Human fertilization

Abstract: Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.

Abstract: IN the natural sequence of mammalian fertilization, the spermatozoa await the arrival of ova in the Fallopian tubes. The time of waiting may vary from 6 hr. (rabbit), 24 hr. (ferret), 20–30 hr. (pig, sheep and cattle) up to perhaps several months (bat)1. Since the transportation of sperm from the vagina to the upper part of the tube is very rapid (a few minutes to 6 hr.1), and since the number of spermatozoa present at the site of fertilization is very small1, this time interval is not necessarily for the accumulation of a large number of spermatozoa to ensure fertilization. The following experiment demonstrates that such a period of time in the female tract is required for the spermatozoa to acquire their fertilizing capacity.

Abstract: A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed. IVF frequency of in vitro matured oocytes was increased by a swimup sperm separation procedure (P=0.01) or treatment of sperm with the glycosaminoglycan heparin (P=0.0001), but the two factors did not interact (P=0.23). Heparin was the most important factor in increasing IVF frequencies. The fertilization frequency was not affected by the batch of oocytes used (P=0.38), but bull effects were present (P<0.05). Within a bull, the IVF system was highly repeatable and varied between trials no more than +/- 12% in fertilization frequency with an overall fertilization frequency of 299 379 (79%) on four trials over four bulls. In vivo matured oocytes fertilized in vitro were transferred to ewe or heifer oviducts. Morulae or blastocysts were recovered from ewes after four to five days, while conceptuses were present in the bovine after 25 days (diagnosed by ultrasound). Embryonic development from the IVF system either pre- or postimplantation was normal.

Abstract: This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm to fertilize ova in vitro. During 20 mm exposure of sperm cells to defined media of increasing NaCI concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic components was initiated and completed as reflected by an immunological assay at approximately 300 and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or alteration of these components as determined by the immunological assay during the first 5 mm of sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration of some but not all of the antigenic sperm coating seminal plasma components detectable by this means during 20 mm of exposure. Sperm within the perivitelline apace, presence of pronuclei. and 2- and 4-cell cleavage stages were taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg) medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent (SI of 69 ova) when results were considered according to individual male sperm donors. No large differences were found by the immunological assay that could be linked to variability of fertilizing ability of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested as a likely reason for differences in fertilizing ability of sperm from different bucks. In experiments using sperm and ova from the same sources, no differences in fertilization results were apparent following 305 and 380 mOsm/kg sperm treatments, Initiation, but not completion, of removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm penetration into ova occurred in some cases before 7-10 h after insemination (4 of 54 ova penetrated), but actively motile sperm were found within the perivitelline apace of other ova as late as 25 h after insemination. One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does. Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and represents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and embryo transfer in the rabbit.

Abstract: 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.