Waltraud Eggert-Kruse

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.

Cytomegalovirus (CMV) infection—related to male and/or female infertility factors?

OBJECTIVE To screen for cytomegalovirus (CMV) infection in male and female partners of subfertile couples and to analyze a potential relationship with clinically relevant parameters of male and female factor fertility. DESIGN Prospective study. SETTING Outpatient infertility clinic of a university-based hospital. PATIENT(S) Randomly selected male and female partners of asymptomatic subfertile couples. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Screening for CMV in the semen of the male partner (using nested polymerase chain reaction) and, at the same time, in the endocervical material of the female partner; same-day CMV serology (anti-CMV IgG and IgM class antibodies [Ab]); medical history and clinical examination; evaluation of semen quality, including sperm functional capacity and detection of antisperm Ab (ASA) and seminal white blood cells (WBC) in aliquots of the same ejaculates; bacterial screening of both partners; and detailed examination of the cervical factor and other variables of female subfertility. RESULT(S) The presence of CMV in semen was not significantly related to semen quality including sperm functional capacity, local antisperm Ab, or seminal WBC. CMV in endocervical material was not associated with a reduced quality of the cervical mucus or with other female infertility factors. CMV in semen was not associated with the presence of CMV in the endocervical material of the female partners. CMV infection was not significantly associated with other microorganisms of the lower genital tract. CMV serology (IgG and/or IgM Ab) did not sufficiently reflect CMV presence in semen or the cervix. CONCLUSION(S) CMV presence in the genital tract of subfertile patients is considerable, but findings do not suggest that sexual transmission is a frequent route of infection or that CMV infection is a significant cause of infertility.

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.

Thyrotropin-releasing hormone (TRH) and metoclopramide testing in infertile women.

Thyroid stimulating hormone (TSH) determinations before and after thyrotropin-releasing hormone (TRH) stimulation were obtained in 834 infertile women, from 1982 until 1985. Thyroid function disturbances were seen in 20% of the women, in accordance with the prevalence in South Germany. Postcoital tests were significantly poorer in women with subclinical hypothyroidism than in euthyroid patients. Spontaneous conception was more frequent in euthyroid (16%) than in hypothyroid (6%) women. During the same period, prolactin was determined after TRH stimulation in the early follicular phase and after metoclopramide stimulation in the luteal phase, in 759 women. The pregnancy rate was not improved by administration of dopamine agonists in women with an exaggerated response to TRH or metoclopramide. Our results suggest that subclinical hypothyroidism as well as disorders of prolactin secretion may play a role in infertility. The TRH test is proposed to rule out thyroid dysfunction. Neither the TRH nor the metoclopramide test was useful for the prognostic differentiation of prolactin secretion disorders.

Relationship of Sperm Acrosin Activity to Semen and Clinical Parameters in Infertile Patients/Die Beziehung der Akrosinaktivität zu verschiedenen Sperma- und klinischen Parametern bei infertilen Patienten

Summary: Acrosin activity (aa) was routinely measured in 189 infertile men who attended the hospital between March 1984 and January 1985. The evaluation of the male included semen analysis, microbial screening, postcoital testing (PCT), in vitro sperm penetration test (SPMT), and screening for anti‐sperm antibodies in serum. The strongest positive correlations of aa were found for sperm motility, count, morphology, vitality and volume. In presence of a poor PCT result, aa was significantly reduced, even in patients with good motility in native semen. A similar result was obtained with the SPMT: reduced sperm density and poor sperm motility in the capillary after 2 hours were significantly correlated to reduced aa. Smokers exhibited lower aa in presence of normal sperm count and motility. In the infertile group aa was significantly lower than in the group of men whose spouses conceived. On the basis of our results, the routine determination of aa is not necessary, but it provides additional prognostic information in couples with unexplained infertility.

Are Chlamydial Lipopolysaccachide-directed Antibodies in Seminal Plasma or Serum Clinically Significant During Investigation of Male Infertility?

OBJECTIVES To analyze the clinical relevance of chlamydial serology in seminal plasma (SP)/serum for male fertility. METHODS We determined the IgG, IgA, and IgM class antibodies to Chlamydia trachomatis with a lipopolysaccharide-directed standard enzyme-linked immunosorbent assay in the serum of 173 asymptomatic subfertile couples and in the same-day SP in a prospective study. The semen quality evaluation was done using aliquots of the same ejaculate (eg, sperm analysis, leukocytes, bacterial cultures, sperm functional capacity). The medical history, clinical examination, postcoital testing, and determination of subsequent fertility were also done. RESULTS The antichlamydial antibodies of the 3 classes were significantly interrelated, and SP Chlamydia IgA antibodies were significantly associated with the findings in the serum. The evidence for previous or persistent chlamydial infection was significantly more frequent in the female partners of men with Chlamydia antibodies in the serum and/or Chlamydia IgA antibodies in the SP. The chlamydial serology results in the SP/serum were not associated with the sperm quality, leukocytes, semen culture findings, or sperm functional capacity. CONCLUSIONS The chlamydial serology results (ie, IgG, IgA, and IgM antibodies in SP/serum) determined using a lipopolysaccharide-directed enzyme-linked immunosorbent assay were not indicative of reduced sperm function or subsequent fertilizing capacity.

Hormone Load Tests in Infertile Male Patients

The recognition that discreet hormonal abnormalities may cause ovulation disorders in women suggested that the male partner of infertile women might also suffer from unrecognized hormonal dysfunction amendable to substitution therapy. We obtained a combined stimulation test with gonadotropin-releasing hormone (GnRH), thyreotropin-releasing hormone (TRH), and ACTH in 225 males with childless spouses, when the couple sought to have children for at least one year. The following hormone levels were determined: estradiol (E), thyroid-stimulating hormone (TSH), prolactin, testosterone (T), dihydrotestosterone (DHT), androstenedione(A), 17-OH-pregnenolone (17-OH-Preg), 17-OH-progesterone (17-OHP), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), cortisone (F), and 21-desoxycortisone (21DF). Basal and stimulated, and adrenal-testicular steroids with and without ACTH stimulation failed to demonstrate a relevant relationship to semen parameters. Gonadotropin levels had a significant negative correlation to all important semen parameters (testicular volume, sperm count, motility, morphology, and vitality) and were positively correlated to spermiogenetic defects. Stimulated LH values were more clearly associated with spermiogenetic defects than basal LH. Nonetheless, basal FSH concentrations were more informative than LH. Stimulated prolactin values were positively correlated with both gonadotropin and with sperm morphology. E concentrations had a significant positive correlation with both basal and poststimulation DHEAS values, and showed a highly negative correlation with sperm count, morphology, and vitality. In comparison, good sperm parameters were associated with high poststimulation T concentrations. The results of this study suggest that basal FSH and E concentrations, as well as the stimulated LH, T, and prolactin determinations, should be included in the evaluation of male sterility.