Evaluation of a container for collection and shipment of semen with potential uses in population-based, clinical, and occupational settings.

Large, population-based studies of semen quality are encumbered by the logistics and expense of obtaining semen samples from men who live in a variety of locations. A prototype semen collection and transportation kit, the TRANSEM100, can be distributed to study participants and then directly shipped to a central laboratory for analysis. This study was designed to evaluate the ability of male volunteers to correctly use the kit. Thirty volunteers aged 20 to 44 years with no history of diabetes, recent chemotherapy, fertility problems, or vasectomy were recruited through a newspaper advertisement, interviewed to obtain demographic information, and instructed on the use of the kit. Twenty-six of the initial subjects provided at least 1 semen specimen using the kit and returned the specimens by overnight delivery to the laboratory for analysis, 25 completed a follow-up interview on the use of the collection kit, and 20 submitted a second semen sample using the same method. The average volunteer was white, 27.8 years old, and held at least a college degree. Forty percent of the volunteers were married. In general, participants correctly followed the instructions for collecting, packaging, and shipping the semen samples. Volunteers were instructed to collect samples after at least 2, but no more than 7 days of abstinence. For the first and second samples submitted, participants collected semen samples after an average of 3.3 and 3.9 days of abstinence, respectively. Seventeen (65%) of the samples from the first sampling period and 16 (80%) of the samples from the second period were received in the laboratory the day after they had been collected. In summary, the TRANSEM100 may prove to be useful for collecting human semen in field studies. Further testing of this method is warranted to evaluate preservation of sample quality and use of the kit by men among diverse socioeconomic groups.

Effect of catheter composition on sperm quality.

Bladder catheterization for collection of retrograde ejaculates is commonly practiced, although previous studies have shown that materials used in the manufacture of the catheters may be toxic to sperm. Potential toxicity is particularly relevant to electroejaculation, as sperm from anejaculatory individuals undergoing this procedure characteristically exhibit poor motility and viability. To determine the effect of short-term exposure to various catheter materials on sperm quality, donor semen was diluted with BWW medium and aliquots incubated for 1 and 5 minutes with segments of four different catheters. The catheters were composed of latex, silicone rubber, polytetrafluoroethylene (Teflon), and radio-opaque Teflon, respectively. Following incubation, eosin-nigrosin staining for viability was performed and sperm motility assessed using computer-assisted sperm analysis (Cellsoft, Cryo Resources, Ltd). In the second phase of the study, donor semen was incubated with catheter segments coated with 0.3 ml of a water-soluble, nontoxic lubricant (Cellulosagel, Lederle) to evaluate whether the combination adversely affects sperm. Percent motility and viability for the semen specimens incubated with the four catheters alone did not differ significantly from control values either at 1 or at 5 minutes (P = 0.3, motility; P = 0.6, viability). The addition of lubricant did not change the catheter data significantly, indicating the absence of independent or synergistic toxicity (P = 0.5, motility; P = 0.4, viability). This study provides substantial evidence that brief exposure to conventional catheters, with or without a nontoxic lubricant, does not adversely affect sperm motility or viability.(ABSTRACT TRUNCATED AT 250 WORDS)

The personality and motivation of semen donors: a comparison with oocyte donors.

Seventeen consecutively recruited candidates for semen donation were evaluated by a psychologist with testing and a structured interview. Most men (71%) were motivated by financial compensation. Only 29% would donate semen if records were open to potential offspring. Fifty-nine per cent of the men were rated as excellent candidates from a psychological perspective and 35% were rated as acceptable with slight reservations. One was excluded as a donor. Psychological testing revealed mildly abnormal subscale scores for 35% of donors. Forty-seven per cent had histories of minor depressive or anxiety episodes and 35% had had periods of heavy alcohol use. Compared to oocyte donors at the same institution, the men were less altruistic, more affluent, and more likely to have abused alcohol. Women had more traumatic family and reproductive histories. Psychological evaluation can be a valuable tool in gamete donor selection.

Accuracy and precision of computer-aided sperm analysis in multicenter studies.

STUDY OBJECTIVES: To develop methods of instrument calibration and standardization that enable the use of computer-aided sperm analysis (CASA) technology in multicenter studies of sperm motility. SETTING: Clinical semen evaluation for the andrology laboratory and in vitro fertilization programs. PATIENTS: Semen specimens were selected from the videotape archives of the University of California at Davis Andrology Laboratory and used to produce a videotape reference standard for CASA instrument testing and calibration. Four identical CASA instruments, two each at two sites, were used to analyze the videotape. Machine accuracy was verified by manual analysis of the videotape. RESULTS: The coefficient of variation (CV) for repeated measures was between 1% and 8% for each variable on all CASA instruments. Mean values for CASA parameters varied as a function of the digitization threshold. A minimum CV was obtained at a particular threshold setting, suggesting an optimum for each machine. Because of high within-machine precision, slight differences in mean values between machines were statistically significant for some variables and some specimens; such differences are probably not biologically significant. CONCLUSION: Multicenter standardization of CASA instrumentation is possible, but threshold settings are one source of variation that must be controlled using CVs or another means of objective calibration. One method is to adjust the threshold of each instrument until the count obtained by CASA equals the count obtained by the manual method used to determine the dilution requirements for neat semen.

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.

Laboratory diagnosis in andrology.

Evaluation of male fertility traditionally has relied on analysis of spermatozoa and seminal plasma. Technology has consisted almost exclusively of subjective motility assessment and morphology of stained sperm-cell preparations. Evaluation of seminal plasma components, particularly anti-sperm antibodies, was based on functional properties of sperm immobilization and agglutination. In the last few years, objective measurements of sperm motility have become available, and new methods for immunological evaluation have been applied to andrology laboratory evaluations. This review discusses methods of laboratory diagnosis of male infertility, including many of these new technologies.

In vitro growth of rat bone marrow BFU-E.

Pokeweed mitogen-stimulated rat spleen cells were identified as a reliable source of rat burst-promoting activity (PBA), which permitted development of a reproducible assay for rat bone marrow erythroid burst-forming units (BFU-E). Optimum BPA dose, assay time, cell dose, and erythropoietin requirements for rat BFU-E were identified. A serum-free assay and a method for stimulating endogenous bone marrow BPA were developed. Identification of sources of rat BPA and characterization of the rat BFU-E assay makes this species more useful for hematopoietic studies. The similarity of rat BFU-E to human and mouse BFU-E strengthens the validity of rat models for erythropoiesis.

Causes of donor rejection in a sperm banking program.

During a 2-year period, 93 men between the ages of 18 and 46 years were screened for sperm donation using a three-stage protocol. Thirty (32%) applicants became active donors. Eighty-five percent of the semen specimens from these men were successfully frozen, quarantined, and released for insemination. The three-stage protocol successfully eliminated most uncommitted or unqualified applicants in an efficient, cost-effective manner. Sperm cryopreservation increased the rejection of otherwise acceptable donors by 27%.

Modulation of erythropoietin production by adenosine.

Adenosine is an important regulatory molecule that increases in hypoxic and ischemic tissues and has been proposed to mediate blood flow in response to oxygen availability. The current study ascribes another oxygen-responsive role to adenosine, that of regulating synthesis of the erythropoiesis-stimulating hormone, erythropoietin. When perfused through isolated rat kidneys, exogenous adenosine in nanomolar concentrations increased erythropoietin production, whereas inosine, the deaminated nucleoside, had no effect. In addition, an adenosine antagonist, and adenosine deaminase, diminished erythropoietin titers in renal perfusates. In intact rats, adenosine deaminase injections followed by a hypoxic stimulus slightly reduced erythropoietin serum concentrations, whereas an adenosine deaminase inhibitor sharply increased erythropoietin titers. The results suggest that adenosine may function as a mediator to link oxygen supply with erythropoietin production.

Improved assessment of bioactive erythropoietin in human sera using a modified tritiated-thymidine-incorporation assay.

The reproducibility and utility of an in vitro bioassay for erythropoietin were evaluated, and modifications, including the use of frozen target cells, were made. The assay, based on 3H-thymidine incorporation into phenylhydrazine-treated mouse spleen cells, showed a dose-response curve from 0.2 to 20 mU of sheep plasma or human urinary erythropoietin, with a 50-100-fold increase in 3H-thymidine incorporation. The dose-response curve, using purified, recombinant erythropoietin was identical to that from material obtained from other sources. The use of frozen cells simplified the methodology, reduced the time for assay preparation, and lowered costs. The freezing process resulted in 15% attrition of the cells, but after one year of storage, this level was not increased nor was the response to erythropoietin diminished. The nonspecificity of the assay was reduced by supplementing the cells with critical nutrients, depleting fetal bovine serum of endogenous erythropoietin, and reducing solute variability by ultrafiltration and reconstitution of samples with culture medium. Heat inactivation diminished inhibitors in sera samples, but affinity chromatography using wheat-germ lectin-Sepharose was most effective for extracting erythropoietin and reducing nonspecific effects. The assay is sensitive, can be used to measure multiple samples rapidly, and should prove useful for complementing immunoassay techniques.

Effect of cis-diamminedichloroplatinum on erythropoietin production and hematopoietic progenitor cells.

Studies were conducted to determine whether the progressive development of anemia associated with the antineoplastic drug cis-diamminedichloroplatinum (cDDP) was the consequence of decreased erythropoietin (Epo) production due to cDDP-induced nephrotoxicity or selective inhibition of erythroid progenitor cells. Five days after a single intraperitoneal injection of cDDP, hypoxia-induced Epo production was not decreased in mice and was increased significantly in rats in spite of severe multifocal tubular necrosis. In both species, colony-forming units-granulocyte macrophage (CFU-gm) and colony-forming units-erythroid (CFU-e) were reduced significantly, with a greater decrease in CFU-e. Studies of an anemic patient receiving cDDP also showed elevated Epo and decreased CFU-gm and CFU-e. In vitro exposure of mouse and human bone marrow to cDDP caused a dose-dependent inhibition of CFU-gm and CFU-e in both species, with human CFU-e showing greatest sensitivity. The results indicate that the primary hematologic toxicity of cDDP is directed at the hematopoietic stem cell compartment.

Chronic induction of interferon by continuous infusion of polyriboinosinic:polyribocytidilic acid.

In an attempt to induce circulating interferon (IFN) chronically, C57BL/6 or Balb/cBy mice received daily injections or continuous infusion of polyriboinosinic:polyribocytidilic acid [poly(I:C)] i.p. for 7 days. Significantly higher plasma IFN was present in C57BL/6 mice receiving poly(I:C) via continuous infusion compared with daily injections. Spleen and bone marrow natural killer (NK) cells were elevated only in the group receiving poly(I:C) continuously. Little IFN was induced acutely in Balb/cBy mice and none was present after chronic infusion of poly(I:C). NK cells were not elevated. Continuous infusion of poly(I:C) appears to overcome the hyporesponsiveness to repeated IFN induction in a susceptible mouse strain.

Erythropoietin secretion by isolated rat Kupffer cells.

Hepatic parenchymal and Kupffer cells were isolated from regenerating rat liver and maintained in primary culture in an attempt to identify the cell type responsible for extrarenal erythropoietin (Ep) production. Conditioned media from Kupffer cell cultures were shown to contain Ep. Kupffer cells were cultured in the presence and absence of serum, but only cultures with serum contained bioactive Ep. The addition of latex beads to Kupffer cell cultures increased the amount of Ep secreted. Parenchymal-cell-conditioned media were negative for Ep activity. Isolated liver macrophages might be useful in vitro for the study of Ep elaboration.

Treatment of severe aplastic anemia with antithymocyte globulin.

Eleven patients with acute-onset (less than 2 months), severe aplastic anemia were treated with antithymocyte globulin (ATG) at a total dose between 50 and 420 mg/kg. Median age was 27 (5-74) years. Two additional patients with chronic severe aplastic anemia received ATG but were excluded from analysis after development of bone marrow morphologic and cytogenetic abnormalities suggestive of acute leukemia. Of the 11 analyzed patients, 5 died within 6 months after initial ATG treatment. Six patients, or 54 percent, survived with a minimum follow-up of 24 months and the longest 48 months. Median survival is 42 months. All patients are transfusion-independent although none are completely normal, due to mild thrombocytopenia. The in vitro effect of ATG on pretreatment marrow CFUE was determined in 8 patients and concordance with clinical outcome was observed for only 3 patients. Three patients had no in vitro response and survived, and 2 patients had a positive in vitro response and died. Survival after ATG correlated with maximum percent decrease in absolute lymphocyte count during treatment. No significant correlation was determined for any other parameter. The mechanism of ATG action remains unknown but the clinical response suggests that immune dysfunction may play an important role in the development or prolongation of aplastic anemia, and that this abnormality may be reversible by ATG in some patients.

Erythropoietin-dependent erythrocytosis associated with hepatic angiosarcoma.

Erythropoietin (Ep) dependent erythrocytosis was discovered in a 77-year old man with angiosarcoma involving the liver and spleen. At autopsy, tissue from tumorous and normal sections of liver was obtained and extracts prepared for Ep assay in exhypoxic polycythemic mice. Neither tumor nor normal extracts had Ep activity; however, when the extracts were incubated with normal plasma prior to assay, significant amounts of Ep were generated. The normal liver tissue had three times the activity of the tumorous portion, 0.21 U/ml. These results suggest that the tissue contained an Ep activator, probably erythrogenin. It is proposed that the greater amount of erythrogenin activity in the normal tissue may reflect an attempt at liver regeneration following damage by an invading tumor, as regenerating liver has been identified as a stimulus for extrarenal EP and erythrogenin production.