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The oocyte is the major determinant of embryo developmental competence in women. It delivers half the chromosomal complement to the embryo, but the maternal and paternal genomes are neither symmetrical nor equal in their contributions to embryo fate. Unlike the paternal genome, the maternal genome carries a heavy footprint of parental aging. Indeed, age is the single best predictor of reproductive outcome in women, and the oocyte is the locus of reproductive aging in women. The oocyte transmits not only the mother's nuclear but also her mitochondrial genome to the embryo, and mitochondrial DNA is known to be especially susceptible to aging. Morphological studies of the oocyte and its associated cumulus corona cells provide only marginal value in the assessment of embryo developmental potential. A number of novel technologies, however, have improved the noninvasive assessment of oocyte quality. Moreover, during maturation, the oocyte ejects half its homologous chromosomes into the first polar body and half its chromatids into the second polar body. Polar body DNA is remarkably similar to that of the oocyte, so analysis of polar body DNA provides a window into the oocyte's genome and telomeres, which may enhance prediction of embryo developmental competence.

Objective To develop a method to evaluate spindle dynamics in living oocytes and in karyoplasts during the initial stages of activation and after pharmacological disruption of cytoskeleton. Design Morphological study using a novel microscope. Setting Translational research laboratory at marine biological laboratory. Animal(s) Six-week-old CD-1 or B6C3F1 mice superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Intervention(s) Spindles of living oocytes and karyoplasts were imaged at 5–10 minute intervals using the Pol-Scope during the initial stages of oocyte activation and after pharmacological disruption of cytoskeleton. Main Outcome Measure(s) Assessment of spindle dynamics using Pol-Scope imaging. Result(s) During oocyte activation, spindle mid-region birefringence increased, followed by spindle rotation and second polar body extrusion in both intact oocytes and karyoplasts. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate failed to induce spindle activation in 60% of living oocytes and caused spindle disruption in some oocytes. Inhibition of PKC by a myristoylated PKC pseudosubstrate inhibited metaphase II release in most oocytes evaluated (86.7%). Cytochalasin D inhibited only spindle rotation and separation. Nocodazole disrupted spindles in less than 5 minutes after administration. Conclusion(s) Pol-Scope imaging allows investigation at near real time of spindle dynamics during activation of living oocytes. Spindles also showed evidence of activation even in karyoplasts. The procedure may be useful for detecting functional spindle aberrations in living oocytes. Further studies are needed to determine whether spindle dynamics predict clinical outcome.

The effects of estradiol (E(2)) and progesterone (P(4)) on fluid and sodium regulation may have important clinical implications with respect to cardiovascular and renal disease as well as reproductive syndromes such as preeclampsia and ovarian hyperstimulation syndrome. We tested the hypothesis that sodium excretion is reduced in response to a sodium load during combined P(4)-E(2) treatment, but P(4) administration alone has little effect on sodium regulation. Fifteen women (22 +/- 2 yr) used a GnRH antagonist to suppress endogenous E(2) and P(4) for 9 d; for d 4-9, eight subjects used P(4) (200 mg/d), and seven subjects used P(4) with E(2) (two E(2) patches, 0.1 mg/d each). On d 3 and 9, isotonic saline (0.9% NaCl) was infused [120 min at 0.1 ml/kg body weight (BW).min], followed by 120 min of rest. Compared with GnRH antagonist alone, P([P4]) increased from 1.6 +/- 0.8 to 9.4 +/- 2.3 ng/ml (5.1 +/- 2.5 to 29.9 +/- 7.3 nmol/liter, P0.05) in the P(4) treated group, with no change in P([E2]). In the P(4)-E(2) treated group P([P4]) increased from 1.6 +/- 0.5 to 6.7 +/- 0.6 ng/ml (5.1 +/- 1.6 to 21.3 +/- 1.6 nmol/liter, P0.05 and P([E2]) increased from 17.9 +/- 6.3 to 200 +/- 41 pg/ml (65.7 +/- 23 to 734.6 +/- 150.0 pmol/liter, P0.05). Before isotonic saline infusion, renal sodium and water excretion were similar under all conditions, but during isotonic saline infusion, cumulative sodium excretion was lower in the P(4)-E(2) treated women (34.1 +/- 5.1 mEq) compared with GnRH antagonist (50.2 +/- 11.4 mEq). Sodium excretion was unaffected by P(4) treatment (48.0 +/- 8.2 and 41.2 +/- 5.1 mEq, for GnRH antagonist and P(4)). Compared with GnRH antagonist alone, P(4)-E(2) treatment increased distal sodium reabsorption and transiently decreased proximal sodium reabsorption, whereas P(4) treatment did not alter either distal or proximal sodium reabsorption. Before isotonic saline infusion, the plasma aldosterone (Ald) concentration was greater during P(4) treatment (153 +/- 25 pg/ml; 3883 +/- 1102 pmol/liter) and P(4)-E(2) treatment (242 +/- 47 pg/ml; 6373 +/- 1390 pmol/liter) than during their respective GnRH antagonist alone treatments [96 +/- 13 and 148 +/- 47 pg/ml (2598 +/- 475 and 3284 +/- 973 pmol/liter) for P(4) and combined P(4)-E(2), respectively]. Compared with GnRH antagonist alone treatments, preisotonic saline infusion plasma renin activity was greater only with P(4)-E(2) treatment, whereas the plasma atrial natriuretic peptide concentration was lower only with P(4) treatment. Isotonic saline infusion suppressed plasma Ald under all conditions, but decreased plasma renin activity only with P(4)-E(2) treatment (average decrease, 1.3 +/- 0.5 ng/ml angiotensin I.h; P0.05). In summary, we found that P(4)-E(2) treatment decreased sodium excretion via either renin-angiotensin-Ald system stimulation or direct effects on kidney tubules. P(4) treatment at these plasma concentrations had no independent effect on the renal response to acute sodium loading. These data suggest that E(2) is the more powerful reproductive hormone involved in sodium retention relative to P(4), and that estrogen-induced up-regulation of P(4) receptors is required for the effects of P(4) on sodium regulation.

Egg infertility remains the greatest challenge in the treatment of the infertile couple. As women increasingly delay attempts at childbearing, egg infertility has become more prevalent. Attempts to overcome egg infertility by superovulation and in vitro fertilization have produced an epidemic of multiple gestations, itself a major public health concern. The pathophysiology of egg infertility arises from chromosomal nondisjunction. Cytogenetic analyses of polar bodies and/or blastomeres currently provide the most powerful predictors of egg infertility. Approaches that label all chromosomes (spectral karyotyping and comparative genomic hybridization), or identify predisposition to aneuploidy (spindle imaging, telomere length measurement) are on the horizon. For the foreseeable future, the treatment of egg infertility will be limited to egg donation for severe cases and transfer of the most viable embryos for milder cases. Oocyte reconstitution not only lacks evidence of clinical efficacy, but also biological credibility, given that growing evidence supports the primacy of chromosomes themselves in meiotic nondisjunction.

The transcription factor Oct-4 is expressed in germ cells and also is considered as a marker for pluripotency of stem cells. We first examined dynamics of Oct-4 protein expression during preimplantation development using both Western blot analysis, and immunofluorescence staining. We show that intact Oct-4 protein is not detected in either ovulated mature oocytes, or in zygotes and 2-4-cell embryos, which are the only known totipotent cell types in mammals. This finding is unexpected, since Oct-4 has been proposed to play a role in the control of totipotency. The results suggest that Oct-4 is not indispensable for fertilization and early cleavage. Rather, expression of Oct-4 protein is first detected in the nuclei of 8-16 cell morula, increases in early blastocysts, and declines in late blastocysts, in which most Oct-4 protein is confined to the inner cell mass (ICM) region, consistent with previous findings. We further compared Oct-4 protein expression in diploid and tetraploid blastocysts derived from normal fertilization or parthenogenesis, as well as expression in diploid androgenetic blastocysts. Expression levels and localization of Oct-4 protein are similar in both diploid and tetraploid early blastocysts, regardless of whether blastocysts are derived from fertilization or parthenogenesis. Androgenetic diploid blastocysts also express similar levels of Oct-4. Late blastocysts generated by both fertilization and parthenogenesis show a similar pattern of Oct-4 expression, suggesting that paternal genome activation is not required for Oct-4 expression. Expression of Oct-4 protein does not differ between diploid and tetraploid embryos, indicating that tetraploidy does not influence Oct-4 expression. Thus, expression of Oct-4 protein is initiated at morula stage in preimplantation embryos and completely controlled by a mechanism activated in oocytes. Downregulation of Oct-4 expression coincides with differentiation of trophectoderm. Similar profiles of Oct-4 expression observed in embryos with different ploidy and genome composition, are suggestive of Oct-4 being necessary but not sufficient for developmental potency.

Objective To assess differences in pregnancy and implantation rates as a function of the embryo placement. Design Prospective cohort study. Setting A tertiary care center. Subject(s) All fresh, nondonor IVF cycles performed in 2001. Intervention(s) Alteration in embryo transfer (ET) target location from the fundal region to the middle to lower uterine segment. Main outcome measure(s) Clinical pregnancy rate (sonographic sac evidence/number of transfer cycles), implantation rate (number of sacs/number of embryos transferred), patient age, peak E 2 , and fertilization rate. Result(s) A total of 393 fundal and 273 lower to middle uterine segment ETs were performed. The pregnancy (PR), implantation, and birth rates were significantly higher after a middle to lower uterine segment ET compared with fundal ET (39.6% vs. 31.2%; 21% vs. 14%; and 34.1% vs. 26.2%, respectively). Groups did not differ regarding patient age, basal FSH, peak E 2 , number of intracytoplasmic sperm injection (ICSI) cycles, fertilization rate, embryo quality, or number of embryos transferred. Conclusion(s) Both PR and implantation rates are favorably affected by directing embryo placement to the lower to middle uterine segment. By some unknown mechanism, it appears that this endometrial location provides a more favorable region for embryo deposition.

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR−/−) and wild-type (WT) mice. The MSCs isolated from mTR−/− mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR−/−MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.

Objective To characterize the architecture of the zona pellucida in living human eggs and embryos, noninvasively with the PolScope, a digital polarizing light microscope. Design The PolScope was used to examine zonae pellucida of living human eggs and embryos. Setting Academic IVF clinic. Patient(s) Patients undergoing fresh, nondonor infertility treatment who underwent egg aspiration, fertilization by intracytoplasmic sperm injection or traditional IVF, and cleavage-stage embryo transfer (day 3). Intervention(s) The PolScope imaged the zona of eggs before intracytoplasmic sperm injection and in cleavage-stage embryos before transfer. Main outcome measure(s) Thickness and retardance of three zona layers were measured from eight quadrants. Mean and variance in thickness and retardance were calculated for individual eggs and embryos, between eggs and embryos of a cohort, and across the sample patient population. Result(s) Cleavage-stage (day 3) embryos have thinner zonae (15.2 ± 2.9 μm) than both immature (20.4 ± 2.4 μm) and mature (19.5 ± 2.2 μm) eggs. The zona of embryos is thinner, primarily owing to thinning of the outer layer. The thicker the zona layer, the greater its retardance. Considerable variation exists in the thickness and retardance of zona layers around individual eggs and embryos and between members of a cohort. The zona of eggs and embryos from different patients differ in thickness, retardance, and variation. Conclusion(s) Thickness and organization of zonae pellucida of human eggs and embryos varies considerably and can be quantitatively imaged with the PolScope.

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24–36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 °C for up to 20 min. After heating, oocytes were returned to 37 °C and then imaged for another 20 min at 37 °C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 °C during 40 min of examination and their maximum retardance (1.72–1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 °C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 °C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 °C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 °C and 40 °C, respectively. Heating to 40 °C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 °C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 °C is crucial for normal spindle morphology.

The environmental contaminant arsenic causes cancer, developmental retardation, and other degenerative diseases and, thus, is a serious health concern worldwide. Paradoxically, arsenic also may serve as an anti-tumor therapy, although the mechanisms of its antineoplastic effects remain unclear. Arsenic exerts its toxicity in part by generating reactive oxygen species. We show that arsenic-induced oxidative stress promotes telomere attrition, chromosome end-to-end fusions, and apoptotic cell death. An antioxidant, N-acetylcysteine, effectively prevents arsenic-induced oxidative stress, telomere erosion, chromosome instability, and apoptosis, suggesting that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic-induced chromosome instability and genotoxicity. Embryos with shortened telomeres from late generation telomerase-deficient mice exhibit increased sensitivity to arsenic-induced oxidative damage, suggesting that telomere attrition mediates arsenic-induced apoptosis. Unexpectedly, arsenite did not cause chromosome end-to-end fusions in telomerase RNA knockout mouse embryos despite progressively damaged telomeres and disrupting embryo viability. Together, these findings may explain why arsenic can initiate oxidative stress and telomere erosion, leading to apoptosis and anti-tumor therapy on the one hand and chromosome instability and carcinogenesis on the other.

Objective To study the relationship between IVF-ET pregnancy outcomes and measures of embryo placement. Design Case–control study. Setting Tertiary care center. Patient(s) Twenty-three patients who underwent two ultrasonography-guided ETs, of which one resulted in a clinical pregnancy and the other did not. Main outcome measure(s) Point of embryo placement normalized to the endometrial cavity length (the transfer point), distance from the point of embryo placement to the uterine fundus, time required for ET, contact with the uterine fundus, and evidence of trauma. Videotaped ETs were quantitatively analyzed. Result(s) From February 1, 2000, to March 31, 2001, videotaped ETs from 23 pairs of pregnant and nonpregnant cycles were identified. Embryo placement was more shallow in pregnancy cycles than in nonpregnancy cycles. The groups did not differ in the absolute distance of embryo placement to the fundus, ovarian stimulation, or other features of the ET. Conclusion The transfer point may serve as a better marker of embryo position than does the absolute distance to the uterine fundus.

Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers).

Objective: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. Design: Morphological study. Setting: Academic IVF clinic. Patient(s): Women undergoing oocyte retrieval for ICSI treatment. Intervention(s): Oocytes were examined after in vitro maturation. Main Outcome Measure(s): The study examined meiotic spindle morphologic features and chromosome alignments. Result(s): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. Conclusion(s): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination.

The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37 degrees C is crucial for normal spindle function. As chromosomes#10; are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation#10; efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer.

Objective: To examine the effects of different thermodynamic control systems on the temperature stability of human eggs during in vitro manipulation, with the integrity of meiotic spindles imaged using the LC-PolScope (Cambridge Research & Instrumentation, Inc., Woburn, MA). Design: We performed intracytoplasmic sperm injection (ICSI) and/or imaging of eggs with the temperature regulated by three different systems: thermostated coverslip (system 1), thermostated coverslip combined with objective heater (system 2), and conventional stage warmer (system 3). Setting: Academic in vitro fertilization clinic. Patient(s): Oocytes were aspirated from stimulated ovaries of patients undergoing oocyte retrieval for ICSI. Intervention(s): Measurement of temperature regulation in media surrounding eggs during in vitro manipulation and imaging. Main Outcome Measure(s): Rate of oocytes with spindles, fertilization rates, and clinical pregnancy rates after ICSI. Result(s): We imaged spindles in more oocytes with system 2 (81.2%) than with system 1 (61.4%). Spindles could not be imaged for system 3 because of technical limitations. Fertilization rates were significantly higher when oocytes were imaged and used for ICSI with system 2 (78.8%) than with system 1 (56.7%) or system 3 (64.0%). Most importantly, a significantly higher clinical pregnancy rate was observed when oocytes were manipulated with system 2 (51.7%) than with system 1 (25.0%) or system 3 (23.1%). No differences were found in average ages, number of previous cycles, number of eggs, or day 3 FSH or E 2 levels among groups. Conclusion(s): Imaging meiotic spindles with the PolScope provides an intracellular thermostat during ICSI. Rigorous thermal control during ICSI stabilized spindles and increased the fertilization and clinical pregnancy rates achieved after ICSI. The presence of birefringent spindles in living human eggs served as a monitor for in vitro conditions.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.

Vitamin D plays crucial roles in neuroprotection and neurodevelopment, and low levels are commonly associated with schizophrenia. We considered if the association was spurious or causal by examining the association of Vitamin D with Leukocyte Telomere Length (LTL), a marker of cellular aging. Vitamin D levels in 22 well-characterized schizophrenia cases were examined with respect to symptoms, cognition, and functioning. LTL was assessed using quantitative polymerase chain reaction (qPCR). The results showed that 91% (20) had deficient or insufficient Vitamin D levels, which were associated with excitement and grandiosity, social anhedonia, and poverty of speech. Sex-specific analyses showed strong associations of hypovitamintosis D to negative symptoms and decreased premorbid adjustment in males, and to lesser hallucinations and emotional withdrawal, but increased anti-social aggression in females. In females LTL was furthermore associated with Vitamin D levels. This study demonstrates a relationship of low vitamin D levels with increased cellular aging in females. It is also the first study to demonstrate potential sex-specific profiles among schizophrenia cases with hypovitaminosis.

Telomeres protect and cap linear chromosome ends, yet these genomic buffers erode over an organism’s lifespan. Short telomeres have been associated with many age-related conditions in humans, and genetic mutations resulting in short telomeres in humans manifest as syndromes of precocious aging. In women, telomere length limits a fertilized egg’s capacity to develop into a healthy embryo. Thus, telomere length must be reset with each subsequent generation. Although telomerase is purportedly responsible for restoring telomere DNA, recent studies have elucidated the role of alternative telomeres lengthening mechanisms in the reprogramming of early embryos and stem cells, which we review here.

In industrialized societies the progression of natural selection has been determined and in many cases superseded by social evolution. In the case of reproduction, there has been a decline and delay of childbearing without diminished sexual activity. While this has value for these societies, there are penalties associated with barren cycles. These include increases in endometriosis and breast and genital cancer. There also are associated issues regarding population movements that fill the “vacuums” left by underpopulation. These matters are of more than passing interest as we cope with unintended consequences of Man’s dominance over the environment and other life forms.

Background Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. Methods Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. Results Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. Conclusions These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.

To determine sex differences in osmoregulation of arginine vasopressin (AVP) and body water, we studied eight men (24 ± 1 yr) and eight women (29 ± 2 yr) during 3% NaCl infusion [hypertonic saline infusion (HSI); 120 min, 0.1 ml · kg body wt−1 · min−1]. Subjects then drank 15 ml/kg body wt over 30 min followed by 60 min of rest. Women were studied in the early follicular (F; 16.1 ± 2.8 pg/ml plasma 17β-estradiol and 0.6 ± 0.1 ng/ml plasma progesterone) and midluteal (L; 80.6 ± 11.4 pg/ml plasma 17β-estradiol and 12.7 ± 0.7 ng/ml plasma progesterone) menstrual phases. Basal plasma osmolality was higher in F (286 ± 1 mosmol/kgH2O) and in men (289 ± 1 mosmol/kgH2O) compared with L (280 ± 1 mosmol/kgH2O, P < 0.05). Neither menstrual phase nor gender affected basal plasma AVP concentration (P[AVP]; 1.7 ± 4, 1.9 ± 0.4, and 2.2 ± 0.5 pg/ml for F, L, and men, respectively). The plasma osmolality threshold for AVP release was lowest in L ( x-intercept, 263 ± 3 mosmol/kgH2O, P < 0.05) compared with F (273 ± 2 mosmol/kgH2O) and men (270 ± 4 mosmol/kgH2O) during HSI. Men had greater P[AVP]-plasma osmolality slopes (i.e., sensitivity) compared with F and L (slopes = 0.14 ± 0.04, 0.09 ± 0.01, and 0.24 ± 0.07 for F, L, and men, respectively, P < 0.05). Despite similar Na+-regulating hormone responses, men excreted less Na+ during HSI (0.7 ± 0.1, 0.7 ± 0.1, and 0.5 ± 0.1 meq/kg body wt for F, L, and men, respectively, P < 0.05). Furthermore, men had greater systolic blood pressure (119 ± 5, 119 ± 5, and 132 ± 3 mmHg for F, L, and men, respectively, P < 0.05) than F and L. Our data indicate greater sensitivity in P[AVP] response to changes in plasma osmolality as the primary difference between men and women during HSI. In men, this greater sensitivity was associated with an increase in systolic blood pressure and pulse pressure during HSI, most likely due to a shift in the pressure-natriuresis curve.

The purpose of this study was to determine estrogen (E2) and progesterone (P4) effects on atrial natriuretic peptide (ANP) control of plasma volume (PV) and transcapillary fluid dynamics. To this end, we suppressed reproductive function in 12 women (age 21–35 yr) using a gonadotropin releasing-hormone (GnRH) analog (leuprolide acetate) for 5 wk. During the 5th week, the women either received 4 days of E2administration (17β-estradiol, transdermal patch, 0.1 mg/day) or 4 days of E2 with P4 administration (vaginal gel, 90 mg P4 twice per day). At the end of the 4th and 5th week of GnRH analog and hormone administration, we determined PV (Evans blue dye) and changes in PV and forearm capillary filtration coefficient (CFC) during a 120-min infusion of ANP (5 ng · kg body wt−1 · min−1). Preinfusion PV was estimated from Evans blue dye measurement taken over the last 30 min of infusion based on changes in hematocrit. E2 treatment did not affect preinfusion PV relative to GnRH analog alone (45.3 ± 3.1 vs. 45.4 ± 3.1 ml/kg). During ANP infusion CFC was greater during E2 treatment compared with GnRH analog alone (6.5 ± 1.4 vs. 4.9 ± 1.4 μl · 100 g−1 · min−1 mmHg−1, P < 0.05). The %PV loss during ANP infusion was similar for E2 and GnRH analog-alone treatments (−0.8 ± 0.2 and −1.0 ± 0.2 ml/kg, respectively), indicating the change in CFC had little systemic effect on ANP-related changes in PV. Estimated baseline PV was reduced by E2-P4treatment. During ANP infusion CFC was ∼30% lower during E2-P4 (6.0 ± 0.5 vs. 4.3 ± 4.3 μl · 100 g−1 · min−1mmHg−1, P < 0.05), and the PV loss during ANP infusion was attenuated (−0.9 ± 0.2 and −0.2 ± 0.2 ml/kg for GnRH analog-alone and E2-P4treatments, respectively). Thus the E2-P4treatment lowered CFC and reduced PV loss during ANP infusion.

Objective: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI. Design: The LC polscope was used to examine spindles in an observational study of living oocytes. Setting: Academic IVF clinic. Patient(s): Women being treated for infertility. Intervention(s): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. Main Outcome Measure(s): Spindle structure in living oocytes and fertilization after ICSI. Result(s): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. Conclusion(s): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI.

Leukocyte telomere length (LTL) is longer in association with advanced paternal age, but this association has not been examined along with family history (FH) in schizophrenia. LTL was measured by PCR and compared across cases and controls as part of a study to examine the characteristics of paternal age related schizophrenia. The 53 schizophrenia cases had similar mean LTL as 20 controls, although cases were significantly older than controls and overwhelmingly smoked cigarettes. Multivariate analyses showed that a FH of schizophrenia was associated with longer LTL in both male and female cases. Later paternal age was also related to longer LTL in male cases, but with shorter LTL in female cases. Male cases with older fathers and a FH had the longest LTL. The genetic architecture associated with a familial risk for schizophrenia may include pathways that lengthen LTL. Paternal aging conferred an additional increase in LTL lengthening in male cases, but reduced LTL in female cases. The gender difference in LTL for paternal aging is consistent with the severe illness features reported for female cases with older fathers and could implicate epigenetic alterations in the paternal X chromosomal region with advanced paternal age in association with the risk for schizophrenia.

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.

Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175 microM H(2)O(2) that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P

The self-referencing electrode technique was employed to noninvasively measure gradients of dissolved oxygen in the medium immediately surrounding developing mouse embryos and, thereby, characterized changes in oxygen consumption and utilization during development. A gradient of depleted oxygen surrounded each embryo and could be detected >50 microm from the embryo. Blastocysts depleted the surrounding medium of 0.6+/-0.1 microM of oxygen, whereas early cleavage stage embryos depleted the medium of only 0.3+/-0.1 microM of oxygen, suggesting a twofold increase in oxygen consumption at the blastocyst stage. Mitochondrial oxidative phosphorylation (OXPHOS) accounted for 60-70% of the oxygen consumed by blastocysts, while it accounted for only 30% of the total oxygen consumed by cleavage-stage embryos. The amount of oxygen consumed by non-OXPHOS mechanisms remained relatively constant throughout preimplantation development. By contrast, the amount of oxygen consumed by OXPHOS in blastocysts is greater than that consumed by OXPHOS in cleavage-stage embryos. The amount of oxygen consumed by one-cell embryos was modulated by the absence of pyruvate from the culture medium. Treatment of one-cell embryos and blastocysts with diamide, an agent known to induce cell death in embryos, resulted in a decline in oxygen consumption, such that the medium surrounding dying embryos was not as depleted of oxygen as that surrounding untreated control embryos. Together these results validate the self-referencing electrode technique for analyzing oxygen consumption and utilization by preimplantation embryos and demonstrate that changes in oxygen consumption accompany important physiological events, such as development, response to medium metabolites, or cell death.

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.

The oxidation of cellular sulfhydryl (SH) groups has been implicated in the induction of apoptosis in various types of cells and in the disturbance of the meiotic spindle of murine oocytes during aging. The objective of this study was to determine whether the SH-specific oxidant diamide could inhibit embryo development and induce cell death, and whether the antioxidant dithiothreitol (DTT) could counteract such effects. Exposure of mouse zygotes to diamide for 3 h at 25 or 50 microM (but not 12.5 microM) resulted in cell cycle arrest and cell death with evidence of apoptosis. At higher concentrations (100 or 200 microM), diamide induced necrosis as evidenced by propidium iodide-positive pronuclei within 24 h of treatment. Simultaneous addition of DTT at equimolar concentration prevented these effects. However, when DTT was added later, it was no longer protective. DTT also effectively protected against the thiol-oxidative damage caused by diamide in blastocysts. These results suggest that altering thiol-redox status in zygotes and blastocysts may result in cell cycle arrest, apoptosis, and/or cell death.

To test the hypothesis that estrogen reduces the operating point for osmoregulation of arginine vasopressin (AVP), thirst, and body water balance, we studied nine women (25 ± 1 yr) during 150 min of dehydrating exercise followed by 180 min of ad libitum rehydration. Subjects were tested six different times, during the early-follicular (twice) and midluteal (twice) menstrual phases and after 4 wk of combined [estradiol-norethindrone (progestin), OC E + P] and 4 wk of norethindrone (progestin only, OC P) oral contraceptive administration, in a randomized crossover design. Basal plasma osmolality (Posm) was lower in the luteal phase (281 ± 1 mosmol/kgH2O, combined means, P < 0.05), OC E + P (281 ± 1 mosmol/kgH2O, P < 0.05), and OC P (282 ± 1 mosmol/kgH2O, P < 0.05) than in the follicular phase (286 ± 1 mosmol/kgH2O, combined means). High plasma estradiol concentration lowered the Posm threshold for AVP release during the luteal phase and during OC E + P [ x-intercepts, 282 ± 2, 278 ± 2, 276 ± 2, and 280 ± 2 mosmol/kgH2O, for follicular, luteal (combined means), OC E + P, and OC P, respectively; P < 0.05, luteal phase and OC E + P vs. follicular phase] during exercise dehydration, and 17β-estradiol administration lowered the Posm threshold for thirst stimulation [ x-intercepts, 280 ± 2, 279 ± 2, 276 ± 2, and 280 ± 2 mosmol/kgH2O for follicular, luteal, OC E + P, and OC P, respectively; P < 0.05, OC E + P vs. follicular phase], without affecting body fluid balance. When plasma 17β-estradiol concentration was high, Posm was low throughout rest, exercise, and rehydration, but plasma arginine vasopressin concentration, thirst, and body fluid retention were unchanged, indicating a lowering of the osmotic operating point for body fluid regulation.

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.

Objective: To compare the demographic and psychological characteristics of oocyte recipients and to determine whether the issue of disclosure about the use of a donor is a correlate of the decision to use an anonymous or directed donor. Design: Cross-sectional study. Setting: University teaching hospital. Patient(s): Ninety consecutive recipients of donated oocytes (64 of whom used anonymous donors and 26 of whom used directed donors). Intervention(s): Pretreatment psychosocial evaluation. Main Outcome Measure(s): Recipient opinions and attitudes regarding the choice of donor type and disclosure to others as determined through a semistructured interview. Result(s): There were no statistically significant differences with regard to demographic characteristics between recipients who used anonymous and directed donors. There were statistically significant differences between the groups with regard to the issue of disclosure. Recipients who used directed donors were more likely to have told others about using an oocyte donor and were more likely to indicate that they intended to inform the child about the nature of his or her conception. Conclusion(s): Oocyte recipients who use known donors differ significantly from those who use anonymous donors with regard to the issue of disclosure to others. Further studies are needed to determine the causal direction of this relation.

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction.

Objective: To develop an acceptable model system to study calcium activation of human oocytes. Design: Study of oocyte development and intracellular calcium [ca] i dynamics of activated oocytes. Setting: Research center affiliated with infertility service. Main Outcome Measure: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca] i levels, oocytes were incubated with fura-2 (2μM) for 30 minutes and [Ca] i was determined by ratioing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. Result(s): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca] i , followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. Conclusion(s): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca] i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

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.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.SBP and HR declined across the postpartum and were significantly lower in breastfeeding compared to formula feeding mothers (p0.05). 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.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.

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.