Lead concentrates in ovarian follicle compromises pregnancy.

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.

Mitochondrial dysfunction leads to telomere attrition and genomic instability.

Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies.

Noninvasive imaging of spindle dynamics during mammalian oocyte activation.

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.

Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos.

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 microm) than both immature (20.4 +/- 2.4 microm) and mature (19.5 +/- 2.2 microm) 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.

Cannulation of a resistant internal os with the malleable outer sheath of a coaxial soft embryo transfer catheter does not affect in vitro fertilization-embryo transfer outcome.

OBJECTIVE: To assess the impact of cannulation of a resistant cervical os with the outer malleable sheath of a double-lumen, soft ET catheter on IVF-ET outcomes. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): One hundred forty-two patients undergoing 142 ETs. INTERVENTION(S): Trial ultrasound-guided ET at all transfers, leaving the malleable outer sheath in situ when the soft inner catheter could not negotiate the internal os. MAIN OUTCOME MEASURE(S): Implantation rate and clinical pregnancy rate. RESULT(S): In 102 ETs (71.8%), the soft inner sheath easily negotiated the internal os (group 1). Forty ETs (28.2%) required cannulation of resistant internal ora with the outer sheath of the trial catheter (group 2). Implantation rates (35% vs. 32% in groups 1 and 2, respectively) and clinical pregnancy rates (50% vs. 45%) were not significantly different between groups. Blood was present on the transfer catheter after ET more frequently in group 2 than in group 1 (55% vs. 15%); however, neither the implantation rate nor the clinical pregnancy rate were affected by the presence of blood. CONCLUSION(S): Cannulation of a resistant internal os by the malleable outer sheath and blood on the transfer catheter after ET do not have an adverse effect on implantation rate or clinical pregnancy rate.

Trace element concentrations in follicular fluid of small follicles differ from those in blood serum, and may represent long-term exposure.

OBJECTIVE: To determine the levels of elements in follicular fluid (FF) of patients undergoing IVF and evaluate the relationship between the concentration of elements in FF, follicular volume, and blood. DESIGN: Prospective blinded study. SETTING: University-based IVF center. PATIENT(S): Follicular fluid/blood samples from 6/3 patients, respectively, undergoing IVF. INTERVENTION(S): Single follicular aspirations of 33 follicles were performed. Blood samples ( approximately 5 mL) were drawn at the time of oocyte retrieval from 3/6 patients only. The concentrations 26 elements were measured by inductively coupled plasma mass spectroscopy. MAIN OUTCOME MEASURE(S): Trace elements concentrations in follicular fluid and blood. RESULT(S): [1] Calcium and magnesium were the most abundant, followed by Cu, Zn, Fe, Cr, Rb. The elements V, Sr, Se, B, As, Pb, Al, Mo, Mn, and Cs were found in trace amounts. The elements Li, Be, Ag, Cd, Ba, Ti, Bi, U were not detected. [2] Element concentrations in small follicles frequently differed from those of large follicles. [3] Element concentrations in large follicles more closely resembled those in blood. CONCLUSION(S): Concentrations of elements in FF of small follicles can differ from those of large follicles in the same woman and from those of blood serum. When follicles grow they become filled with fluid of an elemental composition similar to blood. Concentrations of elements in small follicles may represent longer term element exposure, whereas those of growing follicles represents the coincident blood concentrations.

Pregnancy outcome in in vitro fertilization decreases to a plateau with repeated cycles.

Clinical pregnancy rate (CPR) and implantation rate (IR) in 1,177 patients who had 1,788 fresh, nondonor, nonPGD IVF cycles were highest in cycle 1, significantly declined in cycle 2, and reached a plateau for cycles 3-5 at a rate lower than in cycle 2. In patients >38 years of age CPR and IR in cycles 1 and 2 were significantly lower than in younger patients, but there was no decline in CPR or IR with advancing IVF attempts.

Ultrasound-guided miduterine cavity embryo transfer is associated with a decreased incidence of retained embryos in the transfer catheter.

After initiating a policy of ultrasound-guided miduterine cavity embryo transfer (ET) on August 1, 2001, the incidence of retained embryos in the transfer catheter declined significantly from 4.2% (46 of 1,101 ETs) during the time interval January 1, 2000 to July 31, 2001, to 0.7% (6 of 908 ETs) during the time interval August 1, 2001 to December 31, 2002. Blood on the transfer catheter (38% vs. 19%) occurred with significantly greater frequency at ET in which embryos were retained; ETs of > or =4 embryos were more likely to be associated with retained embryos than ETs of <4 embryos (3.7% vs. 2.2%), and both the clinical pregnancy rate (PR) and implantation rate (IR) were lower when embryo retention occurred at ET than they were when embryos were not retained (25% vs. 34% clinical PR; 12% vs. 19% IR).

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

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.

Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation.

Cell shrinkage is an incipient hallmark of apoptosis and is accompanied by potassium release that decreases the concentration of intracellular potassium and regulates apoptotic progression. The plasma membrane K+ channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here we provide evidence that two-pore domain K+ (K(2P)) channels underlie K+ efflux during apoptotic volume decreases (AVD) in mouse embryos. These K(2P) channels are inhibited by quinine but are not blocked by an array of pharmacological agents that antagonize other K+ channels. The K(2P) channels are uniquely suited to participate in the early phases of apoptosis because they are not modulated by common intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K+ channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. We propose that K(2P) channels are the pathway by which K+ effluxes during AVD and RVD and that apoptosis co-opts mechanisms more routinely employed for homeostatic cell volume regulation.

Checkpoint for DNA integrity at the first mitosis after oocyte activation.

Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

Oxidative stress contributes to arsenic-induced telomere attrition, chromosome instability, and apoptosis.

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.