experimental adenomyosis

Best Practice & Research Clinical Obstetrics and GynaecologyVol. 20, No. 4, pp. 503e510, 2006

doi:10.1016/j.bpobgyn.2006.01.003available online at http://www.sciencedirect.com

5

Experimental adenomyosis

Peter Greaves* MBChB, FRCPath

Honarary Senior Lecturer

Ian N.H. White PhD, DSc

Team Leader

MRC Molecular Endocrinology Group, Cancer Studies and Molecular Medicine,

Robert Kilpatrick Building, University of Leicester, Leicester LE2 7LX, UK

Adenomyosis has been reported in a number of different animal species, whereas endometriosisappears limited to humans and non-human primates. This suggests a different aetiology of thetwo conditions. Adenomyosis develops spontaneously in certain strains of laboratory mice.Its incidence in mice can be markedly enhanced by systemic exposure to various hormonalagents, including prolactin, progesterone, synthetic progestins, certain oestrogenic agents, aswell as tamoxifen and toremifene. The precise hormonal changes necessary remain unclear,although the evidence suggests that adenomyosis in this model is not due to a simpleoestrogenic effect. Study of the pathological and molecular alterations in this model indicatesthat disturbances to the uterine stroma, blood vessels and myometrium are also importantfactors in the development of adenomyosis.

Key words: animal model; human disease; growth factor; adenomyosis; endometriosis; mice;oestrogen; prolactin; progesterone; tamoxifen.

Adenomyosis e defined as the benign invasion of endometrial glands and stroma deepand haphazardly into the myometrium e occurs spontaneously with a variable inci-dence in a number of animal species. It has well been characterized in dogs, cats,non-human primates, laboratory rodents and rabbits. Adenomyosis has also been in-duced in some laboratory animal species by experimental means. Whilst studies of allthese animal models provide some insights into the pathology and pathogenesis of thisenigmatic condition, it is the mouse model that has most frequently been used exper-imentally to study adenomyosis. Certain strains of laboratory mice are predisposed toadenomyosis, and its prevalence can be markedly enhanced by hormonal manipulation.Although recently there have been exciting advances in genetic modelling of pelvic

* Corresponding author. Tel.: þ44-116-252-3143.

E-mail address: [email protected] (P. Greaves).

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504 P. Greaves and I. N. H. White

endometriosis in mice carrying latent alleles of an active mutant K-ras oncogene,1,2

this modelling does not facilitate the development of adenomyosis, supporting theconcept that endometriosis is a separate condition with a different aetiology. Despitethe use of in vitro techniques to study the characteristics of the various cells that com-prise adenomyosis, production of a good in vitro model for the complex stromaleglandular interactions in this disorder has proved elusive.

COMPARATIVE PATHOLOGY

Information about the true incidence of adenomyosis among animal species is limitedby the lack of systematic evaluation of the uterus in most series. Nevertheless suffi-cient studies have been conducted to conclude that it is generally prevalent amongmany animal species.

In view of the close similarity of the female reproductive system of non-human pri-mates to that of humans, it is not surprising that monkeys develop uterine adenomyosisnaturally. In a population-based study of 38 laboratory and 17 free-ranging rhesus mon-keys (Macaca mulatta) of at least 4 years of age, uterine adenomyosis and pelvic endo-metriosis were some of the most prevalent findings in the reproductive tract.3

A relationship was observed between the occurrence of adenomyosis, vaginitis,metritis, gravid status, time elapsed since the last pregnancy, number of matings, repro-ductive ability and reproductive status. The overall prevalence of adenomyosis in thispopulation was 20%. Less was found in free-ranging animals compared with the labora-tory monkeys. In another small study of uterine pathology in the same species, out of sixadult laboratory monkeys subject to ovariectomy and given implants containing 200 mgof 17b-oestradiol, one showed adenomyosis alongside endometrial hyperplasia at 16months.4 Although its occurrence was ascribed by the authors to oestrogen administra-tion, the number of animals studied was too small to exclude spontaneous development.

More recently a study of the records from 3827 baboon necropsy and surgerypathology records from between 1986 and 2002 at the Southwest Foundation for Bio-medical Research, Texas, revealed adenomyosis in 37 animals.5 Species included Papioanubis, Papio cyanocephalus, and Papio hamadyra, as well as crosses. A comparison ofreproductive background was made between affected animals and matched controlswithout adenomyosis. The strength of this study was that uterus was examined histo-logically using the same number of tissue blocks and the same pathologist over a 16year period. Although the presence of adenomyosis might have been missed bylimitations of sampling, the figures are derived from a standard sampling procedure.There was a strong association between adenomyosis and life-long infertility in theseanimals. Although the presence of adenomyosis was also strongly associated with thepresence of endometriosis, the association of adenomyosis with infertility remainedeven after cases with co-existing endometriosis were excluded from the analysis.

Non-primate species also develop adenomyosis spontaneously. Both dogs and catsare reported to show adenomyosis in small numbers.6,7 In one study detailed histo-pathological examination of the uterus from 37 dogs and nine cats with uterine disor-ders, out of a total of 52 non-neoplastic and neoplastic disorders only one cat and onedog demonstrated adenomyosis.7 These lesions were considered incidental and hadnot affected reproductive performance, suggesting the relative lack of biological impor-tance of adenomyosis in these species.

Adenomyosis has also been well described in untreated, laboratory rodents. Al-though it is more common in mice than rats, its prevalence is highly strain-dependent.

Experimental adenomyosis 505

However, the exact incidence is often difficult to ascertain because of relatively limitedhistopathological examination of the uterus in many conventional rodent studies. Inthis laboratory, where at least three histological sections are examined from eachuterine horn in life-time studies using conventional CD-1 studies, adenomyosis startsto appear in control mice from about 6 months of age onwards. Over 80% of controlmice are affected by minimal degrees of adenomyosis by 12 months of age, and itsseverity increases thereafter.

In a study of four strains of mice maintained for over 40 generations, the percentage ofmice and the age of onset remained similar within the same strain. In this study it was notedthat all mice of the SHN and SLN strains that developed mammary tumours also hadadenomyosis, and that the strains that did not develop mammary tumours; 43% of theGR/A strain and 100% of the C3H/He strain also had adenomyosis at 20 months of age.8

Adenomyosis has also been reported to occur spontaneously with a high preva-lence in SMXA recombinant inbred mice which are derived from the F2 generationof a cross between A/J and SM/J strains.9

EXPERIMENTAL MODELS

Mouse models

It has long been known that adenomyosis can be produced in experimental animals byexperimental means. The mouse model is one of the most commonly employed forstudy of the condition because various forms of hormonal imbalance have been shownto enhance the development of adenomyosis in this species. It has been linked in miceto hyperprolactinaemia,10,11 prolonged treatment with oestrogenic compounds,12,13

and prolonged progesterone administration.14,15 Unfortunately, because the precisehormonal environment in many of these experiments is not clearly defined, thehormonal patterns that increase the prevalence of adenomyosis are unclear.

Intrauterine implantation of isographs of anterior pituitary glands in mice has beenemployed by Mori and colleagues over a number of years to explore the pathogenesisof adenomyosis.16,17 In this model, mice of the SHN or SLN strain receive a singleisologous graft of an anterior pituitary gland in the lumen of the right anterior uterinehorn at about 50 days of age. The mice are subsequently killed and are subject to a nec-ropsy and pathological examination at various periods after implantation. Almost allmice develop adenomyosis after about 90 days from engraftment in the uterinehorn subject to grafting. However, adenomyosis also develops in the contralateraluterine horn not subject to grafting or when the pituitary gland is put under the renalcapsule.16,18 Nevertheless, the incidence of adenomyosis remains higher in the righthorn, suggesting that the disruption of the myometrium and the ensuing fibroblasticresponse and invasion of blood vessels by the graft exacerbate the development of ad-enomyosis. Mori and colleagues ascribe the high prevalence of adenomyosis primarilyto prolactin. This is because of increased circulating levels of prolactin, its preventionby administration of bromocriptine (a suppressor of pituitary prolactin),10 and thepresence of increased prolactin receptor mRNA in the affected uterus.19 This grouphas also reported that treatment of neonatal mice or young adult mice with prolactinor administration of dopamine antagonists which increase prolactin release also resultsin a high incidence of adenomyosis.10,20

Other studies using this model have shown elevated matrix metalloproteinasemRNA in uterine tissues e mainly luminal and glandular epithelium and myometrial


cells e in affected mice.17 Moreover, these authors also reported an increase in apo-ptosis in myometrial cells in mice with adenomyosis. A reduction in the number ofuterine microvessels with concomitant increase in the numbers of dilated uterinevenules has also been described, suggesting that alterations to blood vessels alongsidedegradation of stroma by matrix metalloproteins might be important factors in thegenesis of adenomyosis.21 Interestingly, probucol, a rarely used cholesterol-loweringdrug, was shown to prevent the development of adenomyosis in this model.18 Thisdrug not only has cholesterol-lowering effects but also antioxidant properties andinhibits endothelial cell proliferation after injury.22

The pituitary graft model is complicated by the ill-defined hormonal interactionsthat can be produced. Other hormonal alterations produced in this model have ledto the suggestion that hormonal imbalance is more important than high levels ofcirculating prolactin alone. Indeed, a number of experiments in adult mice treatedfor long periods with various agents with oestrogenic activity have also been reportedto induce adenomyosis.12,13 It has been suggested that this might also be mediated viaprolactin excess as oestrogen may also elevate prolactin levels in rodents. Unfortu-nately, the interpretation of many of these studies is complicated by the presence ofother oestrogenic effects on the uterus, such as endometrial hyperplasia, squamousmetaplasia and uterine carcinomas.

An older but careful study of the uterine changes in BALB/c mice by Lipschutzand colleagues15 showed a marked and dose-related increase in adenomyosis after12 or 18 months exposure to progesterone. Moreover, the synthetic progestinsnorethindrone and norethynodrel also produced adenomyosis, although to a lesserextent than progesterone. A detailed study of the response of the uterus to neo-natal diethylstilboestrol treatment and subsequent hormonal treatment in ovariec-tomized mice by Ostrander and colleagues14 also showed that adenomyosisdeveloped after prolonged progesterone treatment of both controls and mice givendiethylstilboestrol in the neonatal period. These ovariectomized mice had loweroestrogen levels and probably lower prolactin levels, which reinforces the link be-tween adenomyosis development and a hormonal imbalance involving high levels ofprogesterone.14

More recently another mouse model has been developed whereby oral administra-tion of tamoxifen and toremifene for several days during in the neonatal period givesrise to the development of a high incidence of adenomyosis in adults. Parrott and col-leagues23 produced adenomyosis in the uterus of CD-1 mice by oral dosing thesedrugs for 5 days during the neonatal period. It is worth noting that in the mouse uterusboth tamoxifen and toremifene tend to act as oestrogen agonists. This study showedthat by 3 months a high proportion of mice treated in the neonatal period developedadenomyosis, whereas it was not often present in untreated controls around this age(Figure 1a,b). At later stages the severity of adenomyosis increased greatly andpenetrated up to but not through the serosa of the uterus (Figure 1c). Interestingly,a related compound, raloxifene, administered in a similar way was devoid of this effectalthough the reason for this was unclear. In a mouse uterotrophic assay, raloxifene haslittle or no oestrogen agonist activity compared to tamoxifen or oestradiol.24 How-ever, the study from Parrott and colleagues23 using a similar uterotrophic dose of oes-tradiol administered in a similar manner to the same strain of mice under identicalconditions also did not produce adenomyosis, indicating that the effects were notdue to a simple oestrogenic effect.25 It is perhaps salient to note, in view of thepossible role that progesterone or oestradiol may have in the development of adeno-myosis in this mouse model, that mice treated neonatally with the naturally occurring


oestrogen agonist genestein have normal serum levels of both oestradiol and proges-terone at 6 months after dosing.26

In this model there is a striking derangement of the normal uniform concentriclayers of muscle in the myometrium of treated mice both in the neonatal period beforethe development of adenomyosis as well as in adult mice in association with adeno-myosis (Figure 1b,c). The implication of this is that the disruption to the mesenchymallayers of the uterine wall gives rise to disordered structure and function of the myo-metrium which provides the abnormal architectural framework for aberrant growth ofthe endometrium and its penetration deep into the myometrium. These pathologicalchanges are mirrored by alterations in genes that are believed to be important in theregulation of mesenchymal cells, notably nerve growth factor (NGF). NGF and its low-affinity receptor p75NTR are believed not only to have a role in synchronizing the de-veloping visceral nervous system but also in myogenic differentiation. It was suggestedthat the observed up-regulation of Ngf gene expression might have contributed to therepression of myometrial differentiation in these tissues. Further study using laser cap-ture microscopy demonstrated that Ngf was found primarily in endometrial luminalepithelium of controls, and it was up-regulated in luminal epithelium in treatedmice. The low-affinity receptor was shown to be distributed in both the glands andstroma, suggesting that NGF acts as in a paracrine manner to modify stromal develop-ment.27 It is interesting that NGF has also been demonstrated by immunohistochem-istry in deep adenomyotic and endometriotic nodules in women.28 Moreover, in micetreated neonatally with tamoxifen, gene array time-course studies have shown largenumbers of expression changes during development, consistent with tissueremodelling.29

Additionally intriguing is the fact that whilst Parrott and colleagues observed nouterine carcinoma development in their model in CD-1 mice over observation periodsof up to 2 years, a similar regimen has been reported to produce uterine carcinomas

Figure 1. Mouse model of adenomyosis as reported by Parrott and colleagues23 showing the histological

appearances of a cross-section of normal uterine horn from an adult mouse (a), adenomyosis in a mouse

of about 6 months of age (b), and advanced adenomyosis in a 2-year-old mouse showing penetration of

glands to the serosa (c). Note the clear demarcation of the endometrial stroma from the concentrically or-

ganized myometrial layer (arrow) compared with the disorganized muscle layer in the cases of adenomyosis

(arrowhead). All micrographs at the same magnification, stained with haematoxylin and eosin.


by others. Newbold and colleagues reported neonatal administration of the same doseof tamoxifen to CD-1 mice via the subcutaneous rather than the oral route producesuterine carcinomas rather than adenomyosis.30 Whilst the precise reason for this dif-ference is not clear, the hormonal imbalances in the two studies were different.Parrott and colleagues23 reported that in mice affected by adenomyosis, ovariesshowed the normal pattern with developing follicles and corpora lutea, similar to con-trols and mice undergoing oestrus cycling. In contrast, Newbold and colleagues indi-cated that in their study in mice treated with tamoxifen, ovaries were often cysticand lacking corpora lutea, reflecting perturbed or arrested follicular development.This highlights the crucial nature of quite precise hormonal perturbations in the gen-esis of adenomyosis or uterine cancer in this model.

Rat models

Far few studies on adenomyosis have been performed in the rat than in the mouse,although those that have been performed suggest that similar hormonal perturbationsare important. Whilst the mouse appears to show a predilection to adenomyosis fol-lowing pituitary grafting, the rat also has been shown to develop adenomyosis follow-ing the same procedure.31 Furthermore, treatment of intact but not ovariectomizedrats with fluoxetine, a serotonin reuptake inhibitor, for 14 weeks induced high circu-lating levels of prolactin along with adenomyosis.32

Rabbit models

The rabbit is another animal that has been used as an experimental model for adeno-myosis because it develops the condition spontaneously and its prevalence can be en-hanced by oestrogen administration over 1e2 years.33 However, as in mice, thedevelopment of adenomyosis following oestrogen administration is not ideal for thestudy of adenomyosis as it is complicated by the associated development of endome-trial hyperplasia and endometrial carcinoma.

SUMMARY

The fact that adenomyosis occurs in many animal species and endometriosis is limited toprimates suggests that the two conditions have a different aetiology. This is supportedby the lack of adenomyosis in a novel model of endometriosis in mice carrying latentalleles of active mutant K-ras oncogene. This model suggests that peritoneal endome-triosis is probably initiated by endometrial tissue reflux through the fallopian tubes,whereas adenomyosis is likely to represent a down-growth of endometrium in recep-tive myometrial tissue. Another mouse model also suggests that physical disturbance tothe normal myometrium or the myometrialeendometrial junction in early life is also animportant factor that predisposes to the development of adenomyosis in adulthood.

Research agenda

� use of the mouse model to test ways in which the penetration of endometriuminto other tissues might be inhibited.


A factor common to the development of adenomyosis in rodent models is hor-monal imbalance, notably the presence of high progesterone levels. Whereas high cir-culating prolactin appears important in some mouse models, this does not seem to bean absolute requirement for adenomyosis development. In adenomyosis that developsfollowing neonatal tamoxifen or toremifene administration, the presence of normallyfunctioning corpora lutea appears to be important. Intriguingly, in a similar model sup-pression of normal ovarian function is associated with endometrial hyperplasia andcarcinoma, underlining the crucial importance of the precise nature of the perturbedhormonal balance in the development of endometrial pathology.

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experimental adenomyosis

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