Journal of Medical Virology 87:1749–1753 (2015)
Detection of Cytomegalovirus, Human Parvovirus B19, and Herpes Simplex Virus-1/2 in Women With First-Trimester Spontaneous Abortions Ya Zhou,1 Guohui Bian,1 Qiongxiu Zhou,1 Zhan Gao,1 Pu Liao,2 Yu Liu,1* and Miao He1** 1 2
Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China The Third People’s Hospital of Chongqing, Chongqing, China
The relationship between viral infections and first-trimester spontaneous abortions is not well-understood. The study aim was to investigate the prevalence of cytomegalovirus (CMV), human parvovirus B19 (B19V), and herpes simplex virus-1/2 (HSV-1/2) infection by molecular and serological techniques in women experiencing spontaneous miscarriage in the first trimester of pregnancy. Plasma samples were examined for CMV, B19V, and HSV-1/2 DNA using real-time quantitative polymerase chain reaction (Real-time qPCR), and for specific IgG antibodies against B19V, CMV, and HSV-1/2 using serological assays. The abortion group consisted of women (n ¼ 1,716) with a history of two or more first-trimester spontaneous abortions. Women younger than 30 years possess higher portion to experience spontaneous abortion. No specimens were positive for B19V or CMV DNA. Seven out of the 1,716 specimens were positive for HSV-1/2 DNA. By serology, 47.24% of patients were positive for B19V IgG, 39.66% for HSV IgG, 79.31% for CMV IgG, and 9.31% for B19V IgM. The high rate of positivity for CMV IgG suggests that the majority of women with first-trimester spontaneous abortions are not susceptible to primary CMV infection. The lack of virus DNA in the majority of cases indicates that B19V, CMV, and HSV-1/2 infection is not commonly associated with first-trimester spontaneous abortion. J. Med. Virol. 87:1749–1753, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
cytomegalovirus; Human parvovirus B19; Herpes simplex virus-1/2; first-trimester spontaneous abortion; Real-time qPCR
INTRODUCTION There are several factors related to spontaneous abortion, such as genetic abnormalities and infections [Brown, 2008; Atik et al., 2010]. Recent studies showed viruses such as cytomegalovirus (CMV), herpes simplex virus (HSV-1/2), human parvovirus B19 (B19V), enterovirus, adenovirus, and varicellazoster virus are causative agents of spontaneous abortion [Chow et al., 2006; Zaki and Goda, 2007; Syridou et al., 2008a; Kim et al., 2012; Syridou et al., in press]. It has been frequently reported that spontaneous abortions were caused by CMV, HSV-1/ 2, and B19V [Petrikovsky et al., 1996; Lowden and Weinstein, 1997; Skj€oldebrand-Sparre et al., 2000; Nyman et al., 2002a; Spano et al., 2002a; Penta et al., 2003; Johansson et al., 2008; Kapranos and Kotronias, 2009; Lassen et al., 2012a]. Some of these studies being based on small sample numbers and having no classification of the periconceptional period. Some studies showed a great variation in detection of CMV, HSV-1/2, and B19V DNA or antigens in placenta, amniotic fluid, cord blood, and fetal tissues. Thus, the relationship between CMV, HSV-1/2, and B19V infection and first-trimester spontaneous abortion remains controversial [Zaki and Goda, 2007]. Real-time qPCR provides a sensitive and accurate means to detect and quantify pathogen genomes [Gouarin et al., 2002; Bustin et al., 2009; Saijo et al., Grant sponsor: Natural Science Foundation of China; Grant number: 81300395. Zhou Ya and Bian Guohui contributed equally to this study. Correspondence: Liu Yu, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610052, China. E-mail:
[email protected] Correspondence to: He Miao, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610052, China. E-mail:
[email protected] Accepted 23 March 2015 DOI 10.1002/jmv.24218 Published online 14 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
C 2015 WILEY PERIODICALS, INC.
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in press]. Serologic assays are a useful tool for detecting the status of the pregnancy predisposes to virus [Shah et al., 1980]. In the present study, maternal plasma was examined for the presence of B19V, CMV, and HSV-1/2 DNA using a real-time qPCR assay developed in-house and for specific IgG antibodies against B19V, CMV, and HSV-1/2 by serological assays. The results getting from those assays allowed us to examine the relationship between the viral infection and spontaneous abortion. MATERIALS AND METHODS Sample Collection Blood samples were collected from women (n ¼ 1,716) with a history of two or more spontaneous abortions within 9 gestational weeks [Practice Committee of the American Society for Reproductive Medicine, 2008] after the fetal loss. The majority of the women had no serious systemic disease in their medical history. The samples were collected at the Institute of Blood Transfusion from November 2010 to October 2012. The first trimester was defined as no more than 13 gestational weeks according previous study [Nyman et al., 2002b]. The study was approved by the ethics committee of the Institute of Blood Transfusion. The samples were subsequently examined for the viral genomes of B19V, CMV, and HSV-1/2 using an in-house real-time qPCR system. The presence of specific IgG antibodies against B19V, CMV, and HSV-1/2, and IgM antibody against B19V, was measured by serological assay. IgM antibodies against CMV and HSV-1/2 were not detected, as determined during the routine pregnancy examinations implemented in China. DNA Extraction and Quantification by Real-Time PCR The plasma samples were preserved at 20˚C after collection. DNA extraction was performed with a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The isolated DNA was stored at 80˚C before PCR analysis. Protocols for detection of B19V, CMV, and HSV-1/2 DNA in plasma were developed. For the detection of B19V, the forward primer S-B19-F and reverse primer S-B19-R were used to amplify a 133 bp fragment of the conserved region of the NS1 gene in the B19V genome [Rinckel et al., 2009]. The primers used for the detection of the CMV, termed SHCMV-F and S-HCMV-R, were used to amplify a 136 bp segment from the MIE gene [Lisboa et al., 2012]. HSV-1/2 DNA was detected using the primers SP-HSV-F and SP-HSV-R, which amplified a 112 bp segment located in the most conserved region of the HSV DNA polymerase gene [Kessler et al., 2000]. The detailed primer sequences used are shown in Table I. A reaction volume of 20 ml was used for the realtime qPCR, which contained 10 ml of FastStart Universal SYBR Green Master (Rox) (Roche J. Med. Virol. DOI 10.1002/jmv
Diagnostics, Indianapolis, IN), 0.5 ml of forward primer (10 pmol/ml), 0.5 ml of reverse primer (10 pmol/ml), and 5 ml of extracted DNA as template. Quantitative standard curves generated by serial dilution of plasmids were used to assign values (copies/ml) to the tested samples. Synthetic serial diluted plasmids (1 106, 1 105, 1 104, 1 103, 1 102, 10, and 1 [copies/ml]), which contained the relevant sequences of B19V, CMV, and HSV-1/2, were used in each plate for real-time qPCR detection. Negative controls that used water as template were also included in each run. The cycling conditions were as follows: 1 cycle of 95˚C for 10 min; 45 cycles of 95˚C for 15 sec, 58˚C for 30 sec, and 72˚C for 30 sec; and a final cycle of 95˚C for 15 sec, 60˚C for 15 sec, with a gradual increase to 95˚C in 30 min at a ramp rate of 2% to get melting curves. The sensitivity and stability of the detection system were also tested by serial dilution of each plasmid. The detection limits of the real-time qPCR were 50 copies per reaction volume for all three viruses. The detailed real-time qPCR procedures were previously described [Ke et al., 2011]. Serological Tests We randomly selected 290 cases for serologic evaluation of specific antibodies. The plasma samples were analyzed qualitatively for specific IgG/IgM for B19V (Virion\Serion, Germany), IgG for CMV (Cusabio, USA), and IgG for HSV-1/2 (Cusabio, Barksdale, USA) according to the manufacturer’s recommendations. Samples were initially tested for one round, with a second test being conducted if the initial results were ambiguous. If the ambiguous result still remained, it was taken as the final result. Statistics All statistical analyses were conducted using SPSS 11.5 (SPSS, Inc., Chicago, IL). The x2 test was applied to estimate the association between categorical variants. A P value of less than 0.05 was considered to be statistically significant. RESULTS Demographic Features All women had undergone routine pregnancy testing for specific IgM against toxoplasma, rubella virus, CMV, and HSV-1/2 before sampling. Spontaneous abortions largely occurred from the sixth to eighth week of pregnancy, and not later than the twelfth week. The mean age was 29.83 years, with a range of 16–47 years. The women (n ¼ 1,716) with a history of two or more spontaneous abortions within 9 gestational weeks were divided into three age groups (less than 30, 30 to 40, and above 40) (Table II). Women less than 30 years of age possess higher portion to
Detection of CMV, B19V, HSV-1/2 in Women With First-Trimester Spontaneous Abortions
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TABLE I. Primers Used for Real-Time PCR Primers
S-B19V-F S-B19V-R S-HCMV-F S-HCMV-R SP-HSV-F SP-HSV-R
Sequences
Location
5’-ACCAGTTCAGGAGAATCAT-3’ 5’-CCCACACATAATCAACCC-3’ 5’-GACTATCCCTCTGTCCTCAGTA-3’ 5’-AGACACTGGCTCAGACTTGA-3’ 5’-CCGGAGAGGGACATCCAGGACTT-3’ 5’-GGGCCATGAGCTTGTAATACACCGT-3’
2256–2274 2371–2388 171231–171252 171117–171136 65876–65898 65963–65987
experience spontaneous abortions than women in the other age groups, which was 53.26% of the total study population. Viral DNA Study for B19V, HSV-1/HSV-2, and CMV None of the specimens were positive for B19V or CMV DNA. Seven out of the 1,716 specimens were positive for HSV DNA. Serologic Study for B19V, HSV-1/ 2, and CMV We selected 290 cases randomly and divided them into three age groups (Table III). In the serologic study, CMV IgG had the highest rate of positivity (79.31%), followed by B19V IgG (47.24%) and HSV IgG (39.66%). Thus, specific IgG antibodies against CMV were present at a high rate in the population studied, indicating that a large proportion of the participants had been infected by CMV in the past. Women in the age range of 30–40 years were more likely to have experienced B19V infection than women in other ages. By contrast, past HSV and CMV infections were more likely in the less than 30year-age group (20 and 43.1%, respectively). In addition, 27 out of 290 recurrent spontaneous abortion samples (9.31%) were positive for B19V IgM, which might indicate that the women were undergoing a recent B19V infection. DISCUSSION First-trimester spontaneous abortion occurs in more than 50% of all pregnancies; however, only 15– 20% of all cases are clinically identified [Wilcox et al., 1988]. Several causes were hypothesized, such as genetic abnormalities, structural abnormalities,
Amplicon length (bp)
133 136 112
infection, immune dysfunction, endocrine abnormalities, antiphospholipid syndrome, and inherited thrombophilic disorders [Rai and Regan, 2006]. Causal relationships are difficult to establish, but the detection rate of CMV, B19V, and HSV during pregnancy is an important way to analyze their relationship with first-trimester spontaneous abortion [Syridou et al., 2008b]. Although recent reports show that infections with B19V, CMV, and HSV were frequently associated with second or third recurrent spontaneous abortions based on detection of specific IgM and DNA in sera or placental tissue [el-Sayed Zaki and Goda, 2007; Syridou et al., 2008b], the relationship between first-trimester spontaneous abortion and B19V, CMV, and HSV infection is still controversial. Several studies reported that B19V, CMV, and HSV infection is associated with an increased risk of firsttrimester spontaneous abortion [Miller et al., 1998; Enders et al., 2004; Lassen et al., 2012b]. Using serum samples and serological assays, Lassen et al. examined 2,918 women with fetal loss and found a correlation between acute B19V infection during the first trimester of pregnancy and increased risk of fetal loss [Lassen et al., 2012b]. Spano et al. reported a high rate (75%) of CMV antigens in tissues from first-trimester abortions, which indicated a possible relationship between CMV infection and spontaneous abortion [Spano et al., 2002b]. Recently, Kapranos et al. reported a significant role of HSV in first-trimester spontaneous abortion, based on nested PCR studies indicating that 41 of 95 cases (43.2%) of early spontaneous abortion showed signs of HSV infection compared with 36 of 216 (16.7%) cases of elective pregnancy termination [Kapranos and Kotronias, 2009]. The higher prevalence of B19V, CMV, and HSV1/2 in maternal or abortion samples reported in these
TABLE II. Molecular Study for Parvovirus B19, HSV-1/HSV-2, and CMV in Women With First-Trimester Spontaneous Abortions Maternal age
40 Total
Number of cases (%)
B19V DNA
HSV DNA
CMV DNA
914 (53.26) 749 (43.65) 53 (3.09) 1716 (100)
0 0 0 0
5 1 1 7
0 0 0 0 J. Med. Virol. DOI 10.1002/jmv
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TABLE III. Serologic Study for B19V, HSV-1/HSV-2, and CMV in Women With First-Trimester Spontaneous Abortions Maternal age
Mean
Number of cases (%)
B19 IgG (%)
HSV IgG (%)
CMV IgG (%)
B19 IgM (%)
40 total
25.90 33.98 43.43 29.89
155 (53.45) 128 (44.14) 7 (2.41) 290 (100)
64 (22.07) 67 (23.10) 6 (2.07) 137 (47.24)
58 (20.00) 54 (18.62) 3 (1.03) 115 (39.66)
125 (43.10) 99 (34.14) 6 (2.07) 230 (79.31)
16 (5.52) 11 (3.80) 0 (0) 27 (9.32)
studies suggests a possible relationship between B19V, CMV, and HSV-1/2 infection and the first-trimester spontaneous abortion. In this study, we examined the potential association between infection of CMV, HSV, and B19V and first-trimester abortion in a large number of maternal plasma samples collected in first-trimester spontaneous abortions using molecular and serological techniques. In a total of 1,716 cases, only seven samples were positive for HSV DNA. None were positive for CMV or B19V. These negative results for virus DNA in a large number of cases indicated that firsttrimester spontaneous abortions associated with B19V, CMV, or HSV infection are not common. Maternal sera were simultaneously examined for the detection of specific IgG antibodies against B19V, HSV, and CMV, which indicate past infection [elSayed Zaki and Goda, 2007]. As no B19V IgM was recommended to prenatal screening in China, 27 out of 290 (9.31%) cases were positive for B19V IgM. But interestingly, no B19V DNA was detected in those samples. This situation could be due to the low viral load of B19V in acute infection being easily cleared by the autoimmune system in pregnant women. The positive B19V, CMV, and HSV IgG could be attributed to the fact that primary infection is usually acquired during childhood, which means that the risk of primary infection is lower during pregnancy. Using detection of viral antigens or DNA in samples, not all researchers found a potential pathogenic association between B19V, CMV, or HSV infection and spontaneous abortion in the first trimester [Sifakis et al., 1998a; Nyman et al., 2002a; Matovina et al., 2004; Sarfraz et al., 2009]. One study in Sweden measured B19V DNA in placental tissue and found a lower frequency of parvovirus B19 infection in first-trimester fetal loss (3%) compared with that in second-trimester fetal loss (12%), which indicated that parvovirus B19 infection associated with first-trimester fetal loss is not common (Nyman et al., 2002a). In a larger study in Norway, 2 of 281 (0.7%) women who experienced fetal death and 9 of 957 (0.9%) controls had IgM antibodies against B19V, indicating that maternal B19V infection was not associated with fetal death [Sarfraz et al., 2009]. Based on the detection of CMV using PCR, a study of 102 cases of spontaneous abortion did not detect CMV in the spontaneous abortion samples [Sifakis J. Med. Virol. DOI 10.1002/jmv
et al., 1998b]. Other studies investigating the presence of CMV DNA using PCR in placenta specimens also failed to support a role for CMV infections in miscarriages during the first trimester of pregnancy [Matovina et al., 2004]. In a PCRbased analysis of abortion material from 102 cases of spontaneous abortions at gestational age less than 20 weeks, HSV infection was only detected in three cases, which does not support a role for HSV infection as a major abortion-related factor [Sifakis et al., 1998a]. In a cohort study of pregnant women during the first trimester in Norway, HSV2 was detected serologically in 29.2% of women with fetal death compared with 27% in the controls, which also indicated no evidence of an association between HSV-2 infection during pregnancy and fetal death [Eskild et al., 2002]. Our results demonstrated a negative result for B19V and CMV DNA in maternal plasma, which indicated that a relationship between maternal B19V and CMV infection and spontaneous abortion is not common. To our knowledge, this is the first report to investigate the association between HSV infection in maternal plasma and first-trimester spontaneous abortion by real-time qPCR. Our results indicate the association is not obvious between HSV infection in maternal plasma and first-trimester spontaneous abortion. In this study, the high sensitivity of real-time qPCR was valuable for detecting the low viral load of B19V, CMV, and HSV-1/2 DNA. Real-time qPCR can detect viral loads as low as 50 copies per reaction. Since detection of viral DNA by real-time qPCR and detection of specific IgG/IgM through ELISA are complementary approaches, we used both methods to investigate the relationship between viral infection and spontaneous abortion. Determination of viral DNA in maternal serum through real-time qPCR improves the diagnostic value of the serologic tests and leads us to conclude that both test methods would be effective tools. Finally, the results could be influenced by seasonal and geographic variations in viral spread. We collected samples every month from 2010 to 2013 in Sichuan province to minimize selection bias. Further large case-control studies are required to elucidate the possible relationship between viral infections and pregnancy outcomes in China. DNApositive plasma may not always represent fetal infection because of the placental barrier. Therefore,
Detection of CMV, B19V, HSV-1/2 in Women With First-Trimester Spontaneous Abortions
real-time qPCR should be performed directly using fetal tissues or placenta of DNA-positive women to confirm the causal relationship. Our results may contribute to further research on the biological mechanisms related to first-trimester spontaneous abortions in larger epidemiological studies. ACKNOWLEDGMENT This study was supported by Natural Science Foundation of China (81300395). REFERENCES Atik RB, Hepworth-Jones BE, Doyle P. 2010. Risk factors for miscarriage. Early Pregnancy 9. Brown S2008. Miscarriage and its associations. Paper presented at: Seminars in reproductive medicine. Bustin SA, Benes V, Garson JA, et al. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. Chow S, Craig M, Jacques C, et al. 2006. Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7. J Med Virol 78:747–756. el-Sayed Zaki M, Goda H. 2007. Relevance of parvovirus B19, herpes simplex virus 2, and cytomegalovirus virologic markers in maternal serum for diagnosis of unexplained recurrent abortions. Arch Pathol Lab Med 131:956–960. Enders M, Weidner A, Zoellner I, Searle K, Enders G. 2004. Fetal morbidity and mortality after acute human parvovirus B19 infection in pregnancy: Prospective evaluation of 1018 cases. Prenat Diagn 24:513–518. Eskild A, Jeansson S, Stray-Pedersen B, Jenum PA. 2002. Herpes simplex virus type-2 infection in pregnancy: No risk of fetal death: Results from a nested case–control study within 35,940 women. Brit J Obstet Gynecol 109:1030–1035. Gouarin S, Gault E, Vabret A, et al. 2002. Real-time PCR quantification of human cytomegalovirus DNA in amniotic fluid samples from mothers with primary infection. J Clin Microbiol 40:1767–1772. Johansson S, Buchmayer S, Harlid S, et al. 2008. Infection with Parvovirus B19 and Herpes viruses in early pregnancy and risk of second trimester miscarriage or very preterm birth. Reprod Toxicol 26:298–302. Kapranos NC, Kotronias DC. 2009. Detection of herpes simplex virus in first trimester pregnancy loss using molecular techniques. In Vivo 23:839–842. Kim ID, Chang HS, Hwang KJ. 2012. Herpes simplex virus 2 infection rate and necessity of screening during pregnancy: A clinical and seroepidemiologic study. Yonsei Med J 53:401–407. Ke L, He M, Li C, et al. 2011. The prevalence of human parvovirus B19 DNA and antibodies in blood donors from four Chinese blood centers. Transfusion 51:1909–1918. Kessler HH, M€ uhlbauer G, Rinner B, et al. 2000. Detection of herpes simplex virus DNA by real-time PCR. J Clin Microbiol 38:2638–2642. Lassen J, Jensen AK, Bager P, et al. 2012. Parvovirus B19 infection in the first trimester of pregnancy and risk of fetal loss: A population-based case-control study. Am J Epidemiol 176:803– 807. Lisboa L, Tong Y, Kumar D, et al. 2012. Analysis and clinical correlation of genetic variation in cytomegalovirus. Transpl Infect Dis 14:132–140.
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Lowden E, Weinstein L. 1997. Unexpected second trimester pregnancy loss due to maternal parvovirus B19 infection. Southern Med J 90:702–704. Matovina M, Husnjak K, Milutin N, Ciglar S, Grce M. 2004. Possible role of bacterial and viral infections in miscarriages. Fertil Steril 81:662–669. Miller E, Fairley CK, Cohen BJ, Seng C. 1998. Immediate and long term outcome of human parvovirus B19 infection in pregnancy. Brit J Obstet Gynecol 105:174–178. Nyman M, Tolfvenstam T, Petersson K, Krassny C, Skj€ oldebrandSparre L, Broliden K. 2002. Detection of Human Parvovirus B19 Infection in First-Trimester Fetal Loss. Obstet Gynecol 99:795–798. Penta M, Lukic A, Conte M, et al. 2003. Infectious agents in tissues from spontaneous abortions in the first trimester of pregnancy. New Microbiol 26:329. Petrikovsky BM, Baker D, Schneider E. 1996. Fetal hydrops secondary to human parvovirus infection in early pregnancy. Prenat Diagn 16:342–344. Practice Committee of the American Society for Reproductive Medicine. 2008. Definitions of infertility and recurrent pregnancy loss. Fertility Sterility 90:S60. Rai R, Regan L. 2006. Recurrent miscarriage. The Lancet 368:601–611. Rinckel LA, Buno BR, Gierman TM, Lee DC. 2009. Discovery and analysis of a novel parvovirus B19 genotype 3 isolate in the United States. Transfusion 49:1488–1492. Saijo M, Morikawa S, Kurane I. 2008. Real-time quantitative polymerase chain reaction for virus infection diagnostics. Sarfraz AA, Samuelsen SO, Bruu AL, Jenum PA, Eskild A. 2009. Maternal human parvovirus B19 infection and the risk of fetal death and low birthweight: A case-control study within 35 940 pregnant women. Brit J Obstet Gynecol 116:1492–1498. Shah K, Daniel R, Madden D, Stagno S. 1980. Serological investigation of BK papovavirus infection in pregnant women and their offspring. Infect Immun 30:29–35. Sifakis S, Ergazaki M, Sourvinos G, Koffa M, Koumantakis E, Spandidos DA. 1998. Evaluation of Parvo B19, CMV and HPV viruses in human aborted material using the polymerase chain reaction technique. Eur J Obstet Gynecol Reprod Biol 76:169–173. Sifakis S, Koumantakis E, Koffa M, Ergazaki M, Spandidos D. 1998. Detection of herpes simplex virus (HSV) in aborted material using the polymerase chain reaction technique. Gynecol Obstet Invest 45:109–115. Skj€ oldebrand-Sparre L, Tolfvenstam T, Papadogiannakis N, Wahren B, Broliden K, Nyman M. 2000. Parvovirus B19 infection: Association with third-trimester intrauterine fetal death. Brit J Obstet Gynecol 107:476–480. Spano LC, Fe LP, Mercon-de-Vargas P. 2002. Human cytomegalovirus infection and abortion: An immunohistochemical study. Med Science Monit 8:BR230–BR235. Spano LC, Lima Pereira FE, Gomes da Silva Basso N, Mercon-deVargas PR. 2002. Human cytomegalovirus infection and abortion: An immunohistochemical study. Med Science Monit 8: BR230–BR235. Syridou G, Skevaki C, Kafetzis DA. 2005. Intrauterine infection with parvovirus B19 and CMV: implications in early and late gestation fetal demise. Syridou G, Spanakis N, Konstantinidou A, et al. 2008. Detection of cytomegalovirus, parvovirus B19 and herpes simplex viruses in cases of intrauterine fetal death: Association with pathological findings. J Med Virol 80:1776–1782. Wilcox AJ, Weinberg CR, O’Connor JF, et al. 1988. Incidence of early loss of pregnancy. New Engl J Med 319:189–194. Zaki ME-S, Goda H. 2007. Relevance of parvovirus B19, herpes simplex virus 2, and cytomegalovirus virologic markers in maternal serum for diagnosis of unexplained recurrent abortions. Arch Pathol Lab Med 131:956–960.
J. Med. Virol. DOI 10.1002/jmv
