Stem Cell Reviews and Reports

, Volume 15, Issue 4, pp 558–573 | Cite as

Effects of VEGF + Mesenchymal Stem Cells and Platelet-Rich Plasma on Inbred Rat Ovarian Functions in Cyclophosphamide-Induced Premature Ovarian Insufficiency Model

  • Birol VuralEmail author
  • Gokhan Duruksu
  • Fisun Vural
  • Merve Gorguc
  • Erdal Karaoz


Premature ovarian insufficiency (POI), a fertility disorder affecting women under 40 years of age, is characterized by early loss of ovarian function. This study was aimed to maintain ovarian function in POI animal models by mesenchymal stem cells (MSCs) transplantation with/without the supplementation of platelet-rich plasma (PRP). Adipose tissue-derived MSCs were isolated from inbred rats (Fisher-344), and constitutive expression of both VEGF and GFP were maintained by transfection with plasmids, pVEGF and pGFP-N. PRP was derived from the blood of healthy untreated rats. A total of 60 rats were divided into 5 groups of 12 rats in each. First group was kept as untreated-control (Control), and POI model was induced in Fisher-344 rats by cyclophosphamide in the next four groups. Second group was kept as sham-operated-control (Sham). MSC, PRP and MSC+ PRP-treated groups were transplanted following the validation of POI model in rats. After 2 months following the transplantation, anti-mullerian-hormone (AMH) and oestradiol (E2) blood levels were measured. Follicles were evaluated after hematoxylin-eosin staining, and the immunofluorescence staining and gene expression analyses were performed to show the ovarian regeneration. The follicular count was improved after MSC- and MSC + PRP-treatment to 63% of Control-group and significantly higher than that in Sham-group, but a significant increase was not observed in PRP-group. Higher AMH and E2 levels were measured in MSC + PRP than in Sham-group, and CXCL12, BMP-4, TGF-β and IGF-1 expressions were also increased. This study showed MSCs +/-PRP transplantation after POI supports recovery of the follicular count and function. For ovarian recovery, a single administration of PRP was found not sufficient. Although MSC treatment increased follicular regeneration, better results were obtained in the co-transplantation of MSCs and PRP. These results might be promising for follicular regeneration in POI patients.


Cyclophosphamide Granulosa cells Mesenchymal stem cells Oocyte Platelet-rich plasma Premature ovarian insufficiency Theca cells Vascular endothelial growth factor 





Adipose tissue-derived mesenchymal stem cells


Basic fibroblast growth factor


Bone morphogenetic protein 4


Ciliary neurotropic factor


C-X-C motif chemokine ligand 12


Cytochrome P450 aromatase


Pearson product-moment correlation coefficient




Foetal bovine serum


Glyceraldehyde 3-phosphate dehydrogenase


Growth Differentiation factor 9


Green fluorescent protein


Germinal vesicle


Hematoxylin and Eosin


Insulin-like growth factor 1


Interleukin 10


Interleukin 1b


Keratinocyte growth factor




Leukemia inhibitory factor


Mesenchymal stem cells


Oogonial stem cells




Proliferating cell nuclear antigen


Premature ovarian insufficiency


Platelet-rich plasma


Transforming growth factor beta 1


TNF-related apoptosis-inducing ligand


Vascular endothelial growth factor


Very small embryonic-like stem cells



We thank Experimental Medicine Research and Application Unit (DETAB) at Kocaeli University for their assistance in our experimental work. This work was supported by the Scientific and Research Council of Turkey (TUBITAK) [grant number 114S398].

Compliance with Ethical Standards

Disclosure of Interest

The authors have no conflict of interest.


  1. 1.
    Woad, K. J., Watkins, W. J., Prendergast, D., & Shelling, A. N. (2006). The genetic basis of premature ovarian failure. Australian and New Zealand Journal of Obstetrics and Gynaecology, 46, 242–244.CrossRefGoogle Scholar
  2. 2.
    Shelling, A. N. (2010). Premature ovarian failure. Reproduction, 140, 633–641.CrossRefGoogle Scholar
  3. 3.
    Sheikhansari, G., Aghebati-Maleki, L., Nouri, M., Jadidi-Niaragh, F., & Yousefi, M. (2018). Current approaches for the treatment of premature ovarian failure with stem cell therapy. Biomedicine & Pharmacotherapy, 102, 254–262.CrossRefGoogle Scholar
  4. 4.
    Sukur, Y. E., Kivancli, I. B., & Ozmen, B. (2014). Ovarian aging and premature ovarian failure. Journal of The Turkish-German Gynecological Association, 15, 190–196.CrossRefGoogle Scholar
  5. 5.
    Bedoschi, G., Navarro, P. A., & Oktay, K. (2016). Chemotherapy-induced damage to ovary: Mechanisms and clinical impact. Future Oncology, 2, 2333–2344.CrossRefGoogle Scholar
  6. 6.
    Soleimani, R., Heytens, E., Darzynkiewicz, Z., & Oktay, K. (2011). Mechanisms of chemotherapy-induced human ovarian aging: Double strand DNA breaks and microvascular compromise. Aging, 3, 782–793.CrossRefGoogle Scholar
  7. 7.
    Zhou, L., Xie, Y., Li, S., et al. (2017). Rapamycin prevents cyclophosphamide-induced over-activation of primordial follicle pool through PI3K/Akt/mTOR signaling pathway in vivo. Journal of Ovarian Research, 10(1), 56.CrossRefGoogle Scholar
  8. 8.
    Yuksel, A., Bildik, G., Senbabaoglu, F., et al. (2015). The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells. Human Reproduction, 30, 2926–2935.Google Scholar
  9. 9.
    Galvez-Martin, P., Sabata, R., Verges, J., Zugaza, J. L., Ruiz, A., & Clares, B. (2016). Mesenchymal stem cells as therapeutics agents: Quality and environmental regulatory aspects. Stem Cells International, 2016, 9783408.CrossRefGoogle Scholar
  10. 10.
    Qiu, P., Bai, Y., Pan, S., Li, W., Liu, W., & Hua, J. (2013). Gender depended potentiality of differentiation of human umbilical cord mesenchymal stem cells into oocyte-like cells in vitro. Cell Biochemistry and Function, 31, 365–373.CrossRefGoogle Scholar
  11. 11.
    Bukovsky, A. (2011). Immune maintenance of self in morphostasis of distinct tissues, tumor growth, and regenerative medicine. Scandinavian Journal of Immunology, 73, 159–189.CrossRefGoogle Scholar
  12. 12.
    Pourgholaminejad, A., Aghdami, N., Baharvand, H., & Moazzeni, S. M. (2016). The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells. Cytokine, 85, 51–60.CrossRefGoogle Scholar
  13. 13.
    Caplan, A. I., & Correa, D. (2011). The MSC: An injury drugstore. Cell Stem Cell, 9, 11–15.CrossRefGoogle Scholar
  14. 14.
    Gobbi, A., & Fishman, M. (2016). Platelet-rich plasma and bone marrow-derived mesenchymal stem cells in sports medicine. Sports Medicine and Arthroscopy Review, 24, 69–73.CrossRefGoogle Scholar
  15. 15.
    Sills, E. S., Rickers, N. S., Li, X., & Palermo, G. D. (2018). First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated autologous platelet rich plasma. Gynecological Endocrinology, 2018, 1–5.Google Scholar
  16. 16.
    Wang, S., Yu, L., Sun, M., et al. (2013). The therapeutic potential of umbilical cord mesenchymal stem cells in mice premature ovarian failure. BioMed Research International, 2013, 690491.Google Scholar
  17. 17.
    Liu, T., Huang, Y., Guo, L., Cheng, W., & Zou, G. (2012). CD44+/CD105+ human amniotic fluid mesenchymal stem cells survive and proliferate in the ovary long-term in a mouse model of chemotherapy-induced premature ovarian failure. International Journal of Medical Sciences, 9, 592–602.CrossRefGoogle Scholar
  18. 18.
    Johnson, J., Bagley, J., Skaznik-Wikiel, M., et al. (2005). Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell, 122, 303–315.CrossRefGoogle Scholar
  19. 19.
    Lee, H. J., Selesniemi, K., Niikura, Y., et al. (2007). Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure. Journal of Clinical Oncology, 25, 3198–3204.CrossRefGoogle Scholar
  20. 20.
    Santiquet, N., Vallières, L., Pothier, F., Sirard, M. A., Robert, C., & Richard, F. (2012). Transplanted bone marrow cells do not provide new oocytes but rescue fertility in female mice following treatment with chemotherapeutic agents. Cellular Reprogramming, 14, 123–129.CrossRefGoogle Scholar
  21. 21.
    Wang, Z., Wang, Y., Yang, T., Li, J., & Yang, X. (2017). Study of the reparative effects of menstrual-derived stem cells on premature ovarian failure in mice. Stem Cell Research & Therapy, 8, 11.CrossRefGoogle Scholar
  22. 22.
    Sun, M., Wang, S., Li, Y., et al. (2013). Adipose-derived stem cells improved mouse ovary function after hemotherapy-induced ovary failure. Stem Cell Research & Therapy, 4, 80.CrossRefGoogle Scholar
  23. 23.
    Takehara, Y., Yabuuchi, A., Ezoe, K., et al. (2013). The restorative effects of adipose-derived mesenchymal stem cells on damaged ovarian function. Laboratory Investigation, 93, 181–193.CrossRefGoogle Scholar
  24. 24.
    Demirayak, B., Yüksel, N., Çelik, O. S., et al. (2016). Effect of bone marrow and adipose tissue-derived mesenchymal stem cells on the natural course of corneal scarring after penetrating injury. Experimental Eye Research, 151, 227–235.CrossRefGoogle Scholar
  25. 25.
    Dominici, M., Le Blanc, K., Mueller, I., Slaper-Kortenbach, I., Marini, F., & Krause, D. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315–317.CrossRefGoogle Scholar
  26. 26.
    Ozbek, E., Adas, G., Otunctemur, A., et al. (2015). Role of mesenchymal stem cells transfected with vascular endothelial growth factor in maintaining renal structure and function in rats with unilateral ureteral obstruction. Experimental and Clinical Transplantation, 13, 262–272.Google Scholar
  27. 27.
    Öksüz, S., Alagöz, M. Ş., Karagöz, H., et al. (2015). Comparison of treatments with local mesenchymal stem cells and mesenchymal stem cells with increased vascular endothelial growth factor expression on irradiation injury of expanded skin. Annals of Plastic Surgery, 75, 219–230.CrossRefGoogle Scholar
  28. 28.
    Adas, G., Koc, B., Adas, M., et al. (2016). Effects of mesenchymal stem cells and VEGF on liver regeneration following major resection. Langenbeck's Archives of Surgery, 401, 725–740.CrossRefGoogle Scholar
  29. 29.
    Nagata, M. J., Messora, M. R., Furlaneto, F. A., et al. (2010). Effectiveness of two methods for preparation of autologous platelet-rich plasma: An experimental study in rabbits. European Journal of Dentistry, 4, 395–402.Google Scholar
  30. 30.
    Cakici, C., Buyrukcu, B., Duruksu, G., et al. (2013). Recovery of fertility in azoospermia rats after injection of adipose-tissue-derived mesenchymal stem cells: The sperm generation. BioMed Research International, 2013, 529589.CrossRefGoogle Scholar
  31. 31.
    Sun, X., Su, Y., He, Y., et al. (2015). New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators. Cell Cycle, 14, 721–731.CrossRefGoogle Scholar
  32. 32.
    Flaws, J. A., Abbud, R., Mann, R. J., et al. (1997). Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary. Biological Reproduction, 57, 1233–1237.CrossRefGoogle Scholar
  33. 33.
    Pedersen, T. (1970). Determination of follicle growth rate in the ovary of the immature mouse. Journal of Reproduction and Fertility, 21, 81–93.CrossRefGoogle Scholar
  34. 34.
    Detre, S., Saclani Jotti, G., & Dowsett, M. A. J. (1995). A "quickscore" method for immunohistochemical semiquantitation: Validation for oestrogen receptor in breast carcinomas. Clinical Pathology, 48, 876–878.CrossRefGoogle Scholar
  35. 35.
    Epstein, R. J. (1990). Drug-induced DNA damage and tumor chemosensitivity. Journal of Clinical Oncology, 8, 2062–2084.CrossRefGoogle Scholar
  36. 36.
    Kilic, S., Pinarli, F., Ozogul, C., Tasdemir, N., Naz Sarac, G., & Delibasi, T. (2014). Protection from cyclophosphamide-induced ovarian damage with bone marrow-derived mesenchymal stem cells during puberty. Gynecological Endocrinology, 30, 135–140.CrossRefGoogle Scholar
  37. 37.
    Song, D., Zhong, Y., Qian, C., et al. (2016). Human umbilical cord mesenchymal stem cells therapy in cyclophosphamide-induced premature ovarian failure rat model. BioMed Research International, 2016, 2517514.Google Scholar
  38. 38.
    Kerr, J. B., Brogan, L., Myers, M., et al. (2012). The primordial follicle reserve is not renewed after chemical or gamma-irradiation mediated depletion. Reproduction, 143, 469–476.CrossRefGoogle Scholar
  39. 39.
    Lei, L., & Spradling, A. C. (2013). Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles. Proceedings of the National Academy of Sciences, 110, 8585–8590.CrossRefGoogle Scholar
  40. 40.
    Yuan, J., Zhang, D., Wang, L., et al. (2013). No evidence for neo-oogenesis may link to ovarian senescence in adult monkey. Stem Cells, 31, 2538–2550.CrossRefGoogle Scholar
  41. 41.
    Virant-Klun, I., Skutella, T., Bhartiya, D., & Jin, X. (2013). Stem cells in reproductive tissues: From the basics to clinics. BioMed Research International, 2013, 357102.Google Scholar
  42. 42.
    White, Y. A., Woods, D. C., Takai, Y., Ishihara, O., Seki, H., & Tilly, J. L. (2012). Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nature Medicine, 18, 413–421.CrossRefGoogle Scholar
  43. 43.
    Johnson, J., Canning, J., Kaneko, T., Pru, J. K., & Tilly, J. L. (2004). Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature, 428, 145–150.CrossRefGoogle Scholar
  44. 44.
    Bukovsky, A., Svetlikova, M., & Caudle, M. R. (2005). Oogenesis in cultures derived from adult human ovaries. Reproductive Biology and Endocrinology, 3, 17.CrossRefGoogle Scholar
  45. 45.
    Bukovsky, A. (2011). Ovarian stem cell niche and follicular renewal in mammals. The Anatomical Record, 294, 1284–1306.CrossRefGoogle Scholar
  46. 46.
    Bukovsky, A. (2015). Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases. Reproductive Biology and Endocrinology, 13, 10.CrossRefGoogle Scholar
  47. 47.
    Hayama, T., Yamaguchi, T., Kato-Itoh, M., et al. (2014). Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells. Biology of Reproduction, 91, 89.CrossRefGoogle Scholar
  48. 48.
    Morgan, S., Anderson, R. A., Gourley, C., Wallace, W. H., & Spears, N. (2012). How do chemotherapeutic agents damage the ovary? Human Reproduction Update, 18, 525–535.CrossRefGoogle Scholar
  49. 49.
    Young, J. M., & McNeilly, A. S. (2010). Theca: The forgotten cell of the ovarian follicle. Reproduction, 140, 489–504.CrossRefGoogle Scholar
  50. 50.
    Virant-Klun, I. (2015). Postnatal oogenesis in humans: A review of recent findings. Stem Cells Cloning, 8, 49–60.Google Scholar
  51. 51.
    Eppig, J. J. (2001). Oocyte control of ovarian follicular development and function in mammals. Reproduction, 122, 829–838.CrossRefGoogle Scholar
  52. 52.
    Bukovsky, A., & Caudle, M. R. (2012). Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment and a clinical trial. Reproductive Biology and Endocrinology, 10, 97.CrossRefGoogle Scholar
  53. 53.
    Frese, L., Dijkman, P. E., & Hoerstrup, S. P. (2016). Adipose tissue-derived stem cells in regenerative medicine. Transfusion Medicine and Hemotherapy, 43, 268–274.CrossRefGoogle Scholar
  54. 54.
    Dewailly, D., Robin, G., Peigne, M., Decanter, C., Pigny, P., & Catteau-Jonard, S. (2016). Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Human Reproduction Update, 22, 709–724.CrossRefGoogle Scholar
  55. 55.
    Virant-Klun, I., Skutella, T., Stimpfel, M., & Sinkovec, J. (2011). Ovarian surface epithelium in patients with severe ovarian infertility: A potential source of cells expressing markers of pluripotent/multipotent stem cells. Journal of Biomedicine and Biotechnology, 2011, 381928.CrossRefGoogle Scholar
  56. 56.
    Huda, F., Fan, Y., Suzuki, M., et al. (2016). Fusion of human fetal mesenchymal stem cells with "degenerating" cerebellar neurons in spinocerebellar Ataxia type 1 model mice. PLoS One, 11, e0164202.CrossRefGoogle Scholar
  57. 57.
    Binelli, M., & Murphy, B. D. (2010). Coordinated regulation of follicle development by germ and somatic cells. Reproduction Fertility and Development, 22(1), 12.CrossRefGoogle Scholar
  58. 58.
    Shaikh, A., Anand, S., Kapoor, S., Ganguly, R., & Bhartiya, D. (2017). Mouse bone marrow VSELs exhibit differentiation into three embryonic germ lineages and Germ & Hematopoietic Cells in culture. Stem Cell Reviews and Reports, 13, 202–216.CrossRefGoogle Scholar
  59. 59.
    Xia, X., Wang, T., Yin, T., Yan, L., Yan, J., & Lu, C. (2015). Mesenchymal stem cells facilitate in vitro development of human Preantral follicle. Reproductive Sciences, 22, 1367–1376.CrossRefGoogle Scholar
  60. 60.
    Pangas, S. A. (2012). Regulation of the ovarian reserve by members of the transforming growth factor beta family. Molecular Reproduction and Development, 79, 666–679.CrossRefGoogle Scholar
  61. 61.
    Chen, Y. C., Chang, H. M., Cheng, J. C., Tsai, H. D., Wu, C. H., & Leung, P. C. (2015). Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells. Human Reproduction, 30, 2190–2201.CrossRefGoogle Scholar
  62. 62.
    Al-Samerria, S., Al-Ali, I., McFarlane, J. R., & Almahbobi, G. (2015). The impact of passive immunisation against BMPRIB and BMP4 on follicle development and ovulation in mice. Reproduction, 149, 403–411.CrossRefGoogle Scholar
  63. 63.
    Dunlop, C. E., & Anderson, R. A. (2014). The regulation and assessment of follicular growth. Scandinavian Journal of Clinical and Laboratory Investigation, 244, 13–17.CrossRefGoogle Scholar
  64. 64.
    Park, E. S., Woods, D. C., & Tilly, J. L. (2013). Bone morphogenetic protein 4 promotes mammalian oogonial stem cell differentiation via Smad1/5/8 signaling. Fertility and Sterility, 100, 1468–1475.CrossRefGoogle Scholar
  65. 65.
    Bayne, R. A., Donnachie, D. J., Kinnell, H. L., Childs, A. J., & Anderson, R. A. (2016). BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2. Molecular Human Reproduction, 22, 622–633.CrossRefGoogle Scholar
  66. 66.
    Tan, S., Feng, B., Yin, M., et al. (2017). Stromal Senp1promotes mouse early folliculogenesis by regulating BMP4 expression. Cell & Bioscience, 7, 36.CrossRefGoogle Scholar
  67. 67.
    Polat, I. M., Alçiğir, E., Pekcan, M., et al. (2015). Characterization of transforming growth factor beta superfamily, growth factors, transcriptional factors, and lipopolysaccharide in bovine cystic ovarian follicles. Theriogenology, 84, 1043–1052.CrossRefGoogle Scholar
  68. 68.
    Vural, F., Vural, B., Doğer, E., Çakıroğlu, Y., & Çekmen, M. (2016). Perifollicular blood flow and its relationship with endometrial vascularity, follicular fluid EG-VEGF, IGF-1, and inhibin-a levels and IVF outcomes. Journal of Assisted Reproduction and Genetics, 33, 1355–1362.CrossRefGoogle Scholar
  69. 69.
    Bukovsky, A., Keenan, J. A., Caudle, M. R., Wimalasena, J., Upadhyaya, N. B., & Van Meter, S. E. (1995). Immunohistochemical studies of the adult human ovary: Possible contribution of immune and epithelial factors to folliculogenesis. American Journal of Reproductive Immunology, 33, 323–340.CrossRefGoogle Scholar
  70. 70.
    Jasti, S., Warren, B. D., McGinnis, L. K., Kinsey, W. H., Petroff, B. K., & Petroff, M. G. (2012). The autoimmune regulator prevents premature reproductive senescence in female mice. Biology of Reproduction, 86, 110.CrossRefGoogle Scholar
  71. 71.
    Ye, H., Zheng, T., Li, W., et al. (2017). Ovarian stem cell nests in reproduction and ovarian aging. Cellular Physiology and Biochemistry, 43, 1917–1925.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Birol Vural
    • 1
    Email author
  • Gokhan Duruksu
    • 2
    • 3
  • Fisun Vural
    • 4
  • Merve Gorguc
    • 2
  • Erdal Karaoz
    • 5
  1. 1.School of Medicine, Department of Obstetrics and Gynecology, Reproductive Endocrinology and Infertility and Assisted Reproductive Technology (ART)Kocaeli UniversityKocaeliTurkey
  2. 2.Graduate School of Health Sciences, Department of Stem Cell, Center for Stem Cells and Gene Therapies Research and PracticeKocaeli UniversityKocaeliTurkey
  3. 3.Center for Stem Cells and Gene Therapies Research and PracticeKocaeli UniversityKocaeliTurkey
  4. 4.Department of Obstetrics and GynecologyUniversity of Health Sciences- Haydarpasa Numune Training and Research HospitalIstanbulTurkey
  5. 5.Department of Histology and EmbryologyIstinye UniversityIstanbulTurkey

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