The Effects of Administrated Sildenafil Citrate on Uterine Luminal Epithelium Height Associated with Ovarian Angiogenesis: An Experimental Animal Study.

BACKGROUND: Ovarian angiogenesis (OA) remains in lifetime and normal ovarian function depends to this continual remodeling of a complex vascular system. Endometrial thickness (ET) is one of the strongest predictors of successful implantation and pregnancy. Appropriate OA effects on ET by facilitating of ovarian hormone delivery.
MATERIALS AND METHODS: Thirty adult female mice and twenty adult male mice were purchased. The female mice were divided into three groups: (1) control group without any intervention (n = 10), (2) gonadotropin group: receiving human menopausal gonadotropin (HMG) and human chorionic gonadotropin (n = 10), and (3) gonadotropin and sildenafil citrate (SC) group: receiving HMG and SC administration (n = 10). After mating, animals were deeply anesthetized, and the ovary and uterus was rapidly removed for histology and immunohistochemistry process.
RESULTS: Four days after ovarian induction, all three layers of the uterus with specified thickness can be clearly seen. The heights of endometrial epithelial cells in gonadotropin group were not significantly different than those in control group. In gonadotropin and SC group, heights of the cells were significantly (P < 0.05) shorter than control and gonadotropin groups. ETs in all groups were not significantly deferent from each other (P > 0.05 each). Our results of immunohistochemistry survey for ovarian CD31 demonstrated that administrated SC increased OA but not significantly (P > 0.05 each).
CONCLUSION: It may finally conclude that administration of SC does not cause notable alterations in OA and ET; although for realistic decision about the SC effects on aforementioned parameters, more molecular investigations and longer drug consumption period are necessary.

Introduction

Based on the reproductive medicine sciences, infertility is defined as the inability to conceive after 12 months of unprotected sexual intercourse.[12] Implantation failure is one of the well-known causes of female infertility, and endometrial thickness (ET) is one of the strongest predictors of successful implantation.[3] A series of scientific reports have presented that embryo implantation and pregnancy rates are significantly higher in women with an ET more than 9 mm. Thin endometrium, generally measuring <7 mm, is assumed to be less able to support of implantation.[4] ET is dependent on several factors including woman age, menstrual cycle phase, ovarian hormone (estrogen and progesterone) concentration, ovarian angiogenesis (OA), and density of hormone receptor in endometrial tissue.[56]

The capacity for angiogenesis, creating of new blood vessels from preexisting ones, is the crucial feature of endothelial cells, but the issue is open to discussion.[7] This process occurs in both physiological and pathological conditions. Physiological angiogenesis is a key step in wound healing, tissue remodeling, luteinization of the ovarian follicle, placental development, and pregnancy establishment. In other word, physiological angiogenesis plays an important role in pregnancy and childbearing.[89] In contrast, pathological angiogenesis is a known landmark of some serious disease such as cancer and associated metastasis, diabetic neuropathy, endometriosis, psoriasis, and rheumatoid arthritis.[1011]

Production of female germ cell and synthesis of sexual hormones and growth factors are two major functions of mammalian ovary. Unlike many other parts of the body, OA remains to before menopause, and this continual remodeling of a complex vascular system is necessary for normal ovarian function.[1213] This is important for increasing delivery of the gonadotropins, luteinizing hormone, follicular stimulating hormone, growth factors, oxygen, lipids, and steroid precursors required for folliculogenesis.[14] One of the proposed factors that stimulated OA is hypoxia. In this condition, ovarian endothelial cells respond to hypoxia with increased vascular epithelial growth factor (VEGF) expression as a key signal molecule involved in angiogenesis. VEGF binds to the vascular epithelial growth factor receptor (VEGFR) that located on adjacent endothelial cells. Interaction between VEGF and VEDFR plays an important role in angiogenesis process and induced endothelial cell division, forms vessels by clustering on adjacent capillaries, and increasing hydraulic conductivity of isolated microvessels. Furthermore, VEGF stimulates the release of matrix metalloproteinase-9 for degradation of extracellular matrix and creating efficient space for new blood vessel formation.[151617] Finally, the newly formed endothelial cells adhere together and create tubes and loops that work as functional vascular units. Platelet endothelial cell adhesion molecule or CD31 is a new marker for the detection of angiovasculogenic activity express in endothelial cells with high levels of activity.[18]

Sildenafil citrate (SC) or Viagra is a selective phosphodiesterase type-5 (PDE-5) inhibitor that induces vasodilation by inhibition of the cyclic guanosine monophosphate hydrolysis and increases the accessibility of the nitric oxide (NO).[1920] For the first time, SC developed as an antihypertensive drug, and it was noted during early trials of the drug that PDE-5 inhibition led to an increased response in subjects with erectile dysfunction.[21] In recent years, many studies were performed for investigating the effects of SC on female fertility, for example, some studies indicated the positive role of vaginal SC administration on improved endometrial development.[41922] A clinical survey that performed by Mangal and Mehirishi presented the improving role of SC on ET, blood flow, and pregnancy rates in infertile women undergoing intrauterine insemination.[4] Celik et al. in their study examined the effects of SC on ischemic and reperfusion injury in the rat ovary. The biochemical and histopathological results of their experimental study demonstrated that aforementioned injury is ameliorated by sildenafil consumption.[23]

Due to these fact that sufficient OA effect on ET by facilitating of hormone delivery and current controversies concerning the probable effects of SC on angiogenesis of female genital system, this study aimed to analyze effects of administrated SC on OA and associated ET changes.

Materials and Methods

Study design and experimental groups

All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals of Isfahan University of Medical Sciences, Isfahan, Iran. Thirty adult Naval Medical Research Institute (NMRI) female mice (25–30 g body weight, 3 months old) and twenty adult NMRI male mice were purchased from Isfahan University of Medical Sciences Experimental Animal House (Isfahan, Iran). Animals were housed in individual cages at 22°C ± 2°C with free access to pellet food and water and on a 12 h light/dark cycle. They were fed a regular rat chow. In the present study, the female mice were divided into three groups: (1) Control group without any intervention. (2) Gonadotropin group: 7.5 IU human menopausal gonadotropin (HMG) (Ferring, Germany) was administered intraperitoneally (IP). After 2 days, 7.5 IU human chorionic gonadotropin (HCG) (Ferring, Germany) was injected IP. (3) Gonadotropin and SC group: after injection of HMG, SC administrated in 24, 48, 72 h interval (IP). Then, every two female mice with one male mouse put in one cage for mating. Four days after HMG injection, animals were deeply anesthetized with chloral hydrate (350 mg/kg),[24] and the whole right ovary and uterus was rapidly removed for histology and immunohistochemistry process.

Histological staining

The uterus of the mice was gathered. For fixation, the samples were buffered in 10% formalin. Next, each uterus was dehydrated in graded ethanol solutions and embedded in paraffin. Horizontal serial sections with 4 μm thickness were cut. To be studied microscopically sections must typically be stained or dyed because most tissues are colorless. In the present study, histological sections stained with periodic acid-Schiff for optical microscope analyzing. Furthermore, for morphometric study, we surveyed 25 fields for each group.

Immunohistochemistry staining

To analyze OA, expression of CD31 antigen was evaluated by immunohistochemistry staining technique. For this purpose, we surveyed 25 fields for each group. Fixation of ovarian samples was done by 10% buffered formalin (Sigma, USA) for 24 h, and other steps of tissue processing were done with ascending grade of alcohol and clearness in xylene (Sigma, USA). Finally, they were embedded in paraffin, and horizontal serial sections with 4 μm thickness were cut using microtome. For immunohistochemistry staining, the slices were removed and placed on slides previously covered with poly-L-lysine in all groups. The main basis of this method is interaction of CD31 antigen with anti-CD31 antibody (AbCam, UK); then, the secondary antibody binds to the primary antibody as its antigen for the second level. Antibody-labeled and antibody complex can be exposed using the enzyme substrate. In the final step, due to the reaction of peroxidase enzyme with diaminobenzidine (DAB), DAB is utilized as a peroxidase substrate, and a dark brown product is formed.

Statistical analysis

With using of Motic Image Plus 2.0 (Motic Group Co., Ltd, Hong Cong, China), CD31-positive cell count was performed after creating the connection between computer and microscope with ×40 magnifications. Statistical analysis was performed using the SPSS statistical package, version 15.0 (SPSS Inc., Chicago, IL, USA).and the Kruskal–Wallis test. P < 0.05 was accepted as statistically significant.

Results

Optical microscopic observation and the height of endometrial epithelial

Four days after ovarian induction, all three layers of the uterus including perimetrium, myometrium, and endometrium with specified thickness can be clearly seen. ET which contains stroma, uterine glands, and luminal epithelium is found several times in some part with just a row of epithelium and small amount of stroma. In control group, the height of endometrial epithelial cells was 20.52 ± 2.43 μm. In gonadotropin group, the heights of the cells were 20.85 ± 2.55 μm, which were not significantly different than those in control group. In gonadotropin + SC group, heights of the cells were 17.60 ± 2.49 μm, which were significantly shorter than control and gonadotropin groups (P < 0.05) [Figure 1a and b].

Figure 1

(a) histologic view of luminal epithelium of endometrium (×660). (A): Control group (Ctr). (B): Gonadotropin group (Gnt). (C): Gonadotropin and SC group (Gnt + SC). (b) Data are presented as means ± standard error the heights of endometrial epithelial cells; in Gnt group, the heights of the cells were not significantly different than those in control group. In Gnt + SC group, heights of the cells were significantly (P < 0.05) shorter than Ctr and Gnt groups

Morphometric study associated with endometrial thickness

ET defined the distance between myometrium to top of epithelial cells. Based on our morphometric survey and statistical analyzing, the mean ± standard deviation of ET was 358.05 ± 13.35 μm in control group, 389.84 ± 12.46 μm in gonadotropin group, and 316.81 ± 12.10 μm in gonadotropin + SC group. Finally, the ET in all groups was not significantly deferent from each other [P > 0.05 each, Figure 2a and b].

Figure 2

Results of optical microscopy and morphometric study. (a) PAS staining of mice uterus (×10), 96 h after ovarian induction of ovary. A: Endometrial thickness in control group (Ctr). B: Endometrial thickness in gonadotropin group (Gnt) C: Endometrial thickness in gonadotropin and SC group. (b) Data are presented as means ± standard error. The endometrial thicknesses in all groups were not significantly deferent from each other (P > 0.05 each)

Immunohistochemistry for detection of ovarian CD31-positive cells

According to our findings, the mean of CD31-positive cells was 22 ± 3.42 in control group, 24 ± 1.48 in gonadotropin group, and 25 ± 2.09 in gonadotropin + SC group. The present data showed that angiogenesis parameter can be affected by exogenous factors such as gonadotropins and SC but not significantly (P > 0.05). The utilization of gonadotropins in the ovulation-stimulating process increased OA in this group in comparison with control group. In the other group, the use of gonadotropins + SC leading to increased OA when compared with gonadotropins and control group [Figure 3a and b].

Figure 3

Results of immunohistochemistry study. (a) Immunohistochemistry staining of mice ovary for detection of CD31-positive cells (×40), 96 h after ovarian induction. A: Control group (Ctr). B: Gonadotropin group (Gnt) C: Gonadotropin and SC group. (b) Data are presented as means ± standard error. The number of CD31-positive cells in all groups was not significantly deferent from each other (P > 0.05 each)

Discussion

Some researchers presented the positive role of SC on ovarian function. For example, Taskin et al. demonstrated that pretreatment with SC is useful for protecting the ovaries from cisplatin-induced damage, and this drug can be a choice for fertility preservation after chemotherapy.[25] In another experimental animal study, Celik et al. examined the effects of SC on activities of antioxidant enzyme, lipid peroxidation, and histopathological characteristic of ovarian tissue after ischemia-reperfusion injury. Their results showed that aforementioned injury in the ovary was ameliorated by SC treatment.[23] Our study demonstrated that administrated SC increased OA but not significantly. However, the precise molecular studies are needed to definitively comment.

As previously mentioned, ovarian blood vessel formation through angiogenesis is not limited to embryological period but occurs throughout mammalian life. During the blood vessel formation at adulthood, angioblasts that originated from the mesodermal layer are no longer involved. Instead, endothelial progenitor cells (EPCs) that derived from the bone marrow and circulate through the blood contribute to new blood vessel formation. The bone marrow-derived cells are thought to be enrolled by some factors involved in angiogenesis, such as VEGF and stromal-derived factor-1 (SDF-1). The interaction between VEGF and SDF-1 leads circulating EPCs to the ischemia area where they differentiate into endothelial cells and form some portion of new blood vessels. In addition, OA contributes to the remodeling of the early blood vessels that formed in before birth life.[2627] OA occurs largely in the cortical region because folliculogenesis and luteinization occur in this area.[28] In our study, we investigate the presence of CD31-positive cells in the cortical region of the ovary and around the follicles.

Pregnancy complications represent a main challenge to the National Health Service. Ovarian hyperstimulation is usually used in reproductive medicine.[2930] Ovulation that induced by HMG offers the ability to surveys’ several factors that may contribute to reproductive capacity. One of the most important points, which must be considered in selecting an appropriate animal model, is the age of animals, to reflect the same condition in human.[31] OA is a vital event that performed in successful pregnancy. In the present study, HMG was administered IP at 0 days, HCG was injected at 2 days, and tissue sampling was done in 4 days. Based on our previous surveys that performed on pregnant mice, 96 h or 4 days after HMG injection is the best time of tissue sampling because this time is almost accordance with the time of implantation of mouse embryos but the issue is open to discussion.[1932]

In the present study, we used immunohistochemistry technique for detection of CD31-positive cell. Aforementioned technique is not only an accurate method for recording CD31-positive cells but also a suitable method to observe angiogenesis alteration of tissue, in other word, in immunohistochemistry; CD31 is used primarily to determine the presence of endothelial cells in histological sections.[33] To avoid mistaken identification of CD31-positive cells, we paid special attention to some key features of these cells including; dark brown color of cells (because of reaction with DAB) and having the appearance of simple squamous cells in the inner surface of the lumen of microvasculature.

Endometrial characteristics, such as endometrial cellular pattern, endometrial blood flow, endometrial angiogenesis, and ET, have been described as key prognostic factors for successful pregnancy.[634] In humans, ET can be measured by transvaginal ultrasonography.[35] Some surveys indicated that thin endometrium is related with lower IVF (in vitro fertilization) success rates[3637] while some another studies could not approve this.[38] In spite of all these, ET assessment has become part of routine monitoring during female infertility treatment. Some studies showed that Assisted Reproductive Technology drugs increased ET and the likelihood of pregnancy.[3940] Recently, the focus was on the role of NO as a relaxing factor of vascular smooth muscles and its moderating effects on blood flow of the uterus.[41] As previously mentioned, SC is one of the interesting drugs that may be helpful for the treatment of female infertility, but the issue is open to discussion. Based on our morphometric study, the ET in all groups was not significantly different from each other and SC could not be created a significant change in histologic features of endometrium. These data are in consistent with the results of Rashidi et al. study (2015) that investigated the effects of SC on Ki-67-positive stromal cells of mice endometrium.[41] Their results indicated that SC does not cause a significant change in numbers of Ki-67-positive cells. Our results are in contrast with the results of the study done by Dehghani Firouzabadi1 et al. They examined the effects of oral use of SC on endometrial preparation and outcome of frozen-thawed embryo transfer cycles. Their results indicated that SC increased ET and is a good way to improve the endometrial receptivity.[22]

Conclusion

Taking together, it may finally conclude that administration of SC does not cause remarkable changes in OA and associated ET; albeit for realistic decision about the effect of SC, more molecular investigations, and longer drug consumption period are necessary. SC injection alters the morphometrically features of luminal epithelium of endometrium, which could effect on its maturation.

Financial support and sponsorship

Grant support was provided by Isfahan University of Medical Sciences (MUI) (No. 394919).

Conflicts of interest

There are no conflicts of interest.