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Twins, placentas, and genetics: Acardiac twinning in a dichorionic, diamniotic, monozygotic twin gestation
HUMAN PATHOLOGY
Volume 29, No. 9 (September 1998)
16. Plaut M, Pierce JH, Watson CJ, et al: Mast cell lines produce lymphokines in response to cross-linkage of Fc RI or to calcium ionophores. Nature 339:64-67, 1989 17. Hamid Q, AzzawiM, Ying S, et al: Expression of mRNAfor intefleuldn-5 in mucosal bronchial biopsies from asthma. J Clin Invest 87:1541-1546, 1991 18. Dubucquoi S, Janin A, Desreumaux P, et al: Evidence for eosinophil activated in eosinophilic cystitis.Eur Uro125:254-258, 1994 19. Navarro-Roman L, Medeiros LJ, Kingman DW, et al: Malignant lymphomas of B-ceUlineage with marked tissue eosinophilia: A report of five cases. AmJ Surg Patho118:347-356, 1994
20. Arinaga S, Krimine N, Takamuku K, et al: Correlation of eosinophilia with clinical response in patients with advanced carcinoma treated with low-doserecombinant interleukin-2 and mitomycinC. Cancer Immunol Immunother 35:246-250, 1992 21. Huland E, Huland H: Tumor-associated eosinophilia in interleukin-2treated patients: evidence of toxic eosinophil degranulation on bladder cancer cells.J Cancer Res Clin Onco1118:463-467,1992 22. Enoldhara H, Kajitani H, Nagashima S, et al: lnterleuldn 5 activityin sera from patients with eosinophilia. BrJ Haemato175:458-462, 1990 23. Prin L, Plumas J, Gruart V, et al: Elevated serum levels of soluble interleukin-2 receptor: A marker of disease activityin the hypereosinophilic syndrome. Blood 78:2626-2632, 1991
TWINS, PLACENTAS, AND GENETICS: ACARDIAC TWINNING IN A DICHORIONIC, DIAMNIOTIC, MONOZYGOTIC TWIN GESTATION CHRISTOPHER A. FRENCH, MD, FREDERICKR. BIEBER, PHI), DAVID H. BING, PHD, AND DAVID R. GENEST, MD
We describe a human acardiac twin with associated vascular anastomoses in a dichorionic diamulotic fused twin placenta. A 22-year-old woman delivered a healthy 3,554 g male infant and a fused dianmiotic dichorionic twin placenta with a 230g umbilical cordattached, skin-covered, ovoid mass, consistent with acardiac amorphus. By gross and histological examination, the placental dividing membranes comprised four leaves, one amnion from each placenta, and two centrally fused chorions, diagnostic of dichorioulcity. Placental barium injection of the normal twin's umbilical vein showed an anastomosis with the acardiac twin which traversed the dividing membranes, then supplied major vessels of the acardiac mass via its
5.5 cm umbilical cord. DNA-typing studies of the normal twin's placenta and of the acardiac twin's tissues revealed identical alleles at 11 distinct genetic polymorphic loci, consistent with monozygosity. Our findings demonstrate that vascular anastomoses can occur in dichorionic twin placentas, and that human acardiac twinning is not, as heretofore believed, restricted to monochorioulc placentas. HUM PATHOL29:1028-1031. Copyright © 1998 byW.B. Saunders Company Key words: acardia, twins, placenta, malformation, zygosity. Abbreviations: MoMo, monoamniotic monochorioulc; DiMo, diamulotic monochorioulc; DiDi, dianmlotic dichorionic; UC, umbilical cord; MZ, monozygotic; DZ, dizygotic.
Acardiac twins r e p r e s e n t rare and bizarre malformations f o r m e d in the setting of major placental vascular anastomoses between twins or h i g h e r multiple births. W i t h o u t such vascular anastomoses, acardiac twins would have n o b l o o d flow. Reversed arterial perfusion has b e e n p r o p o s e d as the specific type of placental vascular anastomosis which is causally related to acardiac twinning. 1,2 Extensive placental artery-artery and vein-vein anastomoses reverse the n o r m a l direction of b l o o d flow t h r o u g h the acardiac's umbilical cord vessels such that arterial b l o o d flows toward and venous b l o o d flows away f r o m the acardiac twin. D o p p l e r studies have c o n f i r m e d this reversal of circulation in vivo. 2,3 In m o n o c h o r i o n i c h u m a n twin placentas, the frequency of artery-to-artery vascular anastomoses is 64%, 4 and the incidence of acardia is approximately 1%.5 In contrast, dichorionic h u m a n twins only very rarely f o r m placental vascular anastomoses6,7; it follows that h u m a n dichorionic acardiacs should be exceedingly rare. In fact, to our knowledge no case has b e e n reported. However, in o t h e r animals vascular anastomoses between dichorionic twin placentas have b e e n described, Ls and cases of acardiacs in cytogenetically proven dizygotic bovine twin pregnancies have b e e n reported. 9,1° It is thus conceivable that, t h o u g h exceptional, h u m a n acardiacs m i g h t occur in diamniotic dichorionic (DiDi) placentas. We r e p o r t the gross, light microscopic, immunohisto-
chemical, and genetic features of a h u m a n acardiac twin occurring with superficial vascular anastomoses in a DiDi placenta, showing that vascular placental anastomoses and acardiac twinning are n o t restricted to m o n o c h o r i o n i c placentas.
From the Department of Pathology, Brigham and Women's Hospital, Harvard Medical School; and the CBR Laboratories, Center for Blood Research, Boston, MA. Address correspondence and reprint requests to David R. Genest, MD, Department of Pathology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. Copyright © 1998 by W.B. Sannders Company 0046-8177/98/2909-002358.00/0
CASE R E P O R T A 22-year-old gravida 3 para 1, spontaneous abortus 1 p r e g n a n t w o m a n had an obstetrical ultrasound examination at 13.7 weeks that revealed a twin p r e g n a n c y with p r e s u m e d fetal demise of one twin at 9.5 weeks. A subsequent ultrasound p e r f o r m e d at 41 weeks revealed a n o r m a l fetus and an 8 × 10 cm n o d u l e suggestive o f a leiomyoma, a placental mass, or the p r e s u m e d deceased twin. At 42 weeks, a 3,554g healthy male fetus and a placenta with an encapsulated, "double-fist-sized" mass (acardiac) was delivered vaginally. T h e healthy twin is currently alive and well at 16 months. MATERIALS AND M E T H O D S Gross Placental Examination
This was c o n d u c t e d according to the m e t h o d s described elsewhere. 6 T h e dividing m e m b r a n e s were separated with forceps to reveal the n u m b e r of layers. A Barium solution consisting of 340 m L n o r m a l saline, 10 m L diethylene glycol histological grade (Fisher Scientific Co.), 30 g m Bacto-Gelatin (Difco Laboratories, Detroit, MI), and 260 m L Micropaque (Picker X-Ray Corp., White Plains, NY) was w a r m e d to 98.6°F and injected via a 14-gauge n e e d l e attached to a b u l b e d syringe into the n o r m a l twin's umbilical vein. T h e Barium solution was kept f r o m leaking d u r i n g injection by suturing the umbilical cord a r o u n d the syringe needle, proximal to its o u t p u t bulb.
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CASE STUDIES
Immunohistochemistry Immunohistochemical stains for keratin proteins (AE1AE3, Boehringer Mannheim, Indianapolis, IN) were performed on formalin-fixed, paraffin-embedded tissue sections of the dividing membranes. Antibodies (1:2,500 dilution) were incubated for 37°C for 32 minutes, pretreated with pronase for 8 minutes at 37°C, and processed on a Ventana ES Automated Immunostainer (Ventana Medical Systems, Tucson, AZ) using an avidin-biotin peroxidase detector system.
PCR Analysis DNA extracted from formalin-fixed umbilical cord and cutaneous tissue from the acardiac, and amnion and chorion from the normal twin were typed for allelic polymorphisms at 11 different genetic loci (HLA DQA1, LDLR, GYPA, HBGG, D7S8, Gc, D1S80, CSF1PO, TPOX, THO-1, Amylogenin) using commercially available kits from Perkin-Elmer, Applied Biosystems Division (Norwalk, CT), and Promega Corp. (Madison, WI). RESULTS
Placenta and Acardiac Twin The fused twin placenta (Fig 1) weighed 450 grams. The umbilical cord of the healthy twin was centrally inserted and trivascular; that of the acardiac was marginally inserted and contained two vessels (ie, only a single umbilical artery). The placental tissue associated with the healthy twin (18 X 13 × 2.5 cm) contained a single 0.5 cm intervillous thrombus. The placental tissue associated with the acardiac (11.5 × 5 × 2.5 cm) was grossly unremarkable. An intact, opaque dividing membrane was present, consisting of two amnions and a fused dichorionic layer (Fig 2). A large, superficial vein-to-vein placental anastomosis was present. The umbilical cord of the healthy twin was clamped and its vein injected with barium. The barium passed through the anastomosis into the cord and systemic circulation of the acardiac twin (Fig 3). The acardiac twin (13.5 × 6.5 × 4.5 cm, 230 g) was a pink, skin-covered ovoid mass with multiple dimples and a caudal, 1.9 cm, yellow, multiloculated, fluid-filled cyst. Radiographs and dissection revealed subcutaneous tissue, a spinal column with poorly developed ribs, and an amorphous,
FIGURE !. Fused diamniotic dichorionic twin placenta (after membrane removal) with acardiac fetus a t t a c h e d via its umbilical cord. The fused dichorion of the dividing membrane has been o p e n e d to reveal a superficial venous anastomosis traversing the membranes.
FIGURE 2. The separated placental dividing membrane was composed of two amnions (arrow heads) and two chorions (arrow).
cartilagenous/bony "cephalic" end. No cranium, internal organs, or limbs were identified. This twin is best described as the rare variant of acardiac known as "acardius amorphus." The vascular anatomy of the acardiac twin and its placenta was revealed by radiographs after barium injection of the umbilical vein of the healthy cotwin. The barium-filled umbilical vein bifurcated on entering the acardiac. These vessels eventually converged at a blind-end sac near the center of the acardiac corpus (Fig 3). Histological study of the dividing membranes confirmed the presence of two distinct amnions and two distinct chorions (Figure 4A). Because the amniotic epithelium of the acardiac's sac was extensively debrided (presumably from severe oligohydramnios), immunohistochemical evaluation for keratin proteins (AE1/AE3) was used to confirm residual amniotic epithelium (Figure 4B). Both placentas were histologically mature.
PCR DNA typing revealed allelic identity at each of 11 distinct genetic loci typed from tissues obtained from the twins (see Table 1), as would be expected in monozygotic twins. Identity at the amylogenin locus for both X and Y alleles confirmed the male sex chromosome constitution of both twins.
FIGURE 3. Barium injection of the normal twin's umbilical vein traversed the dividing membranes and anastomosed with the acardiac's vasculature.
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HUMAN PATHOLOGY
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FIGURE 4. (A) Hematoxyiin and eosin histology of the dividing membrane shows two chorions (C) and two amnions (A), but the acardiac's amniotic epithelium (arrow) is poorly preserved, presumably because of oligohydramnios. (B) Immunohistochemistry for AE1/AE3 confirms reactivity of acardiac's debrided amniotic epithelium for keratin (arrow); original magnification ×150, beth panels. DISCUSSION Acardia refers to a broad spectrum of abnormal fetal morphologies ranging from an amorphous mass, to a recognizable fetus with multiple severe anomalies. The most complete description of the various morphotypes is provided in the 1898 text by Schatz. t In general, acardia is characterized by failure of cranial and thoracic (heart and lung) development, 11 with fetal life entirely supported by a reversed nmbili-
TABLE l . identical Allelic Polymorphisms Found at Each of 11 Loci Tested From the A c a r d i a c Twin a n d its Normal Cotwin Gene Locus
Chromosomal Map Location
AllelesDetected
HLA-DQAI LDLR GYPA HBGG D7S8 Gc DIS80 CSF1PO TPOX THO1 Amylogenin
6p21.3 19p13.1-13.3 4@8-31 11p15.5 7q22-31.1 4q11-13 1p36-34 5q33.3-34 2p13 11p15.5 Xp22.1-22.31 ,Ypll.2
0101, 0201 A, B A, B A A B, C 18 11, 12 8, 11 9.3 X, Y
cal blood flow from the cotwin via extensive placental arteryartery and vein-vein anastomoses. 2,3 Although documented DiDi placental anastomoses are exceptional, monoamniotic monochorionic (MoMo) and diamniotic monochorionic (DiMo) placental anastomoses occur in most reported cases. 6 It is believed that acardia develops in the setting of large vascular anastomoses in a twin with a primary anomaly whose circulation becomes dependent on the reversed circulation provided by the eotwin. 1,6,12Evidence which supports this idea includes an extra marker chromosome found in fibroblasts cultured from an acardiac but not its cotwinlS; a triploid female acardiac arising from fertiliza• tion of the polar body of its n o r m a l twin14; and a 46,X,i(X) (pl0) hydropic acardiac whose cotwin's karyotype was normal? 5 However, the karyotypes of both the acardiac and normal twin are most often normal16; in addition, both may have the same cytogenetic abnormality17; or, the acardiac may have a normal karyotype whereas the normal cotwin has an abnormal karyotype, is Benirschke suggests, as the cytogenetic picture is complex and difficult to interpret, and because the reversal of circulation must be a very early event, reversed circulation alone is responsible for the formation of acardiacs. 6 Benirschke's theorizes that circulatory reversal causing acardia is consistent with the hypothesis of original normal-
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CORRESPONDENCE ity. 19'20 This was d e r i v e d f r o m t h e o b s e r v a t i o n t h a t acardiacs o c c u r in f r a t e r n a l b o v i n e a n d ovine twins, w h i c h p r e s u m a b l y were originally n o r m a l , b u t b e c a m e a n o m a l o u s a f t e r vascular a n a s t o m o s e s a n d c i r c u l a t o r y reversal was established. 9,1° To o u r knowledge, t h e p r e s e n t case is t h e only docum e n t e d e x a m p l e o f a h u m a n acardiac arising in f u s e d DiDi, m o n o z y g o f i c p l a c e n t a s with vascular anastomoses. This shows t h e f u n c t i o n a l c o n s e q u e n c e s o f m a j o r vascular a n a s t o m o s e s in this rare setting, a n d raises q u e s t i o n s with r e s p e c t tO o u r c u r r e n t u n d e r s t a n d i n g o f p l a c e n t a l vascularization. T h e t e r r i t o r y o f vascular n e t w o r k f o r m a t i o n i n p l a c e n t a l d e v e l o p m e n t is t h o u g h t to b e limited by t h a t r e g i o n d e m a r c a t e d by t h e c h o r i o n i c m e m b r a n e s . 6 T h u s , M o M o a n d DiMo p l a c e n t a s c o m m o n l y have vascular anastomoses, w h e r e a s DiDi p l a c e n t a s theoretically d o not. T h e diagnosis o f DiDi placentation with vascular a n a s t o m o s e s rests o n t h e histological confirm a t i o n o f two layers of a m n i o n , a n d two layers of c h o r i o n within t h e dividing s e p t u m , a n d t h e d e m o n s t r a t i o n 0 f i n j e c t e d dye t r a n s m i s s i o n f r o m a vessel o n o n e p l a c e n t a i n t o a vessel o n t h e other. U s i n g this m e t h o d o f diagnosis, very rare cases 7 have s h o w n u n e q u i v o c a l DiDi-fused p l a c e n t a s with large vascular anastomoses. T h e s e cases, i n c l u d i n g t h e p r e s e n t one, a n d t h e fact t h a t DiDi p l a c e n t a t i o n with vascular a n a s t o m o s e s regularly occurs in s o m e m a m m a l s , c h a l l e n g e s t h e c o n c e p t t h a t c h o r i o n i c m e m b r a n e s are strict b a r r i e r s to vascular network formation. REFERENCES 1. Schatz F: Die Acardii und ihre Verwandten. Hirschwald, Berlin, 1898 2. Benson CB, Bieber FR, Genest DR, et al: Doppler demonstration of reversed umbilical blood flow in an acardiac twin.J Clin Ultrasound 17:291-295, 1989
3. Pretorius DH, Leopold GR, Moore TR, et al: Acardiac twin-report of Doppler sonography.J Ultrasound Med 7:413-416, 1988 4. Leroy F: Major feta! hazards in multiple pregnancy. Acta Genet Med Gemello125:299-306, 1_976 5. Gillim DL, Hendricks CH: Holoacardius: Review of the literature and case report. Obstet Gyneco! 2:647-653, 1953 6. Benirschke K, Kaufman P: pathology of the Human placenta ed 3. New York, Springer-Verlag,1995, pp 778-786 7. Cameron AH: The Birmingham Twin Survey.R Soc Med Proc 61:229,34, 1968 8. Wislocki GB: Observations on twinning in Marmosets. Am J Anat 64:445-481, 1939 9. Dunn HO, Lein OH, Kenney RM: The cytological sex of a bovine anidian (amorphous) twin monster. Cytogenetics 6:412-19, 1967 10, Dunn HO, Roberts sJ: Chromosome studies of an ovine acephalicacardiac monster. Cornell Vet 62:425-31, 1972 11. Winter RM, Knowles SAS, Bieber FB, et al: The Malformed Fetus and Stillbirth. New York,John Wileyand Sons, 1988, p 227 12. Gruenwald P: Early humar~ twins with peculiar relations to each other and the chorion. Anat Rec 83:267-269, 1942 13. Turpin R, Bocquet L, Grasset J: Etude d'un couple monozygote: fille normale-monstre acardique feminin: Considerations anatoma-pathologiques et cytogenetiques. Ann Genet (Paris) 10:10%113, 1967 !4. Bieber FR, Nance WE, Morton CC, et al: Genetic studies of an acardiac monster; evidence of polar body twinning in man. Science 213:775-777, 1981 15. Wolf H, MacDonald J, Bradford W: Holoacardius anceps with abnormal karyotype and multicystic renal dysplasiawith patent ductus arteriosus in a surviving c0-twin.Mod Pathol 3:10P, 1990 (abstr) 16. Buehler BA, McManus BM, Mrocezek EC: Acardiac monsters: C!inical karyotype and pathologic features. Teratology 33:44C, 1986 (abstr) 17. Ginsberg NA, Applebaum M, Rabin SA, et al: Term birth after midtrimester hysterotomy and selective delivery of an acardiac twin. Am J Obstet Gynecol !67:33-37, 1992 18. Landy HJ, Larsen JW, Jr, Schoen M, et al: Acardiac fetus in a triplet pregnancy. Teratology 37:1-6, 1988 19. Claudius M: Die Entwicklung der herzlosen Missgeburten. Schwers, Kiel, 1859 20. Ahlfe!d F: Beitrage zur Lehre yon den Zwillingen. VI. Die Entstehung der Acardiaci. Arch Gynakol 14:321-360, 1879
CORRESPONDENCE To the Editor:--We r e a d with i n t e r e s t t h e article by Tomashefski a n d associates ! r e g a r d i n g the p a t h o g e n e s i s o f pulm o n a r y i n t r a l o b a r s e q u e s t r a t i o n (ILS). We feel it is u n f o r t u n a t e t h a t a few previously r e p o r t e d i m p o r t a n t studies 2,~ strongly i n d i c a t i n g t h e c o n g e n i t a l o r i g i n o f ILS have n o t received e n o u g h a t t e n t i o n . T h e s e studies m a d e clear t h a t g e n u i n e ILS h a s n o t only a s e p a r a t e arterial system b u t also a c o m p l e t e l y d i f f e r e n t b r o n c h i a l system f r o m t h a t o f t h e r e m a i n i n g n o r m a l lung, t h a t is, t h e b r o n c h i a l system t h a t b e g i n s as a b l i n d - e n d e d b r o n c h u s n e a r t h e e n t r y o f t h e a b e r r a n t elastic artery, b r a n c h i n g f r o m t h e r e intraparenchymal!y. Even l y m p h n o d e s are s e e n close to t h e e n t r y o f t h e a b e r r a n t artery, t h e s e t o g e t h e r f o r m i n g a p u l m o n a r y hilus-like structure. A c c o r d i n g t o t h e study by l s h i d a a n d associates, 3 o f 20 cases o f p u l m o n a r y lesions previously d i a g n o s e d as ILS in t h e i r institution, 12 cases were g e n u i n e ILS h a v i n g t h e b r o n c h i a l system as d e s c r i b e d earlier, w h e r e a s e i g h t cases were false ILS with a b r o n c h i a l tree t h a t was c o n s i d e r e d to b e l o n g to t h e b r o n c h i a l system o f t h e r e m a i n i n g n o r m a l l u n g b u t have only b e e n isolated f r o m it, m o s t p r o b a b l y b e c a u s e o f some k i n d o f o b s t r u c t i o n o r stenosis a n d s u b s e q u e n t i n f l a m m a tion. Cases o f g e n u i n e ILS all h a d a n a b e r r a n t a r t e r y o f t h e elastic type a n d were located in t h e p o s t e r i o r basal s e g m e n t o f t h e lower lobes, w h e r e a s cases o f false ILS h a d a b e r r a n t arteries o f t h e m u s c u l a r type a n d were located in various segments of the lungsP
FIGURE 1. A low-power vtew of intralobar sequestration, Note the complex of a blind-ended bronchus, an aberrant elastic artery and a lymph node forming a hilus-like structure at the entry of me aberrant artery. (H&E stain; original magnification ×7.9.) T h e i r f i n d i n g s strongly s u p p o r t t h e t h e o r y o f accessory l u n g b u d o r i g i n o f g e n u i n e ILS a n d are against t h e t h e o r y that t h e majority o f ILS are p o s t i n f l a m m a t o r y in origin. 4 T h e validity o f t h e i r n e w a p p r o a c h to t h e analysis o f ILS has b e e n
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Volume 29, No. 9 (September 1998)
16. Plaut M, Pierce JH, Watson CJ, et al: Mast cell lines produce lymphokines in response to cross-linkage of Fc RI or to calcium ionophores. Nature 339:64-67, 1989 17. Hamid Q, AzzawiM, Ying S, et al: Expression of mRNAfor intefleuldn-5 in mucosal bronchial biopsies from asthma. J Clin Invest 87:1541-1546, 1991 18. Dubucquoi S, Janin A, Desreumaux P, et al: Evidence for eosinophil activated in eosinophilic cystitis.Eur Uro125:254-258, 1994 19. Navarro-Roman L, Medeiros LJ, Kingman DW, et al: Malignant lymphomas of B-ceUlineage with marked tissue eosinophilia: A report of five cases. AmJ Surg Patho118:347-356, 1994
20. Arinaga S, Krimine N, Takamuku K, et al: Correlation of eosinophilia with clinical response in patients with advanced carcinoma treated with low-doserecombinant interleukin-2 and mitomycinC. Cancer Immunol Immunother 35:246-250, 1992 21. Huland E, Huland H: Tumor-associated eosinophilia in interleukin-2treated patients: evidence of toxic eosinophil degranulation on bladder cancer cells.J Cancer Res Clin Onco1118:463-467,1992 22. Enoldhara H, Kajitani H, Nagashima S, et al: lnterleuldn 5 activityin sera from patients with eosinophilia. BrJ Haemato175:458-462, 1990 23. Prin L, Plumas J, Gruart V, et al: Elevated serum levels of soluble interleukin-2 receptor: A marker of disease activityin the hypereosinophilic syndrome. Blood 78:2626-2632, 1991
TWINS, PLACENTAS, AND GENETICS: ACARDIAC TWINNING IN A DICHORIONIC, DIAMNIOTIC, MONOZYGOTIC TWIN GESTATION CHRISTOPHER A. FRENCH, MD, FREDERICKR. BIEBER, PHI), DAVID H. BING, PHD, AND DAVID R. GENEST, MD
We describe a human acardiac twin with associated vascular anastomoses in a dichorionic diamulotic fused twin placenta. A 22-year-old woman delivered a healthy 3,554 g male infant and a fused dianmiotic dichorionic twin placenta with a 230g umbilical cordattached, skin-covered, ovoid mass, consistent with acardiac amorphus. By gross and histological examination, the placental dividing membranes comprised four leaves, one amnion from each placenta, and two centrally fused chorions, diagnostic of dichorioulcity. Placental barium injection of the normal twin's umbilical vein showed an anastomosis with the acardiac twin which traversed the dividing membranes, then supplied major vessels of the acardiac mass via its
5.5 cm umbilical cord. DNA-typing studies of the normal twin's placenta and of the acardiac twin's tissues revealed identical alleles at 11 distinct genetic polymorphic loci, consistent with monozygosity. Our findings demonstrate that vascular anastomoses can occur in dichorionic twin placentas, and that human acardiac twinning is not, as heretofore believed, restricted to monochorioulc placentas. HUM PATHOL29:1028-1031. Copyright © 1998 byW.B. Saunders Company Key words: acardia, twins, placenta, malformation, zygosity. Abbreviations: MoMo, monoamniotic monochorioulc; DiMo, diamulotic monochorioulc; DiDi, dianmlotic dichorionic; UC, umbilical cord; MZ, monozygotic; DZ, dizygotic.
Acardiac twins r e p r e s e n t rare and bizarre malformations f o r m e d in the setting of major placental vascular anastomoses between twins or h i g h e r multiple births. W i t h o u t such vascular anastomoses, acardiac twins would have n o b l o o d flow. Reversed arterial perfusion has b e e n p r o p o s e d as the specific type of placental vascular anastomosis which is causally related to acardiac twinning. 1,2 Extensive placental artery-artery and vein-vein anastomoses reverse the n o r m a l direction of b l o o d flow t h r o u g h the acardiac's umbilical cord vessels such that arterial b l o o d flows toward and venous b l o o d flows away f r o m the acardiac twin. D o p p l e r studies have c o n f i r m e d this reversal of circulation in vivo. 2,3 In m o n o c h o r i o n i c h u m a n twin placentas, the frequency of artery-to-artery vascular anastomoses is 64%, 4 and the incidence of acardia is approximately 1%.5 In contrast, dichorionic h u m a n twins only very rarely f o r m placental vascular anastomoses6,7; it follows that h u m a n dichorionic acardiacs should be exceedingly rare. In fact, to our knowledge no case has b e e n reported. However, in o t h e r animals vascular anastomoses between dichorionic twin placentas have b e e n described, Ls and cases of acardiacs in cytogenetically proven dizygotic bovine twin pregnancies have b e e n reported. 9,1° It is thus conceivable that, t h o u g h exceptional, h u m a n acardiacs m i g h t occur in diamniotic dichorionic (DiDi) placentas. We r e p o r t the gross, light microscopic, immunohisto-
chemical, and genetic features of a h u m a n acardiac twin occurring with superficial vascular anastomoses in a DiDi placenta, showing that vascular placental anastomoses and acardiac twinning are n o t restricted to m o n o c h o r i o n i c placentas.
From the Department of Pathology, Brigham and Women's Hospital, Harvard Medical School; and the CBR Laboratories, Center for Blood Research, Boston, MA. Address correspondence and reprint requests to David R. Genest, MD, Department of Pathology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. Copyright © 1998 by W.B. Sannders Company 0046-8177/98/2909-002358.00/0
CASE R E P O R T A 22-year-old gravida 3 para 1, spontaneous abortus 1 p r e g n a n t w o m a n had an obstetrical ultrasound examination at 13.7 weeks that revealed a twin p r e g n a n c y with p r e s u m e d fetal demise of one twin at 9.5 weeks. A subsequent ultrasound p e r f o r m e d at 41 weeks revealed a n o r m a l fetus and an 8 × 10 cm n o d u l e suggestive o f a leiomyoma, a placental mass, or the p r e s u m e d deceased twin. At 42 weeks, a 3,554g healthy male fetus and a placenta with an encapsulated, "double-fist-sized" mass (acardiac) was delivered vaginally. T h e healthy twin is currently alive and well at 16 months. MATERIALS AND M E T H O D S Gross Placental Examination
This was c o n d u c t e d according to the m e t h o d s described elsewhere. 6 T h e dividing m e m b r a n e s were separated with forceps to reveal the n u m b e r of layers. A Barium solution consisting of 340 m L n o r m a l saline, 10 m L diethylene glycol histological grade (Fisher Scientific Co.), 30 g m Bacto-Gelatin (Difco Laboratories, Detroit, MI), and 260 m L Micropaque (Picker X-Ray Corp., White Plains, NY) was w a r m e d to 98.6°F and injected via a 14-gauge n e e d l e attached to a b u l b e d syringe into the n o r m a l twin's umbilical vein. T h e Barium solution was kept f r o m leaking d u r i n g injection by suturing the umbilical cord a r o u n d the syringe needle, proximal to its o u t p u t bulb.
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Immunohistochemistry Immunohistochemical stains for keratin proteins (AE1AE3, Boehringer Mannheim, Indianapolis, IN) were performed on formalin-fixed, paraffin-embedded tissue sections of the dividing membranes. Antibodies (1:2,500 dilution) were incubated for 37°C for 32 minutes, pretreated with pronase for 8 minutes at 37°C, and processed on a Ventana ES Automated Immunostainer (Ventana Medical Systems, Tucson, AZ) using an avidin-biotin peroxidase detector system.
PCR Analysis DNA extracted from formalin-fixed umbilical cord and cutaneous tissue from the acardiac, and amnion and chorion from the normal twin were typed for allelic polymorphisms at 11 different genetic loci (HLA DQA1, LDLR, GYPA, HBGG, D7S8, Gc, D1S80, CSF1PO, TPOX, THO-1, Amylogenin) using commercially available kits from Perkin-Elmer, Applied Biosystems Division (Norwalk, CT), and Promega Corp. (Madison, WI). RESULTS
Placenta and Acardiac Twin The fused twin placenta (Fig 1) weighed 450 grams. The umbilical cord of the healthy twin was centrally inserted and trivascular; that of the acardiac was marginally inserted and contained two vessels (ie, only a single umbilical artery). The placental tissue associated with the healthy twin (18 X 13 × 2.5 cm) contained a single 0.5 cm intervillous thrombus. The placental tissue associated with the acardiac (11.5 × 5 × 2.5 cm) was grossly unremarkable. An intact, opaque dividing membrane was present, consisting of two amnions and a fused dichorionic layer (Fig 2). A large, superficial vein-to-vein placental anastomosis was present. The umbilical cord of the healthy twin was clamped and its vein injected with barium. The barium passed through the anastomosis into the cord and systemic circulation of the acardiac twin (Fig 3). The acardiac twin (13.5 × 6.5 × 4.5 cm, 230 g) was a pink, skin-covered ovoid mass with multiple dimples and a caudal, 1.9 cm, yellow, multiloculated, fluid-filled cyst. Radiographs and dissection revealed subcutaneous tissue, a spinal column with poorly developed ribs, and an amorphous,
FIGURE !. Fused diamniotic dichorionic twin placenta (after membrane removal) with acardiac fetus a t t a c h e d via its umbilical cord. The fused dichorion of the dividing membrane has been o p e n e d to reveal a superficial venous anastomosis traversing the membranes.
FIGURE 2. The separated placental dividing membrane was composed of two amnions (arrow heads) and two chorions (arrow).
cartilagenous/bony "cephalic" end. No cranium, internal organs, or limbs were identified. This twin is best described as the rare variant of acardiac known as "acardius amorphus." The vascular anatomy of the acardiac twin and its placenta was revealed by radiographs after barium injection of the umbilical vein of the healthy cotwin. The barium-filled umbilical vein bifurcated on entering the acardiac. These vessels eventually converged at a blind-end sac near the center of the acardiac corpus (Fig 3). Histological study of the dividing membranes confirmed the presence of two distinct amnions and two distinct chorions (Figure 4A). Because the amniotic epithelium of the acardiac's sac was extensively debrided (presumably from severe oligohydramnios), immunohistochemical evaluation for keratin proteins (AE1/AE3) was used to confirm residual amniotic epithelium (Figure 4B). Both placentas were histologically mature.
PCR DNA typing revealed allelic identity at each of 11 distinct genetic loci typed from tissues obtained from the twins (see Table 1), as would be expected in monozygotic twins. Identity at the amylogenin locus for both X and Y alleles confirmed the male sex chromosome constitution of both twins.
FIGURE 3. Barium injection of the normal twin's umbilical vein traversed the dividing membranes and anastomosed with the acardiac's vasculature.
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FIGURE 4. (A) Hematoxyiin and eosin histology of the dividing membrane shows two chorions (C) and two amnions (A), but the acardiac's amniotic epithelium (arrow) is poorly preserved, presumably because of oligohydramnios. (B) Immunohistochemistry for AE1/AE3 confirms reactivity of acardiac's debrided amniotic epithelium for keratin (arrow); original magnification ×150, beth panels. DISCUSSION Acardia refers to a broad spectrum of abnormal fetal morphologies ranging from an amorphous mass, to a recognizable fetus with multiple severe anomalies. The most complete description of the various morphotypes is provided in the 1898 text by Schatz. t In general, acardia is characterized by failure of cranial and thoracic (heart and lung) development, 11 with fetal life entirely supported by a reversed nmbili-
TABLE l . identical Allelic Polymorphisms Found at Each of 11 Loci Tested From the A c a r d i a c Twin a n d its Normal Cotwin Gene Locus
Chromosomal Map Location
AllelesDetected
HLA-DQAI LDLR GYPA HBGG D7S8 Gc DIS80 CSF1PO TPOX THO1 Amylogenin
6p21.3 19p13.1-13.3 4@8-31 11p15.5 7q22-31.1 4q11-13 1p36-34 5q33.3-34 2p13 11p15.5 Xp22.1-22.31 ,Ypll.2
0101, 0201 A, B A, B A A B, C 18 11, 12 8, 11 9.3 X, Y
cal blood flow from the cotwin via extensive placental arteryartery and vein-vein anastomoses. 2,3 Although documented DiDi placental anastomoses are exceptional, monoamniotic monochorionic (MoMo) and diamniotic monochorionic (DiMo) placental anastomoses occur in most reported cases. 6 It is believed that acardia develops in the setting of large vascular anastomoses in a twin with a primary anomaly whose circulation becomes dependent on the reversed circulation provided by the eotwin. 1,6,12Evidence which supports this idea includes an extra marker chromosome found in fibroblasts cultured from an acardiac but not its cotwinlS; a triploid female acardiac arising from fertiliza• tion of the polar body of its n o r m a l twin14; and a 46,X,i(X) (pl0) hydropic acardiac whose cotwin's karyotype was normal? 5 However, the karyotypes of both the acardiac and normal twin are most often normal16; in addition, both may have the same cytogenetic abnormality17; or, the acardiac may have a normal karyotype whereas the normal cotwin has an abnormal karyotype, is Benirschke suggests, as the cytogenetic picture is complex and difficult to interpret, and because the reversal of circulation must be a very early event, reversed circulation alone is responsible for the formation of acardiacs. 6 Benirschke's theorizes that circulatory reversal causing acardia is consistent with the hypothesis of original normal-
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CORRESPONDENCE ity. 19'20 This was d e r i v e d f r o m t h e o b s e r v a t i o n t h a t acardiacs o c c u r in f r a t e r n a l b o v i n e a n d ovine twins, w h i c h p r e s u m a b l y were originally n o r m a l , b u t b e c a m e a n o m a l o u s a f t e r vascular a n a s t o m o s e s a n d c i r c u l a t o r y reversal was established. 9,1° To o u r knowledge, t h e p r e s e n t case is t h e only docum e n t e d e x a m p l e o f a h u m a n acardiac arising in f u s e d DiDi, m o n o z y g o f i c p l a c e n t a s with vascular anastomoses. This shows t h e f u n c t i o n a l c o n s e q u e n c e s o f m a j o r vascular a n a s t o m o s e s in this rare setting, a n d raises q u e s t i o n s with r e s p e c t tO o u r c u r r e n t u n d e r s t a n d i n g o f p l a c e n t a l vascularization. T h e t e r r i t o r y o f vascular n e t w o r k f o r m a t i o n i n p l a c e n t a l d e v e l o p m e n t is t h o u g h t to b e limited by t h a t r e g i o n d e m a r c a t e d by t h e c h o r i o n i c m e m b r a n e s . 6 T h u s , M o M o a n d DiMo p l a c e n t a s c o m m o n l y have vascular anastomoses, w h e r e a s DiDi p l a c e n t a s theoretically d o not. T h e diagnosis o f DiDi placentation with vascular a n a s t o m o s e s rests o n t h e histological confirm a t i o n o f two layers of a m n i o n , a n d two layers of c h o r i o n within t h e dividing s e p t u m , a n d t h e d e m o n s t r a t i o n 0 f i n j e c t e d dye t r a n s m i s s i o n f r o m a vessel o n o n e p l a c e n t a i n t o a vessel o n t h e other. U s i n g this m e t h o d o f diagnosis, very rare cases 7 have s h o w n u n e q u i v o c a l DiDi-fused p l a c e n t a s with large vascular anastomoses. T h e s e cases, i n c l u d i n g t h e p r e s e n t one, a n d t h e fact t h a t DiDi p l a c e n t a t i o n with vascular a n a s t o m o s e s regularly occurs in s o m e m a m m a l s , c h a l l e n g e s t h e c o n c e p t t h a t c h o r i o n i c m e m b r a n e s are strict b a r r i e r s to vascular network formation. REFERENCES 1. Schatz F: Die Acardii und ihre Verwandten. Hirschwald, Berlin, 1898 2. Benson CB, Bieber FR, Genest DR, et al: Doppler demonstration of reversed umbilical blood flow in an acardiac twin.J Clin Ultrasound 17:291-295, 1989
3. Pretorius DH, Leopold GR, Moore TR, et al: Acardiac twin-report of Doppler sonography.J Ultrasound Med 7:413-416, 1988 4. Leroy F: Major feta! hazards in multiple pregnancy. Acta Genet Med Gemello125:299-306, 1_976 5. Gillim DL, Hendricks CH: Holoacardius: Review of the literature and case report. Obstet Gyneco! 2:647-653, 1953 6. Benirschke K, Kaufman P: pathology of the Human placenta ed 3. New York, Springer-Verlag,1995, pp 778-786 7. Cameron AH: The Birmingham Twin Survey.R Soc Med Proc 61:229,34, 1968 8. Wislocki GB: Observations on twinning in Marmosets. Am J Anat 64:445-481, 1939 9. Dunn HO, Lein OH, Kenney RM: The cytological sex of a bovine anidian (amorphous) twin monster. Cytogenetics 6:412-19, 1967 10, Dunn HO, Roberts sJ: Chromosome studies of an ovine acephalicacardiac monster. Cornell Vet 62:425-31, 1972 11. Winter RM, Knowles SAS, Bieber FB, et al: The Malformed Fetus and Stillbirth. New York,John Wileyand Sons, 1988, p 227 12. Gruenwald P: Early humar~ twins with peculiar relations to each other and the chorion. Anat Rec 83:267-269, 1942 13. Turpin R, Bocquet L, Grasset J: Etude d'un couple monozygote: fille normale-monstre acardique feminin: Considerations anatoma-pathologiques et cytogenetiques. Ann Genet (Paris) 10:10%113, 1967 !4. Bieber FR, Nance WE, Morton CC, et al: Genetic studies of an acardiac monster; evidence of polar body twinning in man. Science 213:775-777, 1981 15. Wolf H, MacDonald J, Bradford W: Holoacardius anceps with abnormal karyotype and multicystic renal dysplasiawith patent ductus arteriosus in a surviving c0-twin.Mod Pathol 3:10P, 1990 (abstr) 16. Buehler BA, McManus BM, Mrocezek EC: Acardiac monsters: C!inical karyotype and pathologic features. Teratology 33:44C, 1986 (abstr) 17. Ginsberg NA, Applebaum M, Rabin SA, et al: Term birth after midtrimester hysterotomy and selective delivery of an acardiac twin. Am J Obstet Gynecol !67:33-37, 1992 18. Landy HJ, Larsen JW, Jr, Schoen M, et al: Acardiac fetus in a triplet pregnancy. Teratology 37:1-6, 1988 19. Claudius M: Die Entwicklung der herzlosen Missgeburten. Schwers, Kiel, 1859 20. Ahlfe!d F: Beitrage zur Lehre yon den Zwillingen. VI. Die Entstehung der Acardiaci. Arch Gynakol 14:321-360, 1879
CORRESPONDENCE To the Editor:--We r e a d with i n t e r e s t t h e article by Tomashefski a n d associates ! r e g a r d i n g the p a t h o g e n e s i s o f pulm o n a r y i n t r a l o b a r s e q u e s t r a t i o n (ILS). We feel it is u n f o r t u n a t e t h a t a few previously r e p o r t e d i m p o r t a n t studies 2,~ strongly i n d i c a t i n g t h e c o n g e n i t a l o r i g i n o f ILS have n o t received e n o u g h a t t e n t i o n . T h e s e studies m a d e clear t h a t g e n u i n e ILS h a s n o t only a s e p a r a t e arterial system b u t also a c o m p l e t e l y d i f f e r e n t b r o n c h i a l system f r o m t h a t o f t h e r e m a i n i n g n o r m a l lung, t h a t is, t h e b r o n c h i a l system t h a t b e g i n s as a b l i n d - e n d e d b r o n c h u s n e a r t h e e n t r y o f t h e a b e r r a n t elastic artery, b r a n c h i n g f r o m t h e r e intraparenchymal!y. Even l y m p h n o d e s are s e e n close to t h e e n t r y o f t h e a b e r r a n t artery, t h e s e t o g e t h e r f o r m i n g a p u l m o n a r y hilus-like structure. A c c o r d i n g t o t h e study by l s h i d a a n d associates, 3 o f 20 cases o f p u l m o n a r y lesions previously d i a g n o s e d as ILS in t h e i r institution, 12 cases were g e n u i n e ILS h a v i n g t h e b r o n c h i a l system as d e s c r i b e d earlier, w h e r e a s e i g h t cases were false ILS with a b r o n c h i a l tree t h a t was c o n s i d e r e d to b e l o n g to t h e b r o n c h i a l system o f t h e r e m a i n i n g n o r m a l l u n g b u t have only b e e n isolated f r o m it, m o s t p r o b a b l y b e c a u s e o f some k i n d o f o b s t r u c t i o n o r stenosis a n d s u b s e q u e n t i n f l a m m a tion. Cases o f g e n u i n e ILS all h a d a n a b e r r a n t a r t e r y o f t h e elastic type a n d were located in t h e p o s t e r i o r basal s e g m e n t o f t h e lower lobes, w h e r e a s cases o f false ILS h a d a b e r r a n t arteries o f t h e m u s c u l a r type a n d were located in various segments of the lungsP
FIGURE 1. A low-power vtew of intralobar sequestration, Note the complex of a blind-ended bronchus, an aberrant elastic artery and a lymph node forming a hilus-like structure at the entry of me aberrant artery. (H&E stain; original magnification ×7.9.) T h e i r f i n d i n g s strongly s u p p o r t t h e t h e o r y o f accessory l u n g b u d o r i g i n o f g e n u i n e ILS a n d are against t h e t h e o r y that t h e majority o f ILS are p o s t i n f l a m m a t o r y in origin. 4 T h e validity o f t h e i r n e w a p p r o a c h to t h e analysis o f ILS has b e e n
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