Comparison of the roles of bilateral intraovarian platelet rich plasma (PRP) injection and ovarian mechanical damage during injection on the functional ovarian reserve in rats for which a premature ovarian insufficiency (POI) model was created

OBJECTIVE: Premature ovarian insufficiency (POI); amenorrhea observed under the age of 40 is a gynecological pathology characterized by loss of ovarian functions and residual follicles accompanied by hypergonadotropism – hypoestrogenism. Various methods are used to increase ovarian reserve in premature ovarian failure, which causes many health problems. Intraovarian injection of platelet-rich plasma (PRP), which is known to have effects such as tissue regeneration, wound healing and skin regeneration, is one of the methods claimed to increase ovarian reserve. On the other hand, another method claimed to increase ovarian reserve is iatrogenic mechanical damage to the ovarian tissue. In this method, it is claimed that the result is mediated by the mechanical signals of the primordial follicles and the biochemical factors caused by these signals. Follicle-stimulating hormone (FSH) and anti-mullerian hormone (AMH), the two main regulatory hormones of follicle development, are used clinically as biomarkers about the ovarian follicle. When the levels of these two hormones are irregular, it results in unwanted reproductive disorders. With the POI model created in rats in this study; In one group, intraovarian PRP injection, which is claimed to increase ovarian reserve, and mechanical damage to the ovary during this injection, and in the other group, ovarian mechanical damage caused by the injector used for iatrogenic PRP injection; It was aimed to compare the rates of increasing functional ovarian reserve, FSH and AMH levels measured by the enzyme-linked immunosorbent method (ELISA) in the blood of rats, and the number of oocytes and follicles and the degree of fibrosis in the ovarian tissue by histological examination. METHOD: Our study was carried out at Dokuz Eylül University Experimental Animal Laboratory between July 2023 and October 2023. Approval was received from the Dokuz Eylül University Multidisciplinary Laboratory Animal Experiments Local Ethics Committee. Whistar Albino female rats weighing 180-220 g (8-10 weeks old) were included in our study. Subjects were randomly divided into four groups. Group A control group [1 mL/kg intraperitoneal single dose of physiological saline containing 0.9% sodium chloride (NaCl)], group B premature ovarian failure (POF) group, POF model creation (150 mg/kg intraperitoneal single dose cyclophosphamide); Group C POI+PRP+MOD group, POI model created and intraovarian PRP injection and mechanical ovarian damage (MOD) during injection (550,000 platelets / 200 μl physiological saline), D group POI+MOD group, POI model created and MOD (15th day with insulin injector). ) was created. In rats in which a POI model was created, 15 days were waited after intraperitoneal cyclophosphamide injection for maximum effect. Eight weeks after PRP injection was applied to the rats in group C and MOD was performed on the rats in group D; FSH and AMH levels of rats in all groups were measured and compared. In order to create platelet-rich plasma, blood was taken intracardiac from 3 rats with the help of a syringe. After obtaination, PRP was injected into the rats that would receive bilateral intraovarian injection, with the help of an insulin injector, at a dose of 550,000 platelets/200 μl physiological saline, by four penetrations. The same number of penetrations as the number of penetrations with the insulin syringe were applied to group D rats. Anesthesia of the rats was provided with ether. For analgesia, intraperitoneal parol was used only in rats in groups C and D. After the experiment was completed, blood was taken intracardiacly with the help of a syringe to study serum hormone levels, the animals were sacrificed and histological examination was performed. Tissue sections were evaluated with an Olympus BX 51 light microscope by histochemical and immunohistochemical staining. Grading of fibrosis in the ovarian tissue was made with Masson's Trichrome stain. Avidinbiotin-peroxidase method (ABC method) was used for immunohistochemical examinations. RESULTS: The results of a total of 20 Whistar albino rats were examined. Serum FSH value was found to be significantly different only between the SF group and the EOY group; There was no significant difference between the SF group and the EOY+PRP+MOH, EOY+MOH groups. Again, no significant difference was detected between the EOY+PRP+MOH and EOY+MOH groups. Finally, serum FSH/AMH level ratios did not differ significantly between any groups. When examining the preparations stained with Hematoxylin & Eosin, the number of primordial follicles and secondary follicles do not show a significant difference between the groups. While the number of primary follicles was calculated significantly differently between the EOY group and the EOY+MOH group, it did not show a significant difference between the other groups. While the number of Graafian follicles was calculated to be significantly different between the SF group and the EOY+PRP+MOH group, it did not show a significant difference between the other groups. Atretic follicle numbers; while EOY+PRP+ was calculated significantly different between SF group and EOY group, SF group and EOY+PRP+MOH group, SF group and EOY+MOH group, EOY group and EOY+PRP+MOH group, EOY group and EOY+MOH group, no significant difference was calculated between the MOH group and the EOY+MOH group. A loss of normal histological appearance was observed in the ovarian sections of the EOY group compared to the SF group. When the sections belonging to the EOY+PRP+MOH group are examined; an increase in the number of primordial and primary follicles was observed compared to the EOY group. Although there was an increase in the number of normal-appearing follicles of different numbers and sizes compared to the EOY group, the normal histological appearance was not fully preserved compared to the SF group; an increase in the appearance of corpus luteum and atretic follicles was observed compared to the SF group. A decrease in the number of atretic follicles was observed compared to the EOY group. An increase in healthy follicles at different stages was observed in the ovarian sections of the EOY+MOH group, similar to the EOY+PRP+MOH group. It was observed that there was a decrease in the number of atretic follicles compared to the EOY group. When looking at the H score distribution of Masson Trichrome staining according to groups; the presence of fibrosis in the EOY group was found to be statistically significant compared to the EOY+PRP+MOH and SF groups (p=0.04; p=0.00). Although the collagen increase in the EOY+MOH group was significant compared to the SF group (p=0.06) and fibrosis decreased in the EOY+MOH group compared to the EOY group, this difference was not statistically significant (p=1). Caspase-3 immune reactivity was examined in the SF group. Caspase 3 positivity was observed at basal level in granulosa cells and more specifically in the theca interna. A weak immunopositivity for Caspase-3 was also occasionally observed in primary oocytes located in follicles at different stages of development. A stronger staining was observed in corpus luteum cells than in granulosa cells. When Caspase-3 immune reactivity was examined in the ovarian sections of the EOY group, the number of positive cells increased in all areas; it was observed that immune positivity was evident in the granulosa cells and theca interna, oocytes and corpus luteum of follicles at different developmental stages. However, this increase was not statistically significant (p = 0.15). When Caspase-3 immune reactivity was examined in the ovarian sections of the EOY+PRP+MOH group, a weaker staining was observed in the granulosa and corpus luteum cells and oocytes compared to the EOY group, and this decrease in immune positivity was statistically significant (p=0.00). Compared to the SF group, the number of immune positive cells was similar in the follicles, but was less evident in other areas, but this decrease was not statistically significant (p = 1). In the EOY+MOH group, it was observed that the number of immune positive cells decreased when compared to the EOY group, and immune positivity increased when compared to the SF and EOY+PRP+MOH groups. However, these changes were not statistically significant (p=0.44; p=1; p=0.57). Ki-67 immune reactivity was examined in the SF group. Although more prominent in granulosa cells, immune positivity was also observed in other cells in the cortex. A weaker staining was observed in corpus luteum cells compared to granulosa cells. It was observed that the number of immune positive cells in the EOY group decreased in some samples, and was similar to the SF group in some samples. Again, immune positivity was more evident in the granulosa cells of follicles at different developmental stages compared to other cells. However, this decrease was not statistically significant (p = 1). A stronger staining was observed in the cells in the entire cortical area in the EOY+PRP+MOH group compared to the SF and EOY groups, and this increase in immune positivity was statistically significant compared to both groups (p=0.02; p=0.02). Although there was a tendency for an increase in the number of immunopositive cells in the EOY+MOH group compared to the EOY group, the immunohistochemical findings were similar and there was no statistically significant difference between the two groups (p = 1). Although the number of immunopositive cells decreased significantly compared to the EOY+MOH group and the EOY+PRP+MOH group, this decrease was not statistically significant (p=0.17). DISCUSSION: When rats were compared for the isolated effect of mechanical ovarian damage during intraovarian PRP administration, in the EOY+MOH group compared to the EOY+PRP+MOH group; Although a decrease in the number of primordial follicles, an increase in the number of primary follicles, a decrease in the number of secondary follicles, an increase in the number of graafian follicles, and a decrease in the number of atretic follicles were observed, these differences were not found to be statistically significant (p = 1 in all results). Serum FSH and AMH levels increased but were not found to be statistically significant (p=0.99, p=1). The H score increased in fibrosis scoring with Masson Trichrome, but it was not statistically significant (p = 0.58). Immunohistochemically, proliferation was observed to be less in Ki-67 staining, but it was not found to be statistically significant (p = 0.17). Immunohistochemically, staining with Caspase-3 showed that apoptosis was higher, but it was not statistically significant (p = 0.57).