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Biomedical subjects

R J Swanson

Publications and source records attributed to R J Swanson.

At least 19 recordsLinked to original sources

The YAG laser used in micromanipulation to transect the zona pellucida of hamster oocytes.

PROBLEM: Since there has been no reported use of the YAG laser to micromanipulate oocytes, our purpose was to study whether (1) a YAG laser could be used to open the zona pellucida of hamster oocytes; (2) human sperm could reach the ooplasm and (3) under sperm penetration assay conditions, sperm would bind and penetrate the ooplasm. RESULTS: A YAG 100 laser was used at 10 W and 0.4-sec pulse width to open eight of eight ooplasm oocytes. The opening in the zonae was 0.25 to 1.0 rad (10 to 40 microns). For the initial eight oocytes and two parallel controls, the coarse appearance of the ooplasm was unchanged after 3 days. Next, in 11 of 12 manipulated oocytes, the sperm clustered at the opening of the zona. When 16 more oocytes were opened and exposed to sperm in sperm penetration assay conditions, each ooplasm bound sperm. There was no penetration noted. Each manipulation time was < 1 min. To clarify the laser effect, oocytes were exposed to laser energy then utilized as the interactive surface in the sperm penetration assay. It was found that only 20% bound sperm with no penetration. CONCLUSION: While the time factor compares favourably with other methods of zona opening, further study needs to be performed to minimize effect to the exposed oocyte.

Animals

Fertilization and mouse embryo development in the presence of midazolam.

Mouse embryo in vitro development elucidates the effect of a pharmacologic agent on cellular differentiation. Midazolam provides conscious sedation for patients undergoing egg retrieval for in vitro fertilization and is found in patient follicular fluid. Mouse preimplantation embryo formation and development were evaluated in the presence of midazolam. Midazolam was cocultured with two-cell mouse preimplantation embryos over 72 h and injected systemically just before ovulation and coitus. Concentrations to 12.5 micrograms/mL displayed no significant toxic effects on in vitro two-cell-to-blastocyst development. Doses to 35.0 mg/kg did not prevent or impair in vivo fertilization. Midazolam has no adverse effect on in vitro development of two-cell-to-blastocyst-stage embryos nor on in vivo fertilization and cell division at concentrations approximating and exceeding those that ova are exposed to during clinical anesthesia. Midazolam is recommended for use to induce sedation in human in vitro fertilization where association with gametes and zygotes is probable.

Animals

Hypoosmotic swelling test in the prediction of male fertility.

The hypoosmotic swelling test (HOS) is a simple test to measure the functional competence of human sperm membranes. The question is, does a relationship exist between this competence and the fertilization potential of human spermatozoa? In this study the strongest correlation (r = 0.76) was obtained between sperm swelling and sperm viability (supravital staining). Only a moderate correlation (r = 0.50) was obtained with normal sperm morphology; weaker correlations were also obtained with the sperm penetration assay (r = 0.42) and human IVF (r = 0.24). The results, therefore, indicate that the HOS test has a limited predictive value. Notwithstanding this low concordance between sperm swelling and fertilizing potential, a less than 50% HOS test threshold was seen to be a definite indicator of a male factor.

Cell Survival

Usefulness of sperm penetration assay in fertility predictions.

The competence of the sperm penetration assay (SPA) to predict male fertility, as determined by normal sperm morphology and the fertilizing potential, as shown by human in vitro fertilization (IVF), was investigated. A significant correlation was obtained between normal sperm morphology and the SPA (phi = 0.623). A weaker correlation was however obtained with human IVF (phi = 0.397). Notwithstanding this weak association, a positive SPA (greater than 10%) was highly predictive (95%) of human IVF success. In contrast, a negative SPA (less than or equal to 10%) was associated with a high rate of false-negatives (65%). The SPA does however warn that a male factor may be present, as the mean fertilization rate of this group of patients was markedly reduced. The preincubation period for the spermatozoa did not play a major role in the predictive ability of a SPA outcome.

Female

Extravasation. A serious complication of the split-sheath introducer technique for venous access.

The use of split-sheath introducers to place venous access catheters results in the potential for subcutaneous extravasation and tissue injury or necrosis. We present six cases that demonstrate this complication and illustrate the probable mechanism. The safe use of these catheters requires verification that blood can be aspirated from the catheter and a high index of suspicion for extravasation when symptoms develop.

Adult

Mouse embryo quality control for toxicity determination in the Norfolk in vitro fertilization program.

Ongoing quality control is necessary as part of the maintenance and improvement of a successful human in vitro fertilization (IVF) program. Using a mouse quality-control culture system, several instrument preparation protocols were reevaluated to determine their efficiency in the control or elimination of potential toxicity. Dilute concentrations of urine and endometrial fluid were also tested. Medium rinsed through laparoscope and aspiration needles failed to support embryo development. This effect was reversed in needles that were pretreated with rinses of Dulbecco's phosphate-buffered saline. Endometrial fluid demonstrated no obvious toxic effect, but urine-exposed embryos arrested in the two-cell state. The importance of periodic evaluation of materials and their pretreatment before use in in vitro fertilization of human oocytes is essential to ensure control of potentially toxic substances.

Animals

Predictive value of abnormal sperm morphology in in vitro fertilization.

In patients with acceptable sperm count and motility, two patterns of abnormal morphology, judged with strict criteria, were identified and described. Patients with less than 4% normal forms and less than 30% morphology index (summation of normal and slightly amorphous forms) had a fertilization rate of 7.6% of the oocytes (P pattern, poor prognosis). Patients with normal morphology between 4 and 14% had a significantly better fertilization rate of 63.9% of the oocytes (P less than 0.0001). Cases with greater than 14% normal forms fertilized within the normal range for the laboratory. By evaluating sperm morphology with the proposed strict criteria, its predictive value in in vitro fertilization is enhanced.

Female

Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities.

Sperm morphology evaluated by new, strict criteria is a good predictor of outcome in in vitro fertilization (IVF). This study aimed (1) to determine whether the fertilization rate of preovulatory oocytes in patients with abnormal morphology can be improved by increasing insemination concentration at the time of IVF and (2) to evaluate the pregnancy outcome in patients with abnormal sperm morphology. Three groups were studied: (1) normal morphology, (2) good prognosis pattern, and (3) poor prognosis pattern. All other sperm parameters were normal. Group 3 had a lower overall fertilization rate, lower pregnancy rate/cycle, and lower ongoing pregnancy rate/cycle. Groups 2 and 3 showed a higher miscarriage rate, although not significantly different from group 1. By increasing insemination concentration from 2- to 10-fold, the fertilization rate in group 3 increased from 14.5% to 62.6%. However, pregnancy outcome did not improve. We conclude that patients with severe sperm head abnormalities have a lower ability to establish successful pregnancies, even though fertilization may be achieved.

Abortion, Spontaneous

Abnormal sperm morphology and other semen parameters related to the outcome of the hamster oocyte human sperm penetration assay.

A new method for evaluation of sperm morphology using strict criteria is currently used in the andrology laboratory at the Eastern Virginia Medical School. A prospective study was designed to evaluate the following semen parameters in samples of all patients over a set period of time: sperm concentration and motility, and normal sperm morphology. These factors were correlated with results of the hamster zona-free oocyte/human sperm penetration assay (SPA). One hundred patients with a sperm concentration ranging from 2 to 219 X 10(6)/ml, a motile sperm fraction ranging from 6.9 to 87%, and normal sperm morphology ranging from 1 to 39%, were evaluated. The statistical analysis system general linear model was used to judge the influence of the different variables. There was a statistically significant relationship between the per cent of sperm with normal morphology and penetration rate in the SPA (P = 0.001). Outcome of the SPA was also correlated with in vitro fertilization, retrospectively, in 84 patients. Thirty-eight patients had an SPA less than 10%, with no fertilization in vitro in 13 patients (33.3%) and fertilization in 25 (66.7%). Forty-five had an SPA greater than 10% with fertilization in 37 (82.2%) and no fertilization in eight (17.8%) patients.

Animals

Correlation between sperm morphology, acrosin, and fertilization in an IVF program.

Acrosin, a neutral proteinase, is located within the acrosome. The aim of this study was to evaluate acrosin concentrations in patients with severe damage of the sperm head and to determine whether acrosin concentration could predict the chances of fertilization in an IVF program. Sixty patients were accepted into this study, prospectively. The patients were divided into two groups, those with a normal morphology of less than 14% (group I, n = 33) and those with normal morphology less than 14% (group II, n = 27). All patients had a sperm concentration of less than 20 million sperm/ml and less than 30% progressively motile sperm. The acrosin assays were performed on the semen sample obtained on the day of IVF. Routine IVF insemination procedures were used, and only mature oocytes were considered. The only factor that showed a significant correlation of fertilization was normal morphology (p less that 0.01). The mean acrosin level was 73.4 /+- 38.6 mED/10 million sperm in group I and 70.9 /+- 42.7 mIU/10 million sperm in group II (no significant difference). The fertilization rate in group I was 45.4% and in group II, 77.7% p less than 0.002). Acrosin levels were not significantly different in patients with and without fertilization (72.0 /+- 42.1 and 73.6 /+- mIU/10 million sperm, respectively).

Acrosin

Evaluation of semen parameters by means of automated sperm motion analyzers.

Fresh semen specimens from 46 patients and donors were evaluated for concentration, motility, velocity, and linearity using two different commercially available computerized sperm motion analyzer systems. Although no significant differences in measurement of concentration or motility were observed, significant differences in velocity and linearity were recorded. Fourteen cryopreserved/thawed samples were assessed with the same set-up parameters as fresh specimens. When discrepancies between manual and computer counts were noted, the authors changed the set-up parameters and evaluated 33 additional specimens. Again, no differences in concentration and motility, but significant differences in velocity and linearity were observed. Interlaboratory results must be correlated and standardization of set-up parameters of various analyzers is essential.

Autoanalysis

New method of evaluating sperm morphology with predictive value for human in vitro fertilization.

A prospective study was planned to evaluate sperm morphology as a parameter to predict the fertilization outcome in an in vitro fertilization program. Couples applying to in vitro fertilization were admitted into this project when the sperm concentration was greater than 20 million per mL and motility greater than 30 per cent. Based on new strict criteria for evaluating normal sperm morphology, patients were divided prospectively into 2 groups. In group I (25 patients) normal sperm morphology was less than 14 per cent, and in group II (71 patients) normal sperm morphology was greater than 14 per cent, using a threshold established previously. Multiple regression analysis was used to evaluate different parameters: concentration, motility, and morphology against the dependent variables, fertilization, and cleavage. The only factor which was significantly correlated with fertilization and cleavage was normal sperm morphology (P less than 0.0001). The fertilization rate (per oocyte) and the cleavage rate were 49.4 per cent and 47.6 per cent in group I and 88.3 per cent and 87 per cent in group II (P less than 0.0001). The ongoing pregnancy rate per laparoscopy and per embryo transfer was 4 per cent and 5.5 per cent, respectively, in group I and 18.3 per cent and 18.5 per cent, respectively, in group II (no significant difference). This study demonstrates the value of analyzing sperm morphology using the criteria recommended in terms of predicting fertilization and perhaps pregnancy outcome. Patients can be better counseled and the probability of fertilization or no fertilization can be more accurately established. Furthermore a trend is shown in the pregnancy rate that may indicate the importance of the male genome in establishing a pregnancy.

Embryo Transfer

A quick, reliable staining technique for human sperm morphology.

The evaluation of sperm morphology is still an important parameter in the diagnosis of the infertile male. Most techniques used for staining human sperm are very time-consuming. A routine stain used for determining differential count of leucocytes (Diff-Quik stain) was evaluated against the standard Papanicolaou stain. Morphology results from 20 duplicate semen smears using both staining methods were determined separately by 2 technicians using a blind protocol. No significant differences were observed when comparing the two staining methods (paired Student's t-test). The advantages of the Diff-Quik stain are: a) complete staining-to-reading time under 7 min, b) commercially prepared reagents, and c) case of staining procedure.

Humans

Toxicity testing for human in vitro fertilization programs.

Using a mouse embryo culture system, several procedures and materials associated with human in vitro fertilization protocols were tested for potential toxicity. Also, quality-control assays were performed for media prepared by nine different human in vitro fertilization programs. Detrimental effects upon embryo development were observed when culture media were exposed to the following substances: surgical instruments sterilized with Cidex or Cidex-7 or sterilized with ethylene oxide after packaging in Nest Protector Packs, various brands of surgical gloves, and various synthetic materials being evaluated as possible needle or catheter "liners." Results from comparative testing of media and serum supplements prepared by different in vitro programs indicated a wide range in culture medium quality, as assayed by the ability of the reagents to support mouse embryo development. The importance of an animal model system available to all human in vitro programs for routine quality-control analysis and testing of novel uses of materials and innovative methods is discussed.

Animals

Male factor evaluation in in vitro fertilization: Norfolk experience.

Thirty-three patients from the in vitro fertilization (IVF) program at Norfolk are critically reviewed. A battery of tests was designed and an endocrine investigation was carried out on these patients. The fertilization rate for preovulatory oocytes was lower than in the normal male population (39.6% versus 88.6%). When total concentration of sperm with rapidly progressive motility was less than 6 X 10(5), to fertilize several eggs together the fertilization rate was zero. No fertilization was obtained when the number of sperm with rapidly progressive motility recovered after the separation was less than 1.5 X 10(6). The hamster zona-free oocyte penetration test correlated well with the human IVF system. The other parameters investigated did not show good correlation. When fertilization was achieved, the results of the IVF procedure in the series reviewed rendered a 30.8% pregnancy rate per transfer in 26 transfers. Fifty percent of the pregnancies were normal (either ongoing or delivered). Thirty-seven percent were preclinical miscarriages, and 12.5% were clinical abortions. In the abnormal male population, higher concentrations of sperm per egg should be used for insemination for achievement of optimum fertilization rates. Once fertilization is obtained, the results do not differ substantially from the IVF population at large.

Animals