الفهرس 
Introduction and Aim of workOnly 14 pages are availabe for public view
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Abstract
Diabetes is relatively common throughout the world. According to the World Health Organization reports, more than 150 million people throughout the world suffered from diabetes. It is well established that the fetus of the mother with poorly controlled diabetes mellitus is at increased risk for neonatal morbidity and mortality. Less recognized, and less accepted, are the deleterious effects of diabetes on the reproductive performance at earlier stages of pregnancy.
Implantation is a complex developmental process that involves an intimate cross-talk between the embryo and uterus. Synchronized development of the embryo to the blastocyst stage and the differentiation of the uterus to the receptive state are essential to this process.
In the human endometrium, the vascular endothelial growth factor (VEGF) had been detected at the mRNA throughout the menstrual cycle with maximal expression during the secretory phase. This protein was predominantly localized in the glandular epithelial cells. Moreover, angiogenesis, the prerequisite for embryo implantation, placentation and embryogenesis is regulated by many factors, such as hormones, and growth factors especially the VEGF.
Therefore, the present study was performed to demonstrate the effect of diabetes on the implantation process in rats, with special emphasis on the endometrial angiogenesis accompanying this event via studying the VEGF expression.
Forty adult female albino rats, aged 4-6 months, were used in this study. Rats were divided into 2 groups (n=20), the first group was the control while the second was treated with alloxan to induce experimental diabetes. Rats of the second group were intravenously injected in the dorsal tail vein by alloxan monohydrate (40 mg/kg body weight).
Vaginal smears were collected from each animal every morning. The smear was examined for the presence of sperms. When sperms were identified in the smear this day was designated as D 1of pregnancy. Pregnant rats from the two groups at D 4, 5, 6 and 7 of pregnancy (n=5) were sacrificed by ether inhalation. Before sacrificing, the rats were intravenously injected in the tail vein with 1 ml of 2% Evans blue in 0.9% saline. The leakage of Evans blue dye occurs at areas of increased vascular permeability. The abdominal wall was opened for exposure of the uterine horns. Blue bands were identified along the uterine horns at each implantation site. The areas were the dye was not localized were considered implantation intersites.
Specimens extracted from the uterine horns, at the implantation sites and intersites, were processed and subjected to; light microscopic, scanning electron microscopic and immunohistochemical examinations for the detection of VEGF.
The present work tried to supply the literatures with the detailed changes occurring in the endometrium during the pre-implantation and implantation periods in normal and diabetic albino rats.
A) Non-diabetic (control) group:
The pre-implantation period is the period at which the endometrium prepares itself for implantation. In the control group it included the 4th and 5th days of pregnancy.
The epithelium at the 4th day of pregnancy was formed of simple columnar epithelium displaying regular brush border. At the 5th day, the epithelium varied between columnar in some area to cubical in other area. Using the scanning electron microscope (SEM), the 5th day also showed decreased density of the microvilli which was probably a necessary step to make contact between the trophoblast and the epithelial cells.
Periodic acid Shiff (PAS) stained sections revealed strong reaction of the 5th day luminal epithelium while the SEM displayed secretory vesicles (pinopodes) and numerous mucus patches distributed over the surface of the cells.
The stroma showed preparatory changes for implantation such as increasing density of granulated metrial gland cells (GMGCs), numbers of the uterine glands, and leucocytic infiltrations. The stroma also showed prominent extravasation of red blood cells and slightly increased vascularity of the stroma. Moderate staining for VEGF at this period was confined to the luminal and glandular epithelia.
The endometrium at the 5th day showed variation in the density of collagenous fibers. Markedly dense fibers were evident in area of normal thickness in the endometrium, meanwhile, a thick area of the endometrium displayed less dense collagenous fibers. This finding was evident using the Masson’s trichrome stain and indicated that implantation favors cellular stroma.
The day 6 pregnancy of the control rat was characterized by the settling of the implantated blastocysts in the rat’s endometrium. The implantation sites were mainly located at the antimesometrial side of the uterine horn. Each uterine horn contained from 3 to 5 implantation sites.
The implantation site was covered with either flat apoptotic or completely sloughed epithelium. On the other hand, the columnar covering epithelium of the intersites showed marked hyperplasia, intervening clear cells, and frequent mitotic figures. This was evident in the immunohistochemical study, as the implantation site revealed faint reaction for VEGF at the luminal epithelium while the intersites showed moderate positive staining for VEGF.
The stromal cells change from spindle-shaped fibroblast at the intersite into large polyhedral cells with pale cytoplasm and vesicular nuclei, the decidual cells, at the implantation site. Around the blastocyst, the decidua is differentiated into two zones, a primary avascular zone surrounding the implanted embryo, and secondary highly vascular zone at the periphery.
The primary decidual zone displayed numerous large, PAS positively stained GMGCs, few mitotic figures of the decidual cells and lymphocytic infiltration. The secondary decidual zone on the other hand, showed numerous apoptotic cells. The apoptosis occurred to give vicinity to the implanted blastocyst. This zone was infiltrated mainly by eosinophils.
The subepithelial area of the stroma at the implantation intersite is highly vascularized showing numerous congested blood vessels and eosinophilic infiltration.
The 7th day endometrium at the implantation site was more or less similar to the description of the 6th day except for the increased cellularity of the primary decidual zone and the frequent mitotic figures of the secondary zone.
The obvious immunohistochemical change at the 7th day of pregnancy was the numerous positively stained GMGCs for VEGF among the negatively stained decidual cells.
SEM examination of the 7th day showed that the epithelial cells were devoid of microvilli. The cell borders were raised and well-defined giving the surface a honey comb appearance.
B) The diabetic group:
The pre-implantation period in the diabetic group extended from the 4th, 5th to include the 6th day of pregnancy.
This period of early pregnancy was characterized by increased secretory function evident by the numerous luminal secretory cells, uterine glands lined by low cubical cells with vacuolated cytoplasm, and the strong PAS positive reaction of the brush border of the uterine epithelium. The SEM also revealed high density microvilli of the luminal epithelium together with scattered multiple surface mucus patches and smooth rounded pinopodes of varying size. The 5th and 6th days of pregnancy also displayed moderate positive staining for VEGF of the luminal and glandular epithelia.
The stroma of diabetic group at the pre-implantation period showed prominent cellular infiltration mainly in the subepithelial area, frequent mitotic activity and prominent leucocytic infiltration mainly lymphocytes. Decidual cells made their appearance in the stroma at D5 and some of them were apoptotic with vacuolated cytoplasm and pyknotic nuclei.
The stromal blood vessels at the 4th day were thick walled, dilated and congested. During the 5th day and 6th day there was increased vascularity evident by the numerous minute blood vessels especially in the subepithelial upper zone of the stroma.
Implantation occurred at the 7th day of pregnancy in the diabetic group i.e. delayed implantation. Also, each horn displayed only from one to two implantation sites. This day displayed the strongest positive staining for VEGF at the brush border of the luminal and glandular epithelia of the intersite, while at the implantation site, this reaction was not encountered.
The decidua of the 7th day diabetic pregnant rats was comparably similar to that of the control rats, yet the difference was in the increasing number of degenerated cells with cytoplasmic rarefaction and nuclear shrinkage among the normal decidual cells. Moreover, the stroma showed congestion and extravasation of red blood cells.
The GMGCs were fewer as compared to the control group and located mostly at the periphery of the 1ry decidual zone.
In conclusion, diabetes was associated with an obvious delay in the timing of implantation. The reduced number and abnormal blastocysts might be an element for this delayed implantation. Further, the increased expression of VEGF and the evident angiogenesis observed in the endometrium of diabetic rats might be a compensatory reaction to overcome the hypoxia resulting from diabetic vasculopathy.