Trish Berger

Clinical applications of techniques used in human in vitro fertilization research

Human in vitro fertilization-embryo transfer not only provides an opportunity for pregnancy in women who were previously considered to be sterile, but also provides a unique method by means of which basic reproductive physiology can be investigated. From September, 1981, to September, 1982, 71 women with normal cycles who elected to attempt in vitro fertilization-embryo transfer underwent timed laparoscopy for recovery of oocytes. Oocytes were recovered in 60 patients, with embryo transfer resulting in 50 patients, and normal implantation occurred in nine patients. There was a significant correlation between ultrasound observation of follicle size and serum estradiol levels, thus making ultrasound monitoring of follicular growth during stimulation with clomiphene citrate or human menopausal gonadotropin in anovulatory women clinically useful. The technique of sperm washing employed for in vitro fertilization has now been used with good results for intrauterine insemination in patients with infertility due to a cervical factor or oligospermia. Therefore, the techniques used in human in vitro fertilization-embryo transfer are now clinically applicable for couples with infertility due to causes other than tubal disease.

Factors affecting human sperm penetration of zona-free hamster ova

Abstract The effects of in vivo and in vitro aging of hamster ova, protein supplementation of culture media, sperm concentration, and sperm motility on penetration of zona-free hamster ova by human sperm were investigated. Penetrability of ova was significantly lowered by 2 to 4 additional hours of in vivo aging or by an additional 3 hours of in vitro aging. A comparison was made of the effects of the addition to the media of human preovulatory serum and human serum albumin, and the penetrating ability of human sperm was increased with the addition of 10% human preovulatory serum. Sperm motility was also better maintained in the presence of 10% serum. Maximum penetration occurred after 3 hours of sperm-egg interaction following a 3-hour preincubation period with a sperm concentration of 5 × 10 6 motile sperm per milliliter. When motile sperm concentration was maintained at 1 × 10 7 motile sperm per milliliter, there was no correlation between penetrating ability and percentage of motility. These factors should be controlled to allow reproducible results with the hamster test.

Effect of variable concentration of serum on mouse embryo development

This study was undertaken to determine the optimal concentration of serum necessary for maximal embryo development. Mouse embryos were cultured in vitro with 0% to 30% concentrations of serum of 96 hours. After 72 and 96 hours of culture, embryo growth was improved with 5%, 10%, 20%, and 30% serum supplement when compared with Hams F-10 medium alone. Embryos were then cultured with the same concentrations of serum for 29 hours, following which blastomere number, sister chromatid exchange (SCE), number of micronuclei, and chromosomal aberrations were observed. There was no difference in blastomere number with any concentration of serum supplement studied. All concentrations of serum decreased the number of SCE when compared with Hams F-10 medium alone. The rate of SCE in embryos cultured with 10%, 20%, or 30% serum was also smaller than that of the embryos cultured with 5% serum. The results of these studies indicate that serum should be employed for culturing embryos, and at least 10% serum concentration is necessary to obtain optimal conditions for embryo development.

Correlation of semen transferrin concentration and sperm fertilizing capacity.

To determine whether a correlation exists between semen transferrin and sperm fertilizing capacity, transferrin concentration was determined in the semen of 52 male patients referred for the hamster ova penetration test (group 1) and in 17 men participating in the human in vitro fertilization program (group 2). In both groups 1 and 2 seminal transferrin levels were also compared to semen volume, sperm density, and motility. Serum follicle-stimulating hormone and luteinizing hormone concentrations that were determined in 34 individuals (24 in group 1, 10 in group 2) showed no correlation with seminal transferrin levels. In conclusion, low seminal transferrin levels correlated with low sperm density and with poor fertilizing ability of human oocytes in vitro.

Ibuprofen modulation of human chorionic gonadotropin-induced ornithine decarboxylase activity and ovulation in the rabbit ovary

The activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, increases as granulosa cells proliferate in developing follicles. Both luteinizing hormone and prostaglandins stimulate ornithine decarboxylase activity. Here, we sought to determine the relative contributions of both trophic stimuli to ornithine decarboxylase activity in the preovulatory rabbit ovary. Baseline ovarian ornithine decarboxylase activity, determined by measuring the release of CO2 from (1-14C)-ornithine, was 13.4 +/- 1.27 (mean +/- SD) pmol of carbon dioxide per hour per milligram of protein. Treatment with ibuprofen, a prostaglandin synthetase inhibitor, led to a significant (p less than 0.05) decrease in the baseline ovarian ornithine decarboxylase activity (4.7 +/- 0.29 pmol of carbon dioxide per hour per milligram of protein). Administration of human chorionic gonadotropin (hCG) (50 IU/kg intramuscularly) to adult rabbits (2.5 to 3.5 kg) elicited a 1,200% elevation of ovarian ornithine decarboxylase activity 5 hours after injection; there was a return to baseline by 11 hours after injection. Stimulation with human chorionic gonadotropin led to ovulation in 22.2%, 25%, and 60% of rabbits at 7, 9, and 11 hours after treatment, respectively. Single-dose ibuprofen treatment (5 mg/kg intramuscularly), 7 hours after human chorionic gonadotropin administration inhibited ovulation and elevated ovarian ornithine decarboxylase activity. These results indicate that ibuprofen effectively inhibits ovulation in hCG-stimulated rabbit ovaries in the presence of a significant (p less than 0.001) elevation in ovarian ornithine decarboxylase activity. Thus, different intracellular mechanisms are involved in the prostaglandin modulation of basal and hCG-stimulated cells during the course of preovulatory follicle maturation.

Seminal prolactin concentration and sperm reproductive capacity**Presented in part at the Thirty-First Annual Meeting of the Pacific Coast Fertility Society, October 12 to 16, 1983, Rancho Mirage, California.

Prolactin (PRL) levels in the seminal plasma were determined in 63 men, all partners from infertile marriages. For comparison of seminal PRL concentration and semen quality, a sperm concentration and motility were studied in all men. Also, for determination of the fertilizing capacity of the semen, a zona-free hamster ovum penetration test was done in 49 men, while 14 men were studied by determination of the fertilization of human oocytes in vitro. Significantly higher levels of semen PRL were found in those individuals with low sperm concentration and motility as well as in those individuals that showed an abnormal hamster ovum penetration test. This suggests that high seminal PRL levels have a negative impact on sperm functional capacity.

Comparison of techniques for selection of motile spermatozoa**Supported in part by National Institutes of Health grants 5R23HD16306 and 7R23HD18892 (to T. B.).††Presented in part at the Thirty-First Annual Meeting of the Pacific Coast Fertility Society, October 12 to 16, 1983, Rancho Mirage, California.

Procedures to separate motile sperm with high rates of recovery may have clinical application in in vitro fertilization and intrauterine insemination in increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. A two-step continuous Percoll gradient was an effective means of separating motile sperm which also had enhanced ability to penetrate zona-free hamster ova. However, the requirement for a high-speed centrifuge and rotor makes the procedure impractical in many cases. A one-step discontinuous Percoll gradient was also effective in separating a population of motile sperm. Comparison of the discontinuous Percoll gradient with other techniques for separation of motile sperm indicated the discontinuous Percoll gradient had advantages in terms of recovery, enhancement of motility, and increased ability to penetrate zona-free hamster ova. The velocity of selected sperm was not significantly different among techniques. The one-step discontinuous Percoll gradient appears to have value both for increasing homogeneity of human sperm populations used for basic research and in clinical practice for male subfertility.

The effect of serum fractions on embryo growth**Presented in part at the Thirty-Ninth Annual Meeting of The American Fertility Society, April 16 to 20, 1983, San Francisco, California.††Supported in part by the Ford Foundation.

In order to determine optimal culture conditions for embryos, human fetal cord serum (HCS) small- and large-molecular-weight fractions of HCS, and human serum albumin were employed as media supplements. In the first phase, embryos were cultured with the various protein supplements for 96 hours and observed morphologically every 24 hours. In the second phase, embryos were cultured with the various protein supplements and 5-bromo-2-deoxyuridine for 29 hours. Blastomere number, sister chromatid exchange (SCE), micronuclei, and chromosomal aberrations were observed. Morphologically, any protein supplement was better than no media supplement. Whole HCS and large-molecular-weight fraction provided the best conditions for embryo growth. Blastomere number was increased with whole serum when compared with no supplementation, small-molecular-weight fraction, or human serum albumin. In the analysis of SCE, all supplements had a lower SCE number than no serum supplement, and HCS supplement demonstrated a significantly lower SCE number than the other supplements. No difference in micronuclei or chromosomal aberration was observed in any of the supplements. In conclusion, whole HCS provides optimal conditions for culturing embryos, compared with the various serum fractions, and the beneficial components are primarily found in the large-molecular-weight fraction.