Presence of HSV-1 DNA in semen and menstrual blood

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Abstract

Using a specifically designed diagnostic PCR assay with nested primers the following could be achieved: (1) a group of 22 clinically indistinguishable women attending an infertility clinic, 18 with repeated embryo transfer failure, and asymptomatic for HSV-1 could be divided into two subgroups after testing their menstrual blood. An HSV-DNA positive (50%) and HSV-DNA negative group (50%) could be distinguished. None of the four controls were positive; (2) semen from 154 infertile and 24 fertile men was tested in relation to infertility. In the group of infertile men 37 (24%) were HSV-DNA positive but none of the fertile control (0%) was positive; (3) treatment of both partners with an antiviral drug resulted in two pregnancies; (4) HLA data on four of the couples in which the wife’s menstrual blood was HSV positive was compared to seven HSV negative couples and 22 infertile, as well as 22 fertile couples. Clustering of class I HLA (B61 and Cw3) was found with a significant increase in Cw3 in both partners.

Introduction

Herpes simplex virus 1 (HSV-1) is a double stranded enveloped DNA virus. After initial infection the virus will remain latent and may become reactivated throughout the lifetime of an individual, especially if immunocompromised. The first infection leads to antibody formation. However, even in the presence of antibodies, reactivation still occurs so that the presence of antibodies does not ensure protection against re-infection (Whitley and Roizman, 1997).

There has been a steady increase in the incidence of HSV-1 infection in the genital area, from 21% in 1982, to the present day figure of 50% with an even distribution of lesions caused by HSV-1 and HSV-2. Herpes viruses in general are able to infect a wide variety of hosts from fishes to elephants. In horses, equine herpes virus 1 (EHV-1), also known as equine abortion virus, causes late miscarriages in mares. Using the fast word search algorithm for the presentation of sequence similarity in genomic DNA (Lefèvre and Ikeda, 1994), the genomic sequences of HSV-1 and EHV-1 were found to be very similar (El Borai et al., 1997a).This suggests a possible functional similarity between HSV-1 in humans and EHV-1 in horses.

At present, the most sensitive direct method of demonstrating the presence of HSV can be done by DNA detection using the polymerase chain reaction (PCR). Hence a PCR assay was designed to detect very small concentrations of HSV-DNA in clinical samples to try and determine if HSV-1 may be responsible for some cases of infertility.

To try and demonstrate this, the following was performed using the PCR test: (1) a group of women clinically identical for being both infertile and asymptomatic for HSV were tested; (2) semen was tested in relation to infertility; (3) antiviral treatment was performed and documented; (4) as infertility is the problem of a couple, some of the couples tested for HSV were analysed with available HLA data and compared to other couples both fertile and infertile.

Section snippets

Material and methods

The method for HSV detection was previously published (El Borai et al., 1997b) and is briefly described here with additional technical detail to emphasise precautions.

Viruses

After the first PCR, a positive band of 544 bp could be detected for the control sample which was a semen sample spiked with virus. The virus was added at a concentration of one virus particle per 3000 sperm. Dilution experiments revealed that, at lower dilutions, HSV-1 which could not be detected with the first set of primers could be detected with nested primers.

The nested PCR allows a detection of one virus particle in 30 000 or 300 000 sperm cells.

Semen samples

A total of 154 infertile samples were

Discussion

HSV DNA was detected in menstrual blood and semen of individuals attending an infertility clinic although the individuals were mostly asymptomatic. The diagnostic test is sensitive and can detect low concentrations of HSV DNA. The link between the presence of HSV and infertility was strengthened when after antiviral treatment of two couples, both became pregnant. One couple had twins after in vitro embryo transfer although five previous attempts had resulted in failure (case 9). The other

Acknowledgements

We wish to acknowledge the help and cooperation of: the Department of Obstetrics and Gynaecology, Tokai Univ School of Medicine, especially Professor T. Makino and Dr T. Suzuki; Drs Kurata and Arao of the National Institute of Infectious Diseases for supplying the viruses; Professor Ken-ichi Yamamura for the synthesis of the primers; Dr Bunzo Sato for support. The fathers are kindly thanked for donating sperm. We thank Dr Kim O’Hoy LeFèvre for her constructive comments on the manuscript. This

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