Stereological Analysis of Mature Human Placenta of Pregnant Women of Different Age

Th e normal placentas, regular pregnancies and deliveries were structurally examined. Th e aim of this research was to compare the results and to confi rm if there were some diff erence in the structure of placenta related to the age of pregnant women. We examined  human placentas. Th e examined group of women were divided into two groups: ) pregnant women - years old; ) pregnant women over  years old. Th e stereological method was used. Th e volume density, absolute volume, the surface density and absolute surface of terminal villi of placentas in younger and older pregnant women were not signifi cantly diff erent. Th e volume density, absolute volume, the surface density and absolute surface of the other placentas villi in younger pregnant women compared to older ones, were signifi cantly increased (p<,). Th e volume density of fi brinoid of placentas in older pregnant women compared to younger ones was signifi cantly increased (p<,). Th e surface density, absolute volume and absolute surface of fi brinoid in these two examined groups of pregnant women were not signifi cantly diff erent. Th e volume density of intervillous space of placentas in older pregnant women compared to younger ones was signifi cantly increased (p<,). Absolute volumes of intervillous space of placentas in these two examined groups of pregnant women are not signifi cantly diff erent.


Introduction
The basic functional unit of placenta is composed of chorionic villi with epithelial surface (trophoblast) and of mesenchimal origin stroma in which blood vessels are embedded.Numerous authors () classifi ed types of the chorionic villi inside the villous tree depending on gestational period of their occurrence and on their function in the course of pregnancy.According to the morphological properties, parts of the villous tree of the mature human placenta are: stem villus, mature intermediary villus, and terminal villus.Morphological characteristic of the stem villi is stroma, rich in connective fi bers.Mature intermediary villus is composed of stroma and most of blood vessels.Terminal villi are the fi nal branches of the villous tree.Blood capillaries of these villi are very enlarged, and they occupy more than  of the stroma volume ().Th rough the surface of these villi, maternofetal exchange is done.Fibrinoid is either on the surface of the chorionic villi or among them.Placental fi brinoid is extracellular deposit material.Mass of fi brinoid indicates the gestational placental age.Surface villi fi brinoid replaces worn parts of sintitiotrophoblast ().Two kinds of fi brinoids are evident according to origin, structure and function.Fibrin-tipe of fi brinoid is structured mostly of fi brin and molecules resulting in the course of blood clotting, or in the degeneration processes ().Mayhew and Sampson () established that fi brin-type fi brinoid volume in the pregnancies complicated by diabetes is increased.In the pregnant women smokers placentas increased deposits of perivillous fi brinoid were found compared to non smokers ().Intervillous space is found among chorionic villi and is fi lled by mother's blood.Because ofthe intervillous deposition of fi brinoids, intervillous space volume is physiologically decreasing after th week ().In the available literature we have not found any evidence on the histologic structure of placenta related to the pregnant woman age.However, a great number of investigations focused on how a pregnant woman age impacts on a delivery outcome, i.e. on malformation occurrence in the course of pregnancy.Threatening abortions, preeclampsia and preterm deliveries occurred - times more frequently in the older pregnant women ().Since quntitative researches on placentas of different age pregnant women were not performed stereologically in the Tuzla Canton region, our objectives were to analise placentas of younger and older pregnant women morphologically and quantitatively, to fi nd out if there were statistically signifi cant diff erences among volume densities, absolute volumes, surface densities, and absolute surfaces of the chorionic villas, fi brinoid and intervillous space in the tested placental groups.Morphological researches of placenta enable us to presume conditions for fetus formation and development, especially important in risky pregnancy (EPH gestosis, diabetes mellitus, older mothers).

Material and Methods
Th e research was carried out on  human placentas of normal pregnancy and normal delivery.Th e age of the pregnant women ranged from  to  years.Th ey were divided into two groups: a) - years old pregnant women b) over  years old pregnant women.Gestational age of the tested groups of placentas ( to  weeks) was determined according to the fi rst day of the last menstruation.Amnion and umbilical cord were removed from each placenta.Th e placenta mass was measured by weighting, its volume was determined indirectly by measuring the liquid squeezed out.Tissue samples were taken through the thickness of the entire organ, from chorion to decidua basalis.Th e tissue was fi xed in  water solution of neutral formalin then embedded in paraffi n wax, cut into μm thick sections and stained by hematoxylin-eosin.Stereological analysis was done by multipurpose test-system M, magnifi cation x.Referential space was parenhim of the placenta.Relative variables (are related to one part of tissue -cm) and absolute variables (are related to entire organ) were stereologically examined and compared.Relative variables were determined as follows: ) the volume density of terminal and other villi, fi brinoid, and intervillous space; ) the surface density of terminal and other villi and fibrinoids.Absolute variables were determined as follows: the absolute volume and absolute surface of the cited structures.Signifi cance of diff erent results obtained by the stereological analysis of the diff erent age pregnant women's placentas was estimated by F-test and t-test.

Results
Referential space was made of terminal villi, other villi, fibrinoid and intervillous space (Figure ) in this researches.RELATIVE VARIABLES Volume densities and surface densities of terminal villi (Vvt), other villi (Vvo), fi brinoid (VVf) and intervillous space (VViv) of both, younger and older pregnant women's placenta, are shown in the Table  and .Placentas of older pregnant women had signifi cantly lower proportion of other villi and signifi cantly higher proportion of intervillous space and perivillous fi brinoid in the volume unit, in comparison to placentas in the younger group. and Table .

Discussion
In our research, placentas of older and younger pregnant women had similar values of volume density and surface density of terminal villi.Many authors have determined volume density of fi brinoids( Vvf) so far.Th ey established that it amounts , (),  () in a volume unit.In our researches placentas of the older pregnant women had signifi cantly higher proportion of perivillous fi brinoid (placed on the free surface of the terminal villi) in the volume unit in comparison to placentas in the younger group.It means that lower part of trofoblast surface of terminal villi participates in materno fetal excange ().
Volume density of the intervillous space is , (),  () in the volume unit of placenta.According to our results the diff erence of the volume density of the intervillous space in the placenta of both younger and older pregnant women was statistically signifi cant.It might be the result of: a) decreased number of villi or their size or b) impaired space arrangement of the villous tree in one cm of placental volume ().Th e absolute surface of terminal villi has been frequently researched because it is the surface where the maternal -fetal exchanges take place.Th e absolute surface of the terminal villi in the human placenta was  m ().In our researches placentas of the older and younger pregnant women had similar values of absolute volume and absolute surface of terminal villi, fi brinoid and intervillous space.

Conclusion
Results of our research show that of placenta in one cm of older pregnant women had a statistically signifi cant higher proportion of fi brinoid and intervillous space.However, apsolute volume of terminal villi, fi brinoid and intervillous space had similar values in both tested groups.Th at aditionally explains physiology of placenta and confi rms capability of older pregnant women placenta to induce compensational mechanisms, whose main role is providing the suffi cient maternofetal exchange.
BASIC MEDICAL SCIENCES 2006; 6 (2): 7-10 SUADA RAMIĆ ET AL.: STEREOLOGICAL ANALYSIS OF MATURE HUMAN PLACENTA OF PREGNANT WOMEN OF DIFFERENT AGE ABSOLUTE VARIABLES Absolute volumes and absolute surfaces of the terminal villi (Vt), other villi (Vo), fi brinoid (Vf) and intervillous space (Viv) of both younger and older pregnant women placentas are shown in Table