Roll of Viruses in Male Infertility

Tehran University Of Medical Science Medical Virology Department Dr.Hamidreza Monavari

Sherko Naseri

Introduction
Infertility Inability to conceive after one year of unprotected intercourse (6 months for women over 35?) The distribution of etiological factor for male infertility reveal that apparently 30% of the patients suffer from idiopathic infertility idiopathic infertility , The term used to explain when the cause of infertility cannot be explained Acute and chronic infection may play important role in this patient Infection processes can impair fertility by different mechanism ,for ex: HIV can cause oligoteratoespermy or HSV is detected in spermatozoid nucleus

Etiologies
Idiopathic or unknown

Physical and chemical alteration of semen factors History is important

Female factors

 Infection processes can impair fertility by different mechanisem

Deterioration of Spermatogenesis Impairment of sperm function Effect on accessory gland function Obstruction of seminal tract
For next presentation

History
(Berger et al.,1984) Say That : In up to 60% of patient with acute epididymitis,spermatogenesis at least temporarily impaired (Weidner et al.,1990) continued : after antibiotic therapy semen parameters usually return to normal so that this phenomena is reversible (Jarow et al.,1990) Result in : If The infectious process cause the deterioration in blood-testis barrier,this may lead to significant formation of sperm antibody which can be detected in serum and seminal plasma

Immunological Infertility
The blood-testis barrier formed by tight junction between sertoli cell, prostate late stage testicular germ cell and spermatozoa witch express unique antigens that can stimulate autoimmune response This barrier infiltrate lymphocyte, antibodies and complement from germ cells If germ cells can penetrate to seminiferous tube, they can survive for extended long period of time by escaping from immune system(Anderson and politch,1996)

escaping from immune system

Immunosuppressive factors in semen plasma (effect on B,T,PMN,NK cells) Sperm cells are coated with immunosuppressive carbohydrate sequence that protect from NK cells (HIV seem to carry the same immunosuppressive carbohydrate)
a biantennary N-Linked Glycon carrying the bisecting GlcNAc sequence (Clark et al.,1997)

It suggested with Barratt,1997 that immune response is depend on danger signal that if HIV may activate a danger signal but its subverted

(Clark, Dell et al. 1997)

Or maybe.
APC
inhibit

19-hydroxy PGE

IL-12

stimulate

IL-10

T-cell

Biasing to

inhibit

Th2 NK cell

Viral infection
There is growing evidence that viral infection may cause male infertility Direct effect Indirect effect By causing local inflematory and immunological reaction

The way
Viruses act on Through the urethra and invade the reproductive tract whereas for bacterial infection the most common site are epididymis or prostate

Viral infection are often disseminated and acure in epithelial (eg:HPV)and neuronal cells(eg:HSV) Or even in Wight blood cell eg:CMV & EMV HSV1,2EBVCMVHPVHBVHIVHHV6,AAV

Viral infection
Viral DNA and RNA can be detected in seminal plasma by amplification method(PCR,LCR) By Using this techniques

The most common disorder associated with male infertility is varicocele In a few studies, the relation of viruses such as human papilloma virus and adeno-associated virus to male infertility has been investigated In this study sensitive nested PCR technique for the detection of HSV, cytomegalovirus (CMV) and Epstein-Barr virus (EBV), in the semen of men with fertility problems was used.

MATERIALS AND METHODS

Samples 113 men who were attending the infertility clinic DNA Extraction From Semen Samples
centrifuge at 2,500 rpm for 10 minutes The supernatant was removed DNA extraction was performed using the protocol of EPICENTRE DNA extraction kit (MasterPure DNA Purificationkit, Madison,WI)

PCR Amplification Reactions

All the samples were examined for the presence of HSV,CMV, and EBV DNA by the nested PCR technique using one set of outer primers for primary PCR and one set of inner primers for nested PCR

PCR Amplification Reactions

35 L of extracted DNA of each sample reaction solution of 50 L containing 25 mM MgCl2, 0.25 M of each primer 200 M of each dNTP 1 PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, at 22C) 2.5 U Taq Polymerase (EPICENTRE MasterAmp Taq DNA polymerase) For HSV,EBV,CMV the primary PCR mixture was heated for 5 minutes at 95C and then subjected to 40 cycles of amplification 95C for 45 seconds, 54C for 45 seconds, and 72C for 45 seconds For NESTED PCR The same reaction mixtures were applied for nested PCR using 2 L of the primary PCR product as template

Primers
(Kapranos, Petrakou et al. 2003)

Electrophoresis result
(Kapranos, Petrakou et al. 2003)
1, 3, 4 PCR product from 3 HSVpositive semen samples

1, 3 two CMV-positive semen samples

Electrophoresis result
(Kapranos, Petrakou et al. 2003)

N,2 EBV-positive semen sample

RESULTS
Viral DNA was detected by the nested PCR technique in 64 (56.6%) of 113 semen samples HSV was detected in 56 (49.5%) semen samples EBV in 19 (16.8%) semen samples CMV in 8 (7.1%) semen samples The simultaneous presence of two DNA viruses was detected in 18 (16%) semen samples HSV and EBV DNA were detected in 14 (12.4%) samples HSV and CMV DNA, in 4 (3.6%) samples. DNA of all three viruses was found in only 1 (0.9%) semen sample

Persistent infection with the human helper virusdependent adenoassociated virus (AAV) recently has been detected in female genital tissue and in material from spontaneous abortions(Tobiasch, Rabreau et al. 1994) However, AAV has not been detected in human semen. In an experimental study, infection of pregnant mice with AAV-2 led to fetal death and early abortion(Botquin, Cid-Arregui et al. 1994)

MATERIALS AND METHODS

Ejaculates were obtained from 30 infertile men aged 1941 years Eight men with normal semen analyses who had induced a pregnancy in the past 3 years served as controls One milliliter of each semen sample was fractionated through a Ficoll Hypaque(Sigma, Deisenhofen, Germany) with proteinase K and Tween 20 buffer before DNA extraction To disrupt all cells except mature spermatozoa,cells from the pellet were lysed in a lysis buffer centrifuged at 12,500 * g for 5 minutes Washed Buffer Lysis +ditioterithol Washed For detection of AAV DNA, the primer sets pan1/pan3 and Rep78.1/Rep78.2 were used according to the method of Tobiasch et al. In brief, an initial polymerase chain reaction (PCR) using the primers Rep78.1 and pan3 was followed by a second (nested) PCR using the primers 78.2 and pan1 and 5 mL of the amplified product of the first PCR

RESULTS
Adeno-associated virus DNA was detected in 30% (9/30) of the ejaculates from the infertile men. No AAV DNA was found in the ejaculates from the 8 control subjects In 8 of 9 samples, AAV DNA could be found only in the spermatozoal fraction of the specimen. Seven of 9 semen specimens that contained viralDNA also demonstrated oligoasthenozoospermia. Both AAV and HPV DNA was found in the spermatozoal fraction of 3 of 30 (10%)specimens. HPV-16 or HPV-18 DNA was identified in 8 of 30 specimens from the infertile men and in 2 of 8 specimens from the control subjects

HA 16 cells, HeLa cells with integrated AAV DNA SP 76 and NSC 70 were positive for AAV DNA

(Rohde, Erles et al. 1999)

REFERENCE
Keck C, Gerber-Schfer C, Clad A, Wilhelm C, et al. Seminal tract infections: impact on male fertility and treatment options. Hum Reprod Update. 1998 Nov-Dec;4(6):891-903. Berger,R.E.(1984)Epididymites.In Holmes,K.K.,Mardh,P.A.,Sparling,P.F. and Wiesner,P.J.(eds),Sexually Transmitted Disease.Mcgraw-Hill,Newyork,pp.660-662. Weidner,W.,Garbe, Ch.and Weibbach,L.(1990)Initiale Therapie der akuten einseitigen Epididymitis mit Ofloxacin. Urologe,29,277-280. Jarow,J.P., Kirkland,J.A.and Assismos, D.G.(1990)Association of antisperm antibodies with chronic nonbacterial prostatitis.Urology,36,154-156 Kapranos, N., E. Petrakou, et al. (2003). "Detection of herpes simplex virus, cytomegalovirus, and Epstein-Barr virus in the semen of men attending an infertility clinic." Fertil Steril 79 Suppl 3: 1566-1570 Tobiasch, E., M. Rabreau, et al. (1994). "Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion." Journal of Medical Virology 44(2): 215-222. Botquin, V., A. Cid-Arregui, et al. (1994). "Adeno-associated virus type 2 interferes with early development of mouse embryos." Journal of General Virology 75 ( Pt 10): 26552662. Rohde, V., K. Erles, et al. (1999). "Detection of adeno-associated virus in human semen: does viral infection play a role in the pathogenesis of male infertility?" Fertil Steril 72(5): 814-816.

Clark, G. F., A. Dell, et al. (1997). "Viewing AIDS from a glycobiological perspective: potential linkages to the human fetoembryonic defence system hypothesis." Molecular Human Reproduction 3(1): 5-13.