Faculty Bibliography

In this Journal in 1972, 100 leaders in obstetrics and gynecology published a compelling statement that recognized the legalization of abortion in several states and anticipated the 1973 Supreme Court decision in Roe v Wade. They projected the numbers of legal abortions that likely would be required by women in the United States and described the role of the teaching hospital in meeting that responsibility. They wrote to express their concern for women's health in a new legal and medical era of reproductive control and to define the responsibilities of academic obstetrician-gynecologists. Forty years later, 100 professors examine the statement of their predecessors in light of medical advances and legal changes and suggest a further course of action for obstetrician gynecologists.

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

Abstract Background: There have been recent reports that lactational history is associated with long-term women's health benefits. Most of these studies are epidemiological. If particular cardiometabolic changes that occur during lactation ultimately influence women's health later is unknown. Methods: Seventy-one healthy women participated in a prospective postpartum study that provided an opportunity to study anthropometric, endocrine, immune, and behavioral variables across time. Variables studied were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), C-reactive protein, body mass index (BMI), perceived stress, and hormones. A cohort of women without a change in breastfeeding (N=22) or formula feeding (N=23) group membership for 5 months was used for analysis of effects of feeding status. The data were analyzed using factorial repeated measures analysis of variance and analysis of covariance. Results: SBP and HR declined across the postpartum and were significantly lower in breastfeeding compared to formula feeding mothers (p<0.05). These differences remained statistically significant when BMI was added to the model. Other covariates of income, stress, marital status, and ethnicity were not significantly associated with these variables over time. DBP was also lower, but the significance was reduced by the addition of BMI as a covariate. Stress also was lower in breastfeeders, but this effect was reduced by the addition of income as a covariate. Conclusions: These data suggest that there are important physiological differences in women during months of breastfeeding. These may have roles in influencing or programming later risks for a number of midlife diseases.

STUDY QUESTION: Does resveratrol counteract age-associated infertility in a mouse model of reproductive aging? SUMMARY ANSWER: Long-term-oral administration of resveratrol protects against the reduction of fertility with reproductive aging in mice. WHAT IS KNOWN ALREADY: Loss of oocytes and follicles and reduced oocyte quality contribute to age-associated ovarian aging and infertility. Accumulation of free radicals with age leads to DNA mutations, protein damage, telomere shortening, apoptosis and accelerated ovarian aging. Increasing evidence shows that resveratrol, enriched in certain foods, for example red grapes and wine, has anti-tumor and anti-aging effects on somatic tissues by influencing various signaling pathways, including anti-oxidation, as well as activating Sirt1 and telomerase. We investigated the potential of resveratrol to stave off ovarian aging in the inbred C57/BL6 mouse model. STUDY DESIGN, SIZE, DURATION: Young C57/BL6 females (aged 2-3 months) were fed with resveratrol added to drinking water at 30 mg/l (providing approximately 7.0 mg/kg/day) for 6 or 12 months, and the fertility and ovarian functions were compared among mice treated with or without resveratrol, and young mice served as reproductive controls. Experiments were repeated three times, with an average of 25 females randomly allocated to each treatment group for each repeat. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive performance of female mice was determined by litter size, ovarian follicles and oocyte quantity and quality, and compared with age-matched controls. The impact of resveratrol on telomeres and telomerase activity, and expression of genes associated with cell senescence also was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Young mice fed with resveratrol for 12 months retained the capacity to reproduce, while age-matched controls produced no pups. Consistently, mice fed with resveratrol for 12 months exhibited a larger follicle pool than controls (P < 0.05). Furthermore, telomerase activity, telomere length and age-related gene expression in ovaries of mice fed with resveratrol resembled those of young mice, but differed (P < 0.05) from those of age-matched old mice. Resveratrol improved (P < 0.05) the number and quality of oocytes, as evidenced by spindle morphology and chromosome alignment. Also, resveratrol affected embryo development in vitro in a dose-dependent manner. LIMITATIONS, REASONS FOR CAUTION: The doses of resveratrol and the experimental conditions used by different research groups have varied considerably, and the dosage influences both the effectiveness and toxicity of resveratrol. Fine-tuning the dosage of resveratrol likely will optimize its anti-aging effects on ovarian function. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide a proof of principle of the fertility-sparing effect of resveratrol in female mice. Although depletion of the ovarian reserve of high-quality oocytes also contributes to increased infertility with reproductive aging in women, the data obtained using a mouse model may not extrapolate directly to human reproduction, and more extensive research is needed if any clinic trials are to be attempted. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by MOST of China National Basic Research Program (grant number: 2010CB94500 and 2012CB911200). The authors have no competing interests to declare.

Accumulation of double-stranded DNA breaks and inhibition of DNA repair contributes to reproductive aging by diminishing ovarian reserves in mice and women.

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

BACKGROUND Ovarian aging is associated with declining numbers and quality of oocytes and follicles. Oxidative stress by reactive oxygen species (ROS) contributes to somatic aging in general, and also has been implicated in reproductive aging. Telomere shortening is also involved in aging, and telomeres are particularly susceptible to ROS-induced damage. Previously, we have shown that antioxidant N-acetyl-l-cysteine (NAC) effectively rescues oocytes and embryos from ROS-induced telomere shortening and apoptosis in vitro. Using mice as models, we tested the hypothesis that reducing oxidative stress by NAC might prevent or delay ovarian aging in vivo. METHODS Initially, young females were treated with NAC in drinking water for 2 months and the quality of fertilized oocytes and early embryo development were evaluated. Next, young mice 1-1½ months old were treated for 1 year with NAC added in drinking water, and their fertility was analyzed starting at 6 months, as indicated by litter size, oocyte number and quality. The ovaries were also examined for telomere activity and length and the expression of selected genes related to aging and DNA damage. RESULTS Short-term treatment of mice for 2 months with NAC demonstrated that NAC improved the quality of fertilized oocytes and early embryo development. Mice treated with a long-term low concentration (0.1 mM) of NAC had increased litter sizes at the ages of 7-10 months compared with age-matched controls without NAC treatment. NAC also increased the quality of the oocytes from these older mice. Moreover, the expression of sirtuins was increased, telomerase activity was higher and telomere length was longer in the ovaries of mice treated with NAC compared with those of the control group. CONCLUSIONS These data suggest that appropriate treatment with the antioxidant NAC postpones the process of oocyte aging in mice.

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.

Telomerase and telomeres are important for indefinite replication of stem cells. Recently, telomeres of somatic cells were found to be reprogrammed to elongate in induced pluripotent stem cells (iPSCs). The role of telomeres in developmental pluripotency in vivo of embryonic stem cells (ESCs) or iPSCs, however, has not been directly addressed. We show that ESCs with long telomeres exhibit authentic developmental pluripotency, as evidenced by generation of complete ESC pups as well as germline-competent chimeras, the most stringent tests available in rodents. ESCs with short telomeres show reduced teratoma formation and chimera production, and fail to generate complete ESC pups. Telomere lengths are highly correlated (r > 0.8) with the developmental pluripotency of ESCs. Short telomeres decrease the proliferative rate or capacity of ESCs, alter the expression of genes related to telomere epigenetics, down-regulate genes important for embryogenesis and disrupt germ cell differentiation. Moreover, iPSCs with longer telomeres generate chimeras with higher efficiency than those with short telomeres. Our data show that functional telomeres are essential for the developmental pluripotency of ESCs/iPSCs and suggest that telomere length may provide a valuable marker to evaluate stem cell pluripotency, particularly when the stringent tests are not feasible