Journal of Medical Virology 87:1046–1053 (2015)
Study of Human Cytomegalovirus Replication in Body Fluids, Placental Infection, and Miscarriage During the First Trimester of Pregnancy Xiao Chuan Yan,1 Jian Hua Wang,2* Bo Wang,2 Li Li Huang,2 Li Qin Zhou,3 Bo Zhu,4 and Yun Liang5 1
Department of General Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China Department of Gynaecology and Obstetrics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 3 Department of Medical Microbiology and Molecular Biology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 4 Department of Immunology and Biochemistry, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 5 Department of Pathology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2
Intrauterine infection caused by human cytomegalovirus (HCMV) can lead to embryo, fetal, and neonatal damage. The prevalence of HCMV replication in body fluids (blood, urine, and cervicovaginal secretion) was investigated, and its effects on HCMV vertical transmission and miscarriages in early pregnant women were evaluated. HCMV DNA in body fluids was detected in 1,064 early pregnant women (624 normal pregnancies and 440 miscarriages). There were 101 cases who were HCMV DNA positive in cervicovaginal secretion and the rates were 10.9% (48/440 cases) and 8.5% (53/ 624 cases) in miscarriages and normal pregnancies, respectively (P > 0.05). A total of 101 cases (63 and 38 cases with and without HCMV DNA in cervicovaginal secretion, respectively) were given HCMV DNA detection in placental villi/deciduas. There were five cases (7.9%; two normal pregnancies and three miscarriages) with HCMV DNA in placental villi/deciduas among the 63 cases with HCMV DNA in cervicovaginal secretion, whereas none of the other 38 cases were detected HCMV DNA positive in their placental villi/deciduas. The percentage of HCMV DNA in placental villi/ deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Two cases with a higher HCMV loads in cervicovaginal secretion and placental villi/deciduas had miscarriages. These findings suggest that HCMV replication in cervicovaginal secretion can involve in placental HCMV infection, and high HCMV DNA loads in cervicovaginal secretion and placental villi/ C 2015 WILEY PERIODICALS, INC.
deciduas are associated with miscarriage. J. Med. Virol. 87:1046–1053, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
cytomegalovirus; virus shedding; pregnancy; miscarriage; placental infection
INTRODUCTION Affecting 0.2–2% of all births in developed countries and even higher in developing countries, human cytomegalovirus (HCMV) is the most common cause of congenital infection [Revello and Gerna, 2002; LaMarca et al., 2006). Approximately 40% HCMV vertical transmission occurs in the first trimester [Bode´us et al., 2010]. Pregnant women with HCMV infection rarely have symptoms, however, HCMV intrauterine infection may cause miscarriage, stillbirth, abnormal fetal development, neonatal death, newborn hearing loss, and mental retardation [Wen The authors declare no conflicts of interest. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81170537; 81070535.; Grant sponsor: Education Department Scientific Research Program of Zhejiang Province, China; Grant number: Y201018629. Correspondence to: Jian Hua Wang, MD, Department of Gynaecology and Obstetrics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China. E-mail: [email protected] Accepted 7 January 2015 DOI 10.1002/jmv.24158 Published online 2 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
Placental Cytomegalovirus Infection
et al., 2002; Dollard et al., 2007; Kenneson and Cannon, 2007; Grosse et al., 2008]. In order to examine fetal HCMV infection, routine HCMV screening is necessary for all pregnant women. The positive rates of HCMV DNA in vaginal secretion were statistically similar between non pregnant and pregnant women [Spano et al., 2004]. In an early study, 848 normal 6–12 weeks pregnant women were given HCMV DNA detection in vaginal secretions and the outcomes of pregnancy were followed-up henceforth. Women with HCMV DNA in vaginal secretions had a higher miscarriage rate (6.7%, 4/60) than that of comparable women with negative HCMV (1.2%, 8/788). These results suggested that HCMV replication in genital tract of 6–12 weeks pregnant women predisposed to adverse pregnancy outcomes (miscarriage) [Tanaka et al., 2006]. HCMV replication in placental villi and deciduas was crucial for the occurrence of fetal HCMV infection [Pereira et al., 2005]. The detection of HCMV replication in placental villi and deciduas is an invasive laboratory technique, therefore, it is urgent and valuable to find a non-invasive testing method. The association between HCMV replication in peripheral body fluids and maternal fetal transmission of HCMV during first trimester has not yet been elucidated. Despite HCMV uterine infection has been established, the association of HCMV infection in placental villi/deciduas with miscarriage in first trimester pregnancy has not been fully clarified. Besides, the specific pathogenesis about miscarriage of early pregnancy induced by HCMV replication is not completely clear at this point. This prospective study focuses on the association among HCMV replication (commonly known as shedding) in blood, urine and cervicovaginal secretion samples, maternal fetal interface (placental villi and deciduas) HCMV infection, miscarriage, and anti HCMV antibody reactivity, during the first trimester of pregnancy. This study aimed at clarifying the effects of HCMV infection in blood, urine and cervicovaginal secretion on maternal fetal interface HCMV infection, and pregnancy outcomes during the first trimester of pregnancy. MATERIALS AND METHODS Study Patients The study was approved by the Medical Research Ethics Committee at Women’s Hospital, School of Medicine, Zhejiang University, People’s Republic of China. This prospective cohort study was conducted with written informed consent from each subject who participated in this study. The study patients were constituted by all women with first trimester pregnancy (normal pregnancy and miscarriage) who underwent dilatation and curettage and agreed to accept HCMV serological test and HCMV DNA detection, during the period between August 2011 and August 2012, at inpatient operating room in the
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hospital. Induced abortion during the first trimester is legal in China. The term “normal pregnancy” implies that the pregnancy ended with an intentional abortion. Figure 1 summarizes the protocol of subjects selected and laboratory tests at the hospital. A total of 1,112 subjects were included in the study, among them, 48 with incomplete information were excluded. Diagnosis of HCMV Infection Active HCMV replication (active HCMV infection) was defined as isolation of HCMV or detection of HCMV proteins or nucleic acid from any body fluid or tissue specimen, according to the previously published literatures [Ljungman et al., 2002; Peres et al., 2010; Schoenfisch et al., 2011]. Specimens Processing Before dilatation and curettage, blood, urine, and cervicovaginal secretion samples from each subject were collected as follows. Four milliliter (ml) of venous blood from each subject was obtained, of which 2 ml in a test tube without anticoagulant, and the remaining 2 ml in a test tube with EDTA anticoagulants. The blood in the first tube was centrifuged to separate the serum for anti HCMV antibodies test, and the blood in the second tube was prepared to isolate leukocytes for HCMV DNA detection. First morning urine samples from each subject were collected in sterile specimen containers. The 2 ml of urine was centrifuged and the urinary sediment was tested for HCMV DNA. Cervicovaginal secretion samples were collected from all subjects using sterile cotton swabs. The swabs were placed in contact with the external os of cervix to absorb mucus. The swabs with the secretion were immersed directly in 2 ml of phosphate buffered saline solution and then were centrifuged. The precipitate was detected for HCMV DNA. During dilatation and curettage, placental villi and decidual samples, respectively, were obtained immediately. The placental villi and deciduas from each subject were divided into two portions, respectively. One portion was snap frozen immediately after collection for HCMV DNA analysis, and the second portion was fixed in formalin and embedded in paraffin for histopathological examination. In order to minimize the possibility of abortion tissue contamination with HCMV from cervical secretion, we disinfected vagina, cervix, and cervical tube with iodophor, and then inserted a sterile suction tube into the uterus and took decidua and chorionic villi with vacuum aspiration. Serological Testing for Anti HCMV Antibodies Serum IgM and IgG antibodies to HCMV were detected by using a chemiluminescent microparticle immunoassay (The ARCHITECT CMV IgM and IgG J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Flow diagram of women with first trimester pregnancies (normal pregnancies and miscarriages) undergoing laboratory tests. aThree cases also had positive HCMV DNA in urine, but there was no case with positive HCMV DNA in blood.
assay, Abbott Park, IL), according to the protocols described by the manufacturers. Specimens with concentration values or ¼ 500 copies/ml. HCMV DNA values in cervicovaginal secretion are < 500 copies/ml. NA, not available. d Two case with anti HCMV IgM (þ) and IgG (þ) and HCMV DNA in cervicovaginal secretion displayed that HCMV DNA () in villi and NA in deciduas. e Five cases with HCMV DNA in cervicovaginal secretion and urine showed that negative HCMV DNA in villi and deciduas. a b c
J. Med. Virol. DOI 10.1002/jmv
J. Med. Virol. DOI 10.1002/jmv
30 (90.9) 28 (93.3) 58 (92.1) 3 (9.1) 2 (6.7) 5 (7.9) 22 (91.7) 28 (96.5) 50 (94.3) All patients with HCMV DNA in cervicovaginal secretion. Investigated specimens from miscarriage group and normal pregnancy group. Value, n (%); (þ) positive; () negative. d Five cases with HCMV DNA in cervicovaginal secretion and urine showed that negative HCMV DNA in villi and deciduas. c
b
31 (93.9) 30 (100) 61 (96.8)
2 (6.1) 3 (10) 5 (7.9)
31 (93.9) 27 (90) 58 (92.1)
1 (3.5) 1 (3.3) 2 (3.4)
28 (96.5) 29 (96.7) 57 (96.6)
2 (8.3) 1 (3.5) 3 (5.7) a
(þ)
2 (6.1) 0 (0) 2 (3.2) 0 (0) 0 (0) 0 (0) 33 (100) 30 (100) 63 (100) Miscarriage (n ¼ 33) Normal pregnancy (n ¼ 30) Total (n ¼ 63)
() (þ) () (þ) () (þ) () ()
(þ)d
Decidua(nb ¼ 24/29)
HCMV DNAc
Villi (nb ¼ 29/30)
() (þ) Groupsa
Active HCMV infection (replication) is a characteristic of primary infection, latent infection reactivation, and reinfection with a different HCMV strain. During these events, HCMV is shed in body fluids [Schoenfisch et al., 2011]. After HCMV primary infection, HCMV remains in a persistent state within the host and enters latency. Although the host’s immune system against the HCMV represses the replication of the HCMV, subsequent HCMV reactivation can still take place in the immunocompetent person [Mocarski et al., 2007]. HCMV reactivation or recurrence is defined as a new detection of HCMV replication in a patient previously diagnosed with HCMV infection and in whom HCMV was not detected for an interval of at least 4 weeks during active surveillance [Peres et al., 2010]. All kinds of active HCMV infection can occur in pregnancy and lead to intrauterine HCMV infection. During the first trimester of pregnancy, the developing embryo is susceptible to HCMV infection. Although transplacental transmission (vertical transmission) of HCMV, resulting in fetal damage or death, can occur at any stage during pregnancy, severe fetal symptoms, sequelae, and abnormalities caused by HCMV congenital infection acquired during the first trimester are more common [Pass et al., 2006; Rahav et al., 2007;
Urine (nb ¼ 33/29)
DISCUSSION
IgM
Histopathology displayed a normal placental villi and deciduas in normal pregnancy, and degradation and degeneration of placental villi and deciduas with inflammatory cell infiltration in miscarriage. There was no histopathological significant difference between the placental villi and/or deciduas with and without HCMV DNA. Specific CMV cytopathic effects (CMV inclusions and cell enlargement) had not been found by microscopic examination, so HCMV infection was not confirmed by histopathology.
IgG
Histopathology
Anti-HCMV antibody (nb ¼ 33/30)c
into miscarriage and normal pregnancy groups, retrospectively. The percentage of HCMV DNA in placental villi/deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Only two cases were tested positive of HCMV IgM serology in miscarriage group and all subjects in normal pregnancy group had negative HCMV IgM serology (Table II). The five subjects with HCMV DNA in placental villi and/or deciduas all had positive HCMV IgG serology and HCMV DNA in cervicovaginal secretion, and negative HCMV IgM serology and HCMV DNA in urine. Among the five subjects, three cases had miscarriages, and two cases had normal pregnancies (Table III). Both case 1 and case 2 with a higher HCMV loads in cervicovaginal secretion were from miscarriage patients and had a higher HCMV loads in decidua and villi, respectively.
villi/Decidua(nb ¼ 33/30)
Yan et al. TABLE II. Comparison of Anti HCMV Antibody Reactivity and HCMV DNA in Urine and Placental Villi/Deciduas Between the Two Groups With HCMV DNA in Cervicovaginal Secretion
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Placental Cytomegalovirus Infection
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TABLE III. Clinical and HCMV Related Testing Results of the Five Patients With HCMV DNA in Placental Villi and/or Deciduas HCMV DNA (copies/ml)
Serum antibodies to HCMV
HCMV DNA (copies/ml)
a
Patient Miscarriage Normal pregnancy Miscarriage Miscarriage Normal pregnancy a b
GA (weeks) 10 11 11 12 12
Villi b
NA 250.0 (þ)
IgM (AU/ml) 0.13 0.17 0.16 0.17 0.38
() () () () ()
Urine 15 (or >20)-week stillborn fetuses in the previous reports and with first trimester pregnancy (normal pregnancy and miscarriage) in our study, respectively. In this study, the positivity rate of HCMV DNA in cervicovaginal secretion (9.5%, 101/1064 cases) was similar to that in the study conducted by Tanaka et al. who displayed that vaginal CMV DNA shedding was 7.7% (76/993 cases) [Tanaka et al., 2006]. Previous study in populations of young, low-income, black, CMV-seropositive, and postpartum women showed that 39% of them had CMV-positive urine by real-time PCR for the AD169 strain DNA [Arora et al., 2010]. The higher rate of CMV-positive urine than our report may be due to the differences of study population, the preparation of urine specimen, and experimental method. HCMV DNA detection by real-time PCR is a reliable assay in diagnosis of HCMV infection [Biri et al., 2006]. A previous study revealed that HCMV DNA loads in blood was much lower than that in cervicovaginal lavage, and HCMV shedding in cervicovaginal lavage and blood was associated with markers of immunosuppression in immunodeficiency virus and HCMV seropositive women [Schoenfisch et al., 2011]. Pregnancy is a subtle state of immunosuppression [Singh and Perfect, 2007]. Pregnancy increases the chances of CMV infection or reactivation of latent CMV. A recent study indicated that HCMV could result in symptomatic congenital HCMV infections in HCMV-seropositive women; even HCMV DNA in their serum were negative ( 0.05; Spano et al., 2004]. Our study also showed J. Med. Virol. DOI 10.1002/jmv
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that HCMV DNA in placental villi/deciduas could occur in HCMV seropositive woman with HCMV DNA in cervicovaginal secretion and negative HCMV DNA in blood; negative HCMV DNA in cervicovaginal secretion, urine, and blood might not be able to cause intrauterine HCMV infection. The information also suggested that HCMV DNA loads in blood were much lower than that in cervicovaginal secretion and urine. This study demonstrates that among the 101 subjects with HCMV DNA in cervicovaginal secretion, all 101 subjects were tested positive for anti HCMV IgG antibody, whereas only two subjects, who had a negative HCMV DNA in placental villi and deciduas, were tested positive for both HCMV IgG and IgM serology. The poor correlation between IgM and HCMV replication in cervicovaginal secretion or intrauterine HCMV infection is caused by several factors: the persistence of positive IgM after the end of HCMV infection, HCMV intermittent or continuous shedding in both cervicovaginal secretion and urine throughout pregnancy [Rosenthal et al., 2009], and limited sensitivity and specificity in HCMV IgM assays [Schoenfisch et al., 2011]. Therefore, the study indicates that HCMV IgM antibody, of which the detection is still widely used for monitoring HCMV infection, might be a poor marker for active HCMV replication. Although primary HCMV infection was not documented and distinguished in this study, HCMV replication in cervicovaginal secretion or urine was the most likely explanation for active HCMV infection due to primary HCMV infection, latent HCMV reactivation or reinfection with a different HCMV strain. HCMV replication in placental villi and deciduas is crucial for its spread from infected mother to fetus [Pereira et al., 2005]. The detection of HCMV replication in placental villi and deciduas is invasive and may lead to miscarriage, hence HCMV replication in body fluids (cervicovaginal secretion, urine, and blood) could be a useful surrogate [Schoenfisch et al., 2011]. This study shows in Table III that there were two cases with a higher HCMV loads in cervicovaginal secretion, and both the cases were from miscarriage patients and had a higher HCMV loads in decidua or villi, respectively. It meant that a higher HCMV loads in cervicovaginal secretion may lead to a higher HCMV loads in decidua and/or villi and a higher miscarriage rate. In other words, high HCMV loads increase the risk of miscarriages, and high HCMV DNA loads in cervicovaginal secretion or placental villi/deciduas are associated with miscarriage. Both ascending and hematogenous CMV infection can lead to its interuterine infection. Therefore, the quantification of HCMV DNA in cervicovaginal secretion could be more than a noninvasive method for monitoring of active CMV infection, but also applicable for guiding the further screening of HCMV intrauterine infection in first trimester pregnant women. This study displayed that histopathological study of the placental villi and deciduas added little to J. Med. Virol. DOI 10.1002/jmv
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determining the etiology of CMV mediated tissue changes and potential stillbirth/miscarriage in the first trimester of pregnant women. In conclusion, in immunocompetent first-trimester pregnant women, HCMV DNA level in blood was much lower than that in cervicovaginal secretion and urine, HCMV replication in cervicovaginal secretion could involve in its interuterine infection and vertical transmission, and high HCMV replication in cervicovaginal secretion and placental villi/deciduas might increase the risk of miscarriage. REFERENCES Adler SP, Nigro G, Pereira L. 2007. Recent advances in the prevention and treatment of congenital cytomegalovirus infections. Semin Perinatol 31:10–18. Arora N, Novak Z, Fowler KB, Boppana SB, Ross SA. 2010. Cytomegalovirus viruria and DNAemia in healthy seropositive women. J Infect Dis 202:1800–1803. Bode´us M, Kabamba-Mukadi B, Zech F, Hubinont C, Bernard P, Goubau P. 2010. Human cytomegalovirus in utero transmission: Follow-up of 524 maternal seroconversions. J Clin Virol 47:201– 202. Biri A, Bozdayi G, Cic¸fti B, Din B, Yu¨cel A, Rota S. 2006. The detection of CMV in amniotic fluid and cervicovaginal smear samples by real-time PCR assay in prenatal diagnosis. Arch Gynecol Obstet 273:261–266. Dollard SC, Grosse SD, Ross DS. 2007. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol 17:355–363. Forbes BA, Bonville CA, Dock NL. 1990. The effects of a promoter of cell differentiation and selected hormones on human cytomegalovirus infection using an in vitro cell system. J Infect Dis 162:39–45. Grosse SD, Ross DS, Dollard SC. 2008. Congenital cytomegalovirus (CMV) infection as a cause of permanent bilateral hearing loss: A quantitative assessment. J Clin Virol 41:57–62. Kenneson A, Cannon MJ. 2007. Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev Med Virol 17:253–276. Iwasenko JM, Howard J, Arbuckle S, Graf N, Hall B, Craig ME, Rawlinson WD. 2011. Human cytomegalovirus infection is detected frequently in stillbirths and is associated with fetal thrombotic vasculopathy. J Infect Dis 203:1526–1533. LaMarca HL, Nelson AB, Scandurro AB, Whitley GS, Morris CA. 2006. Human cytomegalovirus-induced inhibition of cytotrophoblast invasion in a first trimester extravillous cytotrophoblast cell line. Placenta 27:137–147. La Torre R, Nigro G, Mazzocco M, Best AM, Adler SP. 2006. Placental enlargement in women with primary maternal cytomegalovirus infection is associated with fetal and neonatal disease. Clin Infect Dis 43:994–1000. Ljungman P, Griffiths P, Paya C. 2002. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis 34:1094–1097. Mocarski ES, Shenk T, Jr, Pass RF. 2007. Cytomegaloviruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins, pp 2702–2772. Mussi-Pinhata MM, Yamamoto AY, Moura Brito, de Lima RM, Isaac M, de Carvalho e, Oliveira PF, Boppana S, Britt WJ. 2009. Birth prevalence and natural history of congenital cytomegalovirus infection in a highly seroimmune population. Clin Infect Dis 49:522–528. Nagamori T, Koyano S, Inoue N, Yamada H, Oshima M, Minematsu T, Fujieda K. 2010. Single cytomegalovirus strain associated with fetal loss and then congenital infection of a subsequent child born to the same mother. J Clin Virol 49:134–136. Nigro G, Adler SP, Parruti G, Anceschi MM, Coclite E, Pezone I, Di Renzo GC. 2012. Immunoglobulin therapy of fetal cytomegalovirus infection occurring in the first half of pregnancy—A case-control study of the outcome in children. J Infect Dis 205:215–227.
Placental Cytomegalovirus Infection Pass RF, Fowler KB, Boppana SB, Britt WJ, Stagno S. 2006. Congenital cytomegalovirus infection following first trimester maternal infection: Symptoms at birth and outcome. J Clin Virol 35:216–220. Pereira L, Maidji E, McDonagh S, Tabata T. 2005. Insights into viral transmission at the uterine-placental interface. Trends Microbiol 13:164–174. Peres RM, Costa CR, Andrade PD, Bonon SH, Albuquerque DM, de Oliveira C, Vigorito AC, Aranha FJ, de Souza CA, Costa SC. 2010. Surveillance of active human cytomegalovirus infection in hematopoietic stem cell transplantation (HLA sibling identical donor): Search for optimal cutoff value by real-time PCR. BMC Infect Dis 13:147–153. Rahav G, Gabbay R, Ornoy A, Shechtman S, Arnon J, DiavCitrin O. 2007. Primary versus nonprimary cytomegalovirus infection during pregnancy, Israel. Emerg Infect Dis 13:1791– 1793. Revello MG, Gerna G. 2002. Diagnosis and management of human cytomegalovirus infection in the mother, fetus and newborn infant. Clin Microbiol Rev 15:680–715. Rosenthal LS, Fowler KB, BoppanaSB Britt, Pass RF, Schmid SD, Stagno S, Cannon MJ. 2009. Cytomegalovirus shedding and delayed sensorineural hearing loss: Results from longitudinal follow-up of children with congenital infection. Pediatr Infect Dis J 28:515–520. Schoenfisch AL, Dollard SC, Amin M, Gardner LI, Klein RS, Mayer K, Rompalo A, Sobel JD, Cannon MJ. 2011. Cytomegalo-
1053 virus (CMV) shedding is highly correlated with markers of immunosuppression in CMV-seropositive women. J Med Microbiol 60:768–774. Singh N, Perfect JR. 2007. Immune reconstitution syndrome and exacerbation of infections after pregnancy. Clin Infect Dis 45:1192–1199. Spano LC, Gatti J, Nascimento JP, Leite JP. 2004. Prevalence of human cytomegalovirus infection in pregnant and non-pregnant women. J Infect 48:213–220. Syridou G, Spanakis N, Konstantinidou A, Piperaki ET, Kafetzis D, Patsouris E, Antsaklis A, Tsakris A. 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. Tanaka K, Yamada H, Minami M, Kataoka S, Numazaki K, Minakami H, Tsutsumi H. 2006. Screening for vaginal shedding of cytomegalovirus in healthy pregnant women using real-time PCR: Correlation of CMV in the vagina and adverse outcome of pregnancy. J Med Virol 78:757–759. Wen LZ, Xing W, Liu LQ, Ao LM, Chen SH, Zeng WJ. 2002. Cytomegalovirus infection in pregnancy. Int J Gynaecol Obstet 79:111–116. Yamamoto AY, Mussi-Pinhata MM, Boppana SB, Novak Z, Wagatsuma VM, Oliveira Pde F, Duarte G, Britt WJ. 2010. Human cytomegalovirus reinfection is associated with intrauterine transmission in a highly cytomegalovirus-immune maternal population. Am J Obstet Gynecol 202:297 e1–e8.
J. Med. Virol. DOI 10.1002/jmv
Study of Human Cytomegalovirus Replication in Body Fluids, Placental Infection, and Miscarriage During the First Trimester of Pregnancy Xiao Chuan Yan,1 Jian Hua Wang,2* Bo Wang,2 Li Li Huang,2 Li Qin Zhou,3 Bo Zhu,4 and Yun Liang5 1
Department of General Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China Department of Gynaecology and Obstetrics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 3 Department of Medical Microbiology and Molecular Biology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 4 Department of Immunology and Biochemistry, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 5 Department of Pathology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2
Intrauterine infection caused by human cytomegalovirus (HCMV) can lead to embryo, fetal, and neonatal damage. The prevalence of HCMV replication in body fluids (blood, urine, and cervicovaginal secretion) was investigated, and its effects on HCMV vertical transmission and miscarriages in early pregnant women were evaluated. HCMV DNA in body fluids was detected in 1,064 early pregnant women (624 normal pregnancies and 440 miscarriages). There were 101 cases who were HCMV DNA positive in cervicovaginal secretion and the rates were 10.9% (48/440 cases) and 8.5% (53/ 624 cases) in miscarriages and normal pregnancies, respectively (P > 0.05). A total of 101 cases (63 and 38 cases with and without HCMV DNA in cervicovaginal secretion, respectively) were given HCMV DNA detection in placental villi/deciduas. There were five cases (7.9%; two normal pregnancies and three miscarriages) with HCMV DNA in placental villi/deciduas among the 63 cases with HCMV DNA in cervicovaginal secretion, whereas none of the other 38 cases were detected HCMV DNA positive in their placental villi/deciduas. The percentage of HCMV DNA in placental villi/ deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Two cases with a higher HCMV loads in cervicovaginal secretion and placental villi/deciduas had miscarriages. These findings suggest that HCMV replication in cervicovaginal secretion can involve in placental HCMV infection, and high HCMV DNA loads in cervicovaginal secretion and placental villi/ C 2015 WILEY PERIODICALS, INC.
deciduas are associated with miscarriage. J. Med. Virol. 87:1046–1053, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
cytomegalovirus; virus shedding; pregnancy; miscarriage; placental infection
INTRODUCTION Affecting 0.2–2% of all births in developed countries and even higher in developing countries, human cytomegalovirus (HCMV) is the most common cause of congenital infection [Revello and Gerna, 2002; LaMarca et al., 2006). Approximately 40% HCMV vertical transmission occurs in the first trimester [Bode´us et al., 2010]. Pregnant women with HCMV infection rarely have symptoms, however, HCMV intrauterine infection may cause miscarriage, stillbirth, abnormal fetal development, neonatal death, newborn hearing loss, and mental retardation [Wen The authors declare no conflicts of interest. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81170537; 81070535.; Grant sponsor: Education Department Scientific Research Program of Zhejiang Province, China; Grant number: Y201018629. Correspondence to: Jian Hua Wang, MD, Department of Gynaecology and Obstetrics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China. E-mail: [email protected] Accepted 7 January 2015 DOI 10.1002/jmv.24158 Published online 2 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
Placental Cytomegalovirus Infection
et al., 2002; Dollard et al., 2007; Kenneson and Cannon, 2007; Grosse et al., 2008]. In order to examine fetal HCMV infection, routine HCMV screening is necessary for all pregnant women. The positive rates of HCMV DNA in vaginal secretion were statistically similar between non pregnant and pregnant women [Spano et al., 2004]. In an early study, 848 normal 6–12 weeks pregnant women were given HCMV DNA detection in vaginal secretions and the outcomes of pregnancy were followed-up henceforth. Women with HCMV DNA in vaginal secretions had a higher miscarriage rate (6.7%, 4/60) than that of comparable women with negative HCMV (1.2%, 8/788). These results suggested that HCMV replication in genital tract of 6–12 weeks pregnant women predisposed to adverse pregnancy outcomes (miscarriage) [Tanaka et al., 2006]. HCMV replication in placental villi and deciduas was crucial for the occurrence of fetal HCMV infection [Pereira et al., 2005]. The detection of HCMV replication in placental villi and deciduas is an invasive laboratory technique, therefore, it is urgent and valuable to find a non-invasive testing method. The association between HCMV replication in peripheral body fluids and maternal fetal transmission of HCMV during first trimester has not yet been elucidated. Despite HCMV uterine infection has been established, the association of HCMV infection in placental villi/deciduas with miscarriage in first trimester pregnancy has not been fully clarified. Besides, the specific pathogenesis about miscarriage of early pregnancy induced by HCMV replication is not completely clear at this point. This prospective study focuses on the association among HCMV replication (commonly known as shedding) in blood, urine and cervicovaginal secretion samples, maternal fetal interface (placental villi and deciduas) HCMV infection, miscarriage, and anti HCMV antibody reactivity, during the first trimester of pregnancy. This study aimed at clarifying the effects of HCMV infection in blood, urine and cervicovaginal secretion on maternal fetal interface HCMV infection, and pregnancy outcomes during the first trimester of pregnancy. MATERIALS AND METHODS Study Patients The study was approved by the Medical Research Ethics Committee at Women’s Hospital, School of Medicine, Zhejiang University, People’s Republic of China. This prospective cohort study was conducted with written informed consent from each subject who participated in this study. The study patients were constituted by all women with first trimester pregnancy (normal pregnancy and miscarriage) who underwent dilatation and curettage and agreed to accept HCMV serological test and HCMV DNA detection, during the period between August 2011 and August 2012, at inpatient operating room in the
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hospital. Induced abortion during the first trimester is legal in China. The term “normal pregnancy” implies that the pregnancy ended with an intentional abortion. Figure 1 summarizes the protocol of subjects selected and laboratory tests at the hospital. A total of 1,112 subjects were included in the study, among them, 48 with incomplete information were excluded. Diagnosis of HCMV Infection Active HCMV replication (active HCMV infection) was defined as isolation of HCMV or detection of HCMV proteins or nucleic acid from any body fluid or tissue specimen, according to the previously published literatures [Ljungman et al., 2002; Peres et al., 2010; Schoenfisch et al., 2011]. Specimens Processing Before dilatation and curettage, blood, urine, and cervicovaginal secretion samples from each subject were collected as follows. Four milliliter (ml) of venous blood from each subject was obtained, of which 2 ml in a test tube without anticoagulant, and the remaining 2 ml in a test tube with EDTA anticoagulants. The blood in the first tube was centrifuged to separate the serum for anti HCMV antibodies test, and the blood in the second tube was prepared to isolate leukocytes for HCMV DNA detection. First morning urine samples from each subject were collected in sterile specimen containers. The 2 ml of urine was centrifuged and the urinary sediment was tested for HCMV DNA. Cervicovaginal secretion samples were collected from all subjects using sterile cotton swabs. The swabs were placed in contact with the external os of cervix to absorb mucus. The swabs with the secretion were immersed directly in 2 ml of phosphate buffered saline solution and then were centrifuged. The precipitate was detected for HCMV DNA. During dilatation and curettage, placental villi and decidual samples, respectively, were obtained immediately. The placental villi and deciduas from each subject were divided into two portions, respectively. One portion was snap frozen immediately after collection for HCMV DNA analysis, and the second portion was fixed in formalin and embedded in paraffin for histopathological examination. In order to minimize the possibility of abortion tissue contamination with HCMV from cervical secretion, we disinfected vagina, cervix, and cervical tube with iodophor, and then inserted a sterile suction tube into the uterus and took decidua and chorionic villi with vacuum aspiration. Serological Testing for Anti HCMV Antibodies Serum IgM and IgG antibodies to HCMV were detected by using a chemiluminescent microparticle immunoassay (The ARCHITECT CMV IgM and IgG J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Flow diagram of women with first trimester pregnancies (normal pregnancies and miscarriages) undergoing laboratory tests. aThree cases also had positive HCMV DNA in urine, but there was no case with positive HCMV DNA in blood.
assay, Abbott Park, IL), according to the protocols described by the manufacturers. Specimens with concentration values or ¼ 500 copies/ml. HCMV DNA values in cervicovaginal secretion are < 500 copies/ml. NA, not available. d Two case with anti HCMV IgM (þ) and IgG (þ) and HCMV DNA in cervicovaginal secretion displayed that HCMV DNA () in villi and NA in deciduas. e Five cases with HCMV DNA in cervicovaginal secretion and urine showed that negative HCMV DNA in villi and deciduas. a b c
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30 (90.9) 28 (93.3) 58 (92.1) 3 (9.1) 2 (6.7) 5 (7.9) 22 (91.7) 28 (96.5) 50 (94.3) All patients with HCMV DNA in cervicovaginal secretion. Investigated specimens from miscarriage group and normal pregnancy group. Value, n (%); (þ) positive; () negative. d Five cases with HCMV DNA in cervicovaginal secretion and urine showed that negative HCMV DNA in villi and deciduas. c
b
31 (93.9) 30 (100) 61 (96.8)
2 (6.1) 3 (10) 5 (7.9)
31 (93.9) 27 (90) 58 (92.1)
1 (3.5) 1 (3.3) 2 (3.4)
28 (96.5) 29 (96.7) 57 (96.6)
2 (8.3) 1 (3.5) 3 (5.7) a
(þ)
2 (6.1) 0 (0) 2 (3.2) 0 (0) 0 (0) 0 (0) 33 (100) 30 (100) 63 (100) Miscarriage (n ¼ 33) Normal pregnancy (n ¼ 30) Total (n ¼ 63)
() (þ) () (þ) () (þ) () ()
(þ)d
Decidua(nb ¼ 24/29)
HCMV DNAc
Villi (nb ¼ 29/30)
() (þ) Groupsa
Active HCMV infection (replication) is a characteristic of primary infection, latent infection reactivation, and reinfection with a different HCMV strain. During these events, HCMV is shed in body fluids [Schoenfisch et al., 2011]. After HCMV primary infection, HCMV remains in a persistent state within the host and enters latency. Although the host’s immune system against the HCMV represses the replication of the HCMV, subsequent HCMV reactivation can still take place in the immunocompetent person [Mocarski et al., 2007]. HCMV reactivation or recurrence is defined as a new detection of HCMV replication in a patient previously diagnosed with HCMV infection and in whom HCMV was not detected for an interval of at least 4 weeks during active surveillance [Peres et al., 2010]. All kinds of active HCMV infection can occur in pregnancy and lead to intrauterine HCMV infection. During the first trimester of pregnancy, the developing embryo is susceptible to HCMV infection. Although transplacental transmission (vertical transmission) of HCMV, resulting in fetal damage or death, can occur at any stage during pregnancy, severe fetal symptoms, sequelae, and abnormalities caused by HCMV congenital infection acquired during the first trimester are more common [Pass et al., 2006; Rahav et al., 2007;
Urine (nb ¼ 33/29)
DISCUSSION
IgM
Histopathology displayed a normal placental villi and deciduas in normal pregnancy, and degradation and degeneration of placental villi and deciduas with inflammatory cell infiltration in miscarriage. There was no histopathological significant difference between the placental villi and/or deciduas with and without HCMV DNA. Specific CMV cytopathic effects (CMV inclusions and cell enlargement) had not been found by microscopic examination, so HCMV infection was not confirmed by histopathology.
IgG
Histopathology
Anti-HCMV antibody (nb ¼ 33/30)c
into miscarriage and normal pregnancy groups, retrospectively. The percentage of HCMV DNA in placental villi/deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Only two cases were tested positive of HCMV IgM serology in miscarriage group and all subjects in normal pregnancy group had negative HCMV IgM serology (Table II). The five subjects with HCMV DNA in placental villi and/or deciduas all had positive HCMV IgG serology and HCMV DNA in cervicovaginal secretion, and negative HCMV IgM serology and HCMV DNA in urine. Among the five subjects, three cases had miscarriages, and two cases had normal pregnancies (Table III). Both case 1 and case 2 with a higher HCMV loads in cervicovaginal secretion were from miscarriage patients and had a higher HCMV loads in decidua and villi, respectively.
villi/Decidua(nb ¼ 33/30)
Yan et al. TABLE II. Comparison of Anti HCMV Antibody Reactivity and HCMV DNA in Urine and Placental Villi/Deciduas Between the Two Groups With HCMV DNA in Cervicovaginal Secretion
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Placental Cytomegalovirus Infection
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TABLE III. Clinical and HCMV Related Testing Results of the Five Patients With HCMV DNA in Placental Villi and/or Deciduas HCMV DNA (copies/ml)
Serum antibodies to HCMV
HCMV DNA (copies/ml)
a
Patient Miscarriage Normal pregnancy Miscarriage Miscarriage Normal pregnancy a b
GA (weeks) 10 11 11 12 12
Villi b
NA 250.0 (þ)
IgM (AU/ml) 0.13 0.17 0.16 0.17 0.38
() () () () ()
Urine 15 (or >20)-week stillborn fetuses in the previous reports and with first trimester pregnancy (normal pregnancy and miscarriage) in our study, respectively. In this study, the positivity rate of HCMV DNA in cervicovaginal secretion (9.5%, 101/1064 cases) was similar to that in the study conducted by Tanaka et al. who displayed that vaginal CMV DNA shedding was 7.7% (76/993 cases) [Tanaka et al., 2006]. Previous study in populations of young, low-income, black, CMV-seropositive, and postpartum women showed that 39% of them had CMV-positive urine by real-time PCR for the AD169 strain DNA [Arora et al., 2010]. The higher rate of CMV-positive urine than our report may be due to the differences of study population, the preparation of urine specimen, and experimental method. HCMV DNA detection by real-time PCR is a reliable assay in diagnosis of HCMV infection [Biri et al., 2006]. A previous study revealed that HCMV DNA loads in blood was much lower than that in cervicovaginal lavage, and HCMV shedding in cervicovaginal lavage and blood was associated with markers of immunosuppression in immunodeficiency virus and HCMV seropositive women [Schoenfisch et al., 2011]. Pregnancy is a subtle state of immunosuppression [Singh and Perfect, 2007]. Pregnancy increases the chances of CMV infection or reactivation of latent CMV. A recent study indicated that HCMV could result in symptomatic congenital HCMV infections in HCMV-seropositive women; even HCMV DNA in their serum were negative ( 0.05; Spano et al., 2004]. Our study also showed J. Med. Virol. DOI 10.1002/jmv
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that HCMV DNA in placental villi/deciduas could occur in HCMV seropositive woman with HCMV DNA in cervicovaginal secretion and negative HCMV DNA in blood; negative HCMV DNA in cervicovaginal secretion, urine, and blood might not be able to cause intrauterine HCMV infection. The information also suggested that HCMV DNA loads in blood were much lower than that in cervicovaginal secretion and urine. This study demonstrates that among the 101 subjects with HCMV DNA in cervicovaginal secretion, all 101 subjects were tested positive for anti HCMV IgG antibody, whereas only two subjects, who had a negative HCMV DNA in placental villi and deciduas, were tested positive for both HCMV IgG and IgM serology. The poor correlation between IgM and HCMV replication in cervicovaginal secretion or intrauterine HCMV infection is caused by several factors: the persistence of positive IgM after the end of HCMV infection, HCMV intermittent or continuous shedding in both cervicovaginal secretion and urine throughout pregnancy [Rosenthal et al., 2009], and limited sensitivity and specificity in HCMV IgM assays [Schoenfisch et al., 2011]. Therefore, the study indicates that HCMV IgM antibody, of which the detection is still widely used for monitoring HCMV infection, might be a poor marker for active HCMV replication. Although primary HCMV infection was not documented and distinguished in this study, HCMV replication in cervicovaginal secretion or urine was the most likely explanation for active HCMV infection due to primary HCMV infection, latent HCMV reactivation or reinfection with a different HCMV strain. HCMV replication in placental villi and deciduas is crucial for its spread from infected mother to fetus [Pereira et al., 2005]. The detection of HCMV replication in placental villi and deciduas is invasive and may lead to miscarriage, hence HCMV replication in body fluids (cervicovaginal secretion, urine, and blood) could be a useful surrogate [Schoenfisch et al., 2011]. This study shows in Table III that there were two cases with a higher HCMV loads in cervicovaginal secretion, and both the cases were from miscarriage patients and had a higher HCMV loads in decidua or villi, respectively. It meant that a higher HCMV loads in cervicovaginal secretion may lead to a higher HCMV loads in decidua and/or villi and a higher miscarriage rate. In other words, high HCMV loads increase the risk of miscarriages, and high HCMV DNA loads in cervicovaginal secretion or placental villi/deciduas are associated with miscarriage. Both ascending and hematogenous CMV infection can lead to its interuterine infection. Therefore, the quantification of HCMV DNA in cervicovaginal secretion could be more than a noninvasive method for monitoring of active CMV infection, but also applicable for guiding the further screening of HCMV intrauterine infection in first trimester pregnant women. This study displayed that histopathological study of the placental villi and deciduas added little to J. Med. Virol. DOI 10.1002/jmv
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determining the etiology of CMV mediated tissue changes and potential stillbirth/miscarriage in the first trimester of pregnant women. In conclusion, in immunocompetent first-trimester pregnant women, HCMV DNA level in blood was much lower than that in cervicovaginal secretion and urine, HCMV replication in cervicovaginal secretion could involve in its interuterine infection and vertical transmission, and high HCMV replication in cervicovaginal secretion and placental villi/deciduas might increase the risk of miscarriage. REFERENCES Adler SP, Nigro G, Pereira L. 2007. Recent advances in the prevention and treatment of congenital cytomegalovirus infections. Semin Perinatol 31:10–18. Arora N, Novak Z, Fowler KB, Boppana SB, Ross SA. 2010. Cytomegalovirus viruria and DNAemia in healthy seropositive women. J Infect Dis 202:1800–1803. Bode´us M, Kabamba-Mukadi B, Zech F, Hubinont C, Bernard P, Goubau P. 2010. Human cytomegalovirus in utero transmission: Follow-up of 524 maternal seroconversions. J Clin Virol 47:201– 202. Biri A, Bozdayi G, Cic¸fti B, Din B, Yu¨cel A, Rota S. 2006. The detection of CMV in amniotic fluid and cervicovaginal smear samples by real-time PCR assay in prenatal diagnosis. Arch Gynecol Obstet 273:261–266. Dollard SC, Grosse SD, Ross DS. 2007. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol 17:355–363. Forbes BA, Bonville CA, Dock NL. 1990. The effects of a promoter of cell differentiation and selected hormones on human cytomegalovirus infection using an in vitro cell system. J Infect Dis 162:39–45. Grosse SD, Ross DS, Dollard SC. 2008. Congenital cytomegalovirus (CMV) infection as a cause of permanent bilateral hearing loss: A quantitative assessment. J Clin Virol 41:57–62. Kenneson A, Cannon MJ. 2007. Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev Med Virol 17:253–276. Iwasenko JM, Howard J, Arbuckle S, Graf N, Hall B, Craig ME, Rawlinson WD. 2011. Human cytomegalovirus infection is detected frequently in stillbirths and is associated with fetal thrombotic vasculopathy. J Infect Dis 203:1526–1533. LaMarca HL, Nelson AB, Scandurro AB, Whitley GS, Morris CA. 2006. Human cytomegalovirus-induced inhibition of cytotrophoblast invasion in a first trimester extravillous cytotrophoblast cell line. Placenta 27:137–147. La Torre R, Nigro G, Mazzocco M, Best AM, Adler SP. 2006. Placental enlargement in women with primary maternal cytomegalovirus infection is associated with fetal and neonatal disease. Clin Infect Dis 43:994–1000. Ljungman P, Griffiths P, Paya C. 2002. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis 34:1094–1097. Mocarski ES, Shenk T, Jr, Pass RF. 2007. Cytomegaloviruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th edition. Philadelphia, PA: Lippincott Williams & Wilkins, pp 2702–2772. Mussi-Pinhata MM, Yamamoto AY, Moura Brito, de Lima RM, Isaac M, de Carvalho e, Oliveira PF, Boppana S, Britt WJ. 2009. Birth prevalence and natural history of congenital cytomegalovirus infection in a highly seroimmune population. Clin Infect Dis 49:522–528. Nagamori T, Koyano S, Inoue N, Yamada H, Oshima M, Minematsu T, Fujieda K. 2010. Single cytomegalovirus strain associated with fetal loss and then congenital infection of a subsequent child born to the same mother. J Clin Virol 49:134–136. Nigro G, Adler SP, Parruti G, Anceschi MM, Coclite E, Pezone I, Di Renzo GC. 2012. Immunoglobulin therapy of fetal cytomegalovirus infection occurring in the first half of pregnancy—A case-control study of the outcome in children. J Infect Dis 205:215–227.
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