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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 2  |  Page : 64-71

Functional and structural assessment of the possible protective effect of platelet-rich plasma against ischemia/reperfusion-induced ovarian injury in adult rats


1 Department of Medical Physiology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department Histology and Cell Biology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission14-Jan-2022
Date of Decision24-Feb-2022
Date of Acceptance25-Feb-2022
Date of Web Publication28-Apr-2022

Correspondence Address:
Dr. Eman Ahmed Allam
Department of Medical Physiology, Faculty of Medicine, Alexandria University, Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_3_22

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  Abstract 


This study aimed to evaluate the possible protective effect of platelet-rich plasma (PRP) on ischemia reperfusion (I/R)-induced ovarian injury in a rat model. Forty adult female albino rats were randomly assigned to four groups: control, ischemia, I/R, and I/R + intraperitoneal PRP. Induction of ischemia was done by bilateral ovarian torsion for 3 h, while reperfusion was done by subsequent detorsion for another 3 h. PRP was injected 30 min before detorsion. Histological assessment and measurement of ovarian anti-Mullerian hormone (AMH) were done to assess the degree of tissue damage and the remaining ovarian reserve. Ovarian malondialdehyde (MDA) and total antioxidant capacity (TAC) levels were measured to evaluate the oxidant-antioxidant balance. Tumor necrosis factor-α (TNF-α) was measured to assess degree of inflammation. Immunohistochemical assessment of ovarian vascular endothelial growth factor-A (VEGF-A) was also done. PRP treated I/R group revealed a significant decrease in MDA (P = 0.007), TNF-α (P = 0.001), and a significant increase in TAC (P = 0.001) and VEGF-A (P = 0.003) in comparison to the untreated I/R group. Furthermore, limited vascular congestion and inflammatory infiltration were observed after PRP treatment. However, no significant difference was detected in AMH after PRP treatment. Our results denoted that PRP may help in preservation of ovarian function and structure during surgical conservative detorsion of the torsioned ovary. These protective effects could be attributed to its ability to reduce oxidative stress, inflammation and also to its high content of growth factors especially VEGF.

Keywords: Anti-Mullerian hormone, ovarian ischemia/reperfusion, oxidative stress, platelet-rich plasma, tumor necrosis factor-α, vascular endothelial growth factor


How to cite this article:
Allam EA, Abdel Moniem RA, Soliman GY. Functional and structural assessment of the possible protective effect of platelet-rich plasma against ischemia/reperfusion-induced ovarian injury in adult rats. Chin J Physiol 2022;65:64-71

How to cite this URL:
Allam EA, Abdel Moniem RA, Soliman GY. Functional and structural assessment of the possible protective effect of platelet-rich plasma against ischemia/reperfusion-induced ovarian injury in adult rats. Chin J Physiol [serial online] 2022 [cited 2022 Sep 26];65:64-71. Available from: https://www.cjphysiology.org/text.asp?2022/65/2/64/344167




  Introduction Top


Ovarian torsion is responsible nearly for 3% of all gynecological emergencies in women in their reproductive age.[1] Hyperlaxity of ovarian ligaments and any cause for ovarian enlargement are common risk factors for this critical condition.[2] This condition can lead to severe tissue necrosis and damage, thus threatening future fertility. Therefore, early diagnosis and treatment is very important to protect ovarian functions. Conservative management with surgical detorsion and reperfusion is the common first-line of treatment.[3] However, reperfusion following a period of ischemia creates a new pathophysiological process that can lead to more tissue damage. This process is named ischemia-reperfusion (I/R) injury.[4]

During reperfusion, oxidative stress and inflammatory condition are induced within the ovarian tissue due to tissue neutrophil infiltration. The increased production of reactive oxygen species (ROS) and depletion of antioxidants corrupt the oxidative-antioxidant balance resulting in oxidative stress. ROS oxidize cell membrane lipids leading to formation of toxic mediators as malondialdehyde (MDA) that which mutate cell membrane conformation and functions.[4],[5] Also in I/R, some inflammatory mediators are formed early in the ovarian tissue. Tumor necrosis factor-α (TNF-α) is one of these mediators.[6]

Anti-Mullerian hormone (AMH), which is a good indicator for ovarian reserve, was found to be diminished following detorsion surgery meaning that conservative treatment alone cannot protect the ovarian reserve.[7] In this context, adopting new therapeutic strategies that aim to overcome the drawbacks associated with reperfusion following ischemic conditions is necessary.

Vascular endothelial growth factor (VEGF), a peptide expressed in all body tissues, stimulates angiogenesis and supports endothelial cells survival. Regarding it role in female reproductive cycle physiology, it supports angiogenesis during follicular development and helps corpus luteum nutritional support.[8] Its protective effect was evident in several types of organ I/R injuries, such as brain,[9] liver,[10] and heart.[11] In such a context, pro-angiogenic therapy using VEGF itself or any product that contain VEGF is believed to be effective in I/R conditions such as our model of ovarian I/R. In this regard, platelet-rich plasma (PRP) is considered as a promising therapeutic approach due to its high content of VEGF.

Nowadays, PRP is widespread used as a non-operative therapy in multiple medical fields such as dermatology, dentistry, and urology. Its high content from growth factors, stored in platelets' granules, may be responsible for its positive effects. PRP aids in tissue healing by accelerating angiogenesis, inflammation control and cell differentiation.[12] Platelets help local tissue repair after minimal trauma to the ovarian epithelium that occurs during ovulation.[13] In recent studies, PRP showed protective effects in I/R models in kidney[14] and testis.[15] In this context, the current study aimed to investigate the potential effect of PRP to preserve ovarian function and structure during I/R-induced ovarian injury in a rat model.


  Materials and Methods Top


Experimental animals

Forty adult Wistar albino female rats weighing 180–200 g were purchased from and housed in the animal research laboratory of Medical Physiology Department, Alexandria Faculty of Medicine, Egypt. They were housed in the following normal circumstances: natural day/night cycle, temperature of 23 ± 3°C, and stable humidity. They had free access to standard rodent chow laboratory diet and tap water. All study procedures were approved by the institutional animal ethics committee at the Faculty of Medicine, Alexandria University (IRB code 00012098-FWA: No. 00018699; membership in International Council of Laboratory Animal science organization, ICLAS).

Study design

After acclimatization to housing conditions, the rats were categorized into four groups (10/each group) randomly. The groups included: sham operated control group, only laparotomy and gentle ovarian manipulation were performed; ischemia group (I), ovaries were exposed to ischemia for three hours; I/R, ovaries were exposed to ischemia for 3 h followed by reperfusion for another three hours, and I/R + PRP, animals were exposed to 3 h of ischemia, followed by another 3 h of reperfusion. PRP was injected intraperitoneally 30 min prior to reperfusion.

Surgical protocol

Vaginal smears were done and examined daily to determine the regularity and phase of rats' estrous cycles. Only rats showed at least two successive regular 4–5 days estrous cycles were included in this study. The experiment was conducted during the estrous phase of the cycle. All procedures were conducted under strict sterile conditions and at suitable ambient temperature. The rats were injected intramuscularly with 10% ketamine hydrochloride (45 mg/kg) and 2% xylazine hydrochloride (5 mg/kg) (Sigma-Aldrich, Egypt). Rats were placed in a dorsally recumbent position, then the abdominal skin was shaved and cleansed with 10% povidone iodine. Two-cm laparotomy midline incision was done in the lower abdomen to visualize the ovaries and adnexa. For induction of ischemia, the  Fallopian tube More Details and ovarian blood vessels were rotated 360° clockwise, and then fixed on the abdominal wall with a 1-0 nylon suture and were left for 3 h.[7] During the waiting period, the abdominal incision was sutured with 3-0 silk suture.

In I/R groups, the abdomen was re-opened after 3 h of ischemia and the fixation sutures were removed. Then, the ovaries were detorsioned and returned to their original positions to be reperfused for another 3 h.[7] In I/R + PRP group, a single dose of activated PRP (1 ml) was injected intraperitoneally 30 min before detorsion. The dose of injected PRP was chosen depending on other experimental studies in different models.[16],[17] The rats in untreated I/R group were injected with 1 ml of saline 30 min before detorsion.

Tissue sampling

At the end of all experimental procedures (after 3 h of ischemia followed 3 h of reperfusion in I/R groups and after 3 h ischemia in ischemia only group), the animals were sacrificed and bilateral ovariectomy was performed. The left ovaries were stored at -80°C for further biochemical analysis. The right ovaries were fixed in 10% formalin solution and processed to obtain 5 μm thickness sections from the prepared paraffin blocks for histological and immunohistochemical assessments.[18]

Preparation of platelet-rich plasma

PRP preparation was conducted at the Clinical Pathology Lab, Alexandria Faculty of Medicine. Age-matched healthy female Wistar albino rats (other than those used in the experiment) were used as PRP donors. Whole blood samples were withdrawn via ocular vein under general anesthesia. Blood samples were collected in citrated test tubes and mixed by inversion. The blood was centrifuged at 1000 g for 15 min at 4°C for separation of plasma containing platelets. The plasma was drawn off the top and was transferred to another tube without anticoagulant. After a rest period for 5 min, another cycle of centrifugation was done at 1000 g for 10 min to have a platelet concentrate. The upper two thirds (platelet poor plasma) were removed. The lower third (PRP) was used. An automatic cell counter was used to determine the platelet concentration in PRP to ensure that the concentration is at least 4 times its concentration in whole blood.[19] PRP harvested from whole blood ranged nearly between 18% and 20% from the whole blood volume. PRP was injected maximally within 60 min of its preparation. Calcium gluconate was added to PRP in a ratio 1:9 in order to activate PRP before its therapeutic use (for each 0.9 ml PRP, 0.1 ml calcium gluconate was added) according to Gkini et al.[20]

Immunohistochemical staining with anti-vascular endothelial growth factor A antibody (VG-1)

Five μm thick tissue sections obtained from the paraffin blocks were mounted on positively charged slides. The sections were incubated with monoclonal anti-vascular endothelial growth factor A (VEGFA) antibody (VG-1) (Abcam 1316) in a concentration of 5 μg/ml for 15 min at room temperature. According to the manufacturer's instructions, heat-mediated retrieval of the antigen was done using citrate buffer pH 6. All sections were counterstained with hematoxylin.[21]

Light microscopic examination and histomorphometric assessment

Hematoxylin and eosin H&E and immunohistochemically-stained sections were examined by light microscope, (Olympus BX41, Tokyo, Japan) equipped with spot digital camera (Olympus DP20) at the center of excellence, CERRMA, Alexandria Faculty of Medicine. H and E-stained sections were examined to assess the changes in ovarian tissue. Vascular congestion, hemorrhage and cellular infiltration were the parameters used to assess I/R injury in H and E sections. Histomorphometric study was performed, using NIH Fiji© program (NIH, USA), to measure area percentage of positive reaction to anti-VEGFA antibody that exhibited brownish colour. Six randomly selected sections from each group, in images at magnification of 100, were used.

Preparation of ovarian homogenate and biochemical analysis

Frozen ovarian tissues were homogenized in PBS; NaCl (100 mM), EDTA (100 mM), Na-deoxycholate (0.5%), Nonidet p-40 (0.5%), Tris, pH 7.5 (10 mM) containing protease inhibitors cocktail. The resulting homogenate was centrifuged at 2000 g for 15 min at 4°C. The supernatant was collected to measure protein concentration using Lowry method[22] to be further used for biochemical analysis. Enzyme-linked immunoassay (ELISA) measurement of TNF-α (MyBiosource Cat# MBS1754207) and AMH (MyBiosource Cat# MBS7612458) was done in these ovarian homogenates according to the manufacturer's instructions. To assess the oxidant-antioxidant balance, MDA (thiobarbituric acid reaction colorimetric method)[23] and total antioxidant capacity (TAC, enzyme-immunoassay, Abcam, Egypt)[24] were also measured in the ovarian homogenates.

Statistical analysis

Statistical Package for Social Sciences (SPSS, version 20; IBM, Chicago, IL, USA) was used. Parametric tests were used as Shapiro–Wilk test to show normal data distribution. One-way analysis of variance followed by Tukey's post hoc tests was done to analyze difference between groups. Results were presented in the form of mean ± standard deviation. Correlation between parameters was tested using linear regression analysis and Pearson's coefficient was calculated. Significant P values were defined as those less than 0.05.


  Results Top


Histological assessment

In comparison to the normal ovarian histological appearance in control group [Figure 1]a and [Figure 1]b, ischemia group showed variable degenerative changes, in the form of degenerated primordial follicles within the cortex exhibiting necrotic oocytes surrounded by remnants of granulosa cells and atretic follicles. Few growing follicles depicted vacuolated granulosa cells with ill-defined and partially detached theca folliculi. A single corpus luteum was seen in one of the examined sections that depicted granulosa lutein cells with vacuolated cytoplasm and dark pyknotic nuclei. Mononuclear cellular infiltration was observed within ovarian stroma [Figure 1]c and [Figure 1]d. I/R group depicted accentuated degenerative changes, where widely spread areas of hemorrhage were evident in different parts of the ovary and within the degenerated follicles. Most of the follicles depicted degenerated oocytes. Massive and diffuse cellular infiltration was observed [Figure 2]a and [Figure 2]b. The PRP treated I/R group demonstrated marked improvement, where limited areas of hemorrhage were noticed. Also, growing and mature Graafian follicles were more frequently encountered within the ovarian cortex. They appeared surrounded by well-defined theca folliculi and exhibited nearly normal granulosa cells. Corpus luteum depicted esinophilic granulosa and theca lutein cells. In addition, limited cellular infiltration was observed [Figure 2]c and [Figure 2]d.
Figure 1: Representative photomicrographs of H and E-stained ovarian sections for control (a and b) and ischemia groups (c and d). (a) Primary multilaminar follicles (oval outline) depicting multiple layers of granulosa cells, secondary follicle (SF) showing follicular spaces (asterisks) and a primary oocyte (arrowhead). A mature Graafian follicle (GF) with evident basement membrane (1) and theca folliculi (2). Corpus luteum (CL) shows eosinophilic granulosa and theca lutein cells. Blood vessels (BV) are observed within the ovarian medulla. (b) A primordial follicle (arrow) is seen. A: antral space; GE: germinal epithelium. (c) Few growing follicles depicting vacuolated granulosa cells and separated theca folliculi (green arrow), atretic follicles (blue outline), cellular infiltration (arrowheads), CL exhibits vacuolated cells with dark shrunken nuclei. (d) Primordial follicles (blue outline) with degenerated oocytes. (a-d) Microscopic magnification x100.

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Figure 2: Representative photomicrographs of H and E-stained ovarian sections for I/R (a and b) and I/R + platelet-rich plasma (c and d) groups. (a and b) Hemorrhagic areas (arrows) within the follicles and theca folliculi (black arrowhead). Marked mononuclear cellular infiltration (white arrowheads). GF: Graafian follicle; CL: corpus luteum. (c) Ovarian cortex depicting mature GF, CL appears formed of eosinophilic granulosa and theca lutein cells. (d) Growing follicles (arrowheads) are observed within the cortex. (a, c and d) Microscopic magnification x100, (b) ×400.

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Histomorphometric results

Areas of positive reaction to anti-VEGFA appeared within the stroma and corpus luteum cells, where VEGF is mostly freely secreted and can be found within tissue matrix or cell associated. Limited positive reaction was noticed in control group. Meanwhile, evidently increased reaction was demonstrated in all other groups. Anti-VEGF-A positive reaction increased significantly in ischemia (P = 0.0013) and I/R (P = 0.001) groups in comparison to control group, with higher expression in ischemia group versus I/R group (P = 0.0036). In addition, VEGF-A was up-regulated in PRP treated group as compared to untreated group (P = 0.003) [Figure 3].
Figure 3: Representative photomicrographs the anti-vascular endothelial growth factor-A immunohistochemically stained sections. (a) (control group) Limited pattern of positive reaction in form of brown color. (b) (Ischemia group) Apparently increased positive reaction within the stroma (arrowhead) and cells of corpus luteum (CL). (c) (IR group) Limited areas exhibit positive reaction. (d) (IR + platelet-rich plasma group) Apparently increased positive reaction is noticed. (a-d) Microscopic magnification ×100. (e) A histogram representing statistical analysis of the morphometric assessment of percentage area of positive reaction. * Significant versus control group, # Significant versus ischemia group; @ Significant versus I/R group; significant difference when P < 0.05 and results are represented as mean ± standard deviation.

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Biochemical results

Oxidant-antioxidant balance

Ischemia and I/R disturbed the oxidant-antioxidant balance in favor of ROS with subsequent oxidative stress. Ovarian MDA increased significantly in ischemia (P = 0.0021) and I/R (P = 0.001) groups in comparison to control group. However, TAC revealed a significant decrease in ischemia group (P = 0.046) and I/R group (P = 0.008) in comparison to control group. Reperfusion accentuated this disturbance with more oxidative stress. This was proved by the significant increase in MDA (P = 0.042) and the significant decrease in TAC (P = 0.003) in I/R group as compared to ischemia group [Table 1].
Table 1: Oxidative stress and antioxidant parameters in all studied groups

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Meanwhile, PRP treatment was able to scavenge ROS and to reduce the impairment in the ovarian antioxidant system. MDA was significantly decreased (P = 0.007) and TAC was significantly increased (P = 0.001) in I/R + PRP group in comparison to the untreated I/R group without return to their basal values in the control group [Table 1].

Inflammatory marker

Ovarian ischemia and I/R processes produced evident inflammatory response. TNF-α was significantly increased in ischemia (P = 0.001) and I/R groups (P = 0.001) in comparison to the control group without any significant difference between both groups (P = 0.2144). PRP treatment was effective to reduce this ovarian inflammatory marker significantly (P = 0.001) as compared to the untreated I/R group. TNF-α value in treated group was significantly lower than its value in the control group (P = 0.031) denoting a strong anti-inflammatory effect of PRP [Figure 4]a.
Figure 4: Statistical graphical presentation for ovarian tissue levels of tumor necrosis factor-alpha (a) and anti-Mullerian hormone (b). * Significant versus control group, # Significant versus ischemia group, @ Significant versus I/R group; significant difference when P < 0.05 and results are represented as mean ± standard deviation.

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Ovarian reserve marker

Ovarian AMH decreased significantly in the ischemia group (P = 0.0021) and I/R group (P = 0.003) in comparison to control group without any significant difference between I and I/R groups (P = 0.231). PRP increased ovarian AMH level in the treated I/R group, however this increase was not statistically significant in comparison to the untreated I/R group (P = 0.101) [Figure 4]b.

Correlation analysis

Regarding the correlation between oxidative stress and ovarian reserve affection, a significant negative correlation was found between MDA and AMH, while a significant positive correlation was found between TAC and AMH. Concerning the link between inflammatory process and ovarian reserve affection, a significant negative correlation was found between TNF-α and AMH [Figure 5].
Figure 5: Pearson correlations between anti-Mullerian hormone with oxidative stress and inflammatory parameters. (a) Negative correlation between anti-Mullerian hormone and malondialdehyde. (b) Positive correlation between anti-Mullerian hormone and total antioxidant capacity. (c) Negative correlation between anti-Mullerian hormone and tumor necrosis factor-α. Statistically significant correlation when P < 0.05.

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  Discussion Top


This study aimed to evaluate the efficacy of PRP to ameliorate the acute I/R-induced ovarian damage in rat model. It included control (sham operated), ischemia, I/R, and I/R + PRP groups. I/R model was done to simulate conditions of ovarian torsion treated surgically by detorsion. According to results from previous studies, 3-h period of torsion followed by a similar period of reperfusion was considered sufficient for I/R induction.[7],[25],[26]

Ovarian ischemia induces production of chemotactic factors that stimulate ovarian neutrophil infiltration with subsequent inflammatory response and oxidative stress. Its conservative treatment with detrosion to restore blood flow is associated with influx of more activated neutrophils thus enhancing the state of oxidative stress and inflammation previously associated with ischemia leading to more tissue damage.[27] This came in agreement with our results that revealed accentuated tissue degenerative changes and massive mononuclear cellular infiltration within the ovarian stroma in I/R group as compared to ischemia group. This was further confirmed biochemically by the significant increase in oxidative stress and inflammatory markers in both I and I/R groups in comparison to control group. Moreover, oxidative stress markers were significantly changed in I/R group in comparison to ischemia group as an indicator for more oxidative damage produced by reperfusion injury. In agreement with the hypothesis of reperfusion injury, previous studies have demonstrated the efficacy of anti-oxidants and anti-inflammatory treatment to ameliorate tissue damage in I/R animal models.[28],[29],[30],[31]

Following ovarian operations, an AMH level-based estimate of ovarian reserve is important for fertility preservation.[7] In this study, AMH level was significantly decreased in ischemia and I/R groups without significant difference between both groups. This came in accordance with our histological results showing apparently decreased growing follicles in ovaries of both groups. The oxidative stress and inflammatory process accompanying I/R are claimed to be the cause of this ovarian reserve affection. This was evident in our results that revealed significant negative correlations between both MDA and TNF-α with AMH, and significant positive correlation between AMH and TAC.

In the present study, AMH level was measured in the ovarian tissue homogenate rather than in serum. This is suggested to be more reliable and accurate during assessment of such acute insult of ovarian ischemia and reperfusion, whereas the changes in serum level of AMH would take a longer duration to occur. Similar studies[7],[32] with the same time scale found that ovarian ischemia reperfusion did not produce significant change in serum AMH and this was our rationale to measure AMH in ovarian homogenate not in serum. The considerable reduction in AMH following ovarian I/R implies that detorsion alone is insufficient to safeguard the ovaries. The use of treatments before or during the conservative surgery may share in ovarian reserve protection.

In the current study, VEGF-A was significantly up-regulated in ischemia and I/R groups when compared to control group; however it was significantly higher in ischemia group in comparison to I/R group. VEGF is well-known for stimulating angiogenesis and controlling endothelial function during ischemic conditions.[33] Hypoxia accompanying ischemic conditions triggers VEGF expression together with its receptors.[34] Accordingly, the encountered increase in our study is justified.

PRP is a source of growth factors that help tissue repair and healing.[12] The main advantage of PRP over other methods of growth factor administration is that it is a low-cost, easy-to-obtain with no risk of rejection or immunological reaction when it is autologous. Furthermore, PRP including leukocytes have been shown to have antibacterial activity, implying a minimal risk of infection.[35] In this study, the rationale behind our choice to inject PRP 30 min before reperfusion is to mimic real clinical situation. Herein, PRP is suggested to be used as a prophylactic measure before detorsion surgery if the patient is suspected of having adnexal torsion. Moreover, Karakaş et al.[36] reported protective effect of intraperitoneal PRP to reduce postoperative peritoneal adhesions which may be of benefit in our current clinical scenario. So, we decided to inject PRP intraperitoneally.

Intraperitoneal injection of PRP prior to reperfusion produced marked preservation of ovarian strucrure and function that was evident histologically and biochemically. Such observed amelioration is consistent with the regenerative power of PRP. During the early stages of an injury, PRP allows for local delivery of high concentrations of protective growth factors. Insulin-like growth factor-1, platelet derived growth factor, epidermal growth factor, and VEGF are all basic growth factors found in the α-granules of platelets.[37] The active secretion of these growth factors by platelets begins within 10 min after its activation by calcium gluconate and is completed within 1 h. Calcium gluconate helps degranulation of platelet granules.[38] This comes in agreement with our immunohistochemical results that revealed a significant increase in VEGF-A expression in the PRP treated group in comparison to the untreated group. VEGF has an important role in decreasing I/R injury in several organs through stimulation of angiogenesis and vasculogenesis.[39] PRP activation may be beneficial in this issue as demonstrated by Margono et al.[40] who found that calcium gluconate activation of PRP increased VEGF-A expression in human dental pulp stem cells.

Our results revealed also a strong anti-inflammatory effect of PRP evidenced by a significantly decreased TNF-α, together with histologically observed amelioration of vascular congestion and inflammatory cellular infiltration, after PRP treatment. The anti-inflammatory properties of PRP can be referred to its ability to suppress nuclear factor kappa enhancer of activated B cells (NF-κB) signaling pathways in macrophages.[41] Our results came in accordance with research work conducted by Rah et al.[42] demonstrating effectiveness of PRP on I/R injury in skin flap to decrease neutrophil recruitment and inflammatory cytokines. Also, Samy et al.[43] found that PRP was effective to lower TNF-α expression in rat testicular tissue in I/R model.

Moreover, our results showed that PRP was effective to reduce oxidative stress and enhance antioxidant defense mechanisms. The exact role of PRP to ameliorate oxidative stress has several explanations. Some earlier studies suggested that PRP may decrease the level of tissue ROS in I/R conditions, while some others referred to its ability to enhance the endogenous antioxidant capacity within the tissues. In accordance with these data, PRP was efficient to attenuate oxidative stress-induced nephrotoxicity by cisplatin in rat model and was found to increase renal GSH availability.[44] In contrast, the administration of PRP had no effect on testicular antioxidant defense mechanisms as stated by Samy et al.[43]

Regarding the effect of PRP on of ovarian reserve, our results revealed increased AMH level in PRP treated group in comparison to untreated group. This may be attributed to the protective effect of PRP against oxidative stress, inflammation and also to its role in angiogenesis. However, this difference did not follow a significant trend. Significant difference in AMH level may need more time to appear; however our study focused only on the acute effect not long-term effects. Another explanation may be that we relied on ELISA measurement of AMH. So, measurement of AMH by other biochemical methods such as reverse transcription polymerase chain reaction may be helpful in future studies. To the best of our knowledge, this is the first work to evaluate the effect of PRP on AMH level and ovarian reserve in ovarian I/R model. Dehghani et al.[45] reported that intraperitoneal injection of PRP in rat model of premature ovarian failure increased the count of healthy small antral follicles, and they suggested that PRP may protect against ovotoxic chemical substances. Our histological results were consistent with AMH assessment results, where growing follicles were more frequently observed in ovarian sections of PRP treated group than in untreated group.

A potential limitation in our experiment is the use of allogenic PRP. In clinical situation, autologous PRP is used. However, the rationale behind this was to provide a proper amount of blood to obtain a sufficient amount of PRP. Another limitation was that we focused only on the acute ovarian changes after I/R with absence of data related to long-term alterations after PRP administration. This may be a recommendation for a future study. Furthermore, we recommend studying effect of PRP when injected by routes other than intraperitoneal and compare the effects of different routes of PRP use.


  Conclusion Top


It can be concluded from the current study that PRP may help in preservation of ovarian structure and function during surgical conservative ovarian detorsion. These protective effects could be attributed to its ability to reduce oxidative stress, inflammation and also to its high content of VEGF. It is important to mention that a previous study[46] investigated the effect of PRP in ovarian I/R model, but it also worth to mention that our study measured more parameters as inflammatory marker and ovarian reserve marker to elucidate more protective effects of PRP which is important during moving from experimental to clinical application. Although many studies suggest that PRP may promote tissue regeneration, most of these studies are basic experimental animal studies with few clinical studies and this must be taken in consideration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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