Abstract
Seminal sperm-agglutinating antibodies along with IgG antibodies against laminin-1 and intraacrosomal sperm proteins were examined in seventy-one men from infertile couples. The direct mixed antiimmunoglobulin reaction test for IgG, IgA, and the commercial ELISA method for detecting IgG antibodies against laminin-1 in seminal plasma were used. Intraacrosomal proteins in the sperm heads were detected by immunofluorescence using monoclonal antibodies. Cellular elements other than spermatozoa, collectively referred to as “round cells” were also examined. In association with a group of oligoasthenospermatic men, positive levels (44%) of antibodies against laminin-1 of the IgG isotype in seminal plasma were found in conjunction with increased cellularity in semen. Interestingly, the elevated levels of anti-laminin-l IgG and sperm agglutinating positivity were not correlated.
The use of antibodies against sperm antigens targeted to adhesive molecules such as laminin-1 contributes to diagnosing reproductive failure. Detection of intraacrosomal proteins very often correlates to the state of semen pathology and inflammation.
| Abbreviations | ||
| ASA | = | sperm agglutinating antibodies/anti-sperm antibodies |
| FSH | = | follicle stimulating hormone |
| LH | = | luteinizing hormone |
| MAR | = | mixed agglutinating reaction |
| a-Ln-1 | = | anti-laminin IgG antibodies |
| TMB | = | tetramethylbenzidine |
| PBS | = | phosphate buffer solution |
Introduction
More than 15% of the European population suffers from infertility. The male factor plays a role in about 40% of these cases. A simple spermiogram assessing the quality of semen may not be sufficient for identifying these individuals. Detailed examination of key players in male fertility is necessary in such cases. For example, this can take the form of using selected immunological probes including sperm antibodies or antibodies against laminin-1, and those that can discern the quality of acrosome. Anti-sperm antibodies (ASA) found in seminal plasma as well as the cervical ovulatory mucus, sera and in peritoneal and follicular fluids can contribute to infertility. ASA in biological fluids manifest their effects through decreased sperm motility, reduced fertility rates, or decreased rates of cell division in the early embryo [e.g. Meinertz [Citation1992]; Ulcova-Gallova and Mardesic [Citation1996]; Chamley and Clarke [Citation2007]]. It has been proposed that since the ASA induce infertility they have the potential to be developed as immunological based contraceptives.
Laminins are multidomain heterotrimers composed of alpha, beta and gamma subunits serving as multifunctional glycoproteins of the extracellular membrane that contains laminins, nidogens, proteoglycans, and collagen IV. They provide the starting point for the development of a basic network of various biological membranes. As such, laminins participate in various biological activities including cell adhesion, migration, cell proliferation and differentiation, cell shape, movement, maintenance of tissue phenotype, and promotion of tissue survival. At present, more than 15 isoforms of laminins exhibiting different patterns of expression in various cell stages of tissue development are known [Colognato and Yurchenco [Citation2000]; Aumailley et al. [Citation2005]; Dirami et al. [Citation1995]; Timpl et al. [Citation2000]]. Recently, novel gamete receptors mediating sperm-oocyte fusion have been described [Shur et al. [Citation2006]].
In males, laminin is found on the surface of sperm cells along with various adhesive molecules including the α subunit of the β1-2 integrins, and fibronectin. It participates in the adhesion of sperm cells to zona pellucida. Active immunization in monkeys with mouse laminin-1 or by laminin-1 peptides is embryocytotoxic resulting in spontaneous miscarriages [Weeks et al. [Citation1989]].
Patients with severe teratozoospermia have a host of sperm biochemical abnormalities. The basis of impaired infertility has not been firmly established. However, decreased levels of adhesive molecules, like the very late antigens VLA α4, VLA α5, and laminin have been associated with low fertilization capacity [Glander and Schiller [Citation1993]; Koch et al. [Citation1999]; Gülkesen et al. [Citation2002]]. The basal membrane of lamina propria of semenoferous canals contains collagen IV and laminin, which are synthesized by the Sertoli cells [e.g., Häger et al. [Citation2005]; Pöllanen et al. [Citation1985]]. In males with Sertoli-cell-only syndrome laminin-1 appears absent, however, collagen IV along with collagen I and III are present [e.g., Pöllanen et al. [Citation1985]; Takaba [Citation1990]; Geipel et al. [Citation1992]].
Routine methods for the detection of intraacrosomal sperm proteins associated with sperm zona-binding [e.g., Peknicova et al. [Citation2005]; Babcova et al. [Citation2006]; Tepla et al. [Citation2006]] are used in the Czech Republic to evaluate the reproductive potential of human sperm cells. Reduced quantities of the intraacrosomal proteins has been associated with impaired male fertility. In this clinical-experimental study, semen samples from men examined and treated for infertility were screened for the presence of sperm antibodies, IgG anti-laminin-1, and intraacrosomal sperm proteins, as a function of the cellularity of semen.
Results
Of seventy-one males examined, only ten were classified by WHO criteria as normospermic [WHO Citation[1993]]. The remaining 61 patients were classified as oligoasthenospermic. Five of the normospermic males had increased cellularity in their semen, IgG, or IgA sperm antibodies. The distribution of intracrosomal proteins was normal along with the levels of laminin-1 with levels of 0.63 IgG IU/ml and 0.80 IU/ml of a-Ln-1, respectively, in all normospermic men.
In comparison the oligoasthenospermic males divided into two subgroups (OA-A, OA-B) according to the level of a-Ln-1. The second group was distinguished by a higher degree of cellularity with increased a-Ln-1 (0.98–1.123 IU/ml), and various Ig isotypes of sperm antibodies. However, both subgroups lacked intraacrosomal enzymes ( and ). There was no apparent correlation between the higher level of a-Ln-1 and the presence of sperm antibodies.
FIGURE 1 Levels of a-ln-1 associated with semen pathology. Results of analysis of semen a-Ln-1- antibodies against laminin-1, increased cellularity >5×106 round cells/ml (macrophages, leucocytes or/and lymphocytes), detection of sperm agglutinating antibodies by direct mixed antiimunoglobulin reaction test (MAR) in IgG, IgA, IgM, and IgE, intraacrosomal proteins exemined by monoclonal antiboides Hs8 (IgG), Hs-14 (IgM) and Hs-36 (IgM).
Results of Analysis of Semen
Discussion
The rise in infertility is a world problem primarily centering in the highly developed countries. In this study seminal antibodies capable of sperm agglutination by heads, tails, or by both heads and tails were examined. Although the majority of reproductive immunology laboratories involved routinely assay sperm antibodies and intraacrosomal proteins, only a few include adhesive molecules as part of their routine analysis.
The laminin α chain has been detected in testis during spermatogenesis suggesting a very important role in spermatozoa [Dirami et al. [Citation1995]; Takaba [Citation1990]; Häger et al. [Citation2005]; Ooba et al. [Citation2008]]. Ooba et al. [[Citation2008]] showed that overabundance of the beta-2-chain of laminin is associated with increased basement membrane thickness related to spermatogenic dysfunction [Vuolteenaho et al. [Citation1990]]. Experiments in animal models [Carey and Klein [Citation1989]; Weeks et al. [Citation1989]; Chambers et al. [Citation1995]; Kondo et al. [Citation2005]] described the effect of anti-laminin-1 antibodies in repeated reproductive failure as direct embryo cytotoxicity, and repeated spontaneous miscarriages in the first trimester. These anti-Ln-1 antibodies associated with early pregnancy failure recognized the G domain of the α1 subunit of laminin-1. IgG antibodies against laminin-1 are present in women during spontaneous abortion and also in ovulatory mucus of infertile patients. It is rarely observed when a female patient is not pregnant, as noted in a single serum sample from a patient with three pregnancy losses [Gruberova et al. [Citation2007]].
Seminal fluid contains various extracellular matrix factors including laminin, hyaluronan and the propeptide of procollagen III at concentrations higher than those observed in sera of infertile men. The concentration of laminin-1 correlates with the number of sperm cells, age of the patient, and acrosin concentration. The low fertilizing capacity of teratozoospermic semen is characterized by a significantly lower percentage (P < 0.01) of spermatozoa with β1-integrins VLA α4 (CDw 49d), VLA α5 (CDw 49e) and VLA α6 (CDw 49f), the β-chain of β2-integrins (CD 18) and the matrix proteins laminin and fibronectin [Glander and Schiller [Citation1993]]. Basement membrane laminin plays a crucial role in Sertoli cell survival in vitro [Dirami et al. [Citation1995]]. In the absence of a basement membrane, FSH and other regulators of Sertoli cell function cannot prevent Sertoli cell apoptosis. It has been suggested [Takaba [Citation1990], Kondo et al. [Citation2005]], that anti-laminin-1 IgG antibodies are clinically associated with the development of autoimmune-mediated reproductive failures. Similarly, pathological levels of autoimmune sperm antibodies with a-Ln-1 were observed in oligoasthenospermic men with increased semen cellularity.
The presence of intracrosomal proteins [Peknicova and Moos [Citation1990]; Peknicova et al. [Citation2005]] serves as a useful fertility marker for couples undergoing in vitro fertilization or for women being readied for insemination with sperm cells prepared by swim-up. In contrast, decreased quantities of intraacrosomal proteins is indicative of intracytoplasmatic sperm injection.
Material and Methods
Patients
All patients from the Department of Obstetrics and Gynecology of Medical Faculty of Charles University and University Hospital in Pilsen were informed of the aim of the study according to the ethical rules. Seventy-one males, aged 27–48 years (mean age 33 years) from infertile couples were examined and treated in the Division of Reproductive Immunology. Sixty-one patients aged 18–27 years (mean age 32.6 years) were classified as oligoasthenospermic; ten normospermic men aged 30–45 years (mean age 35.3 years) provided a control group. All patients were without any health complaints at the time of the study.
Semen was collected by masturbation after four days of sexual abstinence in sterile vessels and characterized according to WHO [Citation[1993]] criteria. We evaluated time of semen liquefaction, color, sperm concentration, morphology, vitality, progression of movement, bacterial semen contamination, and presence of accessory cells such as epithelia, macrophages, leucocytes, called “round cells.” Higher cellularity of semen was estimated as greater than 5×10 6 round cells per ml.
We examined ASA, IgG antibodies against laminin-1, and intraacrosomal proteins. Serum levels of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were within normal values in all patients. Seminal plasma for ELISA was collected and stored frozen at −79°C in our bank until examination. Intraacrosomal proteins were evaluated in the sperm heads from semen in all groups.
Sperm Agglutination by Direct Mixed Antiimunoglobulin Reaction Test (MAR) Using IgG, IgA Antibodies
The direct MAR test examined IgG, IgA. In brief, one microliter of fresh and liquefied semen, one microliter of glutaraldehyde-fixed sheep erythrocytes coated with human IgG, and IgA and one microliter of the corresponding anti-IgG, or anti-IgA antiserum was added. The mixture was covered with a coverslip then incubated in a humid Petri chamber for 5 min at room temperature. Sperm agglutination was observed using the inverted Zeiss Jena microscope at ×200–300 magnification [Ulcova-Gallova and Mardesic [Citation1996]]. A positive MAR test corresponded to greater than 41% of the motile spermatozoa in mixed agglutinates (spermatozoa and sheep erythrocytes coated by corresponding immunoglobulin).
Measurement of Anti-Laminin-1 IgG Antibodies (a-Ln-1)
We used the commercial ELISA method (AESKULISA Laminin Aesku.Diagnostics GmbH, Germany). Seminal plasma (100 μl) was diluted 1:101 then pipetted into designated microwells. After a 30 min room temperature incubation the plates were washed three times with commercial washing buffer, then 100 μl of conjugate was added. Samples were incubated again for 15 min at room temperature, washed three times with 300 μl of washing buffer. A 100 μl aliquot of tetramethylbenzidine (TMB) substrate was added to each well to generate the colorimetric reaction and the mixture incubated for 15 minutes at room temperature in the dark. The reaction was stopped and then the sample measured using the MRX II, Dynex ELISA reader.
The diagnostic informs only when normal serum levels of a-Ln-1 are 15 IU/ml. Accordingly, levels of seminal plasma a-Ln-1 were calculated as a mean of the levels in normospermic semen estimated as 0.762 IU/ml.
Analysis of Intraacrosomal Proteins
Monoclonal antibodies (Hs-8, Hs-14, Hs-36) targeted against intraacrosomal sperm proteins were used to assess acrosome status by immunofluorescence [Peknicova and Moos [Citation1990]; Peknicova et al. [Citation2005]; Tepla et al. [Citation2006]]. The percentage of acrosome-reactive cells was assessed in human sperm samples by the immunocytochemical method (see below) using monoclonal antibodies against intra-acrosomal sperm proteins (pHs-8, pHs-14 and pHs-36).
Smears were dried and fixed with acetone (22°C). They were rinsed with PBS and incubated for 30 min at 37°C with monoclonal antibodies (diluted in PBS to an immunoglobulin concentration of 20 μg/ml). After thorough washing with PBS, the smears were reacted with FITC-conjugated swine anti-mouse immunoglobulins (SEVAC, Prague, Czech Republic), diluted 1:20 in PBS for 45 min at 37°C, washed with PBS and water, and mounted in 50% glycerol in PBS, pH 9.0. For appropriate controls, smears were incubated with nonspecific monoclonal antibody, with the supernatant of myeloma cells, and with the FITC-conjugate only. Samples (200 sperm cells, except the sixth group) from each male were evaluated then viewed with a Nikon Labophot-2 fluorescent microscope equipped with 40×Nikon Plan 40/0.65 lenses and photographed with a COHU4 CCD camera (Japan) with the aid of LUCIA imaging software (Laboratory Imaging, A.S. Prague, Czech Republic). Labelling of greater than 90% of the acrosomes in semen was considered normal.
Acknowledgment
This research was supported by a grant from Ministry of Education MSM 002 162 0812 and Czech Ministry of Health NR-8913-3.
References
- Aumailley M., Bruckner-Tuderman L., Carter W. G., Deutzmann R., Edgar D., Ekblom P., Engel J., Engvall E., Hohenester E., Jones J. C., et al. A simplified laminin nomenclature. Matrix Biol 2005; 24: 326–332
- Babcova K., Ulcova-Gallova Z., Cervenkova Z., Peknicova J., Micanova Z., Bibkova K., Rokyta Z. Inhibin B and intraacrosomal proteins in men from the couples with fertility disorders. Ceska Gynekol 2006; 71(2)111–117
- Carey S. W., Klein N. W. Autoantibodies to laminin and other basement membrane proteins in sera from monkeys with histories of reproductive failure identified by cultures of whole rat embryos. Fertil Steril 1989; 51: 711–718
- Chambers B. K., Klein N. W., Conrad S. H., Ruppenthal G. C., Sackett G. P., Weeks B. S., Kleinman H. K. Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV. Proc Natl Acad Sci USA 1995; 92: 6818–6822
- Chamley L. W., Clarke G. N. Antisperm antibodies and conception. Semin Immunopathol 2007; 29: 169–184
- Colognato H., Yurchenco P. D. Form and function: The laminin family of heterotrimers. Dev Dyn 2000; 218: 213–234
- Dirami G., Ravindranath N., Kleinman H. K., Dym M. Evidence that basement membrane prevents apoptosis of sertoli cells in vitro in the absence of known regulators of sertoli cell function. Endocrinology 1995; 36: 4439–4447
- Geipel U., Kropf J., Krause W., Gressner A. M. The concentration pattern of laminin, hyaluronan, and aminoterminal propeptide of type III procollagen in seminal fluid. Andrologia 1992; 24: 205–211
- Glander H. J., Schiller J. Semen samples with teratozoospermia show a lower percentage of spermatozoa with detectable adhesion molecules (AM). Int Urol Nephrol 1993; 25: 485–489
- Gruberova J., Ulcova-Gallova Z., Bibkova K., Micanová Z., Kralickova M., Novotny Z., Rokyta Z. Are antibodies against laminin-1 significant marker for decreased fertility in women?. Czech Gynecology 2007; 72: 284–286
- Gülkesen K. H., Erdoğru T., Sargin C. F., Karpuzoğlu G. Expression of extracellular matrix proteins and vimentin in testes of azoospermic man: An immunohistochemical and morphometric study. Asian J N Androl 2002; 4: 55–60
- Häger M., Gawlik K., Nystrom A., Sasaki T., Durbeej M. Laminin {alpha}1 chain corrects male infertility caused by absence of laminin {alpha}2 chain. Am J Pathol 2005; 167: 823–833
- Koch M., Olson P. F., Albus A., Jin W., Hunter D. D., Brunken W. J., Burgeson R. E., Champliaud M. F. Characterization and expression of the laminin gamma-3 chain: A novel, non-basement membrane-associated, laminin chain. J Cell Biol 1999; 145: 605–617
- Kondo A., Inagaki J., Kobayashi K., Tsukamoto H., Yamamoto D., Nakatsuka M., Suzuki N., Nomizu M., Amano S., Matsubayashi H., et al. Characterization of a murine anti-laminin-1 monoclonal antibody (AK8) produced by immunization with mouse-derived laminin-1. Clin Dev Immunol 2005; 12: 67–73
- Meinertz H. Anti-sperm antibodies in the male: Detection and clinical impact. Am J Reprod Immunol 1992; 28: 110–116
- Ooba T., Ishikawa T., Yamaguchi K., Kondo Y., Sakamoto Y., Fujisawa M. Expression and distribution of laminin chains in the testis for patients with azoospermia. J Androl 2008; 29(2)147–152
- Peknicova J., Chladek D., Hozak P. Monoclonal antibodies against sperm antigens as markers for detection of physiology and pathology of human spermatozoa and estimation of spermatogenesis. Am J Reprod Immunol 2005; 53: 42–49
- Peknicova J., Moos J. Monoclonal antibodies against boar acrosomal antigens labeling undamaged acrosomes of spermatozoa in immunofluorescence test. Andrologia 1990; 22: 427–435
- Pöllanen P. P., Kallajoki M., Risteli L., Risteli J., Suominen J. J. Laminin and type IV collagen in the human testis. Int J Androl 1985; 8: 337–347
- Shur B. D., Rodeheffer C., Ensslin M. A., Lyng R., Raymond A. Identification of novel gamete receptors that mediate sperm adhesion to the egg coat. Mol Cell Endocrinol 2006; 250: 137–148
- Takaba H. A morphological study of the testes in patients with idiopathic male infertility: Immunohistochemical analysis of collagens and laminin in human testes. Hinyokika Kiyo 1990; 36: 1173–1180
- Tepla O., Peknicova J., Koci K., Mika J., Mrazek M., Elzeinova F. Evaluation of reproductive potential after intracytoplazmatic sperm injection of varied human semen tested by antiacrosomal antibodies. Fertil Steril 2006; 86: 113–120
- Timpl R., Tisi D., Talts J. F., Andac Z., Sasaki T., Hohenester E. Structure and function of laminin LG modules. Matrix Biol 2000; 19: 309–317
- Ulcova-Gallova Z., Mardesic T. Does In Vitro Fertilization (IVF) influence the levels of sperm and Zona Pellucida (ZP) antibodies in infertile women?. Am J Reprod Immunol 1996; 36: 216–219
- Vuolteenaho R., Chow L. T., Tryggvason K. Structure of the human laminin B1 chain gene. J Biol Chem 1990; 265: 15611–15616
- Weeks B. S., Klein N. W., Kleinman H., Fredrickson T., Sackett G. P. Laminin immunized monkeys develop sera toxic to cultured rat embryos and fail to reproduce. Teratology 1989; 40: 47–57
- World Health Organization. (1993) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction. 3rd ed., Cambridge, UK: Cambridge University Press