Indian Journal of Experimental Biology Vol. 38, February 2000, pp. 129- 136

Organogenesis and tissue regeneration of fallopian tube: A desired metaplastic transformation of mesodermal stem cells in live animal model s (dogs) *

B G M atapurkar"t, A Bhargaveb, L Dawsonc, H M S Rehan<~ , B Sonal" & V K Ramtde"

"Department of Surgery. Lok Nayak Hospi tal & M A Medica l College. New Delhi I 000 I. India hDepartment of Paediatrics & Academic Section, S J Hospital Hospital, New Delhi 110023. India

"Institute of Pathology. I C M R, S J H Complex. New Delhi 110023 , lnJia <~Department of Pharmacology, M A Medical College, New Delh i I I 000 I , India

"ICMR Project, M A Medical College, New Delhi 11000 1, India

Received.., !< August 1997, revised 2 August 1999

The capacity of stem cells of peritonium of mesodermal origin to undergo metapl as ti c transformati on and form different ti ssues developed from mesoderm germ layer is exploited with ulterior moti ve to usc it in the m:magement of human diseases. The exc ised fall opian iube was replaced with a tube on a stent constructed from autogenous perit oneum from a suitable donor site. The effect of the surroundings environment of the new ti ssue system to which the peritoneum stem ce lls arc now exposed was studied for 3, 6 and 12 mon ths peri od in li ve animal models. The gross ami histologica l studies revealed development of all the component of the wall of the fall opian tube. The lumen of the constructed peritoneal tube was well preserved in i ts whole k ngth including the anastomotic sites, The sc ientific rationale of the worki ng hypothesis on whic h the work is based. is discussed.

Plast ic surgery of fa ll opian tube has witnessed use of variety of ti ssues indicating interest in the area of infertility in females. Despite varied success rates and

notable improvement in over all results in their use in plastic surgery o f the tube, these substitutes were not integrated in the process of natural wound healing and repair. The ~uita bl e method to repl ace the part or whole of the fa ll opi an tube is still needed to correct

the ex tensi ve damage of the fall opian tube. The regenerative technique to grow tissues and organs in

the body is probabl y the most ideal and des ired

venture to accompli sh. The pluripotent stem cell s

present in the tissues of the body are responsib le for healing and repair of ti ssue in the damaged or injured

region . But when exposed to irritati on or abnormal conditi ons they have the capacity to undergo met<J.plasia. This transformati on is o ften abnormal I pathological in the region. I f stem cell s in the process

of eli fferentiat ion and pro I i fera ti on to replace morphologica ll y different tissue are subj ec ted to

undergo change which is des ired in the region of new

*The paper was presented in inaugural conference of Americ;m College of Surgeons held at AII MS. New Delhi , II OO IIi. lnd1a. January 16 , 1996. t AdJrcss ror correspondence: D 11 /191. West Kid wai Nagar, 1ew Delhi , 110023, India

environment then the des ired n1etapla ~; ia may be responsib le for ti ssue/organ regenerati on. Accordingly

a hypothesis was proposed, based on the success and fa ilure of experimenta l studi es in ex per i.mental li ve animals . The present study has been undertaken to:

(A) T est Matapurkar ' s hypothes is1: and

(A ) Regenerate fa ll op ian tube in t'il'o in dogs . The hypothesis states that i r stem ce ll s or the

autogenous peritoneum f ro m the embryona l conti guous region to urogenital tract are ex posed in vivo to the surroundings and the environment o f

urogenital tract ti ssue system, then these pluripotent

ce ll s may eli fferentiate and pro I i ferate into des ired

ti ssues in the region based on the princ ipl es o f embryonal organogenes is.

Materials and Methods

All the norms laid down by the loca l ethi ca l committee for the conducti on o f the ex periments on animals were stri ctl y foll owed. The ex per imen ts we re attempted under general anes thes ia in 7 rema le Mongrel dogs o f 4-8 years or age h;1ving 7- 10 kg

body weight. The fa ll opian tu be segment measuring 5-7 em size was exc ised (Fi g. i A and C) and replaced by a free peritonea l tube. The tube was cons tructed from the donor peritoneum measur ing 2-~ cm. exc ised

130 INDI AN J. EXP. BIOL., FEBRUARY 2000

from retro vesicu lar pelvic and posterior abdominal wall peritoneum. By suturing the edges of exci sed peritoneum with 5/0 Vicryl eye less suture on a suitable sized infant feeding tube. The stent bearing the peritoneal tube was transferred to exci sed fallopian tube region. The ends of the stent introduced into the cut ends fimbria! and uterine ends of the fallopian tube. The uterine end of the stent tube was brought down through uterus into vagina and secured with the wall of the vagina in its lower part , with a non absorbable suture, to avoid accidental slip (Fig. lB). The peritoneal tube was anastomosed with the spatu lated cut ends of the fall opian tube to avoid

rFal lop1antube~-;;,';

', \

' \ '~ '' \ ) -

)...J.- Exc1sed part '' ' ' I ' '

Fig. 1-(A and B)Technique of regeneration of fa llopian tube. (diagrammatic representat ion). Excision or fallopian tube [dotted line (A)]. Repl acement with per itonea l tu be (B). The stent was brought down through uterus into vagina and secured with a :1011

abso rbab le sutu:-e at the lower end of the vagina. Stent is shown by do tted line inside the fallopian tu be(b; see tex t). (C)-The fallopian tube as seen in the abdomen of a clog at laparotomy. The tube excision is shown in between the arrows.

stricture formation at the site of the anastomos is. The stent was removed after 3 months of pos t operati ve period. The biopsy spec imen we re obtained after observation peri od of 3 month ~: (3 dogs), 6 months (3 dogs) and 12 months ( I clog) after re-ex pl orat ion under general anesthesia. After stud yin g the gross changes in and around the graft region, the grafted peritoneal tube along with normal fa ll opian tube beyond the site of the anastomosis , was exc ised. The patency of the grafted tube was confirmed by injecting saline through one end of the excised specimen and observing the free fl ow of the saline at the other end. The mi crosecti ons and the srai ni ng procedures were followed as pe r standards of the Institute of Pathology , Indian Council of Medi cal Research, New Delhi. The hi stology was studi ed in Eosin and Haernatoxy lene and Masson's Tri chrome stain.

Results

The post operat ive peri od in a ll the 7 clogs was uneventful except in one dog where the stelll was pulled out accidental! y on 3'd post operati ve clay. There was some bleedin g per vagi num in thi s clog which stopped by itself without any interven ti on.

Gross examination: On re-ex plorat ion under general anesthes ia at 3 month s posroperati ve period , the grafted si te revea led easil y separabl e flim sy ad hesions around the graft. The grafted tube showed definite change in co lorat ion from pale ye ll owis h (Fig. 2A) to pink and rosy red (fig. 2B ). The tube was thickened and turgid. It was co mparabl e to the normal fa ll opian tube tex ture. The mucosal su rface was smooth and shinin g. The transve rse sec ti on of the peritonea l tube and the graft turned l'all opian tube were similar and comparable hav ing well presen ed lumen and thi ckening or the wall or the gran (Fig. 2B). At 6 and 12 mont hs period the adhes ions were mo!-e firm but separabl e by blu nt dissec ti on or occasionall y by sharp di ssecti on (Fig. 2C). In one clog where the stent was accide nta lly cl i ~; l odgcd on 3'd postoperati ve day, the tubular co nfi gurati on was lost near the uterine end, but res t of th e graft maintained tubular structure of the graft. The uterine openin g of the fallopian tube was blocked. fn all other cl ogs the tubular structure of the graft was we ll prese rved and maintained. The tube was pate!ll as evi clcncecl by the free flow of the saline injected from the l'imbri al end of the fa ll opian tube in to uteru s. There was no stricture formati on at the anastomos is sites of the graft with the fa llopi an tube.

MATAPURKAR et. a/:, ORGANOGENESIS & TISSUE REGENERATION OF FALLOPIAN TUBE IN DOG I J 1

Fig. 2-Gross exa mination: (A) Peritoneal tube on a stent. (note the pal e yellow colour of th e thin peritoneal membrane. The stent is seen through the peritonea l tube. The suture line is not seen in the pi cture. (B) Peritonea l tube turned fallopi an tube. The longitudinal slit at one end is showing the inner mucosal lining. (note the thickening of the wall and the shini ng mucosa. (Inset ): Transverse cut sections of the peritoneal tube (white co lour) and peritoneal tube turned fallopi an tube (red co lour) are shown for comparison. (C) The graft turned fallopian tube. (6 months post operative). The part of the Uterus (U), The cut end or the contra lateral i·allopian tube (FT). Regenerated fal lopi an tube is shown in between the arrows.

Microscopic examination: H & E Stain: The histology of the graft turned fallopian tube Fig. Ja, 6.3 xi 0, magnification) was comparable to the normal

fallopian tube (Fig. 3b, I Ox I 0 magnification). The components of the wall deve loped were in tl1e same order as normaL The I Ox25 magnifi cation reve al ed mucosa, sub mucosa, muscu lari s mucosa and serosa (Fig. 3c) . Al l the components of the wall of the fallopian tube in the region of the grafted peritoneal tube were well developed. There was chroni c inflammatory cell infiltration in the mucosaal and serosal layer at some places in 6 ou t of 7 cases. The smooth muscles were arranged in two distinct laye rs . The cilia were not demonstrated in 6 out of 7 cases. In one dog where oest rogen was used before tak ing biopsy specimen the cilia could be demonstrated

(Fig. 3d, I Ox40 magnification ). The Masson' s tn chrome stain revealed development of connecti ve tissue in green and musc le tissue in pink co lour (Fig. 3e) .

Discussion The single celled ovum once fe rtili zed acquires a

tremendous capacity to form tis sues and organs of the whole body having different structures and fun ctions. How thi s is tri ggered was probably known durin g ancient period2

*. This task is achieved by the toti ­potent cells of the fertili zed ovum and deve loping embryo. On speciali zation these ce ll s form germ laye r cells of the ecto , meso and endoderm of germ elise ol' the embryo (Fig. 4a). In thi s process of spec iali zati on the toti potency is reduced to pluri potency , that is, now these cell s can form onl y a few tissues that are developed from ecto, endo or mesoderm All the body ti ssues are formed by single germ layer or by th e combination of more than one germ laye r. Once these cells get speciali zed, they loose reversibility to pluri

*From Markandeya Puran Gunam p oor vasya poo r vasya J>ranJJ!I I'CI Il l \'11 lillaro llalwll

Nanaveerrah prllllwkbhutah SUfJ/1'/e swrhliltlll l'tllll II 5\! II Nashaknu van prajah stmshlrlm sa!lragaiiiWI kmlslwslwll

Sayetyanyonya sanyog manyom·ashmrinoshdwll' II(,() I/ Eka sanghat chinhascha sa!npraJ!rl'kwlnr s/{('s/wwh

PHrushadhislllhilla!mclw avvaklllnllgmirmoclw II()! II Mahadadya vislr cshan/a hw urdwllll lf>({( l\'wrtilc

Jatbudbudva lla /ra kra111adl'e vridhi,wgownr II 02 II The seven clement s including th e divine. act :1s per their n:llurc

and remain separate. All these seven powcrrul suhs t:1 nccs when remain separate are unable to gencr:llc li !C processes . ThesL' unite in one and loose identity, th e di vi ne clement 1·c nnini ng unmanifest, form egg. Thi s stays as :1i 1· huhhlc in w:ller and gradually grows.

132 INDIAN J. EXP. BIOL. , FEBRUARY 2000

Fig. 3-M icroscopic examin ation: Hi stology (H & E Stain ): (a) Graft turned fallopian tube 6.3x I 0 m:1gni fic :ll io11 :1 1 .\ months or observation. Lumen is well preserved. Development of all the com ponents or the wall of the fall opian tuht: is well dt: lll :JrL·:ued I L = lumen, E = epitheleum , M =muscle layer, S = serosal layer). (b) Normal fallopi an tube. IOx JO magnifi cation (compare wit h Fi g. :1 :1 ). (c ) Mucosal and smooth muscle layer. 10x25 magnifi cati on . (d) Mucosal cell s with cili a. 10x40 magnification. :uTow indic:Jtcs cili:1 hc .1ring cell s (see text). (e) Masson ' s TriChrome stain. ( IOx JO magnitl catio n) Smooth mu scles (M), in bri ck red co lou1· ami Co nnecti ve tis.,uc (C), in green colour. ( I) Regeneration beyond th e line or anastomosis.(H & E Stain IOx JO magnifi ca ti on). AIT\lll. ··/\·· slw1.vs line or anastomo sis, held between the two sutures S i and S ii. Components of the wall of the fall opian tube are: Lumen 1 L). Epithclculll (E) .. Smooth Muscle layer (M). , and Serosal layer (S).

MATAPURKAR et. a/: , ORGANOGENESIS & TISS UE REGENERATIO OF FALLOPIAN T UBE IN DOG I :n

(a) Intra embryonic mesoderm

Mesonephric duct & tubule (Intermediate cell mesoderm)

-~--\-t-'----

(b) Transverse section of embryo 5 mm

Gonad (c) Structure of nephrogenic cord

(d) Formation of paramesonepric duct

(e) Formation of uero-vaginal canal (stage I)

I I

Utero vaginal canal

(f) Formation of utero-vaginal canal (stage II)

Fig. 4-Embryology of development or fa ll opi~ll t tube and uteru s. (a) . Germ Di sk : Intra embryonic mesoderm showing ectodcrJ1! (E). endoderm (c) and part s or the mesoderm (M ) A. paraxlal mesoderm. B. intermediate ce l l mass mesoderm & C. lateral plate mesoderm with coelomic ctv it y. (h).Transver>c secti on of the 5 111111 . embryo showing intermediat e ce ll nta>S mesoderm-the nephrogenic cord covered with peritoneum which is the l ining of the in tra elnhryonic coelome. (c). Nephrogenic cord structures : showin i,! different structures or the nephrogenic cord. The dark line is the co vering epithcleum of the coelome-the coelomic cpitheleum which is nothing hut peritoneum. (d) Formation of the paramcsonephric duct. The covering epitheleum of the nephrogenic cord the coelomic epitheleu111 invaginatcs into the surrounding mesenchyme and forms the paramesoncphric duct. The cm: lomic epithelcum i> peritonrum of the abdominal cavit y. Therefore in fact peritoneum cell s arc responsible for the fmm ~ ttion of the fallopian tube. (c). Formation or fall opian tube t> tage I ) :tml (I). utero-vaginal canal (stage II ): The paratne>onephric duct form fallopian tube one on each side or the midline. The two tubes unite lower down in mid line to form the uterus and uterovaginal canal.

134 I DIAN J. EXP. BIOL. , FEBRUARY 2000

or toti potency and hence in higher animals and man , the regenerati on capac ity is bare minimum or even

. absent. ln nature the regenerati on exists in pl ants and lower animals. A plant can be grown into full tree from a small cutting of the stem of the plant, if the sma ll cutting is put in a proper soil , environment and proper water, moisture in the so il. Otherwise the cutting will dry off and die. Similarly in lower animals, the eye lens can be grown by newts if amputated, tail of the li za rd ca n be grown if severed, the claws of the crabs and lobsters can be re-grown if injured and l os t '·~ .

The laws of the nature are intricate and camouflaged by the revea led world of nature but have uniform applicability . Th is is clear from the fact that the earl y embryos of fish , bird , dog, pi g, monkey or man, have striking similariti es morphologica ll y that it is difficult to recogni ze, which one of these earl y embryos will become fish, bird , clog, pi g; monkey or man3

. The man is an integral part of the animal kingdome. The regenerati on exists in nature. Therefor regeneration is a possibility in hi gher fo rms of life and the man as well. The pluripotent stem cell s do exist in ti ssues of hi gher forms of full y deve loped animals, the mammals and the man . These ce lls are responsible for repair, healing and replacement of lost ce ll s of tissues and organs of the body. Also these cell s can undergo metaplastic transformati on on new pathways and form a totall y new different ti ssue if exposed to irritation or abnormal conditi ons5

. For example, in chroni c peritonitis cases the perit oneal stem cell s show fo rmat ion of sq uamous nests, papillary projections , smooth muscle formation , cartilagenous changes and even osseous ti ssue formation. This is confirmed by the pathologists as well6

'7

. This new ti ssue formation IS limited to tissues which are formed from mesodermal germ layer of the developing embryo, since peritoneum stem cell s are developed from germ layer mesoderm onl y. By exploiting these facts of embryo logy, metapl astic transformati on capacity of stem ce ll s of the peritoneum to form mesodermal .ti ssues, when environment is altered, the regenerati on of ureter was successfu ll y achieved in dogsx, and monkeys 1. The regenerati on of abdominal wall aponeuros is from stem ce lls of the peritoneum was achieved and utilized in the management of difficult cases of primary and recurrent incisional herni as<J· 10. Based on similar principles the human ailment s of complex genito urinary rec tal fistulae were managed by using peritoneum for urethrop lasty and as an interpos iti onal ti ssue 11 .

The metap las ia of full y d i1Terenti at..:d ti ssues ce lls is often considered as abnormal and path ological. If the stem cell s in the process or differenti ati on and pro I i feration to rep lace morpholof! ica ll y different ti ssues, are subjected to undergo a chan ge whi ch is desired in the new situation and environment the desired metaplasia is a poss ibilit y and may be responsible for regenerati on of ti ss uo:: s and organs. Accordingly the regenerati on of fall opian tu be is attempted in this study.

Even though the scope of thi s experimental study is limited to test the hypot hes is and stud y the ex tent to which the components of the wa ll of the fall opian tube can be grown, the app lied aspect of the result s can be far reaching. Various ti s s u e~. used in tubal plast ic surgery are vermiform appendi x 12, human

b.1. I · 11 . d . 1-1 . " Lllll 1 1ca vem · , ve1n an artery 1mpla111s , a1nnt on , ·1 16 I ·1 I 1· 17 . I . 1 eum , seromuscu ar 1 ea gra ts . Wit 1 van ous success rates. The reason for Lt ilurc could he that these different ti ssue substitut es wen.' not int egrated in the process of natural wound hea ling and ti ssue repail". The biodegradable synthetic microporous arti fi cial vascular grafts show exn:lle nt heal in n charecteristics 1x. Despite notable imp ro vement in ovc~ all result s of tuboplas ty a suitabil' method is still needed for replacement of part or whole of the fallopian tube to correct the ex tensive da nwge 11.

The fallopian tube and pe rit oneum. both arc developed from germ laye r mcsoderm- -4hc intermedi ate cell mass mesoderm and latcr;tl pbtc mesoderm, respecti ve ly (Fi g. 4a). The urogc nit ~tl cord with its component structures is ...:o ve red by the epithelial lining of the coelomic cavit y of lateral plate mesoderm which becomes the peritoneum. pleura and pericardium of the body cavi ty (fig. 4b). The fall opian tube is formed from the paramesoncphri c duct or the urogenital cord of the urogenil<ti ri dge. The paramesonephric duct is formed by the in vagi nat ion or the coelomic epitheleum into t ~e surrounding mesenchyme (Fig. 4c) . Therefore it can he in J"erred that the fallopian tube is developed from the cell s or the peritoneum. But the regional and funct ional needs of the loca l tissue system were differen t. the environment to which these cell s mi grated in embryo we re different and therefore remained different tissue wise and functioned differentl y. Otherwise these peritoneal pluripotent cells had the capacity to form the fal lopian tube. Therefore the perit oneum is usecl in the prese nt experiment.

The fate of the g rm layer cell s in embryo depend s up on intrinsic and extrinsi c cel l f<~ct ors . ce ll

MATAPURKAR el. a /:, ORGANOGENESIS & TISSUE REGENERATION OF FALLOPI AN T UBE IN DOC I ~5

movements and location to which these cell s are exposed in embryo along with remote control mechani sm of surrounding and distant ti ssues. The other factor bei ng the time needed for the development, coupled with funct ional need of the I . I . I I II . 19 10 I t· · · ocat1on to w 11c 1 t 1ese ce s m1grate ·- . n act 1t 1s well known that the vertebrate deve lopment is due to environmental factors rather than the genetic coding ?I ?? - ·--. Therefore an attempt has been made to study the effect of exposure of peritoneal stem cell s, to a different location of fa ll opi an tube tissue system, in thi s experiment.

The peritoneum has single layer of stem cell s on a -- ?\

tough basement membrane - . These stem cell maintain pluripotent character and are capable of undergoing metapl astic transformation to ti ssues of mesodermal ori gin as ex plained above. The fa llopian tube is developed from germ layer mesoderm. Therefore thi s scientific fact has been ex ploited in thi s experiment.

The surgica l technique pro vides the needed cell movement to the new locati on of fallopian tube ti ssue system. The inherent intrinsic ce ll cod ing and creative intelligence of stem cell s of the peritoneum to di fferenti ate and proliferate on a new pathway coupled with forced new functional need of the changed environment triggered the desired events. Probably these factors are responsible fo r inducing desired changes to regenerate components of the wa ll of the fallopian tube.

References of spec ific direct studi es regarding healing of fallopian tube and the possibility of mi gration of cell s from proximal and distal cut ends of the fallopian tube are not available. However the other experimental studies on different tubular organs I. k ?4-'' 6 I ? 7 d d "~ . I . 1 e ureter- - ,.oesop1agus-, uo enum-, vem pe v1s

d I . . . . 1') 1o I I h an pe vmretenc JUnction- ·· , etc. revea t 1at t e healing and tissue fo rmation is possible by mi grati on of cells to a few milimeters onl y from the cut ends but lead to severe stri cture formati on and contracture at the severed site. The entire ti ssue or organ formation has not been observed in the grafted areas in any of these studies. Some of these studies have even used peritoneum from adjacent peritoneal enve lope or peritoneum covered with fascia and transverse abdominis musc le. The mi gration of cell s from cut ends in these experiments could ha ve generated tissues of entire organ. Apart from thi s the experimental failure in regenerat ing tissues using different materials and peritoneum not from conti guous embryonal segment region (Matapurkar,

unpubli shed data) also suggests that the ce ll migration is not responsible for entire organ regeneration. The migrated ce ll s have no capacity to survive till properly implanted in the area and resulted in undue scarring responsibl e for stri cture formati on. On the contrary the stem ce ll s of the perit oneum h;~ vc unique property to differenti ate and proli fe rate and eve n the free peritoneal grafts survive. Not onl y thi s, even the free-floating peritonea l cells also have the capac it y to differentiate and proliferate to help in the hea lin g. By thi s property the peritoneum acquires tremendous

d · h 1· II spee 111 ea mg .

The chronic inflammatory ce ll infiltrati on see n in the mucosal and serosal layers is probab ly the result of the local inflammatory respons\,;.. or the macro phages present in the peritoneum 1 x 'J.III "" . The microsurgica l reconstructi on of the fa ll op ian tube studied by Guawesky el. u!' " 2 showed rege nerati on of mucosa . The ciliogenes is after sa lpingosto my in rabbit has been observed between 211

" and ~'" week31.

These studi es were 111 art ific ia ll y created hydrosalpings by tube occ lusion. The usc of biodegradable synthet ic mi cro porous tubL· as uterine horn graft has been attempted for hcal1n g and regenerati on of fallopian tube and uteru s in rat s17

These produce obstruct ion at the ju nct ion or fall op ian tube and uterus. There is no menti on or cilia I mucosa l regenerati on. The use or' sp lint s co ntributing to the tubal surgery by biodegradab le microporous artificial graft has been suggestecl 11·'" . Absence of ci li a in maj ority of present ex periment s was due to probabl y that there was a long time gap betwee n l'i x <~ti o n and actual secti oning of the biopsy ti ssue whi ch increased the friability of the ti ssue resu lting in shcclcling up of the cilia . However in one dog where oes troge n was used, parentrall y two weeks pri or to obtaining the biopsy tissue, the cilia were observed in the reg ion or the grafted tube (Fig. ~d). In fa ct the tube part wh ich was excised for regenerati on was th e isthmus area whi ch has very scanty cili a hear in g ce ll s in the mucosa . Moreover the population or cili <l IS dependent upon the phase of the oestrous cyc le vv hi ch was also not taken into considerati on 111 thi s

. 1-1- 17 ex pen menr · .

The metaplastic transformati on capac it y of stem ce ll s of the peritoneum has been exp loited. The regenerati on of the components or the wall or the fallopian tube is possible by desired mc t ~ 1pi ~ 1 Sia of the stem ce ll s of the peritoneum in the region of the fa ll opian tube. The sc ientifi c rati onale or the hypothes is and the factors respo nsible ror

136 I 'DIA J. EXP. BIOL. , FEBRUARY 2000

regeneration of the fallopian tube are di scussed . However the authentication requires spec ificall y targeted studies regarding the extrinsic and intrinsic factors responsible for regenerati on of the fallopian tube.

Acknowledgement

Thanks are due to Director general, ICMR, New Delhi for support , Ms. Pramil a Kaushik , Staff nurse, and the staff of the animal house for cooperation and Mr. Yishal for ed itori al formaliti es. Permi ssion by the Dean, Head, Department of Surgery and Medica l Superintendent , Lok Naya k Hospital and M A Medica l Co ll ege, New Delhi , ts acknowledged.

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In dian 1 Urol , 13 ( 1996) I. 2 Sage Markandeya, Ma rkandera pt11Wt, Verses 59 to 62 ,

Chapter 42 , 5'" and 6'" Century. AD, I 54 (ava il able at Library or Indira Gandh i Nati onal Cen tre for Arts, C V Mess, Janpath , New Delhi 11000 I, India) ..

3 Albert s B, Bray D, Lewis J, Rail M, Roberts K & Watso n J D. The molecula r biolog_\' of the cell . 3.-d ed (Garland Publi shing Inc., New York & London) 1994.1037 .

4 Martin A, Hernan E Z. FZ~ ber J L, Pathologv edi ted by E Rubin (J B Li ppincott Co. Londo n, MZ~x i co , ew York , St. Loui s, San Paul o, Sydney) 1998.66.

5 Lu go M & Putong P B. Arclt Path. Lah Med, I OX ( 1984) 185. 6 Schwartz, S I, Principles of surgerr, 6'" ed . (McGraw Hill ,

London, New York ), 1994, 1449. 7 Rosai J, A ckerman 's surgical pathology (C V Mosby Co .. St

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( 1996) 954. 9 Matapurk ar B G, 13hargave A, Dawson L, & Sonal B, World

1 Surg, 23 ( 1999) 446. 10 Matapurk ar 13 G, Gupta A K & Agarwal A K. World 1 Surg,

15 (199 1) 768. II Matapu rkar 13 G & Rchan H S. !ltd ian 1 Urol , 14 ( 1997) 36. 12 Neill L Jr., Ant 1 Ohstet Cynaecol , 95 { 1958) 2 19 1. 13 Ansari A H, Gould K & Turner R J. Obstet G maecol, 60

( 1982) 733.

14 Davids AM & 13 ell win A, Fertil Stnil. 5 ( ; '!S--1) 32:'i. . 15 Ten Berge 13 S & Tic Lok T, Feu if Stnil. 5 ( 19S4) :rw. 16 Carey M L, Cain L C, Da vis H L. Bovin~ B & Jones B W.

Obstet Cynaecol, II ( 195X ) I 56. 17 Charl s G A, Labes E, Co hen 1. Fitch L B & Shoemaker W

C, Obstet Cvnaecol, II ( 1958 ) I 5(1. 18 Jonk man M F, Kauer F M. Neinwenhu1s P & rvlolcn:lar I.

A rt if Organs, I 0 ( 198o ) 475. 19 Harri son R G, Cunninglwnt '.1· text honk o f' OIUIIII IH\'. 12'11

reprint , edited by G.J.Romanes. (0xlill'd Uni vers it y Press. New York. ) 199 1, 15.

20 Singh I 13 , An introduction to t'nthrmlng-'· . ) 111 ed. (Mac Mi ll an Indi a Ltd .) 199 1,380.

2 1 Schmid P, Cox D & 13ilbe G. /Jet •t·lnJ'nt<'nt. I I I (I 9'J I) I 17. 22 Berry, C L, Molecula r hosis of' det ·clnJ •nt!'nl Jlmgu ·ss i11

Jlatlw logy edit ed by Nigel Kirkham & i>l' tcr Jl all . Ch . 7. I (Churchil Levi ngston Med. Di v. Lon~rn :ln· s ~roup Ltd ) (1995) 12 1.

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