Semen Liquefaction Research Articles

Association between kallikrein-related peptidases (KLKs) and macroscopic indicators of semen analysis: their relation to sperm motility

Human kallikrein-related peptidases (KLKs) are a family of proteases, the majority of which are found in seminal plasma and have been implicated in semen liquefaction. Here, we examined the clinical value of seminal KLKs in the evaluation of semen quality and differential diagnosis and etiology of abnormal liquefaction and/or viscosity. KLK1-3, 5-8, 10, 11, 13, and 14 were analyzed, using highly specific ELISA assays. Samples were categorized into four clinical groups, according to their state of liquefaction and viscosity. Data were compared between the clinical groups and in association with other parameters of sperm quality, including number of motile sperms, straight line speed, sperm concentration, volume, pH, and patient age. Seminal KLKs were found to be differentially expressed in the four clinical groups. Combination of KLK2, 3, 13, and 14 and KLK1, 2, 5, 6, 7, 8, 10, 13, and 14 showed very strong discriminatory potential for semen liquefaction and viscosity, respectively. Liquefaction state was associated with several parameters of sperm motility. Finally, KLK14 was differentially expressed in asthenospermic cases. In conclusion, the expression level of several seminal plasma KLKs correlates with liquefaction and viscosity indicators of semen quality and may aid in their differential diagnosis and etiology.

Some preliminary observations on the liquefaction of human semen.

Liquefaction of human semen in vitro was studied by scanning electron microscopy, determinations of total protein, free amino acid and phosphate and by correlation analysis. During liquefaction of the coagulum, its fibers were transformed into globular bodies that fused to form the homogeneous liquefied semen. It was found that the parts of the ejaculate taking more time to liquefy possessed higher concentration of protein than those of the parts liquefied earlier, a relation also appeared to exist with the whole ejaculates. Release rate of phosphate during macroscopic lysis and free reducing sugar level of ejaculates demonstrated significant negative correlations with liquefaction time which could be shortened using reductants.

Activation profiles of human kallikrein‐related peptidases by proteases of the thrombostasis axis

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.

Notice of duplicate publication.

© 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved. Asian Journal of Andrology (AJA) wishes to inform its readers of the following incident of duplicate publication. The article Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen [1] by Wang ZJ, Zhang W, Feng NH, Song NH, Wu HF and Sui YG, published in the Sep 2008 issue of AJA contains substantial data from the two original articles by Wang ZJ et al. [2] and O’Rand MG et al. [3] based on work performed by Dr Wang while in Dr O’Rand’s laboratory. Wang ZJ, et al. [1] used common data and identical text extensively without the permission of Dr. Michael G. O’Rand, the corresponding author of the two original articles [2, 3]. Specifically, Figures 2 and 3 in AJA [1] were same with Figures 6 and 7 in Biol Reprod [2], and Figure 4 in AJA [1] was the same with Figure 4 in Mol Cell Endocrinol [3]. Dr Wang ZJ is a co-author of original articles [2, 3] and cited these two original articles as references in Wang ZJ et al.[1].

Inhibition of Human Sperm Motility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys: Effect on Cyclic Adenosine Monophosphate1

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen.

To study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg). Human Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored. Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin. Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

Major Role of Human KLK14 in Seminal Clot Liquefaction

Liquefaction of human semen involves proteolytic degradation of the seminal coagulum and release of motile spermatozoa. Several members of human kallikrein-related peptidases (KLKs) have been implicated in semen liquefaction, functioning through highly regulated proteolytic cascades. Among these, KLK3 (also known as prostate-specific antigen) is the main executor enzyme responsible for processing of the primary components of semen coagulum, semenogelins I and II. We have recently identified KLK14 as a potential activator of KLK3 and other KLKs. This study aims to elucidate the cascade-mediated role of KLK14 ex vivo. KLK14 expression was significantly lower (p = 0.0252) in individuals with clinically delayed liquefaction. Concordantly, KLK14 expression was significantly (p = 0.0478) lower in asthenospermic cases. Specific inhibition of KLK14 activity by the synthetic inhibitor ACT(G9) resulted in a significant delay in semen liquefaction, a drop in the "early" (30 min postejaculation) "chymotrypsin-like" and KLK1 activity, and an increase in the "late" (90 min postejaculation) chymotrypsin-like activity. Conversely, the addition of recombinant active KLK14 facilitated the liquefaction process, augmented the early chymotrypsin-like activity, and lowered late chymotrypsin-like activity. Given that the observed chymotrypsin-like activity was almost completely attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally. Accordingly, a higher level of KLK3 fragmentation was observed in KLK14-induced coagula, suggesting an inactivation mechanism via internal cleavage. Finally, semenogelins I and II were directly cleaved by KLK14. Semenogelins were also able to reverse KLK14 inhibition by Zn2+, providing a novel regulatory mechanism for KLK14 activity. Our results show that KLK14 exerts a significant and dose-dependent effect in the process of semen liquefaction.

Kallikrein-related peptidases.

Kallikrein 1 (KLK1), a key component of the kallikrein-kinin system, originates from a locus on the long arm of chromosome 19 that contains several related serine endopeptidases. The biological role of these kallikrein-related peptidases is not clear, but emerging evidence suggests that they might be important in several physiological systems, e.g., in male reproduction, skin homeostasis, tooth enamel formation and neural development and plasticity. The kallikrein locus has undergone some major evolutionary events. Most spectacular are relatively recent duplications of KLK1 that have created 13 and 9 functional genes that are unique to the mouse and the rat, respectively. Human paralogs are KLK2 and KLK3: the latter encoding the cancer biomarker prostate-specific antigen. In this review on kallikrein-related peptidases, the focus is on their evolution, their role in skin homeostasis and semen liquefaction, and their utility as cancer biomarkers.

Human Kallikrein-related Peptidase 14 (KLK14) Is a New Activator Component of the KLK Proteolytic Cascade: POSSIBLE FUNCTION IN SEMINAL PLASMA AND SKIN

Human kallikrein-related peptidases (KLKs) are a family of 15 serine proteases mainly known for their biomarker utility in various neoplastic and non-neoplastic diseases. Despite significant progress in understanding their clinical application, little is known about the activation mechanism(s) of this important family of enzymes. Emerging evidence indicates that KLKs are activated in a stepwise manner, which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. Many members of the KLK family have been reported to be active in seminal plasma and/or skin, suggesting their involvement in common proteolytic cascades. KLK14, in particular, is highly active and has recently been proposed as one of the key trypsin-like proteases involved in skin desquamation. This study aims to elucidate a probable cascade-mediated role of KLK14 by 1) examining KLK14-mediated cleavage of a heptapeptide library encompassing activation sites of the 15 KLKs and 2) verifying activation of certain candidate downstream targets of KLK14 (i.e. pro-KLK1, -KLK3, and -KLK11). Heptapeptides encompassing activation motifs of KLK2, -3, -5, and -11 were cleaved with a high (> or =85%) cleavage efficiency. Activation of these candidates was confirmed using full-length recombinant proteins. Pro-KLK11, -KLK3, and -KLK1 were rapidly activated in a concentration-dependent manner. Pro-KLK3 regulation was bidirectional because activation was followed by inactivation via internal cleavage of active KLK3. We are proposing a putative cascade model, operating through multiple KLKs. Identification of novel members of such proteolytic cascades will aid in further defining mechanisms involved in seminal/skin homeostasis.

The GPI‐anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.

Studies on liquefaction and storage of ejaculated dromedary camel ( Camelus dromedarius) semen

The purpose of this study was to evaluate seminal liquefaction and quality of ejaculated camel semen during storage in different extenders at room (23 °C) and refrigeration (4 °C) temperature. Semen was collected using an artificial vagina and diluted immediately (1:1), using a split-sample technique, in five extenders [(1) Tris–tes egg yolk, (2) Tris–lactose egg yolk, (3) citrate egg yolk, (4) sucrose egg yolk and (5) Tris–fructose egg yolk], while one fraction was kept without an extender to act as control. The semen was transported to the lab at 37 °C, in a portable incubator within half an hour, and thereafter liquefaction of semen was monitored every 15 min. After complete liquefaction of the semen it was evaluated for sperm concentration and morphology and then was extended to a final ratio of 1:3. Aliquots of each semen sample were then stored at refrigeration and room temperature. The average volume of an ejaculate was 4.3 ± 0.4 mL and it had a very viscous consistency. The average concentration of spermatozoa was 230.4 ± 10.7 × 10 6 mL −1 and the proportion of spermatozoa with protoplasmic droplets averaged 1.02 ± 0.2, while 2.7 ± 0.6 and 9.7 ± 2.9% had mid-piece and tail abnormalities, respectively. All extended semen samples liquefied within 1.5 h at 37 °C, however, there was slow liquefaction in the sample without an added extender (control). Best liquefaction was observed in Tris–lactose extender followed by Tris–fructose and citrate egg yolk diluents whereas in the other two extenders there was head-to-head agglutination of the spermatozoa. There was no difference in the initial motility of the spermatozoa in extenders 1–5 after its liquefaction, however, after 24 and 48 h of storage a higher proportion of spermatozoa were motile in extenders 1, 2 and 4 ( P < 0.05) at both the temperatures. There was a gradual decline in viability of the spermatozoa in all extenders at both the temperatures, although, a high portion of the spermatozoa had intact acrosomes throughout the storage period. It may be concluded that dromedary semen, when added to an extender (1:1) immediately after collection, liquefies within 60–90 min at 37 °C. It maintains a high proportion of motile and viable spermatozoa that can survive storage up to 48 h in Tris–lactose egg yolk, Tris–tes egg yolk and sucrose egg yolk diluents. However, best liquefaction and progressive sperm motility is achieved in Tris–lactose egg yolk extender.

Prostate-Specific Antigen As A Diagnostic Tool: How Useful and Reliable Is It?

Prostate-specific antigen (PSA), a kallikrein isolated by Wang in 1979 [1], is a physiologic excretory product of the healthy prostatic epithelium, involved in the semen liquefaction process. A small amount of PSA (less than 1 molecule per million excreted) leaks into the bloodstream where its normal level was – arbritarily – fixed more than 2 decades ago at 4 ng/ml and shown to rise in any prostatic pathologic condition associated with alterations of the basement membrane: prostatitis, benign prostatic hypertrophy (BPH) and, of course, cancer. While definitely not a tumor marker, PSA has been used and approved as a marker for the detection of prostate cancer [1] and therefore, extensively promoted as the marker for early detection and eventually, screening. This has induced an unprecedented revolution in urology and oncology, leading to a spectacular increase in the incidence of prostate cancer, inducing a dramatic expansion of surgery and radiation therapy without any spectacular modification of the prostate cancer death rate up to this point in time. However, once initially seen as a ‘magic bullet’, PSA is increasingly confronting clinicians with more questions than they seem able to answer, which this editorial will try to summarize.

Seminal factor VII and factor VIIa: supporting evidence for the presence of an active tissue factor‐dependent coagulation pathway in human semen

Human semen spontaneously coagulates into a semisolid mass and then wholly liquefies in a process that may have some similarity to that of normal blood. This well described phenomenon is referred to as coagulation and liquefaction of semen. Besides other active components of the haemostatic system, semen contains a significant amount of functional tissue factor (TF). However, TF needs factor (F)VII in order to exert it actions. In this study, we assessed human semen for the presence of FVII and FVIIa, and related their levels to conventional fertility parameters. Using a functional, one stage, clotting assay based upon the prolongation of the prothrombin clotting time, using the ACL 300R analyser and an Imubind FVIIa ELISA assay, FVII and FVIIa levels were measured in 97 semen specimens obtained from sub-fertile (sperm counts <20 x 10(6)/mL), normally fertile (sperm counts >or=20 x 10(6) but <60 x 10(6)/mL), fertile sperm donors (sperm counts >or=60 x 10(6)/mL), vasectomized subjects and in a pooled normal semen parameters group (categorization into groups was based on the World Health Organization guidelines on fertility criteria). In addition, conventional semen parameters were analysed on all semen samples. Both FVII and FVIIa were quantifiable in human semen. The mean levels of FVII and FVIIa were 4.4 IU/dL and 12 ng/mL respectively. Despite the observed variations of FVIIa levels in the studied groups they did not meet statistical significance when the groups were tested against each other. However, seminal FVIIa levels showed a significant positive association with semen liquefaction time, sperm motility and semen volume. The anti-sperm antibodies and sperm-agglutination groups were also associated with raised seminal FVIIa levels. We observed no significant relationship between FVIIa levels and total sperm concentration, sperm count per mL (sperm density), sperm progression and days of sexual abstinence. This study demonstrates that human semen contains appreciable amounts of FVII and FVIIa. It is possible to quantify these using commercially available assays. There also appears to be a direct correlation between the levels of these factors and certain seminal parameters. This finding reinforces the concept of an active clotting system in human semen, by establishing the missing link in the activation of a TF-dependent pathway.

Characteristics of human semen by microscopic observation during liquefaction

目的 比较观察精浆内凝胶状物质消失前后精液分析主要指标的差异,了解该现象在判定精液液化中的意义.方法 体外采精后立即充池入精子计数板,置光学显微镜下观察其液化过程中凝胶状物质的形态变化,并于胶状物质消失前后进行精液分析.结果 人精液液化过程中可以通过显微镜观察到精浆中凝胶状物质呈现特征性的形态变化,32份精液分析结果显示,前向运动精子和精子活动率在此形态变化前后分别为(30.3±12.4)%和(45.0±14.9)%、(44.9±12.7)%和(59.0±15.3)%,差异有统计学意义(t=-1.130和5.023,P均＜0.01),而精子密度在液化前后分别为(55.3±33.9)×106/ml和(62.1±32.3)×106/ml,差异无统计学意义(t=1.180,P＞0.05).结论 通过显微镜观察到精液中凝胶状物质呈特征性变化,该特征可作为一种客观和简易可行的判定精液液化的手段。

Semenogelin, the Main Protein of the Human Semen Coagulum, Regulates Sperm Function

Semenogelin (Sg), the main component of the human semen coagulum, is an important and versatile protein acting on several sperm parameters, both as intact or degraded Sg. Sg originates mostly from seminal vesicle and probably is responsible for sperm immobilization in the seminal coagulum. Purified Sg can be cross-linked by transglutaminase or phosphorylated by kinases, but the actual occurrence of these reactions in reproductive physiology is not clear. Experimental evidence demonstrates that prostate-specific antigen (PSA) rapidly cleaves Sg, an event temporally associated with semen liquefaction and initiation of sperm motility. Sg and its degradation peptides participate in various processes including Zn +2 shuttling, antibacterial activity, hyaluronidase activation, and so on. Sg inhibits sperm motility at the concentration found in the coagulum, but the rapid processing by PSA allows initiation of movement. The mechanism of Sg action and its targets are not known, but improper Sg degradation decreases fertility. Sg and its degradation peptides block sperm capacitation and associated events at concentrations much lower than those of seminal plasma and could play important role in preventing premature capacitation. The effects of Sg are dependent on time and proteolysis due to PSA, and any imbalance may affect sperm physiology and fertility.

Purification and Characterization of Human Kallikrein 11, a Candidate Prostate and Ovarian Cancer Biomarker, from Seminal Plasma

Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma. hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry. hK11 is present in seminal plasma at concentrations ranging from 2 to 37 microg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of approximately 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of approximately 20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, alpha1-antichymotrypsin, alpha2-antiplasmin, and alpha1-antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg156. This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

Peculiarities of semen coagulation and liquefaction in males from infertile couples

The results of electrosemengraphy showed frequent absence of postejaculatory semen coagulation (62% of patients examined) and 3 variants of ejaculate liquefaction in males from infertile couples. Semen liquefaction was mostly of a cascade nature (one- and two-cascade types), but viscid ejaculates often liquefied slowly and monotonously (monotonous type of liquefaction).

Changes in osmolality during liquefaction of human semen

The osmolality of 18 liquefying human semen samples from 15 volunteers was measured by vapour pressure osmometry to be low (294 mmol/kg, range 269-311). For each sample the osmolality increased during liquefaction to reach a mean of 312 mmol/kg (280-331) by 30 min at 37 degrees C. These results are at variance with the widely held view that semen osmolality is greater than that of serum, which results from its first being examined after liquefaction in vitro. Thus when sperm are routinely examined after liquefaction they have been subjected to osmotic stresses that are not experienced by spermatozoa entering the female tract at coitus.

UREAPLASMA UREALYTICUMINFECTION RELATED TO SEMINAL PLASMA IMMUNOSUPPRESSIVE FACTORS, SEMEN PH AND LIQUEFACTION DURATION

In this study, semen samples from fertile and unexplained infertile men were explored for relationships between seminal plasma immunosuppressive factors (SPIFs), semen pH, liquefaction duration and infection of ureaplasma urealyticum (Uu). SPIFs activity was measured by way of counteracting complement. PH was detected by exact pH test paper. Liquefaction duration was observed at 37 degrees C. The results showed that Uu infection ratios of semen samples with abnormal SPIFs, pH or liquefaction duration were markedly higher than those of normal semen samples. It is suggested that Uu infection decreases the level of SPIFs, changes the pH in semen and prolongs the semen liquefaction so as to cause spermatic quality decline. The enhancement of SPIFs level may change the male body against Uu infection.

Does human semen contain a functional haemostatic system?

There is already evidence that a few components of the haemostatic system exist in semen. If these comprise a functional system, they may have a role in seminal clotting and liquefaction processes and ultimately may influence fertility. What might be expected in semen as collected from fertility clinics i.e., after having both coagulated and subsequently liquefied is uncertain. It does however still contain significant amounts of Tissue Factor (TF) although its effect on semen quality remains poorly understood. The present study analyses semen for Tissue Factor Pathway Inhibitor (TFPI). Measurements were made in seminal plasma, swim-up sperm and prostasomes and its relationship with conventional fertility parameters assessed. TFPI antigen levels in seminal plasma were measured in a total of 176 subjects using an Enzyme-linked immunosorbent assay (ELISA). These include sub-fertile (n=37), normally fertile (n=40), fertile sperm donor (n=34), vasectomized subjects (n=65) and in a further group defined by normality in several parameters derived from the World Health Organization (WHO) fertility criteria and termed "pooled normal semen parameters" (PNSP). For characterization studies, both TFPI activity and antigen were measured on whole semen, swim-up sperm and prostasome-rich fraction (n=5). TFPI levels were significantly higher in normal men as compared to sub-fertile (P<0.01) or vasectomized subjects (P<0.001). TFPI levels were even higher in the donor quality semen and the PNSP group. TFPI levels also correlated with semen liquefaction time, normal semen viscosity, sperm progression, percentage of motile sperm and sperm counts (density). In conclusion, the present finding substantiates the concept of an active clotting system in human semen. TFPI could regulate the activity of abundant TF, as it does elsewhere. Given a functional set of coagulation factors in semen, the TF/TFPI balance might impinge on its liquefaction and hence on global fertility.