Faculty Bibliography

PURPOSE: Diploidy in embryos developing from single pronucleated zygotes can occur following parthenogenetic activation or by asynchronous inflation of pronuclei following fertilization. To distinguish between these two mechanisms, sexing was performed. RESULTS: The presence of a Y chromosome in the embryo was considered proof that fertilization had occurred. Twenty-one dividing embryos originating from single pronucleated zygotes were analyzed using the polymerase chain reaction or fluorescence in situ hybridization. In total, 43% (9/21) of the embryos showed Y chromosomes. CONCLUSION: It can be extrapolated that more than 80% of them originated from fertilized eggs

Evidence of sexual dimorphism before fetal gonadal differentiation in mammals has been accumulating, suggesting that male embryos develop faster than female ones. The current investigation was performed to evaluate whether the development rate of precompacted human embryos is controlled by sex chromosomes. Sex was determined by polymerase chain reaction and fluorescence in situ hybridisation in 172 arrested embryos derived from in vitro fertilisation. The sex ratio (1.02:0.98) did not differ significantly from 1:1. Although more males appeared to have greater fragmentation, the difference between the sex ratios of highly fragmented and normal embryos (1.08:0.92) was not significant. Arrested female embryos had a tendency to exhibit more than five nuclei and less than 10% fragmentation, but the trend was not statistically significant. The current results suggest that the first developmental block in human embryos occurs prior to and shortly after genomic activation and is not determined by the presence of the Y chromosome

OBJECTIVE: Our objective was to evaluate the presence of asymptomatic Chlamydia trachomatis infection by means of the polymerase chain reaction in male members of couples with previously undiagnosed infertility. STUDY DESIGN: Twenty-eight infertile-couples who had negative cultures or negative results when tested by deoxyribonucleic acid probe for Chlamydia trachomatis in semen and cervical samples were studied. Semen samples were tested for Chlamydia trachomatis by means of the polymerase chain reaction. Sera from both partners were diluted 1:128 and tested for immunoglobulin M antibodies to Chlamydia. Sera and ejaculated sperm were evaluated for the presence of antisperm antibodies. Semen analyses were also performed. RESULTS: Chlamydia trachomatis was identified in semen from 11 (39.3%) of the male partners. Its detection correlated with the presence in the ejaculate of motile sperm containing antisperm antibodies (p < 0.01). Either antisperm immunoglobulin G, immunoglobulin A, or both were located on sperm only from 5 (45.5%) of the 11 men whose results were positive when tested for Chlamydia trachomatis. Similarly, immunoglobulin G or immunoglobulin A antibodies to sperm were only detected in 5 (45.5%) of the spouses of men with Chlamydia in semen. Immunoglobulin M antibody to Chlamydia trachomatis was identified in only one of the men. However, antichlamydial immunoglobulin M antibodies were present in sera from 6 (54.5%) female partners of men with seminal Chlamydia trachomatis but in none of the other 17 women (p < 0.01). CONCLUSION: Although undetected by culture of deoxyribonucleic acid probe of semen samples, Chlamydia trachomatis was nevertheless identified in semen of some symptom-free men by the polymerase chain reaction. This is probably a result of the increased sensitivity of the polymerase chain reaction to detect Chlamydia trachomatis. The increased prevalence of an autoimmune response to sperm in men with this organism in their semen suggests that a subclinical chlamydial infection may activate an immune response to sperm. A similar association between Chlamydia trachomatis in semen and circulating antisperm antibodies in female partners indicates that Chlamydia may also induce an immune response to sperm in women. Infertility in these couples may be the result of a direct inflammatory response in the cervix or endometrium to repeated Chlamydia exposure or of the ability of Chlamydia to evoke an immune response to spermatozoa

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

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

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

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

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)