SPI TEST ™: Detection of intracellular pathogens (HSV1/2, CMV, Chlamydia) inside spermatozoa

The SPI Test ™ (Sperm Pathogen Immunophenotyping) is a new diagnostic method of semen and sperm-cell population analysis, for the detection of intracellular pathogens such as viruses and Chlamydia inside spermatozoa, by immunofluorescence and flow cytometry. It allows etiological investigation of male-factor infertility, early pregnancy failure and recurrent miscarriages due to sperm-originating pathogens in cases of natural conception as well as in assisted reproduction efforts.

The SPI Test ™ succeeds where conventional culture and immunofluorescence methods fail to adequately detect Chlamydia or viruses, while PCR-based methods can neither locate the microorganisms inside cells, nor determine the type of the infected cell.

For the first time, a fast, economical and sensitive test allows the etiological diagnosis and treatment of sperm infections by intracellular pathogens through the use of targeted antibiotic/antiviral therapy.  

SUCCESFUL TREATMENT RESULTS IN:

  • Improvement of semen parameters 
  • Better chances for natural conception & term pregnancy 
  • Higher success rates at assisted reproduction attempts
  • Lower risk of miscarriage

How sperm intracellular infections affect fertility:

A.   Our data indicate that intracellular infections can compromise sperm morphology while several international studies have shown that infection of semen and sperm cells by intracellular pathogens such as viruses and Chlamydia can affect sperm quality and sperm viability both in vivo and in vitro [1,2,3].
B.   Vertical transmission of intracellular pathogens from spermatozoa to zygote, can result in immunogenic embryos and may initiate immunologic rejective procedures in the implantation site, leading to male-factor infertility, early pregnancy failure (often misinterpreted as inability to conceive) or recurrent miscarriage [4,5].


When to use the SPI TEST ™
 
Whenever there is a clinical history of one or more of the following: 
  • Early pregnancy failure
  • Recurrent miscarriages of immunological etiology
  • Oligospermia, asthenospermia or teratospermia
  • Unsuccessful IVF attempts
  • Infertility of unknown etiology
  • Suspicion of male genital tract inflammation with or without infertility
Furthermore, we recommend the use of the SPI Test ™ in combination with spermiograms and semen cultures for preventive healthcare of the male reproductive system, or during diagnosis of infection
 

Sperm Pathogen Immunophenotyping by flow cytometry (SPI Test ™) is a nationally (patent no. 1008033) and internationally patented clinical test (patent WO 2013144662 A1 “Method of intracellular infectious agent detection in sperm cells”). 

Information for clinicians

A.  The SPI Test™ allows diagnosis of chronic, subclinical infections of the genitourinary tract

 
Acute inflammation of the male genitourinary system due to extracellular pathogen infection usually requires immediate therapeutic intervention in order to clear the infection. In contrast, when investigating male factor infertility, chronic, subclinical infection of the genitourinary tract and eventually the accessory glands by intracellular pathogens such as Chlamydia and viruses are of great importance as it can lead to deterioration of sperm characteristics and can compromise the reproductive outcome of both natural conception and assisted fertilization attempts.
 

B.   Sperm intracellular localization can indicate the origin of infection 

 
Classical methods such as direct immunofluorescence and microbial cultures usually fail to detect chronic, subclinical infections due to lack of sensitivity. At the same time, expensive PCR-based methods, fail to distinguish between intra- and extra-cellular infections.
 
An intracellular infection of spermatozoa may indicate that the microorganisms have had enough time to infect and replicate inside sperm cells in some stage of spermatogenesis or storage in the epididymides, while an extracellular infection may indicate an infection of the sperm release pathway (vas deferens, seminal vesicles, prostate, urethra), which can affect the condition of the tract, but not directly the ability of spermatozoa to fertilize.
 

C.   Intracellular Chlamydia, HSV 1/2 and CMV can damage sperm and lower natural and assisted reproduction success rates.

 
  • Chlamydia. Have been associated with a significant decline in numbers of motile sperm and results in premature sperm death [2,3]. Our data also indicate that intracellular infections can lead to sperm midpiece anomalies.
 
  • HSV 1/2 and CMV. Intracellular CMV infection of immature germ cells develop to mature CMV-carrying spermatozoa. A considerable decrease in the number of immature germ cells indicates that CMV produces a direct gametotoxic effect and can contribute to male infertility [1]. HSV 1/2 and CMV persist even after density gradient centrifugation for IVF purposes [6]. This indicates a risk of oocyte infection during  fertilization, after insemination by IVF or intracytoplasmic sperm injection. Further studies are required to determine whether any correlation exists between their presence, implantation rate and the outcome of pregnancy.
 

D.  Vertical transmission of viruses

 
Intracellular presence of viruses such as HSV1/2 and CMV may result in vertical transmission from sperm to zygote during fertilization. The incorporation of viral DNA into the embryonic genome may result in one of two things:
 
  • In case of fetal survival, there is a strong possibility that T cell clones, which normally recognize viral antigens, will be deleted according to the self-tolerance theory during thymic education. As a result, the fetus would be tolerized against these pathogens and therefore susceptible to them in the future.
 
  • Expression of viral antigens by the embryonic cells and mainly by the cells of the intermediate trophoblast may provoke NK (Natural Killer) cell tropism against these cells, promoting necrosis at the implantation site, disruption, and immunological rejection of the fetus. Such very early miscarriages of immunological etiology, can often be misinterpreted as difficulty of conception when occurring before pregnancy is confirmed by biochemical testing.

 

Data supporting the SPI Test™

A.   Correlation between presence of sperm intracellular pathogens and couple infertility.

 
 
Using the SPI test ™ 886, 762 and 634 sperm samples from the male partners of infertile couples were tested for the presence of semen intracellular Chlamydia, Cytomegalovirus (CMV) and Herpes simplex virus (HSV) 1/2 respectively. 
 
Fig.1 Infectious agent detection on sperm samples using the SPI Test ™.
 
Our results showed that 55% of sperm samples were positive for intracellular Chlamydia, 32% were positive for CMV and 37% were positive for HSV 1/2. 
 
 

B.   In addition to reduced sperm motility and viability [2, 3], our data indicate that intracellular Chlamydia trachomatis infection can lead to sperm midpiece anomalies. 

 
Fig.2 Mean of percentage (%) of semen analysis morphological abnormalities before and after treatment of Chlamydia trachomatis.
 

SPI Test ™ protocol, results report & Interpretation

A.    SPI Test ™ flow cytometry protocol

 

The SPI Test ™ uses a simple and fast protocol that produces reliable, comprehensive and reproducible results. 
  • Sperm liquefaction 
  • Fixation
  • Optional long term storage at 4°C and capability of international shipment of samples
  • Membrane permeabilization 
  • Incubation with monoclonal antibodies against intracellular pathogens such as Chlamydia trachomatis, HSV, CMV and HPV
  • Acquisition of data in a flow cytometer 
 

B.    SPI Test ™ results report

 
SPI Test ™ flow cytometer data acquisition screenshots. 

  • Upper Left Panel: Selection of spermatozoa in the sample.
  • Lower Left Panel: Negative control.
  • Lower Right Panel: Percentage of spermatozoa positive for intracellular Chlamydia.
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

The development of the SPI Test™

A.   The discovery of this novel test was based on a series of scientific observations and research data from studying infertile couples in our fertility clinic since 1996.

 

1.    Professional women with everyday exposure to crowded environments such as educators, exhibit higher rates of infertility. Such environments are characterized by high content of airborne viruses and microbes which over-stimulate the immune system and may lead to mother to embryo hostility [7]

2.    Since 1995, high blood NK lymphocytes have been recognized as a risk factor during the first trimester of pregnancy. In 2003 we showed that high blood NK are associated with early pregnancy failure in couples with infertility or difficult conception [8].

3.    In 2004 and 2005, we described a correlation between high blood ΝΚ lymphocyte numbers in women with a history of infertility and/or miscarriages and the presence of subclinical herpes viremia (HSV1-2, EBV, CMV, HHV6, HHV7) [9,10].

4.    At the same time we did not observe any correlation between female genital tract infection and increased peripheral blood NK cell levels (data not shown). For that study we used Real-Time PCR analysis on menstrual tissue samples in order to have a complete picture of the upper genital tract (what is now commercially known as the Hidden-C Test ® ) which provided higher sensitivity compared to cervicovaginal secretion testing by both culture and PCR-based methods [11].  

5.    Histological testing of 1st trimester abortive material from women with high peripheral blood NK cells routinely shows a high aggregation of NK cells on the border of a necrotic area in the implantation site. 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, NK cell tropism against the embryo and against embryonic cells of the intermediate trophoblast, eventually leading to immunological rejection of the fetus. 

6.    Finally we showed that sperm enrichment prior to assisted reproduction resulted in selective rather in complete elimination of viruses [6]

The above results strongly indicated that viral antigens can be transmitted from sperm to embryo during fertilization, triggering an immunological response from the mother. In order to be able to be transmitted in this fashion, microorganisms must be able to parasitize and replicate with embryonic cells which, in turn, express the exogenous antigens thus triggering immunological response. Such microorganisms include viruses such as HSV 1/2 and CMV. 

Regarding other viruses like HPV, commonly detected inside spermatozoa by the SPI Test ™, the presence of which has not been associated with any known immunological effects, further investigation is required, to elucidate their clinical significance.

 

IVF Clinics & Sperm Banks

A.    The SPI Test ™ guarantees infection-free sperm samples for use in assisted reproduction

 
  • The fact that intracellular presence of viruses may lead to miscarriage, makes the use of the SPI Test ™ by sperm banks invaluable since some viruses persist inside cells even after density gradient centrifugation for IVF purposes [Fig.3]. This indicates a risk of oocyte infection during fertilization, after insemination by IVF or intracytoplasmic sperm injection.
  • By using the SPI Test ™, sperm banks and IVF clinics both 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.
 
 Fig.3. Number of samples infected by each virus before and after PureSperm treatment
 

Awards & Distinctions

A.   National & International Patent

  • Sperm Pathogen Immunophenotyping by flow cytometry (SPI Test ™) is a nationally (patent no. 1008033) and internationally patented clinical test (patent WO 2013144662 A1 “Method of intracellular infectious agent detection in sperm cells”). 

B.    Awards

  • “Greece Innovates!”  2nd contest of Applied Research and Innovation by SEV (Hellenic Federation of Enterprises) and Eurobank. Final phase of competition - Top10 innovations.
  • “SFEE Innovation Project” by the Hellenic Association of Pharmaceutical Companies (SFEE). Final phase of the competition - Top10 innovations.

 

References

  1. Naumenko VA. et al. (2011). Detection of human cytomegalovirus in motile spermatozoa and spermatogenic cells in testis organotypic culture. Herpesviridae. Jun 28;2(1):7
  2. Eley A. et al. (2005). Can Chlamydia trachomatis directly damage your sperm? Lancet Infect Dis. Jan;5(1):53-7.
  3. Eley A. et al. (2003). Chlamydia trachomatis is bad for your sperm! Microbiol. Today May;Vol30.
  4. AD Gritzapis et al., J Antivir Antiretrovir 2014, 6:2 (4th World Congress on Virology October 06-08, San Antonio, USA, 2014
  5. AD Gritzapis et al., Andrology October 2014, 2:2 (Proceedings of the 8th congress of  European Academy of Andrology), ECA Barcelona, Spain 2014
  6. Michou et al. (2012). Herpes virus infected spermatozoa following density gradient centrifugation for IVF purposes. Andrologia. Jun;44(3):174-180.
  7. Perros G. et al. (2011). Are Educators at High Risk of Sub-fertility? A Multicenter Study. Am J Reprod Immunol.Apr;65(4):415-20. 
  8. Mihou et al. (2003). The Fraction of Peripheral Blood CD56 + /CD16 - /CD3 - Subpopulation on Total Natural Killer Cells as an Indication of Fertility-Infertility. Fertil Steril., 80 Suppl 2:691-7.
  9. Thomas D. et al. (2004). The Effect of Valacyclovir Treatment on Natural Killer Cells of Infertile Women. Am. J. Reprod. Immunol., 51(3), 248-255
  10. Thomas D. et al. (2005). Altered Immunophenotypic Parameters in Infertile Women. Possible Role of Herpes Viremia. Am. J. Reprod. Immunol. 54(2), 101-11.).
  11. Michou et al. (2014). Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility. J Obstet Gynaecol Res. Jan;40(1):237-42. doi: 10.1111/jog.12165. Epub 2013 Oct 7.
 
 
© LOCUS MEDICUS SA, 2015
 
Corresponding author Dr. Panagiotis Venieratos PhD, Molecular Biologist, Locus Medicus SA, Athens, Greece
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