Measurement of periimplantational relaxin concentrations in the macaque using a homologous assay.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Molecular target of endocrine disruption in human luteinizing granulosa cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin: inhibition of estradiol secretion due to decreased 17alpha-hydroxylase/17,20-lyase cytochrome P450 expression.

Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.

Enhanced ovarian steroid secretion before implantation in early human pregnancy.

Previous studies have compared ovarian steroid production in the luteal phase of nonconceptive and conceptive cycles. Some investigators reported higher preimplantational levels of progesterone in conceptive cycles vs. nonconceptive cycles, but other studies have found no differences. Many of these results were difficult to interpret because the studies included infertile women and/or women who received exogenous hormones. In this study we have characterized the profiles of gonadotropin secretion and ovarian steroid response during early pregnancy in a population of spontaneously ovulating women and compared them to those in nonconceptive cycles of recently fertile women. Blood samples were collected daily during the luteal phase from 24 women during 51 cycles of artificial insemination with donor semen. Cycles were segregated to those from women who had a successful term pregnancy (normal group) and those having an early spontaneous abortion (SAB group) and were also classified as nonconceptive or conceptive based on measurements of hCG. Serum LH and FSH did not show marked differences between nonconceptive and conceptive cycles in the periimplantation period in either the normal or SAB group. In the normal group, estradiol concentrations were significantly higher in conceptive cycles than in nonconceptive cycles beginning 6 days after the LH peak and continuing through the end of the cycle, while differences in progesterone concentrations bordered on or exceeded significance during the same time period. In the SAB group, preimplantation differences in pituitary gonadotropin and ovarian steroid secretion were not observed, whereas the postimplantation hCG concentrations in the SAB group were significantly lower than those in the normal group. It is reasoned that embryos with defective post-implantation hCG secretion may have had this defect before detection of hCG in serum, thus accounting for the lack of stimulation of steroid secretion in these pregnancies. These findings suggest that the enhanced ovarian steroid secretion in conceptive cycles may be due to a gonadotropic stimulus from the preimplantation embryo.

The role of relaxin in glycodelin secretion.

Glycodelin is a glycoprotein named for its unique carbohydrate structure. Glycodelin is produced by the secretory endometrium during the late luteal phase and returns to baseline during menses of the ensuing cycle, whereas in conceptive cycles it rapidly increases. Although progesterone and possibly estradiol are required for glycodelin production, they are not directly involved in the synthesis and release of this protein. Their role may be development of the endometrial secretory glandular elements, whereas other factors are required to initiate and maintain glycodelin secretion. The pattern of relaxin secretion during the luteal phase and early pregnancy is similar to that of glycodelin, but their profiles have not been determined simultaneously. To investigate the relationship of relaxin and glycodelin, two studies were conducted. In the first study, relaxin, glycodelin, and ovarian steroids were measured in daily serum samples from nonconceptive and conceptive natural cycles. Profiles of relaxin and glycodelin were closely associated, with the onset of relaxin preceding glycodelin secretion by 1-2 days in nonconceptive cycles, and the pregnancy-associated increases in each hormone differing by about 2 days. The second study tested the hypothesis that relaxin stimulates glycodelin secretion. Samples were obtained from patients injected with human relaxin for 28 days. In subjects demonstrating ovarian cyclicity, glycodelin secretion was elevated, but it was not detected in subjects without ovarian cyclicity or in placebo-treated control subjects. This study reveals a close temporal and quantitative relationship between relaxin and glycodelin profiles in the late luteal phase and early pregnancy. It also demonstrates that relaxin administration can stimulate glycodelin production from a developed endometrium. This is the first report of a nonsteroidal ovarian factor that controls glycodelin secretion, and these results suggest a function for relaxin during early pregnancy. Glycodelin is a potent inhibitor of sperm zona pellucida binding by virtue of its extensive carbohydrate structure, but it is normally at a nadir in the periovulatory period. The data demonstrate that relaxin can stimulate glycodelin secretion throughout the menstrual cycle, including the periovulatory period, when relaxin-induced glycodelin secretion could have a contraceptive effect.

Relaxin in the peri-implantation period.

The time of appearance of relaxin in peripheral blood was determined in conceptive and non-conceptive cycles using a sensitive and specific double-antibody enzyme-linked immunoassay for human relaxin. For study of relaxin in early pregnancy, daily plasma samples were collected from women receiving artificial insemination of donor semen. The day of ovulation was determined by daily LH monitoring and ultrasound observation. In three conceptive cycles, relaxin was significantly elevated over baseline 9-10 days following the LH peak. Relaxin concentrations quickly rose over the next 15 days of observation to over 800 pg/ml. Relaxin was observed to increase 1 to 2 days prior to the first detectable increase in plasma hCG as measured by enzyme-linked immunosorbent assay. To compare the relaxin profile in conceptive cycles with normal luteal phase concentrations, relaxin was also measured in daily plasma samples collected from women contracepting with barrier methods, bilateral tubal ligation, or abstinence. A small but consistent rise in relaxin in the late luteal phase was observed in nine of eleven women, which began 6-9 days after the LH peak, averaged approximately 50 pg/ml, and was declining by the next menses. It is concluded that a small but measurable rise in plasma relaxin is associated with the normal luteal phase and that relaxin secretion is accelerated around the time that hCG is first detected in conceptive cycles. This acceleration of relaxin secretion which is associated with the onset of hCG may provide additional evidence for identification of transient early pregnancy.

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling.

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.

Biomarkers for assessing human female reproductive health, an interdisciplinary approach.

Identification of environmental hazards to reproductive health and characterization of the adverse outcomes necessitate a multidisciplinary approach. Epidemiologic studies are required for the identification of adverse health effects in human populations and then to confirm that specific exposures are responsible. Clinical studies are required to develop assays for reproductive biomarkers and to validate these assays prior to their application in the field. Assays for field use must be formatted and streamlined for large-scale applications and, whenever possible, computer algorithms should be developed to interpret biomarker data. Appropriate animal models must be identified, biomarker assays validated for that model, and animal experiments conducted to identify the mode of action and target organ of a putative reproductive toxicant. Finally, in vitro studies at the level of the cell and cell organelle are essential for mechanisms for toxicity to be clearly identified and understood. In this article we describe the interdisciplinary approach that we have developed for study of the effects of environmental agents on female reproductive functions. This effort requires specific skills of toxicologists, epidemiologists, physicians, biochemists, and physiologists.

Total urinary follicle stimulating hormone as a biomarker for detection of early pregnancy and periimplantation spontaneous abortion.

Total concentrations of follicle stimulating hormone (FSH) were evaluated in daily urine samples from conceptive and nonconceptive menstrual cycles by measurement of the FSH beta subunit following treatment of the samples to dissociate the FSH heterodimer. Samples were self-collected by normal subjects during cycles in which daily blood samples also were obtained. Daily blood and urine specimens were collected prospectively from 10 subject in conceptive cycles, which led to normal pregnancies, and from 10 subjects with bilateral tubal ligations to provide control samples form nonconceptive cycles. Mean serum and urinary FSH concentration profiles wer parallel in both groups following ovulation and during he first 9 days of the luteal phase. Mean values for both serum and urinary FSH rose significantly above the postovulatory baseline by 10-12 days following the midcycle luteinizing hormone (LH) peak in nonconceptive cycles, but did not rise at any time following ovulation during conceptive cycles. Following regression analysis of the changing FSH concentration between days 9-14 post-LH surge in conceptive cycles, a slope of

Identification of anovulation and transient luteal function using a urinary pregnanediol-3-glucuronide ratio algorithm.

The sensitivity and specificity of a urinary pregnanediol-3-glucuronide (PdG) ratio algorithm to identify anovulatory cycles was studied prospectively in two independent populations of women. Urinary hormone data from the first group was used to develop the algorithm, and data from the second group was used for its validation. PdG ratios were calculated by a cycles method in which daily PdG concentrations indexed by creatinine (CR) from cycle day 11 onward were divided by a baseline PdG (average PdG/Cr concentration for cycle days 6-10). In the interval method, daily PdG/CR concentrations from day 1 onward were divided by baseline PdG (lowest 5-day average of PdG/CR values throughout the collection period). Evaluation of the first study population (n = 6) resulted in cycles with PdG ratios > or = 3 for > or = 3 consecutive days being classified as ovulatory; otherwise they were anovulatory. The sensitivity and specificity of the PdG ratio algorithm to identify anovulatory cycles in the second population were 75% and 89.5%, respectively, for all cycles (n = 88); 50% and 88.3% for first cycles (n = 40) using the cycles method; 75% and 92.2%, respectively, for all cycles (n = 89); and 50% and 94.1% for first cycles (n = 40) using the interval method. The "gold standard" for anovulation was weekly serum samples < or = 2 ng/ml progesterone. The sensitivity values for all cycles and for the first cycle using both methods were underestimated because of apparent misclassification of cycles using serum progesterone due to infrequent blood collection. Blood collection more than once a week would have greatly improved the sensitivity and modestly improved the specificity of the algorithm. The PdG ratio algorithm provides an efficient approach for screening urine samples collected in epidemiologic studies of reproductive health in women.

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides.

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay.

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.

A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice.

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Sperm motility assessment by videomicrography.

A technically simple, inexpensive method is described for measuring objective parameters of sperm motility. The instruments involved are commercially available, home-oriented videotape equipment. Quantitative measurements of sperm motility are made directly from the video image and are facilitated by use of an analysis transparency that is applied as an overlay to the screen of the television monitor. A protocol is given for describing the motility of a suspension of human spermatozoa in terms of percentage motility, mean swimming speed, and the percentage of progressive sperm. A complete analysis can be done in 20 minutes or less. Examples are presented of videomicrographic assessment of the motility of human and bull spermatozoa.

Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro.

In an in vitro assay human spermatozoa penetrated the zona pellucida of 38.8% of 773 human oocytes recovered from the ovaries of cadavers. Zona penetration was not observed until 8 hours had elapsed. Oocytes examined with the electron microscope were surrounded mainly by sperm with intact acrosomes, but contained sperm in the zona and perivitelline space which had lost the outer acrosomal membrane and acrosomal contents. Sperm entry into the ooplasm was never observed. Spermatozoa from 11 of 16 patients with suspected infertility penetrated the zona, although the penetration rate was lower than that with sperm from fertile donors (12.9% versus 46.4%). When oocytes were incubated with mixed suspensions containing equal numbers of motile sperm from donors and patients, the donor sperm (identified by a fluorescent label) penetrated 50.0% and the patient sperm only 12.7%. These data suggest that human sperm penetrate the zona pellucida of nonliving human oocytes and mature living ova in a similar manner. This method is a potential diagnostic and investigative tool which avoids the ethical and technical problems associated with human in vitro fertilization.

Effects of antibodies to sperm surface fertilization antigen-1 on human sperm-zona pellucida interaction.

OBJECTIVE: To investigate the effects of antibodies to well-defined sperm surface antigens (the fertilization antigen [FA-1] and germ-cell antigen [GA-1]) and nuclear antigen (protamine) on human sperm-zona interaction. DESIGN: Number of total and acrosome-reacted human sperm bound to the human zona pellucida and the sperm movement characteristics assessed by computer-aided sperm analysis were evaluated after incubation of sperm with the antibodies. SETTING: Academic research environment approved by the Institute Review Board. PATIENTS: Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. INTERVENTIONS: Human oocytes were stored in salt solution at -80 degrees C until used. Spermatozoa were treated with the antibodies to various sperm antigens. MAIN OUTCOME MEASURES: Total and acrosome-reacted sperm bound to zona pellucida and sperm movement characteristics were evaluated after 3 to 5 hours of incubation of the antibodies with human sperm. RESULTS: Anti-FA-1 antibodies significantly reduced human sperm fusion with zona-free hamster oocytes and sperm binding to the human zona pellucida but did not affect binding of acrosome-reacted sperm and sperm movement characteristics. Anti-GA-1 and antiprotamine antibodies did not affect sperm-oocyte interaction, acrosomal reaction, or sperm motility. CONCLUSIONS: Antibodies to FA-1 but not to GA-1 and protamine inhibit human sperm-zona interaction.

The importance of seminal plasma for sperm penetration of human cervical mucus.

Experiments were carried out in which semen samples were diluted 1:1 with Tyrode's solution or with their own seminal plasma (obtained by centrifuging another semen aliquot) as a control. Each experiment consisted of a paired comparison of these two sperm suspensions, using a quantitative cervical mucus penetration test with aliquots of the same mucus sample. Videomicrography was used to measure the swimming speeds of spermatozoa in the semen and in the mucus. The spermatozoa swam faster in Tyrode's diluted seminal plasma than in whole seminal plasma, but their swimming speeds in cervical mucus were similar after mucus penetration. Significantly more of the collisions between spermatozoa and the mucus resulted in successful penetration in tests where the sperm were suspended in whole seminal plasma than in tests where they were suspended in Tyrode's diluted seminal plasma. These observations indicate that components of the seminal plasma are important for efficient entry of human spermatozoa into cervical mucus.

A standardized test for visual analysis of human sperm morphology.

OBJECTIVES: To develop a method to train and test simultaneously a large number of observers in the practice of visual sperm morphology analysis. DESIGN: Photographs of fixed and stained sperm were prepared. Fields of suitable sperm images were selected and individual images were numbered on each negative. Two tests, which contained a total of 100 sperm images, were created. Thirty images in each test consisted of three repeats of 10 images, while 70 images in each test were unique. The tests were administered to individuals participating in an American Fertility Society postgraduate course. Sperm images were projected on a screen and participants classified each sperm using the method that was used in their own laboratory. SETTING: Postgraduate course of The American Fertility Society. RESULTS: The majority of individuals participating in the tests used some version of the World Health Organization method. The group using the Strict method reported a lower value for the percentage of normal sperm than the groups using the other methods. The variability for the percentage of normal sperm was highest for the Strict method. The degree of classification reversal, i.e., classifying a sperm as normal during one repeat and then reversing the classification during another repeat, was high for all groups (26% to 44% of the classifications). Some degree of improvement was seen from test 1 to test 2. CONCLUSIONS: It is possible to develop efficient and inexpensive methods to train observers to perform the sperm morphology assay. Such methods also enable the objective measurement of the acquisition of proficiency, comparison between different classification methods, and identification of specific differences between observers. Such methods will become more important with implementation of the Clinical Laboratory Improvement Act.

Application of multivariate cluster, discriminate function, and stepwise regression analyses to variable selection and predictive modeling of sperm cryosurvival.

OBJECTIVE: To develop a mathematical model that predicts sperm cryodamage based on the kinematic characteristics of seminal sperm as detected by computer-aided sperm analysis (CASA). DESIGN: Computer-aided sperm analysis was performed on donor semen before and after freezing. An iterative multivariate statistical analysis technique was developed to identify sperm subpopulations and to select the best variables for modeling. Stepwise, multivariate regression was performed on the selected subpopulations to predict the post-thaw percentage of motile sperm from prefreeze kinematic values. SETTING: Andrology laboratories, IVF laboratories, and sperm cryobanks. PARTICIPANTS: Semen donors in an academic research environment. MAIN OUTCOME MEASURES: Identification of predictive kinematic variables; number of sperm subpopulations per sample; number of kinematic variables per subpopulation; prediction error for subpopulation membership; and an equation for prediction of post-thaw percentage of motile sperm from prefreeze CASA variables. RESULTS: The number of subpopulations for each specimen was predicted by 3 to 5 kinematic variables. Straight-line velocity (VSL) and linearity were the most commonly predictive primary variables, whereas curvilinear velocity and amplitude of lateral head displacement were the most commonly predictive secondary variables. The best linear model predicted the post-thaw percentage of motile sperm from the difference in VSL between the subpopulation with the highest value and the subpopulation with the lowest value in each prefreeze specimen. CONCLUSIONS: A small number of consistent kinematic variables accurately described physiologic subpopulations of sperm in prefreeze and post-thaw specimens from different men. An equation based on the characteristics of these subpopulations predicts the post-thaw percentage of motile sperm (i.e., sperm recovery) from simple prefreeze kinematic variables. This equation could improve specimen screening by eliminating the requirements for freezing and thawing in order to identify a specimen's vulnerability to cryodamage.

Sperm-immunobead binding decreases with in vitro incubation.

OBJECTIVE: To characterize the stability of the sperm-immunobead association over time. DESIGN: Prospective evaluation of sperm-immunobead binding, using direct and indirect assays. SETTING: Male Infertility Clinic, University of California, Davis, Davis, California. PATIENTS: Eleven men with sperm surface antibodies and 25 men with serum antisperm antibodies volunteered. MAIN OUTCOME MEASURES: Repeated assessment of sperm-immunobead binding over time. RESULTS: Serum immunoglobulin (Ig)G decreased a mean of 42.6% over 30 minutes, and serum IgA decreased a mean of 22.7% over 30 minutes. Semen-derived IgG binding fell a mean of 59.9% and semen-derived IgA fell a mean of 27.0% over 25 to 40 minutes. CONCLUSIONS: Sperm-immunobead coincubation results in a decrease in the number of sperm bound to immunobeads.