Luis Blasco

Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay

Human spermatozoa were incubated in culture medium containing human serum albumin (HSA) to promote capacitation, which was monitored by a rapid chlortetracycline (CTC) fluorescence assay. Four CTC fluorescence patterns were readily distinguished, one of which appeared to be correlated with capacitated sperm. When capacitated sperm were treated with either ionophore A23187 or acid-solubilized mouse zonae pellucidae to induce the acrosome reaction, the CTC assay identified acrosome-reacted sperm by lack of fluorescence on the head. Fresh sperm would not undergo the induced acrosome reaction. The percentages of acrosome-reacted sperm identified by the CTC assay in induced and control populations were the same as those identified by the presently used indirect immunofluorescence and triple stain assays.

Impact of stage iii–iv endometriosis on recipients of sibling oocytes: matched case-control study

OBJECTIVE To evaluate the impact of severe endometriosis on IVF-ET outcome in women receiving oocytes from the-same donor. DESIGN A matched case-control study. SETTING Oocyte donation program at the Instituto Valenciano de Infertilidad. PATIENT(S) Fifty-eight recipients were included in a matched case-control study of IVF-ET in our oocyte donation program. Twenty-five patients were diagnosed by laparoscopy with stage III-IV endometriosis (group I), while the remaining 33 were free of the disease (group II). On the day of retrieval, oocytes from a single donor were donated to recipients from both groups. Some of the donors supplied oocytes for more than 2 patients. Recipients received steroid replacement therapy for endometrial preparation. INTERVENTION(S) Ovarian stimulation and oocyte retrieval in donors. Uterine embryo transfer (ET) in recipients after appropriate exogenous hormone replacement therapy (HRT). MAIN OUTCOME MEASURE(S) Pregnancy, implantation, miscarriage, and live birth rates. RESULT(S) The number of oocytes donated and fertilized, as well as the number of available and transferred embryos, was not statistically different between the two groups. Pregnancy, implantation, and miscarriage rates were not affected by stage III-IV endometriosis when compared with the control group. The live birth rate was 28.0% in the group with endometriosis and 27.2% in the control group. CONCLUSION(S) These results show that implantation is not affected by stage III-IV endometriosis. Given the contemporary methods of endometrial preparation for transfer of embryos derived from donor oocytes, any potential negative effect of severe endometriosis on the uterine environment is undetectable.

Effect of follicular size on oocyte retrieval, fertilization, cleavage, and embryo quality in in vitro fertilization cycles: a 6-year data collection

OBJECTIVE To determine the effect of follicular size, including the size of the leading follicle, on oocyte retrieval, fertilization, cleavage, and embryo quality in IVF cycles based on a large data collection. DESIGN Retrospective analysis of 1,109 IVF cycles between 1987 and 1993 at the Hospital of the University of Pennsylvania including 606 patients ranging in age from 23 to 49 years. RESULTS Follicles with a volume < or = 1 mL show a significantly lower oocyte recovery rate than follicles with a volume of > 1 mL. The highest recovery rate (83.5%) was found in follicles with a volume of 3 to 4 mL. Above a follicular volume of 7 mL, the oocyte recovery drops below that observed for follicles between 1 and 7 mL. Fertilization and cleavage rates were also higher in oocytes obtained from follicles > 1 mL compared with follicles < or = 1 mL. Although fertilization rates were fairly stable above volumes of 1 mL, cleavage rates continued to rise to a peak percentage of 92% with volumes between 6 and 7 mL. Leading follicle size did not have an effect on fertilization and cleavage rates of cohort oocytes. Embryo quality was not influenced significantly by follicular volume. CONCLUSION Based on this evaluation of a large number of follicles, follicular size is a useful indicator of oocyte recovery, fertilization, and cleavage in IVF cycles. For optimal results, the follicular fluid volume in gonadotropin- and hCG-stimulated cycles should be > 1 mL, which corresponds to a follicle diameter of > 12 mm, and not larger than 7 mL (24 mm). For timing of hCG administration, the number of adequate size follicles appears to be more important than the size of the leading follicle(s).

Turbidimetric analysis of human sperm motility.

A turbidimetric method has been developed for determining rapidly the fraction of sperm in human ejaculates which show the most vigorous motility. The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette. This results in a time-dependent increase in turbidity in the medium which is recorded spectrophotometrically as an increase in absorbance. The determination requires 10 minutes and yields both the fraction of rapidly moving sperm, FRM, and their average velocity, VRM. Examination of 25 samples yielded FRM values of 10% or lower, whereas values for VRM averaged about 100 microns/second. These vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity. It is suggested that this simple turbidimetric test be used in evaluation of human semen as a possible indicator of fertilizing capacity.

The zona-free hamster egg penetration assay as a prognostic indicator in a human in vitro fertilization program *

The present study was designed to test the validity of the hamster egg penetration assay as a prognostic indicator of male fertility in 54 patients undergoing in vitro fertilization. Human oocyte fertilization, cleavage, and pregnancy were compared with the results of this bioassay. Good correlation was found between hamster egg penetration and oocyte fertilization. Conversely, a definite lower limit of hamster egg penetration to define absolute male infertility could not be established because human oocyte fertilization, cleavage, and even pregnancy occurred in spite of low hamster egg penetration.

Human cervical mucus. I. Rheologic characteristics.

The viscoelastic properties of human cervical mucus were subjected to detailed characterization by microrheometry. An introduced to the range of parameters that can be measured and the sensitivity of microroheometry to variations in mucus viscoelasticity was presented, along with an interpretation of results obtained by this approach. Fresh cervical mucus samples were shown to be rheologically heterogeneous, reflecting compositional differences in situ. Mucas viscoelasticity was stable for several hours when samples were stored at ambient temperature in the microrheometer sample holder. Mucus viscoelasticity was strongly dependent upon nondialyzable solids (NDS) concentration. In order to eliminate (NDS) as a variable in comparative studies of mucus, procedures were developed for reconstitution of mucus at identical (NDS) or for normalization of viscoelasticity data derived from fresh samples to 2.5% NDS. The validity of this approach was then examined by applying these procedures to mucus collected during the follicular, ovulatory, and luteal phases of the normal menstrual cycle.

Human Cervical Mucus. II. Changes in Viscoelasticity During the Ovulatory Menstrual Cycle

Quantitative viscoelasticity measurements were made on individual human cervical mucus samples by microrheometry. Increases in mean values for mucus spinnbarkeit, ferning, and wet weight were associated with the ovulatory phase of the menstrual cycle, while no significant differences versus time were noted for mean values for sample pH or nondialyzable dry weight. A nadir in mucus nondialyzable solids (NDS) concentration and in visoelasticity was seen at or near midcycle. Substantial, highly reproducible, variations in mucus viscoelasticity were observed when mucus from different donors was compared. When the contribution of (NDS) to viscoelasticity was minimized by data normalization or by sample reconstitution, a significant increase in viscoelasticity was associated with the ovulatory phase of the cycle, suggesting the occurrence of a relative increase in mucin concentration or a compositional change in the mucus.

Dose of human menopausal gonadotropin influences the outcome of an in vitro fertilization program

This study compares outcomes of in vitro fertilization (IVF) in two groups of 57 patients when either 2 (group 1) or 3 (group 2) ampules of human menopausal gonadotropin (hMG) were administered daily. Treatment began on day 3 of the cycle and was discontinued when at least 2 follicles attained diameters greater than or equal to 1.5 cm. Human chorionic gonadotropin (hCG) was given either 24 or 48 hours after the last dose of hMG. Although serum estradiol levels were lower in group 1, the average number of oocytes retrieved (3.2 versus 2.9), fertilized (1.9 versus 2.0), and cleaved (1.7 versus 1.8) per completed cycle did not differ between groups 1 and 2. Likewise, the number of oocytes that fertilized abnormally was similar in both groups (0.5 versus 0.3/cycle). However, the number of atretic oocytes (0.03 versus 0.5/cycle) and the percent of oocytes recovered from the cul-de-sac (0 versus 7.2%) were significantly (P less than 0.05) lower in group 1. In group 1, administration of hCG 48 hours after the last dose of hMG was associated with a higher number of cleaving embryos (2.1 versus 1.5/cycle) and a higher pregnancy rate (34.8 versus 14.7%; P less than 0.05) when compared with injection at 24 hours. In group 2, the interval between hMG and hCG did not influence these results. Together, the associations between fewer oocytes that were atretic or recovered from the cul-de-sac, and a trend toward a higher pregnancy rate, suggest that follicular recruitment with 2 ampules of hMG is more appropriate than 3 ampules in an IVF program.

Zona-free hamster eggs and human sperm penetration capacity: a comparative study of proven fertile donors and infertility patients

An in vitro penetration assay utilizing human sperm and zona-free hamster eggs was employed to evaluate human sperm fertilizing capacity for 36 patients from the infertility clinic and 9 donors of proven fertility. The infertility patients were grouped according to the normality of their semen analyses. Test results were different for the three groups: a mean penetration level +/- standard deviation of 81% +/- 26% was obtained for proven fertile donors, while a value of 14% +/- 17% was observed for infertile couples with an abnormal semen analysis. A mean +/- standard deviation penetration level of 48% +/- 33% was associated with infertile couples in which the semen analysis was normal. These groups were statistically different (P less than 0.02) when inseminations were conducted at equivalent concentrations of motile sperm. No correlation was obvious between penetration test results and any of the parameters of the semen analysis; however, penetration test results did not correlate positively with the survival index (sperm survival at the end of insemination). These results are discussed in relation to further clinical application of the test.

Follicular maturation parameters associated with the failure of oocyte retrieval, fertilization, and cleavage in vitro

Follicular maturation parameters predictive of decreased success in human in vitro fertilization were evaluated in 104 gonadotropin-stimulated cycles. Falling estradiol (E2) levels correlated with decreased fertilization (1.9 oocyte/cycle) and cleavage (1.4 oocyte/cycle), when compared with cycles with increasing E2 (2.4 and 2.1 oocyte/cycle, respectively). Likewise, polyspermic fertilization was higher in the former group. Falling E2 levels after human chorionic gonadotropin (hCG) administration correlated with similar trends. Delaying hCG (24 to 96 hours) relative to the last dose of gonadotropin did not affect the total number of oocytes fertilized per cycle. However, polyspermic fertilization and cancellation rates were higher. Leading follicles growing beyond 2.3 cm (determined by ultrasound examination) were associated with decreased recovery, fertilization, and cleavage, and increased polyspermic fertilization. We conclude that ultrasonography and E2 monitoring can predict decreased fertilization and cleavage and should also be monitored after hCG administration.