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DOI: 10.1177/1933719107309646 2008 15: 59Reproductive Sciences

Anthony S.-Y. Leong, Jane E. Norman and Roger SmithVascular and Myometrial Changes in the Human Uterus at Term

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by CARLOS RENGIFO on October 11, 2012rsx.sagepub.comDownloaded from

Vascular and Myometrial Changes inthe Human Uterus at Term

Anthony S.-Y. Leong, MD, Jane E. Norman, MD, and Roger Smith, MD, PhD

At term, amniotic fluid contents may mediate the onset of labor through the activation of amniotic fluidmacrophages and their migration into the myometrium. To test this concept, the authors examine thehistological changes that occur in myometrial biopsies at term prior to (n = 53) and during (n = 15)labor. Biopsies were stained with an antimacrophage antibody, anti-CD34 (endothelial cells), andanticaspase 3 (apoptotic cells). The samples showed a variable inflammatory infiltrate of neutrophils andmacrophages, with a greater infiltrate in the samples obtained during labor (P < .001, Fisher exact test).Prior to labor, there were prominent changes in the myometrial fibers that reflected shearing, shrinkage,edema, and particularly apoptosis; endothelial cells of thin-walled vessels prominent in the biopsies dis-played marked nuclear biotinylation, and the vascular lumen contained fibrin and platelet thrombi,microparticles, desquamated endothelial cells, amniotic squamous cells, and mucoid material. Thesechanges were also present in samples obtained during labor. In an additional 10 patients in labor withmale fetuses, myometrial samples were examined for the presence of macrophages carrying a Y chromo-some indicative of a fetal origin, but none were observed. These findings suggest that endothelial cell dam-age and amniotic fluid embolism are very common at term prior to clinical labor and provide a mechanismby which surfactant protein A and phospholipids present in the amniotic fluid may access myometrial cellsand provoke the inflammatory response that occurs during parturition. The authors studies give no sup-port to the suggestion that fetal macrophages might invade the human myometrium at term.

KEY WORDS: Labor, myometrium, histology, inflammation, macrophages.

functional withdrawal of progesterone has been postu-lated to occur through several different mechanisms,including altered expression of different forms of theprogesterone receptor, changes in progesterone receptorcofactor expression, and functional antagonism of theprogesteroneprogesterone receptor interaction by thetranscription factor nuclear factor B.1-3 Analysis of geneexpression changes in the myometrium at the time oflabor suggests that inflammatory factors drive the proges-terone withdrawal process.4

Recent morphological studies suggest that labor inthe human is an inflammatory process and that inflam-matory cells, predominantly neutrophils andmacrophages, infiltrate the myometrium during sponta-neous labor at term.5 In the mouse, it is suggested thatfetal lung maturation leads to an increased release of sur-factant protein A into the amniotic fluid, activating fetalmacrophages,which in turn migrate into the myometriumand provoke an inflammatory response.6-10 However,studies of human myometrial samples obtained at labor fromwomen pregnant with a male child using polymerase

The mechanisms that regulate the onset of labor inhumans remain unknown. In most mammals,labor is precipitated by a fall in maternal plasmaprogesterone; in contrast, no such fall in circulating prog-esterone concentrations occurs in humans. Recently,evidence has been presented that labor in humans is ini-tiated by a functional withdrawal of progesterone.1 The

From Hunter Area Pathology Service, University of Newcastle, Newcastle,Australia (ASYL); University of Glasgow, Division of DevelopmentalMedicine, Glasgow Royal Infirmary, Glasgow, United Kingdom (JEN); andMothers and Babies Research Centre, Hunter Medical Research Institute, JohnHunter Hospital, Faculty of Health, School of Medicine & Public Health,University of Newcastle, Newcastle,Australia (RS).

Address correspondence to: Roger Smith, PhD, Mothers and Babies ResearchCentre, Endocrine Unit, John Hunter Hospital, Faculty of Health, School ofMedicine & Public Health, University of Newcastle, Locked Bag 1, HunterRegion Mail Centre, NSW 2310, Australia. E-mail: [email protected]

Reproductive SciencesVol. 15 No. 1 January 2008 59-65DOI. 10.1177/1933719107309646 2008 by the Society for Gynecologic Investigation

59 by CARLOS RENGIFO on October 11, 2012rsx.sagepub.comDownloaded from

chain reaction (PCR) and chromogenic in situ hybridiza-tion (CISH) to identify Y chromosome material failed toidentify any fetal macrophages.11

At present, it is unknown if the inflammatory infil-trate in human myometrium at term is related to fetallung maturation and whether amniotic fluid factorsmediate the observed inflammatory state in the humanmyometrium at term. The present studies were under-taken to determine the integrity of the barriers betweenthe amniotic fluid and the myometrium at term in thehuman. In this study, we examine the morphologicalchanges in the lower uterine segment samples obtained atterm from patients undergoing cesarean delivery eitherbefore or after the clinical onset of labor.

MATERIALS AND METHODS

Following permission from both institutional ethics com-mittees and written informed consent, biopsies wereobtained from the anterior lower uterine segment atterm from 68 patients delivered by cesarean delivery atterm pregnancy at John Hunter Hospital, Newcastle,Australia (n = 37, all prior to the onset of labor) or atGlasgow Royal Infirmary, Department of Obstetrics andGynaecology, University of Glasgow, Scotland (n = 31, 15in labor and 16 prior to the onset of labor).The biopsieswere fixed in 10% buffered formalin. Five-micron hema-toxylin and eosinstained sections from all cases wereexamined by one of the authors (A.S.-Y.L.). All caseswere also stained with an antimacrophage antibody(CD68, 1:50; DakoCytomation, Sydney, Australia), anti-CD34 to endothelial cells, and anticaspase 3 to labelapoptotic cells (1:200; Cell Signalling Technology,Genesearch Pty Ltd, Arundel, Queensland, Australia),employing a standard strepavidin-peroxidase techniquefollowing microwave antigen retrieval at 98C in 10mmol citrate buffer at pH 6.0, as previously described.12

An additional 10 samples from women in labor witha male fetus from the Glasgow center were studied. CISHwas performed to detect the presence of the Y chromo-some by employing a recently described enhancedmethod of detection.13,14 The Spot-Light centromeredetection kit (Zymed Labs, San Francisco, CA) wasapplied with a probe for the Y chromosome (84-1600;Zymed Labs), essentially following the manufacturersmethodology,with the difference that the heat pretreatmentstep was substituted with microwave irradiation in citratebuffer 10 mmol/L at pH 6.0 at 120C for 10 minutes

under pressure and the procedure was repeated after therecommended enzyme digestion step.14

All sections were observed for the following: inflam-matory response including neutrophilic infiltration,macrophage infiltration based on immunostaining withanti-CD68, and the vascularity of the section based onCD34 immunoexpression by endothelial cells.The neu-trophilic infiltration was scored on a semiquantitativebasis, in which 0 = no or infrequent perivascular neu-trophils; 1+ = scattered or 0 to 5 neutrophils present in aperivascular location, 2+ = moderate or more than 5 neu-trophils present in a perivascular location, and 3+ = neu-trophils present in perivascular locations and extendinginto the interstitium and myometrium.The infiltration ofmacrophages was scored in an identical manner usingCD68 as a marker of these cells. Apoptotic cells wereidentified by nuclear staining of caspase 3.

RESULTS

In the samples taken prior to the onset of labor, theintensity and extent of inflammatory changes were lessthan in samples taken after the onset of labor (Table 1),and significant increases with labor were observed forboth neutrophils and macrophages (P < .001, using theFisher exact test for both neutrophils and macrophages).Among the 37 not-in-labor Newcastle samples, neu-trophilic infiltration was absent in 20 cases, and only2 cases displayed infiltration of the interstitium andmyometrium; the remaining cases showed neutrophilsonly in a perivascular location. Similarly, the not-in-laborGlasgow samples showed only low levels of neutrophilinfiltration. Macrophage infiltration, as identified bystaining with CD68, was revealed only as scattered cellsin the not-in-labor samples from both centers, while

60 Reproductive Sciences Vol. 15, No. 1, January 2008 Leong et al

Table 1. Neutrophil and Macrophage Score in MyometrialBiopsiesa

Neutrophils Macrophages

0 1++ 2++ 3++ 0 1++ 2++ 3++

Newcastle cases Not in labor (n = 37) 20 10 5 2 22 12 3 0

Glasgow casesIn labor (n = 15) 0 3 6 6 0 6 6 3Not in labor (n = 16) 5 7 4 0 8 8 0 0

a0 = no cells; 1+ = 5 cells inperivascular location; 3+ = interstitial and myometrial infiltration.


in-labor samples showed increased macrophage infiltration(Figure 1).

While the not-in-labor samples had only modest lev-els of inflammatory infiltrate, many other unexpectedchanges were observed in these samples from both cen-ters. There was a variable degree of edema in themyometrium, with focal hemorrhage and prominentmyometrial damage in the form of pallor, variability ofcytoplasmic eosinophilia, and fibrillar shredding; in sev-eral cases, shrunken hypereosinophilic fibers were inter-spersed between swollen pale staining fibers (Figure 2).The myometrial fibers displayed brisk apoptotic activity,

confirmed by staining for caspase 3 (Figure 3).Almost allsamples from both centers showed marked vascularity.The thin-walled vessels were congested and often linedby large endothelial cells with prominent biotinylatednuclei that mimicked viral nuclear inclusions (Figure 4).These cells were occasionally shed into the vessel lumen.In addition, the vessels often contained fibrin, plateletthrombi, microparticles, and amniotic fluid squamouscells and occasional hair (Figure 5) as well as variousunidentified cellular and acellular debris and basophilicmucinous material (Figure 6); the latter was also found inthe interstitium. Fibrin, blood, microparticles, and cellular

Vascular and Myometrial Changes in the Human Uterus at Term Reproductive Sciences Vol. 15, No. 1, January 2008 61

Figure 1. Staining for macrophages with anti-CD68. (left) A 2+ score in a case not in labor compared with (right) a 3+ score in a case in labor.

Figure 2. Extensive myometrial damage in cases in labor (left) and not in labor (right).There is variable eosinophilia of the muscle fibers thatvary in thickness. Many fibers, especially in panel b, show pallor and loss of fibrillary striations, while hypereosinophilic shrunken fibers with con-certina outlines are present between the swollen fibers in both images (black arrows). Note the fibers with fragmented nuclei (white arrows).


debris were often admixed. Focal hemorrhage and fibrindeposition were observed in the connective tissue sur-rounding the vessels, but an inflammatory response wasseldom present.

In 10 patients carrying male fetuses who under-went cesarean delivery after the onset of labor, evidencefor fetal macrophages in the myometrium was soughtusing CISH for the Y chromosome. No Y chromo-somestaining macrophages were identified in any of the10 samples. Nearby trophoblast cells stained clearly for

the Y chromosome, demonstrating the effectiveness of thetechnique for identifying cells of fetal origin (Figure 7).

DISCUSSION

One of the aims of this study was to determine ifhistological examination of the term myometrium couldshed light on the processes leading to the leukocyte infil-tration observed at term in the human. It has been


Figure 3. Caspase 3 immunostaining highlighting many apoptoticmyometrial cells in the field.

Figure 4. A not-in-labor case from Glasgow showing abundant debrisin the vessel lumen. Mucoid material is mixed with amphophilicmicroparticles and dense eosinophilic material. Note the swollen hob-nail endothelial cells projecting into the lumen. Nuclear vacuolationfrom biotinylation is evident in endothelial cells (inset).

Figure 5. Intravascular fetal squames are present in this not-in-laborcase.

Figure 6. Not-in-labor case from Newcastle showing intravascularmucoid material mixed with cellular and acellular debris. Theendothelial nuclei are enlarged and hyperchromatic, and many shownuclear vacuolation. Note the presence of shed endothelial cells in thevessel lumen.


recently suggested that myometrial infiltration by acti-vated fetal macrophages may play a role in the onset oflabor. In addition, previous studies have demonstratedincreased macrophages in the human myometrium at thetime of labor.

In the mouse, surfactant protein A secreted by the fetallung into the amniotic fluid activates the fetal macrophagesresident in the amniotic fluid and promotes their migra-tion into the myometrium of the mother, where they pro-mote an inflammatory reaction that initiates labor.6 Morerecently, PCR and CISH studies of human myometriumat term have failed to identify evidence of fetal macrophagesas part of the inflammatory infiltrate,11 and this negativefinding was confirmed in our own studies. It thereforeseems that the human does not follow the mouse patternin this regard. It remains possible that fetal lung maturationand the consequent production of surfactant proteins andphospholipids may still play a role in human parturition,although not by stimulation of fetal macrophages tomigrate into the myometrium.

Interestingly, although there was neutrophilic infiltra-tion in a portion of our term not-in-labor cases, this wasextensive in only a small number, and macrophage infiltra-tion was not considered to be significant.Neutrophils,whenpresent, were located mostly around vessels, with only 2cases showing extension into the interstitium by theseinflammatory cells.Macrophage infiltration was scanty.Withthe onset of labor, the neutrophil and macrophage infiltrateincreased, as has previously been reported.5

In contrast to the limited inflammatory infiltratesobserved in our samples prior to the onset of labor, therewere other striking changes in the morphology of thenot-in-labor myometrium. There was evidence ofmyometrial damage in the lower uterine segment. Thistook the form of shredding and shearing tears of themyometrial fibers with shrinkage and variable stainuptake and hypereosinophilia. There was focal hemor-rhage and edema, and such changes were present in boththe samples that displayed inflammation as well as thosedevoid of inflammatory infiltration.These changes wereunexpected given that, in most cases, the myometrialfibers in the lower uterine segment had yet to be sub-jected to the extreme stresses that occur during estab-lished labor.There was also extensive apoptosis involvingmyometrial fibers.The presence of apoptotic bodies andthanatosomes15 suggests the active initiation of apoptosis;this may be an early stage of the uterine involution thatfollows labor and may be due to changes in the hormonalenvironment, perhaps including functional progesteronewithdrawal. Cell-derived microparticles that are mostlikely the result of cytoplasmic blebbing, which occurs asone of the earliest changes in the apoptotic process, arenow recognized as mediators of inflammation and coag-ulation and may have a number of other functions,including the transmission of hormones and toxins.16

The second major finding prior to the onset of laborwas clear evidence of the presence of amniotic fluidwithin the interstitium and within vessel lumens in theform of squames,mucoid material, and even hair; this fea-ture was widely present in samples from both Newcastleand Glasgow. The intravascular location of the amnioticfluid material makes it unlikely that this is an artifact gen-erated by surgery at the 2 recruiting sites.

A striking feature observed in most biopsies was thepresence of vascular morphologic changes. Abundantthin-walled vessels lined by prominent endothelial cellswere present in almost all biopsies from cases both not inlabor and in labor. In many cases, the endothelial cells hadenlarged vacuolated nuclei, with an appearance mimick-ing viral inclusions.These appearances have been shownto be due to nuclear accumulation of biotin17 that occursin the gestational endometrium and is present in smallamounts in specific organs such as the kidney, liver,pancreas, mammary gland, adipose tissue, and skeletalmuscle.17 Nuclear accumulation of biotin imparts aground-glass appearance that has been mistaken for viralinclusions, such as in herpes virusinfected cells, and it


Figure 7. Chromogenic in situ hybridisation for the Y chromosome.The single brown dots representing the labeled Y chromosome are seenin trophoblasts that, in this case, carried a male fetus, whereas the non-trophoblastic elements (namely, the myometrial cells) are not staining.


causes a false-positive reaction with immunohistochemicaldetection systems that employ biotin, as confirmed in ourcases.When stained with a biotin-free system (employingthe Vision Bond Max polymer system), the positive label-ing obtained with a streptavidin-biotin peroxidase systemwas eliminated (data not shown).We are not aware of anyprevious description of similar biotin accumulation inendothelial cells in myometrial vessels in gestation. Morerecently, biotin-rich intranuclear inclusions have beenobserved in tumors that are associated with squamousmorules and are reported to be associated with nuclearaccumulation of -catenin and biotin/biotin-bindingenzymes,13 although the mechanism of migration of suchsubstances to the nucleus remains unclear.

Large numbers of the thin-walled sinusoidal vesselscontained fibrin and platelet thrombi as well as otherunidentified cellular and acellular debris.There were alsohemorrhage and fibrin thrombi in the perivascular tis-sues. In addition, there were squamous cells andbasophilic mucoid material within vessels that was mostlikely to be of amniotic fluid origin. Evidence existed ofdesquamated endothelial cells within the vascular lumenand other unidentifiable material, the latter often associ-ated with fibrin thrombi. It is suggested that the biotiny-lated endothelial cells are fragile and more prone todamage and account for the frequent presence of desqua-mated nucleated cells within the vessel lumen. Thesechanges occurred in the absence of an inflammatoryinfiltrate in the surrounding tissue.

The changes in the lower segment myometrial tissueobtained prior to the onset of labor were dramatic andunexpected. To our knowledge, such observations havenot been previously reported, and we are unaware ofreports on the morphology of the myometrium at partu-rition in the contemporary literature. Our observationssuggest that the vascular endothelium may be an impor-tant source for inflammatory factors that are increased atthe time of labor. Potent vasoactive substances that aresynthesized by endothelial cells include nitric oxide,endothelin 1, and prostacyclin, and these are known tohave established roles in the modulation of myometrialcontractility.The presence of amniotic fluid emboli mayalso be a contributing factor to the inflammatorychanges, and the striking biotinylation of endothelial cellsmay contribute to their fragility. Our observation of thepresence of cell-derived microparticles is in keeping withthe prominent apoptosis present in the gestationalmyometrium. Possible sources of these microparticlesinclude endothelial cells, platelets, and myometrium. It is

speculated that these microparticles may contribute tothe escalation of inflammation in the myometrium. Itseems likely that myometrial contractility that increasesduring the process of parturition leads to shearing andtearing of myometrial fibers that perhaps promotes theirapoptosis.The damage to the myometrial cells may play arole in promoting an inflammatory infiltrate in some sub-jects.We postulate that the shear forces within the uterusgenerated by contractions lead to damage and sheddingof endothelial cells that is facilitated by increased fragilitydue to an increased content of biotin. Endothelial shed-ding will expose the underlying basement membraneproteins that will provoke a vigorous inflammatoryresponse. The increased intrauterine pressure and thedamaged vascular beds will facilitate the entry of amni-otic fluid containing surfactant protein A and proinflam-matory phospholipids into the vascular spaces of themyometrium. It may be that these processes help to pro-mote the accelerated phase of human labor through thedevelopment of positive feed-forward processes. Giventhe extensive evidence of damage in the humanmyometrium prior to the onset of active labor, it remainsunclear whether inflammatory genes expressed at thetime of labor are a cause of labor or a consequence of thistraumatic process.

REFERENCES

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2. Condon JC, Jeyasuria P, Faust JM, Wilson JW, MendelsonCR. A decline in the levels of progesterone receptor coacti-vators in the pregnant uterus at term may antagonize proges-terone receptor function and contribute to the initiation ofparturition. Proc Natl Acad Sci U S A. 2003;100:9518-9523.

3. Allport VC, Pieber D, Slater DM, Newton R, White JO,Bennett PR. Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expres-sion and is involved with the functional progesteronewithdrawal. Mol Hum Reprod. 2001;7:581-586.

4. Bisits AM, Smith R, Mesiano S, et al. Inflammatory aetiologyof human myometrial activation tested using directed graphs.PLoS Comput Biol. 2005;1:132-136.

5. Thomson AJ,Telfer JF,Young A, et al. Leukocytes infiltrate themyometrium during human parturition: further evidencethat labour is an inflammatory process. Hum Reprod. 1999;14:229-236.



6. Condon JC, Jeyasuria P, Faust JM, Mendelson CR. Surfactantprotein secreted by the maturing mouse fetal lung acts as ahormone that signals the initiation of parturition. Proc NatlAcad Sci U S A. 2004;101:4978-4983.

7. Mackler AM, Iezza G, Akin MR, McMillan P, Yellon SM.Macrophage trafficking in the uterus and cervix precedes par-turition in the mouse. Biol Reprod. 1999;61:879-883.

8. Mackler AM, Green LM, McMillan PJ, Yellon SM.Distribution and activation of uterine mononuclear phago-cytes in peripartum endometrium and myometrium of themouse. Biol Reprod. 2000;62:1193-1200.

9. Mackler AM, Ducsay TC, Ducsay CA,Yellon SM. Effects ofendotoxin and macrophage-related cytokines on the contrac-tile activity of the gravid murine uterus. Biol Reprod. 2003;69:1165-1169.

10. Yellon SM, Mackler AM, Kirby MA. The role of leukocytetraffic and activation in parturition. J Soc Gynecol Investig.2003;10:323-338.

11. Kim CJ, Kim JS, Kim YM, et al. Fetal macrophages are notpresent in the myometrium of women with labor at term. AmJ Obstet Gynecol. 2006;195:829-833.

12. Leong AS. Microwave techniques for diagnostic laboratories.Scanning. 1993;15:88-98.

13. Gamachi A, Kashima K, Daa T, Nakatani Y, Tsujimoto M,Yokoyama S. Aberrant intranuclear localization of biotin,biotin-binding enzymes, and beta-catenin in pregnancy-relatedendometrium and morule-associated neoplastic lesions. ModPathol. 2003;16:1124-1131.

14. Leong AS-Y HZ, Clarke M. Microwave enhancement ofCISH for HER2 oncogene.Appl Immunohistochem Mol Morphol.2007;15:88-93.

15. Papadimitriou JC, Drachenberg CB, Brenner DS, Newkirk C,Trump BF, Silverberg SG. Thanatosomes: a unifying mor-phogenetic concept for tumor hyaline globules related toapoptosis. Hum Pathol. 2000;31:1455-1465.

16. Horstman LL, Jy W, Jimenez JJ, Bidot C,Ahn YS. New hori-zons in the analysis of circulating cell-derived microparticles.Keio J Med. 2004;53:210-230.

17. Cooper K, Haffajee Z, Taylor L. Comparative analysis ofbiotin intranuclear inclusions of gestational endometriumusing the APAAP, ABC and the PAP immunodetection sys-tems. J Clin Pathol. 1997;50:153-156.



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