Cytomegalovirus Immunity in Pregnancy in South of Iran

Problem statement: Fetal loss and abortion are responsible for signifi cant emotional distress for couples desiring children. There are m any documents which support the role of some certain asymptomatic infections such as Cytomegalov irus (CMV) in spontaneous abortion. This study was aimed to evaluate the prevalence of seropositiv ity for CMV IgG and IgM in women with abortion and compare it with normal women with no previous h istory of abortion. Approach: This case-control study was carried out in Shariatee hospital of Horm ozgan University of medical sciences, during 20032004. A number of 250 women with definite diagnosis of previous abortion and 200 matched women with normal full term delivery and negative history of miscarriage as controls were studied as case an d control groups. Serum samples were obtained from pa rticipants and a demographic questionnaire was also filled for each of them. .All serum samples we re then tested using ELISA method for detection of anti-CMV IgM and IgG Antibodies. Data was analyzed, using SPSS software (chi square and t-test). Results: The average age was 25.6±7.6 years in case group a nd 25.3±6.5 years in control group. The number and percentage for CMV-IgG was 235(94%) and 150(75%) in case and control groups, respectively (p-value = 0.0001). In case group 13(5 .2%) women were positive for CMV-IgM, while no positive sample was detected in control group (p-va lue = 0.0001). Conclusion: This study showed higher prevalence of seropositivity for CMV in wome n with spontaneous abortion comparing to women with normal full term delivery and suggest th at cytomegalovirus plays a significant role in abortion.

Up to 15% of intrauterine CMV infections result in symptomatic congenital disease at birth and 10 to 15% of those born with asymptomatic congenital CMV will develop significant clinical sequelae in infancy (Boppana et al., 1992;Dahle et al., 2000;Fowler and Boppana, 2006). Some researches have conducted to determine the role of (CMV) infection in pregnancy and some of them showed significant relation between this infection and spontaneous abortion (Enders et al., 2001;Cook et al., 1993;Luerti et al., 1983). There are also evidences which suggest that CMV will lead to complicated pregnancies (Hammouda et al., 1993).
The usefulness of prenatal testing for CMV has been questioned due to the absence of clearly effective intervention (Hagay et al., 1996) however, there is a need for a low-risk, noninvasive diagnostic test. Laboratory methods are required to diagnose acute CMV infections since most present nonspecific symptoms.
The presence of CMV-specific Immunoglobulin M (IgM) may not be indicative of primary infection, since it is also produced during reactivation and re infection (Nielsen et al., 1988). IgG antigen avidity has been used to clarify primary or non primary infections by measuring the binding affinity of IgG antibodies. IgG of low avidity are produced at the onset of infections and subsequent maturation of the antibody increases its avidity over time. The use of IgG testing has been shown to be useful for distinguishing primary and non-primary CMV infections (Bodeus et al., 1998;Grangeot-Keros et al., 1997;Lazzarotto et al., 1997).
The aim of this study was to determine the seroprevalence of CMV in our local pregnant population to see the effect of age, previous abortion and parity on its seroprevalence.

MATERIALS AND METHODS
We conducted a case-control study in the delivery population of University Hospital (Hormozgan province, Iran) between Feb. -Jun. 2004, to investigate whether prevalence of AntiCytomegalovirus antibodies (IgM and IgG) in two groups of women with spontaneous abortion and without history of abortion to determine a relation between the role of seropositivity for this virus and abortion as well as factors which might have an influence in the pathogenesis of these infections.
Cases were 250 women who were identified with spontaneous abortion by gynecologist during the study period and control group consisted of 200 asymptomatic women with no history of abortion and full term delivery who were referred to Hormozgan University Hospital.
All subjects gave written consent for obtaining their blood samples according to research purposes. Whole blood samples were collected from all women in both groups. Serum separation was done by centrifuging of whole blood samples at 2000 × g for 20 min.
A structured interview using a standard maternal questionnaire was administered by trained interviewers with the women at their first visit. Questions were asked about the following: age, parity, gynecologic and medical history of abortion, residence and socioeconomic status.
The ELISA technique was performed using kits intended for estimating concentration of specific CMV-IgM and CMV-IgG markers. The kits were purchased from Sigma Diagnostics (USA), the techniques were performed according to the manufacturer's instructions.
Data analysis: For assessment of risk factors for CMV infection (exposure), characteristics of case patients and control subjects were examined using a two-sample Student t test. Cross-tabulation and chi-square or Fisher exacts tests were used to examine the relationship between variables using a 95% confidence interval as a measure of association.
Also, univariate odds ratios 0.19 and 95% Confidence Intervals (CIs) using the exact method were calculated.
All data analyses were performed using SAS 8 statistical software (SAS Institute, Inc, Cary, NC).

RESULTS
The mean age of participants was 25.6±7.6 and 25.3±6.5 years in the pregnant women with abortion and with full term delivery, respectively. The mean gestational age was 8 weeks and the mean parity was 2.4 children in abortion group. The mean gestational age was 38 weeks and the mean parity was 2.1 children in full term group. There was no significant difference between age and parity in two groups (p = 0.60) ( Table 1).
According to prevalence of seropositivity for CMV antibodies, about 88.8% of the subjects were divided in abortion and 75% with full term delivery as it is shown in (figure1) and the results of serologic assay we can divide participants in to 4 groups. Group 1: women with negative serology for IgG and IgM antibodies, we had only (6%) in abortion and (25%) in full term delivery group. Group 2 women with IgM seropositivity without positive serology for IgG antibody, abortion group had only 5.2%, and IgM was not detected in the full term delivery group. Group 3: women with positive serology for IgG and negative for IgM antibodies, we had detected in (88.8%) of abortion groups and in (75%) of full term delivery group. Group 4; women with positive serology for IgG and IgM antibodies, while at the same time that no one was in abortion group and full term delivery group (Table 2).  There was no statistically significant relationship between CMV IgM or IgG seropositivity and parity neither in patients with previous abortion nor with age of the patients with exception in women who had a recent abortion. Women in seronegative IgG group were younger in comparison with seropositive IgG (p = 0.04).

DISCUSSION
CMV is the most common congenital infection and its incidence has been estimated to be between 0.2-2.2% of all live births in different parts of the world (Ross et al., 2006). This study found the prevalence of seropositivity in 75% of asymptomatic women with no history of abortion.
According to the results of serologic assay we can divide participants in to 4 groups.
Group 1: Women with negative serology for IgG and IgM antibodies, they were not contaminated with CMV and were susceptible to infection (primary infection). In this situation there is a risk for transmission of the virus to the fetus during the pregnancy (Enders et al., 2001). It is indicated that prevalence of CMV antibodies was high in pregnant women in south of Iran and a small percent (6% in abortion group and 25% in full term delivery group) of these women were sensitive to CMV infection till reproductive ages that in other studies it was related to some extent to socioeconomic status and geographic location.
Group 2: Women with IgM seropositivity without positive serology for IgG antibody. These women were considered acutely contaminated with CMV. They were also asymptomatic and indicated the substantial prevalence of infection in the local population (Wreghitt et al., 2003). Only 5.2% were IgM positive, and the full term delivery group was not positive for IgM. Abortion group and full term delivery groups were significantly different in IgM seropositivity (p = 0.00001), also primary infection was suspected in abortion group but microbiologic and histologic studies were needed to confirm the infection in fetuses (Collient et al., 2004).
Group 3: Women with positive serology for IgG and negative for IgM antibodies, these women were considered immune and their primary infection with CMV was assumed to have been taken place before the current pregnancy. CMV IgG antibody was detected in (88.8%) of abortion groups and (75%) of full term delivery group without IgM seropositivity. This revealed that most of patients in abortion group and full term delivery group were immune against primary CMV infection.
Group 4: Women with positive serology for IgG and IgM antibodies, at the same time. These women were considered to be possibly infected with CMV during the current pregnancy or a chronic infection which can be confirmed by IgG avidity test because antibody binds to the antigen with less avidity during acute infection than chronic infection (Wreghitt et al., 2003), that no one was in abortion group and full term delivery group.
According to these results we understood prevalence of CMV seropositivity in south of Iran was high but we couldn't conclude there was a relationship between CMV infection and spontaneous abortion.
Most of the patients in abortion group and full term delivery group were immune against CMV and only 6% in abortion group and 25% in full term delivery group were sensitive to CMV infection and CMV maternal seropositivity being associated with less severe fetal involvement and maternal immunity plays a protective role in this setting (Fowler et al., 2003). A cohort study has shown maternal immunity prior to conception is highly protective against congenital CMV infection and usual acquired immunity in course of CMV results in 69% reduction of the risk of congenital CMV infection in future pregnancies (Fowler et al., 2003).
Also primary CMV infection in this study only 5.2% in abortion group but some surveys demonstrate that IgM can be found frequently in the serum of normal pregnant women without any influence on the pregnancy outcome (Lazzarotto et al., 2004), and there was cross reactivity of about 3.3% for IgM positivity with other viral infectious (EBV, measles, herpes simplex varicella-zoster influenza vaccine) (Maine et al., 2000). Although primary infection in any stage of pregnany presents a risk for intrauterine infection from 30-50% but congenital infection in seropostive mothers is only from 0.2 to 1.5 percent, and that it needs more microbiological and histological confirmation (Stagno, 1990). The results of Tanaka et al., (2006) suggested that latent CMV infection predisposes to adverse pregnancy outcomes (Tanaka et al., 2006).
In comparison with other studies, we have these results: In a study which was performed by Munro et al. (2005), rate of CMV IgG seroprevalence in blood donors was shown to increase with age from (34.9-72.4%) during 30 years but our study showed no relation between age and infection with CMV (Munro et al., 2005;Mathur et al., 1981).
In our study the prevalence of seropositivity for CMV was higher than Western Europe, America and Australia (Munro et al., 2005) but our findings were similar to a study in China with prevalence IgG (95.67%) in pregnant women (Guo 1992). In India serological surveys have shown that the prevalence of CMV antibodies in adult population is about (80-90%) (Mukundan et al., 1977).

CONCLUSION
We can diagnose high risk pregnancy even with serological tests in areas with insufficient equipment, and in South of Iran most pregnant women were immune against primary CMV infection but some studies recommended that childbearing age women should be screened for CMV antibodies but we suggest that confirmation of congenital CMV infection is difficult in this area and also there is high immunity for CMV in pregnant women and congenital infection is rare in seropositive mothers (Collient et al., 2004), therefore we recommend pregnant women should be attentive of disease prevention guidelines on personal hygiene during pregnancy, especially hand washing after handling diapers or oral secretions. But we recommend high risk pregnant women for example: mothers that working in day care center or health care worker should be screened for CMV serological tests during pregnancy.

ACKNOWLEDGEMENT
This study was completely financed by Hormozgan University of Medical Sciences. The authors are grateful to the patients and the control individuals who accepted to enter this study.