Congenital cytomegalic inclusion disease

OBSTETRICS Congenital cytomegalic Observations

in a macerated

including

a study

ANGEL

QUAN,

LOTTE

STRAUSS,

New

York,

Neal

inclusion disease

fetus with

congenital

defect,

of the placenta

M.D M.D.

York

T H E transmission of viruses from the mother to the fetus in utero is well-documented. The subject has recently been reviewed with special reference to cytomegalic inclusion disease (CID) .I The salivary gland viruses are among those known occasionally to produce serious disease in the fetus or newborn infant. In contrast to infections in later life, a great tendency toward a generalized, highly fatal disease characterizes intrauterine infection of the fetus. The mother may or may not show any sign of clinical illness, although it is generally believed that she must suffer a viremia in order to transmit virus across the placenta. Among reports of well-documented instances of intrauterine viral infections, few observations on the placenta and fetal membranes are available. Absence of placental pathology has been mentioned in intrauterine infection with Coxsackie virus’ and poliomyelitis.3 With respect to CID, Wyatt and co-workers4 briefly mention the absence of inclusions from the placentas in 2 of their 6 cases where these were examined. A study

of the placenta and membranes in a case of congenital CID was reported recently from France.“* G In view of the paucity of observations concerning the placenta in this disease, an instance of intrauterine CID is reported in which the placenta and membranes were studied. The diagnosis was based on histologic findings in the macerated fetus. Case

report

A female infant was born to a 32-year-old white gravida vi, whose blood was Group 0, Rh positive. Blood Wassermann test was negative. Previous pregnancies were entirely normal, resulting in 5 healthy children who are alive and well. The prenatal course was uneventful until one month prior to delivery when the mother had an upper respiratory infection. The fetal movements were last felt 2 days prior to the spontaneous onset of labor in the thirty-eighth week. A macerated stillborn infant was delivered without complications. Gross necropsy Cmlings. The body was that of

a macerated white female infant, weighing 3,300 grams and measuring 47 cm. in length. The abdomen was protuberant. Features of mongolism were noted, such as brachycephaly, prominent

From the Department of Pathology, Division of Pediatric Pathology, the Mount Sinai Hosfiital. Supported in part by- United States Public Health Service Grant RG6894.

nuchal fat pad, epicanthal folds, flat bridge of the nose, protruding tongue, and angulation of the helix of both ears. Two small defects were 1240

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encountered heart, one branaceum tum. The

Congenital

in the ventricular septum of the in the region of the septum memand the other in the muscular sepliver weighed 220 grams and was

markedl>enlarged and diffusely icteric. The extrahepatic bile ducts were patent. The spleen weighed 13.8 grams and was enlarged. Many lymph nodes had a hemorrhagic appearance. The adrenal glands were bilaterally enlarged, weighing together 17.7 grams. Radiologic examination of the body in the anteroposterior view revealed no definite skeletal abnormalities and no intracranial calcifications. Microscopic findings. In view of the macerated condition of the infant, all the organs were not preser\.ed for histologic examination. Description jvill therefore be limited to a few organs. The lungs showed diffuse interstitial pneumonia with numerous areas of septal mononuclear infiltration, consisting mostly of lymphocytes and plasma cells. The alveolar lining cells were enlarged, and the alveoli contained many exfoliated cells intermingled with aspirated amniotic sac contents, inflammatory cells, and occasional multinucleated foreign body giant cells. Bacterial stains revealed no microorganisms. Numerous nucleated red cells were found in the lumen of the pulmonary vessels. Among the cells lying free in the alveolar lumen, several large oval or polygonal ~~11s with intranuclear and cytoplasmic inclusions were noted. Where these cells were \vcll-preserved, their appearance was typical of the cytomegalic cells associated with salivary gland virus (SGV) disease (Fig. 1) . The nucleus was enlarged and eccentrically placed sho\+ing margination of the chromatin. In the center of the nucleus, an inclusion 8 to 10 p, in diameter with amphophilic staining properties was found; this was surrounded by a clear zone. The basophilic cytoplasmic inclusions were granular or dustlike and concentrated on one side of the cytoplasmic body opposite the nuclear pole of the cell. Relatively few intact inclusion-bearing cells were found, but an intensivc search disclosed numerous cells in varying stages of disintegration. In the kidneys, cytomegalic inclusions in tubular epithelial cells were found focally, unassociated with reactive inflammation (Fig. 2). Some of these cells were very well-preserved, others showed degenerative changes. The parasitized cells appeared to be less affected by the changes of maceration than the uninvoll.rd cells of the kidney. The spleen

cytomegalic

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1241

and the lymph nodes showed diffuse extramedullary blood formation. The liver was unsuitable for microscopic analysis. Extensive extramedullary hematopoiesis could, however, be discerned. In the adrenal glands the fetal cortex contained numerous large cells with deeply stained enlarged nuclei up to three or four times the size of normal nuclei. Some of these were deeply stained with hematoxylin, appearing pyknotic, while others showed a distinct chromatin structure and several nucleoli. An occasional nucleus contained a large round vacuole resembling an inclusion, but was distinguished from that by the absence of margination of the nuclear chromatin. The cytoplasm was eosinophilic and often vacuolated or granular. No mitoses were noted. These cells were limited to the fetal cortex and were not associated with any inflammatory rcaction. Cells resembling those described in the lungs or kidneys were not found in the adrenal glands. Many of the adrenal cortical cells were too macerated to permit histologic study. Extramedullary blood formation was present in many organs. In summary, the findings were: ( 1) diffuse interstitial pneumonia with cytomcgalic inclusion cells, (2) cytomegalic inclusions in renal tubular cells, (3) hepatomegaly with icterus of liver, (4) splenomegaly with extramrdullary hematopoiesis, and (5) extramedullary hrmatopoiesis of lymph nodes and generalized erythroblastosis. On the basis of these findings, a diagnosis of general&d cytomrgalic inclusion disease was made.

Fig. 1. foliated nuclear of SGV

Lung, microscopic section showing exlarge cell in alveolar lumen, with intraand cytoplasmic inclusions characteristic disease. (Azure blue. x950; reduced I/4.)

1242

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and

Strauss

.h.

Fig. 2. Kidney, microscopic section showing ular epithelial cells containing intranuclear cytoplasmic incIusions. (Hematoxylin and ~600: reduced $4.)

tuband eosin.

Additional features were mongolism, congenital cardiac defect, and anaplasia of fetal adrenal cortical cells. Placenta. Gross description. The placenta was roughly round and measured 16.5 cm. in largest diameter and weighed 500 grams together with the membranes. The attached segment of umbilical cord was 30 cm. in length and inserted eccentrically in the placenta. It was thick and macerated and had a corkscrew appearance. A flattened segment was seen about midway bctwccn the two ends. The fetal surface of the placenta showed a green discoloration. The ~cssels in the chorionic pIat? were estrcmrly engorged. Between them could

Fig. 3. Placenta, microscopic showing bulky chorionic villi creased, somewhat cellular (This picture resembles that hydrops fetalis.) (Hematoxylin eosin. x150; reduced w,.)

section with instroma. seen in and

May 1, IY62 J. Obst. & Gynec.

be seen numerous flat, white plaques within the chorion. On the maternal surface the cotyledons were somewhat indistinct. Numerous small foci of calcification were noted. On section the placcntal tissue appeared firmer than usual and somewhat pale. No infarcts wert> noted and the appearance of the placental substance Ivas Imiform throughout. The membranes had an opaque surface and appeared thicker than normal. Multiple tiny whitish flecks wcrc seen in the membrane beneath the amnion which could IW stripped with ease from the underlying chorion. The placenta was fixed i~z toto in 10 per cl,nt buffered formalin. Histologic drscription. Multiple tism: blocks \Y~I‘C cxanlincd including the placenta proper, ~hc decidua, the chorionic plan,. the un~bili~al cord, and the amniotic sac. These Ivere stained with hematoxylin and eosin. Selected ulides were also stained by special methods such as bacterial stains, PAS technique, phosphotungstic acidhrmatoxylin, toluidine blue, and stains for connective tissue and iron pigment. The (.horionic. villi had a11 ill{ W;IVYI tliartl~~~er They were bulky and richly \-nsculnrized, the capillaries being distributed throughout their c-ore (Fig. 3). The villous stron1,t ~vns abundant and rich in fibers disposed in n network about the capillaric,s. Th,> numl)(-~~ of ctromal cr>lls was variable though generallv increased when compared to normal placentas in the third trimester. The rndothelial cells ~vere slightly promincnt. An occasional villus shon-cd round ccl1 infiltration. r\n intrnsixre search in num,‘rnrls blocks

Congenital

of placental tissue did not disclose cells containing the characteristic inclusions. The lining cells of the villi were well-preserved, consisting mainly of syncytial trophoblast. Large numbers of nucleated red cells were noted in the lumen of the chorionic vessels (Fig. 4). Small granules of fragmented nuclear material were scattered here and there in the villi. Deposition of placental fibrinoid was seen in the usual locations, in small clumps between chorionic villi, in the placental scpta, and bcncath the decidua and the chorionic plate. The walls of blood vessels showed no significant changes, except for marked engorgement of the larger ramifications of the umbilical vessels, especially in the chorionic plate. Stain for hemosiderin showed focally distributed granular deposits in the basement membrane of the trophoblast and fine granular deposits in some capillary endothelial cells and stromal cells of the villi. The umbilical cord was free from inflammatory changes. The chorionic plate was for the most part denuded of its amniotic lining. Whcrc present, this appeared normal, consisting of low columnar or cuboidal cells with uniform small nuclei and vacuolated cytoplasm. The layer of homogcmeous ground substance immediately bcneath the amnion was edematous as was the chorionic plate which showed a mild infiltration with polymorphonuclrar leukocytes. More striking alterations were observed in sections of the reflected portion of the amniotic sac (Fig. 5). The amniotic epithelium was partially macerated. Where recognizable, its appearance was not unusual. The subepithelinl layer of IOOW mcxsenchymal tissue was very edematous and intiltratcd by widely scattered polymorphonuclear leukocytes. Denser focal collections of inflammatory cells were also seen, especially in the deeper portion of this layer. These collections seemed to be concentrated about cellular or nuclear debris. The most severe changes were noted in the chorion laeve which consisted of sc*veral layers of closely packed chorionic cells, Many of these cells had undergone complete n<,crosis accompanied by a severe exudative reaction resulting in collections of necrotic debris intermingl(~d with infln&matory cells (Figs. 5 and 6). This zone of inflammatory exudation and cc,llular breakdown appeared almost continuous. with focal arc’as of larger, abscess-like collections bctwc-cn the chorion and the decidua. .Zn intl.nsivc search for inclusions in the chorionilcells !\-a$ rmcucccssful. Either thrar cells

cytomegalic

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Fig. 4. Placental villus, higher magnification. Note particularly the large number of nucleated red blood cells in chorionic capillaries. (Hematoxylin and eosin. x400; reduced I/!.) appeared normal or they were reduced to debris. Bacterial stains revealed no microorganisms. The decidua capsularis showed no noteworthy changes. In summary, the placenta revealed definite, though nonspecific diffuse changes which involved both the villous mesenchyma and the membranes, particularly the chorion laeve. The histopathologic changes of the chorionic villi resembled those of severe hemolytic disease of the newborn infant. Inflammation was seen predominantly in the amnion and chorion and was minimal in the villous mesenchyma. Comment The

diagnosis

at autopsy

by

the

characteristic

by

the

cells or

the liver

comparatively and

CID

is often

cells.

during

Diagnosis

biopsy”

has

few

more

gained

the

secretionql*

been

reported

Since

tissue

from

the

culture human

information distribution

of life,

inclusion-bearing gastric

cases. in

cretions,*“-14 about

of in

agent

only

in tissues

urine,7-10

propagation

pathogenic

made

demonstration

demonstration

in by

of the

in

isolation of

tissues

a cytoor

has of the

se-

been virus

in

1244

Quan

May 1. lY62 -\rn. .I. Obst. & Cynec.

and Strauss

Fig. 5. Reflected portion of amniotic sac: showing acute chorioamnionitis, with edema and abscesslike

collections

of

necrotic

matory sells. (Hematoxylin &Iced ~~ 1

chorionic

and

inflam-

and eosin. x100; re-

humans and its role in producing disease in humans. It has been shown that subjects without clinical SGV disease may excrete virus in the saliva or urine.‘” This supports the view that human SGV is widely disseminated and that its presence in the tissues does not necessarily lead to serious illness. The “accidental” discovery of the characteristic cells in one or more organs in routine autopsies of patients dying of unrelated causes is familiar to pathologists. Reported analyses of autopsy material for the incidence of SGV infection include mostly young infants and children and relatively few stillborn infants or newborn infants under 2 weeks of axe. The incidence is varyingly reported as from 10 to 18 per cent in routine autopsies in thr pediatric a,qe Lgroup. “,-‘!’ These series include few newborn infants 3 days old or less. and no stillborn babies. Potter’ states that she had not observed a single case of CID in over 8,000 autopsies on stillborn and newborn infants. Among 89.5 consectltive autopsies, compris-

ing 450 fetal and 445 neonatal deaths, this is the only instance of SGV disease observed in this laboratory. Rowe and associatrs’ \vcrc unable to isolate SGV from saliva or llrinc of 108 normal newborn infants under 5 days. On the other hand: among children between 8 months and 4 years who had complement fixing antibody, 63 per cent were found to excrete virus, without clinical c\idcncc of disease. These obser\,ations suggest that intrauterine infection with SGV is rare. ‘r’his may be explained by tilts high degree of immunity in the adult population and the passive transfer of protective antibodies across the placenta. It may be that the fetus only becomes infected when the mother has not been previously imrnlmi/ed and contracts a new infection during prcgnanq. or under conditions where her ilnrnlinity breaks down, leading to viremia. IIIelusion-bearing cells in stillborn or newborn infants are usually associated with generalization of the infection1 It is felt that the fetus under discussion had a significant infection with the SGV and that this was probably responsible for death in utero. While it is true that the pathognomonic cells were encountered in only two organs, the lungs and the kidneys, one must make allowance for difficulties in the histologic examination. Corroborating evidence for a severe generalized disease associated with SGV infection in this fetus consists in (I 1 the se\-erity of the pneumonia, (21 hepatosplenomegaly. ( 3 ) crythroblastosis in the absence of blood group isoimmunization. and (4) diffuse alterations in the placenta. It is ~~11 known that intrauterine infections such as SGV disease, toxoplasmosis, or syphilis may manifest themsclv~s as hydrops fetalis or hemolytic disease’, “‘9 ‘I and thus simulate isoimmunization disease. Since it is likely that dissemination of SGV takes place by way of the blood strram. OIW would expect to find the characteristic inclusions in celIs of the placenta. It appears that SGV has a special affinity for epithelial cells: however, mesenchymal cells may be in\-aded as well, both in \ivo and in tissue culture. In the placenta studied by Le-Tan-

Congenital

Vinh,‘; rare cells containing cytomegalic inclusions were found in a single villus, apparently only after a prolonged search, since these had not been reported in an earlier paper by Lepage and Schramm’ referring to the same case. These authors described definite though nonspecific alterations in the placenta and fetal membranes strikingly similar to those observed by us. While we did not observe plasma cells in the stroma of chorionic villi, focal infiltration of villi with mononuclear cells resembling lymphocytes was noted. Infiltration of the fetal part of the placenta with plasma cells is rare; it has been observed mostly in conditions seemingly not associated with specific fetal infrctions.~‘-~ ~ The appearance of plasma cells in the fetal lung is noteworthy since inflammation in fetal life is not usually associated with plasmocytic infiltration, but more commonly with proliferation of other mesenchymal cells an d with extramedullary hematopoiesis.‘” The absence of plasma cells from fetal inflammation appears to be in accordance with the inability of the fetus and newborn infant to produce antibodies. As a rule, plasma cells are not found in aseptic inflammation until the third month of postnatal life, although exceptions seem to existz5 Examination of lungs in cases of diffuse intrauterine pneumonia of presumably bacterial origin at \.arious gestational ages has not revealed

Fig.

6.

Chorion

laeve,

higher

tnagnification showing inflammatory infiltration and nuclear debris. x400;

(Hematoxylin reduced ye.)

and eosin.

cytomegalic

inclusion

disease

1245

plasma cells. The observation of mature plasma cells in this case of SGV pneumonia suggests that, with appropriate stimulation, this type of inflammatory response and perhaps antibody production may occasionally be evoked during intrauterine life. A rise in circulating antibodies after antigenic stimulation has been reported in infants as young as 1 weekzc The coexistence of SGV infection and socalled cytomegaly” or anaplasia’” of cells in the fetal adrenal cortex is considered fortuitous. Although a resemblance of the cytologic alterations in the fetal adrenal cortex to SGV inclusions has been mentioned,‘? there is no indication that these phenomena are related. Craig and Landing” state the absence of SGV disease in 37 instances of adrenal cortical anaplasia which they analyzed. In 10 out of 11 instances of adrenal cytomegaly, observed by one of us in autopsies on fetuses and newborn infants up to 71/2 weeks of age, no evidence of SGV infection was found. The simultaneous occurrence of developmental anomalies and CID has heen noted by others. Seifert and Oehme” report congenital malformations in 16 per cent of 76 cases of CID in infants. Additional cases have been reported by others.?“, x Since the Australian experience with rubella, attention has been focused on the role of maternal viral infection in the genesis of dcvelop-

1246

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and

Strauss Am.

mental anomalies in the fetus. In contrast to the fetal anomalies following maternal rubella, the malformations described in association with CID have not followed any characteristic pattern. Furthermore, it is not possible to time the infection of the mother in the absence of clinical disease. Thus the causal relationship between infection and fetal anomaly in this disease remains highly problematic, and it would be premature to speculate on this. The fallacy of such speculation is perhaps well-illustrated by our case. Since the discovery of a chromosomal abnormality in mongoloid subjects,31, 32 the concept that this anomaly may result from an acquired insult to the early embryo is no longer tenable. It may be, on the other hand, that fetuses or newborn infants with developmental abnormalities are more prone to a generalized infection than normal subjects. The alterations in the placenta closely resemble those described by Lepage and Schramm” who also encountered features of erythroblastosis as well as a nonspecific inflammatory reaction with formation of abscesses in the chorion laeve. The absence of inclusions from the stromal as well as the chorionic cells does not necessarily imply that the inflammation in the chorion is not due to the action of the salivary gland virus. In chronic experimental infection with mouse salivary gland virus, it was found that inclusions disappeared from the salivary glands after the acute stage, while virus could still be recovered from the saliva.33 Arey9 observed few cytomegahc inclusions at the autopsy of an infant who had died about 12 months after a diagnosis of SGV infection had been made. It is conceivable that the observed changes in the chorion represent a relatively advanced phase of the infection with complete breakdown of the involved cells, giving rise to the conspicuous inflammatory reaction. It is usually thought that the fetus acquires the disease by transplacental transition are not fully understood. Viruses, as mission, although the pathways of the infecwell as larger organisms such as protozoa,

hlay 1, 1962 J. Obst. Fi Gynrc.

presumably can cross the placental barrier without producing specific structural changes in that organ. In some well-documented cases of congenital toxoplasmosis, the only changes observed in the placenta have been those usually associated with hydrops fetalis.“’ ‘I, 34 These changes, which have likewise been observed in the few placentas studied in SGV infection of the fetus. must be considered as nonspecific and not diagnostic unless the characteristic inclusions can he found. It is not possible to delineate clearly the pathways of infection of the fetus and membranes on the basis of histopathologic observations. The duration of the infection is not known unless one considers the influenzaIike symptoms of the mother one month prior to the onset of labor as evidence of a transient dissemination of the infection which led to its transmission to the fetus. In the light of the structural alterations in the fetal membranes and in the fetal lung, it seems interesting to re-evaluate the mamrer in which the infection took place and spread in the fetal tissues. It cannot be denied that the character of the inflammation of the membranes bears a resemblance to the membranitis seen in nonspecific bacterial infections. Recently Benirschke35 has pointed out that while intrauterine bacterial infections usually represent amniotic infections transmitted to the fetus by swallowing or inhalation, viral infections tend to take the hematogenous route across the maternal-fetal blood stream. The absence of placental inflammation in some of the known viral diseases, together with the wide dissemination of viral infections in the fetus, seems to bear out this assumption. On the other hand, it is not impossible that viruses may be introduced into the fetus in a manner similar to that of the common bacterial infections and that hematogenous spread in the fetus occurs as a secondary step in the evolution of the disease. This possibility is suggested by the histopathologic findings in the 2 cases of CID of the fetus in which the placenta has heen studied in greater detail. The inflammatory changes appear to be most advanced

Congenital

and severe in the reflected portion of the fetal membranes, more particularly in the chorion laeve where many of the chorionic cells have been completely destroyed. The accompanying amnionitis is far less severe, being characterized mostly by edema and a less massive polymorphonuclear infiltration. The seemingly selective destruction of chorionic cells may indicate affinity of the infecting agent for these cells in preference to other placental tissue components, and the advanced changes in this area point to it as a possible primary localization of the infection. With exception of the diagnostic cells and the more striking interstitial inflammation in the lung, the pneumonia has much the characteristics of the diffuse aspiration pneumonia of fetal bacterial infections secondary to chorioamnionitis. The finding of inclusions in endothelial or other mesenchyma1 cells of the placental villus is no proof of the hematogenous route of intrauterine infection of the fetus, since the placenta may become invaded after hematogenous dissemination has taken place in the fetus. It is quite likely indeed that the described changes in the placental viIIi are accompaniments of the fetal disease, namely, enlargement of the villi, focal inflammatory infiltrations, and erythroblastosis. On the other hand. initial infection of the fetus by the hematogenous route cannot be excluded. If this be the case, the membranes would be damaged after excretion of virus into the amniotic fluid with the fetal urine and thus the infection reintroduced into the lungs by inhalation of the virus-containing amniotic fluid. Up until now, too few observations on the placenta in this disease are available to permit full evaluation of the evolution of the patholo$c changes.

cytomegalic

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1247

Diagnosis of SGV infection of the fetus during pregnancy or at birth is theoretically feasible, considering that the amniotic fluid is likely to contain the virus. Because of the rarity of the disease, a search for this infection on a larger scale during pregnancy is hardly warranted. On the other hand, examination of the placenta may be a valuable aid in finding instances of congenital infection with this virus. A combination of hydropic changes of the villi, erythroblastosis, and necrotizing chorionitis, should direct the search toward SGV infection in the newborn infant.

Summary

and

conclusions

A case of intrauterine infection with SGV associated with fetal death presented the rare opportunity to study the placenta in congenital CID. Diffuse alterations in the placenta and fetal membranes were observed. These were nonspecific and resembled those seen in erythroblastosis fetalis, with the exception of a diffuse necrotizing inflammation in the chorion laeve and an acute nonspecific amnionitis. Cytomegalic inclusions were found in the fetal tissues but were not found in the placenta. The association of SGV infection with congenital defect (mongolism, cardiac anomaly) and with so-called adrenal cytomegaly is critically discussed. This case emphasizes the value of thorough anatomical study of cases of unexplained fetal death, even when maceration has set in. Examination of the placenta may be a valuable aid to the pediatrician in directing his search toward unsuspected and unusual infection in the newborn infant.

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Lepage, F., and Schramm, obst. 57: 273, 1958. Le-Tan-Vinh: Publ. Centre fance, Paris 1: 243, 1959. Fetterman, G. H.: Am. J. 424, 1952. Mercer, R. D., Luse, S., and Pediatrics 11: 502, 1953.

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