Relationship between Semenogelins bound to human sperm and other semen parameters and pregnancy outcomes

Globally, approximately 15% of couples of reproductive-ages do not achieve pregnancy within 1 year of regular unprotected sexual intercourse and seek fertility assessment [1]. Among these couples, eventually 5% remain unwillingly childless. Male infertility-associated factor, together with abnormal semen parameters, contributes to 50% of involuntarily childlessness in couples. Semen analysis is the most widely used biomarker to predict the male fertility potential. Its result gives a wide array of information on the functional status of the whole male sex system, such as hormone axis, seminiferous tubules, epididymis, and accessory sex glands. On the other hand, as it is a complex test, semen analysis should ideally be conducted in andrology laboratories by experienced technicians in the presence of internal and external quality controls; validation of test systems; and quality assurance during all testing processes, some of which are often difficult to implement in practice [2]. These difficulties often prevent comparative studies of multicenter data. Additionally, their ability to predict male fertility potential has been questioned, unless the parameters truly are at extremely low levels. For example, among infertile men, semen analysis results are normal in up to 40% of cases [3].

To overcome these insufficiencies of routine semen analysis for diagnosis, specialized andrology tests have emerged to account for sperm dysfunction. Formerly, particularly before the advent of intracytoplasmic sperm injection (ICSI), tests that assessed antisperm antibodies, sperm hyperactivation, and acrosome reaction; sperm binding; and penetration to the human zona pellucida were used to investigate males with unexplained infertility [4]. These tests hold the capability to reveal problems that may exist in each step of conventional in vitro fertilization (IVF), and help to predict the fertilizing potential of sperm in this procedure. However, treatments for many of these potential issues are often extremely difficult or do not exist. Furthermore, the efficacy of assisted reproductive techniques (ART), particularly ICSI, is essentially unaffected by these problems; thus, clinical usage of these tests is highly restricted. Based on the increased knowledge regarding the molecular mechanisms that regulate sperm function, oxidative stress levels and nuclear DNA integrity in the determination of the functional competence of human spermatozoa have been emphasized. These parameters seem to better correlate with male fertility than does standard semen analysis, and tests to measure them have become clinically available. However, to be used routinely, these tests should meet the following requirements, including standardization of protocols, validation of results in larger trials, and cost-effectiveness analyses.

Semenogelins (SEMGs) are major components of human seminal vesicle secretions and are comprised of two closely-related proteins: SEMG 1 and 2 [5]. These two proteins and their degradation byproducts hold a variety of physiological roles [6]. Ejaculated semen immediately turns into a gelatinous meshwork, in which cross-linking SEMGs are major components and sperms are entrapped. During liquefaction, SEMGs are degraded into low molecular mass proteins by a prostate-specific antigen (PSA), and sperms start to move [7]. However, it was shown that seminal hyperviscosity had no relation to the degree of SEMG degradation [8], thereby indicating that SEMGs do not inhibit sperm motility through seminal viscosity. One of the fragments of the degraded SEMGs, a seminal plasma motility inhibitor (SPMI), inhibits the motility of both demembranated spermatozoa and intact spermatozoa. SEMGs have an inhibitory effect on the acrosome reaction induced by fetal cord serum ultrafiltrate. At present, SEMGs and their degradation peptides are considered decapacitation factors that prevent human sperm capacitation. The mechanism underlying these physiological roles of SEMGs and their degradation fragments are still unclear. Interestingly, our group recently reported the association between SEMGs and spermatozoa in infertile men with asthenozoospermia [9].

Based on the clinical relevance of SEMGs to male factor infertility and their putative physiologic role, we hypothesized that SEMGs might be targets for developing new sperm function tests that can guide couples with male factor infertility to select the most appropriate treatment. In this study, we examined the condition of SEMGs bound to sperm from infertile patients and retrospectively analyzed the pregnancy outcomes of these patients’ partners using questionnaires. These results can provide information on the new sperm function tests using SEMGs by indicating whether they are practical for the evaluation of sperm function beyond motility.

Previously, our group reported the association between SEMGs and spermatozoa in infertile men with asthenozoospermia [9]. A significant negative correlation was found between sperm motility and the proportion (R = − 0.68) and intensity (R = − 0.38) of anti-SPMI labeling, implying that the SPMI, and eventually SEMGs, binding to the sperm surface might account for some disorders in sperm motility that are observed in infertile men with asthenozoospermia.

In this study, we expanded the pool of potential subjects to include infertile patients whose infertility was caused not only by asthenozoospermia but also by oligozoospermia and normozoospermia (cryptogenic male infertility). We further explored the possibility of SEMGs bound to spermatozoa being a predictor of infertility treatment for such a variety of infertile patients in the future. We confirmed a negative correlation between sperm motility and the proportion of SEMG+ in this study, although it was a comparatively weak correlation since the subjects included in this study were not restricted to those with asthenozoospermia (Table 2). For this reason, one might assume that a normal subject bears a lower proportion of SEMG+, since their sperm motility is normal. However, this is not necessarily true since the proportion of SEMG+ is often high, even in an infertile subject whose sperm motility is normal. Therefore, it is possible that the proportion of SEMG+ is an indicator of normal fecundity of the spermatozoa independently of their motility.

The proportion of SEMG+, as well as any other parameters, including sperm motility, did not show a significant difference between the pregnancy and non-pregnancy groups, which indicated that it might not be a general predictor of ability to bear children in an infertile subject. Naturally, the link between SEMGs and the success of pregnancy requires further support. This is because, in addition to factors associated with the male, factors associated with the female, the fertilized egg, and the early development of the embryo are all involved in the success of pregnancy. To conduct an analysis while considering these confounding factors, it is necessary to further investigate increasing the number of specimens.

Next, based on the hypothesis that the condition of SEMGs bound to spermatozoa differs between pregnancy achieved through IUI or spontaneous pregnancy and that achieved through IVF or ICSI, we focused on the result of 36 cases in which conception was achieved. When the summed data of spontaneous pregnancy and IUI was compared to the summed data of IVF and ICSI, the total SEMG− count was apparently higher in the former. Such a tendency was not observed in any of the other parameters, including the proportion of SEMG+. Although the precise molecular mechanism underlying the controlling fertilizing capacity of sperm in vivo have not been revealed, the role of SEMGs on sperm were well studied and proved to control motility and capacitation [8, 16–18]. Moreover, according to our analysis in knockout mice, seminal vesicle secretion 2 (SVS2), an orthologue of SEMGs, is essential for in vivo fertilization [13]. SVS2 is thought to be a protector against spermicidal agents in the uterus, and to be removed from the sperm’s surface after passing through the isthmus [14]. Coincidently, the removal of SVS2 induces the decrease of cholesterol from the sperm membrane, thereby resulting in the ability of sperm to fertilize the egg [15]. The data in this study indicate that a patient with a larger number of spermatozoa with removed SEMGs can be involved in a successful pregnancy via in vivo fertilization. The removal of SEMGs from the surface of spermatozoon under normal conditions allows for sperm to fertilize an egg. Furthermore, the number of such spermatozoa is important for in vivo fertilization.

While results from this study provide a minimum-requirement analysis of the SEMGs bound to spermatozoa and conventional semen analysis focused on pregnancy outcomes, this study also provides the need for further large-scale studies to establish diagnostic certainty. In addition to this study having a small patient population, this study has a limitation that the characteristics of the female partners, such as age and oocyte quality, are not considered. Pregnancy outcomes of a couple are usually strongly affected by the condition of the female partner. Thus, it is possible that the influence of male factors on pregnancy outcomes is importantly obscured under such uncontrolled conditions of the female. Surprisingly, SEMGs were found to be potential biomarkers that can guide a couple to select a suitable method of assisted reproductive technology. They were also found to be particularly valuable for the decision to use IVF or ICSI rather than IUI. Usually, natural pregnancy or IUI using the timing method are used in infertile couples with no particular female factors when the male partner is normozoospermic or mildly oligozoospermic or asthenozoospermic. According to the findings of this study, it is likely that even such a couple should consider early step-up using the data of total SEMG− count. More data must be collected to evidence this hypothesis and to determine any cut-off value. In the future, SEMGs bound to sperm may be used to determine whether IUI, IVF, or ICSI treatment should be administered promptly, without waiting for spontaneous pregnancy, in case of couples who desire to have their own child.

Regarding the partner of patient pregnancy subgroup, the total SEMG− count tended to be high in patients whose partners had spontaneous pregnancy or who underwent IUI treatment, compared with partners of patients who used other treatments. This result suggests that SEMGs inhibit ectopic capacitation before sperm reach the fertilization site and that the number of total SEMG-unbound sperm is a parameter directly linked to the possibility of in vivo fertilization. It is possible that total SEMG− count might be a tool for determining whether IUI, spontaneous conception, IVF, or ICSI should be used for the partners’ treatment of ART. It is necessary to perform further large-scale studies to establish the diagnostic certainty of SEMGs bound to spermatozoa.