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A comparative study between bilateral ligation of the ovarian and the uterine arteries on the structure and function of the ovary of adult white rabbit

histological and immunohistochemical study

Kallini, Dalia F.; Abdelmalik, Sherin W.; Desouky, Ahmed M.; El-Beshbishy, Rana A.

Author Information
The Egyptian Journal of Histology: June 2013 - Volume 36 - Issue 2 - p 300-311
doi: 10.1097/01.EHX.0000428966.85110.1a
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Abstract

Introduction

Abnormal uterine bleeding constitutes one-third of all gynecological consultations and this proportion increases to more than two-third when perimenopausal and postmenopausal women are considered 1,2. Until recently, hysterectomy with or without salpingo-oophrectomy was the surgical treatment that was widely used 3. However, physicians evaluated the impact of removal of the ovaries and the subsequent induction of menopause 4. Therefore, when ovaries are healthy and viable, preservation may be possible to ensure that the natural hormonal benefits can be gained by the patient, thus avoiding surgical menopause 5.

Unsurprisingly, even hysterectomy is not the best option, especially in unmarried and nullipara women. Thus, many researchers investigated other technological therapies for benign gynecological diseases. Fortunately, it was reported by a previous study 6 that bilateral uterine artery embolization (UAE) is an interventional radiology technique successfully used in the management of gynecological or obstetrical hemorrhage. Recently, it was reported that UAE is an effective therapy replacing surgery in many cases especially for the treatment of postpartum hemorrhage and fibroids 7. One of the main goals of UAE is to preserve the uterus and therefore fertility, pregnancies, menses, and ovarian reserve. Some researchers 8 suggested that patients managed with UAE can expect normal menses, preservation of future fertility, and successful pregnancies. However, conflicting results such as negative effects on ovarian function have been reported 9,10. Although UAE is considered a safe, life-saving procedure in fibroid and postpartum hemorrhage, its long-term effect on menstruation and fertility is unclear 11.

Therefore, the aim of the present work was to study the effect of bilateral uterine artery ligation on the ovarian structure and function in comparison with bilateral ovarian artery ligation in white New Zealand rabbits using light and scanning electron microscopes with immunohistochemistry and hormonal assessment. The present work is an attempt to provide valuable data for clinical practice on the long-term assessment of ovarian integrity following the widely used UAE especially in young women.

Materials and methods

Animals

Twenty-eight adult female rabbits of the New Zealand white strain weighing 2.5–3 kg were used from the Medical Research Center, Faculty of Medicine, Ain Shams University. The rabbit was chosen in the present study as an animal model because its ovaries have a dual blood supply, including ovarian and uterine arteries as in humans 10 as well as a sufficiently large vascular anatomy 12. Rabbits were housed individually at 20°C in metallic cages under normal conditions with free access to food and water and were isolated for 1 month from males in order to exclude pregnancy. The animals were divided into four groups, seven rabbits each:

  • Group A: the control group, in which the animals were not subjected to any procedure.
  • Group B: the sham-operated group.
  • Group C: the animals were subjected to bilateral ovarian artery ligation.
  • Group D: the animals were subjected to bilateral uterine artery ligation.

Experimental design

The rabbits in groups B, C, and D were fasted for 12 h, and then anesthetized using propofol 1% (Diprivan, Astra Zeneca, UK) by an intravenous infusion through the veins of the rabbit’s ear 13. The dose was adjusted according to the animal’s heart rate and level of consciousness. The rabbits were then placed in a supine position, with the hind limbs restrained in extension. The anterior abdominal wall was opened by an incision (2–3 cm) in midline between the umbilicus and the cranial rim of the symphysis pubis; the viscera were retracted to one side to explore the ovaries. The ovarian and uterine arteries were explored. No arterial ligation was performed in the sham-operated group B. In group C, the ovarian arteries were bilaterally ligated ahead of the ovaries using silk ligature. In group D, the uterine arteries were bilaterally ligated close to the splitting region of the uterine horns. The viscera were rearranged in their place; the anterior abdominal wall was closed. The rabbits were left to regain consciousness and received postoperative care of augmentin antibiotic (GlaxoSmithKline) and Cataflam (Novartis).

The rabbits of all groups were housed for 2 months individually; all the rabbits received a single dose of human chorionic gonadotrophin 50 IU subcutaneously to induce ovulation, as ovulation does not occur spontaneously in the rabbit 14. Forty-eight hours later, the animals of all groups were sacrificed by a lethal dose of anesthesia according to the protocol of the Animal Care and Use Committee of Ain Shams University. The following techniques were performed.

Hormonal assay

The anterior wall of the thorax was opened by a midline incision and blood samples were collected by an intracardiac puncture from each rabbit to measure serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (17-β-estradiol) to evaluate the ovarian function.

Gross morphology

The anterior abdominal wall was opened; both the uterine and the ovarian arteries were dissected. The effect of ovarian and uterine arteries’ ligation was photographed using a Nikon Coolpix p500 camera (Nikon).

Ovarian weight

Both ovaries in each rabbit were dissected and extracted. Each ovary was washed in saline, and then left to dry on filter paper before weighing using a sensitive digital laboratory balance.

Microscopic study

All ovarian specimens were processed and subjected to the following techniques:

Light microscopy

Extracted ovarian specimens were fixed in 10% neutral-buffered formalin for 1 week. Then, they were dehydrated, cleared, and embedded in paraffin wax. Sections (4–5-μm thick) were cut and stained with H&E 15. The sections were examined using an Olympus light microscope and photographed.

Immunohistochemistry (TUNEL technique)

This protocol was used for the detection and quantification of apoptosis (programmed cell death) at the single cell level on the basis of labeling of DNA strand breaks (TUNEL technology). Cleavage of genomic DNA during apoptosis may yield double-stranded as well as single-strand breaks, which can be identified by labeling the free 3′-OH terminal with modified nucleotides in an enzymatic reaction. Primary antibody: apoptosis protease activating factor 1 antibody: rabbit polyclonal provided by Novocastra Labs (UK) in a 1:20 dilution. The incubation time was 60 min at room temperature. Blocking was performed by 5% normal serum to reduce unspecific background staining. Three percent H2O2 was then added to block endogenous peroxidase activity. Avidin/biotin was used to block endogenous biotin activity. Then, counterstaining was carried out using Mayer’s hematoxylin. The staining pattern was cytoplasmic in the form of brownish coloration 16.

Scanning electron microscopy

Specimens were washed twice with PBS and fixed in a mixture of 1% glutaraldehyde and 2% paraformaldehyde in PBS at room temperature (pH 7.4) for 24 h. The specimens were then washed twice in buffered sucrose for 5 min each (0.1 mol/l PBS, 5% sucrose solution) and then post-fixed in phosphate-buffered 2% osmium tetroxide at 4°C for 60 min. The specimens were dehydrated in ascending grades of ethanol, followed by further dehydration in an ethanol–acetone solution (1:1), and then in absolute acetone. They were dried in the critical point, mounted on stubs, and coated with gold. The specimens were examined and photographed using Philips Scanning Electron Microscopy XL 3 at 30 kV 17.

Statistical analysis

Student’s t-test was used to compare the different measurements of different groups. The P value was calculated using the SPSS program. The significance of the data was determined by P value (P>0.05 was considered insignificant, P≤0.05 was considered significant, and P≤0.001 was considered highly significant) 18.

Results

Hormonal assay

Groups A and B had almost similar serum hormonal FSH, LH, and estradiol (17-β-estradiol) concentration values. These were considered the normal control biochemical range in the present study. Serum FSH and LH showed a statistically significant increase in both groups C and D. Although the serum 17-β-estradiol level was significantly decreased following ovarian artery ligation (group C), its values in uterine artery ligation (group D) showed a nonsignificant decrease (Table 1).

T1-4
Table 1:
Mean values and SD of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol in the studied groups

Gross morphology

In groups A and B, the ovarian artery was found to originate from the abdominal aorta and enter the hilum of the ovary after dividing into several branches. The uterine artery was observed to run in a tortuous course along the uterine horns and fallopian tubes (Fig. 1). However, in group C, ligation of the ovarian artery away from the ovary resulted in darkened dull ovary and there was an apparent increase in the tortuosity of the uterine artery (Fig. 2). Ligation of the uterine artery away from the ovary in group D resulted in an apparent engorgement in the proximal part of the uterine artery and relative expansion of the ovarian artery with loss of ovarian luster (Fig. 3).

F1-4
Figure 1:
Photograph of the posterior abdominal wall of a control New Zealand white rabbit showing the ovarian artery (double arrow) and the uterine artery (arrows). Note the ovary (O) and uterine horn (T).
F2-4
Figure 2:
Photograph of the posterior abdominal wall of a New Zealand white rabbit in group C showing a ligated ovarian artery (double arrow). Note the tortuous uterine artery (arrow), darkened dull ovary (O), and the uterine horn (T). C, colon.
F3-4
Figure 3:
Photograph of the posterior abdominal wall of a New Zealand white rabbit in group D showing a ligated uterine artery (arrow head) with engorgement and tortuosity proximal to the ligation (arrows). There is a relatively dilated ovarian artery (double arrow) and loss of ovarian luster (O). T, uterine horn.

Ovarian weight

The weights of the ovaries in groups A and B were almost the same and were considered the normal range for this work. Following ovarian artery ligation in group C, the ovaries showed a significant decrease in their weight compared with the control. Meanwhile, after ligation of uterine arteries in group D, the ovaries showed a nonsignificant decrease in their weight (Table 2).

T2-4
Table 2:
Mean values and SD of ovarian weight (g) in the studied groups

Histological results

Ovaries taken from both control and sham-operated groups, groups A and B, respectively, showed normal morphology with many follicles in various stages of development ranging from primordial follicles to mature graafian follicles (Fig. 4). The primordial follicles were located in the cortex just beneath tunica albuginea and were composed of one layer of flattened follicular cells surrounding the oocyte. In primary follicles, the oocyte was surrounded by cuboidal or columnar epithelium, which would become the granulosa cells. The continued proliferation of these cells resulted in the formation of a stratified epithelium with a distinct zona pellucida surrounding the oocyte (Fig. 5). Parenchymal cells of the ovary surrounding the growing follicle became organized into concentric sheaths, the theca folliculi (Fig. 5). When small fluid-filled spaces became visible between the granulosa cells, growing follicles were formed (Fig. 5). These spaces enlarged and fused to form large follicular antrum, which was the defining feature of the mature graafian follicles. The oocyte became eccentrically located in the follicle enclosed in the cumulus oophorus and surrounded by granulosa cells (Fig. 6). Corpora lutea were also observed, composed of lightly stained lutein cells separated by minimal connective tissue stroma (Fig. 7).

F4-4
Figure 4:
A photomicrograph of a section in the ovary of the control group A showing follicles of different sizes and developmental stages ranging from primordial follicles (arrow) to mature graafian follicles (double arrow). H&E, ×40.
F5-4
Figure 5:
A photomicrograph of a section in the ovary of the control group A showing secondary follicle with a primary oocyte (O) surrounded by zona pellucida (Z). Note the antral spaces containing liquor (A) and the start of cumulus oophorus formation (C) between the zona pellucida and the antrum. Note also the granulosa cells (G) showing mitotic divisions (arrow) and theca cells (T). H&E, ×400.
F6-4
Figure 6:
A photomicrograph of a section in the ovary of control group A showing a mature graafian follicle. The primary oocyte (O) appears surrounded by zona pellucida (arrow), cumulus oophorus (C). Note the large antrum containing liquor folliculi (A) and granulosa cells (G). H&E, ×400.
F7-4
Figure 7:
A photomicrograph of a section in the ovary of control group A showing a corpus luteum composed of lightly stained granulosa lutein cells (arrow) separated by minimal connective tissue stroma. H&E, ×400.

Examination of the ovaries following bilateral ovarian artery ligation in group C showed an apparent marked decrease in all types of follicles, except for a few peripherally situated atretic follicles. There were several nodules composed of acidophilic cells forming interstitial glands (Figs 8 and 9). Corpora lutea were also observed and appeared to be invaded by blood vessels and connective tissue between lutein cells (Fig. 10).

F8-4
Figure 8:
A photomicrograph of a section in the ovary of group C subjected to bilateral ovarian arteries ligation showing a few peripherally situated atretic follicles (arrow). Note the nodules of acidophilic cells forming interstitial glands (double arrow). H&E, ×40.
F9-4
Figure 9:
A photomicrograph of a section in the ovary of group C subjected to bilateral ovarian arteries ligation showing a few peripherally situated atretic follicles (arrow). Note the nodules of acidophilic vacuolated cells forming interstitial glands (double arrow). H&E, ×100.
F10-4
Figure 10:
A photomicrograph of a section in the ovary of group C subjected to bilateral ovarian arteries ligation showing a corpus luteum. The lutein cells appear to be invaded by blood vessels (arrows) and connective tissue (double arrow). H&E, ×400.

Furthermore, after uterine artery ligation in group D, the majority of the ovaries showed different segments; some showed aggregation of follicles whereas others showed nodules of acidophilic cells forming the interstitial luteinizing glands (Fig. 11). No mitosis was detected in granulose cells; however, these cells were found to be dark, degenerated, and detached from each other, filling the follicular antrum with cell debris indicative of atresia of their follicles. Some of the detected atretic follicles were devoid of ova, zona pellucida, and cumulus oophorus whereas others had apparently normal ova surrounded by zona pellucid (Figs 12–14). Apparently hypertrophied theca was detected, with blood vessel invasion separating it from the granulosa cells (Figs 13 and 14). There was detachment of the follicles from the surrounding interstitial tissue (Fig. 14). The lutein cells in the interstitial tissue appeared to be markedly vacuolated with dark pyknotic nuclei (Fig. 14).

F11-4
Figure 11:
A photomicrograph of a section in the ovary of group D subjected to bilateral uterine arteries ligation showing aggregation of follicles (double arrow). Note the atretic follicles (arrow) and the interstitial glands (*). H&E, ×40.
F12-4
Figure 12:
Higher magnification of the previous section showing some atretic follicles devoid of primary oocytes, zona pellucida, and cumulus oophorus (double arrows). Note the presence of some apparently normal primary oocytes surrounded by zona pellucida in some atretic follicles (arrow). H&E, ×100.
F13-4
Figure 13:
Higher magnification of the previous section showing signs of follicular atresia; the antrum appears to be devoid of oocytes, zona pellucida, and cumulus oophorus with accumulation of some cell debris (*). The surrounding granulosa cells appear to have pyknotic nuclei (double arrow). Note the apparently normal primary oocyte (O) in atretic growing follicles (arrow). Note also the relatively hypertrophied theca (T). H&E, ×400.
F14-4
Figure 14:
A photomicrograph of a section in the ovary of group D subjected to bilateral uterine arteries ligation showing the detached and degenerated granulosa cells (double arrow) with cell debris in the antrum (*). The apparently hypertrophied theca is separated from granulosa cells by blood vessel invasion (arrow) and the atretic follicle is separated from the surrounding interstitium (**). Note the markedly vacuolated lutein cells surrounding atretic follicles (arrow head). H&E, ×400.

Immunohistochemical staining for apoptosis in the ovaries of the control and sham-operated group was only positive for a few granulosa, theca, and interstitial cells (Figs 15 and 16). Positive brownish staining for apoptosis was markedly evident in group C in granulose, theca cells, and corpus luteum (Fig. 17). However, in group D, there was extensive brownish coloration especially in the zona pellucid, granulosa cells, and theca cells. In group D, when zona pellucid were detected, they appeared to be indented and partially collapsed (Fig. 18).

F15-4
Figure 15:
A photomicrograph of a section in the ovary of control group A showing positive immunostaining for apoptosis in growing follicles localized in a few granulose (G), theca (T), and interstitial cells (arrow). ×400.
F16-4
Figure 16:
A photomicrograph of a section in the ovary of control group A showing positive immunostaining for apoptosis in mature follicles localized in a few granulosa (arrow) and theca cells (T). ×400.
F17-4
Figure 17:
A photomicrograph of a section in the ovary of group C subjected to bilateral ovarian arteries ligation showing markedly evident positive immunostaining for apoptosis in granulosa cells (arrow), theca cells (T), and corpus luteum (double arrow). ×400.
F18-4
Figure 18:
A photomicrograph of a section in the ovary of group D subjected to bilateral uterine arteries ligation showing extensive positive immunostaining for apoptosis especially of the zona pellucida (double arrow), granulosa cells (arrow), and theca cells (T). Note the indented zona pellucid and the apparently hypertrophied theca. ×400.

Scanning electron microscope examination of the ovaries in both control and sham-operated groups showed follicles in all developmental stages ranging from primordial follicles to mature follicles (Figs 19 and 20). Following ovarian artery ligation in group C, the ovaries appeared almost devoid of follicles (Fig. 21). However, after uterine artery ligation in group D, the follicles seemed to be reduced in number compared with the control group, with irregular zona pellucida and detached granulosa cells that were separated from the surroundings (Figs 22 and 23).

F19-4
Figure 19:
Scanning electron micrograph of an ovary of control group A showing follicles in different developmental stages ranging from primary follicles (arrow) to large follicles (double arrows). ×232.
F20-4
Figure 20:
Scanning electron micrograph of an ovary of control group A showing a growing follicle consisting of regular zona pellucid (double arrow) surrounding the oocyte (O), granulosa cells (G) enclosed by theca cells (T). ×273.
F21-4
Figure 21:
Scanning electron micrograph of an ovary of group C subjected to bilateral ovarian arteries ligation showing ovaries nearly devoid of follicles. ×70.
F22-4
Figure 22:
Scanning electron micrograph of an ovary of group D subjected to bilateral uterine arteries ligation showing an apparent decrease in follicles (arrows) compared with the control group. ×119.
F23-4
Figure 23:
Scanning electron micrograph of an ovary of group D subjected to bilateral uterine arteries ligation showing an atretic follicle with irregular zona pellucida (double arrow). The granulosa cells are detached (arrow) and separated (*) from the surrounding theca cells. ×477.

Discussion

UAE, as a treatment for fibroids, leiomyomas, and postpartum hemorrhage, has become a widely accepted and effective alternative to surgery 19,20. The use of selective devascularization procedures, such as UAE, mainly depends on the anastomosis among the uterine vessels and the ovarian, vaginal, rectal, and vesical systems 21. One of the main goals of UAE is to preserve the uterus and therefore fertility including pregnancies, menses, and ovarian reserve 7. However, the long-term effect of UAE on menstruation and fertility is unclear and negative effects on ovarian function have been detected by previous studies 9,10. As the histopathologic changes after embolization in New Zealand white rabbits resemble those in humans, rabbits are the appropriate model for experimental UAE 22. Therefore, the present work studied the effect of bilateral uterine artery ligation on the structure and function of the ovary in comparison to ovarian artery ligation using New Zealand white rabbits.

In the rabbit, ovulation does not occur spontaneously, but it has to be induced through a neurohormonal reflex, which is initiated during mating; hence, in the absence of a male, ovulation has to be induced by artificial gonadotropins 23. In the present work, ovulation was induced with human chorionic gonadotrophin 2 days before scarifying the animals. This also ensured that all the rabbits were at the middle of their estrous cycle at the time they were scarified.

In the current work, the ovaries of group C were dark and dull, whereas those of group D showed loss of luster. There was apparent increase in the tortuosity of the uterine arteries in group C, whereas there were engorged ovarian arteries in group D. The presence of ovariouterine anastomosis, which provides the ovary with an alternative source of blood supply in case of ovarian artery obstruction, has been reported previously 24. Similar anastomosis occurs in case of uterine artery occlusion 24. This phenomenon was explained by describing ovarian resistance arteries that might aid the regulation of ovarian flow magnitude 25. These resistance arteries, together with veins and lymphatics, constitute an important alternative way for double regulation between the ovary and the uterus.

The present work found a significant increase in serum FSH and LH in group C, whereas serum 17-β-estradiol level and ovarian weight showed a statistically significant decrease, indicating ovarian failure following ovarian artery ligation. These findings were in contrast to those reported by previous researchers 24. However, following uterine artery ligation in the current work (group D), there was a statistically significant increase in serum FSH and LH, with a nonsignificant decrease in both serum 17-β-estradiol level and ovarian weight. The increase in both FSH and LH recorded in groups C and D of the present work could be correlated to the decreased estrogen found in these groups, resulting in loss of its negative feedback on the pituitary gland. These findings reflect the presence of a countercurrent pathway for cross-regulation between the ovary and the uterus. Similarly, it was reported that women treated with uterine artery occlusion may show a significant increase in FSH level in the first month after the operation, which might be an indication of decreased ovarian function 26. The acquisition of an adequate vascular network is consequent to an upregulation of angiogenesis, important for maturation of ovarian follicles, and essential for sufficient metabolic support to fertility 27.

Light and scanning electron microscopic examination of ovaries following ovarian artery ligation in group C showed a marked decrease in all types of follicles with the presence of multiple nodules of acidophilic cells forming interstitial glands and vacuolated lutein cells with dark nuclei. Interstitial glands have an epitheloid appearance and secretory activity dispersed in stroma as new cells are added to them from the regressing follicles 28. These glands consist of hilar cells; epithelioid androgen-secreting cells; and this androgen is converted into estrogen by the effect of aromatase enzyme produced by granulosa cells 28. In bilateral ovarian ligation, the ovary is formed mainly of interstitial glands in the form of closely packed epithelial cells with an acidophilic vacuolated cytoplasm resulting in virilization, which is common in postmenopausal women 29.

Simultaneously, in group D, following uterine artery ligation, the ovaries showed some areas devoid of follicles whereas others showed follicular aggregation. No mitosis was detected in granulosa cells, but they appeared to be dark, degenerated, and detached from each other and from the surrounding theca cells, indicating atresia of their follicles. Some of the atretic follicles showed the absence of oocyte and zona pellucida whereas others had apparently normal ovum and zona pellucida. In addition, there was apparently thickened theca with blood vessel invasion separating granulosa from theca cells. Previously, many authors have summarized the stages of follicular degeneration as cessation of mitosis within the granulosa cells, sloughing of the granulosa cells into the antrum of the follicle, vascularization of the connective tissue surrounding the granulosa layer, and hypertrophy of the theca interna cells 30. Thus, the current work proves that uterine artery occlusion is associated with atresia of ovarian follicles. In addition, the appearance of the unaffected ova in atretic follicles found in group D could be explained by Ross and Pawlina 30, who reported that in atresia of growing follicles, the degeneration of the mature oocyte is delayed and appears to be secondary to degenerative changes in the follicular wall. This delay indicates that once the oocyte achieves its maturity and competence, it is no longer sensitive to the same stimuli that initiate the atresia in granulosa cells 29. Moreover, in group D, the lutein cells in the interstitial tissue appeared markedly vacuolated with dark pyknotic nuclei with the presence of interstitial glands. In this respect, a previous study 10 found significant hypo-ovulation with increased lipid foci in the ovaries of rabbits after bilateral uterine artery ligation. It was reported that the interstitial lutein cells normally assume a characteristic steroid-secreting appearance and contain numerous small lipid vesicles. In case of advanced degenerative changes, these vesicles coalesce into alveolar-type vacuoles and the nuclei involute 31. Therefore, it could be assumed that bilateral uterine artery ligation is associated with atresia of ovarian follicles and the appearance of interstitial glands; however, there is a delay in this atresia compared with that found following bilateral ovarian artery ligation.

It was found that premenopausal hysterectomy accelerated the ovarian dysfunction and early menopause 32,33. In addition, it was reported previously that hysterectomized women, with ovarian preservation, showed a subsequent 26–39% ovarian failure 34. This might be attributed to the fact that the uterine artery is an important source of blood flow to the ovaries in some species such as rodents, cycling rhesus monkeys, and humans 35,36. The presence of utero-ovarian anastomosis was found by angiography only in 38% of cases 36 and in only 104 of 202 women, where it was observed to be unilateral in 66 cases and bilateral in 38 cases 37. Moreover, the blood supply to the uterus through the ovarian arteries was evaluated using aortography, in 290 patients who underwent UAE, where no utero-ovarian anastomosis was visualized in 70% of cases 38. Therefore, it might be assumed that the ovarian changes that were detected in the current work following uterine artery ligation were not only because of defects in uterine artery supply to the ovary as was predicted, but also as a result of hormonal alterations after occlusion of the uterine ovarian arterial axis.

Thus, different methods that could disturb the correlation between the uterus and the ovary such as hysterectomy, UAE, and tubal ligation might lead to a decrease in the ovulation rate and hormonal imbalance. Although earlier studies considered the desire of childbearing when choosing embolization or surgery 6,8, the current work reflect a suspect on future fertility after UAE.

It was stated that the ovary provides a unique model for studying the hormonal regulation of apoptosis 39. Apoptosis (a marker of cell death) can be used to study the growth and selection of follicles and to correlate the granulosa cells and oocyte quality 40,41. Monitoring of apoptotic DNA fragmentation represents a quantitative and sensitive endpoint to study the hormonal regulation of atresia in the ovarian follicles 42. In the current study, examination of immunohistochemically treated specimens showed markedly evident positive brownish staining for apoptosis, especially in the zona pellucida, granulosa cells, and interstitial cells in both groups C and D. This might be attributed to the fact that granulosa cells have endogenous pathways to trigger apoptosis that are inhibited in the presence of survival factors 43. In addition, apoptosis occurs at the highest frequency in proliferating rather than quiescent tissues 44. Zona pellucida are known for their importance in fertilization and their strong immunogenicity 45. In addition, in group D of the current work, zona pellucida appeared indented and partially collapsed, especially when detected by immune staining. This might be explained by Ross and Pawlina 30, who reported that the zona pellucid is resistant to autolytic changes occurring in atresia and becomes folded and collapsed before being broken down. Therefore, in the present work, it could be suggested that, following bilateral arterial ligation, there was marked follicular atresia accompanying apoptosis of granulosa cells and zona pellucida.

Finally, the present study found that ovarian artery ligation causes ovarian dysfunction and atresia; however, these findings develop much later after uterine artery ligation. Therefore, it could be concluded that although the ovarian artery is the main blood supply to the ovaries, the uterine artery shares in the blood supply of the ovary and there is a countercurrent pathway for cross-regulation between the ovary and the uterus. Thus, selective devascularization procedures such as UAE could anticipate ovarian failure and early menopause.

T3-4
Table:
No title available.

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

function; ligation; ovarian artery; ovary; structure; uterine artery

© 2013 The Egyptian Journal of Histology