TITLE OF THE INVENTION
DIAGNOSIS OF MALE INFERTILITY BY MEASURING
TISSUE FACTOR PATHWAY INHIBITOR
FIELD OF THE INVENTION
[0001] The invention relates to determination of tissue factor pathway inhibitor (TFPI) levels in sperm and sperm plasma to diagnose and treat male infertility. This application claims the benefit of United States provisional patent application no. 60/622,166 filed October 26, 2004.
BACKGROUND
[0002] The procoagulant activity of seminal plasma added to human blood plasma was first recognized in 1942.1 Seminal plasma diluted up to 10 000-fold significantly decreased the re-calcification clotting time of blood plasma. The molecular basis for this activity remains to be elucidated. Semen forms a so-called coagulum immediately after ejaculation, embracing the sperm and finally semen liquefies spontaneously after 5 to 20 minutes after in vitro emission.2'3 This coagulation and liquefaction of semen, may play a major role in sperm capacitation.4 The interaction between the High Molecular Weight Seminal Vesicle (HMW-SV) proteins and Prostate Specific Antigen (PSA) are considered responsible for the coagulation and liquefaction of semen. A limited number of studies have addressed the potential functions of the haemostatic enzymes and inhibitors in semen5"7 but their potential role remains speculative. It is well documented that Tissue Factor (TF) is present in large quantities in human semen8'9 and it has been suggested that seminal TF originates from the prostate and is associated with prostasomes,9 a vesicular product secreted by acinar cells of the prostate gland.10
[0003] Elsewhere in the body, TF complexes with FVII/FVIIa and initiates blood coagulation by the proteolytic conversion of FIX to FIXa and FX to FXa. Neither FVII/FVIIa or TF alone possess this activation capability.11'12
The action of this complex on its substrates is abrogated in a FXa-dependent fashion by a circulating endogenous inhibitor of the TF-dependent pathway of blood coagulation, known as Tissue Factor Pathway Inhibitor (TFPI), which consists of three multivalent Kunitz-type plasma serine protease inhibitors, with an acidic amino-terminal region and basic carboxy-terminal end.13'14 Initially, TFPI binds to FXa via its Kunitz domain 2, followed by a second step in which the TFPI/FXa complex binds to the TF/FVIIa complex via its Kunitz domain 1, forming an inactive quaternary TFPI/FXa/TF/FVIIa complex. In this way, and in a Factor (F) Xa dependent fashion, TFPI prevents further production of FIXa and FXa. by the TF/FVII/FVIIa complex. Thus, additional FIXa and FXa can only be generated through the lower part of the intrinsic pathway, which involves the activation of FX by the FVIIIa-FIXa complex, where FIXa results from the initial action of TF-FVII/FVIIa and its supplemental generation by FXIa.
[0004] Seminal TF may serve to limit bleeding and consequent vascular access by semen-born agents as a result of tissue damage which may occasionally arise during intercourse and may also contribute to the anti¬ inflammatory properties attributed to prostasomes.9'15 On the other hand, TF function in semen appears to be independent from its role in blood coagulation, as semen contains a limited amount of FVII7 and lacks FX7 (the natural substrate for TF-FVII/FVIIa complex). It is therefore possible that TF in semen may have entirely different actions, which as yet remain largely unidentified. Evaluation of semen TF in infertility patients showed a 16-fold variation in TF- FVII activity and no relationship was found between TF and the number of days of abstention before sampling, specimen pH, sperm counts or sperm motility.7 The potential physiological role of other seminal clotting/fibrinolytic factors in the male reproductive system has been discussed.16
[0005] Tissue Factor Pathway Inhibitor (previously called Lipoprotein Associated Coagulation Inhibitor, Extrinsic Coagulation Pathway Inhibitor or Tissue Factor Inhibitor) is an endogenous inhibitor of the TF-dependent pathway of blood coagulation that belongs to the serine protease inhibitors. The mature protein of TFPI has 276 amino acid residues and consists of three multivalent Kunitz-type plasma serine protease inhibitors, with an acidic amino-terminal region and basic carboxy-terminal end.13'14 It is well known to
play an important role in the control of thrombogenesis at both cellular and plasmatic sites.
[0006] Although the seminal coagulum is composed of fibrin-like material18 it has rarely been the subject of thorough investigation of components related to the haemostatic system. TFPI has been found to be present in semen from sub-fertile, fertile and vasectomised subjects. Most of this TFPI is found in the seminal plasma, although both swim-up sperm cells and prostasomes showed a detectable level. Prostasomes can be seen (by electron microscopy) attached to the heads of sperm19 - these organelles certainly bear TF9 and may carry TFPI with them into the swim-up fraction. It has also been shown that seminal plasma levels of TFPI correlate in a general fashion with male fertility (see Thyzel et al., Thrombosis Research 109(56) pgs. 329-332, March 15, 2003. However, there has not been any previous showing of a correlation between seminal TFPI levels and specific causes of infertility.
SUMMARY OF THE INVENTION
[0007] The invention relates in one aspect to a method of diagnosis of male infertility in mammalian subjects, preferably human, due to one or more of insufficient seminal agglutination, low sperm motility or low sperm count (in terms of sperm density namely numbers of sperm per ml.). The method comprises the steps of obtaining a sample of semen from the subject and measuring the content of TFPI therein. The measurement may be carried out using a known assay. TFPI may be measured in either whole semen or plasma. Preferably, the measurement is carried out within about 4 hours of emission of the sample and still more preferably within 1 hour, although it is also possible to refrigerate or freeze the sample for a delayed assay.
[0008] The level of TFPI within the sample determines the likelihood that the subject is infertile. The critical level, below which the subject is likely infertile or nearly infertile, will vary depending on whether the assay is carried out on fresh vs. frozen/refrigerated sample and whether the assay is carried out on whole vs. fractioned semen.
[0009] The invention also relates to a kit for carrying out the above analysis, comprising an assay kit and written instructions describing the method described in this patent specification.
[0010] We have discovered different levels of TFPI in seminal plasma from sub-fertile, fertile and vasectomy subjects. Most of the seminal TFPI was found in the seminal plasma. It was also detected in the swim-up sperm and prostasome fraction. Furthermore, the invention relates to the role of seminal TFPI as a marker for impaired fertility by relating its level to conventional fertility parameters.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGURE 1 is a chart comparing TFPI levels in fertile, sub-fertile and vasectomised men. The figure shows seminal TFPI antigen levels plotted against fertility expressed as sperm count (density). Results are shown as Box and Whisker plots. The bottom and top of the "box" represents the 25th and 75th centile, whilst the line within the box represents the median value. The "whiskers" represent the range.
[0012] FIGURE 2 is a chart comparing TFPI levels in sperm samples with different levels of motility, showing seminal TFPI antigen levels plotted against forward progression of sperm. Results are shown as Box and Whisker plots.
[0013] FIGURE 3 is a chart comparing TFPI levels in sperm samples with different levels of agglutination, showing seminal TFPI antigen levels plotted against seminal agglutination. Results are shown as Box and Whisker plots.
[0014] FIGURE 4 is a graph showing the relationship between detected TFPI levels and sperm density.
DETAILED DESCRIPTION
[0015] In normally fertile men, total seminal TFPI level was 4.5+/-2A ng/ml compared to total blood plasma of 68.7+/-14.1 ng/ml.22 TFPI levels
were significantly higher in fertile men as compared to sub-fertile men (P<0.01) or to the vasectomised subjects (P<0.001). TFPI levels were even higher in the high sperm-count donor group. Again, a significant difference was observed when this group was compared to the sub-fertile or the vasectomy groups (P<0.001; respectively). The PNSP group showed comparable levels with the donor group, suggesting a potential value for TFPI
- 4a -
as a marker for infertility, especially as 15% of infertile men have normal semen parameters according to the WHO criteria, but are unable to produce a pregnancy in spite of having a normal partner.23 Furthermore, the conventional parameters do not correlate well with each other in predicting outcome.
[0016] As the sub-fertile men and the vasectomised subjects showed significantly lower levels of seminal TFPI, the difference might be due to the number of sperm. As indicated above, although TFPI was detected in sperm the vast amount of seminal TFPI is found in the plasma and all these measurements were performed free of sperm. The site of production of this form of TFPI is not yet established. Transudation from the blood plasma is possible but by no means certain, as the levels of TFPI obtained in semen, which necessarily must have both coagulated and liquefied before it can be measured, are high - 6.6% of the concentration in blood plasma, considering that only about 1% of albumin or IgG, which have much smaller molecular weights, are recovered in seminal plasma after extravasations in the prostate.24
[0017] TFPI levels positively correlated with seminal liquefaction time. Semen of normal viscosity showed higher median and IQR then the abnormal group. This is important as good semen quality correlates with normal coagulation.3'25 Sperm progression (forward movement), is also correlated with increased TFPI. Indeed, the group with good sperm progression showed a significant increase in TFPI levels when compared to the poor progression group (P<0.001). Progressive motility is important in male factor fertility26 and is also necessary for intrauterine insemination sucess.27 TFPI levels also positively associated with the percentage of motile sperm.
[0018] There was a significant positive correlation between TFPI levels and sperm counts per ml (Λ?=82; r=31; P<0.05; Fig. 4). However, no such significant correlation was observed with the total sperm count, semen volume and days of abstention. The latter results agree with those reported earlier for seminal TF.7 Sperm appear to bear TFPI (albeit perhaps through adherent prostasomes) but whether this is related to the sperm membrane
- 5 -
modification required to fulfil future functional activity in the female genital tract remains to be established.
[0019] Autoantibody formation against sperm in men and women are important causes of infertility.28"30 In the current study subjects positive for ASA showed higher TFPI levels compared to those who did not show ASA. Likewise, the number of agglutinating sperm correlated positively with higher TFPI levels, particularly so when samples with many clumps were tested against samples that show no clumping (P<0.01) or sperm-free vasectomy samples (P<0.001). As high TFPI levels are overall associated with normal fertility increased TFPI in these minority groups may be a response to coagulum dissolution or decreased agglutination of sperm. TFPI levels are increased in inflammation31 or may be counteracting the effect of TF- dependent pathway of blood coagulation32 which is pro-inflammatory.33 Local delivery of recombinant TFPI may also act as an anti-inflammatory agent.34 In addition, reduction of circulating IL-6 levels by TFPI treatment in E.coli induced septicaemia,35 suggests that it may have a direct anti-inflammatory role. If TFPI is carried by prostasomes as is seminal TF, increased TFPI in ASA subjects may be related to the anti-inflammatory properties attributed to these organelles.9'15
[0020] The data presented herein indicates the presence of almost a complete set of coagulation factors in semen and provides a sound basis for predicting that TFPI is important in providing a favourable milieu for spermatozoa to attain fertilizing capability. Thus therapeutic treatment with TFPI would enhance male fertility in mammals. Furthermore, TFPI levels can be used as a marker for male factor infertility. Male infertility is mostly evaluated and defined on the basis of sperm counts, which may not be sufficient to determine outcome as not all subjects with high counts are fertile in real life and not all subjects with low counts are infertile. Clumping and antisperm antibodies are also associated with fertility problems. There is no clear correlation between these observations, suggesting independence of action. In this circumstance an additional diagnostic marker that bridges the discrepancies might be of value.
- 6 -
[0021] One method of determining the probable fertility of a male subject due to one or more of insufficient seminal agglutination, low sperm motility or low sperm count consists of the following steps:
(a) obtaining a sample of semen from the subject;
(b) subjecting the whole semen sample to an analysis to determine the level of TFPI; and
(c) comparing the detected TFPI level with a standard;
[0022] The subject will be considered to be sub-fertile if the TFPI level in whole semen is about 2.5 ng/ml or less.
[0023] The sample should be tested rapidly upon taking of the sample. Alternatively, the sample may be frozen in liquid nitrogen for carrying out the assay at a later time. If maintained at room temperature, the sample should be assayed within about 60 minutes.
[0024] A similar procedure may also be carried out on a fraction of the semen, preferably the plasma. For example, the sample may be separated by centrifuge and the plasma or sperm cells withdrawn for testing. The comparable level of TFPI antigen in each case range between 0.1 -0.7 ng/ml.
[0025] The information derived from the diagnostic procedure may then form by itself the basis for a diagnosis of male infertility. Alternatively, the information may be combined with other information about the subject such as sperm count to yield a more accurate diagnosis.
[0026] A diagnostic kit comprises a container for receiving a sperm sample, a kit for determining TFPI levels in the sample, and written instructions for the diagnostician to enable him or her to correlate the detected level of TFPI in the sample with a diagnosis of likely or infertility.
[0027] The kit may optionally also include a carrier to hold the sperm sample container in a frozen state in order that the assay step may be carried out at a different time and/or location.
- 7 -
[0028] Preferably, the testing system comprises an assay for measuring the ability of TFPI to inhibit the catalytic activity of the TF/FVIIa complex to activate FX to FXa. One such testing system is the Actichrome™ TFPI activity assay sold by American Diagnostica.
Example 1
Stratification of subjects
[0029] Seminal TFPI was measured in a total of 181 subjects with an age range of 20-64. In five of these TFPI activity and antigen were measured on whole semen, seminal plasma, sperm and prostasomes-rich fraction. To compare subject groups we followed the classification of the 4th edition of the WHO guidelines on semen analysis.17 Sub-fertile subjects have sperm counts <20xl06/ml (n = 37), normal fertility implies sperm counts >20<60xl06/ml (n=40). Subjects suitable for semen donation (labelled below as "donors") in Southampton University Hospitals have sperm counts >60xl06/ml (n=34). Vasectomised subjects were also studied (n=65). A further group was defined with all semen parameters being normal according to the WHO criteria (volume of 2-6 ml, a sperm concentration of >20 X 106 /ml, sperm morphology of >15% normal forms, above 40% of the total sperm population is motile, and a sperm progression of 2.5 or more with 4 being the maximum level of progression).17 This is referred to below as "pooled normal semen parameters" (PNSP).
Processing of semen samples
[0030] Semen samples were fractionated into seminal plasma, sperm and a prostasome-rich fraction. Viable intact sperm were isolated in IVF medium (Medi-Cult (UK) Ltd, Surrey, UK) by what is known as the "swim up" technique according to the product instructions. Seminal plasma and prostasomes were prepared by differential centrifugation. Fresh (untreated) semen was spun after liquefaction in 1.5ml Eppendorf tubes at 2000-x g for 10 minutes at room temperature in Heraeus Biofuge 28 RS centrifuge (Jencons, Leighton Buzzard, UK). This low speed supernatant constitutes the
- 8 -
plasma fraction. The prostasome-rich fractions were collected after further high speed centrifugation at 51,000-x g for 180 minutes at 4°C. The supernatants from this spin were discarded and the pellets re-suspended in PBS (Sigma Chemical Company, Poole, UK). Both intact sperm and prostasomes were lysed in 100 μl buffer containing; 10 mM Tris/HCI pH 7.4, 1% (v/v) Triton X-100 before assay. The seminal plasmas were deep-frozen at -72 0C for batch-wise measurement.
Semen analysis
[0031] The conventional fertility parameters were measured according to the WHO guidelines as described in the WHO "golden" laboratory manual for the examination of human semen and sperm-cervical mucus interaction.17
Antisperm antibody assays
[0032] Antisperm antibodies (ASA) were assayed using the Direct SpermMar Anti-Sperm Antibody Tests for the detection of IgA and IgG ASA according to the manufacturer's instruction (Conception Technology, San Diego, USA). In principle, fresh semen is mixed with latex particles that have been coated with human IgA or IgG. The formation of agglutinates between particles and motile sperm indicates the presence of IgA or IgG antibodies on the sperm.
TFPI assays
Activity assay
[0033] The activities of TFPI in fractionated semen specimens were measured using the Actichrome® TFPI activity assay [# 848]. The assay was performed as instructed by the manufacturer (American Diagnostica Inc., Stamford, USA). In principle, the assay measures the ability of TFPI to inhibit the catalytic activity of the TF/FVIIa complex to activate FX to FXa. Following incubation of the samples with TF/FVII and FX, the residual activity of the TF/FVII complex is measured using spcetroxyme FXa, which is a highly
- 9 -
specific chromogenic substrate cleaved only by FXa generation in the assay system (American Diagnostica Inc., Stamford, USA), releasing a para- nitroanilin (pNA) chromophore into the reaction solution. The absorbance of the pNA is then read at 405 nm and compared to those values obtained from a standard curve.
Antigen assay
[0034] Total seminal TFPI antigen levels were measured using commercially available kits; Imubind® Total TFPI ELISA assay [# 849] according to the manufacturer's instructions (American Diagnostica Inc., Stamford, USA). In principle, into duplicate wells of a microtitre plate pre- coated with capture antibody, the test samples were added. TFPI is then detected using a biotinylated monoclonal antibody specific for the TFPI Kunitz domain 1. The subsequent binding of the streptavidin conjugated horseradish peroxidase completes the formation of the antibody enzyme detection complex. The addition of TMB substrate and its subsequent reaction with HRP provides a blue colour. The reaction was stopped with sulphuric acid, and the absorbance read at 450 nm. The values were than calculated automatically from a standard curve after subtraction of background values from blank wells.
[0035] The Imubind® Total TFPI ELISA assay recognises native and recombinant human TFPI in complex and truncated forms. No significant cross-reactivity or interference was observed for the assay (American Diagnostica Inc., Stamford, USA). The lower limit of detection for the assay was 0.18 ng/ml. The intra and inter assay coefficient of variations for 5 ng/ml TFPI were 6.5% and 5.5%, respectively (American Diagnostica Inc., Stamford, USA).
[0036] Human TFPI levels can also be measured with commercially available kits.
Statistical analysis of monoclonal antibody assay systems for detecting free TFPI and TFPI complexes
- 10 -
[0037] Results were entered in a database and analyzed by the STATGRAPHICS™ statistical software system. Data were not normally distributed, and summary statistics were expressed as medians and inter¬ quartile ranges (IQR). The median value is the 50th centile. The 25th and 75th percentiles of the data specify the values covered by the IQR. Differences between two or more groups were assessed by either Mann-Whitney U-Test or Kruskal-Wallis One-Way Analysis by Ranks. Correlations were determined using Spearman's Rank Correlation Test. All assay results are expressed per original sample volume, unit/ml or ng/ml for TFPI activity or antigen, respectively.
Results
Seminal TFPI
[0038] There was an abundance of TFPI activity (Table 1) and antigen (Table 2) in human semen. Most of the TFPI in semen was found in the seminal plasma. It was also detected in the swim-up sperm and the prostasome fraction.
TFPI levels in the studied groups
[0039] TFPI levels were significantly higher in fertile men compared to sub-fertile men (P<0.01) or to vasectomised subjects (P<0.001). The sub- fertile group and vasectomised subjects were not significantly different (Fig. 1). TFPI levels were even higher in the donor group (>60xl06/ml). This group showed significantly higher levels compared to sub-fertile (P<0.001) or vasectomised subjects (P<0.001; Fig. 1). There was no significant difference between the donor and the fertile groups. The PNSP group showed comparable levels with the donor group (Fig. 1).
Liquefaction time
[0040] There was a positive and significant correlation between TFPI levels and the seminal liquefaction time (n=82; r=0.2; P<0.02).
- 11 -
Seminal viscosity
[0041] The normal viscosity semen group showed higher median and IQR values for TFPI (n=21; median = 5; IQR=3-8.1) compared to the abnormal (n = ll; median=3.9; IQR=2.1-8.6). The difference between the two groups was not statistically significant.
Sperm progression
[0042] A significant difference was seen for TFPI levels between sperm with poor and good progression (P<0.001); the good group showing the higher TFPI values (Fig. 2).
Sperm motility
[0043] TFPI levels were higher with a greater percentage of motile sperm (n=61; median = 5; IQR=2.3-8.2) than the lower percentages (n = 30; median = 3.9; IQR=2.9-6.0). However, the levels above and below the conventional clinical cut-off point (50% motile sperm) were not statistically significant.
The degree of seminal agglutination
[0044] There was a significant increase in the number of clumps correlated with a higher increase in TFPI levels. This was statistically significant when samples with many clumps were compared to those showing no clumps (P<0.01) or vasectomised samples (P<0.001). Although the TFPI levels were high when compared to samples having few clumps; statistically, the differences were not significant (Fig. 3).
Sperm counts per ml. total sperm counts, semen volume and days of abstention
[0045] There was a positive and significant correlation between TFPI levels and sperm density (n=82; r=0.31; P<0.05; Fig. 4). No such a significant correlation was seen with the total sperm count (n=82; r=0.2;
- 12 -
P>0.05), semen volume (n=82; r=0.21; P>0.05) and days of abstention (n=82; r=0.1; P>0.05).
Sperm morphology
[0046] The abnormal morphology group showed higher TFPI levels and wider IQR (n=6; median=6.7; IQR=3-9.7) compared to the normal group (n=86; median=4.3; IQR=2.3-6.6). The difference between the two groups was not significant.
Antisperm antibodies
[0047] Patients with antisperm antibodies (ASA) showed higher TFPI levels (n=12; median=5.7; IQR=4.7-8.4) compared to those without antisperm antibodies (n=18; median=4.3; IQR=2.5-9.0). The difference between the two groups did not reach statistical significance.
[0048] The method described above would be suitable for detection of infertility in male mammalian subjects and in particular human subjects. It is contemplated that a kit may be provided which instructs the user to collect whole semen from a subject and to carry out the assay for TFPI levels therein immediately upon collection and in any event within 60 minutes.
[0049] It is also contemplated that male subjects may be treated for infertility by administration of TFPI. Such administration may take any suitable route of administration and for this purpose the active substance may be admixed with suitable carriers, adjuvants etc. A sufficient dosage may be supplied to bring levels of TFPI present within the subject to exceed the levels described above in connection with lack of fertility.
[0050] A further method contemplated is an in vitro method for enhancing the fertility of a sperm sample for use on an in vitro or in vivo artificial insemination process, by adding sufficient TFPI to the sample which is otherwise deficient in TFPI to elevate the TFPI level to that of a normal subject prior to the fertilization step.
- 13 -
[0051] Methods and products may be provided which depart in a manner which would be apparent to one skilled in the art, from the methods and products which have been described herein by way of the examples and detailed description, as well as in the general description of the invention and the patent claims presented herein. It will be understood that any such variations, departures or equivalent methods or products may remain within the scope of the present invention and that no part of the present patent specification is intended to limit the scope of the present invention claimed herein.
- 14 -
TABLES
Table 1: TFPI activity in seminal plasma, swim-up sperm, prostasome-rich fraction, and neat semen, results are shown as median and IQR.
Groups n Median IOR
(unit/ml) Lower Upper
Whole semen 5 1.58 0.9 1.6
Seminal plasma 5 0.30 0.12 0.70
Swim-up sperm 5 0.19 0.18 0.20
Prostasomes 5 0.08 0.073 0.10
Table 2: TFPI antigen in seminal plasma, swim-up sperm, protasome-rich fraction, and neat semen, results are shown as median and IQR.
Groups n Median IOR
(ng/ml) Lower Upper
Whole semen 5 2.21 0.69 2.31
Seminal plasma 5 2.35 1.23 4.85
Swim-up sperm 5 0.75 0.01 1.55
Prostasomes 5 0.35 0.01 0.99
- 15 -
References:
1. Huggins C, Neal W. Coagulation and liquefaction of semen. Proteolytic enzymes and citrate in prostatic fluid. J Exp Med 1942; 76: 527-541.
2. Tauber PF, Zaneveld LJD, Propping D, Schumacher GFB. Components of human split ejaculates. II. Enzymes and proteinase inhibitors. J Reprod Fertil 1976; 46: 165-171.
3. Mandal A, Bhattacharyya AK. Grouping the ejaculates according to the degree of coagulation and the relationship to the levels of choline and cholinesterase. Int J Androl 1986; 9: 407-415.
4. Masaki J. Biochemistry of semen. In: Spermatology. Mohri H, Morisawa M, Hoshi M (Eds). Tokyo: University of Tokyo Press, 1992; ppI21-137.
5. van Wersch JWJ, Ubachs JHM, Dalaere KPJ. Fibrinolysis and coagulation markers in seminal plasma before and after vasectomy. Fibrinolysis 1993; 7: 135-138.
6. Espana F, Gilabert J, Estelles A, Romeu A, Aznar J, Cabo A. Functionally active protein C inhibitor/plasminogen activator inhibitor-3 (pCI/PAI-3) is secreted in seminal vesicles, occurs at high concentrations in human seminal plasma and complexes with prostate-specific antigen. Thromb Res 1991; 64:309-320.
7. Carson SD, De Jonge CJ. Activation of coagulation factor X in human semen. J Androl 1998; 19: 289-294.
8. Fareed J, Callas DD, Hoppensteadt D, Bermes EW. Tissue factor antigen levels in various biological fluids. Blood Coag Fibrinol 1995; 6: 32-36.
- 16 -
9. Frenandez JA, Heeb MJ, Radtke K-P, Griffin JH. Potent blood coagulant activity of human semen due to prostosome-bound tissue factor. Biol Reprod 1997; 56: 757-763.
10. Brody I, Ronquist G, Gottfries A. Ultra structural localisation of the prostasome-an organelle in human seminal plasma. Upsala J Med Sci 1983; 88: 63-80.
11. Nemerson Y, Bach R. Tissue factor revisited. Prog Hemost Thromb 1982; 6: 237-261.
12. Nemerson Y. Tissue Factor: Then and Now. Thromb Haemost 1995; 74: 180- 184.
13. Broze GJ. The role of tissue factor pathway inhibitor in a revised coagulation cascade. Semin In Hematol 1992; 29: 159-169.
14. Broze GJ. Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coag Fibrinol 1995; 6: 7-13.
15. Skibinski G, Kelley RW, Harkiss D, James K. Immunosupression by human seminal plasma-extracellular organelles (prostosomes) modulate activity of phagocytic cells. Am J Rerod Immunol 1992; 28: 97-103.
16. Polak B, Daunter B. seminal plasma biochemistry. IV: enzymes involved in the liquefaction of human seminal plasma. Int J Androl 1989; 12: 187-194.
17. Ronquist G, Nilsson BO. Prostasomes. Wennergren International Serious. London: Portland Press, 2002; pp 1-157.
18. Matsuda Y, Shimokawa K, Katayama M, Shimizu H, Umeda T, Oshio S, Chiba R. Blood coagulation factor X (FX) in human seminal plasma. Arch Androl 2002; 48: 295-300.
- 17 -
19. Yamada M, Gentry P. Hemostatic profile of bovine ovarian follicular fluid. Can J Physiol Pharmacal 1995; 73: 624-629.
20. Mandal A, Bhattacharyya AK. Relationship between the coagulation- liquefaction property of human ejaculates and their volume, sperm count and motility Clin Reprod Fertil 1987; 5: 367-371.
21. De Jonge CJ, Mack SR, Zaneveld LJD. Inhibition of the human sperm acrosome reaction by proteinase inhibitors. Gamete Res 1989; 23: 287-297.
22. Marshburn PB, Kutteh WH. The role of antisperm antibodies in infertility. Fertil Steril 1994; 61 : 799-811.
23. Meinertz H, Hjort T. Antisperm antibodies in male. In Frontiers in Endocrinology, Immunocontraception, vol. 12. Ares-Serono Symposia Publications, 1995, pp 151-159.
24. Hjort T, Meinertz H. Antisperm antibodies in women. In Frontiers in Endocrinology, Immunocontraception, vol. 12. Ares-Serono Symposia Publications, 1995, pp 83-98.
25. Mazumdar S, Levine AS. Antisperm antibodies. Etiology, pathogenesis, diagnosis and treatment. Fertil Steril 1998; 70: 799-810.
26. Mahony MC, Alexander NJ. Sites of antisperm antibody action. Human Reprod 1991; 6: 1426-1430.
27. D'Cruz OJ, Haas GG Jr, Wang B, De Bault LE. Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9- mediated immune injury to human sperm. J Immunol 1991; 146: 611-620.
28. Rooney IA, Oglesby TJ,. Atkinson JP. Complement in human reproduction. Immunol Res 1993; 12: 276-294.
- 18 -
29. Ronquist G, Nilsson BO, Hjerten S. Interaction between prostasomes and spermatozoa from human semen. Arch Androl 1990; 24: 147-157.
30. Arienti G, Carlini E, Palmerini CA. Fusion of human sperm to prostasomes at acidic pH. J Membrane Biol 1997; 155: 89-94.
- 19 -