Faculty Bibliography

Our objective was to evaluate and compare the efficacy and mechanisms of co-culturing mouse embryos with Vero cells in both Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) and human tubal fluid (HTF) culture medium. Two-cell CB6F1 mouse embryos were cultured either in the presence of Vero cells (group A) or in culture medium alone (group B). In DMEM/F12 significantly more morulae developed in group A than in group B on day 3 (91 versus 23%; P less than 0.01). In contrast, the mouse embryos grew rapidly in HTF and significant differences were noted only in later embryonic stages (on day 5; 86% and 50%, P less than 0.01 of group A and B respectively, hatching or hatched). Similar experiments using DF1 and ICR mouse strains also revealed enhanced embryo development in the presence of Vero cells. To determine whether the embryo-enhancing effects of Vero cells were due to the removal of toxins or to the secretion of embryotrophic factors, ICR mouse embryos were cultured in fresh media with cells (group A), without cells (group B) and in cell-free conditions using cell-conditioned media which were obtained in the presence (group C) or absence (group D) of embryos. These results demonstrated that completion of hatching was highest (52%; P less than 0.01) in group A after 6 days in culture. There were no significant differences between groups B, C and D (rates of total hatching 18, 17 and 17%, respectively). It is concluded that Vero cells improve the development of mouse embryos and this is likely to be due to removal of substances inhibitory to development.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE: To evaluate the effect of baseline ovarian cysts at the onset of controlled ovarian hyperstimulation for in vitro fertilization (IVF) on cycle outcome. DESIGN, PATIENTS: A review of 82 IVF cycles in 29 women in which each patient served as her own control. The stimulation regimen for each patient remained constant over time. Each woman had at least one cycle in which an ovarian cyst measuring 14 to 53 mm was present at baseline and one cycle in which no such cyst was present. SETTING: The In Vitro Fertilization Program at Yale University School of Medicine. RESULTS: There was no statistically significant difference in cycle cancellation rates, baseline serum estradiol (E2), peak serum E2, number of follicles present at retrieval, number of oocytes retrieved, or fertilization rate between groups. Stimulation regimen, cyst size, and age were unrelated to outcome. The number of cysts present at baseline correlated positively with the number of follicles present at retrieval. CONCLUSION: Baseline ovarian cysts in the setting of a low baseline E2 level do not affect the clinical response to controlled ovarian hyperstimulation in IVF cycles

OBJECTIVE: Our objective was to determine whether impaired ovarian function induced by short-term creation of a galactosemic state in the rat might be prevented by the coadministration of an aldose reductase inhibitor. STUDY DESIGN: Prepubertal Sprague-Dawley rats were fed four different diets including (1) control, (2) 40% galactose, (3) 40% galactose and an aldose reductase inhibitor, and (4) an aldose reductase inhibitor with the control diet. Percentage germinal vesicle breakdown, postovulatory oocyte quantities, hormonal parameters, ovarian histologic evaluation, and ovarian galactitol concentrations were determined. RESULTS: The galactose-fed animals (group 2) had decreased germinal vesicle breakdown (47%) versus control (69%, p < 0.05). Galactose-exposed animals had significantly decreased quantities of postovulatory eggs (6.4 per animal) after menotropin ovarian stimulation in comparison with controls (14.1, p < 0.01). In rats exposed to high dietary levels of galactose (group 2) ovarian galactitol concentrations were significantly higher (protein 42.12 mumol/gm versus 0.0 for controls, p < 0.005). When galactose-fed animals received the aldose reductase inhibitor, ovarian accumulation of galactitol was significantly reduced and the observed detrimental effects on the oocyte were prevented. CONCLUSION: Galactitol accumulation or metabolic flux through aldose reductase in galactosemic rodents may be involved in the demonstrated ovarian dysfunction

BACKGROUND: Many couples undergo in vitro fertilization due to occlusion of the fallopian tubes. Chlamydia trachomatis infections are a major cause of this tubal damage. Since this organism has also been associated with poor pregnancy outcome, we investigated whether a past exposure to C. trachomatis was associated with spontaneous abortion following in vitro fertilization and embryo transfer. METHODS: Sera from 145 women undergoing IVF were diluted 1:128 and tested for IgG antibodies to C. trachomatis by an immunoperoxidase assay, using infected cells fixed to slides. All subjects and their partners were negative for C. trachomatis by culture or by DNA hybridization. RESULTS: Serological evidence of a past chlamydial infection was observed in 33.8% of the women. The incidence of antichlamydial IgG was greater (P less than 0.001) in women whose infertility was due to known tubal disease (37 of 78; 47.4%) than in women whose infertility was due to other causes (12 of 67; 17.9%). Spontaneous abortions after embryo transfer occurred in 20% of the subjects. The incidence of antichlamydial IgG in aborting women (20 of 29; 69.0%) was greater (P less than 0.001) than the incidence in either women with successful pregnancies (9 of 38; 23.7%) or women who did not become pregnant (20 of 78; 25.6%) after IVF. No relation was observed between antichlamydial antibody status and maternal age, the number of oocytes aspirated, the number of oocytes fertilized, and the number of embryos transferred. CONCLUSIONS: A previous infection with C. trachomatis may increase susceptibility to subsequent spontaneous abortion, even in the absence of a detectable current infection

In situ hybridization allows one to directly visualize DNA sequences of interest for which probes are available. Fluorescent signals can be detected in single cells in either metaphase or interphase nuclei, thus obviating the need for cell synchronization. The potential to use this technology in conjunction with in vitro fertilization techniques to genetically analyze an embryo before transfer could offer patients at genetic risk an alternative. Currently, these patients wait until 9 to 16 weeks' gestation to find out about the genetic makeup of their pregnancy. As such, sexing the embryos of patients at risk for transmitting sex-linked disorders or for performing numerical chromosome analysis could be performed before embryo transfer. Techniques for applying in situ hybridization to single cells obtained from embryos are presented. Hybridization of specific probes to blastomeres of mouse and human embryos as well as sperm is demonstrated

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n = 200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n = 116 cycles) failed to fertilize in a previous cycle. Group B (n = 40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n = 94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently

Human ovarian tissue and bovine ovarian stroma and follicles bound tritiated oxytocin and tritiated arginine vasopressin with similar affinity, whereas bovine corpora lutea bound tritiated oxytocin only. Competition for the binding of tritiated oxytocin by various agonists and antagonists was suggestive of receptor function. The number of oxytocin binding sites varied cyclically in all tissues. In bovine ovarian stroma and corpora lutea the concentrations were lowest on day 14 and highest on days 17, 19, and 21 after ovulation, with a striking peak in the luteal concentration on day 19. In human ovarian tissues the concentrations also were highest in samples obtained in late luteal phase. In large follicles the concentration of oxytocin binding sites was highest on the day of estrus and lowest on day 7. In bovine ovary the number of arginine vasopressin binding sites was approximately 50% lower than oxytocin binding sites and the cyclic variations were not significant. Human ovarian tissue had similar numbers of oxytocin and arginine vasopressin binding sites. Because bovine ovaries produce oxytocin and arginine vasopressin the results suggest a paracrine or autocrine role for these neuropeptides in luteolysis and ovulation. Although their synthesis in human ovaries is still controversial the presence of binding sites suggests a physiologic role in the regulation of human ovarian function as well.

We developed a method for the biopsy of preimplantation mouse embryos (preembryos) at the four- to eight-cell stage, which uses partial zona pellucida dissection. The preembryos were collected in calcium- and magnesium-free phosphate-buffered saline solution with 0.01% ethylenediaminetetraacetic acid, 0.1 mol/L sucrose, and 4 mg/ml of bovine serum albumin to facilitate removal of blastomeres. This allows entry of a fine micropipette into the perivitelline cavity with subsequent removal of a single blastomere by gentle suction. The majority of embryos (75%) from which biopsy specimens were obtained in this fashion developed to the blastocyst stage. The blastomeres obtained were mainly intact and they were fixed to glass slides. After permeabilization, in situ hybridization was performed with chromosome X- and chromosome 3-specific probes. Human unfertilized eggs and blastomeres from human polyspermic embryos also have been analyzed by in situ hybridization with chromosome specific probes. The combination of nondestructive embryo biopsy and in situ hybridization is a possible approach for preimplantation genetic diagnosis

Interstitial pregnancy which resulted in a term, live infant was found in association with pathologic torsion of the uterus. Antenatal sonograms revealed a hypervascular area anterior to the uterus

Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain. On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia, and two (6.9%) had Neisseria and Chlamydia, whereas in 15 (48.3%) patients no pathogen was isolated. Interferon-gamma was present in significantly more sera (p less than 0.025) from patients with pelvic inflammatory disease (65.5%) than from women without pelvic inflammatory disease (15.8%). Sera from 10 healthy women lacked detectable interferon-gamma. In patients with only Neisseria, seven (87.5%) had circulating interferon-gamma; three (60%) of the women with only Chlamydia, one (50%) woman with Neisseria and Chlamydia, and eight (57.1%) with no identified pathogens were also positive for interferon-gamma. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida-induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon-gamma by anti-interferon-gamma antibody but not by treatment with anti-interferon-alpha antibody. The persistence of interferon-gamma in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage