Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis

Background: The pregnancy hormone human chorionic gonadotropin (hCG) and its free subunits (hCG alpha, hCG beta) are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha. Methods: Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29) versus normozoospermic men (n = 16). The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation. Results: hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/191 vs. 2753 +/533 ng/ml, P = 0.022). Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/0.022 vs. 0.203 +/0.040 ng/ml, P = 0.028). Using massspectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under nonreducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app) of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones. Conclusions: The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The alternative glycosylation pattern on the free large hCG alpha in seminal plasma might reflect a modified function of this subunit in the male reproductive tract. Background Male fertility abnormalities are diagnosed by physical examination, endocrine parameters and assessment of semen quality. Endocrine analyses include hormones of the pituitary testicular axis, i.e. pituitary-derived gonadotropins luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone (TT) and free/bioavailable testosterone. Other hormones, such as prolactin (hPRL), estrogen, or stress hormones, are also important parameters of the male fertility workup. Apart from serum hormone levels, investigators sought molecular markers of spermatogenesis in seminal plasma. Such markers were anti-Mullerian hormone, inhibin B [1], and transferrin. Although a few attempts have been made to correlate human chorionic gonadotropin (hCG) and hCG-like molecules to spermatogenesis [2-9], the molecular heterogeneity of molecular hCG species in various body fluids, the lack of appropriate standards of hCG-variants, undefined molecular recognition profiles of immunoassay analyses and interpretation of marker profiles has, until recently, been too poor to do so [10] (the molecular structures of hCG and its variants are schematically depicted in Figure 1). The International Society of Oncodevelopmental Biology and * Correspondence: peter.berger@oeaw.ac. at Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, 6020 Innsbruck, Austria Full list of author information is available at the end of the article Zenzmaier et al. Reproductive Biology and Endocrinology 2011, 9:114 http://www.rbej. com/content/9/1/114 © 2011 Zenzmaier et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine (ISOBM) has initiated and completed, as part of its Tissue Differentiation (TD) program, the international TD-7 Workshop on antibodies to hCG and hCGrelated molecules and recommended epitope combinations for 2-site immunoassays for the measurement and determination of members of the molecular hCG family [11]. With our selective panel of immunofluorometric assays (IFMAs) based on the reference monoclonal antibodies (mAbs) of the TD-7 Workshop [12], we previously investigated the abundance of hCG and hCGlike molecules in seminal plasma of healthy fertile men and found significant differences between the hCG marker profile and that of other hCG-containing physiological body fluids [13]. During pregnancy, the a/bheterodimer hCG is present in vast molar excess in serum, as is hCGb/hCGbcf (hCGb core fragment) in urine, but in seminal plasma, the subunit hCGa was by far the most prominent marker of the hCG molecular family, with concentrations as high as that found during pregnancy in extraembryonic coelomic fluid, and approximately 10,000-fold higher than in normal male serum. hCGb and holo-hCG concentrations were found to be 1000-fold and 10,000-fold less than hCGa, whereas hCGbcf levels were mostly undetectable in seminal plasma [13]. In the present study, the local profile of endocrine parameters, i.e. hCG-like substances and the family of protein hormones prolactin (hPRL), growth hormone (GH) and placental lactogen (PL), was analyzed in seminal plasma of men with abnormal semen analysis findings and compared to normozoospermic men to clarify a possible pathophysiological role of these hormones in spermatogenesis disorders. An important question was whether the highly abundant hCGa is genuine hCGa or a moderately-defined hCGa-like substance. Thus, the glycosylation pattern of hCGa, which has two N-glycosylation sites at Asn and Asn [14], was purified from seminal plasma and the isolated protein analyzed by mass-spectrometry.