The Effects of Leukocyte- and Platelet-Rich Plasma (L-Prp) and Pure Platelet-Rich Plasma (P-Prp) an a Rat Endometriosis Model

Our aim was to investigate the effect of platelet-rich plasma (PRP) derivatives, which can be produced from the patient’s own blood and have minimal side effects, on endometriosis. To the best of our knowledge, this is the first study in the literature that studies the relationship between PRP and endometriosis. Endometriosis foci were created in the first operation. In the second operation (30th day) groups were formed. Group 1 (n= 8) was administered saline, group 2 (n= 7) leukocyte- and platelet-rich plasma (L-PRP), and group 3 (n= 8) pure platelet-rich plasma (P-PRP). Group 4 (n= 10) was used to obtain PRP. In the last operation (60th day), the endometriotic foci were measured, and then excised. There was no statistically significant difference between the pre and post volumes of the endometriotic foci, between their volume differences and volume difference rates (p > .05). However, it was observed that existing implant volumes in all groups decreased statistically significantly within their own groups by the end of the experiment compared to the previous volumes (p < .05). When the implants were assessed through histopathological scoring in terms of edema, vascular congestion, inflammatory cell infiltration, hemorrhage, epithelial line, and hemosiderin accumulation and immunohistochemical staining in terms of VEGF, there was no significant difference in the comparison between the groups. Although L-PRP and P-PRP generated more reduction in the endometriosis foci, they did not create any statistical differences.


Introduction
Endometriosis, which is described as the presence of endometrial gland and stroma 49 outside the uterine cavity, is an important women's health problem seen in 6-10% of women 50 that causes degradation in the quality of life with clinical effects, such as infertility, 3 51 dysmenorrhea, dyspareunia and chronic pelvic pain [1][2][3][4] . Its pathophysiology has not yet been 52 fully resolved, and an effective treatment for it has not yet been found 5,6 . 53 Research has shown that cytokine levels rise in the peritoneal fluid of endometriosis 54 patients 7 . In patients with endometriosis, an angiogenetic activity of peritoneal fluid and 55 increased levels of vascular endothelial growth factor (VEGF) are observed 8,9 . In various 56 experimental studies in the treatment of endometriosis, endometriotic foci have been found to 57 shrink and VEGF levels have been found to decrease 10,11 . 58 The healing properties of platelet-rich plasma (PRP) and platelet-and leukocyte-rich 59 plasma in tissues have also been subject to numerous research studies in recent years. This 60 plasma contains a high proportion of platelets. Platelets are also known to contain many 61 growth factors. Platelet-derived growth factor (PBGF), transforming growth factor beta (TGF-62 B), epidermal growth factor (EGF), insulin-like growth factor (IGF) and vascular endothelial 63 growth factor (VEGF) can be counted among these factors 12, 13 . With such features, PRP can 64 show positive effects on many systems. Such effects of it include many systems such as scalp, 65 skin, heart, bones, cartilage, tendons, liver, kidney, genital tract, ovaries, endometrium and 66 infertility treatments [14][15][16][17][18][19][20] . PRP can be in two different forms: L-PRP (i.e., leukocyte-and 67 platelet-rich) and P-PRP (leukocyte-poor or pure platelet-rich). Although they are similar 68 products, their contents such as cytokines and growth factors are different. L-PRP has a 69 higher proportion of leukocyte, TNF-a and IL-1β concentration 21 . To the best of our 70 knowledge, there is no study in the literature investigating whether PRP administration 71 increases or decreases endometriosis. 72 Our aim was to investigate the effect of two forms of PRP (L-PRP and P-PRP) on 73 endometriosis, which had never been administered in endometriosis, but was known to be 74 effective in many areas. Turkey), and each abdomen was entered through a 5-cm vertical incision. As defined by 87 Vernon and Wilson, foci were formed by implanting the part taken from the rat uterus to the 88 abdominal wall through a surgical intervention using Uygur's modification 22,23 . To do that, a 89 .5 cm section of the left uterine horn was excised at a distance of 1 cm from the ovary. The 90 remaining uterine horn was sutured with 2/0 polyglactin absorbable suture, and the bleeding 91 was controlled. The tissue that was taken was cut longitudinally and sutured without 92 separating the myometrium into the right abdominal peritoneal inner surface with 5/0 93 polypropylene non-absorbable suture by placing the endometrial portion inward, and an 94 implantation was achieved (to ensure the endometriosis model) (Fig 1). The implants were  The rats were divided into 3 groups with random selection:   Group 3: Pure platelet-rich P-PRP group (n= 8): 0,1 cc P-PRP was applied on the implant.

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A total of 10 rats were decapitated after the blood was drawn for the preparation of the 118 heterologous PRP. All the injections were applied once on the lesion in all groups. After that, 119 the abdomen was closed by suturing the peritoneum, fascia and skin with 4/0 polyglactin. A 120 rat in Group 2 died 3 days later, and there were 7 rats remaining in the group. A laparotomy was performed for the third and last time, for final assessments 1 month 123 later. In the last 5 days, vaginal smears were sampled from all rats to assess estrogen cycle.

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The cycle status was assessed by microscopic examination through the Papanicolaou staining 125 method. The vaginal smears were taken in the form of swabs from the vaginal wall by using a 126 cotton brush. The estrogen cycle was determined by the cornification of the cells formed by 127 the estrogen effect and loss of leukocytes 24 . The rats that were in their cycles were selected.

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The pre-operative anesthesia and cleaning were performed as before. The abdomen was 129 entered through the previous incision line. The endometriosis foci were measured by the same 130 researcher using the same method (the prolate ellipsoid formula) as stated above, blindly by 131 not knowing which group the foci were in. After that they were excised. Then, the rats were 132 decapitated (cardiac excision) and were destroyed by red medical waste bins. The tissues that 133 were excised were sent to the laboratory within 10% formaldehyde for histopathological and  whole blood from five rats was centrifuged at room temperature for 10 minutes at 250 g, and 7 147 it was ensured that the blood was separated into three layers: Erythrocytes at the bottom, 148 buffy coat in the middle (rich in platelets, leukocytes and fibrinogen), and plasma containing 149 platelets at the top. The platelets-containing plasma and buffy coat were later transferred into 150 a new tube. A large portion of the platelets, leukocytes, and fibrinogen was re-centrifuged for 151 10 minutes at 1000 g to form precipitate. The supernatant plasma was thrown away, and the 152 precipitated platelets were re-suspended in the residue plasma to obtain L-PRP 25,26 .  Immunoreactive cells were recorded during the immunohistochemical examination for 197 VEGF with the following scoring: 0=negative staining, 1= < 33% positive staining, 2= 33-198 66% positive staining, 3= > 66% positive staining (Fig 2). The immunohistochemical staining 199 was evaluated by the same histologist blindly by a semi-quantitative method using the H-  Table 1. Considering this table, there is no statistically significant difference between 210 the groups (p > .05). However, it was observed that existing implant volumes in all groups 211 decreased statistically significantly within their own groups by the end of the experiment 212 compared to the previous volumes (p < .05) (Fig 3). When the implants were assessed through 213 histopathological scoring in terms of edema, vascular congestion, inflammatory cell 214 infiltration, and fresh hemorrhage, there was no significant difference in the comparison 215 between the groups in terms of the total score that was obtained (Table 1). The data are given as average ± standard deviation. *Total score: Edema + vascular     Endometriosis, which has an important place in female infertility, and whose treatment where estrogen was not used, the volume after the treatment was significantly less compared 261 to the control group in resveratrol and leuprolide acetate groups 10 . We also found in our study,

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where we did not administer estrogen, that implant volumes in PRP groups were significantly 263 decreased after the treatment (p < .05). However, this significant decline was similarly present 264 in the control group, and the rate of volume change did not show any significant difference 265 between the groups (Table 1).

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Another parameter that is compared in endometriosis studies is the histopathological  (Table 2). However, the point is that in the L-PRP group, the epithelial score was 0. There 273 were no cells. L-PRP had almost destroyed the endometrial foci. Nevertheless, this 274 circumstance had not been reflected in the accumulation of hemosiderin. It was similar in 275 every group (Table 2). Hemosiderin is a significant indicator in the assessment of 276 endometriosis 33,34 . In other words, it seems that the endometrial focus examination should not 277 be carried out based on a single factor. We also assessed the inflammatory changes in 278 histopathological scoring as a total score in our study but did not observe any differences 279 between the groups (Table 1). administered has been shown to decrease 25  with different L-PRPs and P-PRPs 52,53 . In our study, although the foci got smaller with P-PRP 315 and L-PRP, we cannot say that PRP has a therapeutic effect on endometriosis since this 316 reduction was also seen in the control group, and the difference in the reduction of volume simultaneously application of them with repeated doses at certain intervals could result in 323 different and effective results. We can think of a limitation of our study that we did not 324 histopathologically examine the similar implants in the first month in which the foci were 325 found to have been formed. They have not been examined in many studies, either. We also 326 performed an endometrial implantation, as in most past studies 22,23 . To the best of our 327 knowledge, our current study is the first study of the relationship between PRP and 328 endometriosis, which can be considered as a preliminary study. Although our results were not 329 significant, it was promising that the PRP foci did not grow, and they did not stay the same - In conclusion, the endometriosis foci were shrinking over time. This reduction was 335 observed in all groups and was significant. However, the shrinking of endometriosis foci did 336 not show any statistically significant difference among the groups. Moreover, there was no 337 difference between the groups in terms of epithelial score, hemosiderin deposits, VEGF and 338 total score. In other words, although both L-PRP and P-PRP generated more reduction in the 339 endometriosis foci, they did not create any statistical differences.