Faculty Bibliography

Parthenogenetic embryonic stem cells (pESCs) have been generated in several mammalian species from parthenogenetic embryos that would otherwise die around mid-gestation. However, previous reports suggest that pESCs derived from in vivo ovulated (IVO) mature oocytes show limited pluripotency, as evidenced by low chimera production, high tissue preference and especially deficiency in germline competence, a critical test for genetic integrity and pluripotency of ESCs. Here, we report efficient generation of germline-competent pESC lines (named as IVM pESCs) from parthenogenetic embryos developed from immature oocytes of adult mouse ovaries following in vitro maturation (IVM) and artificial activation. In contrast, pESCs derived from IVO oocytes show defective germline competence, consistent with previous reports. Further, IVM pESCs resemble more ESCs from fertilized embryos (fESCs) than do IVO pESCs on genome-wide DNA methylation and global protein profiles. In addition, IVM pESCs express higher levels of Blimp1, Lin28 and Stella, relative to fESCs, and in their embryoid bodies following differentiation. This may indicate differences in differentiation potentially to the germline. The mechanisms for acquisition of pluripotency and germline competency of IVM pESCs from immature oocytes remain to be determined

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos

Cigarette smoke is associated with high risk of lung, cardiovascular, and degenerative diseases, reduced fertility, and possibly the health of newborns. Cigarette smoke contains many components and exerts its genotoxicity in part by generating reactive oxidative stress. Telomeres consist of repeated 'G' rich sequences and associated proteins located at the chromosomal ends that maintain chromosomal integrity. We tested the hypothesis that telomere shortening and dysfunction are implicated in smoke associated oxidative damage and chromosomal instability using early mouse embryos in vitro and short-telomere mouse model. Mouse embryos exposed to smoke components, cigarette smoke condensate (CSC) at the concentration of 0.02 mg/ml continuously or 0.1mg/ml for 20 h, or cadmium at 5-100 microM, exhibited increased oxidative stress and telomere shortening and loss, associated with chromosomal instability, apoptosis, and compromised embryo cleavage and development. Remarkably, reduction of oxidative stress by an antioxidant N-acetyl-L-cysteine (NAC) greatly reduced these toxicities. Notably, cadmium led to more severe oxidative damage and telomere dysfunction, which could be more effectively rescued by antioxidant treatment, than did CSC. Moreover, short telomeres predisposed embryos to smoke component-induced oxidative damage. These data further extend our understanding of mechanisms underlying smoke-induced oxidative damage to include telomere dysfunction and chromosomal instability

Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres

OBJECTIVE: To investigate whether or not fertility is reduced in women carrying mutations in the BRCA genes (BRCA1 and BRCA2), compared with noncarrier family members. DESIGN: Matched case-control study. SETTING: Academic. PATIENT(S): A total of 2,254 BRCA carriers and 764 noncarrier controls who were from the same families as the carriers, but tested negative for the BRCA mutation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): History of fertility problems (yes/no) and prior use of fertility medications (yes/no) in cases and controls, obtained through administered questionnaire. RESULT(S): There was no difference in mean parity between carriers (1.9) and noncarriers (1.9). CONCLUSION(S): This study, which is the first epidemiologic study to specifically investigate the effect of carrying a BRCA mutation on parity and fertility, suggests that there is likely little or no effect of the BRCA gene mutation on fertility. To our knowledge previous epidemiologic studies have not investigated the effect of carrying a BRCA mutation on parity or fertility. However, we expect that if fertility differences exist, they would be toward the limits of reproductive life, thus larger studies with age-stratified analyses are needed to definitively answer this question.

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients

Young women diagnosed with cancer have the option of preserving their fertility by using assisted reproductive technology (ART) techniques prior to undergoing cancer treatment. This article presents a composite case of a young woman with cancer who had many unanswered emotional and ethical questions about her future as a parent. Fertility preservation techniques, including preimplantation genetic diagnosis (PGD), and related patient education are described. Current literature regarding reproductive counseling for cancer survivors is reviewed. Resources for providing psychosocial support for decisions about fertility preservation are lagging behind the rapid pace of scientific advancements in cancer treatment and ART. As more young women are surviving cancer and taking steps to preserve fertility, there is great need for the provision of psychologic support services and the establishment of ethical guidelines to aid them on this path. Women's health care providers can provide support to cancer survivors facing fertility and parenting issues by becoming knowledgeable about the long-term aspects of decision making and developing educational materials and guidelines for these patients

Most patients inadvertently miss an occasional dose of antihypertensive therapy, and hence drugs that provide sustained blood-pressure (BP) reduction beyond the 24-h dosing interval are desirable. The primary objective of this study was to compare the 24-h mean ambulatory BP reductions from baseline after a simulated missed dose of the direct renin inhibitor aliskiren, irbesartan or ramipril. In this double-blind study, 654 hypertensive patients (24-h mean ambulatory diastolic BP (MADBP) >/=85 mm Hg) were randomized 1:1:1 to once-daily aliskiren 150 mg, irbesartan 150 mg or ramipril 5 mg. Doses were doubled after 2 weeks. At day 42, patients were again randomized equally within each group to receive 1 day of placebo ('missed dose') on either day 42 or day 49. Patients with a successful 24-h ambulatory BP measurement at baseline and on day 42/49 were included in the analyses. The 24-h mean ambulatory systolic BP (MASBP)/MADBP reductions from baseline after a missed dose of aliskiren 300 mg (9.3/7.0 mm Hg) were similar to irbesartan 300 mg (9.5/7.3 mm Hg) and significantly larger than ramipril 10 mg (7.1/5.0 mm Hg, P</=0.008). Loss of BP-lowering effect with aliskiren in the 24 h after a missed dose (1.0/0.7 mm Hg for 24-48-h vs 0-24-h MASBP/MADBP) was significantly lower than with irbesartan (3.6/2.2 mm Hg, P<0.01) or ramipril (4.0/2.6, P<0.0001). This equates to maintenance of 91/91% of the MASBP/MADBP-lowering effect with aliskiren, greater than irbesartan (73/77%) or ramipril (64/65%). The incidence of adverse events was similar across treatments (32.9-36.0%), although ramipril treatment was associated with an increased incidence of cough (ramipril, 6.1%; aliskiren, 0.5%; irbesartan, 1.8%). Aliskiren 300 mg provided a sustained BP-lowering effect beyond the 24-h dosing interval, with a significantly smaller loss of BP-lowering effect in the 24-48 h period after dose than irbesartan 300 mg or ramipril 10 mg.Journal of Human Hypertension advance online publication, 21 May 2009; doi:10.1038/jhh.2009.38

PURPOSE: Cancer survival rates are improving, and the focus is moving toward quality survival. Fertility is a key aspect of quality of life for cancer patients of childbearing age. Although cancer treatment may impair fertility, some patients may benefit from referral to a specialist before treatment. However, the majority of studies examining patient recall of discussion and referral for fertility preservation (FP) show that less than half receive this information. This study examined the referral practices of oncologists in the United States. METHODS: This study examined oncologists' referral practice patterns for FP among US physicians using the American Medical Association Physician Masterfile database. A 53-item survey was administered via mail and Internet to a stratified random sample of US physicians. RESULTS: Forty-seven percent of respondents routinely refer cancer patients of childbearing age to a reproductive endocrinologist. Referrals were more likely among female physicians (P = .004), those with favorable attitudes (P = .043), and those whose patients routinely ask about FP (odds ratio = 2.09; 95% CI, 1.31 to 3.33). CONCLUSION: Less than half of US physicians are following the guidelines from the American Society of Clinical Oncology, which suggest that all patients of childbearing age should be informed about FP