects on the release of elastase and IL-8, the respiratory burst in response to fMLP and PMA, on TNFalpha-induced NFkappaB activation and on the migration across an endothelial-epithelial bilayer were investigated. RESULTS: PMA and fMLP-induced neutrophil respiratory burst and chemotaxis were lower after 14 days of NAC intake but not after 2 h. In vitro incubation with NAC inhibited release of elastase (p < 0.05), IL-8 (p < 0.05), respiratory burst and NFkappaB activation at 10 mM but not at lower concentrations. This was accompanied by a decrease in cellular ATP content. CONCLUSIONS: Our results suggest that acute in vitro addition of NAC can modulate neutrophil activity only when used at the high concentrations which directly affect cellular metabolism. On the other hand, the lower doses of NAC given in vivo might require longer times in order to achieve sustained effects on the cellular thiols which lead to changes in the redox status.

Cell-mediated LDL oxidation: the impact of transition metals and transferrin.

Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.