The Effect of Erythropoietin on Ovarian Congestion during Ischemia Reperfusion Injury in Rats
C. Τsompos1*, C. Panoulis2, K Τοutouzas3, A. Triantafyllou4, G. Ζografos5 and A. Papalois6
1Consultant A, Department of Obstetrics & Gynecology, Mesolongi County Hospital, Etoloakarnania, Greece
2Assistant Professor, Department of Obstetrics & Gynecology, Aretaeio Hospital, Athens University, Attiki, Greece
3Assistant Professor, Department of Surgery, Ippokrateio General Hospital, Athens University, Attiki, Greece
4Associate Professor, Department of Biologic Chemistry, Athens University, Attiki, Greece
5Professor, Department of Surgery, Ippokrateio General Hospital, Athens University, Attiki, Greece
6Director, Exprerimental Research Center, ELPEN Pharmaceuticals Co. Inc. S.A., Greece
*Corresponding author: Tsompos Constantinos, Department of Obstetrics & Gynecology, Mesolongi County Hospital, Nafpaktou Street, Mesolongi 30200, Etoloakarnania, Greece, Tel: 00302631360237 & 00306946674264, Fax: 00302106811215, E-mail: Constantinostsompos@yahoo.com
Int J Womens Health Wellness, IJWHW-2-010, (Volume 2, Issue 1), Research Article; ISSN: 2474-1353
Received: December 28, 2015 | Accepted: January 22, 2016 | Published: January 25, 2016
Citation: Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos G, et al. (2016) The Effect of Erythropoietin on Ovarian Congestion during Ischemia Reperfusion Injury in Rats. Int J Womens Health Wellness 2:010. 10.23937/2474-1353/1510010
Copyright: © 2016 Τsompos C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Background: This experimental study examined the effect of erythropoietin (epoetin alfa) on rat model and particularly in an ovarian ischemia reperfusion (IR) protocol. The effects of that molecule were studied pathologically using mean ovarian congestion (OC) lesions.
Materials and methods: 40 rats of mean weight 247.7 g, 16-18 weeks old, were used in the study. OC lesions were evaluated after 45 min ischemia, at 60 min (groups A and C) and at 120 min (groups B and D) of reperfusion. Erythropoietin was administered only in groups C and D.
Results: Epo administration kept non-significantly increased the OC scores without lesions (p = 0.2954). Reperfusion time kept non-significantly increased the OC scores without lesions (p = 0.8063). However, Epo administration and reperfusion time together kept non-significantly increased the OC scores without lesions (p = 0.3882).
Conclusions: Epo administration, reperfusion time and their interaction kept non-significantly increased the OC scores. Epo short-term restored the congestive lesions from significant to non-significant level.
Ischemia, Erythropoietin, Ovarian congestion, Reperfusion
Permanent or transient damage with serious implications on adjacent organs and certainly on patients’ health may be due to tissue ischemia and reperfusion (IR). Important progress has been made regarding the usage of erythropoietin (Epo) Epoetin alfa [EPREX (151118) API] of Janssen-Cilag Pty Ltd with CAS Registry Number: 113427-24-0 which is human erythropoietin produced in cell culture using recombinant DNA technology, authorized by the European Medicines Agency on 28 August 2007. Although Epo managed this kind of damages, satisfactory answers have not been given yet to fundamental questions, as, by what velocity this factor acts, when it should be administered and at what dosage. The particularly satisfactory action of Epo in stem blood cells recovery has been noted in several performed experiments. However, just few relative reports were found concerning Epo trial in IR experiments, not covering completely this particular matter. A meta-analysis of 18 published seric variables, coming from the same experimental setting, tried to provide a numeric evaluation of the Epo efficacy at the same endpoints (Table 1). Furthermore, several publications addressed trials of other similar molecules of growth factors to which the studied molecule also belongs to.
The aim of this experimental study was to examine the effect of Epo on rat model and particularly in an ovarian IR protocol. The kind of effects that molecule provokes, were studied by evaluating mean ovarian congestion (OC) lesions. Kim J et al. experienced  an extremely rare case of acute abdomen pain induced by OC triggered by the fallopian tube accompanying a paratubal cyst coiling around the utero-ovarian ligament. Kaido Y et al. revealed  that an elongated right fallopian tube accompanied by a paratubal cyst coiling tightly 2.5 times round the right ovary, caused apparent congestion and enlargement of the right ovary in a pregnant woman for her right lower abdominal pain at 30 weeks of gestation. Soon after the congested right ovary was released from the coiling of the fallopian tube, the congestion subsided. Yassin S et al. associated  OC changes with injectable contraceptives and related them with cycle control.
Materials and Methods
This experimental study was licensed by Veterinary Address of East Attiki Prefecture under 3693/12-11-2010 & 14/10-1-2012 decisions. Everything needed for the study including consumables, equipment and substances were a courtesy of Experimental Research Center of ELPEN Pharmaceuticals Co. Inc. S.A. at Pikermi, Attiki. Accepted standards of humane animal care were adopted for Albino female Wistar rats. Normal housing in laboratory 7 days before the experiment included ad libitum diet. Post-experimental awakening and preservation of the rodents was not permitted, even if euthanasia was needed. They were randomly delivered to four experimental groups by 10 animals in each one. Ischemia for 45 min followed by reperfusion for 60 min (group A). Ischemia for 45 min followed by reperfusion for 120 min (group B). Ischemia for 45 min followed by immediate Epo intravenous (IV) administration and reperfusion for 60 min (group C). Ischemia for 45 min followed by immediate Epo IV administration and reperfusion for 120 min (group D). The molecule Epo dosage was 10 mg/Kg body weight of animals, diluted in 2 ml water of injection.
The detailed preceded pre narcotic and general anesthesiologic techniques of animals are described in related references [4-6]. Oxygen supply, electrocardiogram and acidiometry were continuously provided during whole experiment performance.
The protocol of IR was followed. Ischemia was caused by laparotomic forceps clamping inferior aorta over renal arteries for 45 min. Reperfusion was induced by removing the clamp and reestablishment of inferior aorta patency. The molecules were administered at the time of reperfusion, through catheterized inferior vena cava. The OC evaluations were performed at 60 min of reperfusion (for groups A and C) and at 120 min of reperfusion (for groups B and D). The mean weight of the forty (40) female Wistar albino rats used was 247.7 g [Std. Dev: 34.99172 g], with min weight ≥ 165 g and max weight ≤ 320 g. Rats’ weight could be potentially a confusing factor, e.g. the more obese rats to have higher OC score lesions (Table 2). This suspicion was investigated. Also, detailed pathological  study and grading of OC findings was performed by scores, this is: 0 lesions were not found, 1 mild lesion was found, 2 moderate lesions were found and 3 serious lesions were found. The previous grading was transformed as follows: (0-0.499) without lesions, (0.5-1.499) mild lesions, (1.5-2.499) moderate lesions and (2.5-3) serious lesions damage, because the study concerns score ranges rather than point scores.
Table 2: Weight and ovarian congestion (OC) score mean levels and Std. Dev. of groups. View Table 2
Model of ischemia-reperfusion injury
Control groups:20 control rats (mean mass 252.5 g [Std. Dev: 39.31988 g]) experienced ischemia for 45 min followed by reperfusion.
Group A: Reperfusion lasted for 60 min (n = 10 controls rats) mean mass 243 g [Std. Dev: 45.77724 g], mean moderate OC lesions score 1.6 [Std. Dev: 1.074968] (Table 2).
Group B: Reperfusion lasted for 120 min (n = 10 controls rats) mean mass 262 g [Std. Dev: 31.10913 g], mean moderate OC lesions score 1.9 [Std. Dev: 0.9944289] (Table 2).
Erythropoietin group: 20 Epo rats (mean mass 242.9 g [Std. Dev: 30.3105 g]) experienced ischemia for 45 min followed by reperfusion in the beginning of which 10 mg Epo /kg body weight were IV administered.
Group C: Reperfusion lasted for 60 min (n = 10 Epo rats) mean mass 242.8 g [Std. Dev: 29.33636 g], mean moderate OC lesions score 2.1 [Std. Dev: 0.5676462] (Table 2).
Group D: Reperfusion lasted for 120 min (n = 10 Epo rats) mean mass 243 g [Std. Dev: 32.84644 g], mean moderate OC lesions score 2 [Std. Dev: 0.8164966] (Table 2).
Weight comparison of everyone from 4 rats groups initially was performed with each other from 3 remained groups applying statistical paired t-test (Table 3). Any emerging significant difference among OC scores lesions was investigated whether owed in the above probable significant weight correlations. OC scores lesions comparison of everyone from 4 rats groups initially was performed with each other from 3 remained groups applying Wilcoxon signed-rank test (Table 3). The application of generalized linear models (glm) with dependant variable the OC scores lesions and independent variables the Epo administration or no, the reperfusion time and their interaction were followed. Inserting the rats weight as independent variable at glm, a non significant relation turned on with OC scores lesions (p = 0.7774), so as to further investigation was not needed.
Table 3: Statistical significance of mean values difference for groups after statistical paired t test application for weight and Wilcoxon signed-rank test for scores. View Table 3
The glm resulted in: Epo administration kept non-significantly increased the OC scores by 0.3 without lesions [-0.256345 - 0.856345] (p = 0.2819). This finding was in accordance with the results of Wilcoxon signed-rank test (p = 0.3090). Reperfusion time kept non-significantly increased the OC scores by 0.1 without lesions [-0.4640457 - 0.6640457] (P = 0.7217), also in accordance with the Wilcoxon signed-rank test result (P = 0.8909). However, Epo administration and reperfusion time together kept non-significantly increased the OC scores by 0.1454545 without lesions [-0.1918887 - 0.4827978] (p = 0.3882). Reviewing the above and table 3, table 4 and table 5 sum up concerning the alteration influence of Epo in connection with reperfusion time.
Table 4: The restorating influence of erythropoietin in connection with reperfusion time. View Table 4
Table 5: Concise presence of the restorating influence of erythropoietin in connection with reperfusion time. View Table 5
The following situations show the association between ischemia and congestion in ovaries. Akdemir A et al. found  congestion among histopathological findings in torsion groups after induced ovarian IR injury in rats. Sapmaz-Metin M et al. assessed  congestion as apparent among histopathological changes following IR injury in rat ovaries. Aslan MK et al. examined  ovarian and periovarian congestion after IR injury. Aran T et al. evaluated  high vascular congestion scores after ovarian torsion in female adult Sprague-Dawley rats. Coskun A et al. indicated  a gradually increased congestion correlated with respective increased ischemic times for tied ovaries in rats. Kart A et al. observed  severe congestion on ovary IR injury in rabbits. Cigremis Y et al. observed  severe congestion in the torsion-detorsion female rabbits group. Smorgick N et al. imaged  the pathological series of increased ovarian congestion by ultrasound at twisted IR ovaries and necrosis in menstrually cycling women. Kazez A et al. assessed congestion  in both ovaries after delayed ovarian torsion detorsion in female Wistar albino rats. Uguralp S et al. showed  congestion of contralateral ovaries in different degrees after unilateral ovarian IR in albino Wistar rats. Taskin O et al. showed  prominent congestion on all sections 36 h after detorsion of IR adnexa in cycling female rats. Cavender JL et al. paralleled  congestion chronologically by evidence of ischemia during ovulation in ewe.
The following situations show the association between Epo and ischemic ovaries. Mahmoodi M et al. found  that Epo reduced IR injury and free radical production, increasing follicle survival and function in transplanted ovarian tissue. Sayyah-Melli M et al. determined  that Epo was effective in reducing the oxidative damage of ovarian torsion in operated patients, 18-35 years old, with signs and symptoms of ovarian torsion. Karaca M et al. evaluated  the Epo administration as effective in reversing tissue damage induced by IR in ovaries of adult female rats. Suzuki H et al. demonstrated  that administration of a sialo Epo could effectively enhance the survival of the follicles of transplanted cryopreserved ovaries in frozen-thawed canine ovarian xenotransplantation. However, David RB et al. did not detect  expression of Epo mRNA in porcine ovaries. Kristiansson B et al. concluded  that females with carbohydrate-deficient glycoprotein syndrome type I have primary ovarian failure, but the syndrome does not affect the terminal charged carbohydrate portion in Epo. Hyttinen JM et al. generated  a transgenic calf from in vitro produced bovine embryos microinjected with a gene construct consisting of genomic sequences encoding human Epo. Kamiński M claimed  that apoptosis regulates the atrophy of completely developed organs, e.g. thymus, and the hormonal restructuring of ovaries and others, but on the other hand, the development of apoptosis is arrested by so called "survival factors" as Epo.
Epo administration, reperfusion time and their interaction kept non-significantly increased the OC scores. Epo short-term restored the congestive lesions from significant to non-significant level. This may contribute to relief in subsided congestion at situations as decoiled paratubal cysts or side effects of contraceptives.
This study was funded by Scholarship by the Experimental Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece. The research facilities for this project were provided by the aforementioned institution.
Kim J, Park D, Han WB, Jeong H, Park Y (2014) Acute abdomen due to ovarian congestion caused by coiling of the fallopian tube accompanied by paratubal cyst around the utero-ovarian ligament. Obstet Gynecol Sci 57: 338-341.
Kaido Y, Kikuchi A, Kanasugi T, Fukushima A, Sugiyama T (2013) Acute abdomen due to ovarian congestion: a fallopian tube accompanied by a paratubal cyst, coiling tightly round the ovary. J Obstet Gynaecol Res 39: 402-405.
Yassin S, El-mahgoub S, Ammar R, Karim M (1971) Ovarian morphological changes with injectable contraceptives and their relation to cycle control. Ain Shams Med J 22: 497-501.
C. Tsompos, C. Panoulis, K. Tutouzas, G. Zografos, A. Papalois (2015) The Effect of Erythropoietin on Creatinine Levels during Ischemia Reperfusion Injury in Rats. Literati Journal of Pharmaceutical Drug Delivery Technologies 1: 1-6.
C Tsompos, C Panoulis, K Toutouzas, G Zografos, A Papalois (2015) The effect of erythropoietin on alanine aminotransferase during ischemia reperfusion injury in rats. Acta Chirurgica Iugoslavica 62: 33-39.
C Tsompos, C Panoulis, K Toutouzas, G Zografos, A Papalois (2015) The effect of erythropoietin on γ-glutamyltransferase during ischemia reperfusion injury in rats. International Journal of Advances in Pharmaceutics 4: 88-92.
Osmanaqaoalu MA, Kesim M, Yuluq E, Mentese A, Karahan SC (2012) Ovarian-protective effects of clotrimazole on ovarian ischemia/reperfusion injury in a rat ovarian-torsion model. Gynecol Obstet Invest 74: 125-130.
Akdemir A, Erbas O, Gode F, Ergenoglu M, Yeniel O, et al. (2014) Protective effect of oxytocin on ovarian ischemia-reperfusion injury in rats. Peptides 55: 126-130.
Sapmaz-Metin M, Topcu-Tarladacalisir Y, Uz YH, Inan M, Omurlu IK, et al. (2013) Vitamin E modulates apoptosis and c-jun N-terminal kinase activation in ovarian torsion-detorsion injury. Exp Mol Pathol 95: 213-219.
Aslan MK, Boybeyi Ö, Şenyücel MF, Ayva Ş, Kısa Ü (2012) Protective effect of intraperitoneal ozone application in experimental ovarian ischemia/reperfusion injury. J Pediatr Surg 47: 1730-1734.
Aran T, Guven S, Unsal MA, Alver A, Mentese A, et al. (2010) Serum ischemia-modified albumin as a novel marker of ovarian torsion: an experimental study. Eur J Obstet Gynecol Reprod Biol 150: 72-75.
Coskun A, Coban YK, Ciralik H (2009) Critical ischemic time for the rat ovary: experimental study evaluating early histopathologic changes. J Obstet Gynaecol Res 35: 330-334.
Kart A, Cigremis Y, Ozen H, Dogan O (2009) Caffeic acid phenethyl ester prevents ovary ischemia/reperfusion injury in rabbits. Food Chem Toxicol 47: 1980-1984.
Cigremis Y, Kart A, Karaman M, Erdag D (2010) Attenuation of ischemia-reperfusion injury with Marrubium cordatum treatment in ovarian torsion-detorsion model in rabbits. Fertil Steril 93: 1455-1463.
Smorgick N, Maymon R, Mendelovic S, Herman A, Pansky M (2008) Torsion of normal adnexa in postmenarcheal women: can ultrasound indicate an ischemic process? Ultrasound Obstet Gynecol 31: 338-341.
Kazez A, Ozel SK, Akpolat N, Goksu M (2007) The efficacy of conservative treatment for late term ovarian torsion. Eur J Pediatr Surg 17: 110-114.
Uguralp S, Bay Karabulut A, Mizrak B (2005) Effects of pentoxifylline and vitamin E on the bilateral ovary after experimental ovarian ischemia. Eur J Pediatr Surg 15: 107-113.
Taskin O, Birincioglu M, Aydin A, Buhur A, Burak F, et al. (1998) The effects of twisted ischaemic adnexa managed by detorsion on ovarian viability and histology: an ischaemia-reperfusion rodent model. Hum Reprod 13: 2823-2827.
Cavender JL, Murdoch WJ (1988) Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 39: 989-997.
Mahmoodi M, Soleimani Mehranjani M, Shariatzadeh SM, Eimani H, Shahverdi A (2014) Effects of erythropoietin on ischemia, follicular survival, and ovarian function in ovarian grafts. Reproduction 147: 733-741.
Sayyah-Melli M, Rashidi MR, Kaseb-Ganeh M, Rashtchizadeh N, Taghavi S, et al. (2012) The effect of erythropoietin against oxidative damage associated with reperfusion following ovarian detorsion. Eur J Obstet Gynecol Reprod Biol 162: 182-186.
Karaca M, Odabasoglu F, Kumtepe Y, Albayrak A, Cadirci E, et al. (2009) Protective effects of erythropoietin on ischemia/reperfusion injury of rat ovary. Eur J Obstet Gynecol Reprod Biol 144: 157-162.
Suzuki H, Ishijima T, Maruyama S, Yanagimoto Ueta Y, Abe Y, et al. (2008) Beneficial effect of desialylated erythropoietin administration on the frozen-thawed canine ovarian xenotransplantation. J Assist Reprod Genet 25: 571-575.
David RB, Blom AK, Sjaastad OV, Harbitz I (2001) The porcine erythropoietin gene: cDNA sequence, genomic sequence and expression analyses in piglets. Domest Anim Endocrinol 20: 137-147.
Kristiansson B, Stibler H, Wide L (1995) Gonadal function and glycoprotein hormones in the carbohydrate-deficient glycoprotein (CDG) syndrome. Acta Paediatr 84: 655-659.
Hyttinen JM, Peura T, Tolvanen M, Aalto J, Alhonen L, et al. (1994) Generation of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitro. Biotechnology (N Y) 12: 606-608.
Kaminski M (1994) [Processes of cell necrosis--apoptosis--and their modification by toxic substances]. Med Pr 45: 267-277.