Faculty Bibliography

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

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or 'unwind' mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different