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Liposomes consisting of negatively charged phospholipids interact almost exclusively with the equatorial segment (ES) of human spermatozoa provided the cells have undergone the acrosome reaction (AR) [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently tagged liposomes, this interaction can be observed by fluorescence microscopy, showing either a diffuse fluorescence in the ES region (pattern ESd, presumably reflecting membrane-incorporated lipids as a result of fusion) or a punctate fluorescence (pattern ESp, representing adhering liposomes). These distribution patterns remain unchanged during prolonged incubation, up to 40 min. Not only do these observations suggest the existence of fairly specific liposomal binding sites, associated with the ES region, but also that a barrier to lipid lateral diffusion seems to exist in the ES membrane. Using liposomes that contain fluorescent lipid analogues in either both leaflets or in the inner leaflet only, we demonstrate that this putative barrier entails both membrane leaflets. Treatment with EDTA caused fluorescence to spread from the ES towards other membrane domains. Since only spermatozoa displaying pattern ESd were affected by the chelator, the randomization was not caused by EDTA-induced fusion activity. Therefore, this observation provides further evidence that in spermatozoa displaying pattern ESd the fluorescent lipid analogues were incorporated in the ES membrane as a result of fusion. Furthermore, these experiments support the view of the existence of a transmembranous block to lipid lateral diffusion in the ES, the stability of which may be governed by bivalent cations.

Evidence on Chlamydia trachomatis causing male infertility is conflicting. We therefore collected data on epidemiological and clinical correlates of chlamydial infection and male fertility in 184 males visiting our Fertility Unit. Antibodies against Chlamydia trachomatis in serum and semen were also determined. Significant correlations were demonstrated between current chlamydial urethral infection and semen immunoglobulin (Ig) A, serum IgA and serum IgG. These parameters, however, were neither related to a history of sexually transmitted disease nor to lifetime number of sexual partners. Why, in the male, serology does not correlate with chlamydial infection in a more remote past is explained. Our data support, on epidemiological as well as serological grounds, the conclusion that chlamydial infection probably does not contribute significantly to male infertility.

Human sperm nuclei show an exceptional variability; compared with sperm nuclei of other Eutherian mammalian species. The variable stability is caused by a variable content of chromatin stabilizing disulphide bridges, which in turn is determined by differences in composition of basic proteins: protamine types and subtypes, histones, and intermediate forms. An abnormal state of the chromatin can be related to an abnormal DNA configuration or an abnormal DNA content: diploid instead of haploid. Abnormality of the nuclear chromatin is probably one of the causes of morphological aberrations of the sperm head. A relationship between abnormal chromatin and male infertility has been reported repeatedly. Recently available evidence suggests that living spermatozoa with abnormal chromatin, leading to abnormal morphology, have a strongly reduced capacity to fertilize an oocyte. In addition, if these spermatozoa fertilize oocytes, the embryonal development will most probably be abnormal.

The hypo-osmotic sperm swelling (HOSS) test was performed on semen samples of five normospermic men from couples with prolonged infertility. Previously, the men had negative results of the zona-free hamster oocyte (ZFHO) test on two different ejaculates and the wives subsequently had become pregnant by donor insemination. A high swelling percentage (at least 60%) was found in all five men. It would appear that the HOSS test does not assess fertilizing capacity.

Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001–1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using anti-sperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step.

Objective To develop a method to detect acrosome-reacted spermatozoa on human zonae pellucidae using only commercially available reagents and without need for sperm fixation. Design Sperm head labeling with biotinylated soybean trypsin inhibitor (SBTI-biotin) was compared with results of a known method using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin. The SBTI-biotin method was applied to sperm bound to human zonae pellucidae. Setting and Subjects Healthy sperm donors with normal semen characteristics were recruited by the Laboratory for Reproductive Medicine in a university medical center. Main Outcome Measures Soybean trypsin inhibitor-biotin binding patterns on nonfixed spermatozoa were visualized with avidin-Texas Red. The development in time of various patterns upon induction of acrosome reaction (AR) in suspension with Ca 2+ -ionophore A23187 was noted and compared with P. sativum agglutinin FITC labeling patterns. Soybean trypsin inhibitor-biotin labeling patterns of spermatozoa bound to human zonae pellucidae were determined. Results Soybean trypsin inhibitor-biotin bound specifically, via the SBTI-moiety, to an acrosomal factor as soon as AR started. Sperm-head labeling patterns could be assigned to defined stages of the AR process. The results were highly correlated to those obtained with P. sativum agglutinin FITC. The end point of the AR in suspension and on zonae pellucidae was SBTI-biotin binding confined to the equatorial segment. Conclusion The SBTI-biotin method can be used to detect nonfixed acrosome-reacted spermatozoa both in suspension and on zonae pellucidae.

The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long-term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135-185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.

Relationships were studied between correlates of sexually transmitted diseases and chlamydial antibodies versus cervical mucus abnormalities and tubal abnormalities. An estimate was also attempted of the baseline prevalence of endogenous tubal abnormalities.Prospective, descriptive.The outpatient department of the Fertility Unit of the Department of Obstetrics and Gynaecology of the Groningen University Hospital.Females (n = 184) of infertile couples consecutively visiting the department for evaluation and treatment.None.Sexually transmitted disease correlates, cervical chlamydial infection, cervical and serum chlamydial antibodies, cervical mucus qualities according to World Health Organization criteria, tubal abnormalities.Of the 175 cervical mucus samples studied, subnormal quality was because of imperfect timing of collection of 22 of the 23 subnormal samples. Tubal abnormalities correlated with more than five lifetime sexual partners (P less than 0.035), as well as with serum immunoglobulin G antibodies (P less than 0.05), which could be because of interaction between both risk factors.No correlation was found between subnormal cervical mucus and chlamydial infection. Approximately two thirds of the tubal abnormalities seem unrelated to exogenous factors, although further study seems warranted.

Results of the zona-free hamster oocyte test were compared with pregnancy rates after coitus and after artificial insemination with donor semen (AID) in 322 primary infertile couples. Artificial donor insemination was offered if in the test the percentage of hamster oocytes with at least one decondensed sperm head (oocyte-sperm interaction rate) was less than 20. If the rate was less than 5, the pregnancy rate after coitus was significantly lower than in the group with a rate of greater than or equal to 5 to less than 20. In the former group, the pregnancy rate by AID was significantly higher than the pregnancy rate by coitus. We conclude that the zona-free hamster oocyte test is useful to predict the chance to achieve pregnancy in couples with unexplained infertility and to determine whether AID can increase this chance.

The presence of IgG and IgA on motile spermatozoa from normal semen donors and men from infertile couples was studied with mixed antiglobulin reaction (MAR) tests. The percentage of motile spermatozoa with IgG (IgG MAR%) was found to be related to the circulating antispermatozoal IgG. No direct relation could be detected between the IgG MAR% and the sperm agglutinating activity in seminal plasma (SP) or the percentage of motile spermatozoa showing the shaking phenomenon (S%) in the sperm cervical mucus contact (SCMC) test. The percentage of motile spermatozoa with IgA (IgA MAR%) showed no direct relation to the sperm agglutinating activity in serum and SP, but was roughly proportional with the S%. It was discerned that the shaking phenomenon in the SCMC test was probably due to presence of IgA on the motile spermatozoa. Previously it had been demonstrated that also the sperm agglutinating activity in SP is caused by IgA that is probably locally produced in the male genital tract. In conclusion it was thought that the reduced ability of penetration into cervical mucus by spermatozoa from infertile men is caused by locally produced IgA.

The presence of the tumor marker CA 125 was studied in the cervices of healthy women. Immunohistochemical staining of normal cervical tissue demonstrated the presence of CA 125 in the tall columnar cells of the endocervical epithelium but not in the ectocervical squamous epithelium. We measured very high levels of CA 125 in liquefied cervical mucus from women with regular menstrual cycles. At midcycle, levels ranged from 14,200 to 153,000 U/ml (n = 13) in cervical mucus, while normal levels less than 35 U/ml were found in the corresponding serum samples. Levels of CA 125 in cervical mucus are comparable to the high levels found in cyst fluids from ovarian tumors (median 24,600 U/ml, n = 25). When secretion of cervical mucus was stimulated by ethinyl estradiol, equally high levels were found (7900 to 138,000 U/ml, n = 10). We conclude that the tumor marker CA 125 is synthesized and secreted by normal endocervical cells. Apparently an effective barrier exists between the endocervical mucosa and the circulation.

Donor spermatozoa with good motility were pretreated with four sera containing high titers of sperm-agglutinating IgG, one serum without sperm-agglutinating activity, the IgG fractions from these five sera, F(ab)2 and Fragment antigen binding (Fab) fragments from these sera and from the IgG fractions, and one seminal plasma sample with a high titer of sperm-agglutinating IgA. With mixed antiglobulin reaction tests the percentage of motile pretreated spermatozoa sensitized with IgG Fab or IgG Fragment crystalline (Fc) parts was determined. Spermatozoa sensitized with intact antispermatozoal IgG showed a strong reduction in their capacity to penetrate cervical mucus. The reduction of the penetration capacity was determined by estimation of the percentage of motile spermatozoa rapidly shaking (S%) in the sperm-cervical mucus contact (SCMC) test. Removal of the Fc parts resulted in a decreased S%. Treatment of spermatozoa, on which Fab fragments were present, with intact antibodies to IgG Fab fragments, resulted in a recurrence of a high S%. A decrease of the S% was also found if Fab fragments from antibodies to IgG Fc fragments were added to spermatozoa sensitized with intact antispermatozoal IgG. Similarly, it was found that a decrease of the S% occurred when IgA sensitized spermatozoa were treated with Fab fragments from antibodies to human IgA. In the sperm penetration meter test the IgA sensitized spermatozoa treated with Fab fragments from anti-human IgA antibodies showed a better penetration than untreated IgA-sensitized spermatozoa.

The relation between presence of antispermatozoal antibodies in infertile men and the inhibition of the in vitro sperm penetration into cervical mucus (CM) was studied with the sperm cervical mucus contact (SCMC) test. The tests were performed with semen from infertile men and from semen donors. The CM used permitted good penetration of normal spermatozoa. The so called “shaking phenomenon”, the result of a specific interaction of spermatozoa and CM, was expressed in the shaking percentage (S%). The S% did not change beyond the experimental error within 30 min after mixing semen and CM. The S% was 30 at the most in 194 out of 198 SCMC tests with normal donor semen and normal pre-ovulatory CM. Significant negative correlations (P < 0.005) were found between the readings of the sperm penetration meter (SPM) test on one hand and the S%, the sperm agglutination titer in the serum and the sperm agglutination titer in the seminal plasma (SP) on the other hand. Significant positive correlations (P < 0.005) were found between the S% and the sperm agglutination titer in the SP. The sperm agglutination titer in serum and in SP correlated significantly better (P < 0.02) with the S% in the SCMC test than with the readings of the SPM test. It was concluded that: 1. A high S% is highly specific for the presence of antispermatozoal antibodies in infertile men, 2. The SCMC test is more suited than the SPM test for studying the effect of antispermatozoal antibodies on the penetration and migration of spermatozoa into CM

The interaction between spermatozoa and cervical mucus in the presence of antispermatozoal antibodies was studied in the SCMC-test and in the Miller-Kurzrok test. A high correlation was found between: 1. The "shaking phenomenon" in the SCMC-test; 2. sperm penetration into cervical mucus; 3. the sperm agglutination titre in seminal plasma and in cervical mucus. The authors performed investigations which indicate that antispermatozoal IgA in semen and in cervical mucus is probably responsible for the "Shaking phenomenon" in the SCMC-test and for the reduced penetration of spermatozoa into cervical mucus. Experiments with the Miller-Kurzrok test make it probable that antispermatozoal antibodies in semen, which are responsible for the "shaking phenomenon", are directed against the surface antigens of the sperm tails. Antispermatozoal antibodies in cervical mucus, which are responsible for the "shaking phenomenon", are probably directed against the surface antigens of the sperm acrosome.

Supravital staining of human spermatozoa using trypan blue was compared with eosin Y staining. The dyes were applied for determination of the percentage of "dead" spermatozoa in semen samples and for determination of complement-dependent antisperm antibody activity in serum. Supravital staining percentages with trypan blue were significantly lower than with eosin Y.

Sera containing sperm-agglutinating and/or complement-dependent sperm-immobilizing activity were tested for their ability to induce a shaking phenomenon in the sperm-cervical mucus contact (SCMC) test. Donor spermatozoa were treated with the sera in a one-step incubation and washing procedure. The percentage of motile pretreated spermatozoa showing the shaking phenomenon (S%) was determined in the SCMC test. In addition, the immunoglobulin class of antibodies present on the pretreated spermatozoa was determined in mixed antiglobulin reaction tests for IgG, IgM, and IgA. An S% of at least 80 was always observed with spermatozoa pretreated in sera with a sperm agglutination titer of at least 32, provided that IgG was detected on more than 90% of the motile spermatozoa. The high S% was also obtained with spermatozoa pretreated in purified IgG from sera containing antispermatozoal antibody activity. It was concluded that the reduced cervical mucus penetration capacity of spermatozoa pretreated with sera containing antispermatozoal IgG could be ascribed, at least partially, to the occurrence of a shaking phenomenon.

The in vitro swelling of human sperm nuclei in the presence of sodium dodecyl sulphate (SDS) was studied by counting swollen sperm nuclei in a microscopic preparation and by measuring the decrease of optical density at 600 nm (OD600). At pH 8.0 or lower, OD600 showed a linear relationship to the sperm count. At higher pH values a fraction of the nuclei became swollen and OD600 decreased proportionally, although the sperm count did not change in the first 2 hr. The fraction of swollen sperm nuclei increased with time and by raising the pH or temperature. The swelling occurred only in the presence of SDS and was the consequence of a binding of SDS to the hydrophobic regions of the nuclear proteins.

Infertility in men and women with spermagglutinins is the result of disturbed penetration and migration of spermatozoa in the cervical mucus. In ejaculates with partial spermagglutination caused by autoimmunization, the progressive propUlsion of the sperm was changed into stationary, shaking movement the moment the sperm came into contact with cervical mucus. The same alteration in spermatozoal motility pattern also occurred when spermatozoa from a normal, fertile ejaculate came into contact with cervical mucus of a woman whose serum contained sperm antibodies. This shaking phenomenon was visualized in a simple test, the sperm-cervical mucus contact test. We demonstrated that sensitized spermatozoa exhibit the shaking phenomenon after contact with the glycoprotein fraction of the cervical mucus and not after contact with the aqueous fraction. Therefore, the hypothesis is introduced that the shaking phenomenon is due to an interaction between sensitized spermatozoa and the glycoprotein micelles in cervical mucus.

Factors related to semen improvements and fertility after high ligation of the left internal spermatic vein were investigated in 97 men from infertile couples. Changes in some semen characteristics after operation suggest a relationship between varicocele size and semen improvements and an inverse relationship between preoperative semen values and semen improvements. An inverse relationship seemed to exist between varicocele grades and deterioration of semen values. Fertility after operation was related to age and to semen improvements. Preoperative semen characteristics had no prognostic value for postoperative fertility.

Previous authors reported high incidences of complement dependent sperm immobilizing antibodies in cervical mucus (CM) and found a correlation with poor postcoital tests. In all those investigations the CM samples were tested after dilution with a salt solution. In the present study we investigated 50 CM samples from infertile couples who had during the routine fertility investigation poor postcoital tests. All CM samples were tested after dilution with an equal volume of normal human serum; only one sample was found positive. On the other hand, if the CM samples were diluted with an equal volume of a buffered salt solution, many positive results were obtained. With some guinea pig serum batches, however, all tests with buffer-diluted CM samples were negative, despite good reaction of the positive control serum. We concluded that the positive results with buffer-diluted CM samples were due to a factor in the animal serum used as complement preparation and that complement-dependent sperm immobilizing antibodies in CM are an unlikely cause of a poor postcoital test.

This study concerns the sexual functions of 101 patients who had undergone bilateral retroperitoneal lymph node dissection for stage I or II nonseminomatous testicular cancer between 1969 and 1982. All patients were without evidence of disease after at least 4 years of follow-up. Antegrade ejaculation was present in 12 patients, while 89 patients experienced "dry ejaculation." Urine collected after intercourse or masturbation from 75 patients with dry ejaculation showed retrograde ejaculation in 55 and lack of ejaculatory emission into the urethra in 20 patients. Regarding other sexual functions, 17 patients had a diminished sexual desire (especially those patients who had received radiotherapy), 12 experienced difficulty reaching organism, and 6 complained of erectile dysfunction. The incidence of a contralateral hydrocele developing after retroperitoneal lymph node dissection seems to correlate with ligation of the contralateral spermatic vessels and their lymphatics. A review of the literature is presented comparing the types of dissection with the incidence of sexual disorders after retroperitoneal lymph node dissection. Since preserving normal ejaculation and fertility is important, a modified or unilateral retroperitoneal lymph node dissection, when required, is advocated. In patients, with stage I disease the therapy may be limited to an orchiectomy without lymph node dissection. In patients with retroperitoneal lymph node metastases combination chemotherapy with cisplatin and tumor excision gives good results. Patients with true retrograde ejaculation can be treated with alpha-sympathomimetic drugs such as imipramine HCl, and thus be offered the chance of fatherhood by coitus.

Nineteen men with asthenozoospermia were treated with oral Kallikrein (600 U daily for 12 weeks) and sperm motility evaluated in 16 of these patients. No quantitative or qualitative improvement motility or the duration of motility was found in semen samples examined 4, 12, and 24 weeks after starting the treatment. Two pregnancies occurred 11 and 40 weeks after discontinuation of the Kallikrein therapy.

In 14 patients by bilateral retroperitoneal lymph node dissection for nonseminoma tumors of the testes, several sexual functions before and after node dissection were compared. All patients had normal sexual function before the operation. After the operation, antegrade ejaculation was found to have disappeared completely in 12 patients, retrograde ejaculation was demonstrable in ten of these patients. In ten patients, antegrade ejaculation was restored after treatment with 25 mg imipramine twice daily by mouth. In the two patients who still showed antegrade ejaculation, albeit diminished, imipramine medication led to an increased number of spermatoza. During imipramine medication, the partners of five patients became pregnant. The preliminary conclusion from these findings is that ejaculation disorders, which most males develop after bilateral retroperitoneal lymph node dissection, can be successfully treated with daily oral doses of 25-50 mg imipramine in the majority of patients who still desire children.

Inhibition of sperm penetration by a strong shaking phenomenon was investigated in 5 infertile women (of 46 initially examined) whose cervical mucus produced such a reaction. Cervical mucus was pretreated with bromelin which greatly facilitates determination of anti-sperm antibodies and high sperm agglutination titers (at least 32) were found in the cervical mucus of all 5 women each showing the shaking behavior in more than 75% of local sperm. The sperm agglutinating activity was completely absorbed with anti-immoglobulin A antiserum. The presence of anti-sperm immunoglobulin A at least partly of the secretory subclass and anti-sperm immunoglobulin G were demonstrated with indirect mixed anti-globulin reaction tests. Preliminary experiments indicated that the shaking action and therefore sperm penetration inhibition was caused by action of anti-sperm antibodies of the immunoglobulin A class.