A TEST THAT DIVES DEEPER INTO THE PROBLEMS OF MALE REPRODUCTIVE HEALTH

The SPI™ TEST (Sperm Pathogen Immunophenotyping Test) is a new, patented, award-winning diagnostic test that for the first time allows detection of intracellular pathogens such as viruses (e.g. CMV, HSV 1/2) and Chlamydia inside spermatozoa, by immunofluorescence and flow cytometry.

The SPI™ TEST allows etiological investigation of male factor infertility, early pregnancy failure and recurrent miscarriages due to sperm-originating pathogens even in cases of chronic, subclinical infections where conventional methods such as cultures, immunofluorescence and PCR fail to detect them.

It is the most sensitive test commercially available (more sensitive than PCR-based commercial tests), while at the same time it provides extreme specificity, allowing correlation between infection and infected cell type.

THE SPI™ TEST ALLOWS DIAGNOSIS OF CHRONIC, SUBCLINICAL INFECTIONS OF THE GENITOURINARY TRACT

Acute inflammation of the male genitourinary system due to infection usually requires immediate therapeutic intervention and it is easily diagnosed. In contrast, when investigating male factor infertility, or chronic recurrent infections of the male genitourinary tract and eventually of the accessory glands may go unnoticed through conventional testing. For this reason, a method of extremely high sensitivity and specificity is required such as the SPI Test.

The SPI Test can be combined with Sperm DFI (DNA Fragmentation Index) and oxidative stress levels analysis in the same sample, offering a more comprehensive evaluation of each patient’s sperm profile.

EARLY PREGNANCY FAILURE AND RECURRENT MISCARRIAGES MAY ORIGINATE FROM THE SPERM - A closer look inside spermatozoa reveals why.

INTRACELLULAR INFECTIONS CAN DAMAGE SPERM AND LOWER NATURAL AND ASSISTED REPRODUCTION  SUCCESS RATES

Intracellular infections can affect the viability and morphology of the spermatozoa in different ways, ultimately compromising sperm quality. Problematic parameters of the spermiogram such as low concentration, motility, viability, bad morphology or high DFI (or other types of DNA damage) can be attributed (at least in part) to these microorganisms and eventually improve after treatment. Infections by intracellular pathogens are of great importance as they can lead to deterioration of sperm characteristics and can compromise the reproductive outcome of both natural conception and assisted fertilization attempts.

CHLAMYDIA INFECTIONS

International studies have shown that infection of semen and sperm cells by Chlamydia spp. and intracellular pathogens can affect the quality, motility and viability of the sperm [1, 2]. In men, Chlamydia can lead to acute or chronic genital inflammation (epididymitis, epididymo-orchitis), occasionally to sexually-acquired reactive arthritis (SARA) while they have been implicated with atheromatiosis. In both men and women, Chlamydia may produce proctitis while individuals with Chlamydia are at increased risk of acquiring or transmitting HIV [3]. From the extensive laboratory research of LOCUS MEDICUS SA, there is strong evidence that intracellular Chlamydial infections can lead to spermatozoa midpiece anomalies and can compromise sperm morphology.

HSV 1/2 AND CMV INFECTIONS

Intracellular CMV infections of immature germ cells develops to mature CMV-carrying spermatozoa and have been shown to induce a gametotoxic effect that potentially may lead to male infertility [4]. The persistence of HSV 1/2 and CMV even after density gradient centrifugation for IVF purposes [4], indicates a risk of oocyte infection during fertilization, after insemination by IVF or intracytoplasmic sperm injection.

In this way it can be assumed that such viral infections could contribute to immunogenicity and antigenicity of the embryo after fertilization.

VERTICAL TRANSMISSION OF VIRUSES FROM SPERMATOZOA TO ZYGOTE CAN LEAD TO MISCARRIAGE

Vertical transmission of intracellular pathogens and viruses such as HSV1/2 and CMV may occur from sperm to zygote during fertilization. The incorporation of viral DNA into the embryonic genome may result in expression of viral antigens by the embryonic cells. This can provoke Natural Killer (NK) lymphocyte tropism against them, leading to immunological rejection of the fetus. Even in the case of fetal survival after vertical transmission of viruses, there is a possibility that T-cell clones, which normally recognize viral antigens, will be deleted during thymic education. As a result, the fetus would be tolerized against these pathogens and therefore will be susceptible to them in the future with unpredictable consequences.

Vertical transmission of viruses from spermatozoa to zygote, can result in the creation of immunogenic embryos and may initiate immunologic  response from the mother at the site of fetal implantation, especially in cases where the NK blood levels of the mother are already abnormally high due to idiopathic or other environmental reasons. This will usually result in early pregnancy failure often misinterpreted as difficulty of conception when it occurs before pregnancy and is confirmed by biochemical testing, or recurrent miscarriages of immunological etiology.

PUBLISHED LABORATORY DATA CONFIRMS THE INCREASED SENSITIVITY, SPECIFICITY AND EFFECTIVENESS OF THE SPI™ TEST

More than 20 years of extensive research in the laboratories of LOCUS MEDICUS and a huge clinical experience with more than 10,000 infertile couples led to the development and introduction in clinical practice of the SPI™ TEST: A patented non-invasive test of increased sensitivity for the diagnosis of both active and asymptomatic infections by intracellular pathogens such as Chlamydia, Cytomegalovirus (CMV), Herpes simplex virus (HSV) types 1 and 2, HPV infections and EBV in spermatozoa.

Our evidence strongly supports a role of sperm intracellular pathogens in couple infertility through the vertical transmission of viral genetic information from sperm to embryo during fertilization, thus triggering an immunological  response from the mother during pregnancy. Towards this direction, the following studies from LOCUS MEDICUS research group have been published:

1. ENVIRONMENT, MICROBES AND INFERTILITY

It has been shown that women exposed to crowded occupational environments (such as teachers) with high content of airborne viruses and pathogenic microbes which over-stimulate the immune system exhibit higher rates of infertility compared to the general population [24].

2. CHLAMYDIA AFFECTS SPERM MORPHOLOGY

C. trachomatis infections induce reduced sperm motility and viability as well as sperm midpiece anomalies [1, 2].

3. NK CELLS AND INFERTILITY

During 2003 we showed that high blood NK cells are associated with early pregnancy failure in couples with infertility or difficulties in conception [25]. A correlation has been also

shown between high levels of blood ΕΕ cells in women with a history of infertility and/or miscarriages and the presence of subclinical herpes viremia (HSV1/2, EBV, CMV, HHV6, HHV7) [26, 27]. Interestingly enough, as observed using The Hidden-C® Test, a lack of correlation was identified between female genital tract infection and increased peripheral blood NK cell levels [6] indicating indirectly the role of infected sperm in immunological response of the mother during pregnancy.

4. IMMUNOLOGICAL REJECTION OF THE FETUS

Histological testing of 1st trimester abortive material from women with high peripheral blood NK lymphocytes routinely shows a high aggregation of NK cells on the border of a necrotic area in the implantation site intermingled with embryonic trophoblast cells. The same was observed in abortive material of women whose blood NK cell levels were normal. This can be explained by embryo immunogenicity due to expression of sperm-originating viral antigens resulting in NK cell tropism against the embryo and against embryonic cells of the intermediate trophoblast which eventually leads to immunological rejection of the fetus.

SPI™ TEST REVEALS PREVIOUSLY UNDIAGNOSED INFECTIONS IN THE SPERM OF MEN, IN INFERTILE COUPLES

The SPI™ testing of 886, 762 and 634 semen samples from male partners in infertile couples, revealed that 55%, 32% and 37% of sperm samples were positive for intracellular Chlamydia, CMV and HSV 1/2 respectively. These results showed a statistically significant correlation between the presence of intracellular infection in sperm and couple infertility.

Moreover, the extremely high prevalence of infection in the tested population proves the exceptional sensitivity and specificity of the SPI™ Test compared to all other methods of testing.

ADVANTAGES OF THE SPI™ TEST COMPARED TO OTHER CONVENTIONAL DIAGNOSTIC TESTS

1. IT DETECTS INTRACELLULAR SPERM PATHOGENS AND QUANTIFIES INFECTION

The SPI™ TEST succeeds where conventional culture, immunofluorescence and PCR- based methods fail to localize the microorganisms inside cells discriminating between extra and intracellular infections. Furthermore, other commercially available methods cannot determine the type of the infected cell correlating cell-type and infection thus achieving relative quantification of the result.

2. IT HAS INCRESED SENSITIVITY COMPARED TO CONVENTIONAL TESTS

In conventional tests, the urethral swabs for Chlamydial culture may be painful because it requires deep insertion, and trying to limit the procedural pain can have a negative effect on specimen quality while at the same time culture for Chlamydia is now regarded as slow, labor intensive, and hence quite costly [5]. Furthermore, enzyme immunoassay (EIA) tests on non-invasive specimens such as urine, exhibit lower rates of detection than urethral swabs cultures for both sensitivity and specificity [6]. Moreover, a skilled and experienced microscopist is required for optimal performance and even then compared to cultures, even the most advanced EIAs or direct fluorescence (DIF) methods show lower sensitivity and specificity [7]. When nucleic acid amplification testing (such as PCR) was used on urine samples, the difference between urethral swab and urine specimens was reduced [8 9, 10]. However, it has been known for a while that PCR inhibitors can be present in many clinical specimens, and there is some controversy on the extent of PCR inhibition in urine [11, 12, 13].

Alternatively, Chlamydia testing in semen specimens might provide additional information as to whether there is a Chlamydial infection of the upper genital tract [5]. It has been suggested that protocols be developed for the optimal detection of C. trachomatis in semen. There are suggestions that semen might provide additional information on infections of the upper genital tract that may not be detected in urine [14-22]. However, semen testing for Chlamydia by PCR would suffer from the same limitations with urine PCR due to the presence of PCR inhibitors naturally present in the samples.

On the other hand, The SPI™ Test uses flow cytometry technology which, can be applied to semen samples without fear of reduced sensitivity due to polymerase inhibitors in the samples.

Experimental evidence from LOCUS MEDICUS research, as well as published bibliography, show significantly higher sensitivity of methods using monoclonal anti- MOMP antibodies (Major Outer Membrane Protein) for detection of Chlamydia like the one used in the SPI™ TEST, compared to commercial PCR protocols that target Chlamydial rRNA [23].

The same advantages of SPI™ Test apply for the detection of viruses such as Cytomegalovirus and Herpes Simplex Virus 1/2, especially since viruses cannot be cultured from semen samples, while compared to PCR testing the SPI™ Test is more informative, as it reveals intracellular viral infection, which is determining factor for the risk of vertical transmission during fertilization.

RECOMMENDATIONS IN THE USE OF THE SPI™ TEST:

1. IN CASES OF COUPLES FACING UNEXPLAINED INFERTILITY, DIFFICULTY OF CONCEPTION, EARLY AND RECURRENT MISCARRIAGE (Recommented together with The Hidden-C® Test)

2. IN SUSPICION OF MALE GENITAL TRACT INFLAMMATION FOR DIAGNOSIS OF CHRONIC, SUBCLINICAL INFECTIONS WITH OR WITHOUT INFERTILTIY

3. IN DIAGNOSIS AND TREATMENT OF WEAK SPERM WITH OLIGOSPERMIA, ASTHENOSPERMIA OR TERATOSPERMIA

4. IN ROUTINE ANDROLOGICAL SCREENING

We recommend the integration of the SPI™ Test in basic andrological examination together with spermiograms and semen cultures, for preventive healthcare of the male reproductive system, diagnosis of infection or investigation of male factor infertility.

5. FOR DONOR SPERM SCREENING BY IVF CLINICS & SPERM BANKS

Our published data revealed a selective rather complete elimination of CMV, HSV and EBV in infected spermatozoa following density gradient centrifugation prior to IVF purposes (Fig. 4) [28]. The use of infected spermatozoa, increase the risk of oocyte infection during fertilization after insemination by IVF or intracytoplasmic sperm injection, and the possibility of a miscarriage. The necessity to fully identify the intracellular microbiological profile of the sperm is, therefore, essential not only in natural but also in assisted reproduction. By using the SPI™ Test, IVF clinics and sperm banks can guarantee to their clients healthy sperm clear of viruses for better success rates either by IUI (Intrauterine Insemination), IVF or some other method of assisted reproduction.

THE SPI™ TEST AT A GLANCE

  • Allows detection of the male and couple infertility – associated pathogens inside spermatozoa
  • High sensitivity
  • Self-collected sperm sample
  • Safe storage and shipment to the reference laboratory by post
  • Easy to repeat procedure for monitoring antibiotic/antiviral therapy results treatment
  • Fast turnaround time of results
  • Low cost

SUCCESSFUL TREATMENT RESULTS

  • Improvement of semen parameters
  • Higher success rates at assisted reproduction attempts
  • Better chances for natural conception & term pregnancy
  • Lower risk of miscarriage
  • Lower risk of transmitting infection to the female partner

 REFERENCES

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  2. Eley A. et al. (2003). Chlamydia trachomatis is bad for your sperm! Microbiol. Today. May;Vol30.
  3. Fleming DT, Wasserheit JN (1999) From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect;75:3-17.
  4. Michou et al. (2012). Herpes virus infected spermatozoa following density gradient centrifugation for IVF purposes. Andrologia. Jun;44(3):174-180.
  5. Eley, A. (2011), How to Detect Chlamydia trachomatis in Males?. Journal of Andrology, 32: 15–22. doi: 10.2164/jandrol.110.010363).
  6. Sellors J, et al. (1991) Rapid, onsite diagnosis of chlamydial urethritis in men by detection of antigens in urethral swabs and urine. J Clin Microbiol;29:407–409.
  7. Black CM (1997) Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin Microbiol Rev.;10:160–184.
  8. Carroll KC et al. (1998) Evaluation of the Abbott LCx ligase chain reaction assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and genital swab specimens from a sexually transmitted disease clinic population. J Clin Microbiol;36:1630–1633.
  9. Cook RL et al. (2005) Systematic review: noninvasive testing for Chlamydia trachomatis and Neisseria gonorrhoeae. Ann Intern Med;142:914–925.
  10. Michel CE et al. (2007) Chlamydia trachomatis load at matched anatomic sites: implications for screening strategies. J Clin Microbiol;45: 1395–1402.
  11. Mahony J et al. (1998) Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcriptionmediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol;36:3122–3126.
  12. Toye B et al. (1998) Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis testing. J Clin Microbiol;36:2356–2358.
  13. Van der Pol B et al. (2001) Multicenter evaluation of the BDProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol;39:1008–1016.
  14. Eggert-Kruse W et al. (1997) Chlamydial serology in 1303 asymptomatic subfertile couples. Hum Reprod;12:1464–1475.
  15. Bornman MS et al. (1998) Chlamydial infection in asymptomatic infertile men attending an andrology clinic. Arch Androl;41:203–208.
  16. Fujisawa M et al. (1999) Chlamydia trachomatis detected by ligase chain reaction in the semen of asymptomatic patients without pyospermia or pyuria. Arch Androl;42:41–44.
  17. Rosemond A et al. (2006) Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples. Pathol Biol (Paris);54:125–129.
  18. Gdoura  R et al. (2008) Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples. J Androl;29:198–206.