Paper on Infertility

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Presence of HSV-1 DNA in semen and menstrual blood
Nadia El Borai1, Christophe LeFèvre2, Masato Inoue3, Elena N Naumova4, Kaoru Sato5, Shihoko Suzuki6,Kimiyoshi Tsuji7, Masaichi Yamamura6.

1Department of Biological Chemistry and Biologicals, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158, Japan, 2Gentech, Biot, France, 3Sanno Hospital, Akasaka, Tokyo, 4Department of Family Medicine and Community Health,Tufts University School of Medicine, Boston,  Massachusetts 0211,USA, 5Cell Transplantation Center, 6Department of Molecular Life Sciences 1, 7Dept. Transplantation Immunology,Tokai University School of Medicine, Isehara, Kanagawa, Japan.

Dept.Mol.Life Science 1, Tokai University School of Medicine, Bohseidai, Isehara 259-11, Kanagawa, Japan
 * correspondence to: N.El Borai

This work was funded by the Osaka Foundation for the Promotion of Clinical Immunology

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 7 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.

Key Words: HSV-1, infertility, PCR, HLA-Cw3, semen, menstrual blood

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 & 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 & Ikeda 1996), the genomic sequences of HSV-1 and EHV-1 were found to be very similar (El Borai et al. 1997).Thus suggesting a possible functional similarity between HSV-1 in humans and EHV-1 in horses. At present, the most sensitive direct method to demonstrate 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.

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

Viruses Viruses were obtained from the National Institute of Infectious diseases in Tokyo from Drs. Kurata & Arao Department of Pathology. The HSV-1 virus, was theTomioka strain. All viruses were cultured on Vero cells.

Control Negative control: The control was a semen sample which had tested negative and belonged to a father of three. This internal control was used throughout the experiments as a negative control. Positive control: The positive control was the negative semen control sample to which a known amount of virus was added before DNA extraction.

DNA extraction The sodium iodide method for DNA extraction (WAKO kit), was slightly modified to allow the precipitation of viral DNA by  adding glycogen  to the lysis solution. Furthermore, incubation time with the protease was increased to 2 hours at 37oC to allow better breakdown of cellular material.

Primers All primers were selected from the DNA polymerase region of the HSV-1 genome. This region was chosen as it is well preserved in viral DNA. The first set of primers were 544 base pairs apart. Two sets of nested primers were selected  within this region. The sequence of the primers was already published (El Borai et al. 1997).

PCR method DNA(2μl) and 25pmoles of primers(2μl) were pipetted into 0.5 ml Robins tubes, heated at 95oC for 15min and placed on ice immediately to keep the DNA  single stranded. The reaction mixture consisting of : water, buffer, dNTPs 25 μmoles mixture (Pharmacia), MgCl2 4mM, 3% DMSO(Sigma), 0.25units of Taq polymerase, was added to the DNA and primers to a final volume of 50μl. The Taq polymerase, buffer and MgCl2 were from Perkin Elmer. The PCR was run in a Techne PHC-3 Thermal cycler for 43 cycles as follows: denaturation at 95oC for 1min; annealing at 65oC for 1min and elongation at 72oC for 1min with a final elongation at 72oC for 10min.

Nested PCR Using the product from the first PCR as the template DNA and the nested primers, a second PCR was performed. The conditions were the same as described for the first PCR.

Detection of PCR product To detect the PCR product, 8µl of product was mixed with bromophenol blue dye in sucrose and applied to an agarose gel containing ethidium bromide and electrophoresed at 100V in a MUPID electrophoresis apparatus. The gel was visualised under UV light and photographed with a polaroid camera.

Semen samples Semen samples were obtained from husbands attending an infertility clinic at the Department of Obstetrics & Gynaecology of Tokai University Hospital. The DNA was extracted from a pellet of the semen samples after the samples were washed and fertility testing was performed. Test for semen samples: volume, concentration, motility, speed, number of round cells, hamster oocyte  zona pellucida sperm test (ZPST) (Inoue et al., 1994). Then DNA was extracted and HSV PCR performed.

Control semen samples Control samples were kindly donated by fathers who have at least two children. Semen samples were frozen and DNA extracted from 0.5ml of the frozen sample.

Menstrual blood samples Menstrual blood was collected by aspiration. The samples contained endometrial tissue, were taken generally after failed in vitro embryo transfer. Samples 21 and 22 were menstrual blood samples collected by the women themselves.(Table1) DNA was extracted as detailed above. Control samples were from volunteer women who collected menstrual blood themselves.

HLA typing: The microcytotoxicity test for tissue typing was performed using the NIH standard method for class I and DNA typing for HLA class II (NIAID 1976-1977).

RESULTS
Viruses

After the first PCR a positive band of 544bp 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 samples were tested, 37 were positive and 117 were negative (Table 1). None of the fertile males were positive. The samples were screened with primers 297- and 297-2, and 24  were additionally tested with primers 154-1 and 154-2. The significance of this test for infertility was calculated by the chi square test, X2=7.59 (p<0.05), and was found to be significant. Six semen samples were tested twice at time intervals between one and 4 months and the same result was obtained. Positive semen samples were again tested after antiviral treatment  4 out of 5 became negative. The one that remained positive was from a patient who had had a kidney transplant, and whose test was positive even with the first PCR indicating that the viral load was high. A positive HSV test and the parameters used for semen analysis such as motility, number ,volume, ZSPT were statistically analysed to find any correlation between any single or a combination of parameters, however none were found.

Menstrual blood samples

Of the 22 samples tested 11 were positive (Table 1,2). All were tested with both primers. The control samples were all negative(4/4). The clinical picture of 22 infertile women was as follows: a) women with various etiologies .b) Mean age 35.6 years (range 30-43). c) Infertility period mean 9.5 years (range 4-18). d) Primary infertility in 20 and secondary infertility in 2. e) IVF failure 18 cases, mean 6.1 times. f) Unsuccessful IVF was in the range of 1 to 18. History of herpes infection was known in one case. Out of 22 husbands 8 semen samples were tested for the presence of herpes. In 3 cases the positive result in the menstrual blood corresponded with the husband positive semen. Also one case of negative menstrual blood corresponded to a negative semen sample. In four cases the semen sample was positive while the menstrual blood negative.

HLA The HLA data of 10 women was compared to the HLA types of their spouses. These women had their menstrual blood tested for HSV DNA. Also compared were the HLA of three men who had HSV positive semen samples with the HLA of their wives, two wives had tested negative for HSV-1 and one had not been tested. All the women whose menstrual blood had tested positive had HLA Cw3 (4/4). Furthermore, 3 out of the 4 husbands had the same HLA Cw3. In the negative samples none of the couples had HLA Cw3 but three of the husbands had HLA Cw3 one of them had tested positive for HSV (Table3). As the number of samples was very small, data from parents of family studies were used as a comparison. It was found that amongst 22 couples, three couples (14%) had HLA Cw3 in both partners. In infertile couples not tested for HSV but whose  tissue type was known 7 out of 22 couples (32%) were found to have HLA Cw3  (Table 4). The couples, where the wife's menstrual blood tested HSV positive were compared with other couples. A significant increase in the incidence of Cw3 was demonstrated when couples with the HSV positive wife were compared to infertile couples (Chi square=6.41, p<0.05) the infertile couples. A  significant difference was also found when the infertile couples with HSV-1 positive females were compared to control parents (Chi square = 19 p<0.05). No significant difference was found between the infertile and the fertile couples. The results suggest an association of an HSV positive test with incidence of Cw3 in both partners.

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 previously  five attempts had resulted in failure (case 9). The other one had to have the pregnancy terminated because it was ectopic (case 21). Antiviral treatment was first given to twelve infertile couples by Kundsin et al., 1987 for six cycles and in 6 out of 12 couples successful pregnancies occurred. The treatment here was only for 10 -14 days. Before treatment could have begun one couple, whose wife had tested positive, became spontaneously pregnant, but this pregnancy ended in miscarriage.

An HSV-DNA positive test could be correlated to infertility, as only in the infertile group could a positive semen sample be detected. Specific treatment with an antiviral drug can then be given, in contrast to other fertility tests available now (Gudzick,1995).

In the four HSV positive couples, a common HLA class I antigen, HLA Cw3 was found. HLA data for the other couples tested for HSV were not available.

It is surprising that the HSV positive women would have partners with the same HLA type. This predominance was neither found in the negative women nor in the women whose husbands had HSV positive sperm. It seems that HLA B61 and Cw3 may play some role in the immunity to HSV. HLA class I molecules play a role in the presentation of viral antigens to the subclass of CD8 T cells, and also in the protection from natural killer cell-mediated lysis (Zappacosta et al. 1997).

The unique property of herpes viruses to recur in the presence of antibodies can be compared to the situation in  HIV. In both cases humoral immunity does not provide complete protection  as people can develop disease symptoms even with antibodies present in the blood. There is no way of predicting the time of reactivation or the reason, although stress, lowered immunity and UV light have been implicated. Two factors may be considered in the case of infertility and HSV, one would be the immunological constituency of the individual, here it may be the presence of Cw3. The other may be hormonal stimulation due to pregnancy in which dramatic hormonal changes occur, these hormonal changes may be a trigger in reactivating a latent virus. It would also explain individual differences to treatment and outcome as is now being shown for AIDS (O'Brien & Dean 1997).

Since Goodpasteur described that herpes simplex lies dormant in ganglia, this site has been assumed to be the only one. We have been able to detect the virus although asymptomatic in most cases in the semen and in menstrual blood after in vitro embryo transfer. Early vaccines which were not screened as rigorously as now, may have harbored adventitious viruses and could be the source of HSV. In the past decade, in Japan, there has been a general trend in the reduction of HSV-1 antibody titers from 80% to about 50-60% (Kawana 1995). Japan has one of the lowest prevalence of HSV antibodies in prenatal screening, less than 50% compared to other countries where the values are over 80-90% (Whitley 1996,1997). Therefore it could be assumed that infertility caused by HSV may be higher in other populations.

Infertility is not only a medical but also a social problem, in extreme cases e.g. in Tanzania women who were unable to bear children during their lifetime will not be allowed burial in the village cemetry (Mgalla & Boerma 1997). Antiviral therapy could be prescribed in desperate cases where advanced technologies are not available.

In conclusion, clinically indistinguishable women, infertile and asymptomatic for HSV could be divided into an HSV positive and an HSV negative group. Treatment with an antiviral drug was successful. The HSV test could also identify infertility in men and could be treated. Finally an HSV positive test in the woman was correlated with the serological determination of Cw3 in both couples, suggesting an association of Cw3 with HSV.

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.