This study was funded by and conducted in the diagnostic and research laboratories of Locus Medicus SA, Athens, Greece Speaker: Dr. Angelos Gritzapis,

INTRACELLULAR DETECTION

OF INFECTIOUS PATHOGENS

IN SPERM CELLSThis study was funded by and conducted in the diagnostic and research laboratories of Locus Medicus

SA, Athens, Greece

Speaker: Dr. Angelos Gritzapis, Biologist - Immunologist

A.D. Gritzapis1 PhD, K. Makarounis2 MD, M. Leventopoulos1 PhD, E. Nossi1, D. Nikolopoulos1, G. Georgoulias3 MD, V. Kapetanios4 MD, V Tsilivakos1 MD, PhD

1: Dept. of Cellular Biology and Immunology, Locus Medicus SA, Athens, Greece2: Urology clinic, Locus Medicus SA, Athens, Greece 3: Dept. of Microbiology, Locus Medicus SA, Athens, Greece 4: Gynecology & Obstetrics clinic, Locus Medicus SA, Athens, Greece

THE THREE MAIN GOALS OF THIS STUDY:

1. Treatment of male factor subfertility.

2. Prevention of miscarriages due to immunogenic embryos from infected spermatozoa.

3. Prevention of vertically-inherited congenital infections from spermatozoa.

OBSERVATIONS LEADING TO THE PRIMARY CONCEPTUAL IDEA

Every Day Observations in a subfertility clinic• Miscarriages occur slightly more often during winter.

• Women that interact professionally with many people such as teachers, are dominant in the subfertility group.(Perros et al. 2011)

Data from bibliography• Increment of NK percentage in the peripheral blood of women

with a history of miscarriages (Coulam et al. 1995).

• The main cell population that makes implantation (trophoblasts) is potential targets of NK cells as they conventionally lack MHC-I expression.

• NK cells are the first line of defense against virally infected cells.

QUESTION IWhat is the consequence of NK cell increment?• Difficulty in conception?

• Miscarriages of the first trimester?

• Both?

Michou et al., Fertility & Sterility 2003

Group I. Consecutive spontaneous aborters [n=25; mean age, 30.4 ±4.1 years; median (range) of abortions, 3 (2–5); mean infertility duration, 2.4±1.35 years]

Group II. Sporadic spontaneous aborters [n=30; mean age,34.7±5.3 years; median (range) of abortions, 2 (1–3); mean infertility duration, 5.03±2.81 years]

Group III. Infertile (n=33; mean age, 35.4±5.4 years; mean infertility duration, 9.32 ±4.65 years)

Group IV. Fertile controls (n=11; mean age, 32.5±3.9 years)

0

100

200

300

400

500

Total Peripheral blood NK cell concentration (n/μl)

Group II:Sporadic Aborters

Group III:Infertile

Group IV:Fertile

Group I:Consecutive aborters

217.0 138.8

299.8 173.5

272.2 115.7

131 63

Group I Group II Group III Group IV

Groups I, IV Groups II, III

190.8 126.4

285.3 145.5

Groups I, IV Groups II, III

PANOVA=0.003

CONCLUSION

An absolute number of peripheral blood NK cells could be used rather as an indicator of difficulty of conception than as a risk factor for miscarriage.

QUESTION II• Which is the causative force of NK increment in the peripheral

blood of women with a history of subfertility?• Are there genital microbial infections?• Are there subclinical viral infections?

Thomas D. et al., Αm J Reprod Immunol 2005

Herpesviruses related to the increase of NK cell concentration in the peripheral blood in women with a history of subfertility.

0

200

400

600

800

Total Peripheral blood NK cell concentration (number/μl)

NK c

ell c

once

ntr

atio

n (n/μ

l)

105.0 50.0

183.0 95.0

313.0 156.0 320 156

tripleviremia

377.0 209.0

doubleviremia

singleviremia

herpes -control

p<0.01

p<0.01

p<0.01

CONCLUSION

Assuming that all women under study remained asymptomatic, these data suggest that subclinical herpesvirus viremia may be an important cause of immunostimulation in women with a history of subfertility by increasing the concentration of NK cells in the peripheral blood.

But why are NK cells destructive at the level of the decidua?

Cells of the internal trophoblast diffuse in the endometrial stroma at the implantation site. Furthermore, they emigrate into the lumen of endometrial vessels in order to regulate blood pressure and subsequently the diffusion of oxygen towards the embryo (endothelium is red)

Intermediate trophoblast invasion of decidual vessels and stroma. Anti–cytokeratin 8–18 immunoperoxidase staining for intermediate trophoblastic cells (brown), anti–CD34 for vessels and surface epithelium. Immunoalkaline phosphatase (red), x40.

Cells of the internal trophoblast diffuse in the endometrial stroma at the implantation site. Furthermore, they emigrate into the lumen of endometrial vessels in order to regulate blood pressure and subsequently the diffusion of oxygen towards the embryo (endothelium is red)

Necrosis of decidua. Immunostaining anti–cytokeratin 8–18. Immunoalkaline phosphatase x40.

In the case of first trimester miscarriages due to immunologic factors necrosis presents

Necrosis of decidua. NK cells with cytotoxic activity. Immunostaining with anti–CD16. Immunoalkaline phosphatase x40.

At the border of necrosis in a mixture with the cells of intermediate trophoblast, NK cells are diffuse (anti CD16+) and have a tropism against cells of the intermediate trophoblast.

The border of decidual necrosis. NK cells with Anti–CD16+ , Immunoalkaline phosphatase, x40.

HISTOLOGICAL EVALUATION OF MISCARRIAGES OF THE FIRST TRIMESTER OF GESTATION

NK tropism against embryonic cells of the intermediate trophoblast and correlation with necrosis at the implantation sites characterizes the histologic appearance of miscarriage for immunological reasons. Surprisingly, the same phenomenon appears even in cases with normal NK cell concentration in peripheral blood!

Consequently, embryonic cells could be immunogenic!

DO THEY CONTAIN VIRUSES?

Couple HSV 1-2 EBV CMV1 + - + + - + + - +2 + - + - - - + - +3 + + - - + + - + -4 - - + - - + + - +5 - - + - - + + - +6 - + - - - - + + +7 - - - - - - + + +

Yellow: Chorionic villusRed: Decidua without embryonic cellsGreen: Washed spermatozoa

PCR Herpesviruses evaluation in chorionic villi, decidua distant from the implantation site, and sperm

HSV EBV CMV HHV6 HHV7 HSV EBV CMV HHV6 HHV7

0

20

40

60

Number of samples infected by each virus before and afterPureSperm treatment

32.0

19.0

49.0

11.0

47.0

42.0

9

04.0

0

WASHING PROCEDURE FOR REMOVAL OF INFECTED SPERM CELLS

DIRECT DETECTION OF VIRUSES IN SPERM

Viruses are present in sperm Viruses are still present even after sperm

enrichment

Liarmakopoulou M: “The presence of herpes viruses in the semen of infertile couple, following two density gradient method’’ Masters, University of Leeds, 2004 (DISTINCTION, Financial award)

Michou et al., ANDROLOGIA 2012

Regarding the localization of pathogens, the PCR assay can not distinguish between seminal plasma and sperm cells or identify the infected cells.

This is very important as the interior of the spermatozoa but not the membrane, flows into the ovule.

So, a direct and high throughput assay is needed, as for example HBV is already detected in sperm cells by FISH (Huang et al, 2003).

FLOW CYTOMETRY PROTOCOL

Sperm liquefaction

centrifugation

Fixation with 4% PFA.

Membrane permeabilization with saponin and DMSO.

DNase I treatment for 30 min at 37oC as the DNA in sperm cells is very dense.

Incubation with monoclonal antibodies against intracellular pathogens (i.e. Chlamydia trachomatis, clone: Herpes Simplex Virus-HSV, Cytomegalovirus-CMV, Human Papiloma Virus-HPV).

Acquisition in a flow cytometer.

ADNANTAGES OF FLOW CYTOMETRIC METHOD OVER PCR Evaluation of the intracellular presence of

pathogens.

Greater sensitivity as Taq inhibitors should be suppressed by dilutions (Garolla et al. 2013). This can explain the discrepancy between PCR and flow cytometry.

Information on the identity of infected cells.

Lower cost.

Copyright 2012 Malamis & Malamis

54.63% CHLAMYDIA +

Total=886

32.15% CMV +

Total=76237.22% HSV +

Total=634

PREVALENCE OF PATHOGENS IN SUBFERTILE POPULATION

Apr-12 Jul-12 Oct-12 Jan-13 May-13 Aug-13 Nov-13 Mar-140.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00% Seasonal fluctuation cCT

CMV

HSV

Month

Pe

rce

nta

ge

of

po

sit

ive

in

fecti

ve

ag

en

ts

There is no correlation between virus presence and sperm morphology.

But there is correlation between sperm abnormalities and Chlamydia trachomatis presence in the interior of sperm cells. The

abnormalities in the midpiece of sperm cells, are statistically reduced after treatment with

antibiotics.

Head Abnormalit

ies

Mid Piece Abnorm.

Tail Abnorn.

0

10

20

30

40

50

60

70

80

90 83.67

28

8.83

Mean and S.E.M. of the % of spermcount morphological abnormalities before and after treatment of Chlamydia

trachomatis BEFOREAFTER

Spermcount morphological parameters

Perc

en

tag

e (

%)

*

0

20

40

60

80

100

Results after Chlamydia therapy (n=93)

% p

erce

nta

ge

of pat

ient 67.74

16.13

reduction unchanged increment

16.13

cCT0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Mean (S.E.M.) of the % of positive Chlamydia trachomatis in sperm cells, before and after treatment

BEFORE

AFTER

Intracellular pathogen

Pe

rce

nta

ge

(%

) o

f p

osit

ive

Ch

lam

yd

ia

in s

pe

rm c

ell

s

*

Series10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

Mean and S.E.M of the ΤΖΙ parameter, before and after treatment of Chlamydia trachomatis

BEFORE

AFTER

TZI

Va

lue

*

POTENTIAL CONSEQUENCES OF THE EXISTENCE OF VIRUS IN THE INTERIOR OF SPERMATOZOA

Incapability of conception due to a) abnormalities of spermatozoa.b) very early rejection of the fetus before the next

menstruation, is misinterpreted as infertility.

Early miscarriage of the fetus with the histologic image described previously, with or without NK increase in the periphery.

Because of the neurotropic properties of Herpesviruses, it is possible that congenital anomalies occur due to them.

In case of fetal survival, there is a strong possibility that T and B cell clones, which normally recognize virus antigens, will be deleted according to the self-tolerance theory during thymic education.

As a result, the newborn organism would be tolerized against intracellular pathogens and therefore more susceptible to them in the future.

SPERM BANKS

The fact that miscarriage is possible due to the intracellular presence of virus, makes their detection by sperm banks necessary.

In addition, the seasonal fluctuation of their presence, makes testing of every sample given by the same donor equally necessary.

Artificial Insemination Techniques (ART) – In vitro Fertilization (IVF)

The high economic and psychological cost of a miscarriage after either ART or IVF, makes intra Sperm Pathogen Immunophenotyping (SPI test) of great importance.

Thank you for your attention.