Comparative Study of Bone Marrow and Adipose Tissue-Mesenchymal Stem Cells In The Treatment of Cyclophosphamide Induced Ovarian Damage In Adult Female Albino rats.

Talaat M. Mohamed, Gamal Elsayed Abd- Salam, Kamal M. Kamal, Eman El Bana, Mohammed A. Gebba Anatomy and Embryology Department, Faculty of Medicine, BenhaUniversity, Egypt [email protected]

Abstract: Background: Cyclophosphamide (CLP), used as an anticancer therapy , One of its side effects in women is through inducing ovarian toxicity and consequently infertility Aim of work: to evaluate the impact of BM- MSCs&AT-MSCs in the overcome of cyclophosphamide-induced ovarian damage in rats. Materials&Methods: Forty female adult Albino rats were randomly divided into four groups. Group I (control group). Group II (cyclophosphamide treated group) were subjected to intra-peritoneal injection of 150 mg/kg- B.W, followed by a dose of 8 mg/kg every week, the rats were scarified after 3 weeks. Group III (BM-MSCs treated group) were treated by cyclophosphamide in the same manner as in group II and after five weeks from the second CLP injection 100 μl of BM-MSCs mixture (2.5x106cells) in 100 µl PBS was injected intra-venous. Group IV(AT-MSCs treated group) were treated by CLP as in group II and after 5 weeks from the second CP injection, 100 μl of AT-MSCs mixture (2.5x106cells) in 100µl PBS was injected intra-venous, Then 30 days after cell therapy the rats were scarified, Ovarian sample sections were stained with different stains, H& , M on T ic o e n Immunohistochemical stains for anti-PCNA antibody. The average number of primordial germ follicles, % of fibers, area % of +ve immune-reactivity area for PCNA were determined by histomorphometric tests and analyzed statistically. Results: Histological examination of the of CLP treated group revealed severe degeneration of ovarian follicles with sloughing of granulosa cells. The ovarian follicles treated with BM-MSCs showed a normal appearance of in comparison to the cyclophosphamide-group. The AT-MSCs treated group rats showed relative improvement of oogenesis but no graafian follicle was detected. Conclusion: BM-MSCs and AT-MSCs were impressive in the therapy of ovarian failure in albino rat model where they repair the ovarian structure and protected against CLP-induced toxicity. However BM-MSCs more effective [Talaat M. Mohamed, Gamal Elsayed Abd-Salam, Kamal M. Kamal, Eman El Bana, Mohammed A. Gebba.Comparative Study of Bone Marrow and Adipose Tissue-Mesenchymal Stem Cells in The Treatment of Cyclophosphamide Induced Ovarian Damage In Adult Female Albino rats. Nat Sci2019;17(12):267-283]. ISSN 1545-0740 (print); ISSN 2375-7167 (online).http://www.sciencepub.net/nature. 33. doi:10.7537/marsnsj171219.33.

Keywords: Bone marrow, Adipose tissue, Mesenchymal stem cells, Cyclophosphamide, Premature ovarian failure. 1. Introduction Furthermore, metabolic diseases represented in Cyclophosphamide is a chemotherapeutic classic galactosemia, mumps, toxins, virus anticancer agent widely used in the treatment of many infections[e.g. Human Immunodeficiency types of malignant tumors, however it has a potent Virus(HIV)], and lifestyle factors(e.g. cigarette ovarian toxic effect in the form of: gonadal damage, smoking)which are accompanied with the initiation of ovarian follicular fibrosis associated with insufficiency POF (7) Though, the maximal of cases still idiopathic in the that leads to a significant (8). reduction in the total number of ovarian follicles (1) of A suitable therapy for some women suffering the most serious side effects of cyclophosphamide is from menopausal symptoms is the hormonal premature ovarian failure (2). replacement therapy (9) (Tan et al., 2010), however Hypergonadotropic secretion from the gonads is its risks that include cardiovascular disease, stroke, the main cause of premature ovarian failure (POF) breast and endometrial cancer (10). accompanied with cessation of menses early before Introduction of stem cell therapy is a promising 40years old(3).About 0.01 % of women experience tool for overcoming of sever disordered particularly, POF before age of 30, while 1% - 3% of women mesenchymal stem cells (MSCs) which play an practice POF before 40years old (4). important role in the field of regenerative medicine Infertility remains a significant issue for most where it characterized by their capability for multiline women undergoing POF (5) 25% of all POF forms age differentiation and self-renewal (11). MSCs are nowadays, can be divided as iatrogenic and connected characterized as undifferentiated cells, with high to tumor therapy such as radiotherapy or proliferative capacity, and possess a mesodermal chemotherapy (6). differentiation potential (12).

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Another alternative source of stem cell is the t wit Dulbecco’ o ifie gle’ e iu adipose tissue (AT), which can be harvested easily , (DMEM, GIBCO/BRL) supplemented with 10% fetal non-invasive technique and with sufficient amounts bovine medium (GIBCO/BRL). Nucleated cells were than bone marrow (BM) (13). Some authors isolated with a density gradient [Ficoll/Paque established that AT encloses stem cells comparable to (Pharmacia)] and resuspended in complete culture BM-MSCs (14). medium supplemented with 1% penicillin The most generally applied origins of MSC is streptomycin (GIBCO/BRL) (22). from BM and AT , due to ease of harvest and potential Cells were incubated at 37°C in 5% humidified autologous application (15),which type of cells is CO2 for 12-14 days as primary culture or until more preferable and appropriate for stem cell formation of large colonies. The resulting cultures treatment is still a remarkable question which waiting were referred to as first-passage cultures. MSCs in for answered (16). culture are characterized by their adhesiveness and Several researches have demonstrated that AT- fusiform shape. On day 14, the adherent colonies of MSCs and BM-MSCs possessing the same characters , cells were identified as being MSCs by their involving the expression and the morphologic pictures morphology and adherence (23). of cell surface antigens (17), while some researchers 4) Isolation and culture of rat adipose-tissue recorded an important differences in the biological derived mesenchymal stem cells: activity regarding their differentiation capacities and Rats were anesthetized by injection of Xylazine proliferation rates (18), whereas, some investigators and Ketamine; 1-2cm³ of preperitoneal adipose tissue contradict results, such as AT-MSCs is more efficient was removed. The yellowish white tissue was minced concerning possibility of the clinical application than and enzymatically digested in DMEM (Dulbecco’ BM-MSCs, however other authors established that o ifie gle’ e iu ) containing 0.075% BM-MSCs are preferable greatly than AT-MSCs (19). collagenase2 at 37ºc for 60 min. The cell suspension 2. Materials and Methods was filtered with 70 µm sieve. The cells were A-Material: resuspended in MEM medium supplemented with 1% 1) Albino rats: penicillin/streptomycin and 15% FBS (fetal bovine The experimental study was conducted on 40 serum). Erythrocytes and other non –adhesive cells adult female albino rats 10 weeks old, their average were removed from the culture (24). weight was 200-220 grams, they were divided into The cells were collected by centrifugation, and four groups. The rats were kept in experimental animal subcultured at 1:3- 1:4 ratio and were counted by unit, Kasr El Einy University and they were trypan blue by their shape (a typical -like maintained in a controlled environment with access to spindle shape). The adherent AD-MSC were expanded food and water. The rats were obtained from Holding and either assessed for gene expression and flow company for biological products and vaccines cytometry, or induced to differentiate toward the (Vacsera), Handling of the animals will be approved chondrogenic, adipogenic and osteogenic lineages. by the care of experimental animal committee of The blue staining of the cells after mixing (1:1) will be Benha medical university and is in compliance with used as indicator for cell death (25). the national research council's guideline. Ethical 5)Stem cell preparation and examination were done at protocols for animal treatment will be followed. Unit of Biochemistry and Molecular Biology, 2) Drug and chemicals: Biochemistry Department, Faculty of Medicine, Cairo Cyclophosphamide (Endoxane; Baxter; Deerfield, University. Illinois, USA): (B) Methods: Used as 1 gram vial (dry powder), the required Forty adult albino female rats were divided into dose for each rat weighed with the drug being equal four groups, each group include ten rats as dissolved in 0.9% saline; the female rats were injected following: intraperitoneally with cyclophosphamide, each rat was 1) Group I (Control Group) included 10 rats held in one hand in the dorsal position, that were subdivided into: cyclophosphamide was injected into the caudal - Group Ia(negative control group):included abdominal cavity using a sterile needle (20) Each rat of 5 rats that were chosen randomly, and not received was injected with single dose of cyclophosphamide any drugs. intraperitoneal in a dose of 150 mg/kg -b.w, followed - Group Ib(positive control group): included by a dose of 8 mg/kg every week, for 3 weeks (21). of 5 rats that were injected with PBS for sacrificed 3) Isolation and culture of rat bone marrow after 3 weeks. derived mesenchymal stem cell: 2) Group II (Cyclophosphamide group): Bone marrow was harvested by flushing the included 10 rats were injected with single dose of tibiae and femurs of 6 weeks old male white albino cyclophosphamide intraperitoneal in a dose of 150

268 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ mg/kg b.w of rats., followed by a dose of 8 mg/kg antibodies. CD44 antibody is a mouse monoclonal every week for 3 weeks., the rats were sacrificed to antibody clone 2A4 (30). obtain their ovaries for histopathological examination CD44-positive cells (20) showed brown (21). cytoplasmic deposits (Lab Vision Corporation, 3) Group III (BM-MSCs group) bone Neomarkers Laboratories, Westing House, and marrow mesenchymal stem cells group: included 10 Thurmont, California, USA). rats were injected with cyclophosphamide as group II. IV- Morphometric study (By Image analysis) After 3 weeks, these rats were injected with BM- Slides were examined using computerized image MSCs intravenous through the tail veins in a single analyzer (Lecia Imaging System Ltd., Cambridge, dose (5× 106 cells/ml) in a volume of 0.3 ml of England). Images were captured live on the screen 0.1mol/l PBS (pH 7.4).30 days after injection of stem from sections under a light microscope (Olympus BX- cells, (26) the rats were sacrificed after 30 days from 40, Olympus Optical Co. Ltd., Japan) with a fixed cell therapy. video camera. From H &E. sections of different 4) Group IV (AT-MSCs group) adipose tissue experimental groups the number of different types of mesenchymal stem cells group: included 10 rats ovarian follicles was counted (24). were injected with cyclophosphamide as group II. 1) The Primordial follicles: were counted in After 3 weeks, these rats were injected with AT-MSCs each slide using the interactive measuring menu with intravenous through the tail veins in a single dose an objective lens of magnification 20 i.e. of total (5× 106 cells/ml)in a volume of 0.3 ml of 0.1mol/l magnification 200. Ten readings were obtained for PBS (pH 7.4).30 days after injection of stem cells, the each specimen of all subgroups&the mean values were rats were sacrificed (24). obtained.(26). At end of the experiment, The rats were 2) The other types of follicles (primary, anesthetized with 50mg/kg ketamine and 10mg/kg secondary, graafian and atretic follicles): were Xylazine before scarification by oblique incision in counted in each slide using the interactive measuring lower abdominal wall to reach both ovaries by menu with an objective lens of magnification 10(26). removal of broad of the ,. In each rat, 3) Mean area% of collagen fibers: was the right ovary was removed in its entirety, weighed, e u e in t e M on’ T ic o e-stained sections fixed in 10% formaldehyde, for histomorphometrical using the color detect menu. The image analyzer was evaluation and the whole left ovary was preserved for used to measure the area of collagen fiber content and the biochemical studies. Paraffin sections were was expressed in an area in relation to a standard prepared by dehydrating ovaries in ascending grades measuring frame. This was done in 10 microscopic of alcohol (50%, 70%, 90% and 100%). Three samples fields for each rat and their mean was obtained. i.e. of of (5 ml each) were mixed, pooled and total magnification 100(24). centrifuged at 4000 g for 5 min. The supernatant was 4) Mean area % of PCNA: was measured in filte e (0.22 μ po e ize) to eli in te cont in ting the immunostained sections were counted in each slide cells potentially present in follicular fluid, aliquoted using the interactive measuring menu with an n to e t −20°C until use (27). objective lens of magnification 20(24). ♦Histopathological examination: V-Statistical analysis: After the slides deparaffinized, and both ovaries The mean values of the data obtained from the were obtained and prepared for: image Analyzer were calculated and compared 1- Light microscope examination: by using statistically using the statistical package for social hematoxylin and eosin stain (H&E) was used for sciences (Windows, version 9; SPSS Inc., Chicago, routine histological examination (28) Masson's Illinois, USA). Difference between groups under study Trichrome stain for detection of collagen fibers (29). were examined for statistical significance for the 2- Immunohistochemical stain: staining for various parameters using the analysis of variance test. PCNA (Proliferating cell nuclear antigen) using the This test is used to find a significant difference avidin–biotin peroxidase complex technique for between more than two groups. A P value less than detection of specific nuclear proteins (29). 0.05 was considered significant. Data were tabulated 3- CD44: the marker for mesenchymal stem and represented graphically (31). cells (20) The (Avidin-Biotin Complex) ABC 3. Results: technique was carried out as follows for the I- Histological results: expression of CD44. Paraffin sections were Haematoxyline and Eosin (Hx. &E.) stain: deparaffinized and hydrated. After blocking the Group I (control group): endogenous activity of peroxidase using10% hydrogen Examination of rat ovarian specimens of the peroxide, the sections were incubated with primary subgroups control female rats showed normal histological structures .The surface of the ovary was

269 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ covered with germinal epithelium consisting of single vacuolated cytoplasm were seen, they were covered by layer of simple squamous epithelium; dense normal germinal epithelium (fig. 6). Sections showed layer and tunica albuginea beneath also degenerated unilaminar primary follicle. It had the epithelium. The cortex that was full of growing degenerated oocyte with pyknotic nucleus and follicles and . of ovary between vacuolated cytoplasm (Fig. 5). the follicles had dense appearance consisting of loose Group III (BM-MSCs group): connective tissue which contained fibroblast like cells Ovarian sections of these rats revealed good (Fig. 1). Primordial follicles appeared under tunica improvement as indicated by relatively normal ovarian albuginea, the oocytes are enclosed by one layer of structure. Sections showed multiple relatively normal flattened squamous follicular cells (Fig. 1). Primary growing ovarian follicles, mature graafian follicle and follicles consisted of an enlarged oocyte with zona corpora lutea within the cortex of ovary. In the central pellucida surrounding it. Granulosa cells which are zone of ovary, medulla appeared consisting of loose polyhedral cells surrounding the oocyte. These cells connective tissue containing blood vessels (Figs. 7,8). were arranged in several layers forming multilaminar Some degenerated ovarian follicles still appear (Fig. primary follicle. Flattened cells of 7). Mature Graafian follicle consisted of normal appeared surrounding the follicle (Fig.1,3). Secondary oocyte surrounded by corona radiata cells. The follicle (Antral) follicles appeared consisting of an oocyte was surrounded by zona granulosa cells. The oocyte surrounded with well-defined . Several was attached to the wall of the follicle by cumulus layers of polyhedral cells surrounding the oocyte, oophorus. The cavity within the follicle was full of forming granulosa cell layer. These follicles had wide liquer folliculi. Flattened cells of theca externa cavities full of liquor folliculi (Fig. 2,3). surrounded the follicle (Fig.7). Sections also showed Mature Graafian follicles appeared consisting relatively normal multilayered primary follicle. It of an enlarged oocyte surrounded by clear zona consisted of an enlarged oocyte surrounded by well- pellucida present at one side of the follicle. Single defined zona pellucida, follicular granulosa cells layer of columnar cells was surrounding the ovum which were formed of multilayer of polyhedral cells forming corona radiata. Wide cavity full of liquor and peripheral fusiform theca folliculi cells (Fig. 8). folliculi appeared within the follicle. The wall was Group IV (AT-MSCs group): lined by multilayer of polyhedral cells of granulosa Ovarian sections of this group revealed mild cells. The ovum was connected to the wall of the improvement, and the degenerative changes were still follicle by .The flattened cells of evident. Sections showed single relatively normal theca externa were seen surrounding the follicle (Fig. growing follicle and corpus luteum (fig.9). Sections 4) . Corpus Luteum appeared polygonal shaped also showed relatively normal primordial germ vesicle granulosa lutein cells which contain eosinophilic each composed of an oocyte enclosed by one layer of cytoplasm surrounded by Theca lutein cells (smaller in flattened follicular cells (fig. 9). Other section showed size), the vascularity is normal (Fig. 1,3), the medulla relatively normal secondary follicle. It consisted of contained loose connective tissue with and relatively normal oocyte surrounded by clear zona few smooth muscle cells. No congestion or dilatation pellucida and relatively normal granulosa cells. Some of blood vessels and lymphatics were found in the granulosa cells were still vacuolated. Cavities medulla. appeared within the follicle (fig. 10). Group II (cyclophosphamide treated rats): Masson's Trichrome stain (M.T): Ovarian sections of these rats revealed marked Group I (Control group) showed degenerative changes. They revealed multiple Ovarian sections of both subgroups control rats markedly degenerated follicles within the cortex, and revealed very low collagen fibers appearing green in medulla appeared degenerated having multiple color surrounding the ovarian follicles and within the vacuoles with markedly congested blood vessels. ovarian stroma (Figs.11, 12). Group II Deposition of hyaline material in the interstitial tissue (cyclophosphamide treated group): ovarian sections of was noticed (Figs.5,6). The degenerated follicles had the control subgroups revealed massive increase of degenerated oocytes with Pyknotic nuclei and collagen fibers surrounding the ovarian follicles and vacuolated cytoplasm. Granulosa cells of these around congested blood vessel. Some vacuoles (V) degenerated follicles appeared vacuolated, appeared within ovarian stroma (Fig.13). Ovarian disorganized with pyknotic nuclei. Also stroma cells sections of Group III (BM-MSCs) revealed marked appeared vacuolated (fig.5,6). Some of these granulosa decrease of collagen fibers surrounding ovarian cells were exfoliated within the center of the follicles. follicle and within the ovarian stroma (Fig.14). Interstitial hemorrhage (Fig.6). The cortex showed Ovarian sections of Group IV (AT-MSCs) of this disfigurement of follicles which are markedly group revealed mild to moderate decrease of collagen degenerated primordial follicles with faint nuclei and

270 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ fibers surrounding ovarian follicles and within the Sections of BM-MSC treated ovarian tissue have ovarian stroma (Fig.15). shown positive cell membranous reaction for CD 44 in Proliferating cell nuclear antigen (PCNA) the form of brown pigmentation of cell membrane and immunostaining: cytoplasm of granulosa cells (Fig.21). Sections of AT- Ovarian sections of the control subgroups MSC treated ovarian tissue have shown positive cell showed positive expression of PCNA which appeared membranous reaction for CD 44 in the form of brown brown to black color in mostly all nuclei of granulosa pigmentation of cell membrane and cytoplasm of cells (Figs.16,17).Ovarian sections of Group II granulosa cells (Fig.22). (cyclophosphamide treated group) revealed negative Histomorphometrical results: expression for PCNA in some nuclei of granulosa cells Mean values of number of primordial follicles which appeared blue in color massive increase of (Table 1&Graph 1): collagen fibers surrounding the ovarian follicles and The mean value of the number of primordial around congested blood vessel. Some vacuoles (V) follicles has highly significantly decreased (P< 0.01) appeared within ovarian stroma (Fig.18). Ovarian in cyclophosphamide treated group as compared to sections of Group III (BM-MSCs group) revealed control group. In BM stem cell treated group the mean positive expression for PCNA in most of nuclei of value of the number of primordial follicles has granulosa cells which appeared brown to black color highly improved with highly significant difference and negative expression for PCNA in some nuclei of when compared to cyclophosphamide treated granulosa cells which appeared blue in color (Fig.19). group(P< 0.01).and it has significantly decreased(P < Ovarian sections of Group IV (AT-MSCs group) 0.05) in comparison with control ovary. In addition, showed positive expression of PCNA in most of nuclei the average value of number of primordial follicles has of granulosa cells. Some nuclei showed negative highly significantly increased in AT stem cell treated expression of PCNA (Fig.20). group as compared to cyclophosphamide group (P< CD 44immunostaining: 0.01) but it has significantly decreased when compared to control treated gro up (P < 0.05) and insignificantly decreased when compared to BM stem cell treated group (P>0.05)

Table 1: Comparison between all groups as regarding number of primordial follicles. Group Mean±SD Comparison Sig (p) Cyclophosphamide treated 0.000** Control 11.1±3.73 Control BM stem cells treated 0.837 AT stem cells treated 0.548 Control 0.000** Cyclophosphamide treated 1.4±1.07 Cyclophosphamide treated BM stem cells treated 0.000** AT stem cells treated 0.000** Control 0.837 BM mesenchymal stem cells 10.8±2.62 BM stem cells treated Cyclophosphamide treated 0.000** AT stem cells treated 0.625 Control 0.548 AT mesenchymal stem cells 10.2±2.78 AT stem cells treated Cyclophosphamide treated 0.000** BM stem cells treated 0.625 P value > 0.05 means "non significant".

number of primary follicles has highly significantly decreased (P< 0.01) in cyclophosphamide treated group as compared to control group. In BM stem cell treated group the mean value of the number of primary follicles has highly improved with highly significant difference when compared to cyclophosphamide treated group(P< 0.01).and it has significantly decreased (P < 0.05) in comparison with control ovary Also, the average value of number of

Graph (1): Comparison between all groups as primary follicles has highly significantly increased in regarding number of primordial follicles. AT stem cell treated group as compared to Mean values of number of primary cyclophosphamide group (P< 0.01) but it has follicles(Table 2, Graph 2): The mean value of the significantly decreased when compared to control treated group (P < 0.05) and insignificantly decreased

271 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ when compared to BM stem cell treated group (P>0.05).

Table 2: Mean values of number of primary follicles: in the studied groups Group Mean±SD Comparison sig Cyclophosphamide treated 0.000** Control 4.2±1.033 Control BM stem cells treated 0.245 AT stem cells treated 0.029* Control 0.000** Cyclophosphamide treated 1.2±0.63 Cyclophosphamide treated BM stem cells treated 0.000** AT stem cells treated 0.000** Control 0.000** BM mesenchymal stem cells 3.7±0.82 BM stem cells treated Cyclophosphamide treated 0.000** AT stem cells treated 0.237 Control 0.000** AT mesenchymal stem cells 3.3±0.63 AT stem cells treated Cyclophosphamide treated 0.000** BM stem cells treated 0.237

decreased (P< 0.01) in cyclophosphamide treated group as compared to control group. In BM stem cell treated group the mean value of the number of secondary follicles has highly improved with highly significant difference when compared to Cyclophosphamide treated group (P< 0.01).and it has significantly decreased(P > 0.05) when matched with control ovaries. Too, the average value of number of secondary follicles has highly significantly increased in AT stem cell treated group as compared to Cyclophosphamide group (P< 0.01) but it has Graph 2: Mean values of number of primary follicles: significantly decreased when compared to control in the studied groups treated group (P > 0.05) and insignificantly decreased when compared to BM stem cell treated group Mean values of number of secondary follicles (P>0.05). (Table 3&Graph 3): The mean value of the number of secondary follicles has highly significantly

Table 3: Mean values of number of secondary follicles in the studied groups Group Mean±SD Comparison sig Cyclophosphamide treated 0.000** Control 4.0±0.67 Control BM stem cells treated 0.425 AT stem cells treated 0.065 Control 0.000** Cyclophosphamide Cyclophosphamide treated 1.1±0.73 BM stem cells treated 0.000** treated AT stem cells treated 0.000** Control 0.425 BM mesenchymal stem cells 3.7±0.95 BM stem cells treated Cyclophosphamide treated 0.000** AT stem cells treated 0.431 Control 0.065 AT mesenchymal stem cells 3.4±0.7 AT stem cells treated Cyclophosphamide treated 0.000** BM stem cells treated 0.431

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decreased (P< 0.01) in Cyclophosphamide treated group as compared to control group. In BM stem cell treated group the mean value of the number of graafian follicles has highly improved with highly significant difference when compared to Cyclophosphamide treated group(P< 0.05).and it has significantly decreased (P > 0.05) when matched with control ovaries. In addition, the average value of number of Graafian follicles has highly significantly increased in AT stem cell treated group as compared Graph 3: Mean values of number of secondary to Cyclophosphamide group (P< 0.01) but it has follicles in the studied groups significantly decreased when compared to control treated group (P < 0.05) and highly significantly Mean values of number of graafian follicles decreased when compared to BM stem cell treated (Table 4&Graph 4): The mean value of the number group (P<0.01). of primordial follicles has highly significantly

Table 4: Mean values of number of graafian follicles in the studied groups. Group Mean±SD Comparison sig Cyclophosphamide treated 0.009** Control 1.4±0.52 Control BM stem cells treated 0.131 AT stem cells treated 0.000** Control 0.009** Cyclophosphamide treated 0.6±0.69 Cyclophosphamide treated BM stem cells treated 0.049* AT stem cells treated 0.013* Control 0.131 BM mesenchymal stem cells 1.1±.30 BM stem cells treated Cyclophosphamide treated 0.049* AT stem cells treated 0.000** Control 0.000** AT mesenchymal stem cells 0.0±0.0 AT stem cells treated Cyclophosphamide treated 0.013* BM stem cells treated 0.000**

significantly decreased when compared to cyclophosphamide treated group (P<0.01) and significantly increased when compared to control group (P> 0.05) and it has insignificantly decreased when compared to BM stem cell treated group (P>0.05).

Graph 4: Mean values of number of graafian follicles in the ovarian sections of the studied groups

The number of atretic follicles (Table 5&Graph 5): has highly significantly increased (P< 0.01) in cyclophosphamide treated group as compared to control group. In BM stem cell treated group, the number of atretic follicles has increased significantly (P> 0.05)in case of when matching with control group Graph5: Mean values of number of atretic follicles in it has highly significantly decreased when compared to the ovarian sections of the studied groups. cyclophosphamide treated group (P<0.01). Also, the number of atretic follicles AT has highly

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Table 5: Mean values of number of atretic follicles in the studied groups. Group Mean±SD Comparison Sig Cyclophosphamide treated 0.000** Control 0.50±0.53 Control BM stem cells treated 0. 230 AT stem cells treated 0. 264 Control 0.000** Cyclophosphamide treated 2.30±0.95 Cyclophosphamide treated BM stem cells treated 0.002** AT stem cells treated 0. 000** Control 0. 230 BM mesenchymal 0.90±0.87 BM stem cells treated Cyclophosphamide treated 0.002** stem cells AT stem cells treated 0.771 Control 0. 264 AT mesenchymal 0.80±0.63 AT stem cells treated Cyclophosphamide treated 0. 000** stem cells BM stem cells treated 0.771

The Mean area% of collagen fibers (Table significantly decreased when compared to AT stem 6&Graph 6): it was increased high significantly (P< cell treated group (P>0.05). Also, the Mean area% of 0.01) in cyclophosphamide exposed group as collagen fiber has highly significantly decreased in compared to control one. In BM stem cell treated AT stem cell treated group as compared to group. The Mean area% of collagen fiber has cyclophosphamide treated group (P< 0.01) and non markedly improved with highly significant increase significantly increased when compared to BM stem when compared to control group (P< 0.01) and it has cell treated group (P> 0.05) but it has increased high highly significantly decreased when compared to significantly (P< 0.01)when matched with control cyclophosphamide treated group (P<0.01) no treated rats .

Table 6: Mean values of Mean±SD of collagen fiber content in the studied groups. Group Mean±SD Comparison Sig Cyclophosphamide treated 0.000** Control 5.1 ±0.4 Control BM stem cells treated 0.000** AT stem cells treated 0.000** Control 0.000** Cyclophosphamide treated 16.2 ±4.5 Cyclophosphamide treated BM stem cells treated 0.000** AT stem cells treated 0.002** Control 0.000** BM mesenchymal 9.6 ±1.4 BM stem cells treated Cyclophosphamide treated 0.000** stem cells AT stem cells treated 0.125 Control 0.051** AT mesenchymal 10.80±1.9 AT stem cells treated Cyclophosphamide treated 0.002** stem cells BM stem cells treated 0.125

Mean values of Mean area% of proliferating cell nuclear antigen immunoreactivity(Table 7&Graph 7): The Mean area% of proliferating cell nuclear antigen immunoreactivity highly significantly decreased (P< 0.01) in cyclophosphamide treated rats in comparison with control un-treated rat group. In BM stem cell treated group The Mean area% of proliferating cell nuclear antigen immunoreactivity showed marked improved with significant decrease

Graph 6: Mean area percent of collagen fiber content when compared to control group and non significant in the ovarian sections of the studied groups increased when compared with AT stem cell treated group (P>0.05) and it has highly significantly

274 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ increased when compared to cyclophosphamide treated group (P< 0.01) and non significantly treated group (P>0.01).Also, the mean area% of decreased when compared to BM stem cell treated proliferating cell nuclear antigen immunoreactivity group (P> 0.05) but it has significantly has highly significantly increased in AT stem cell decreased(P<0.05) when parallel with control treated treated group as compared to cyclophosphamide group.

Table 7: showed Mean values of Mean area% of proliferating cell nuclear antigen immunoreactivity in all groups. Group Mean±SD Comparison sig Cyclophosphamide treated 0.000** Control 5.1±0.8 Control BM stem cells treated .038 AT stem cells treated .032* Control 0.000** Cyclophosphamide treated 0.5±0.3 Cyclophosphamide treated BM stem cells treated 0.000** AT stem cells treated 0.000** Control .038 BM mesenchymal 4.3 .8 BM stem cells treated Cyclophosphamide treated 0.000** stem cells ± AT stem cells treated .647 Control 0.051** AT mesenchymal 4.1 1.1 AT stem cells treated Cyclophosphamide treated 0.000** stem cells ± BM stem cells treated .647

Fig. (2): A Photomicrograph of subgroup Ia(negative control Graph7: Mean area percent of proliferating cell nuclear antigen group): rat ovary showing a secondary follicle. It consists of (PCNA) immunoreactivity in the ovarian sections of the studied an enlarged oocyte (O) with well-defined zona pellucida groups (arrow) and surrounded by follicular granulosa cells (G) formed of multilayer of polyhrdral cells and peripheral fusiform theca folliculi cells (T). Two cavities (A) full of liquor folliculi appear within the follicle. (Hx.&E. X400).

Fig. (1): A photomicrograph of subgroup Ia(negative control group): adult rat ovary showing wide cortex which is full of primordial follicles (F), primary follicles of different stages (PF), secondary follicles (SF)a, corpus luteum (CL) and Fig. (3): A photomicrograph of an adult rats of subgroup single atretic follicle (AF). Stroma cells (ST) in between the Ib(positive control group): showing cortex (C) which is full follicles are clearly seen.(Hx.&E. X100). of different growing follicles, primordial follicle (F), primary follicles (PF) and secondary follicles (SC) and multiple corpora lutea (CL) with stroma cells (ST) in between.(Hx.&E. X100).

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Fig. (4): A photomicrograph of an adult rats of subgroup Fig. (7): A photomicrograph of an adult rat ovary of group Ib(positive control group): showing relatively normal mature III (BM-MSCs group) showing relatively normal mature graafian follicle. It consists of normal oocyte (O) surrounded graafian follicle (GF) and normal corpus luteum (CL). by normal zona pellucida (arrows). The oocyte is attached to Mature graafian follicle consists of an oocyte (O) present at the wall of the follicle by cumulus oophorus (CO). Normal one side, its wall is formed of relatively normal cells of cells of zona granulosa (G) and theca folliculi (T) form the granulosa cells (G) and liquor folliculi appears within the wall of the follicle. Liquor folliculi appears within the cavity of the follicle (A), The oocyte was attached to the antrum (A) of the follicle. Notice, Stroma (ST) cells are wall of the follicle by cumulus oophorus. (CO). Notice, so seen. (Hx.&E. X400). degenerated follicles (DF) still present in the cortex. (Hx&E x100).

Fig. (5): A photomicrograph of an adult rat ovary of CLP treated group II (CLP group) showing cortex containing multiple markedly degenerated follicles (DF) devoid of Fig. (8): A photomicrograph of an adult rat ovary of group oocytes which contain exfoliated granulosa,pyknotic III (BM-MSCs group): showing relatively normal granulosa cells (arrow head). Notice, hyaline material multilayered primary follicle surrounded by multiple deposition (H), congested blood vessels (BV), presence of growing primordial follicles (F). It consists of an enlarged vacuoles (V) due to degeneration of stroma cells (ST). notice oocyte (O) surrounded by well-defined zona pellucida degenerated corpus luteum (CL). (Hx&E 100). (arrows), follicular granulosa cells (G) which is formed of multilayer of polyhedral cells and peripheral fusiform theca folliculi cells (T). Notice stroma (ST) cells are arranged in clusters.(Hx&E x400).

Fig. (6): A Photomicrograph of an adult rat ovary of CLP treated group II (CLP group) showing a degenerated multilayered follicle (DF) with degenerated oocyte (O). The granulosa cells appear disorganized with pyknotic nuclei Fig. (9): A photomicrograph of an adult rat ovary of group (arrow) and vacuolated cytoplasm (V). Interstitial IV (AT-MSCs group): showing relatively normal growing hemorrhage (H) inside congested blood vessels, cellular ovarian follicles (F). Two relatively normal corpora lutea infilteration (I) and multiple different size vacuoles (V) (CL) also appear. Notice: degenerated follicle (DF) and found inbetween stroma cells (ST). Notice, another multiple different size vacuoles (V) inbetween stroma cells (ST) with congested blood vessels (BV). (Hx&E x 100). primordial follicle contain degenerated oocyte (arrow head).

(H&E 400)

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Fig. (10): A photomicrograph of an adult rat ovary of group IV (AT-MSCs group): showing relatively normal growing secondary follicle. It consists of relatively normal oocyte (O) Fig. (13): A photomicrograph of an adult rat ovary of group surrounded by clear zona pellucida (arrow head), relatively II (CLP group) showing marked increase in the amount of normal granulosa cells (G), theca folliculi (T)&two cavities collagen fibers surrounding degenerated ovarian follicles (A) appear within the follicle. But still some granulosa cells and inbetween stroma cells (arrows).(Masson's are vacuolated (V)Notice, multiple different size vacuoles TrichromeX100). (V) inbetween stroma cells (ST) with dilated blood vessels (BV), degenerated follicle (DF).(Hx&E. x400).

Masson's Trichrome Groups

Fig. (14): A photomicrograph of an adult rat ovary of group III (BM-MSCs group): showing minimal amount of collagen fibers (arrow) surrounding and within the Fig. (11): A photomicrograph of an adult rat ovary of ovarian stroma indicating marked improvement similar to subgroup Ia(negative control group): showing minimal control group.(Masson's TrichromeX100). collagen fibers (arrow) surrounding ovarian follicle. Also few collagen fibers inside stroma (arrow) (Masson's TrichromeX100).

Fig. (15): A photomicrograph of an adult group IV (AT- MSCs group): showing moderate amount of collagen fibers (arrow) surrounding ovarian follicle and within the ovarian stroma, indicating moderate improvement of this Fig. (12): A photomicrograph of an adult rat ovary of group.(Masson's TrichromeX100) subgroup Ib (positive control group)showing: minimal collagen fibers (arrow) surrounding ovarian follicle. Also few collagen fibers (arrow) appear within the ovarian stroma similar to the control group.(Masson's TrichromeX100).

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PCNA Groups

Fig. (16): A Photomicrograph of subgroup Ia (control group) Fig. (19): A photomicrograph of an adult rat ovary group III adult rat ovary showing different types of ovarian follicles (BM-MSCs group): showing positive expression for PCNA with positive expression of PCNA (arrow) in mostly all in most of nuclei (brown to black color - arrow) and negative nuclei of granulosa cells(brown to black color of nuclei). expression for PCNA in some nuclei (blue color) of (PCNAX200). granulosa cells (arrow head). (PCNAX200).

Fig. (20): A Photomicrograph of an adult rat ovary of group IV (AT-MSCs group): showing multiple growing ovarian

Fig. (17): A Photomicrograph of an adult rat ovary of follicles with positive expression of PCNA in nuclei of subgroup Ib (positive control group)showing :different types granulosa cells which appear brown to black colored nuclei of ovarian follicles with positive expression of PCNA (arrow), some nuclei (blue nuclei) show negative expression (arrows) in mostly all nuclei of granulosa cells(PCNAX200) of PCNA (arrow head) (PCNAX200).

Figure (21): A photomicrograph of a section in the rat ovary Fig. (18): A photomicrograph of an adult rat ovary group II group III (BM-MSCs group): showing positive cell (CLP group) showing multiple degenerative follicles with membranous reaction for CD44 antibody expressed as negative expression for PCNA in mostly all nuclei of brown pigmentation of cell membrane and cytoplasm of granulosa cells(blue colored nuclei). (PCNAX200). granulosa cells(black arrow). (CD44 expressed as X400).

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(33).Meirowet al., 2010, postulated that the toxic influence of CLP can be attributed to the releasing of active metabolites as acrolein and phosphoramide mustard from CLP. They proposed that these metabolites can bind to DNA, subsequently leading to failure in synthesis of DNA and cell apoptosis (36).In addition to the previous hypothesis, metabolites of CLP released reactive oxygen species (ROS) via ligation with glutathione , which consequently leads to suppression of the antioxidant defense mechanism of the ovaries (37). It is found that some chemical substances may Figure (22): A photomicrograph of a section in the rat ovaries injected by group IV (Ad-MSCs group): showing interfere with interaction amide the oocyte and positive cell membranous reaction for CD 44 antibody granulosa cells(GCs), where GCs are considered one expressed as brown pigmentation of cell membrane and of the most important constituents of ovary as cytoplasm of granulosa cells (black arrow). (CD 44X400). folliculogenesis; and playing an important vital roles inside the follicles such as enhancing the processes of 4. Discussion differentiation ,proliferation and apoptotic process.It is Cyclophosphamide (CLP) is widely used as a proposed that the impact of degeneration of GC on potent alkylating antitumor drug in patients suffering oocytes is in form of liberation of reactive oxygen from tumor. CP induces DNA damage in tumor cells, species (ROS) [38]. yet, it possessing some adverse side effects through It has been clearly observed in the current study affecting on the patient fertility, where it causing extensive interstitial degeneration of ovarian stroma in permanent or temporary amenorrhea, reduction of the the form of vacuoles with mononuclear cellular follicles in ovary makes reduced fertility, or infertility infiltration. marked congested dilated of the blood (32,33) vessels of the CP-treated group, some dispersed areas In the present study, examination of the CLP of interstitial hemorrhage and these vascular changes treated ovaries has shown massive loss of follicles, in accordance with study of [24](Omar et al.,2016), markedly shrunken, extensively distorted with the and the study of [39](Hamzeh et al,2018) in his study presence of many atretic follicles. Some follicles received a single dose of 150 mg/kg of CP, IP, for showed disorganization, as granulosa cells undergo 5days . apoptosis and slough from their attachment, the ovum, It is postulated that vascular changes and local is anatomically surrounded by the zona pellucida, ischemia lead to destruction parts of the normal swimming in the follicular fluid. The zona pellucida ovarian cortex which consequently leads to and ovum also are subjected for degeneration and degeneration cortical follicles [33]. necrosis during the growth of ovum. The germinal Our results confirmed by the morphometric and epithelium has shown dispersed areas of degeneration. statistical results which have proved that the mean These results run with that published by (34),who value of the number of the ovarian follicles stated that histological examinations of ovaries in CLP (primordial, primary, secondary and graafian) has treated group after two months revealed degenerative highly significantly decreased(P< 0.01)in group changes such as tertiary follicles atrophy, degenerative treated with CLP as matched with control rat group, and necrotic changes of the zona pellucida and ovum, The average level of the collagen area % in the ovary these results run with that published by (35) who between the cortical follicles has highly significantly stated decreased thickness of germinal epithelium of increased (P < 0.01) in CLP treated group as compared the ovarian cortex and oogenesis was completely to control group. The graafian follicles diameter has destroyed, and (24),2016who administrates 2 types of highly significantly decreased in CLP treated group chemotherapy (CLP and busulfan) to rats on ovary and relative to control group (P < 0.01), These statistical stated substantial degeneration of follicles, marked results agreed with (38) who reported that the average contracted with irregular shape of the ovary . number of the ovarian follicles response (primordial, The mode of action of chemotherapeutic that primary, secondary and Graafian) of the control group leads to degeneration of follicles until now not known were higher than that in the CLP testes (P<0.05), also well. Some investigators postulated that this loss of the value of the collagen area % of the follicles may be due to hastening of the natural ovarian cyclophosphamide group was high significantly aging manner, which attributed to a direct cytotoxic (P<0.05) in comparison with the control testes and impact on oocytes integrity and apoptosis of a part of (35) who found that the Graafian follicles was less primordial follicles, damaging the processes of ovarian t n cont ol (P˂0.05).

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The Proliferative cell nuclear antigen (PCNA) BM-MSCs treated and control groups were larger than functions as an important factor in several necessary in the CLP treated testes (P<0.05), also The number cellular procedures, like DNA repair, DNA of atretic follicles has highly significantly increased replication, DNA damage avoidance, sister-chromatid (P<0.01) in CLP treated group as compared to control cohesion, cell survival and cell cycle control, in group. addition to regulating function in the development of In BM stem cell treated group, The mean value follicles in the ovary (40) . of the collagen area %in the ovary between the cortical Immunhistochemical study of ovarian section by follicles was markedly improved and it has highly PCNA stain in the present study stated that CLP significantly decreased when compared to treated ovaries, showed degeneration of the germinal cyclophosphamide treated group (P<0.01) and this epithelium in most ovarian follicles with negative agreed with result of Boa et al. (2016) (44) reaction in the nuclei of their oogonia. However, few The BM-MSC-treated ovarian tissue have shown oogonia in the remaining follicles revealed positive positive cell membranous reaction for CD 44 which nuclear reaction and the area percent of PCNA means that transplanted BM-MSCs have differentiated immunoreactivity has highly significantly decreased into these oogonia as BM-MSCs are CD44 positive. (P< 0.01)in group treated with cyclophosphamide as These findings agreed with (45) who found that BM- paralleled with control group. These findings in MSCs treated testes were positive for CD 44. accordance with (41) and with that published by (9) In the current study, Histological analysis of the who demonstrated that starting from one week ovaries of rats after AT-MSCs transplantation has following CLP therapy,the apoptotic–positive follicles shown mild restoration of architecture and outline of percentage elevated, while at 4 weeks , there are a ovarian follicles but still less than normal. The primary suppression in proliferation of cell nuclear antigen and secondary follicles have shown early stages of expression. oogenesis but no Graafian follicles were detected, In this work, BM-MSCs transplantation in rat these results are in agreement with those of other ovaries treated by CLP has shown restoration of investigators who showed that AD-MSC transplanted architecture and outline of ovarian follicles nearly groups demonstrated an affinity to an elevation in the similar to normal. The ovarian follicles are densely numbers of early growing ovarian follicles, not packed and lined with stratified granulosa cells. The reached to differentiate into Graafian follicles directly, adherence of granulosa cells to zona pellucida and Similar findings were recognized by (40) who reported has been also restored, vacuoles have the presence of oogonia in the primary and secondary nearly disappeared, however the congestion of the follicles with AT-MSCs transplantation. They blood vessels has mild improved. These findings demonstrated that injected AT-MSCs differentiated to agreed with (42) who reported that BM-MSCs, ovarian germinal cells. transplanted into ovaries of a cyclophosphamide- In the present study post ADSCs treatment a treated female mouse model, appeared to differentiate vascular alterations was noticed in blood vessel supply into germ cells, granulosa cells and stroma cells, the of the cortex in ovarian sections which reported also in study of (17) showed that transplanted BM-SCs could the work of some authors (25). The scientists try to differentiate into germinal cells (immature oocyte) in illustrate the increase in blood vessels by the ovarian follicles of rats and other germ cells of hypothesis that stem cells are responsible for releasing primary and secondary oocyte, in some ovarian of special mediators which work as a paracrine gland follicles of recipient rats. Also reported that after to augment angiogenesis this was illustrated as a side treatment of cyclophosphamide treated infertile group effect of stem cell therapy as they may lead to with BM-SCs, the structure of the ovarian follicles hemorrhage and veno-occlusive disorders (25, 46). restored. This has been supported by the morphometric The findings of this study have been supported and statistical results which have proved that the mean by the morphometric and statistical results which have value of the number of the ovarian follicles proved that in BM stem cell treated group the number (primordial, primary, secondary) was decreased of the ovarian follicles (primordial, primary, significantly(P<0.05) in contrast with control secondary and graafian) significantly increased in BM untreated group and highly significantly increased stem cell treated group as compared to CLP group (P relative to cyclophosphamide treated group (P< 0.01)., < 0.05) and the number of atretic follicles has Similar findings were reported by (47) who found that significantly increased when compared to control mean number of the primordial, primary and group (P> 0.05), and highly significantly decreased secondary ovarian follicles in rats with AT-MSCs when compared to CLP treated group (P<0.01). transplantation did not have difference with normal These findings were in agreement with (44) who rats and both groups were more than the treated rats proved that the number of the ovarian follicles in the with CLP, Another study conflicted by (24) proved

280 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ that a significant elevation in the average counts of hypotheses try to explaining this, 1)the differentiation primordial follicles after ADSCs treatment, as of MSCs into the target cells via proper generation matched with chemotherapy-treated rat group. conditions (48), and 2)the release of growth factors by Comparable data was recorded in other investigations MSCs into culture medium to diminish germ cell and in which the total ovarian response (follicles and death and to augment folliculogenesis corpora lutea) was elevated post ADSCs treatment throughout enhancements in the microenvironment (26). (49), that excite the neighborhood oogonial stem cells The Mean area% of collagen fiber has highly to reestablish the utility of the host cells (50). The last significantly decreased in AT stem cell treated group mode of action is the integration of MSCs with as compared to cyclophosphamide treated group (P< endogenous follicular cells to improve the damaged 0.01) and non-significantly increased when compared tissue function. to BM stem cell treated group (P> 0.05) and these In this study, we show the BM-MSCs have high finding was recorded in the study of (40) potency in improving the ovarian function and In the current work Immunohistochemical morphology in rats with chemotherapy-POF. It examination of AT-MSC treated ovarian sections appears to be greater than the efficiency of AT-MSCs stained by PCNA has shown mild positive nuclear in the restoring the function of ovary, in agreement reaction in most oogonia in the form of dark brown with other researchers (44) who used BM-MSCs to pigmentation of the nucleus, which has indicated more restore the ovaries in mice of chemotherapy-POF. The regeneration and proliferation of cells and moderate transplanted BM-MSCs continued and transferred to reaction in the nuclei of other granulosa cells. This ovary of mice ,then differentiated into granulosa cells agreed with (24). These results have been supported occupying all different types of ovarian follicles and by the morphometric and statistical results which have also corpora lutea. But our results have been conflicted proved that the Mean area% of proliferating cell with (51)who found that AT-SCs were preferable than nuclear antigen immunoreactivity has highly BM-SCs, regarding their ability for maintenance of significantly increased in AT stem cell treated group proliferating process, however had analogous as compared to cyclophosphamide treated group (P< phenotype and morphology, did not expose 0.01) and non-significantly decreased when compared differentially expressed adipogenic or osteogenic to BM stem cell treated group (P> 0.05) but it has related genes between BM-SCs and AT-SCs. significantly decreased when compared to control A new option to treat female infertility or to treated group (P< 0.05). results are in agreement with restore ovarian function is the application of MSC those of some authors(15) who showed that AT-MSC treatment. Several reports have verified the defensive transplanted groups established an affinity for impact of ovarian function resulting from elevation in the numbers of early growing ovarian administration of MSCs harvested from different cell follicles, which didn`t reach to transformed to origins in POF animal models .this is the first report Graafian follicles, yet it may continue in the depending on our knowledge, dealing with the interstitium, playing significant secondary characters comparative study of BM-MSCs and AT-MSCs in the in the microenvironment nearby the ovarian oocytes. remedy of ovarian dysfunction, but several preceding But another conflict by (24) who reported that trials using BM-MSCs were performed in the same the follicular populations of at different stages of issu. development and ovulation significantly elevated following adipose tissue-derived stem cells , this study References not agreed with 26) who reported that human amniotic 1. Huong DL, Amoura Z. and Duhaut P. (2002): Risk of membrane stem cells (HAMsc) have higher efficacy ovarian failure and fertility after intravenous than AT-SC in treatment of cyclophosphamide– cyclophosphamide. A study in 84 patients. J induced ovarian dysfunction . Rheumatol; 29:2571-76. AT-MSC treated ovarian tissue have shown 2. Meirow D, Dor J, Kaufman B, Shrim A, Rabinovici J. positive cell membranous reaction for CD44 in the and Schiff E. (2007): Cortical fibrosis and blood- vessels damage in human ovaries exposed to form of brown pigmentation of cell membrane and chemotherapy. Potential mechanisms of ovarian cytoplasm of oogenic cells which means that injury. Hum Reprod;22:1626–1633. transplanted AT-MSCs have differentiated into these 3. Goswami, D. and Conway, GS. (2005): Premature oogonia as CD44 characterizes MSCs. This agreed ovarian failure. Human Reproduction Update 11 391– with (24) who reported expression of MSC surface 410. markers CD44 in ATMSCs. 4. Jacobsen BK, Heuch I. and Kvale G. (2003): Age at These current finding and with respect to other natural menopause and all-cause mortality: a 37-year data, may illustrate the retrieval procedures of follow-up of 19,731 Norwegian women. Am J oogenesis post MSCs transplantation. There are two Epidemiol;157(10):923–9.

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5. Ye H, Li X, Zheng T, Hu C, Pan Z, Huang J, Li J, Li 19. Elman JS, Li M, Wang F, Gimble JM. And W. and Zheng Y. (2017): The Signaling Pathway Parekkadan B. (2014): A comparison of adipose and Regulates Ovarian Function via the Proliferation of bone marrow-derived mesenchymal stromal cell Ovarian Germline Stem Cells. Cell Physiol Biochem; secreted factors in the treatment of systemic 41: 1051-1062. inflammation. J Inflamm.;11:1. 6. Torino F, Barnabei A, De Vecchis L, Appetecchia M, 20. Afifi, M. and Noah, O. (2013): Role of mesenchymal Strigari L. and Corsello SM. (2012): Recognizing stem cell therapy in restoring ovarian function in a rat menopause in women with amenorrhea induced by model of chemotherapy-induced ovarian failure: a cytotoxic chemotherapy for endocrine-responsive early histological and immunohistochemical study The breast cancer. Endocr Relat Cancer; 19: R21–R33. Egyptian Journal of Histology, 36:114-12611. 7. Jin M, Yu YQ. and Huang H. (2012): An update on 21. Nese AY, Orhun S, Erdin I, Aygen C, Gulbuz S, primary ovarian insufficiency. Sci China Life Sci; Ahmet M, Ugur D. and Fehime A. (2013): Effects of 55:677–686. Spirulina on Cyclophosphamide-Induced Ovarian 8. Kalu E. and Panay N. (2008): Spontaneous premature Toxicity in Rats: Biochemical and Histomorphometric ovarian failure: management challenges. Gynecol Evaluation of the Ovary Biochemistry Research Endocrinol; 24:273–279. International Volume, Article ID 764262, 6 pages. 9. Tan S, Yeh Y, Shang W, Wua G. and Liu J. (2010): 22. Jiang Y, Jahagirdar BN. And Reinhardt RL. (2002):. Protective effect of a gonadotropin-releasing hormone Pluripotency of mesenchymal stem cells derived from analogue on chemotherapeutic agent-induced ovarian adult marrow. Nature 418:419. gonadotoxicity: a mouse model. Europ J Obstet 23. Alhadlaq A and Mao J. J; (2004): Mesenchymal stem Gynecol Reprod Biol 149:182–185. cells: isolation and therapeutics. Stem Cells Dev.; 10. Schover, LR. (2008):. Premature ovarian failure and its 13(4): 436-448. consequences: vasomotor symptoms, sexuality, and 24. Omar, F. R., Afifi, N. M., Elsherif, H. A., and fertility. J Clin Oncol; 26:753–758. Mohamed, D. H. (2016): Role of Adipose-Derived 11. Zou JP, Huang S, Peng Y, Liu HW, Cheng B. and Fu Stem Cells in Restoring Ovarian Structure of Adult XB. (2012): Mesenchymal stem Albino Rats with Chemotherapy-Induced Ovarian cells/multipotentmesenchymal stromal cells (MSCs): Failure: A Histological and potential role in healing cutaneous chronic wounds. Int ImmunohistochemicalStudy, JCarcinog Mutagen, 7:1. J Low Extrem Wounds;11:244–53. 25. Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T. and 12. Mabuchi. Y, and Matsuzaki Y. (2016): Multiline H ii K. (2017):“Cell-assisted lipo-transfer for agepotential of adult human mesenchyme stem cells. cosmetic breast augmentation: supportive use of International Journal of Hematology, Volume 103, adipose-derived stem/ stromal cells, Aesthetic Plastic Issue 2, pp 138–144. Surgery, vol. 32, no. 1, pp. 48–55. 13. Riqiang Bao, Ping Xu, Wang, Jing, Li Xiao, Gang Li 26. Fouad H, Sabry D, Elsetohy K. and Fathy N. (2016): and Chunping Zhang. (2017): Bone marrow derived Therapeutic efficacy of amniotic membrane stem cells mesenchymal stem cells transplantation rescues and adipose tissue stem cells inrats with chemically premature ovarian insufficiency induced by induced ovarian failure. J Adv Res; 7:233-41. chemotherapy Pages 1-7. 27. Lai D, Wang F, Chen Y, Wang L. and Wang Y. 14. Zuk PA, Zuh M. and Ashjian P. (2002):Human (2013): Human amniotic fluid stem cells have a adipose tissue is a source of multipotent stem cells. potential to recover ovarian function in mice with Mol Biol Cell2002; 13:4279–429. chemotherapy-induced sterility. BMC Dev Biol 13: 34. 15. Takehara Y, Yabuuchi A, Ezoe K, Kuroda T, 28. Kiernan, JA. (2001): Histological and histochemical Yamadera R. and Sano C. (2013): Theraputic effects methods: theory and practice. 3rded. New York: of adipose-derived mesenchymal stem cells on Arnold publisher;. pp. 111–162. damaged ovarian function. Lab Invest; 93:181–193. 29. Bancroft JD, Cook HC. and Turner DR. (2008): 16. Vishnubalaji R, Al-Nbaheen M, Kadalmani B, Immunocyto- chemistry. In: Manual of histological Aldahmash A. and Ramesh T. (2012): Comparative techniques and their diagnostic applications. 6th ed. investigation of the differentiation capability of bone- London: Churchill Livingstone; Pp150-180. marrow- and adipose-derived mesenchymal stem cells 30. Young, H. E.; Steele, T. A.; Bray, R. A.; Hudson, J.; by qualitative and quantitative analysis. Cell Tissue Floyd, J. A.; Hawkins, K.; Thomas, K.; Austin, T.; Res.; 347:419–27. Edwards, C.; Cuzzourt, J.; Duenzl, M.; Lucas, P. A. 17. Lee HJ, Selesniemi K. and Niikura Y. (2007): Bone and Black, A. C. (2001): Human reserve pluripotent marrow transplantation generates immature oocytes mesenchymal stem cells are present in the connective and rescues long-term fertility in a preclinical mouse tissues of skeletal muscle and dermis derived from model of chemotherapy-induced premature ovarian fetal, adult, and geriatric donors. Anat Rec, 264:51-62. failure. J Clin Oncol;25:3198–204. 31. Armitage P and Berry G; (2008): Medical research 18. Puissant B, Barreau C, Bourin P, Clavel C, Corre J. statistical methods. In: Armitage P, Berry G, editors. and Bousquet C. (2013): Immunomodulatory effect of Medical research. 4th ed. London: Blackwell Scientific human adipose tissue-derived adult stem cells: Publications; pp. 12–48. comparison with bone marrow mesenchymal stem 32. Green DM, Kawashima T, Stovall M, Leisenring W, cells. Br J Haematol.; 129:118–29. Sklar CA, Mertens AC, et al. Fertility of female

282 Nature and Science 2019;17(12) http://www.sciencepub.net/natureNSJ

survivors of childhood cancer: a report from the chemotherapy-induced ovarian damage. Cytotherapy; childhood cancer survivor study. J Clin Oncol. 2009; 10:353–63. 27:2677–2685. 43. Salooja N, Szydlo RM, and Socie G,. (2001): 33. Meirow D, Nugent D. The effects of radiotherapy and Pregnancy outcomes after peripheral blood or bone chemotherapy on female reproduction. Hum Reprod marrow transplantation: a retrospective survey. Update. 2001; 7:535–543. Lancet.;358(9278):271–276. 34. De Vos M, Devroey P, Fauser BC. (2010): Primary 44. Riqiang Bao, Ping Xu, Yishu Wang, Jing Wang, Li ovarian insufficiency. Lancet; 376:911–921. Xiao, Gang Li. and Chunping Zhang. (2017): Bone 35. Sevtap K, Ferda P, Candan O, Nicel T, Gulce N S. and marrow derived mesenchymal stem cells Tuncay D. (2014): Protection from cyclophosphamide- transplantation rescues premature ovarian induced ovarian damage with bone. marrow-derived insufficiency induced by chemotherapy Pages 1-7. mesenchymal stem cells during puberty Gynecol 45. Kimbrel EA, Kouris NA, Yavanian GJ, (2014): Endocrinol,; 30(2): 135–140. Mesenchymal stem cell population derived from 36. Fleer R, Brendel M. Toxicity, interstrand cross-links human pluripotent stem cells displays potent and DNA fragmentation induced by immunomodulatory and therapeutic properties. Stem ‘ ctiv te ’cyclop o p i e in ye t: co p tive Cells Dev.; 23(14):1611-1624. studies on 4-hydroperoxy-cyclophosphamide, its 46. Bollard C, Krance RA and Heslop HE (2011) monofunctionalanalogon, acrolein, phosphoramide Hematopoietic stem celltransplantion in pediatric mustard, and nor-nitrogen mustard. Chem Biol oncology. In: Pizzo PA, Poplack DG (eds.) Principles Interact. 1982;39:1–15. and Practice of Pediatric Oncology. (6thedn) 37. Tsai-Turton M, Luong BT, Tan Y, Luderer U. Philadelphia: Lippincott Williams & Wilkins 16: 467- Cyclophosphamide-induced apoptosis in COV434 490. human granulosa cells involves oxidative stress and 47. Yasser H., Ahmed H., Soad J. T., Dina H. M., and glutathione depletion. Toxicol Sci. 2007; 98:216–230. Marwa M. (2018): Effect of Adipose-derived stem 38. Bukovsky A, Caudle MR (2012): Immunoregulation cells versus clomiphen on treatment of experimental of follicular renewal, selection, POF, and menopause polycystic ovary in rats: Histological and in vivo, vs. neo-oogenesis in vitro, POF and ovarian Immunohistochemical study1110-0559, Vol. 41, No.4. infertility treatment, and a clinical trial. Reprod Biol 48. Kode JA1, Mukherjee S, Joglekar MV, Hardikar AA Endocrinol 10: 97-102. (2009): Mesenchymal stem cells: immunobiology and 39. Hamzeh M, Hosseinimehr S J, Mohammadi H R, role in immunomodulation and tissue regeneration. Beklar S Y, Dashti A, and Amiri F T, Atorvastatin Cytotherapy 11: 377-391. attenuates the ovarian damage induced by 49. Mohamed SA, Shalaby SM, Abdelaziz M, Brakta S, cyclophosphamide in rat: An experimental study Int J Hill WD, Ismail N, (2018): Human mesenchymal stem Reprod Biomed (Yazd). 2018 May; 16(5): 323–334. cells partially reverse infertility in chemotherapy- 40. Sun S. Y., Zhang W., et al., (2014): Cell proliferation induced ovarian failure. Reprod Sci; 25:51-63. and apoptosis in the fetal and neonatal ovary of guinea 50. Abd-Allah SH, Shalaby SM, Pasha HF, El-Shal AS, pig ‖. Genet. Mol. Re . 13 (1): 1570-1578. Raafat N, et al. (2013): Mechanistic action of 41. Picut CA, Remick AK, Asakawa MG, Simons ML. mesenchymal stem cell injection in the treatment of and Parker GA. (2014):. Histologic features of chemically induced ovarian failure in rabbits. prepubertal and pubertal reproductive development in Cytotherapy 15: 64-75. female rats. Toxicol Pathol.;42:403–13. 51. Wagner W, Wein F. and Seckinger A. (2005): 42. Fu X, He Y, Xie C. and Liu W. (2008): Bone marrow Comparative characteristics of mesenchymal stem mesenchymal stem cell transplantation improves cells from human bone marrow, adipose tissue, and ovarian function and structure in rats with umbilical cord blood. ExpHematol;33:1402–1416.

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