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ORIGINAL ARTICLES
NUMBER 1 YEAR 2003
Microscopic and Vascular Morphometry Aspects at the Level of the Myometrium
1 Department of Anatomy
2 Pius Brânzeu Center of Laparoscopic Surgery and Microsurgery
3 Department of Histology
Victor Babes University of Medicine and Pharmacy

Correspondence to:
Andrei Motoc, MD
Victor Babes University of Medicine and Pharmacy
RO – 1900 Timisoara, Piata Eftimie Murgu 2, e-mail: amotoc@umft.ro
ABSTRACT
Experimental research as preparing stages for uterus transplantation are not very frequent in the literature, because the intent to perform this modern procedure has not yet been stated by many gynecologists or surgeons. Since good results in experimental models were already reported, the data regarding the microscopical anatomy of the myometrium and vessels of uterus are valuable to interested scientists.
Although the structure and function of the uterine muscle has been the subject of several studies, one can emphasize that detailed anatomical knowledge of the myometrium and its relationship to other components of the uterus is essential to the understanding of its function. The paper compares microscopic anatomy and vascularization in rats (useful for experimental models) with similar aspects in humans.
Introduction and Objective

Anatomical knowledge of the myometrium and its relationship to other components of the uterus is essential for the understanding of its function. The structure and function of the uterine muscle has been the subject of several studies.1-3
Our objective was the study of the angio- and micro-architecture of the uterus, as well as studies of vascular morphometry performed on animal and human tissue harvested during surgical procedures, between 1998-2000. These studies represented an intermediary phase in perfecting transplantation technique, in orthotopic and / or heterotopic block of the uterus - fallopian tubes - ovaries complex, at the Pius Branzeu Centre of Laparoscopic Surgery and Microsurgery of the University of Medicine and Pharmacy, Timisoara, during 1999-2001.

Material and method

Histological preparations of uterus and fallopian tube were set up for:
• Sprague Dawley rats, from the animal house of the of the Pius Brinzeu Center:
- 16 samples of pregnant uteruses
- 32 samples of fallopian tubes
•  15 human samples harvested during surgical procedures at the Dumitru Popescu Obstetrics and Gynecology Clinical Hospital, during 1998-2001.
The histological processing techniques implied the insertion into paraffin and the sectioning of the samples, HE stain, Masson trichrome and the LSAB immuno-histochemical technique (1A4 DK antiactinic clone).
The vascular morphometry was assed with a Olympus optical microscope, which allowed the computerized performing of an impressive number of measurements (thousands of vessels) within a very short period of time.
Prior to preparing the samples for histological examination, we carried out macroscopic studies of the vascularization of the genital apparatus in
the female rat in order to highlight the vascular pe-dicles.
The processing of the vascular morphometry data was carried out with the help of MS EXCEL software, and for each vascular category the average (M), standard deviation (SD) and average standard error (SM) was calculated.

Results and Discussions

Macro- and microscopic aspects of the genital apparatus in Sprague Dawley rats
The female rat presents cylindrical fallopian tubes, which slope obliquely from infero-medial to supero-lateral, having as vascular sources the uterine artery inferiorly and the ovarian artery superiorly. These are anastomosed and give out perpendicular ramifications on the whole length of the uterine tube 4-6 Figure 1.
The uterus is a single hollow organ, with its base oriented towards the top. It is vascularized by the uterine artery, which is oriented in an inferior to superior direction and is parallel to the lateral margin of the uterus, and then inflects laterally to form the tubal vascular arcade.
The study of histological preparations has generated data regarding the length of three categories of vessels from the uterus's and the fallopian tubes- circulatory network - capillaries, meta-arterioles and venules, and small-caliber vessels. A number of 309 capillaries (diameter smaller than 15 micrometers) were studied, as well as 300 meta-arterioles and venules (diameter between 15 and 40 micrometers) and 405 small-caliber vessels (diameter between 41 and 100 micrometers).Figure 2.
Figure 1. Non-pregnant uterus of a Sprague Dawley rat in vivo study, preparing period for uterus transplantation.
Figure 2.

Microscopic and vascular morphometry aspects in humans
The arterial vascularisation of the uterus has three source directions: the uterine artery, the ovarian artery and the round ligament artery (these arteries have branches that are often anastomosed among each other), but the inferior mesenteric artery, the medium sacral artery, the inferior epigastric artery and the external pudendal artery can also contribute. 7
The arterial anastomoses are well represented and can be encountered both at perivisceral and myometrium (intramural) level. Therefore, the uterus is a very well vascularised organ, but its vascularization depends on its functional state and age.
The myometrium has a heterogeneous compo- sition, as it consists predominantly of muscle fibers and fibroblasts, blood and lymphatic vessels, immune cells and rich connective tissue. The myometrium presents two layers: one with longitudinal muscle fibers, generally oriented in the longitudinal axis of the uterus,8,9 and one with circular muscle fibers, arranged around the longitudinal axis of the uterus in a more disorderly manner than the longitudinal layer.Vascular morphometric studies (Figure 6) were performed on 15 samples of human uterus, harvested during hysterectomies, between 1998-2000. The harvested samples maintained to a great extent the vascular pedicles, which allowed us to evaluate the aspect and dimension of the main vessels (artery and uterine vein). Seven uteri were fibromatous, which made the harvesting and processing more difficult. The harvesting for the histological and morphometry studies was performed from several areas of the same uterus, along the myometrial microcirculatory device.Our aim was to determine the number of vessels on to the surface unit, at the level of the uterus's three distinct parts - body, base, and cervix. For this purpose ten myo- metrial fragments were harvested from each of the 15 uteri (in the case of the samples with fibromas, the fibro- matous areas were avoided), in order to study ten areas for vascular counting, in the case of each pre- paration separately: 4 fields for the corporeal area, 3 for the fundus area and 3 for the isthmian/cervix area .Figure 7.
Figure 6.
Figure 7. Computerized morphometry. Comparative representation of the number of vessels in three uterine areas.
The counting allowed to make estimations regarding the vascularization of the above-mentioned uterine areas. The preparations on which the counting was performed were HE coloured, thus allowing a simultaneous histological evaluation of the myometrial structure.The analysis of the 150 fields, of which 60 harvested at body level, 45 at uterine fundus level, and 45 at isthmian level, revealed the following aspects about the vascularisation of the myometrium: a richer vascularisation at isthmian level (50 % of the total number of vessels in a sample), an average situation at body level (30 % of the total) and the smallest number of vessels at the level of uterine fundus.

Conclusions

1. The microscopic study performed on HE samples, Masson stains and the LSAB immuno-histochemical technique (Figure 3, Figure 4, Figure 5) have allowed the monitoring of the way sanguine vessels across the myometrium in their trajectory towards the lamina propria of the endometrium, and also the presence in the myometrium of thick-walled arteries with the middle tunic made of layers of smooth muscle cells. Regarding the Masson coloration, a well represented fiber-like component present in both the interstice and the walls of the sanguine vessels was highlighted at cervix level. The highlighting of the muscle cells was performed using a 1A4 DK antiactinic clone.
2. The vascular morphometry studies, performed on myometrium preparations from the 15 human samples, included 10 fragments for each of the 15 samples, summing up a total of 10 fields studied for the counting (4 fields for the body, 3 for the cervix and 3 for the uterine fundus). At myometrial level there is a richer vascularisation of the isthmus (50 % of the total number of vessels), in comparison with the body (30 %) and the uterine fundus (20 %), thus indicating a microcirculatory pattern superposable with the macrovascular one. At the level of the isthmian inflexion of the uterine artery, there are branches more voluminous than the ones at uterine base level, which confirms that the uterine artery is the main source of myometrial vascularisation.
3. An important aspect for the experimental field (also confirmed by the present study) is that nowadays the rat can offer major models in the study of transplantation of tissue fragments from vascularized organs.10,11 Besides their bio-availability, their high resistance to disease and poor representation of parametrial tissues, there is also a great similarity between their vascular device and the human one - many reasons for choosing the rat as the lab animal for the study of experimental uterus transplantation.
4. The present study has also been the database for the quantification of hystopathological modifications following uterus transplantation.
Figure 3. Artery from the middle layer of the myometrim with thick walls, having its media made up of multiple layers of smooth muscle cells (longitud [...]
Figure 4. Detail from the myometrium, revealing the longitudinal fiber-like character of the muscle tissue, Masson's.

Figure 5. Immuno-histochemistry. Internal layer of the myometrium. In the lamina propria and the endometrium one can notice an arteriole and several v [...]
REFERENCES

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11. Ionac M. Aplicatii experimentale si clinice de microchirurgie vasculara si nervoasa, Teza de doctorat, Timisoara, UMF Victor Babes, 2001.



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