Michael J. Zinaman

Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination

Semen samples from 77 men were used to estimate the accuracy and precision of measurements of sperm density, percent motility, and motion characteristics using a new, fully automated, computer-assisted semen analyzer (CASA). Results are compared with traditional semen analysis methodology. Acceptable precision for count and percent motility was obtained using three to nine random fields of observation when there were more than 10 cells per high-power microscopic field. A highly significant correlation (P less than 0.01) was found between the two methods in the assessment of sperm concentration and percent motility, but CASA overestimated concentration significantly (P less than 0.01) by about 30% when there were less than 60 cells per high power field. This overestimation seemed to be due to the number of nonsperm particles in semen that are confused with spermatozoa by the program. Linear velocity determination was influenced by the number of tracking points as well as by the concentration of cells present in the counting chamber. More representative linear velocity estimations in semen were obtained when sperm concentration was less than 40 x 10(6) cells/ml and 20 tracking points were used in the determination. For semen specimens containing higher cell density, sperm concentration needed to be reduced by diluting the semen with the patients own sperm-free seminal plasma before measuring motion characteristics.

LABORATORY METHODS FOR ASSESSING HUMAN SEMEN IN EPIDEMIOLOGIC STUDIES: A CONSENSUS REPORT

It is clear that additional methodologic work needs to be performed. Some data gaps described above are being actively investigated. Other standards were not addressed at this meeting; statistical handling of the data, differences among CASA machines, and factors to consider as potential confounders in analysis are just a few. These may be the subject of future workshops, which will also review progress made in the existing knowledge base. For now, this effort represents a first attempt to share information and to use it to encourage investigators in different laboratories to employ similar methods. In this way more direct comparisons among studies can be made, and our collective data base can be strengthened.

Factors associated with withdrawal bleeding after administration of oral micronized progesterone in women with secondary amenorrhea

OBJECTIVE To compare two dosages of oral micronized progesterone (P) and placebo for withdrawal bleeding and side effects. DESIGN Prospective, randomized, double-blind. SETTING Academic institution. PARTICIPANTS Out of 190 screened with oligomenorrhea/amenorrhea, 60 who qualified completed the study. INTERVENTIONS A 10-day course of (1) oral micronized P 300 mg, (2) oral micronized P 200 mg, or (3) placebo. MAIN OUTCOME MEASURES Withdrawal bleeding, side effects, and changes in lipids. Endogenous estradiol (E2) concentrations at baseline and P concentrations during treatment were correlated with bleeding response. RESULTS Withdrawal bleeding occurred in 90% of women taking 300 mg, 58% of women taking 200 mg, and 29% of women taking placebo (P less than 0.0002 for 300 mg versus placebo). Side effects occurred similarly among the groups (P = not significant). Lipid concentrations were unchanged. Endogenous E2 and treatment P concentrations were of limited predictive value for withdrawal bleeding. CONCLUSIONS Progesterone 300 mg induced significantly more withdrawal bleeding than placebo, with similar side effects. Bleeding response cannot be predicted reliably from E2 and P concentrations.

Calcium metabolism in postpartum lactation: the effect of estrogen status * †

Postpartum lactation represents a unique state of increased calcium demand in which women are also hyperprolactinemic and hypoestrogenic. This is associated with increased calcium mobilization from bone and bone loss. To better understand the effect of estrogen (E) status on calcium metabolism during lactation, we studied 10 long-term lactating women at 12 weeks postpartum when they were hypoestrogenic and again at 37.4 +/- 3.4 (+/- SD) weeks during the midfollicular phase of their second ovulatory cycle. Urinary and serum markers of calcium metabolism were measured at these intervals. The results revealed that when E was low, osteocalcin and hydroxyproline were increased with a lower circulating parathyroid hormone (PTH) level, whereas reciprocal changes were noted when E was increased. The findings suggest that E status can modulate PTHs ability to mobilize ones stores of calcium.

The effect of nonsteroidal anti-inflammatory drugs on ovulation: a prospective, randomized clinical trial☆

OBJECTIVE To assess the effect of ibuprofen, a nonspecific inhibitor of prostaglandin synthesis, on ovulation. DESIGN Prospective, randomized, double-blind, placebo-controlled cross-over study. SETTING University Medical Center. PATIENT(S) Twelve normally cycling women between ages 20 and 40. INTERVENTION(S) Subjects were randomized to either oral ibuprofen (800 mg) or placebo three times per day, beginning when the maximum diameter of the leading follicle reached 16 mm by ultrasound, and continuing for 10 days total. The second cycle was a washout period, and in the third cycle, the subjects were crossed over to the alternate regimen from the first cycle. The probability of delayed follicular collapse was determined using the binomial distribution, and changes in P levels were compared using the paired t test. MAIN OUTCOME MEASURE(S) Urinary LH surge, follicular collapse by serial transvaginal ultrasonography, and serum midluteal P levels. RESULT(S) Eleven of 12 subjects detected an LH surge with both ibuprofen and placebo. Five of 11 women demonstrated a >or=2-day increase in time interval from detection of the LH surge to follicular collapse, and 3 of those 5 had been randomized to ibuprofen. This represents a 27% (3 of 11; 95% confidence limits: 1%, 53%) rate of delay for follicular collapse for ibuprofen. There was no difference in average midluteal P levels for ibuprofen or placebo. CONCLUSION(S) If ibuprofen inhibits follicular collapse, this effect is seen in a small group of study subjects, and this information should be clinically reassuring to patients who take nonsteroidal anti-inflammatory drugs. Serum midluteal P levels were unaffected by administration of ibuprofen.

Effects of vehicle supplementation on total estradiol absorption from a transdermal estradiol delivery system

Objective To evaluate the effects of vehicle supplementation on serum estradiol (E 2 ) delivery pharmacokinetics from the Ciba-Geigy (Summit, NJ) 0.1-mg Estraderm Patch. Design Postmenopausal women were randomized to a 28-day crossover treatment protocol separated by a 14-day wash out period. Setting Normal human volunteers were studied in an academic research environment. Patients, Participants The subject pool included eight healthy postmenopausal women between 32 and 60 years of age. Interventions In treatment A, a 0.1-mg Estraderm Patch was worn for 7 days; in treatment B, an identical patch was worn into which 0.6mL of ethanol was injected on day 3 of use. Main Outcome Measures Serum E 2 levels were measured in both groups. Results Although E 2 absorption showed characteristic interpatient variability, addition of ethanol significantly extended the mean time for serum E 2 levels to return to baseline, without increasing peak absorption. The mean extension was 50 hours. Conclusion The addition of ethanol to the Estraderm Patch increased the duration of elevated serum E 2 levels measured in menopausal women, thus potentially increasing the effective life span of the transdermal therapeutic system.

Incidence and implications of altered semen quality on family planning.

Alterations in the expression of the human genome, or interference with its products, can be induced in the male reproductive system by chemicals mimicking or antagonizing naturally occurring hormones. Opportunities exist for disruption at the hypothalamus, pituitary and testis levels. Recent concerns generated by the increased incidence of testicular cancer, congenital anomalies of the male genitalia and possible alterations in human semen quality have been linked to the environment.The report by Carlsen in 1992 [1] suggested that semen quality has deteriorated over the past six decades. More recent reports suggest that the decline may be globally non-uniform and regional in nature. The effects of any such declines upon overall pregnancy rates are generally unknown, although some studies have attempted to address them. A preliminary review of the impact of a small decrease in sperm concentrations suggests that a directly measurable reduction in fecundity does not occur, but that future problems could be anticipated. Decrements in semen quality will alter the epidemiological probabilities of pregnancy due to coitus on different cycle days and may thereby change the duration of the fertile time. Current understanding of the implications of altered semen quality on relative fertility is not sufficient to change our current teaching and practice of NFP.

Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342**Supported in part by a subcontract SBIR 2R44 ES0562 to Cornell University, Ithaca, New York, from The Small Business Innovative Research Program, National Institutes of Health, Bethesda, Maryland.††Hoechst 33342, Sigma Chemical Company, St. Louis, Missouri.

OBJECTIVE To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA). DESIGN Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm. SETTING Normal human volunteers in an academic research and medical environment. PATIENTS Selected healthy student volunteers. INTERVENTIONS Delivered semen to the laboratory within 1 hour of collection. MAIN OUTCOME MEASURE Semen quality measured by CASA. RESULTS Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent. CONCLUSIONS The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.

Enhanced follicle regulatory protein levels accompany reinitiation of ovulatory function after parturition

OBJECTIVES We examined the changes in follicle regulatory protein, estrone-3-glucuronide, pregnanediol-3-glucuronide, and luteinizing hormone levels in first-morning urine samples from postpartum, fully breast-feeding women to characterize the reemergence of these urinary hormones after pregnancy ovarian quiescence and early postpartum period and to determine whether follicle regulatory protein could be used prospectively to predict the return of fertility. STUDY DESIGN Twenty-five hundred urine samples collected from six postpartum women were evaluated. Daily urine samples collected from normally cycling women were used to establish normal urinary hormone and hormone metabolite cyclicity. Luteinizing hormone, estrone-3-glucuronide, and pregnanediol-3-glucuronide levels were measured by radioimmunoassay. Follicle regulatory protein level was assayed with a double-antibody enzyme-linked immunosorbent assay. RESULTS Although follicle regulatory protein levels were found to be very low or undetectable in early postpartum urine, they began to rise with episodes of estrone-3-glucuronide and pregnanediol-3-glucuronide secretion. A chi 2 analysis suggests that increasing urinary follicle regulatory protein levels are most closely associated with the luteal phase of the first menstrual cycles in postpartum women. CONCLUSIONS These results suggest that follicle regulatory protein is of little value in predicting either the onset of renewed ovarian activity or the fertile period.