Gerhard Rohr

Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male fertility

OBJECTIVE To differentiate round cells in semen samples of subfertile men and evaluate the clinical significance during infertility investigation. PATIENTS One hundred and eight randomly chosen couples with a median duration of infertility of 4 (range, 1 to 20) years presenting at the outpatient infertility clinic of the University of Heidelberg, Germany. MAIN OUTCOME MEASURES Differentiation of round cells in semen by means of monoclonal antibodies (mABs) and a streptavidin-biotin system for staining. Correlation of results with medical history, outcome of clinical examination, sperm analysis, microbial screening of both partners, evaluation of sperm functional capacity in vivo by means of the postcoital test (PCT) and in vitro with the standardized crossed sperm-cervical mucus penetration test (SCMPT) and the subsequent fertility in a prospective study. RESULTS The method used for differentiation of round cells proved to be practical and suitable for routine use. The percentage of leukocytes ranged from 0% to 58% with a median of 3%. Number of round cells and percentage of leukocytes did not differ markedly with regard to andrologic history, clinical findings, for example, varicocele, results of standard sperm analysis, and microbial colonization of semen samples. However, high rates of leukocytes of the round cells correlated with reduced sperm count and morphology and results of PCT. Leukocyte-positive (> 15% leukocytes) specimens were also significantly more frequent in case of inadequate SCMPT and reduced sperm penetration ability in vitro. CONCLUSIONS In asymptomatic patients (in terms of genital tract infection), the majority of round cells consist of immature germ cells and < 5% are white blood cells. The streptavidin-biotin system and the mABs used in this study proved to be useful to identify patients with elevated rates of leukocytes in semen possibly reflecting subclinical genital tract infection with influence on sperm functional capacity and subsequent fertility. Thus the procedure can be recommended to be included in a comprehensive evaluation of male fertility.

The pH as an important determinant of sperm-mucus interaction*

OBJECTIVE To determine the clinical significance of endocervical mucus pH on sperm-mucus interaction during infertility investigation. PATIENTS AND MATERIAL Two hundred sixteen couples with a median duration of infertility of 4 years (range, 1 to 19 years) presenting at the infertility unit of the Womens University Hospital of Heidelberg, Germany. MAIN OUTCOME MEASURES Determination of endocervical pH by colorimetric and electrometric measurement and correlation of results with the outcome of postcoital testing (PCT) and other parameters of infertility investigation (semen and cervical mucus [CM] quality, microbial colonization of cervix and ejaculates, medical history, hormonal status, and specific medication) and the subsequent fertility in a prospective study. In vitro experiments with the sperm-cervical mucus penetration test (SCMPT) used as biological model. RESULTS The colorimetric determination of endocervical mucus pH is an easy method, suitable for routine clinical use, correlating significantly with electrometric measurement of pH. Median pH was 7.0 (range, 5.4 to 8.2). The mucus pH was significantly related with the results of PCT, even when mucus and semen variables were taken into account. No significant relationship was seen between the cervical index and mucus pH and the microbial colonization of cervix and ejaculates. The pH of endocervical secretions correlated with the peripheral hormonal status: low pH levels were significantly more frequent in patients with hyperandrogenemia, indicated by high testosterone and/or dehydroepiandrosterone sulfate levels before medication was started, and in hyperandrogenemic patients treated with dexamethasone than in the other women. Oral administration of estrogens led to a subtle alkalinization of the CM. With regard to subsequent fertility 6 months after pH testing, the pregnancy rate was significantly lower in women offering reduced mucus pH on occasion of the PCT in the group of couples with primary infertility and in couples with oligozoospermia of the male partner. The significant influence of pH on sperm-mucus interaction was confirmed in vitro with the SCMPT. CONCLUSIONS The results indicate that the pH of the CM, easily determined with pH indicator paper, is an important parameter of mucus quality with significant influence on spermatozoal viability in CM, which correlates with peripheral hormonal status and can be affected by oral medication with estrogens. Therefore the routine determination of pH on occasion of the PCT is recommended during infertility investigation.

Are zinc levels in seminal plasma associated with seminal leukocytes and other determinants of semen quality

OBJECTIVE To evaluate a potential association of zinc levels with seminal leukocytes, the outcome of semen cultures; and semen quality and sperm fertilizing capacity. DESIGN Prospective study. SETTING Outpatient infertility clinic of a university hospital. PATIENT(S) Two hundred fifty-six randomly chosen asymptomatic males from subfertile couples. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Determination of zinc in seminal plasma by flame atomic absorption spectroscopy. In aliquots of the same ejaculates the following tests were performed: immunocytochemical round cell differentiation to determine leukocyte counts and ratios, microbial screening, and comprehensive evaluation of semen quality (sperm analysis, biochemical parameters, antisperm antibody testing, and in vitro examination of sperm ability to penetrate cervical mucus). The patients underwent medical history, clinical examination, and postcoital testing. Subsequent fertility was determined (controlled for female infertility factors). RESULT(S) The concentration of zinc in seminal plasma did not correlate in a statistically significant way with leukocytes in semen, nor was it associated with bacterial colonization. There was no statistically significant relationship of zinc in seminal plasma or serum with semen quality parameters nor with local antisperm antibody testing of the IgG or IgA class. Zinc levels did not influence sperm capacity to penetrate cervical mucus in vitro or in vivo, and did not affect subsequent fertility. CONCLUSION(S) The zinc level in seminal fluid and serum is not associated with silent male genital tract infection (indicated by seminal leukocytes); nor is it related to semen cultures in asymptomatic individuals. The lack of association with other semen quality parameters indicates that the routine determination of zinc levels during infertility investigation is not recommended.

Induction of immunoresponse by subclinical male genital tract infection

OBJECTIVE To determine the relationship of subclinical infection or inflammation of the male genital tract, as evaluated with seminal markers, with local antisperm antibodies as potential parameter of immunoresponse. PATIENTS One hundred ninety-one randomly chosen males of subfertile couples who were asymptomatic in terms of genital tract infection. SETTING Outpatient Infertility Clinic of the University of Heidelberg, Germany. MAIN OUTCOME MEASURES Determination of leukocytes rates in semen using an immunocytochemical method for differentiation of round cells and measurement of polymorphonuclear (PMN) granulocyte elastase concentration in seminal plasma in addition to semen cultures as screening for subclinical infection of the male genital tract. Determination of local antisperm antibodies (Ab) with the mixed antiglobulin reaction ([MAR] immunoglobulin [Ig] G and IgA) in aliquots of the same ejaculates. RESULTS Leukocyte rates of the round cells ranged from 0% to 93%, leukocytospermia was found in 6.8%. This was not related significantly to the presence of local antisperm antibodies of the IgG or IgA class. There was also no significant association of antisperm Ab with the concentration of PMN granulocyte elastase in seminal plasma and the outcome of semen cultures. CONCLUSIONS The results of this prospective study suggest that when the rate or number of leukocytes or the concentration of PMN elastase in semen are taken as markers for subclinical infection or inflammation of the male genital tract, this is not associated significantly with the production of local antisperm Ab of the IgG or IgA class as indicator of immunoreaction.

Screening for subclinical inflammation in ejaculates

OBJECTIVE To determine the clinical significance of albumin determination in ejaculates by means of an easy office test to screen semen samples for subclinical infection-inflammation. PATIENTS One hundred fifty-nine randomly chosen males of couples with longstanding infertility (median duration of infertility 4 years (range 1 to 19 years) without clinical signs or symptoms of genital tract infection. SETTING Outpatient Infertility Clinic of the University of Heidelberg, Germany. MAIN OUTCOME MEASURES Screening of ejaculates for subclinical infection-inflammation by means of a ready-to-use kit for semiquantitative detection of albumin in addition to determination of leukocytes rates by means of monoclonal antibodies for differentiation of round cells and measurement of granulocyte elastase concentration in semen samples. Evaluation of sperm quality by means of standard sperm analysis including determination of local antisperm antibodies with the mixed antiglobulin reaction, evaluation of sperm functional capacity in vitro with the standardized sperm-cervical mucus (CM) penetration test, and semen cultures. All tests were performed from aliquots of the same ejaculates. RESULTS Screening of semen samples for elevated albumin with the modified paper strips proved to be very easy, quick, and suitable for routine use. Positive results were not related markedly to medical history and outcome of clinical examination as well as to standard parameters of sperm analysis and were not influenced by local antisperm antibodies of the immunoglobulin (Ig)G and/or IgA class and microbial colonization. However, albumin-positive semen samples were significantly less frequent in case of very good outcome of the sperm-CM penetration test. A significant relationship was found with high rates of leukocytes of the round cells in semen samples (total range 0% to 96%) and the concentration of granulocyte elastase (total range 1 to 880 micrograms/L). CONCLUSIONS The results of this prospective study suggest that the determination of albumin in semen samples with ready-to-use test kits might be a valuable additional marker for subclinical infection-inflammation of the male genital tract and therefore suitable for screening during infertility investigation.

Fate of Radioactive Exocrine Pancreatic Proteins Injected Into the Blood Circulation of the Rat:Tissue Uptake and Transepithelial Excretion

[35S]methionine or [35S]methionine-labeled exocrine pancreatic proteins were injected into the bloodstream of conscious rats. Samples of blood, urine, bile, and pancreatic juice were collected at varying intervals through 7 h. Injection of [35S]methionine resulted in the appearance of trichloroacetic acid--soluble radioactivity [( 35S]methionine) in bile and urine within 4 min and trichloroacetic acid-insoluble radioactivity in blood, bile, and pancreatic juice after 20 min. Analysis of these body fluids by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis and fluorography indicated that rat serum, biliary, and pancreatic proteins were labeled, respectively. After the injection of [35S]methionine-labeled pancreatic proteins, half of the trichloroacetic acid-insoluble radioactivity disappeared from the serum in 10-15 min. Radioactive proteins appeared after 5 min in urine and bile, and, over the course of the experiment, accounted for 1%-2% and 0.3%-0.5% of the injected radioactivity, respectively. Analysis of individual radioactive proteins excreted into bile by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis indicated preferential transhepatic transport of negatively charged pancreatic proteins. The majority of pancreatic proteins (approximately 97%) were taken up by a variety of body tissues, particularly kidney, liver, spleen, and lung. Trichloroacetic acid-soluble radioactivity, largely representing [35S]methionine, appeared sequentially in serum, urine, and bile within 2-12 min. At later experimental time points (greater than 60-90 min), radioactive rat serum, biliary, and pancreatic proteins appeared in blood, bile, and pancreatic juice, respectively. After the injection of 35S-labeled guinea pig pancreatic proteins into the blood circulation of the rat, trichloroacetic acid-insoluble radioactivity, observed in pancreatic juice after 60-90 min, exclusively represented rat exocrine pancreatic proteins as judged by the two-dimensional gel procedure. These studies indicate that pancreatic proteins are removed from the blood circulation by at least three separate pathways: (a) uptake and degradation by a variety of tissues in the body (approximately 97% of injected radioactivity), (b) excretion of intact proteins into urine (1%-2%), and (c) transport of intact proteins into bile (0.3%-0.5%). Transport of exocrine pancreatic proteins from the blood circulation to pancreatic juice could not be demonstrated.

High resolution proton magnetic resonance spectroscopy of human cervical mucus

High resolution 1H NMR spectroscopy is a powerful method for qualitative and quantitative analysis of the highly viscous human cervical mucus (CM). Up to 23 compounds could be identified in this study and can be observed in the 1H NMR spectra of native mucus without the need of any complicated preparative chemistry. Storage conditions could be excluded as a possible reason for variations observed between different samples. pH values decreased after freezing and storing at 253 K. For NMR studies, lyophilization proved to be most useful, allowing the determination of the water content, replacement of H2O by D2O, and most importantly, absolute quantification of low molecular mass compounds. In a small collective of women the concentrations of some of the small constituents of the CM are strongly correlated; an example is the mutual positive correlation of taurine, citrate and creatinine. In conclusion, high resolution 1H NMR spectroscopy is a valid method to investigate mucus composition and to determine absolute concentrations of low molecular mass compounds in CM.

Evidence in vivo of asynchronous intracellular transport of rat pancreatic secretory proteins

SummaryLabeled proteins which appeared in pancreatic juice after the intravenous (i.v.) injection of [35S]methionine into conscious rats with chronic pancreatic duct fistulae were separated by gel electrophoresis and measured by determination of the radioactivity of each of the separated bands. Radioactivity appeared in the secreted proteins 20 min after injection of the label. In the subsequent 10 min, 6.44% of the radioactivity was found in trypsinogen, whereas 100 min later only 3.4% of the radioactivity was associated with this enzyme. The values at 10 and 100 min for amylase were 10.85% and 21%, respectively, showing an earlier appearance of labeled trypsinogen than of amylase. Chymotrypsinogen behaved similarly to trypsinogen. Early secretion of labeled proteases was also demonstrated by separation of pancreatic proteins by two-dimensional gel electrophoresis followed by fluorography. In pancreatic duct cannulated rats, zymogen granules were prepared 30 and 60 min after injection of the labeled methionine. Determination of the radioactivity of the individual proteins demonstrated a similar time course of the labeling pattern in the zymogen granule fraction to that in pancreatic juice. The results of the experiments suggest an asynchronous secretion of newly synthesized rat pancreatic proteins.

Influence of secretagogues on asynchronous secretion of newly synthesized pancreatic proteins in the conscious rat.

The secretion of newly synthesized pancreatic enzymes was studied in pancreatic duct cannulated rats after intravenous injection of 100 μCi of [35S]methionine. Secretion rate was stimulated by intravenous infusion of either cerulein (0.2 μg/kg h) or carbachol (10 nmol/kg h) starting simultaneously with or 180 min before the injection of the labeled methionine. Secretory proteins were analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis or by nondenaturing gel electrophoresis followed by determination of the radioactivity associated with the individual proteins. Similar to unstimulated controls in all experiments, an early secretion of newly synthesized trypsinogen and chymotrypsinogen was found, whereas amylase and lipase were secreted only after a certain lag period. The results suggest that the intracellular transit of endoproteases is faster than that of other enzymes, irrespective of whether or not secretagogues were applied.