Faculty Bibliography

The ovary contains oocytes within immature (primordial) follicles that are fixed in number at birth. Activation of follicles within this fixed pool causes an irreversible decline in reproductive capacity, known as the ovarian reserve, until menopause. Premenopausal women undergoing commonly used genotoxic (DNA-damaging) chemotherapy experience an accelerated loss of the ovarian reserve, leading to subfertility and infertility. Therefore, there is considerable interest but little effective progress in preserving ovarian function during chemotherapy. Here we show that blocking the kinase mammalian/mechanistic target of rapamycin (mTOR) with clinically available small-molecule inhibitors preserves ovarian function and fertility during chemotherapy. Using a clinically relevant mouse model of chemotherapy-induced gonadotoxicity by cyclophosphamide, and inhibition of mTOR complex 1 (mTORC1) with the clinically approved drug everolimus (RAD001) or inhibition of mTORC1/2 with the experimental drug INK128, we show that mTOR inhibition preserves the ovarian reserve, primordial follicle counts, serum anti-Mullerian hormone levels (a rigorous measure of the ovarian reserve), and fertility. Chemotherapy-treated animals had significantly fewer offspring compared with all other treatment groups, whereas cotreatment with mTOR inhibitors preserved normal fertility. Inhibition of mTORC1 or mTORC1/2 within ovaries was achieved during chemotherapy cotreatment, concomitant with preservation of primordial follicle counts. Importantly, our findings indicate that as little as a two- to fourfold reduction in mTOR activity preserves ovarian function and normal birth numbers. As everolimus is approved for tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer, these findings represent a potentially effective and readily accessible pharmacologic approach to fertility preservation during conventional chemotherapy.

Increasing percentages of children are being born to older fathers. This has resulted in concerns about the potential adverse effects of advanced paternal age. To help clinicians counsel couples, a systemic review was performed to attempt to address questions that these couples may ask: Should routine sperm testing be performed in older males? Should preimplantation genetic diagnosis (PGD) be performed? How do providers counsel patients about risk? Should young males freeze sperm if they plan to delay paternity? Using the terms "advanced paternal age", "semen testing", "preimplantation genetic diagnosis/screening", and "cryopreservation", a comprehensive search was performed in PubMed and the Cochrane Library, and numerous international societal guidelines were reviewed. In total, 42 articles or guidelines were reviewed. There were no limits placed on the timing of the articles. Thirty articles were found to be relevant and beneficial to answering the above questions. Each question was answered separately by the supporting literature. While primary research exists to support the role of semen testing, PGD/preimplantation genetic screening, and sperm banking in males who may be affected by advancing age, comprehensive studies on the possible clinical benefit of these interventions have yet to be performed. As a result, societal guidelines have yet to incorporate distinct best-practice guidelines on advanced paternal age.

Implantation rate decreases and miscarriage rate increases with advancing maternal age. The oocyte must be the locus of reproductive aging because donation of oocytes from younger to older women abrogates the effects of aging on fecundity. Nuclear transfer experiments in a mouse model of reproductive aging show that the reproductive aging phenotype segregates with the nucleus rather than the cytoplasm. A number of factors within the nucleus have been hypothesized to mediate reproductive aging, including disruption of cohesions, reduced chiasma, aneuploidy, disrupted meiotic spindles, and DNA damage caused by chronic exposure to reactive oxygen species. We have proposed telomere attrition as a parsimonious way to explain these diverse effects of aging on oocyte function. Telomeres are repetitive sequences of DNA and associated proteins, which form a loop (t loop) at chromosome ends. Telomeres prevent the blunt end of DNA from triggering a DNA damage response. Previously, we showed that experimental telomere shortening phenocopies reproductive aging in mice. Telomere shortening causes reduced synapsis and chiasma, chromosome fusions, embryo arrest and fragmentation, and abnormal meiotic spindles. Telomere length of polar bodies predicts the fragmentation of human embryos. Telomerase, the reverse transcriptase capable of reconstituting shortened telomeres, is only minimally active in oocytes and preimplantation embryos. Intriguingly, during the first cell cycles following activation, telomeres robustly elongate via a DNA double-strand break mechanism called alternative lengthening of telomeres (ALTs). Alternative lengthening of telomere takes place even in telomerase-null mice. This mechanism of telomere elongation previously had been found only in cancer cells lacking telomerase activity. We propose that ALT elongates telomeres across generations but does so at the cost of extensive genomic instability in preimplantation embryos.

OBJECTIVE: The architecture and structure of the meiotic spindle influence embryo development(1) and risk of aneuploidy in women(2). The factors that disrupt the meiotic spindle remain incompletely understood. Dysfunctional mitochondria produce reactive oxygen species (ROS), which may directly perturb spindles(3). ROS also can disrupt spindles by inducing telomere attrition, since telomeres are essential for spindle formation and are especially susceptible to ROS(4). We studied the impact of ROS, produced by uncoupling mitochondria, on the area and retardance (measure ofmolecular order) ofmeiotic spindles and on mean telomere length of individual human oocytes. DESIGN: Prospective, randomized, paired research laboratory intervention. MATERIALS AND METHODS: 44 germinal vesicle (GV) stage oocytes were accessioned from 16 patients undergoing IVF/ICSI. GVs from each patient were randomly assigned to control or treatment group. Oocytes in the treatment group were cultured in media containing Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM) for one hour to induce mitochondrial stress. Control oocytes were cultured in media without FCCP. GVoocytes from both groups were further cultured to permit meiotic maturation, as indicated by extrusion of the first polar body. Spindles were imaged non-invasively with an orientation independent polarized light microscope (Oosight, Hamilton Thorne, MA, USA). Spindle area and mean retardance were measured with software from the Oosight Imaging System. Mean oocyte telomere length was measured by single cell qPCR. Data were analyzed by Chi-square test and independent t test. RESULTS: Maturation rates of oocytes in the control and FCCP groups were 66.67% and 56.52% respectively. 71.4% of MII oocytes in the control group and 61.5% in the FCCP group had a detectable birefrigent spindle. FCCP decreased the area of the spindles compared to controls (19.84+/-2.11 sq. microns versus 37.77+/-4.79, P=0.011). Mean spindle retardance in the FCCP group also was significantly lower than that of controls (1.45+/-0.11 nm versus 2.19+/-0.16 nm, P=0.003). Telomere length (T/R ratio) did not differ between treatment and control groups (1.11+/-1.56 versus 1.29+/-0.19, P>0.05). Telomere length of oocytes with and without birefringence spindle within the treatment group also did not differ significantly (P>0.05). CONCLUSIONS: Meiotic maturation itself is relatively resistant to ROS, consistent with prior studies showing limited cell cycle check point control during oogenesis. However, ROS produced by acute mitochondrial stress disrupts spindle retardance and size. Reactive oxygen, at least acutely, does not induce telomere attrition in human oocytes

OBJECTIVE: Telomere attrition may mediate some of the effects of aging on reproductive function in women. Mice null or haploinsufficient for telomerase phenocopy the profile of reproductive aging in women, with progressive infertility caused by numerous defects in their reproductive cells. Telomeropathies, such as Dyskeratosis Congenita, provide a natural experiment to test the Telomere Theory of Reproductive Aging in women. This study attempts to extensively characterize the reproductive function in women with telomeropathies for the first time. DESIGN: Blood samples, cumulus cells, and arrested embryos were collected following the cycle of A 30 year old woman with a precocious aging syndrome and aplastic anemia, attributed to a telomeropathy (AMH=0.3 and AFC=8). She underwent, controlled ovarian stimulation with E2 prime protocol and 600 IU/day of gonadotropin, using mixed protocol and GnRH antagonist for 18 days. MATERIALS AND METHODS: Monochrome multiplex quantitative polymerase chain reaction (qPCR) assay (Cawthon 2009) measured telomere length in leukocytes extracted from whole blood as well as, cumulus cells stripped from retrieved follicles. Telomere (T) amplification was normalized to a single copy gene (S), resulting in a T/S ratio proportional to average telomere length in the population. Single-Cell Amplification of Telomere Repeats (SCATR) PCR (Wang 2013) was used to measure telomere length in discarded embryo blastomeres. Telomere (T) amplification was normalized a reference gene (R), producing a T/R ratio . One-Way ANOVA test was used to determine statistical significance. RESULTS: Hyperstimulation resulted in only 7 oocytes and 1 euploid blastocyst. Over the treatment course, leukocyte telomere length increased from T/S ratio= 0.192+/-.0157 to 0.234+/-.0306 and there was a statistically significant (p= .0256) linear increase during the treatment. Further, telomere length in a retrieved parthenogenetic, 2-cell embryo was (T/R average= .169+/-.021) and that in cumulus cells (T/S= 0.586+/-.0147). Telomere lengths in all assayed cell types were shorter than those from age matched controls. CONCLUSIONS: Young woman with reduced ovarian reserve, poor response to ovarian stimulation and a high percentage of arrested embryos and aneuploid embryos was still able to generate one euploid blastocyst with high dose controlled ovarian stimulation, demonstrating the promise of ART for fertility preservation in women with telomeropathies. Intriguingly, controlled ovarian hyperstimulation increased her leukocyte telomere length. Presumably, the supraphysiologic levels of estrogen activated telomerase activity, consistent with prior studies reporting an estrogen response element in the TERT gene. Future studies should examine whether women with telomeropathies may benefit from estrogen supplementation

OBJECTIVE: Reactive oxygen species (ROS) are a major cause of aging in all tissues studied, including reproductive tissues. Recent studies demonstrate extensiveDNA damage repair capacity in oocytes (1), and genetic variation in DNA damage repair pathways is associated with reproductive lifespan in women (2). We hypothesized that MII oocytes are more resistant to oxidative stress than other stages of development. DESIGN: Prospective, randomized study of a biologic intervention on mouse oocytes and embryos. MATERIALS AND METHODS: 80 MII oocytes and 40 cleavage embryos from B6C3F1 mice (Embryotech Laboratories, Inc, USA) were thawed and exposed to oxidative stress induced by Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM), which generates ROS by uncoupling mitochondrial electron transport and disrupting mitochondrial function. Oocytes and embryos were randomized into untreated controls, or 5, 10 or 20 hour exposure to FCCP. DNA damage was determined by immuno- fluorescent staining for gamma-H2AX, or by mean telomere length, measured by single-cell qPCR. Data were analyzed by chi-square test and one-way ANOVA. RESULTS: 20 hours of exposure to FCCP is lethal for all embryos. Embryos exposed to FCCP for 5 and 10 hours of FCCP show increased g- H2AX staining (5 hours- 87.5% vs. 25% positive cells P<0.05; 10 hours- 100% vs 25%, P<0.05). However, these same doses and durations of FCCP do not increase DNA damage in oocytes- there is no significant increase of gamma-H2AX positive MII oocytes compared to controls (30% in the control group, 40% in the 5-hour group and 30% in the 10-hour group, P>0.05). Significantly higher numbers of gamma-H2AX positive MII oocytes are found only in the 20-hour group (100%, P<0.05), a dose which is uniformly lethal to embryos. As previously demonstrated, 45 minutes of 750nM FCCP treatment shortens telomeres in cleavage stage mouse embryos (3). However, MII oocytes exposed to 750nM FCCP for 5, 10, or 20 hours show no statistically significant telomere shortening compared to controls (P>0.05). CONCLUSIONS: MIIoocytes are more resistant to oxidative stress than cleavage embryos

A schizophrenia phenotype for paternal and maternal age effects on illness risk could benefit etiological research. As odor sensitivity is associated with variability in symptoms and cognition in schizophrenia, we examined if it was related to parental ages in patients and healthy controls. We tested Leukocyte Telomere Length (LTL) as an explanatory factor, as LTL is associated with paternal age and schizophrenia risk. Seventy-five DSM-IV patients and 46 controls were assessed for detection of PEA, WAIS-III for cognition, and LTL, assessed by qPCR. In healthy controls, but not schizophrenia patients, decreasing sensitivity was monotonically related to advancing parental ages, particularly in sons. The relationships between parental aging and odor sensitivity differed significantly for patients and controls (Fisher's R to Z: chi2 = 6.95, P = 0.009). The groups also differed in the association of odor sensitivity with cognition; lesser sensitivity robustly predicted cognitive impairments in patients (<0.001), but these were unassociated in controls. LTL was unrelated to odor sensitivity and did not explain the association of lesser sensitivity with cognitive deficits.Parental aging predicted less sensitive detection in healthy subjects but not in schizophrenia patients. In patients, decreased odor sensitivity strongly predicted cognitive deficits, whereas more sensitive acuity was associated with older parents. These data support separate risk pathways for schizophrenia. A parental age-related pathway may produce psychosis without impairing cognition and odor sensitivity. Diminished odor sensitivity may furthermore be useful as a biomarker for research and treatment studies in schizophrenia. (c) 2015 Wiley Periodicals, Inc.

Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3beta inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.

INTRODUCTION: PI3K/AKT/mTOR up-regulation results in accelerated activation of the primordial follicle (PMF) pool and has been implicated in cyclophosphamide (CY)-induced 'follicular exhaustion'. We compared follicular dynamics in murine models of CY-induced POI and age-related DOR, hypothesizing that 'follicular exhaustion' may occur similarly in both groups. METHODS: C57BL/6 female mice aged 8 wks (n=25) housed in identical conditions were assigned to 5 groups: 150mg/kg CY intraperitoneal (IP) x1, 75mg/kg CY IP weekly x3, control (8 wks and 11 wks), and DOR (13 months). Blood was extracted by terminal cardiac puncture for anti-mullerian hormone (AMH). Ovaries were paraffin-embedded, sectioned, and H&E stained; blinded follicle counts were confirmed by two reviewers. Follicle counts are presented as follicles/section area (mm2); data presented as mean+/-SEM. One-way ANOVA and student's t-test were used for statistical analysis (p<0.05). 'Follicular exhaustion' was calculated as the ratio of total growing follicles (primary, secondary, antral) to PMFs. RESULTS: A DOR murine model was confirmed by low AMH in 13-mth-old mice compared to controls (7.2+/-0.1 vs. 12.1+/-0.5, p<0.05) and fewer PMFs/mm2 (0.6+/-0.2) compared to 8-wk (7.7+/-1.2) and 11 wk controls (6.0+/-1) (p<0.05). A CY-induced POI model was confirmed by lower AMH in 150mg/kg CY-exposed mice compared to age-matched controls (9.6+/-0.9 vs. 12.1+/-0.5) and fewer PMFs/mm2 (2.5+/-0.4 vs. 7.7+/-1.2, p<0.05). CY-treated mice had more primary & secondary follicles/mm2 compared to controls (p<0.05), suggesting increased follicle activation. PMF counts were similar in POI [75mg/kg CY (2.4 +/- 0.5) and 150mg/ kg CY (1.9+/-0.3)] compared to DOR (0.6+/-0.2) despite an 11-mth age difference. Importantly, similar rates of 'follicular exhaustion' were seen in CY-exposed POI mice (75mg/kg CY ratio 1.9; 150mg/kg CY ratio 2.6) and DOR mice (ratio 2.4). In contrast, low 'follicular exhaustion' ratios were seen in both control groups (8-wks ratio 0.64; 11-wks ratio 0.89) signifying maintenance of follicles as PMFs. CONCLUSIONS: Accelerated PMF activation and subsequent follicular exhaustion occur similarly in murine models of CY-induced POI and age-related DOR. Future studies should investigate if PI3K/AKT/mTOR up-regulation is also involved in DOR, suggesting possible implications for prevention

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.