Normal and Abnormal Transformation of the Spiral Arteries During Pregnancy

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J. Perinat. Med. 34 (2006) 447–458 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/JPM.2006.089

Normal and abnormal transformation of the spiral arteries during pregnancy

Jimmy Espinoza1,2, Roberto Romero1,3,*, Yeon Keywords: Atherosis; immunohistochemistry; impe- Mee Kim1,4, Juan Pedro Kusanovic1, Sonia dance to blood flow; integrin; physiologic transformation Hassan1,2, Offer Erez1, Francesca Gotsch1, of the spiral arteries; placental bed. Nandor Gabor Than1, Zoltan Papp5 and Chong Jai Kim1,4 1 Perinatology Research Branch, NICHD, NIH, DHHS, Anatomy and physiology of the uterine Detroit MI 48201, and Bethesda MD 20892, USA circulation 2 Department of Obstetrics and Gynecology, Wayne The blood supply of the uterus is provided by the uterine State University School of Medicine, Detroit MI 48201, and ovarian arteries w102x. After entering the myome- USA trium, the uterine arteries give rise to the ‘‘arcuate arter- 3 Center for Molecular Medicine and Genetics, Wayne ies,’’ which branch into the ‘‘radial arteries’’ w102x. The State University School of Medicine, Detroit MI 48201, radial arteries divide into the ‘‘basal arteries,’’ which sup- USA ply the basal portion of the endometrium (critical for 4 Department of Pathology, Wayne State University endometrial regeneration after menstruation) and the School of Medicine, Detroit MI 48201, USA ‘‘spiral arteries,’’ which continue toward the endometrial 5 First Department of Obstetrics and Gynecology, surface w95x. The term ‘‘spiral arteries’’ reflects the coiled Semmelweis University, Budapest, Hungary appearance of these vessels, whose role is to supply blood to the upper functional layer of the endometrium, thought to be shed during menstruation w95x. The spiral Abstract arteries branch into a subepithelial capillary plexus w95x. Markee reported that each spiral artery supplies approx- This article reviews the anatomy and physiology of the imately 4–9 mm2 of endometrial surface w71x. Blood from uterine circulation, with emphasis on the remodeling of the intervillous space is drained through the utero-pla- spiral arteries during normal pregnancy, and the timing cental veins, whose openings are on the floor of the inter- and anatomical pathways of trophoblast invasion of the villous space. The venous endothelium lines part of the spiral arteries. We review the definitions of the placental floor of the intervillous space w62x. The uteroplacental bed and basal plate of the placenta, their relevance to veins undergo less dramatic changes than the arteries. the study of the physiologic transformation of the spiral Trophoblast may or may not invade the venous wall, even arteries, as well as the methods to obtain and examine though free floating endoluminal trophoblast has been placental bed biopsy specimens. We also examine the found in these vessels w36x. role of the extravillous trophoblast in normal and abnormal pregnancies, and the criteria used to diagnose failure Endometrial bleeding during menstruation and of physiologic transformation of the spiral arteries. Final- hemostasis ly, we comment on the use of uterine artery Doppler velocimetry as a surrogate marker of chronic uteroplacental The endometrium has evolved to meet the needs of ischemia. menstruation, implantation, placentation, as well as post- partum hemostasis. The specific mechanisms responsible for endometrial bleeding (during menstruation) and *Corresponding author: hemostasis (after menstruation) are relevant to the study Roberto Romero, MD Chief, Perinatology Research Branch of pregnancy, since uterine bleeding during pregnancy Intramural Division, NICHD, NIH, DHHS (i.e., hemostatic failure) is a risk factor for adverse preg- National Institute of Child Health nancy outcome w75, 101x. and Human Development Perinatology Research Branch The current understanding of the vascular changes Hutzel Women’s Hospital–4th Floor that occur during menstruation is based upon classic 3990 John R physiologic studies conducted by Markee, who exam- Detroit, MI 48201/USA ined the behavior of endometrium from rhesus monkeys Tel.: q1 (313) 993-2700 Fax: q1 (313) 993-2694 implanted into the anterior chamber of the eye of non- E-mail: [email protected] human primates w71x. The explants underwent a reduc- Article in press - uncorrected proof

448 Espinoza et al., Normal and abnormal transformation of the spiral arteries during pregnancy

tion in size of 25–75%, 2–6 days before the onset of bleeding. The spiral arteries became more coiled, and blood flow either slowed down or came to a complete stop. Red blood cells were reported not to move for 60–90 seconds w71x. Menstrual bleeding was considered to occur when vessels vasodilated after a period of intense vasoconstriction. Five mechanisms were proposed to account for the bleeding: (1) blood could pass through a damaged arteriole and form a hematoma, which would then rupture into the uterine cavity; (2) blood would pass directly from a ruptured arteriole into the uterine cavity; (3) red blood cells would extravasate by crossing the wall of the arterioles; (4) venous bleeding, which may occur either through the damaged venous wall or drainage following extravasation from arterioles; and (5) secondary bleeding may happen following violent Figure 1 Microscopic demonstration of physiologic transfor- movement. Markee proposed that 75% of bleeding mation of the myometrial segment of the spiral artery in normal derived from arterioles, and 25% from the venules. Ces- pregnancy at term. The placental bed biopsy specimen was sation of bleeding is thought to be due to vasoconstric- immunostained for cytokeratin-7 to locate trophoblasts (arrow- tion w71x. heads) infiltrating the arterial wall, and additional Periodic Acid Schiff (PAS) staining was performed to identify fibrinoid (arrows). The fact that menstrual fluid generally does not clot Spiral arterial lumen is dilated, and fibrinoid replaces the media w x 94 should not be taken as an indication that coagulation and intima. L: spiral aterial lumen (Cytokeratin 7-PAS staining, is not important for uterine hemostasis. For instance, =100). patients with Von Willebrand disease have excessive menstrual blood loss w29x. During normal menstruation, platelets and fibrin plugs are formed within the lumen of erally stains positive on Periodic Acid Schiff (PAS) stain- w x the spiral vessels w23x. Consumption of platelets and ing 16 (Figure 1). Brosens et al. proposed that these fibrinogen is thought to result in decreased concentra- structural changes, particularly the destruction of muscle tions of platelets and fibrinogen in menstrual fluid w28x. in the media, would lead to loss in vasomotor control w x Lockwood et al. proposed that tissue factor and plas- 16 . Collectively, these changes are thought to maximize minogen activator inhibitor-1 (PAI-1) are implicated in the delivery of maternal blood to the intervillous space both decidual hemostasis during implantation and bleed- by making the arterial lumen wider, as well as reducing ing during menstruation w67x. Tissue factor would induce the responsiveness of these vessels to vasoconstrictor coagulation and prevent hemorrhage during implanta- agents. Invasion of the utero-placental veins has been tion, while PAI-1 would prevent extracellular matrix deg- implicated as a mechanism responsible for the lateral w x radation and fibrinolysis. At the end of the menstrual placental growth 25 . Much less is known, however, cycle, progesterone withdrawal would reduce tissue fac- about changes in the morphology of the utero- w x tor and PAI-1 production and facilitate menstruation w67x. placental veins during pregnancy 36 .

Timing and anatomical pathways of Changes in the uterine circulation during trophoblast invasion of the spiral arteries pregnancy Robertson et al. w86x and Pijnenborg et al. w82x proposed The spiral arteries in the placental bed are normally trans- that trophoblast invasion occurs in two stages: (1) the formed into large dilated vessels undergoing dramatic transformation of the decidual segment of the spiral structural changes in their vessel wall. The spiral arteries arteries by a ‘‘wave’’ of endovascular trophoblast migra- are also known as the utero-placental arteries w16x. The tion in the first trimester; and (2) the transformation of the terms ‘‘physiologic changes’’ or ‘‘physiologic transfor- myometrial segments of the spiral arteries in the second mation’’ of the spiral arteries were first introduced by trimester w82, 86x. In 1972, Brosens and colleagues w17x Brosens, Robertson and Dixon in 1967 to emphasize that reported that in patients with preeclampsia and SGA, the these changes were part of normal pregnancy. The key physiologic transformation of spiral arteries is limited to findings of physiologic transformation are: (1) dilatation the decidual segment of the uteroplacental arteries, and of the lumen; (2) trophoblast invasion of the vessel wall Robertson proposed that this process is due to inhibition which includes both the media and endothelium; and (3) of the second ‘‘wave’’ of endovascular trophoblast migra- replacement of the muscular and elastic tissue of the tion that occurs from about 16 weeks onwards w51x. arterial wall by a thick layer of fibrinoid material that gen- However, Lyall et al. have concluded that morphological Article in press - uncorrected proof

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poses that trophoblast cells gain access to the uteroplacental arterial lumen via the point of entrance to the intervillous space, or close to it w48x. Thereafter, the cells migrate along the arterial lumen retrograde to the blood flow by adhering to and replacing the endothelium, forming intraluminal trophoblastic plugs. Some of these cells were thought to leave the arterial lumen and invade the media as well as adventitia (Figure 2A) w8, 48, 82, 83x. In contrast, the intravasation model is based on studies in human tissues and proposes that endovascular trophoblast represents differentiated interstitial trophoblast (Figure 2B) w27, 34, 48x. According to this model, the trophoblast invades the arterial wall from the surrounding junctional zone and migrates inside the arterial lumen w27, 34, 48x. More recently, a combination of both hypotheses was proposed by Kam et al. w47x.

Number and location of spiral arteries supplying the placental bed

A comprehensive assessment of the vascular supply to the placental bed can only be determined by examination of cesarean section hysterectomy specimens. Indeed, Ivo Brosens studied 15 cesarean section hysterectomy specimens, including three cases in which the placenta had been left in situ. He estimated that there were 120 arterial openings in the placental bed, with each artery containing only one opening into the intravillous space w15x. However, the estimates have ranged widely in studies reported during the past century w36x. The number of arteries and openings in the placental bed have been the subject of disagreement. Some investigators report more openings than arteries, while others propose that the number of openings equals that of utero-placental arteries. Moreover, other investigators have emphasized the difficulty in distinguishing arterial from venous openings w36x. The density of the spiral artery opening at term varies from 0.5–1 per centimeter square w36x. Brosens et al. reported that spiral artery openings are topographically adjacent to the placental septae, and 96% of spiral arteries from normal pregnancies have findings of physiologic transformation. Spiral arteries in the center of the placenta are more likely to have physiologic transformation Figure 2 Intravasation (Figure 2A) and extravasation (Figure than those located on the periphery w16x. 2B) routes of trophoblast invasion. Reproduced with permission from Kaufmann P et al. Biology of Reproduction 2003; 69: 1–7. Definition of placental bed and basal plate evidence of this proposal is unclear and that the molecular mechanisms to control such a process would be The term ‘‘placental bed’’ refers to part of the uterus very complex w68x. Moreover, recent studies have sug- (decidua and myometrium) lying underneath the placen- gested that trophoblast invasion of the spiral arteries is tal insertion site w36x. The maternal-fetal junction com- a continuous process w68, 69x. prises the basal plate of the placenta, which is composed The anatomic pathways taken by the endovascular tro- of the tissues normally detached with the placenta and phoblast (either the extravasation or intravasation model) the placental bed, which is what is left in the uterus (Fig- have also been a matter of controversy w48x. The extra- ure 3) w36x. The term ‘‘placental bed’’ should not be con- vasation model, which is based on animal studies, pro- fused with the ‘‘basal plate of the placenta,’’ which is the Article in press - uncorrected proof

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technique a ‘‘placental bed biopsy’’ w31x. The authors were clear in understanding that the biopsy specimen had to include not only the decidua but also the myometrium w31x, since the pathology observed in preeclampsia consisted of failure of physiologic transformation of the myometrial segment of the spiral arteries. This technique has been used extensively to study the placental bed in patients with complications of pregnancy w37, 87x. Rob- ertson et al. published a review of the experience of placental bed biopsies from three European centers and concluded that the technique was safe, and contributed to the understanding of the biology of this interface w87x. An important limitation of placental bed biopsies is the potential for sampling problems. The number of vessels present in one biopsy is limited and the biopsy may not Figure 3 Microscopic feature of the fetomaternal junction (bas- be representative of the lesions present in the entire uter- al plate and placental bed) showing distal end of the spiral artery us. Obtaining a placental bed biopsy can be carried out (arrows) emptying into the intervillous space (IVS). Inset shows either at the time of cesarean section or by using a trans- a longitudinally running spiral arterial segment at a lower mag- vaginal method under ultrasound guidance. nification. BP: basal plate, CV: chorionic villi, M: myometrium (Cytokeratin 7-PAS, =40 and =100). Transabdominal placental bed biopsies floor of the intervillous space. Its surface is visible with The transabdominal approach is used at the time of the naked eye on the maternal surface of the placenta, cesarean section. It is of the utmost importance that the and is composed of the part of the maternal-fetal junc- placental location be determined by ultrasound exami- tion adhering to the delivered placenta w7x. nation immediately before the procedure and then recorded. Robertson et al. recommended that the sur- geon identifies the true center of the placenta, which may not be underneath the umbilical cord insertion w87x. One Methodologies to obtain placental bed biopsy approach is to deliver the uterus during the cesarean specimens section after delivery of the fetus, identify the placental center, and place the digit of one of the operators on the A comprehensive and accurate study of the placental serosal surface of the uterus. The placenta is then deliv- bed requires access to the post-partum uterus, knowl- ered and the biopsy taken from this place. Robertson et edge of the site of placental implantation, and systematic al. recommended taking biopsies of 1.5 cm in diameter sectioning of the specimen. Cesarean section hysterec- w87x. The specimen needs to contain myometrium; this tomy specimens were used several decades ago to gain is easily achieved, as only a few millimeters of depth understanding of the microscopic morphology of the pla- (5 mm) would include this tissue w87x. When the placenta cental bed. However, these operations, once performed is implanted in the fundus biopsies are difficult to obtain. as a method of sterilization, are no longer in use, as the In this case, after uterine contraction, reaching the proper morbidity associated with the procedure is higher than site for the biopsy may not be possible. that of other sterilization techniques (e.g., tubal ligation). The instruments used for the biopsy could be a curved Thus, uteri for these types of studies become available scissors and a scalpel. It is recommended that non- generally in the instance of post-partum hemorrhage toothed forceps be employed in order to avoid crushing refractory to medical and surgical maneuvers (e.g., uter- the specimen w87x. Another alternative is to use derma- ine artery ligation), or when hysterectomy is clinically indi- tologic punch biopsies, as they provide an easy method cated (such as early cervical or ovarian cancer diagnosed to standardize the procedure. Robertson et al. indicated during pregnancy or after maternal death). The underly- that an experienced individual using this technique can ing pathology, which was the indication for hysterectomy, sample the true placental bed in 70% of cases w87x. introduces potential biases in these studies. For exam- Hemostasis may sometimes be required, and this can be ple, some patients with intractable post-partum hemor- accomplished by using absorbable sutures. rhage to conventional treatment may have placenta accreta, and to consider them representative of a phys- Transvaginal approach iologic state may not be appropriate. Dixon and Robertson introduced in 1958 a new tech- The first transvaginal approach, reported in 1981, con- nique for obtaining tissue in order to study the placental sisted of introducing forceps through the cervix after bed at the time of cesarean section. They called this delivery of the placenta to obtain placental bed biopsies Article in press - uncorrected proof

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w37x. This procedure was subsequently modified by cytoskeleton and karyoskeleton of eukaryotic cells w40x. Michel et al., who performed ultrasound examinations to The term ‘‘intermediate’’ is used because their diameter determine the location of the placenta. The authors used (7–22 nm) is between that of actin (5–7 nm) and tubulin cervical biopsy forceps under ultrasound guidance to (22–25 nm) w40x. The anti-cytokeratin 7-specific antibody sample the placental bed in women who were diagnosed OV-TL 12/30 has been reported to be the only one that with a blighted ovum (or anembryonic pregnancies) and binds all trophoblast populations (and uterine glands) underwent first trimester termination of pregnancy w72x. without any reactivity to mesenchymal stromal cells w11x. Khong et al. were successful in retrieving placental bed Thus, anti-cytokeratin 7 is considered to be the most biopsies using curettage under ultrasound guidance in useful antibody to identify trophoblast cells w56x. While patients with spontaneous abortions w52x. Robson et al. the choice of marker to purify trophoblast in vitro is the modified this technique, and reported that placental bed subject of debate, there is general acceptance of the val- biopsies (containing a spiral artery in 50% of cases) ue of anti-cytokeratin 7 for histologic studies w11, 56x. could be obtained using a transvaginal approach. The forceps they employed were 310 mm long, with a 5 mm- Does the specimen contain more than one wide movable jaw with a cutting edge. The goal was to myometrial spiral artery? take four biopsy specimens from each case. This procedure was attempted in first trimester terminations of Spiral arteries in the myometrium have a luminal diameter pregnancy (ns201), missed abortions (ns104), cesarean of approximately 200 mm (Boyd and Hamilton, 1970, cit- sections at term (ns95), and after a vaginal delivery ed in Frank et al. w36x). However, as the arteries travel (ns13) w88x. Overall, in 67% (281/417) of the cases the through the decidua to the intervillous space, they widen placental bed was sampled, and in 55% at least one spi- and can attain a diameter of 500–1000 mm w91, 92x. ral artery was present. The authors did not encounter any Close to the ostia or the opening of the artery in the inter- significant complications attributable to the procedure villous space, the diameter may reach 2000 mm w36x. w88x. Arteries with a lumen of -120 mm within the myometrium generally represent ‘‘basal’’ or straight arteries, and should not be confused with spiral arteries. Several dec- Immunohistochemical evaluation of a placental ades ago, some authors proposed that the spiral arteries bed biopsy have a sphincteric mechanism close to the ostia to regulate the entry of blood into the intervillous space (Span- The four key questions when examining a biopsy are: (1) ner 1935, Debiasi 1963, cited in Frank et al. w36x). does the specimen come from the placental bed?; (2) Subsequent investigations w36x have disproved this con- does it contain more than one myometrial spiral artery?; cept. Biopsy specimens that have segments of a spiral (3) is there physiologic transformation of the myometrial artery within the decidua, but not the myometrium, are segment of the spiral arteries?; and (4) is there atherosis? of limited value for the diagnosis of failure of physiologic Placental bed biopsies are generally fixed in formaline, transformation. and then stained with cytokeratin 7 and PAS to answer these questions. Is there physiologic transformation of the spiral arteries? Does the specimen come from the placental bed? The characteristics of physiologic transformation include: Evidence that the biopsy derives from the placental bed (1) a dilated myometrial segment with a luminal diameter (rather than the non-placental bed or decidua parietalis) of greater than 120 mm; (2) infiltration of the vessel wall requires identification of interstitial trophoblast. Interstitial by trophoblast; (3) destruction of the media (i.e., smooth trophoblast may be difficult to distinguish from maternal muscle and elastic tissue); and (4) fibrinoid deposition in cells in the decidua and basal plate w83x. Immunohisto- the media, recognized as PAS positive material w16x. chemistry is therefore used to detect trophoblast. The Therefore, physiologic transformation of the spiral arter- most widely used antibody to accomplish this procedure ies can be documented by staining the placental bed is anti-human cytokeratin 7. This type of antibody rec- biopsy specimens with PAS and an anti-cytokeratin anti- ognizes epithelial cells (trophoblast has epithelial char- body (Figure 1). acteristics). In placental bed biopsy material, this antibody would identify both trophoblast cells and glan- dular epithelium of the endometrium. We have used anti- Identification of fibrinoid The term ‘‘fibrinoid’’ has cytokeratin 7 (DAKO, OV-TL 12/30), which reacts with the been used to refer to an acellular, homogeneous material, 54 kDa cytokeratin intermediate filament protein w26x. which binds to acid stains such as eosin and PAS. Two Cytokeratins are types of ‘‘intermediate filament proteins’’ types of fibrinoid material have been identified: fibrin- belonging to a supergene family that includes tubulin type and matrix-type w49x. These subtypes cannot be polymers and actin microfilaments, and are part of the distinguished with hematoxyllin eosin stain. However, Article in press - uncorrected proof

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Figure 4 Atherosis of the spiral arteries and decidual arterioles in the basal plate (A) and decidua of the fetal membrane (B), respectively. Arrows indicate vessels affected by atherosis. The hallmark of atherosis is fibrinoid necrosis of the wall (arrows) and the presence of lipid-laden macrophages. (H&E, A: =40, B: =100). matrix-type fibrinoid contains trophoblast cells, while patients with chronic glomerulonephritis with super- fibrin-type does not w49x. With trichrome staining, fibrin- imposed preeclampsia. However, Zeek and Assali sub- type fibrinoid stains red to orange, whereas the matrix- sequently proposed that the lesion was confined to type has a blue to light green appearance. Lang has also preeclampsia w104x. Acute atherosis may be seen in the called attention to a usefulness of a modified paralde- myometrial or decidual segments of the spiral arteries, hyde fuchsin stain in differentiating two types of fibrin- and it is believed to occur only in vessels that have failed oids w61x. Fibrin-type stains green/orange, while to undergo physiologic transformation (i.e., trophoblast matrix-type stains blue, violet or green w49x. The type of invasion of the arterial wall). The lesion resembles those fibrinoid found in the vessel wall is fibrin-type (Figure 1). observed in atherosclerosis w100x, and has been reported Thought to be derived from the deposition of fibrin, the in other complications of pregnancy (such as growth fibrin-type is therefore generated by activation of the restriction w50, 93x, intrauterine fetal demise w52x, diabe- coagulation cascade w49x. This observation is based tes mellitus w6, 57, 58x, systemic lupus erythematosus upon immunohistochemistry studies demonstrating that (SLE), discoid lupus erythematosus w2, 59x, and anti- the material stains with antibodies against fibrin, which phospholipid antibody syndrome) w64, 76, 97x. The fre- do not cross-react with fibrinogen w42x. quency of this lesion in the ‘‘great obstetrical sydromes’’ Physiological transformation of the spiral arteries is not was recently described by Kim et al. w55x. an ‘‘all or none’’ phenomenon, since it may vary among spiral arteries in the same patient as well as within the same artery. In some of them, even the decidual segment is not transformed w51, 60x, while in others the myome- The role of extravillous trophoblast invasion trial segments may be only partially transformed w51x. Thus, a more objective assessment of the degree of Does decidual spiral artery remodeling begin before physiologic transformation of the spiral arteries may be or after trophoblast invasion? achieved by referring to the proportion of the artery that Several authors have proposed that maternal vessels is transformed in the placental bed biopsy specimens may undergo changes before interstitial trophoblast inva- w53, 54x. Physiologic transformation is more likely at the sion w14x (Harris and Ramsey 1966, Boyd and Hamilton center of the placental bed than on the periphery w15, 68, w x 87x. Therefore, the determination of the true center of the 1970, as cited in Craven et al. 24 ). Craven et al. provid- placenta, by ultrasound or during the cesarean section ed evidence that vasodilatation and some degree of vas- before the biopsy, may reduce sampling bias w87x. cular remodeling (expression of VCAM-1 by endothelial cells) could be detected in very early intrauterine preg- Is there atherosis? nancies, in the endometrium of patients with ectopic ges- tations, as well as in the decidua parietalis of two patients ‘‘Acute atherosis’’ of the spiral arteries was first described who underwent hysterectomies at 5 and 11 weeks of in 1945 by Herting in patients with preeclampsia w41x. gestation. The control group consisted of endometrial The typical findings are: (1) fibrinoid necrosis of the ves- samples from patients in the secretory phase of the men- sel wall; (2) accumulation of lipid-laden macrophages in strual cycle. The authors suggested that the arterial the vessel wall; and (3) a mononuclear perivascular infil- changes observed in early pregnancy may be maternal trate w85x (Figure 4). Sexton et al. coined the term ‘‘athe- responses associated with early endometrial deciduali- rosis’’ w90x and reported that it could be observed in zation w24x. Article in press - uncorrected proof

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Following these initial changes, further structural mod- migration has been demonstrated through in vitro exper- ifications have been described, such as reduction of the iments in which blocking antibodies against integrins smooth muscle in the vascular media and deposition of results in loss of trophoblast cell adhesion to extracellular fibrinoid material before infiltration of the media by tro- matrix w45x. phoblast w48x. The trophoblast invasion of the arterial wall The following integrins have been detected in extravil- induces further dilation of the spiral arteries w48x. lous trophoblast w12x:

1) a b , which is the receptor for laminin; Invasion of extravillous trophoblast 3 1 2) a6b4, which binds to laminin and through it to colla- According to Kaufmann and Huppertz, the invasion of the gen IV; extravillous trophoblast requires: (1) a source of prolifer- 3) a5b1, a fibronectin receptor; ating cells; (2) expression of extracellular matrix recep- 4) a1b1, a receptor for laminin and collagens I and IV; tors, which will facilitate adhesion of invasive cells to the 5) avb3 and avb5, which are the receptors for vitronectin, extracellular matrix of the uterus; (3) the presence of inva- as well as fibronectin and osteopontin. sive cells, which produce extracellular matrix; (4) matrix degrading enzymes to form channels in the uterine matrix for migration; (5) cellular motion; and (6) arrest of The extravillous trophoblast in normal pregnancy invasion. undergoes a gradual switch from the proliferative to the invasive phenotype. This phenotypic change is also Proliferation The stem cell for extravillous trophoblast associated with a switch in integrin expression w36x. The invasion is considered to be attached to the basement extravillous trophoblast with proliferative phenotype, membrane of anchoring villi. These cells stain positive for located on or close to the basal lamina of the anchoring w x proliferation markers such as H thymidine, Ki-67 (MIB-1) villi, expresses predominantly a6b4 and, in part, a3b1 36 . and antibodies against proliferating cell nuclear antigen The integrin a6b4 is a laminin receptor that functions to (PCNA) and, thus, have a proliferative phenotype w9, 18x anchor epithelial cells to the basal membrane w19x. This (Muhlhauser, 1993, Konsake, 1994, in Frank et al. w36x). function may be aided by the long intracytoplasmic tail w x Upon departure from this location, cells abandon the cell of the subunit b4 19 . Cells that express this integrin type cycle and acquire an invasive or migratory phenotype also express proliferative markers, exhibit polar secretion w36x. However, in pathologic conditions such as chorio- of extracellular matrix or absence of visible matrix secre- carcinoma w103x, ectopic pregnancy (Ketschanska, 1998, tion, and do not have an invasive behavior w36x. In con- Kemp 1999 in Frank et al. w36x), and placenta accreta/ trast, invasive trophoblast in the deciduas expresses w x percreta 36 , invasive trophoblast retains its proliferative interstitial integrins (such as a5b1, a1b1, avb3 and avb5), capacity. While this is easy to understand in the case of which bind to fibronectin, show apolar secretion of the a neoplasia, the dissociation in ectopic pregnancy and extracellular matrix, and upregulate certain metallopro- placenta accreta suggests that factors intrinsic to the teinases (e.g., MMP-2, MMP-11) w36x. decidua play a role in altering the proliferative/migratory Inadequate trophoblast migration and shallow tropho- switch w36x. blast invasion of the decidua and spiral arteries, as seen in preeclampsia, have been associated with failure to Integrin switch Cells interact with extracellular matrix down-regulate the b4 integrin subunit and upregulate the components through a set of transmembrane proteins w x a1 integrin 105 . This suggests that changes in the a6b4 linking the cytoskeleton of the cell to the extracellular and a1b1 integrins may be important in the trophoblast matrix components such as fibronectin, laminin and col- migration and invasion during pregnancy w19x. These w x lagen 4 . Integrins are a family of proteins that bind to findings, however, have not been confirmed by other 6 9 their ligands with a lower affinity (Ka 10 –10 liter/mol) studies w30x. and are present in higher concentrations (10–100-fold) than receptors for hormones and other soluble signaling molecules w4x. These properties allow a cell to explore its The role of matrix degrading enzymes in trophoblast environment without becoming irreversibly attached to it invasion Trophoblast invasion requires the degradation w4x. Integrins are important as they are the main mole- of extracellular matrix in the decidua and spiral arteries cules allowing cells to bind and respond to extracellular and the proximal third of the myometrium w38x. Proteo- matrix. Integrins are formed by two non-covalently asso- lytic enzymes involved in the extracelluar matrix degra- ciated transmembrane glycoprotein subunits called alpha dation include serine proteases of the plasmin system and beta. The matrix binding domain projects more than w43, 74x and matrix metalloproteinases (MMPs) w43, 98x. 20 nm from the plasma membrane w4x. The intracellular Immunohistochemical studies demonstrated that MMP- end of the dimers binds to the actin cytoskeleton (via 1 w43, 65x, MMP-2 w10, 43x, MMP-3 w43, 98x, MMP-9 w43, proteins such as talin and alpha actinin) w12x. The func- 65x and its inhibitors TIMP-1 w43, 44x and TIMP-2 w43, tional importance of integrin bindings in trophoblast 44x are present in the extravillous trophoblast. Article in press - uncorrected proof

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Figure 5 Comparison of transformed and non-transformed spiral arteries in the myometrium. Contours of the two arteries are in stark contrast. (A) A transformed spiral artery is characterized by the presence of intramural trophoblasts (arrowheads) and fibrinoid degeneration (arrows) of the wall. (B) Absence of intramural trophoblasts, fibrinoid degeneration, and intact arterial contour mark spiral artery with failure of physiologic transformation (Cytokeratin 7-PAS, =200).

Control of invasion Huppertz et al. have proposed that myometrial segment in normal pregnancy, the key feature trophoblast invasion can be controlled by several mech- of ‘‘failure of physiologic transformation of the spiral anisms, including: (1) a change from a proliferative to an arteries’’ in preeclampsia was the lack of invasion of the invasive phenotype. Unlike neoplastic cells, which prolif- trophoblasts into the spiral arteries of the myometrial erate and invade tissue, invasive trophoblast is not segment w17x (Figure 5). Subsequent studies using spec- proliferative except under special circumstances (e.g., imens from cesarean hysterectomies from patients with ectopic pregnancy and placenta accreta); (2) pro- preeclampsia and intrauterine growth restriction demon- grammed cell death or apoptosis; (3) endoreduplication; strated that physiological transformation of the spiral and (4) syncytial fusion (personal communication). After arteries is not an ‘‘all or none’’ phenomenon and varies delivery, trophoblast cells in the uterine wall undergo among spiral arteries in the same patient. In some spiral apoptosis generally within weeks. However, some tro- arteries from these patients, even the decidual segment phoblast cells have been identified in hysterectomy was not transformed w51x. More recently, failure of phys- specimens several years after pregnancy (personal com- iologic transformation of the spiral arteries has also been munication). It is uncertain whether some trophoblast reported in spontaneous abortion w52x, placental abrup- cells may persist for months or even years. tion w32x, preterm labor and intact membranes w53x,as well as preterm PROM w54x.

Is interstitial trophoblast invasion ‘‘shallow’’ in preeclampsia? The relationship between Doppler of the uterine artery and ‘‘failure of physiologic trans- Kadyrov et al. w46x reported a study in which the depth formation’’ of trophoblast invasion was examined in hysterectomy specimens from patients with normal pregnancies, early Failure of physiologic transformation of the spiral arteries onset preeclampsia and anemia. The depth of invasion in placental bed biopsies has been associated with high was determined by identifying the endometrial-myome- impedance to blood flow in the uterine arteries, as meas- trial junction by actin staining (smooth muscle). Com- ured by Doppler velocimetry in patients with preeclamp- pared to normal pregnancies, trophoblast invasion was sia w1, 66, 79, 99x, intrauterine growth restriction w1, 66, shallower in patients with preeclampsia and deeper in 70, 79x, preterm labor with intact membranes, and pre- patients with anemia. In contrast, apoptosis was term PROM w33x. decreased in both anemia and preeclampsia w46x. Pulsed Doppler ultrasound has been utilized since its Redline et al. proposed the diverging view that preeclam- introduction by Campbell et al. w21x for the non-invasive psia is associated with an excess in proliferative imma- assessment of impedance to blood flow in the uterine ture intermediate trophoblast w84x. arteries w3, 5, 13, 22, 39, 63, 80, 81x. High impedance to blood flow in the uterine arteries between 20 and Failure of physiologic transformation of the spiral 24 weeks, defined as bilateral uterine artery notches and arteries and pregnancy complications a pulsatility index or resistance index above the 95th per- Brosens and Robertson, using specimens from placental centile w3, 13, 39x, has been associated with high risks bed biopsies and cesarean hysterectomies, described of developing early onset preeclampsia, delivering an that while physiological changes of the spiral arteries SGA neonate and perinatal death w3, 39, 63, 80x. Indeed, extended both to the decidual segment and 1/3 of the 80% of patients developing preeclampsia before Article in press - uncorrected proof

Espinoza et al., Normal and abnormal transformation of the spiral arteries during pregnancy 455

34 weeks w3, 39x (as opposed to only 27% of those who Note added at proof: The authors would like to recommend develop the disease after 34 weeks w3x) have a high to the interested reader the article entitled ‘‘The Uterine Spiral impedance to blood flow in the uterine arteries between Arteries in Human Pregnancy: Facts and Controversies’’ by Rob- 22 and 24 weeks. ert Pijnenborg published in Placenta 2006;26:939–958. This article examines in-depth the controversial issues on the subject. Bilateral diastolic notches in the uterine arteries represent very high impedance to blood flow References Patients with bilateral notches in the Doppler waveform of the uterine arteries at 23–24 weeks are at in- w1x Aardema MW, H Oosterhof, A Timmer, R van, I JG Aar- creased risk for adverse pregnancy outcome (preeclam- noudse: Uterine artery Doppler flow and uteroplacental psia, SGA, and perinatal death) w20x. Studies in which vascular pathology in normal pregnancies and pregnan- gelfoam has been used to embolize the uterine circula- cies complicated by pre-eclampsia and small for gestational age fetuses. Placenta 22 (2001) 405 tion in pregnant animals have shown that the develop- w2x Abramowsky CR, ME Vegas, G Swinehart, MT Gyves: ment of uterine artery notches are associated with high Decidual vasculopathy of the placenta in lupus erythe- impedance to blood flow w77, 78x. Indeed, progressive matosus. N Engl J Med 303 (1980) 668 embolization of the spiral arteries in pregnant animals w3x Albaiges G, H Missfelder-Lobos, C Lees, M Parra, KH produces a diastolic notch only when the uterine blood Nicolaides: One-stage screening for pregnancy compli- flow has been reduced to approximately one third, and cations by color Doppler assessment of the uterine arteries at 23 weeks’ gestation. Obstet Gynecol 96 (2000) 559 the vascular resistance has been increased three to four w4x Alberts B, D Bray, J Lewis, M Raff, K Roberts, JD Watson: w x times compared to its normal values 77, 78 . Cell junctions, cell adhesion, and the extracellular matrix. Longitudinal studies in uncomplicated pregnancies In: Molecular biology of the cell. 1994 indicate that diastolic notches in uterine arteries disap- w5x Arduini D, G Rizzo, C Romanini, S Mancuso: Utero-pla- pear at 24 weeks of gestation w35, 89x. Therefore, per- cental blood flow velocity waveforms as predictors of sistence of uterine artery notches in the third trimester pregnancy-induced hypertension. Eur J Obstet Gynecol may indicate that high impedance to flow and the con- Reprod Biol 26 (1987) 335 w6x Barth WH Jr., DR Genest, LE Riley, FD Frigoletto Jr., BR sequent uteroplacental ischemia are chronic in nature. Benacerraf, MF Greene: Uterine arcuate artery Doppler The pathophysiology of the uterine artery diastolic notch and decidual microvascular pathology in pregnancies is not completely understood. However, two theoretical complicated by type I diabetes mellitus. Ultrasound Obstet computer models have been used to explain the physio- Gynecol 8 (1996) 98 pathology of uterine artery diastolic notches. Mo et al. w7x Benirschke K, P Kaufmann: Microscopic survey. In: proposed that the notch is the result of a wave reflection Benirschke, K. P Kaufmann: Pathology of the Human Pla- due to a high placental bed resistance w73x. In contrast, centa. Springer, Springer 1999, pp 16–28 w8x Blankenship TN, AC Enders, BF King: Trophoblastic inva- Talbert et al. put forward the idea that the uterine artery sion and the development of uteroplacental arteries in the diastolic notch is due to increased arterial wall compli- macaque: immunohistochemical localization of cytokera- ance w96x. During labor there is a high impedance to tins, desmin, type IV collagen, laminin, and fibronectin. blood flow in the uterine arteries. However, labor is not Cell Tissue Res 272 (1993) 227 generally associated with uterine artery notches w96x. w9x Blankenship TN, BF King: Developmental expression of Ki- 67 antigen and proliferating cell nuclear antigen in macaque placentas. Dev Dyn 201 (1994) 324 w10x Blankenship TN, BF King: Identification of 72-Kilodalton Acknowledgements Type-Iv Collagenase at sites of trophoblastic invasion of macaque spiral arteries. Placenta 15 (1994) 177 w x The authors would like to acknowledge the substantial contri- 11 Blaschitz A, U Weiss, G Dohr, G Desoye: Antibody reaction butions of Professor Peter Kaufmann of the University of patterns in first trimester placenta: implications for tropho- Aachen, Germany, Professor John Kingdom of the University of blast isolation and purity screening. Placenta 21 (2000) 733 Toronto, Professor Robert Pijnenborg of the University Leuven, w12x Bowen JA, JS Hunt: The role of integrins in reproduction. Belgium, Professor Judith N. Bulmer of the University of New- Proceedings of the Society for Experimental Biology and castle Upon Tyne, United Kingdom, and Professor Susan Fisher Medicine 223 (2000) 331 of University of California, San Francisco. Each of these distin- w13x Bower S, K Schuchter, S Campbell: Doppler ultrasound guished scientists contributed to our interest in the study of the screening as part of routine antenatal scanning: prediction placental bed, visited the Perinatology Research Branch of of pre-eclampsia and intrauterine growth retardation. Br J NICHD and were generous with their ideas, talent and expertise. Obstet Gynaecol 100 (1993) 989 This review and other scientific work of our Branch would not w14x Brettner A: On the behavior of the secondary wall of ute- have been possible without their guidance and the inspiration roplacental blood vessels during decidual reactions. Acta they provided to us. Anat (Basel) 57 (1964) 366 This work was supported by the Division of Intramural w15x Brosens IA: The uteroplacental vessels at term – The dis- Research of the National Institute of Child Health and Human tribution and extend of physiological changes. Trophoblast Development, NIH, DHHS. Research 3 (1988) 61 Article in press - uncorrected proof

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