FREQUENCY OF CYTOMEGALOVIRUS, RUBELLA, AND HERPES SIMPLEX VIRUS IN EMBRYONIC TISSUES OF WOMEN WITH MISSED ABORTION

Background and objectives: Spontaneous abortion (SA), also known as miscarriage, is considered as one of the most frequent problems a woman may face during early pregnancy, is not only related to morbidity or mortality, but also has an obvious social and psychological impact on women. An abortion is the removal or expulsion of an embryo or fetus from the uterus, resulting in \or caused by its death, the loss of a pregnancy occurs within the first 2024 weeks of gestation, after which, fetal death is known as a stillbirth. This study was first aimed to assess the frequency of, Cytomegalovirus, Rubella and Herpes simplex virus in embryonic tissues of women with abortion. Next to find out the sociodemographic characteristics of the studied populations. Methods: This cross-sectional prospective study was carried out from February 2019 through January 2020.This study prospectively deal with tissues from conceptus after abortion which collected from maternity teaching hospital in Erbil city, Iraq for the diagnosis of frequency of CMV, rubella and herpes simplex virus. RT-PCR were used in tissue analysis. SPSS version 25 was used for data entry and analysis. Results: Out of 72 cases with spontaneous abortion 8 (11.1%) were due to human Cytomegalovirus,2 (2.8%) were due to Rubella virus and 1case (1.4%) was due to Herpes simplex virus, the age range of participants was 17-45 years with mean ± Sd of (31.29 ± 6.11) years. The highest percentages of participants were in their second and third decades of life (40.5% and 37.5% respectively) and about 86.1% of them were un-employed (housewives) and majority about 57% of them attending primary school, and more than half percentage (63.8%) of them have intermediate economic state. Histopathological analysis shows that out of 72 cases only 9 samples (16.6%) show histopathological abnormalities. A statistically significant association was found between Rubella infection and histopathological abnormality. Conclusions: Cytomegalovirus infection was more prevalent among the study samples, followed by Rubella infection then Herpes simplex virus infection by real-time polymerase chain reaction, on other hand 16.6% of samples shown nonspecific histopathological abnormality.


INTRODUCTION:
Spontaneous abortion (SA), also known as miscarriage, is considered as one of the most frequent problems a woman may face during early pregnancy, is not only related to morbidity or mortality, but also has an obvious social and psychological impact on women. It was estimated that 6-15% of all clinically detected pregnancies end with spontaneous abortion, there are many more pregnancies that end with abortion prior to being clinically recognized. It is estimated that about 30% of all conceptions result in a live birth (Rouse et al., 2017).
Early miscarriage is defined as pregnancy loss during the first trimester of pregnancy (less than 12 weeks of gestation) and occurs in up to one in five pregnancies. Late miscarriage occurs during the second trimester (12-24 weeks of gestation) and is less common, occurring in 1-2% of pregnancies (Giakoumelou et al., 2016). As many as 50% of all pregnancies end in miscarriage, mostly before the woman realized that she is pregnant and about 15-25% of recognized pregnancies will end in an abortion. Early pregnancy loss, which occurs in the first trimester, is the most

Design of the study:
A cross sectional prospective study in which tissues from conceptus after abortion are collected from maternity teaching hospital in Erbil city, Iraq.

Setting and time:
This study carried out in emergency

3.Embedding:
In which the specimens are placed in an embedding center and placed in wax-filled molds, the specimen "block" is now allowed to solidify on a cold surface and the cassette now filled with wax and forming the block.

4.Sectioning:
Sections are cut on a precision instrument called a "microtome" using extremely fine steel blades. Paraffin

Data Analysis and Interpretation:
The sample is considered positive

Fig.2: proportions of HCMV, Rubella and HSV infection among study population by PCR.
This study found that out of 72 sample of abortion only 9 (16.6%) of them showed histopathological abnormality in form of villitis and only 2 (22.2%) of these villitis were PCR positive for HCMV, which was statistically nonsignificant as showed in Table 3. This study found that out of 72 sample of abortion only 9 (16.6%) of them showed histopathological abnormality and only 1 (11.1%) of these histopathological abnormalities were PCR positive for Rubella which was statistically significant as showed in Table 4.

DISCUSSION:
Abortion is major women's health problems and is the most common type of pregnancy loss, according to the American College of Obstetricians and Gynecologists (ACOG). Studies showed that about 10-20% of all clinically recognized pregnancies will end in miscarriage, by studying the underline causes and appropriate management will help to reduce miscarriage and provide a healthier babies and healthier mothers (Dugas and Slane, 2020).
Abortion is known as a main cause of maternal mortality, life threatening complications such as hemorrhage, fever, and infection and psychological disorders such as sadness, guilt, and even suicide (Pourreza and Batebi, 2011), Pregnant women are at greatest risk of viral infection, due to that they have changed in immune response (Silasi et al., 2015). This is first study done in Erbil in order to find viral causes of miscarriage using novel technique in form of polymerase chain reaction with high specificity and sensitivity, the idea was to investigate the viral infection rate among pregnant women with spontaneous abortion. The current study included 72 women with spontaneous miscarriages, among them eight cases (11.1%) were found to have HCMV infection. This finding is nearly in consistent with other studies done in our region; like in Bagdad city a study among 108 women with abortion, 15.7% had CMV specific IgM antibodies (S. Khalf et al., 2012).
And other study done in three provinces Baghdad, Babylon and Al-Najaf at teaching hospitals, the study involved 90 case of abortion between first trimester and second trimester the results of real time-PCR (RT-PCR) test for aborted women showed that only 6 (6.7%) cases positive for Cytomegalovirus infection (Al-Roubaey, 2018) , while in another study done in Turkey found that CMV was positive in two patients out of 134 (1.4 %) (Dinc et al., 2010), and according to another study done in Mansoura University, Egypt, it's found that about 12% of patient with abortion (el-Sayed Zaki and Goda, 2007).
While in another study done in Tehran found that 5.2% of women with abortion including repeated abortion are due to HCMV infections (Sotoodeh jahromi et al., 2010), the difference might be related to regional variations and sample size differences.
Data from this study suggest that two (2.8%) out of 72 cases were due to rubella and none of them were due to herpes simplex virus, and these findings are nearly in consistence with other studies done in our region ,according to a study done in three provinces Baghdad, Babylon and Al-Najaf at teaching hospitals, the study involved 90 aborted women between first trimester and second trimester the results of real time-PCR (RT-PCR) test for aborted women showed that only 12 (13.3%) cases were positive for Rubella virus (Al-Roubaey, 2018).
In a study was carried out on 57 pregnant women with abortion and they were attending Al-Zahra Teaching Hospital, in Al-Najaf province, Iraq, the Real Time PCR results showed only 6 out of 57 cases (10.5%) were positive for Rubella virus (Mahmood et al., 2015). On other hand the results of this study are similar to a study done in Mwanza City, Tanzania, where a total of 268 women with spontaneous abortion were involved in the study for detection of rubella specific IgM antibodies using indirect Enzyme Linked Immunosorbent Assay (ELISA) and the prevalence of acute rubella virus infection was found to be 9/268 (3.7%) cases (Lulandala et al., 2017).
In this study approximately 1.4% of abortion samples are due to Herpes Simplex Virus which identified by PCR-technique, which is similar to study done in Al-Batool Teaching Hospital for Maternity and Children in Baqubah city, in which there were 2% incidence of HSV type1 and 2 in pregnant women (Hussein et al., 2017). On other hand these results are comparable with a study done in Azadi Teaching Hospital, Kirkuk, Iraq, in which incidence of HHSV by ELISA test was 2.6% (Obaid and Juma, 2017).
While in a study done by Sifakis et al, in Greece in which 102 women with spontaneous abortion was analyzed for the presence of HSV DNA applying the PCR technique, in which only 2 out of 102 cases (1.9%) were positive for HSV (Salman, 2017), the difference might be contributed to different technique of detection and sample size difference.
Data from this study suggest that abortion cases are more between age 17-30 years and 31-40 years about (40.5%) and (37.5%) respectively, while; slight decline observed in age group 41-50 years, although high maternal age is a significant risk factor for spontaneous abortion (Andersen et al., 2000), this difference might be due to decrease pregnancy rate after 40 years of age.
In the current study the percentage of abortion among those with low and intermediate economic level were (93.0%) and most of them about (72.3%) were low educational level (non-educated and primary school level), these findings are similar to that reported in a study done in China, revealed that women with high income had a decreased risk of spontaneous abortion when compared with that of women with low income, also a comparison between women in low educational level, women in higher educational level had a lower prevalence of spontaneous abortion (Zheng et al., 2017).
Regarding gestational age at time of abortion, it found that (88.9%) were at first trimester, and majority of them having second and third abortion about (78.2%) with (55.6%) of them were multiparous which is consistent with other studies done in USA and Europe (Cunningham et al., 2013).
Regarding abnormal histopathological finding we found that (22.2%) of them were due to HCMV by Polymerase Chain Reaction which is nonsignificant, on other hand there are statistically significant association between Polymerase Chain Reaction outcome and histopathological result for Rubella, in which (1.4%) were positive. This is agreed with study done by Chen and Roberts (Chen and Roberts, 2018).
In the current study, there is no significant correlation between the HCMV, Rubella and Herpes Simplex Virus infection in aborted pregnant women with age, education level, occupation, economic level, residence, gestational age, history of abortions and parity, so these cannot be considered as risk factors for infection. The differences between the results of the formerly mentioned studies and the current study could be related to many factors, like the methodology in the current study used molecular technique while other study may use serological methods, sample size, difference of studied population from one area to another, the duration of infection, individual's immune status, demographic and geographical variations, season and etc.

LIMITATION OF THE STUDY:
many cases of spontaneous abortion are not attending the public hospital and if they do so, they treated medically rather than surgically and most of them are expelling the product of conception at home.

CONCLUSIONS:
The present study shows that about 11.1% of study samples are due to HCMV infection, 2.8% are due to Rubella infection and only 1.4% are due to HHSV infection by RT-PCR study, and there were around 16.6% of the samples shows abnormalities in histopathological analysis with 83.4% shows no abnormalities.

RECOMMENDATIONS:
Establishment of a center with high facility for performing PCR study for research purpose. Suggestion for pregnant woman or those planning to become pregnant to be tested for TORCH infections, and vaccinated against Rubella, Cytomegalovirus and Herpes simplex virus to grantee healthy pregnancy outcome.