Joseph E. Sokoloski

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.

Composition and function of human cervical mucus

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, b ut not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Laboratory Details in an In Vitro Fertilization and Embryo Transfer Program

In this chapter, the details of the IVF-ET program at UT-Houston are presented. This is done not only to introduce the complexities of IVF-ET but also with the objective of moving towards a standardization of protocols. Initially, standardization requires a complete description of the techniques, equipment, and supplies employed. Even with the most minute attention to detail, duplication of results by other laboratories will not necessarily be obtained. The protocols described here are certainly not unique to the UT program and credit as well as appreciation is expressed to Dr. Alex Lopata and co-workers (Lopata et al., 1980). Dr. Lopata visited Houston in the very early phases of our program and was kind enough to examine our protocols and modify them where appropriate. Additionally, references have been added throughout the chapter where modifications can be directly attributed to the contributions of others. IVF-ET is an exceedingly complex undertaking and the parameters which may contribute to success are virtually infinite.

FERTILITY POTENTIAL EVALUATION WITH THE ZONA-FREE HAMSTER EGG BIOASSAY

The evaluation of male fertility traditionally has centered on semen analysis describing semen volume, sperm density, morphology, motility and progression. Unfortunately, results from such analyses correlate poorly with fertility except in the extreme cases of azoospermia or asthenospermia. This poor correlation has forced attention to direct measurements of sperm function. Presently several bioassays are available for this purpose including in vivo sperm penetration of cervical mucus (the post coital or Sims-Huhner test), the in vitro capillary tube penetration test (with bovine or mid-cycle human cervical mucus) and sperm-egg interaction systems. At least two in vitro sperm-egg interaction assays have been described in addition to the fertilization of human eggs. In one, sperm are incubated with non-living human oocytes and the ability of sperm to penetrate the zona pellucida is measured (Overstreet and Hembree, 1976). The other, the zona-free hamster egg bioassay, utilizes the living naked egg of the hamster and sperm-egg fusion is the measured parameter (Yanagamachi et al., 1976).

A Practical, Objective Approach to the Evaluation of Sperm and Cervical Mucus in Humans**Supported by United States Public Health Service Contract N01-HD-4-2838 and the Booth-Ferris Foundation Fund.

Human sperm fertility potential, expressed as a quality index (QI), was evaluated objectively from considerations of sperm velocity, percentage of motile forms, sperm density, and ejaculate volume. Turbidimetry was applied in the quantitation of sperm velocity. High QIs (700) were characteristic of semen samples that were capable of penetrating cervical mucus in capillary tube penetration tests, while low QIs (80) were associated with specimens that did not penetrate mucus. The rate of decline in QI as a function of time postejaculation was determined for samples stored at 37 degrees C and at ambient temperature, providing correction factors for the comparative evaluation of semen samples analyzed at different times. The penetrability of cervical mucus was determined by capillary tube penetration testing. Subsequent chemical analysis of sperm-penetrable and impenetrable samples indicated that the concentrations of mucus nondialyzable solids (NDS), mucins, and soluble proteins were significantly higher in impenetrable specimens. These characteristics were also observed in impenetrable mucus obtained from patients with cervical factor infertility. These studies indicate that it is now feasible to employ mucus [NDS] in the diagnosis of cervical factor infertility and turbidimetry in the evaluation of human sperm quality.

Human Cervical Mucus. III. Isolation and Characterization of Rheologically Active Mucin**Supported by United States Public Health Service Grant NO1-HD-4-2838.

Several common mucolytic agents were screened for their ability to solubilize cervical mucus without incurring irreversible losses in mucus viscoelasticity. Sodium thiocyanate was selected on the basis of its effectiveness and handling convenience, and solubilized mucus was fractionated by gel filtration on Sepharose columns. The excluded mucin fraction was characterized by a high neutral hexose to protein ratio and contained all of the elasticity of thiocyanate-treated whole mucus. The concentration of mucin in individual mucus samples collected throughout the ovulatory menstrual cycle was determined from column elution profiles. An increased relative amount of mucin was associated with samples collected during the ovulatory phase of the cycle, and a strong correlation was obtained when the percentage of mucin in individual samples was plotted against their respective storage moduli. These results indicate that the cyclic fluctuations in mucus viscoelasticity seen previously at constant concentrations of nondialyzable solids can be attributed to variations in mucin concentration. Evidence was also obtained, however, for compositional differences in mucus derived from different patients.