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Clinical and Experimental Obstetrics & Gynecology  2020, Vol. 47 Issue (3): 383-390    DOI: 10.31083/j.ceog.2020.03.5297
Original Research Previous articles | Next articles
Mechanistic study of vitamin C attenuation of endometriotic fibrosis
X.J. Shi1, 2, L.B. Shi1, S.Y. Zhang1, *()
1Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, P. R. China
2The First Affiliated Hospital of Jiaxing University, Jiaxing, Zhejiang, P. R. China
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Background: Endometriosis is a common disease in females that seriously affects quality of life. The principal pathological process of endometriosis is pelvic inflammation, and local and peripheral fibrosis. Treatment of endometriosis requires both pharmacological and surgical approaches. Vitamin C can scavenge oxygen free radicals and thus accelerate repair of damaged endometrium. This aim of this study was to investigate whether vitamin C can reduce fibrosis in endometriotic lesions. Methods: After establishing a rat model of endometriosis, vitamin C solution (vitamin C group) or physiological saline solution (control group) was injected into the abdominal cavity. We compared the indices of fibrotic endometriotic lesions between the two groups. Results: The volume of endometriotic lesions and degree of fibrosis observed in rats within the vitamin C group was significantly reduced compared with those observed in the control group. Immunohistochemistry showed that transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), α-SMA, and collagen type I staining in lesions of the vitamin C group was significantly less than that observed in lesions from the control group (P < 0.05). Quantitative, real-time PCR (RT-PCR) determined that relative mRNA expression levels of TGF-β1, CTGF, α-SMA, and collagen type I in lesions obtained from the vitamin C group were significantly lower than levels measured in lesions obtained from animals in the control group. Conclusion: Vitamin C can reduce the volume of endometriotic lesions and inhibit fibrosis of lesions in rats. This study supports the use of vitamin C in the treatment of endometriosis.

Key words:  Vitamin C      Endometriosis      Fibrosis     
Submitted:  18 June 2019      Accepted:  03 September 2019      Published:  15 June 2020     
81671435/National Natural Science Foundation of China
2017C03022/Key Research and Development Program of Zhejiang Province
*Corresponding Author(s):  SONGYING ZHANG     E-mail:

Cite this article: 

X.J. Shi, L.B. Shi, S.Y. Zhang. Mechanistic study of vitamin C attenuation of endometriotic fibrosis. Clinical and Experimental Obstetrics & Gynecology, 2020, 47(3): 383-390.

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Figure 1.  — Construction of rat endometriosis model by endometrial transplantation. (A) Macroscopic features of transplanted endometrium. (B) Autologous endometrium transplanted on the abdominal wall of rat. (C) Endometriotic lesion formed after 2 weeks of transplantation.

Figure 2.  — Representative photomicrograph of the cystic walls from rat endometriotic lesions after Masson’s staining (A: 100 ×; B: 200 ×).

Table 1  — Comparison of body weight between the two experimental groups
Control Group (n = 7) Vitamin C Group (n = 7) P Value
Body Weight (g) Before perfusion 232.02 ± 17.44 231.94 ± 16.54 > 0.05
Body Weight (g) After perfusion 233.85 ± 17.64 234.28 ± 16.37 > 0.05
Table 2  — Comparison of endometriotic lesions between two experimental groups
Control Group (n = 14) Vitamin C Group (n = 14)
Mean volume of endometriotic lesions (mm3) 246.00 40.917
P Value < 0.05 < 0.05
Figure 3.  — Size comparison of endometriotic lesions between vitamin C and control groups. (A and B) Representative endometriotic lesion of vitamin C group. (C) Representative endometriotic lesion of control group.

Figure 4.  — Comparison the size of endometriotic lesions between control group and vitamin C group.

Table 3  — Comparison of fibrosis scores in endometriotic lesions in vitamin C and control group rats
Control Group (n = 5) Vitamin C Group (n = 4)
Fibrosis score 2.40 1.83
P Value < 0.05
Figure 5.  — Comparison of the degree of fibrosis between the two experimental groups by Masson’s staining. (A and B) and (C and D) represent Masson’s staining of endometriotic lesions in control group and vitamin C group respectively. (A, C: 100 ×; B, D: 200 ×).

Figure 6.  — Comparison the expression of Collagen I, α-SMA, CTGF and TGF-β1 between control and vitamin C groups by immuniohistochemistry. (A and B) Collagen I staining of control group; (C and D) Collagen I staining of Vitamin C group. (E and F) and (G and H) represent α-SMA staining of control group and vitamin C group respectively. (I and J) and (K and L) demonstrate CTGF staining of control group and vitamin C group, respectively. (M and N) and (O and P) represent TGF-β1 staining of control group and vitamin C group, respectively. (A,C,E,G,I,K,M,O: 100×; B,D,F,H,J,L,N,P: 200×).

Figure 7.  — Relative mRNA expression levels of the fibrotic markers Collagen I, α-SMA, CTGF, TGF-b1 in endometriotic lesions from control and vitamin C group.

[1] Marschalek J., Ott J., Husslein H., Kuessel L., Elhenicky M., Mayerhofer K., Franz M.B.: “The impact of GnRH agonists in patientswith endometriosis on prolactin and sex hormone levels: a pilotstudy”. Eur. J. Obstet. Gynecol. Reprod. Biol., 2015, 195, 156.
doi: 10.1016/j.ejogrb.2015.10.016 pmid: 26546756
[2] Robin B., Planeix F., Sastre-Garau X., Pichon C., Olesen T.K., Gogusev J., Ghinea N.: “Follicle-Stimulating Hormone Receptor Expressionin Endometriotic Lesions and the Associated Vasculature: AnImmunohistochemical Study”. Reprod Sci., 2016, 23, 885.
doi: 10.1177/1933719115623647 pmid: 26704526
[3] Crosignani P., Olive D., Bergqvist A., Luciano A.: “Advances in themanagement of endometriosis: an update for clinicians”. Hum. Reprod.Update, 2006, 12, 179.
[4] Giudice L.C.: “Clinical practice. Endometriosis”. N. Engl. J. Med., 2010, 362, 2389.
doi: 10.1056/NEJMcp1000274 pmid: 20573927
[5] Zorbas K.A., Economopoulos K.P., Vlahos N.F.: “Continuous versuscyclic oral contraceptives for the treatment of endometriosis: asystematic review”. Arch. Gynecol. Obstet., 2015, 292, 37.
doi: 10.1007/s00404-015-3641-1 pmid: 25644508
[6] Omwandho C.O., Konrad L., Halis G., Oehmke F., Tinneberg H.R.: “Role of TGF-betas in normal human endometrium and endometriosis”. Hum. Reprod., 2010, 25, 101.
doi: 10.1093/humrep/dep382 pmid: 19892717
[7] Keenan J.A., Williams-Boyce P.K., Massey P.J., Chen T.T., Caudle M.R., Bukovsky A.: “Regression of endometrial explants in a ratmodel of endometriosis treated with the immune modulators loxoribineand levamisole”. Fertil. Steril., 1999, 72, 135.
doi: 10.1016/s0015-0282(99)00157-0 pmid: 10428162
[8] Anaf V., Simon P., El Nakadi I., Fayt I., Buxant F., Simonart T., et al.: “Relationship between endometriotic foci and nerves in rectovaginalendometrioticnodules”. Hum. Reprod., 2000, 15, 1744.
doi: 10.1093/humrep/15.8.1744 pmid: 10920097
[9] Matarese G., De Placido G., Nikas Y., Alviggi C.: “Pathogenesis ofendometriosis: natural immunity dysfunction or autoimmune disease”. Trends Mol. Med., 2003, 9, 223.
doi: 10.1016/s1471-4914(03)00051-0 pmid: 12763528
[10] Hart R.J., Hickey M., Maouris P., Buckett W.: “Excisional surgeryversus ablative surgery for ovarian endometriomata”. CochraneDatabase Syst. Rev., 2008, 2, CD004992.
[11] Zhang C., Gao L., Yi Y., Han H., Cheng H., Ye X., et al.: “AdenosineTriphosphate Regresses Endometrial Explants in a Rat Model of Endometriosis”. Reprod. Sci., 2016, 23, 924.
doi: 10.1177/1933719115625847 pmid: 26887426
[12] Hagemann I.S., Hagemann A.R., LiVolsi V.A., Montone K.T., Chu C.S.: “Risk of occult malignancy in morcellated hysterectomy: a caseseries”. Int. J. Gynecol. Pathol., 2011, 30, 476.
doi: 10.1097/PGP.0b013e3182107ecf
[13] Vercellini P., Crosignani P.G., Abbiati A., Somigliana E., ViganÚ P., Fedele L.: “The effect of surgery for symptomatic endometriosis: theother side of the story”. Hum. Reprod. Update, 2009, 15, 177.
doi: 10.1093/humupd/dmn062 pmid: 19136455
[14] Donnez J., Nisolle M., Gillet N., Smets M., Bassil S., Casanas-Roux F.: “Large ovarianendometriomas”. Hum. Reprod., 1996, 11, 641.
doi: 10.1093/humrep/11.3.641 pmid: 8671283
[15] Zhang Q., Duan J., Liu X., Guo S.W.: “Platelets drive smooth musclemetaplasia and fibrogenesis in endometriosis through epithelial-mesenchymaltransition and fibroblast-to-myofibroblasttransdifferentiation”. Mol. Cell. Endocrinol., 2016, 428, 1.
doi: 10.1016/j.mce.2016.03.015 pmid: 26992563
[16] Kitajima M., Defrère S., Dolmans M.M., Colette S., Squifflet J., VanLangendonckt A., Donnez J.: “Endometriomas as a possible cause ofreduced ovarian reserve in women with endometriosis”. Fertil.Steril., 2011, 96, 685.
[17] Kim J.Y., Jee B.C., Suh C.S., Kim S.H.: “Preoperative serum antimullerianhormone level in women with ovarian endometrioma andmature cystic teratoma”. Yonsei Med. J., 2013, 54, 921.
doi: 10.3349/ymj.2013.54.4.921 pmid: 23709427
[18] Shi L.B., Zhou F., Zhu H.Y., Huang D., Jin X.Y., Li C., et al.: “Transforminggrowth factor beta1 from endometriomas promotes fibrosisin surrounding ovarian tissues via Smad2/3 signaling”. Biol. Reprod., 2017, 97, 873.
doi: 10.1093/biolre/iox140 pmid: 29136085
[19] Darling A.M., Chavarro J.E., Malspeis S., Harris H.R., Missmer S.A. “A prospective cohort study of Vitamins B, C, E, and multivitamin intakeand endometriosis”. J. Endometr., 2013, 5, 17.
doi: 10.5301/je.5000151 pmid: 24511373
[20] Mier-Cabrera J., Genera-GarcÌa M., De la Jara-DÌaz J., Perichart-Perera O., Vadillo-Ortega F., Hernndez-Guerrero C.: “Effect of vitaminsC and E supplementation on peripheral oxidative stressmarkers and pregnancy rate in women with endometriosis”. Int. J. Gynaecol. Obstet., 2008, 100, 252.
doi: 10.1016/j.ijgo.2007.08.018 pmid: 18005966
[21] Erten O.U., Ensari T.A., Dilbaz B., Cakiroglu H., Altinbas S.K., Çay dere M., Goktolga U.: “Vitamin C is effective for the prevention andregression of endometriotic implants in an experimentally inducedrat model of endometriosis”. Taiwan J. Obstet., Gynecol., 2016, 55(2):251-7.
doi: 10.1016/j.tjog.2015.07.004
[22] Dooley S, ten Dijke P.: “TGF-β in progression of liver disease”. CellTissue Res., 2012, 347, 245.
[23] Pinzani M., Macias-Barragan J.: “Update on the pathophysiology ofliver fibrosis”. Expert Rev. Gastroenterol. Hepatol., 2010, 4, 459.
doi: 10.1586/egh.10.47 pmid: 20678019
[24] Zhou F., Shi L.B., Zhang S.Y.: “Ovarian Fibrosis: A Phenomenon ofConcern”. Chin. Med. J. (Engl.), 2017, 130, 365.
[25] Lee H.J., Kim H., Ku S.Y., Kim S.H., Kim J.G.: “Transforminggrowth factor-β1 gene polymorphisms in Korean women with endometriosis”. Am. J. ReprodImmunol., 2011, 66, 428.
[26] Kong W., Yang H., He L., Zhao J.J., Coppola D., Dalton W.S., Cheng J.Q.: “MicroRNA-155 is regulated by the transforming growth factorbeta/Smad pathway and contributes to epithelial cell plasticity bytargeting RhoA”. Mol. Cell. Biol., 2008, 28, 6773.
doi: 10.1128/MCB.00941-08 pmid: 18794355
[27] Mason R.M.: “Fell-Muir lecture: Connective tissue growth factor(CCN2) — a pernicious and pleiotropic player in the development ofkidney fibrosis”. Int. J. Exp Pathol., 2013, 94, 1.
doi: 10.1111/j.1365-2613.2012.00845.x
[28] Romão L.F., Mendes F.A., Feitosa N.M., Faria J.C., Coelho-Aguiar J.M., de Souza J.M., et al.: “Connective tissue growth factor(CTGF/CCN2) is negatively regulated during neuron-glioblastomainteraction”. PLoS One, 2013, 8, e55605.
doi: 10.1371/journal.pone.0055605 pmid: 23383241
[29] Alfaro MP, Deskins DL, Wallus M, DasGupta J, Davidson JM, Nanney LB., et al.: “A physiological role for connective tissue growthfactor in early wound healing”. Lab. Invest., 2013, 93, 81.
doi: 10.1038/labinvest.2012.162 pmid: 23212098
[30] Lai K.B., Sanderson J.E., Yu C.M.: “The regulatory effect of norepinephrineon connective tissue growth factor (CTGF) and vascularendothelial growth factor (VEGF) expression in cultured cardiac fibroblasts”. Int. J. Cardiol., 2013, 163, 183.
doi: 10.1016/j.ijcard.2011.06.003
[31] Chang J.Z., Yang W.H., Deng Y.T., Chen H.M., Kuo M.Y.: “EGCGblocks TGFβ1-induced CCN2 by suppressing JNK and p38 in buccalfibroblasts”. Clin. Oral Investig., 2013, 17, 455.
doi: 10.1007/s00784-012-0713-5 pmid: 22415218
[32] Rodrigues da Silva M., Schapochnik A., Peres Leal M., Esteves J., Bichels Hebeda C., Sandri S., et al.: “Beneficial effects of ascorbicacid to treat lung fibrosis induced by paraquat”. PLoS One, 2018, 13, e0205535.
doi: 10.1371/journal.pone.0205535 pmid: 30395570
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[15] P. Fuchs, I. Czech, A. Fuchs, J. Sikora. Use of neutral argon plasma in the treatment of endometriosis initial findings[J]. Clinical and Experimental Obstetrics & Gynecology, 2019, 46(3): 434-436.
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