The blood supply of myomatous uteri collected upon autopsy was examined.
The uterine vascular beds were perfused via afferent vessels with fixative followed
by Mercox resin and corroded after polymerisation of the resin. The vascular
casts thus obtained were examined using scanning electron microscopy.
The vascular system of the uterine fibroids was also examined using immunohistochemical
analysis (FVIII, factor VIII-related antigen)

Miodoński, Adam J.

Skrzat, Janusz

Stachura, Jerzy

Szczepański, Wojciech

Walocha, Jerzy A.

English

Via Medica Journals

Folia Morphol. Vol. 63, No. 1, pp. 37–41Copyright © 2004 Via MedicaISSN 0015–5659www.fm.viamedica.pl O R I G I N A L  A R T I C L E37Two types of vascularisation of intramuraluterine leiomyomata revealed by corrosioncasting and immunohistochemical studyJerzy A. Walocha1, Adam J. Miodoński2, Wojciech Szczepański3, Janusz Skrzat1,Jerzy Stachura31Department of Anatomy, Collegium Medicum, Jagiellonian University, Kraków, Poland2Laboratory of Scanning Electronic Microscopy, Kraków, Poland3Department of Pathomorphology, Collegium Medicum, Jagiellonian University, Kraków, Poland[Received 5 November 2003; Accepted 25 November 2003]The blood supply of myomatous uteri collected upon autopsy was examined.The uterine vascular beds were perfused via afferent vessels with fixative fol-lowed by Mercox resin and corroded after polymerisation of the resin. The vas-cular casts thus obtained were examined using scanning electron microscopy.The vascular system of the uterine fibroids was also examined using immunohis-tochemical analysis (FVIII, factor VIII-related antigen).Key words: fibroids, corrosion casting, leiomyoma,immunohistochemistry, vasculatureAddress for correspondence: Jerzy A. Walocha, Department of Anatomy, Collegium Medicum, Jagiellonian University, ul. Kopernika 12,31–034 Kraków, Poland, tel/fax: +48 12 422 95 11, e-mail: jwalocha@poczta.onet.plINTRODUCTIONUterine fibroids or leiomyomata are the mostcommon solid tumours in women, affecting at least50% over 30 years of age [16]. Their high incidence,associated symptoms (pelvic pain, menorrhagia) andside-effects (infertility, loss of pregnancy) presenta serious clinical problem. Very little is known of theaetiology of uterine leiomyomas. It is known thatthere are a number of risk and protective factors forand against fibroid growth related to oestradiol[9, 15] and that there are an increased numbers ofsex steroid receptors in fibroids compared with my-ometrium [2]. Other factors that may be associatedwith fibroid aetiology include peptide growth fac-tors, such as the insulin-like growth factor (IGF), fam-ily and epidermal growth factor (EGF) as well as pos-sible chromosomal and subsequent gene changes[14] within these tumours. The corrosion castingtechnique combined with scanning electron micros-copy (SEM) is the best method currently availablefor morphological examination of the vascular net-works. SEM offers a high resolution and quasi-three-dimensional image. SEM was also used to study thevascular system of intramural leiomyomata [17]. FVIIIforms part of the von Willebrand factor complex,plays a part in the coagulation cascade and its ex-pression is reduced in smaller, less mature bloodvessels [7]. The vascular staining for this factor is nodifferent from that for other vascular markers, i.e.CD 31, CD34 [3]. The present study has been under-taken to compare the results of studies of the vascu-lar network of human uterine fibroids using SEM andimmunohistochemistry.MATERIAL AND METHODS30 uteri were obtained upon autopsy of womenaged 25–56 years, deceased due to causes unrelat-ed to disorders of the reproductive system. The studywas approved by the Ethics Committee of the Jagiel-lonian University Medical College. The material wascollected 6–24 h after death. Each uterus togetherwith the ovaries and cervical portion of the vagina38Folia Morphol., 2004, Vol. 63, No. 1was removed in such a way that relatively long frag-ments of uterine and ovarian vessels (arteries andveins) were retained.Immediately after removal, 22 uteri were perfusedvia the afferent arteries with prewarmed (37oC), he-parinised saline (12.5 IU/ml heparin, Polfa, Poland)containing 3% dextrane (70 kDa) and 0.025%lidocaine (Lignocaine, Polfa), until the fluid outflow-ing via the veins was completely transparent (~5 min).Perfusion was then continued using a solution of0.66% paraformaldehyde/0.08% glutaraldehyde (Sig-ma) in 0.1 mol/l cacodylate buffer, pH 7.4 supplement-ed with 0.2% lidocaine. Finally, the vascular systemwas injected with 60–80 ml of Mercox CL-2R resin(Vilene Comp. Ltd. Japan) containing 0.0625 mg/mlmethyl acrylate polymerisation initiator (VileneComp. Ltd.) and the uteri were left in a warm waterbath (56oC) for several hours to allow polymerisa-tion and tempering of the resin.When the polymerisation was completed, theuterine tissues were macerated for 5–6 days by re-peated baths in 10% potassium hydroxide at 37oCfollowed by washing with warm (50–55oC) runningtap water. The vascular casts obtained were washedfor the next 4–5 days in multiple changes of distilledwater under mild vacuum conditions, cleaned in 5%trichloroacetic acid for 1–2 days, washed again indistilled water for 2–3 days and freeze-dried in a lyo-philiser (Liovag G2; Aqua Fina, Germany).The freeze-dried casts were examined macroscop-ically, gently dissected [18] to expose the vasculatureof myomata and stored in an exiccator containingphosphorus pentoxide before microscopic examina-tion. They were then mounted onto copper plates us-ing colloidal silver and “conductive bridges” and coatedwith gold. The casts were examined using a JEOL SEM35-CF scanning electron microscope at 20–25 kV.The vascular beds of the next 10 uteri were per-fused with saline and then injected with a solutionof acrylic emulsion (Liquitex R, Binney and Smith,USA) [19]. The specimens were collected mainly fromlarge fibroids > 3 cm in diameter. All tissue speci-mens were fixed in 10% formalin and embedded inparaffin wax. The tissue blocks were sectioned (4 µm)and section-mounted on aminopropyltriethoxysilane(APES)-coated slides. The endothelial cell marker wasfactor VIII-related antigen (FVIII). Next the tissueblocks were deparaffinised and hydrogen peroxidase(3%) in methanol was applied to the tissue sectionsas an endogenous peroxidase block for 10 min. Theprotein blocking steps included the application of10% normal rabbit serum prior to the application ofthe primary antibody (anti Human von Willebrandtfactor, Dako) in 20% foetal calf serum. After incuba-tion with primary antibody a biotynyled secondaryantibody was applied, followed by horseradish per-oxidase-streptavidin conjugate and visualisation bymeans of chromogen 3-amino 9-ethylchlorcarbazole(AEC, Zymed, USA), which identifies tissue antigenswith a red-brown stain. Serial sections were immun-ostained using the endothelial cell marker. The neg-ative controls used substitution of the primary anti-body with the non-immune serum for the polyclonalfactor VIII antibody.RESULTSAmong 30 uteri prepared for corrosion casting,only 7 yielded casts of acceptable quality. The ob-servation was limited to intramural fibroids, sincethe vascular casts of subserosal tumours were alwaysconsiderably damaged.Two systems of blood supply to intramural uter-ine leiomomata were observed. Some of the fibroidswere centrally almost avascular, although their pe-ripheral vessels were usually arranged in the form ofa dense network composed mostly of capillaries ormultiple smaller and shorter arteries and veins (Fig. 1),forming a so-called “vascular capsule”. The otherswere supplied by 2 or 3 larger arteries running incompany with veins, which penetrated the centralareas of the lesion, giving relatively few side branches(Fig. 2). In both patterns the veins were flattened,which was probably a result of compression of thetumour.Very commonly the vessels penetrated the con-nective tissue septa, which separated the small tu-mours from each other (Fig. 3).In routine histological sections the vessels seemto follow the course of the septa and are not visi-ble in the centre of the tumour. By using immun-ostaining with a vascular marker (FVIII) large ves-sels can be observed following the septa and smallvessels (capillaries) penetrating the substance of thetumour. Here two types of vascularisation of theuterine leiomyomata can be distinguished, one in theform of dense peripheral “vascular capsule” (Fig. 4)and the other made up of a few vessels with sideramifications, penetrating the lesion (Fig. 5).DISCUSSIONThe vasculature of fibroids has not been studiedin detail and has rarely been studied in a quantita-tive manner [3, 8]. Earlier structural studies [4, 5,11, 12] using similar methods of pigment or radio-39Jerzy A. Walocha et al., Two types of vascularisation of intramural uterine leiomyomataFigure 2. Corrosion cast — bundle of vessels (160–230 µm)penetrating small uterine leiomyoma. SEM. Bar = 1000 µm.opaque dye injection demonstrated that the arterialvessels of uterine leiomyoma were typically increasedin size, especially in larger tumours, whereas thedegree of arterial vascularity was variable. It was alsoobserved that small uterine fibroids were usually, butnot always, less vascular than the surrounding myo-metrium, while larger leiomyomas were usually morevascular than the myometrium. Farrer-Brown et al.[4, 5] also demonstrated that there was no intrinsicvascular pattern in uterine fibroids but rather thatthe arrangement appeared to represent a localisedexpansion of the myometrial vasculature with thevessels within these tumours oriented in the direc-tion of the muscle cell bundles. They also showedthere were changes in vasculature distant to the tu-mour, with dilation and congestion of vessels con-tralateral to the site of the uterine fibroids.Blood flow studies have been performed in uter-ine fibroids. The vasculature of uterine leiomyomawas investigated using colour Doppler ultrasound[10, 13]. Their results concurred with the earlier workof Sampson [11, 12], showing that the vascularisa-tion of these tumours was largely dependent on thetumour size, position and the extent of secondarydegenerative changes.It is thought that the growth of a tumour is lim-ited by its blood supply, with tumours dependentupon the ingrowth of capillary sprouts from sur-rounding tissues for growth beyond a diameter ofa few millimetres [1]. The differences in vascular den-sity between myometrium and uterine leiomyomasmay represent a difference in angiogenesis and vas-cular remodelling in these vascular beds. These dif-ferences in angiogenesis may be the result of changesin the balance between the factors that promote orinhibit the angiogenesis.Faulkner [6] in his classic report described thevascular system of the leiomyomata as “a mass ofFigure 1. Arteries (*) and veins (   ) of the “vascular capsule” of small uterine leiomyoma— SEM. Bar = 1000 µm.40Folia Morphol., 2004, Vol. 63, No. 1proliferating arteries”. The technique used byFaulkner did not visualise the capillaries and our stud-ies have shown that arteries are not the dominantvessel type in uterine fibroids. The vascular densityof myomata demonstrated by SEM and immunohis-tochemistry seems to be either lower than or similarto that of unchanged myometrium. In a recent im-munohistochemical/morphometric study, Casey et al.[3] reported significantly higher microvascular den-sity in the adjacent myometrium than in small andlarge leiomyomata. SEM demonstrated the perma-nent feature of all myomata, which had the form ofa “vascular capsule”. The immunostaining for FVIIIalso proved the significantly increased density ofperipheral vessels of uterine leiomyomata, thus form-ing a dense “vascular capsule”.Knowledge of vascular differences may provideinformation on basic biology, aid understanding ofthe variability of symptoms reported by women withfibroids, better define the mode of action of someof the current medical managements (gonadotro-phin releasing hormone agonists [GnRHa] and RUFigure 3. Corrosion cast. Vessels of connective tissue septa. SEM. Bar = 1000 µm.Figure 4. Arteries (*) of leiomyoma injected with acrylic emulsion (Liquitex R, Binney and Smith, USA).Numerous empty arteries running through the fibroid septa. Immunostaining for FVIII. Bar = 500 µm.41Jerzy A. Walocha et al., Two types of vascularisation of intramural uterine leiomyomata486) and, finally, provide knowledge of the futureutility of angiogenesis inhibitors for treatment.REFERENCES1. Alberts B, Bray D, Lewis J (1994) Molecular Biology ofthe Cell, 3rd ed. Garland, New York.2. Anderson J (1998) Factors in fibroid growth. Bailliére’sClin. Obstet. Gynecol, 12: 225–243.3. Casey R, Rogers PAW, Vollenhoven BJ (2000) An im-munohistochemical analysis of fibroid vasculature.Hum Reprod, 15: 1469–1475.4. Farrer-Brown G, Beilby JOW, Rowles PM (1970) Mi-crovasculature of the uterus: an injection method ofstudy. Obstet Gynecol, 35: 21–30.5. Farrer-Brown G, Beilby JOW, Tarbit MH (1970) Thevascular patterns in myomatous uteri. J Obstet Gynecol,77: 967–975.6. Faulkner RL (1944) The blood vessels of the myoma-tous uterus. Am J Obstet Gynecol, 47: 185–197.7. Fay PJ (1993) Factor VIII structure and function. ThrombHaemostat, 70: 63–67.8. Hague S, Zhang L, Oehler MK, Manek S, MacKenzie IZ,Bicknell R, Rees MCP (2000) Expression of hypoxicallyregulated angiogenic factor adrenomedullin correlateswith uterine leiomyoma vascular density. Clin Canc Res,6: 2808–2814.9. Hong T, Shimada Y, Uchida S, Itami A, Li Z, Ding Y,Kaganoi J, Komoto I, Sakurai T, Imamura M (2001)Expression of angiogenic fasctors and apoptotic fac-tors in leiomyosarcoma and leiomyoma. Int J MolMed, 8: 141–148.10. Kurjak A, Kupesic-Urek S, Miric D (1992) The assessmentof benign uterine tumor vascularization by transvaginalcolor Doppler. Ultrasound Med Biol, 18: 645–649.Figure 5. Large arteries (*) injected with acrylic emulsion (Liquitex R, Binney and Smith, USA)present in the centre of the myoma. Small empty vessels (≠) — capillaries observable in the centre,O — margin of the myoma. S — septa. Immunohistochemical staining for FVIII. Bar = 500 µm.11. Sampson JA (1912) The blood supply of uterine myo-mata. Surg Gynecol Obstet, 14: 15–234.12. Sampson JA (1912) The influence of myomata on theblood supply of the uterus, with special reference toabnormal uterine bleeding. Surg Gynecol Obstet, 16:144–180.13. Sosic A, Skupski DW, Sterlzoff J, Yun H, Chervenak FA(1996) Vascularity of uterine myomas: assessment bycolor and pulsed Doppler ultrasound. Int J GynecolObstet, 4: 245–250.14. Tsibris JCM, Segars J, Coppola D, Mane S, Wilbanks GD,O’Brien WF, Spellacy WN (2002) Insights from genearrays on the development and growth regulation ofuterine leiomyomata. Fertil and Steril, 78: 114–121.15. Vollenhoven B (1998) Introduction: the epidemiologyof uterine leiomyomas. Bailliére’s Clin Obstet Gynecol,12: 169–176.16. Wallach EE (1992) Myomectomy. In: Thompson JD,Rock JA (eds.). Te Linde’s operative gynecology, 7th ed.JB Lippincott, London, pp. 647–662.17. Walocha JA, Litwin JA, Miodoński AJ (2003) Vascularsystem of intramural leiomyomata revealed by corro-sion casting and scanning electron microscopy. HumReprod, 18: 1088–1093.18. 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Two types of vascularisation of intramural uterine leiomyomata revealed by corrosion casting and immunohistochemical study

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Two types of vascularisation of intramural uterine leiomyomata revealed by corrosion casting and immunohistochemical study

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