European Journal of Cardiovascular Medicine

Spontaneous abortion(miscarriage) is defined as spontaneous loss of product of conception before 20th week of gestation. Approximately 23 million spontaneous abortions are registered yearly around the world.1 It has been found that infections account for 15% of early miscarriage and 66% for late miscarriages respectively.2 Though there may be innumerable causes of spontaneous abortion, viral infections are considered as one of the important causes associated with adverse pregnancy outcomes. Viral infections disrupt trophoblast functions and influence implantation and mating abnormalities.3 Viruses like cytomegalovirus (CMV), herpes simplex virus -2(HSV-2), rubella virus can affect both fetus and mother at any week of gestation. Primary infections with these viruses in pregnant women elevate risk of miscarriage and other adverse pregnancy otcomes.4 Specific immunoglobulin IgM against the viruses can be determined to identify primary maternal infections.

The ubiquitous nature of CMV makes it the most common viral infection affecting pregnant women. Primary infection in mother can affect the fetus in 40-45% of cases. Mothers can get affected by sexual transmission or by infected body fluids like saliva. CMV can result in hearing loss, vision loss, and mental health issues in infants.5

Herpes simplex viruses can cause wide variety of illness in humans. Herpes simplex virus-1 (HSV-1) is transmitted primarily by contact with infected saliva whereas Herpes simplex virus -2 (HSV-2) is the most prevalent sexually transmitted viral infection in the world. About 30% to 60% of antenatal women suffer from asymptomatic herpes making the newborn susceptible to severe herpes disease and neonatal infection.6

Rubella infection in mothers can exert teratogenic effect in the embryo. Fetus may develop neurological disorder, cardiovascular disorders, visual and hearing disorders. The risk to the fetus is maximum if the mother gets infected in the first 12 weeks of gestation and gradually decreases thereafter.7Clinical diagnosis during pregnancy is difficult as 50% of the mothers remain asymptomatic.8

Physiological changes during pregnancy predispose pregnant women to infectious diseases. SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) named COVID-19 has been in circulation since 2019. Studies report adverse pregnancy outcome with higher risk of intensive care admission in pregnant women.9 As Angiotensin converting enzyme 2 (ACE2) is the binding site for COVID-19 entry and ACE2 receptor is present in female reproductive organs it may cause malfunction of the reproductive organs.10Pregnant women rarely get screened for CMV, HSV-2, rubella and SARS-CoV-2 viral infections until and unless any complication arises.

Considering the importance of these viral infections in the exaggeration of abortive conditions our study aims to find out the prevalence of these viruses in women presenting with recent history of abortion in our region and determine the sociodemographic factors related to it.

A prospective case control study was carried out in the department of Microbiology in our tertiary care centre based on simple random sampling over a period of 6 months from January 2022 to June 2022. A total of 150 antenatal mothers attending the gynaecology department of the tertiary care centre were included. Study subjects were divided into two groups: study group and control group. Study subjects consisted of antenatal mother presenting with recent miscarriages and control group consisted of healthy post-partum women(38-40weeks) with normal pregnancy. Inclusion criteria: Antenatal mother with recent history of miscarriages who agreed to participate were included in the study. Exclusion criteria: Study subjects with medical conditions like diabetes milletus, hypothyroidism, chronic renal disease, uterine anomaly, Rh incompatibility were excluded. Study and control group equally comprised of 75 subjects each. 8-10ml of blood samples were collected from 150 antenatal women under aseptic condition. Serum was separated and stored at -20ᴼC till testing was done. IgM antibodies against CMV, HSV-2 and Rubella were obtained using commercially available ELISA kits (Xema and Diesse, Italy) Results were interpreted as per manufacturer’s instructions. Results were read at 450nm in ELISA reader. A positive IgM was an indication of recent infection. COVID 19 RTPCR was done for all the samples to rule out any association of miscarriage to SARS-CoV2 virus. Informed consent was taken from all the subjects and a detailed proforma was filled up which consisted detailed clinical history and other relevant details. Ethical permission was obtained from Institutional Ethics Committee (IEC/2021/03a). Mean and standard deviation of the quantitative variables were calculated. Chi-square test was employed to compare between the qualitative variables. p-values were obtained using SPSS software and value less than 0.05 at 95% confidence interval (CI) was considered significant. Logistic regression analysis was done to show association between dependant and independent variables for CMV and HSV-2 seropositive subjects respectively.

A total of 150 antenatal women were recruited in the study of which 75 antenatal women presenting with spontaneous history of abortion were included in study group and another 75 healthy postnatal women were included in control group. The mean of the age of the participants in study group was 26.12±6.02 years while in the control group it was 26.46± 5.54 years. 62.66% (47) women were from rural areas and 37.33% (28) were from urban areas. Out of 75 study subjects, 10(13.33%) had repeated history of abortion while 86.66% (65) had no previous history. Out of 150 women tested, CMV IgM seroprevalence was found among 8% (6) subjects in study group and it was 0% in the control group. IgM CMV levels were significantly higher in study group as compared to control group(p=0.01). HSV-2 IgM positivity was 1.33% (1) in the study group with no seropositivity (0%) in the control group. Rubella IgM was not detected in either group. COVID-19 RTPCR was positive in 1.33% (1) women in control group with absence in study group (Table 1).

Both CMV IgM and HSV-2 IgM seroprevalence was found to be highest in 21-25 years of age. (Table 2). In this study IgM positivity for both the viruses were not found above 25 years of age indicating absence of current infection. Odds ratio for CMV (OD 24.14, P=0.03) and HSV-2 (OD 2.92, P=0.51) shows higher association of primary infection in women less than 25 years (Table 3).

Women living in rural areas showed higher seroprevalence of CMV IgM (12.76%) as compared to study subjects living in urban areas (0%) and it was significant(p=0.04). However, IgM HSV-2 seropositivity was higher in urban study subjects (3.57%) as compared to rural subjects (0%) and the difference was also not found to be significant (p=0.) Also, IgM seropositivity to CMV was the most for study subjects in 2^nd trimester of gestation (6.66%) as compared to women in 1^st trimester (1.33%). Crowding also played a significant role in IgM seroprevalence of CMV and HSV-2 (Table 4).

Both CMV IgM and HSV-2 IgM were higher in housewives and in women with primary level education. On univariate analysis it was seen that increased gestational age (OR:9.38,95%CI:1.03-85.19, p=0.04) was significantly associated with IgM CMV positivity. For HSV-2, univariate analysis showed no significant association with variables though odds ratio was more than 1 for type of residence, occupation and gestation age (Table 5) No study subjects were positive for rubella IgM and COVID-19 by RTPCR. The control positive subject for COVID-19 PCR had uneventful pregnancy. The woman was 27 years of age and had mild symptoms, baby born was healthy and can be considered an incidental finding.

Table 1: Seropositivity to CMV, HSV-2, Rubella and molecular detection of COVID-19 in study and control subjects

TABLE 2: Age wise positivity of the viruses in study group

Table 3: Odds ratio of CMV and HSV-2 IgM in women less than 25 years of age

Table 4: IgM positivity of CMV and HSV 2 in relation to sociodemographic variables in study subjects

Table 5: Logistic regression analysis of factors associated with positivity to CMV and HSV-2 in study subjects

A significant number of miscarriages can be attributed to infections during pregnancy. Infections with viral, bacterial parasitic agents may act as significant contributor in abortion. Considering the insufficient information of viral infections in pregnancy associated with adverse outcomes in this part of the region our study was undertaken to explore the serological prevalence of IgM antibodies against CMV, HSV-2, rubella and molecular prevalence of SARS-CoV2 in this region of West Bengal. In this study, out of 75 study subjects IgM CMV antibody was detected in 8% (6) and HSV-2 IgM antibody was detected in 1.33% (1). However, rubella IgM was absent in the study subjects. In the control group, IgM antibodies were not detected (0%) for any of the viruses. SARS-CoV2 detected by RT PCR was absent in study group but it was found in 1.33% of control group.

CMV infection during pregnancy is the most complex one as it can be transmitted to the foetus in spite of maternal immunity. In our study IgM CMV prevalence in study subjects was 8% which is less than the findings reported by Singh et al from Mumbai (12%)11 and Denoj Sebestian et al from Kerala (28.2%).12 Global prevalence of CMV in women with bad obstetric history varies from 1.4% to 60.2%.13 Abbas et al reported IgM seropositivity of 7.33% among aborted women which is in line with our study.14. Our study subjects had significantly more (p<0.05) IgM antibodies for CMV virus than the control group indicating recent exposure to CMV which might be a risk factor towards abortion and congenital infection. Positive IgM is a signal towards recent infection and indicates acute infection. Difference in rate of prevalence in different studies might be due to epidemiology of CMV in different groups of people and due to variation in sample size of the study. CMV seroprevalence significantly varies with age. In this study, odds of recent CMV infection (0D=24.14, P=0.03) was more in women <25 years of age as compared to women more than 25 years. This may be due to accumulation of CD 28 effector cytotoxic T lymphocytes which protects the elderly women against CMV infection.15 However, there is considerable debate regarding this as some previous studies have reported elderly women to have higher rate of CMV infection while some others have reported complete absence or more prevalence in younger age group.15,16

In our study current CMV infection was found to be prevalent only in rural women (8%) which is consistent with previous studies.16,17 This may be due to favourable environmental factors. Our study results indicate residence (x2=3.88, p=0.04), crowding (x2=0.02, p=0.02) and gestation (x2=4.40. p=0.03) to be significant risk factors as compared to occupation and education associated with recent CMV infection. Sayar et al18 also reported that increased gestational age was associated with higher IgM CMV positivity. Our study reported highest number of IgM CMV positive cases in women with primary education (5.33%) which is consistent with previous finding by Kolo et al.16 Rural residence was significantly related to CMV infection and this may be due to environmental factors, poor health, socioeconomic status.

Primary infection with HSV-2 may cause placental infection and affect the neonate with different morbidities.19The current study reports prevalence of IgM HSV-2 antibody to be 1.33%(1/75) among the study subjects which is quite low and echoes with a study done by Mohsen et al from Iraq20 and Nyawale et al from Tanzania.21Our finding contradicts with studies done  by Shweta et al22 and Haider et al19 from different parts of India where a higher prevalence of 6% and 15.11% was reported respectively among women with history of abortion.22The findings of our study also reveals that seroprevalence of HSV-2 IgM is not significantly related to age, residence, crowding, occupation and gestational age and is consistent with a study by Alanen et al23.This difference in prevalence and characteristics within and across the countries may be due to geographical variation or presence of some ecological factors which needs to be looked upon.

Rubella virus causes teratogenic effects in pregnancy and can cause congenital defects and abortion of the foetus. Our study found that seroprevalence of rubella virus IgM antibody was absent in both study and control subjects thus indicating absence of recent infection. A study done in Orissa24 also reported similar absence of Rubella IgM antibodies and is in line with our study. Since rubella vaccination has not been introduced in India in a programmatic manner, regional serological data describing the epidemiology of the disease will help the policy makers in designing an intervention program based on its prevalence.

It was hypothesized that COVID-19 affects the granulosa cells and ovarian tissue and thus increases chances of abortion hampering the ovarian function.25Since the study was done in a period when omicron variant of COVID-19 was in circulation, our study intended to find out the impact of COVID-19 in causing abortion. This study reports absence of COVID-19 in study subjects while a positive case of COVID-19 was detected among the control group and it was not statistically significant (p=0.31). This indicates that COVID-19 has insignificant role in causing abortion. The COVID-19 positive subject in the control group had an uneventful pregnancy. Our study finding is similar to a study done by Rashidi et al26 who also did not find any association of COVID-19 to abortion but is in contrast with a UK meta-analysis which supports the hypothesis that COVID-19 affects pregnancy outcomes causing abortions.27

Limitation: This study was done in a district of West Bengal and may not be representative of the total burden of infection of the state. We could not perform IgG avidity assay along with IgM to affirm the diagnosis of primary infection due to unavailability of kit for the viruses which would have increased the sensitivity.

Our study highlights the impact and association of viral infections with spontaneous abortion cases. Though it always may not be necessary to screen the unexpected infections, awareness regarding the local prevalence of rare infections will help in preventing many adversities. As most of the viral infections remain asymptomatic in mothers, a local insight regarding the same would help healthcare providers in opting diagnostic facilities for early screening and thus early detection and timely intervention. Acknowledgement: We would like to acknowledge Principal of the institution Prof (Dr) Panchanan Kundu and staffs of VRDL for helping us to carry out the study. Financial support: The researchers did not receive any financial support from anywhere. It is a self-funded study. Conflicts of interest: The authors hereby declare that there are no conflicts of interest involved in the study.

1. The Lancet. The Lancet Miscarriage: Worldwide Reform of Care Is Needed. Lancet.2021;397:1597. DOI: 10.1016/S0140-6736(21)00954-5

2. Kourtis AP, Read JS, Jamieson DJ. Pregnancy and infection. N Engl J Med.2014; 370:2211–8. DOI: 10.1056/NEJMra1213566

3.Chow SS, Craig ME, Jacques CF, Hall B, Catteau J, Munro SC, et al. Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7. J Med Virol. 2006;78(6):747-56. DOI: 10.1002/jmv.20618

4.Baghel, S., and S. A. Inamdar. “TORCH Infection and Its Influence on High‐Risk Pregnancy.” Journal of South Asian Federation of

Obstetrics and Gynaecology.2020; 12(6): 377.

 5. Paschale, M. Agrappi, C. Manco, T. Paganini, A. and Clerici, P. Incidence and Risk of Cytomegalovirus Infection during Pregnancy in an Urban Area of Northern Italy. Infectious Diseases in Obstetrics and Gynecology. 2009:206505 DOI: 10.1155/2009/206505

6. LeGoff, J. Pere, H. and Belec, L. Diagnosis of genital herpes simplex virus infection in the clinical laboratory. Virology J. 2014; 11:83. DOI: 10.1186/1743-422X-11-83

7. Miyakawa M, Yoshino H, Yoshida LM, Vynnycky E, Motomura H, Thole H, et al. Seroprevalence of rubella in the cord blood of pregnant women and congenital rubella incidence in Nha Trang, Vietnam. Vaccine. 2014;32(10):1192–8. DOI: 10.1016/j.vaccine.2013.08.076

8.Usonis V, Anca I, André F, Chlibek R, Čižman M, Ivaskeviciene I, et al. Rubella revisited: Where are we on the road to disease elimination in Central Europe? Vaccine. 2011; 29(49):9141-7. DOI: 10.1016/j.vaccine.2011.09.104

9. Sharma, I.; Kumari, P.; Sharma, A.; Saha, S.C. SARS-CoV-2 and the reproductive system: Known and the unknown!! Middle East Fertil. Soc. J. 2021; 26(1):1. DOI: 10.1186/s43043-020-00046-z

10. Liu, C.; Mu, C.; Zhang, Q.; Yang, X.; Yan, H.; Jiao, H. Effects of Infection with SARS-CoV-2 on the Male and Female Reproductive Systems: A Review. Med. Sci. Monit. 2021; 27: e930168. doi: 10.12659/MSM.930168

11. Singh N, Anant Dattatray Urhekar, Sareena Rao. Clinico-Microbiological study of CMV in pregnant females With Bad Obstetric History (BOH). International Journal of Life Sciences, Biotechnology and Pharma Research.2024;13(3): 567-574 DOI:10.47583/ijpsrr.2024.v84i05.004

12. Denoj Sebastian, K.F.Zuhara and K. Sekaram. Influence of TORCH infections in first trimester is carriage in the Malabar region of Kerala. African Journal of Microbiology Research.2008;2:056-059.

13.Aljumaili ZKM, Alsamarai AM, Najem WS. Cytomegalovirus seroprevalence in women with bad obstetric history in Kirkuk, Iraq. Journal of Infection and Public health.2014;7:277-278.

14. Abbas, Abdul Baset, et al. “Seroprevalence of cytomegalovirus antibodies among aborted women in Ibb city-Yemen.” Journal of Human Virology and Retrovirology.2022; 9 (2): 49-52 DOI: 10.15406/jhvrv.2022.09.00248

15. Kolo RL, Umoh JED, Ella EE et al.Seroprevalence of cytomegalovirus among antenatal patients attending primary health centres in some parts of Kaduna State, Nigeria.2013;3: 43-48. https://doi.org/10.38150/sajeb.3(1).p43-48

16. Dollard SC, Staras SAS, Amin MM et al. National prevalence estimates for cytomegalovirus IgM and IgG avidity and association between high IgM antibody titer and low IgG avidity. Clinical Vaccine Immunolo.2011;18(11):1895-1899. doi:10.1128/CVI.05228-11

17. Khalf MS, Ahmad DW, Ibraheem KA. The seroprevalence of IgM among Iraqi aborted women infected with human cytomegalovirus. Iraqi postgrad Med J. 2012; 11:123-129.

18. Sayar MS, Cag Y, Yilmaz N et al. Evaluation of cytomegalovirus prevalence in pregnant women: a multicentric study. Meditter J Infect Microb Antimicrob.2025;14(1):11-19.

19. Haider M, Rizvi M, Khan N, Malik A. Serological study of herpes virus infection in female patients with bad obstetric history. Biology and Medicine.2011;3(2):284-290.

20. Mohsen SM, Abboud AA. Prevalence of IgM and IgG Against Herpes Simplex Virus (HSVI, II) in the serum of abortion women in Diyala province. Iraqi Journal for Applied Science (IJAS).2024;1(1):65~71.https://doi.org/10.69923/IJAS.2024.010107

21. Nyawale HA, Chibwe E, Mujuni F, et al. Herpes Simplex Virus Type 2 (HSV-2) and Cytomegalovirus (CMV) among Women with Macerated Stillbirth: A Cross-Sectional Hospital-Based Study from Mwanza, Tanzania. J Pregnancy. 2022; 2022:2156835. doi:10.1155/2022/2156835

22.Shweta B, Nupur Shweta Shweta B, Nupur A, Archana A et al. The serological profile of herpes virus amongst patients with bad obstetric history. Apollo medicine volume.2015;12(3):7-10. https://doi.org/10.1016/J.APME.2015.02.005

23. Alanen A, Kahala K, Vahlberg T, et al. Seroprevalence of prenatal infection and reliability of maternal history of varicella zoster virus, cytomegalovirus, herpes   simplex   virus   and   parvovirus   B19   infection   in   south –western Finland.  Brit J Obst Gynecol.2004;112(1):50-60.https://doi.org/10.1111/j.1471-0528.2004.00320.x

24. Sahoo PK, Sabat J, Subhadra S et al. Burden of Rubella virus infection among females attending tertiary care hospitals of Odisha, India: a need for adult women vaccination. Human Vaccine Immunotherapy.2021;17(100):3757-3760. DOI: 10.1080/21645515.2021.1935168

25. Gomes C, Rodrigues da Rocha MD, Da Rocha Coelho FA et al. Alterations

of the male and female reproductive systems induced by COVID-19. Wien. Klin. Wochenschr. 2021; 133: 966–972. doi: 10.1007/s00508-021-01875-2.

26. Rashidi BH, Bandarian F, Bandarian M. Maternal and neonatal outcomes of pregnancies of infertile women during the COVID-19 pandemic: A realworld evidence. JBRA Assist. Reprod. 2022; 26: 594–598. DOI: 10.5935/1518-0557.20210119

27. Aho Glele LS, Simon E, Bouit C et al. Association between SARS-CoV-2infection during pregnancy and adverse pregnancy outcomes: A re-analysis of the data reported by Wei et al. (2021). Infect. Dis. 2022; 52:123–128. DOI: 10.1016/j.idnow.2022.02.009