Faculty Bibliography

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 (2 microM) for 30 minutes and [Ca]i was determined by rationing 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

Uterine cervical stenosis of either congenital or acquired etiology is a contributing factor in fertility. In such patients it is technically difficult to traverse the tortuous or stenotic cervical canal, precluding diagnostic procedures such as endometrial biopsy and hysterosalpingography, as well as therapies such as in vitro fertilization and embryo transfer (IVF-ET) or insemination. The standard method of dilatation with successively larger dilators may be difficult and traumatic, causing false channels or perforation of the uterus. Fifteen patients were referred for cervical dilatation because of inability to gain access to the uterine lumen. Under fluoroscopic guidance, the cervix was cannulated and the endocervical canal dilated with an angioplasty balloon. Five women had simultaneous fallopian tube recanalization. Only one woman had mild postoperative vaginal bleeding that subsided spontaneously at 48 hours. No patients experienced pain requiring narcotics, and no infections occurred. Five women conceived, one after IVF-ET, two with intrauterine inseminations, and two spontaneously. In those who did not conceive, the cervix was easily cannulated after the procedure. Cervical dilatation may provide options for treatment that would otherwise not be available to a select group of infertile women with cervical stenosis

PURPOSE: To assess efficacy of uterine cervical dilation performed with fluoroscopic guidance to treat patients with infertility who have cervical stenosis, false channels within the endocervical canal, or both. MATERIALS AND METHODS: Fifteen patients in whom infertility was diagnosed were referred because the uterine lumen could not be accessed. Three of the patients had endometriosis. With fluoroscopic guidance, the cervix was cannulated and the endocervical canal was dilated with an angioplasty balloon or with dilators. Five patients underwent simultaneous fallopian tube recanalization. Five of 15 patients who underwent dilation subsequently underwent in vitro fertilization for embryo transfer (IVF-ET) or intrauterine insemination. RESULTS: Four patients became pregnant. Of those four, one underwent IVF-ET and one underwent intrauterine insemination. Two patients became pregnant spontaneously. In the five patients who underwent IVF-ET or intrauterine insemination and in the remaining eight patients, the cervix could be easily cannulated up to 7 months after dilation. CONCLUSION: Dilation of the uterine cervix may provide options for treatment in selected patients with infertility. The effect of dilation on patients with other sequelae of cervical obstruction such as endometriosis remains uncertain

OBJECTIVE: To determine whether oocytes from women harbor deletions in mitochondrial DNA (mtDNA) and whether deleted mtDNA is more common in oocytes from older women than oocytes from younger women. DESIGN: A polymerase chain reaction (PCR)-based strategy, which depends on deletions approximating otherwise widely separated primers to demonstrate mtDNA deletions in individual oocytes, was used. SETTING: Yale In Vitro Fertilization Clinic and Laboratory at Yale University School of Medicine. MAIN OUTCOME MEASURES: Primers flanked a region of the mitochondrial genome in which long direct repeated sequence predispose to deletions. The primers identified the 0.5-kb 'common' deletion. Deleted mtDNA was represented by a 0.5-kb band when primers separated by 5 kb were used. Control reactions used primers that amplify mtDNA outside the deletion hotspot. Positive controls included brain and/or muscle from aged individuals, and negative controls included fetal tissue and DNA-free blanks. Nested primers confirmed the specificity of the deleted product. RESULTS: Unfertilized oocytes, muscle, and brain tissue contained PCR products consistent with deleted mtDNA. Fetal tissue lacked the mtDNA deletion product. Deleted mtDNA was detected in single oocytes. Oocytes from older women were more likely to contain deleted mtDNA than oocytes from younger women. CONCLUSION: Deleted mtDNA in unfertilized oocytes may serve as a marker of oocyte senescence

PURPOSE: To compare anonymous and directed candidates for oocyte donation within a single program. METHODS: 75 consecutive candidates for oocyte donation (49 anonymous and 26 directed) were studied using a semistructured interview to collect data on demographics, psychosocial history, motivation, and candidate views on disclosure to a potential child. RESULTS: Donor groups were similar with regard to race, religion and education. Anonymous donors (mean age 27.33 years, SD 4.17) were significantly younger than directed donors (mean age 37.54 years, SD 4.94), (t = -4.83, df = 73, p < 0.001). Marital duration was significantly longer for directed donors (6.92 years, SD 5.64) versus (2.57 years, SD 3.96) for anonymous donors (t = -3.50, df = 38.47, p = .001). Forty-one percent of anonymous and 69% of directed donors had previous pregnancies (X2 = 4.60, p < .05). A greater percentage of anonymous donors (34.7%) felt that the potential child should be informed of his or her origins, compared to 19% of directed donors, but this difference fell short of statistical significance. CONCLUSIONS: There were more similarities than differences among groups of potential donors with the exception of age, marital status, and previous pregnancies. Differing views about disclosure were suggested in both groups with anonymous donors tending to opt for disclosure

OBJECTIVE: Pharmacologic disruption of microtubule function may provide effective therapy for advanced epithelial ovarian cancer, as has been observed in clinical trials using taxol. However, the limited availability of taxol and taxol's side effects emphasize a need to develop alternative antimicrotubule agents. Estramustine (EM) inhibits binding of microtubule-associated proteins (MAPs) to microtubules, promotes microtubule disassembly, disrupts spindle formation, and induces metaphase arrest in human prostate carcinoma and glioma cells in culture. We studied the effect of EM on DNA synthesis and on the cell cycle in four human ovarian carcinoma cell lines and examined the cell lines for evidence of MAP-like immunoreactivity. METHODS: The effect of EM on DNA synthesis and on the cell cycle was determined using [3H]thymidine incorporation assays and flow cytometry, respectively. Microtubule-associated protein-like immunoreactivity was determined using monoclonal antibodies directed against MAP 1A, MAP 1B, and MAP 2(2A + 2B) for Western analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: We demonstrated a dose-dependent inhibitory response to EM in BIXLER, DK2NMA, and SKOV3. BIX3 showed a dose-dependent inhibitory response to EM concentrations from 25 micrograms/mL to 100 micrograms/mL, but a stimulatory response at 10 micrograms/mL. Estramustine inhibited exponentially growing cells by causing mitotic arrest with subsequent accumulation of cells in G2/M phase of the cell cycle in all four cell lines. We found MAP 1A, MAP 1B, and MAP 2-like immunoreactivity in all four cells lines studied. CONCLUSIONS: These results are consistent with a MAP-microtubule mechanism of action for EM in ovarian carcinoma cells and provide reason to conduct further study of EM for potential use in the treatment of human epithelial ovarian cancer

Progressive arcuate glial peroxidase activity is associated with reproductive aging of the female rat. We tested the hypothesis that age-related increase of glial peroxidase activity within the arcuate nucleus and posterior periventricular area is due to endogenous estrogen and could be reduced by an antiestrogen, keoxiphene, or a gonadotropin-releasing hormone super agonist, goserelin acetate (Zoladex), or both. At 3 months of age, 4-5-day regularly cycling female rats were divided into four groups. One group served as a baseline 3-month-old control and was killed by perfusion-fixation followed by serial sectioning of the hypothalamus and staining with 3,3'-diaminobenzidine tetrahydrochloride (DAB). Other groups consisted of animals receiving keoxiphene, goserelin acetate depot (Zoladex), or sham implants for 6 months. Two months after removal of the implants, at 11.25 months of age, the animals were killed by perfusion-fixation, and serial hypothalamic sections were stained with DAB. Sections from each of the four groups were examined in a fixed-size standard test area using a computerized image analyzer to assess the surface density of the DAB reaction. Overall, periventricular peroxidase reaction of the 11.25-month keoxiphene group showed a 63.5% increase in surface density of DAB reaction over the 3-month control group (5,834.6 +/- 101.0 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)), whereas the 11.25-month Zoladex group showed a 42.4% increase in surface density of DAB reaction over the 3-month control group (5,078.5 +/- 114.6 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)). This was in sharp contrast to the 11.25 month sham control group, which showed a 94% increase over the 3-month control group (6,926.6 +/- 121.3 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)), p < 0.0001. The differences in increase of the surface density of DAB in zone of reaction in keoxiphene-treated and Zoladex-treated rats were not equally distributed throughout the periventricular brain: the greatest difference was noted in the anterior arcuate (p < 0.0001), followed by the posterior arcuate (p < 0.001), with no difference noted in the posterior periventricular area. These results demonstrate than an antiestrogen and functional hypogonadotropic hypogonadism induced by a gonadotropin-releasing hormone agonist reduce the increase in hypothalamic glial peroxidase activity in the arcuate nucleus of maturing (aging) female rats but not in the posterior periventricular area. They support the hypothesis that estrogen secreted during the ovarian cycle contributes causally to hypothalamic aging in the female rat and indicate the presence of an estrogen-sensitive anterior region and an estrogen-insensitive posterior periventricular zone of age-dependent glial reaction. The failure of blockade by Zoladex further indicates that hypothalamic aging in the posterior periventricular area may be indifferent to the presence of the ovary. $$: