Accepted Manuscript

Title: Investigation of the effects of unilateral total

salpingectomy on ovarian proliferating cell nuclear antigen
and follicular reserve: experimental study

Author: Remzi Atilgan Tuncay Kuloğlu Abdullah Boztosun

Uğur Orak Melike Baspinar Behzat Can Ekrem Sapmaz

PII: S0301-2115(15)00063-9
DOI: http://dx.doi.org/doi:10.1016/j.ejogrb.2015.02.028
Reference: EURO 8924

To appear in: EURO

Received date: 15-6-2014

Revised date: 12-2-2015
Accepted date: 19-2-2015

Please cite this article as: Atilgan R, Kuloğlu T, Boztosun A, Orak U, Baspinar M,
Can B, Sapmaz E, Investigation of the effects of unilateral total salpingectomy on
ovarian proliferating cell nuclear antigen and follicular reserve: Experimental study,
European Journal of Obstetrics and Gynecology and Reproductive Biology (2015),
http://dx.doi.org/10.1016/j.ejogrb.2015.02.028

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Condensation (one sentence summary)

Condensation

Unilateral total salpingectomy procedure decreased ovarian follicular reserve, increased

apoptosis, and decreased follicular proliferating cell nuclear antigen (PCNA) staining in rat
ovary.

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 Investigation of the effects of unilateral total salpingectomy on ovarian proliferating cell
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2 nuclear antigen and follicular reserve: Experimental study
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7 Remzi ATILGAN1, Tuncay KULOĞLU2, Abdullah BOZTOSUN3, Uğur ORAK1, Melike
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10 BASPINAR1, Behzat CAN1, Ekrem SAPMAZ1
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1Firat University School of Medicine, Department of Obstetrics and Gynecology, Elazig,

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17 Turkey.
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22 3Akdeniz University School of Medicine, Department of Obstetrics and Gynecology,
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32 Corresponding Author
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34 Remzi Atilgan
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37 Postal address: Firat University School of Medicine, Department of Obstetrics and

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39 Gynecology, 23119, Elazig, Turkey
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Phone: +90 5336295981
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44 E-mail: remzi_atilgan@hotmail.com
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 Condensation
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2 Unilateral total salpingectomy procedure decreased ovarian follicular reserve, increased
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4 apoptosis, and decreased follicular proliferating cell nuclear antigen (PCNA) staining in rat
5 ovary.
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 ABSTRACT
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2 Objective: We aimed to investigate the effects of unilateral total salpingectomy procedure on
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5 ovarian follicular reserve, apoptosis, and proliferating cell nuclear antigen (PCNA) staining in
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7 this study.
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Study Design: Fourteen female Wistar Albino rats of 12 weeks were randomly divided into

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16 Group 1(G1) (n = 7): Group in which only the abdomen was opened and closed,
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20 Group 2(G2) (n = 7): Group that underwent right total salpingectomy
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22 After 1 month, abdomens of all rats were opened. Ovaries were macroscopically evaluated.
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Right ovarian tissue was quickly removed, fixed in 10% formaldehyde, and paraffin blocks

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29 Follicles were microscopically classified and counted. The prevalence of cytoplasmic
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32 immune staining and TUNEL staining was scored semi-quantitatively.
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34 Statistical analysis: SPSS 17.0 software was used for the statistical analysis of data. First,
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Kruskall-Wallis variance analysis was conducted, and then Mann-Whitney U test was utilised
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39 for inter-group dual comparisons for parameters found as p<0.05.
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Results: While the number of CL was found out dramatically high, secondary follicle count
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44 was found out to be significantly low in G2. Also ın G2, although the number of atretic
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46 follicle and fibrosis were found out significantly increased, and the score of the angiogenesis
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49 was found to be significantly decreased in CL. When compared PCNA immunoreactivity in
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51 granulosa cells with the control group, there was a significant decrease in G2. When
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54 compared the malondialdehyde (MDA) immunoreactivity with G1 a significant increase was
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56 established in G2. Apoptosis score of ovarian follicles in granulosa cells was significantly
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59 higher in G2.
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 Conclusions: In this experimental study, the decrease in the ovarian reserve and PCNA
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2 staning of granulosa cells, an increase in apoptosis, fibrosis and the number of atretic follicles
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5 in unilateral total salpingectomy operation were analyzed in rats. We found out significantly
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7 higher MDA staining rates in G2 in comparison to in G1. According to the study, the
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10 unilateral total salpingectomy procedure can damage to the same side ovarian tissue by means
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15 Keywords: salpingectomy; ovarian reserve; PCNA; apoptosis; malondialdehyde.

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 INTRODUCTION
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2 Salpingectomy is a kind of surgical procedure which is also used frequently for illnesses
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5 damaging ovarian artery blood support such as ectopic pregnancy, hydrosalphinx, and
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7 pyosalpinx (1,2).
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10 The effects of salpingectomy procedure on ovary are under debate. Since, while Dar et al (3)
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laparoscopic salpingectomy due to ectopic pregnancy. Chan et al (4) suggested that

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17 laparoscopic unilateral salpingectomy had negative impact on ovarian function in ectopic
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19 pregnancy cases.
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23 It was reported that tubal surgical procedures have negative effects on ovarian blood
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circulation and ovarian reserve due to the damage inflicted on ovarian blood vessels in
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humans and rats (4-6). In addition, some authors suggest that uterine and tubal lymphatics
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30 may also be damaged by tubal surgery (7,8). Pepler et al. (9) established in two separate
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studies that both uterine artery ligation and hysterectomy disrupted ovarian blood circulation
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35 and thus negatively affected ovulation in rats. Zackrisson, et al. (10) demonstrated in their
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37 experimental study that both ovarian artery ligation and uterine artery ligation had negative
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impacts on ovarian blood circulation and ovarian functions.
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42 Anti-proliferating Cell Nuclear Antigen (PCNA) expression is an essential regulator of cell
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45 cycle. It appears as an indicator in follicular growth. In some cases, it leads as the primary
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47 indication of granulosa cell expansion and oocyte growth (11). PCNA was also suggested to
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be a key regulator throughout the ovarian follicle development (12).
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53 Objective: We aimed to investigate the effects of unilateral total salpingectomy procedure on
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55 ovarian follicular reserve, apoptosis, and PCNA staining in this study.
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59 MATERIALS AND METHODS
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 Animals: Twenty eight adult Wistar Albino rats with regular estrus cycle, weighing 200-240
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2 grams, and 14 weeks of age were kept in 12-hour light (08:00-20:00) and 12-hour dark
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5 photoperiod, at a fixed room temperature of 21-23C°, and fed with standard pellet and city
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7 water. Permission for this study was granted by the Ethics Committee of the Medical Faculty
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10 of Firat University. This experimental study was conducted in Experimental Animals
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Experimental Design: Oral feeding of rats was stopped 18 hours before the experiment; only

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17 water was allowed. In order to provide rats with anaesthesia, Ketamine (Ketalar, Eczacıbaşı
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19 Warner- Lambert, Istanbul, Turkey) 60mg/kg and Xylazine (Rompun, Bayer, Istanbul,
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22 Turkey) 7 mg/kg were administered intramuscularly to left anterior foot muscle. Rats were
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placed on their backs on the operation table; antisepsis was provided by washing the surgical

27 area with 10% povidone iodine solution, and abdomen midline incision was conducted.
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29 Rats were randomly divided into two prospective groups.
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Group 1 (n = 7): Group in which only the abdomen was opened and closed,
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34 Group 2 (n = 7): Group that underwent right total salpingectomy
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36 Abdominal layers were closed in a continuous manner with 3/0 silk suture. After 1 month,
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39 abdomens of all rats were opened at estrus phase with transverse subcostal incision under
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41 anaesthesia. Ovaries were macroscopically evaluated. The presence of cyst and the
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44 dimensions of the existing cysts were measured in mm and recorded.
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46 Histological preparation of tissues and evaluation of fibrosis: Right ovarian tissue was
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49 quickly removed, fixed in 10% formaldehyde, and paraffin blocks were prepared for
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51 histological and immunohistochemical examinations. Sections of 5 µm taken from paraffin
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blocks were stained with Masson's trichrome stain. Under light microscopy, fibrosis was
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56 evaluated by the same histologist and semi-quantitatively scored. An ordinal scale was created
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 for remission in angiogenesis and presence of fibrosis in CL (no= 0p, with= 1p, too much=
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2 2p) created.
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5 Microscopic classification for follicular development: Method by Mazaud et al. (13) was
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7 utilised. In the light microscopic examination of ovary sections belonging to all groups,
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10 follicle classification was carried out according to characteristics noted below.
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13 Primordial follicle: Oocyte was surrrounded either partially or completely by granulose
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Primary follicle: Follicle in which a single layer of cubic granulose cells was observed around

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21 oocytes,
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Antral (secondary) follicles: Follicle in which oocyte was covered with more than two layers

27 of granulosa cells and in which antrum formation commenced,

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30 Tertiary (Graafian) follicle: Follicle that possesses a single and big space (antrum), in which a
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33 decreasing number of granulosa cells surround an antrum full of follicular fluid, and that
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35 oocyte surrounded by some granulosa cells (cumulus cells).
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38 Atretic antral: Degenerated oocyte along with pyknosis in granulosa cells, low number of
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41 pyknotic granulosa cells, and hypertrophic thec interna.
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44 For each rat, the total number of atretic follicles was calculated (14).
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47 Immunohistochemical Examination: Sections of 5-6mm thickness obtained from paraffin
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49 blocks were taken to glass slides with polylysine. Deparaffinised tissues were passed through
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graded alcohol series and boiled in citrate buffer solution at pH6 in microwave oven (750W)
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54 for 7+5 minutes for antigen retrieval. In order to prevent surface staining, tissues were
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56 incubated with primary antibodies (Monoclonal Mouse Anti-Proliferating Cell Nuclear
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59 Antigen, M0879, Dako, Baltimore, MD, USA, Anti-ab6463 antibody, Malondialdehyde
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 Abcam, Cambridge, UK) for 60 minutes following treating with Ultra V Block (TA-125-UB,
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2 the Lab Vision Corporation, USA) solutions. After the application of primary antibodies,
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5 tissues were incubated with secondary antibodies (30 minutes) (biotinised anti-mouse/rabbit
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7 IgG, Diagnostic BioSystems, KP 50A, Pleasanton, USA), Streptavidin Alkaline Phosphatase
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10 (30 minutes) (TS-060-AP, the Lab Vision Corporation, USA) and Fast Red Substrate System
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staining with Mayer's haematoxylin were treated with PBS (Phosphate Buffered Saline) and

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17 distilled water, and then, closed with the appropriate shutdown solution. The preparations
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23 Extensity of the staining was taken as the basis when evaluating immunohistochemical
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staining. The extensity of cytoplasmic immune staining was semi-quantitatively scored from
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0 to + 3 points (0: none, 1: mild, + 2: medium, + 3: severe).
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31 TUNEL Staining: Sections in 5-6 mm thickness obtained from paraffin blocks were taken to
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33 glass slides with polylysine. In accordance with the manufacturer's instructions, cells
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36 undergoing apoptosis were established by using ApopTag Plus Peroxidase In Situ Apoptosis
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38 Detection Kit (Chemicon, cat no: S7101, USA). Tissues deparaffinised with Xylene were
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41 treated with graded alcohol and washed with phosphate buffered saline (PBS). Incubated with
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43 0.05% proteinase K, tissues were then incubated for 5 minutes with 3% hydrogen peroxide in
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order to prevent endogenous peroxidase activity. After washing with PBS, tissues were
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48 incubated with 6-minute Equilibration Buffer and again incubated at 37°C in a humid
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50 environment with study solution (70% µl Reaction Buffer + 30% TdT Enzyme) for 60
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53 minutes. Maintained in Stop/Wash Buffer for 10 minutes, tissues were then treated with Anti-
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55 Digoxigenin Peroxidase for 30 minutes. Apoptotic cells were imaged with Diaminobenzidine
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58 (DAB) substrate. Sections that were contrast stained with Harris haematoxylin were shut
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60 down with appropriate solution. Breast tissue was used for positive control. The preparations
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 were examined and evaluated under test microscope (Olympus BX-50) and then
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5 haematoxylin were assessed as normal, and cells demonstrating brown nuclear staining were
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7 established as apoptotic. Extensity of the staining was taken as the basis when evaluating
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10 TUNEL staining. The extensity of TUNEL staining were semi-quantitatively scored from 0
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test microscope (Olympus BX-50) and then photographed.

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18 Statistical analysis: SPSS 17.0 software was used for the statistical analysis of data. First,

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RESULTS
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Histological findings: As a result of the examination of staining with Masson's trichrome
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appearance (Figure 1a, 1b, 1 c). A significant difference was non-existent between the two
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35 groups in terms of total follicular count, primordial, primary, and tertiary follicular counts
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37 (p>0.05, MWU Test). While the number of CL was found as significantly increased in G2,
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secondary follicle count was established as significantly reduced in G2 (p<0.05). There was a
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42 significant increase in atretic follicular count in G2, fibrosis and atretic follicular count
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47 (p<0.05) (Table) (Figure 1 d, 1e, 1f).
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Immunohistochemical findings: As a result of the examination of immunohistochemical
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52 staining conducted for PCNA (Proliferating Cell Nuclear Antigen) immunoreactivity under
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54 light microscope, a significant decrease was found in PCNA immunoreactivity in granulosa
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57 cells in TS group compared to the control group (p<0.05) (Table) (Figure 2a and Figure 2b).
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59 As a result of the examination of immunohistochemical staining conducted for MDA
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 immunoreactivity under light microscope, a significant increase was established in MDA
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5 and Figure 2d).
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10 determining apoptotic cells under light microscopy (Figure 3a, 3b, and Figure 3c, 3d),
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(p<0.05) (Table), (Figure 3a, 3b, and Figure 3c, 3d).

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17 COMMENTS
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20 In our experiment, we established a decrease in PCNA staining in ovarian reserve and
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23 granulosa cells, and increase in fibrosis, apoptosis, and atretic follicles thanks to unilateral
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total salpingectomy. We found the tissue MDA staining score to be significantly higher in the
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group that underwent salpingectomy compared to the control group. This finding showed us
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30 that total salpingectomy procedure may harm ovarian tissue on the same side with ischemia
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and reperfusion damage.

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36 In some clinical and experimental studies, it was demonstrated that ovarian blood
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38 support decrease deteriorated ovarian functions and that follicular reserve was not
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41 significantly affected in interventions carried out by preserving ovarian blood support
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43 (14,15,16). Hypoxia occurred in ovary since we disturbed the blood flow in utero-ovarian
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anastomosis hypoxia (12). We found the tissue MDA score to be significantly higher in the
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48 group that underwent total salpingectomy compared to the control group. This finding is a
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50 histological indication that total salpingectomy procedure causes ischemia and reperfusion
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53 damage in the same-side ovarian tissue. Hypoxic environment causes regression and
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55 apoptosis in follicles, and as a result, increase in atretic follicles and decrease in follicular
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58 reserve (17). The reason for an increase in the number of follicles and fibrosis in G2 might be
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60 due to the apoptotic effect of chronic hypoxia (18). In addition, we established that a
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 reduction in the antral follicle count was higher considering other follicles in G2 compared to
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2 the control group. This is also another indication that ovarian reserve decreased (19).
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6 VEGF increased as a result of hypoxia helps in angiogenesis, rise in vascular
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8 permeability, normal course of folliculogenesis in ovaries, follicular cyst development in
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ovary, and fibrosis development over fibroblast growth factor-1 in long term (from week 3)

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When a cell DNA is exposed to a severe physical or chemical mutagenic agent, p53

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21 protein concentration increases as a result of p53 gene expression. As a result of this protein
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activating the p21 gene, p21 protein amount rises, and either cyclin D kinase protein

formation is suppressed or PCNA activity is supressed and DNA synthesis is inhibited. Thus,
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36 apoptosis known as the programmed death occurs (22,23). It was shown in essential processes
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41 cell cycle control, and cell life that PCNA has a key role (24,25). When compared the group
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43 that underwent total salpingectomy with the control group in our experiment, we established a
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significant increase in apoptosis in granulosa cells in follicles compared to the control group.
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48 This increase in apoptosis may be accepted as an indication of DNA damage that induced
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50 ischemia and reperfusion. Also, we may conclude in the light of the information above that
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53 PCNA activity is suppressed.
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56 Ischemia and reperfusion damage induced by oxygen-related free radicals and lipid
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59 peroxidation cause primordial follicle loss (26). Soleimani et al (27) demonstrated that
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 sphingosine-1-phosphate (SIP) decreased ischemia and reperfusion damage in transplants. In
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2 the same study, they established that ovarian stromal cell proliferation in animals treated with
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5 SIP stained with PCNA at a higher degree compared to the control group. We, too, can
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7 evaluate the significant decrease in PCNA staining in granulosa cells on the side that
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10 underwent total salpingectomy in our experiment compared to the control group as a result of
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PCNA was established as a sensitive marker of early events in follicular growth.

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17 Increased PCNA expression is correlated with the earliest indication of granulosa cell
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19 proliferation. Additionally, oocytes begin to express PCNA in early follicular growth before
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22 starting their expansion. PCNA immunoreactivity maintains to be significant in granulosa and
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theca cells of follicles, and it progressively decreases along with advanced atresia in

27 proceeding phases of follicular growth (11). The fact that PCNA immunoreactivity decreases
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reason for that was that we established the atretic follicle count as significantly higher in total
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37 In conclusion, total salpingectomy procedure may cause a reduction follicular

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reserve by damaging the same-side ovarian tissue. Operation must be carried out by
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42 preserving vascular structures as much as possible during tubal surgical procedures.
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ACNOWLEDGEMENT: None
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49 Conflicts of interest: The authors declared no conflicts of interest in this work.
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4. Chan CC, Ng EH, Li CF, et al. Impaired ovarian blood flow and reduced antral follicle

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 Figure Legends
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3 FIGURE 1: Red star: follicles in different types, black star: fibrosis areas, CL; corpus luteum,
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Table

Table: Histological and immunohistochemical staining parameters belonging to both groups

Parameters Group 1 (n=7) Group 2 (n=7) p value

Total follicle count 49,85+7,49 43,8+3,53 0,109 ns

Primordial follicle count 13,71+5,58 13+4 0,847 ns

Primary follicle count 25+10 24,85+5,49 0,898 ns

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Secondary follicle count 8,14+2,11 3,14+0,69 0,001 **

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Tertiary follicle count 3+1,15 3,57+0,97 0,272 ns

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CL count 3,28+0,75 5,28+1,7 0,041 **

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Atretic follicle count 0,28+0,48 1,42+0,97 0,02 **

Angiogenesis in CL 1,71+0,48 0,42+0,44 0,003 **

Fibrosis score 0,28+0,48

an 1,57+0,78 0,005 **

Apoptosis score 0,42+0,53 2,28+0,75 0,002 **

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MDA score 0,71+0,48 1,85+0,69 0,07 **

PCNA painting score 2,71+0,48 1,28+0,48 0,002 **

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*Values were demonstrated as mean ± SD and n as (%). ** = p< 0.05, Mann Whitney U-test.
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Ns = p>0.05, Mann Whitney U-test. CLA= Remission in angiogenesis in CL.

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