Faculty Bibliography

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

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

OBJECTIVE: Telomeres are DNA repeats which cap and protect chromosome ends, facilitate homologue pairing and chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen to mediate aging. Early germ cells contain telomerase, a reverse transcriptase which adds telomeres to 3-prime DNA ends, but telomerase activity declines in oocytes, fixing telomere length earlier during development. Experimentally induced telomere shortening in mice disrupts meiosis, impairs chiasmata formation, halts embryonic cell cycles, and promotes apoptosis in embryos, a phenotype which mimics reproductive senescence in women. Ethical constraints limit study of human embryos to nondestructive assays, such as morphologic evaluation under transmission optics, but cytoplasmic fragmentation is a reliable marker of apoptosis. STUDY DESIGN: Study design consisted of observational study of effect of telomere length in human eggs on cytoplasmic fragmentation, and on other morphologic features of preimplantation embryos. To test the hypothesis that telomere shortening triggers apoptosis in human embryos, we evaluated telomere length as a predictor of cytoplasmic fragmentation in embryos from women undergoing in vitro fertilization. RESULTS: Telomere length negatively predicted fragmentation in day 3 preimplantation embryos, after controlling for patient age and basal follicle stimulating hormone level. Telomere length did not predict other features of preimplantation embryo morphology. CONCLUSION: The finding that telomere length in human eggs predicts cytoplasmic fragmentation in embryos provides evidence that telomere shortening induces apoptosis in human preimplantation embryos, consistent with a telomere theory of reproductive senescence in women

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, P < 0.05) 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, P < 0.05 and P([E2]) increased from 17.9 +/- 6.3 to 200 +/- 41 pg/ml (65.7 +/- 23 to 734.6 +/- 150.0 pmol/liter, P < 0.05). 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; P < 0.05). 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

The effect of light on neuroendocrine functions is thought to be mediated through retinal inputs to the circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). The present studies were conducted to provide experimental evidence for this signaling modality in non-human primates. In the St. Kitts vervet monkey, anterograde tracing of SCN efferents revealed a monosynaptic pathway between the circadian clock and hypothalamic neurons producing luteinizing hormone-releasing hormone (LHRH). Using a variety of tracing techniques, direct retinal input was found to be abundant in the SCN and in other hypothalamic sites. Strikingly, in hypothalamic areas other than the SCN, primary visual afferents established direct contacts with neuroendocrine cells including those producing LHRH and dopamine, neurons that are the hypothalamic regulators of pituitary gonadotrops and prolactin. Thus, our data reveal for the first time in primates that light stimuli can reach the hypothalamo-pituitary-gonadal axis, directly providing a pathway independent of but parallel to that of the circadian clock for the photic modulation of hormone release

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

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects

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