evaluation of effects of repeated sevoflurane exposure on rat testicular tissue and reproductive...

2013

http://informahealthcare.com/ihtISSN: 0895-8378 (print), 1091-7691 (electronic)

Inhal Toxicol, 2013; 25(4): 192–198! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2013.773109

RESEARCH ARTICLE

Evaluation of effects of repeated sevoflurane exposure on rat testiculartissue and reproductive hormones

Ziya Kaya1, Erkan Sogut2, Sevil Cayli3, Mustafa Suren1, Semih Arici1, Serkan Karaman1, and Fikret Erdemir4

1Department of Anesthesiology and Reanimation, 2Department of Biochemistry, 3Department of Histology and Embryology, and 4Department of

Urology, Gaziosmanpasa University School of Medicine, Tokat, Turkey

Abstract

Context: Evaluation of inhalation anesthetics on sperm and reproductive hormones areextremely important.Objective: Investigation of the effects of sevoflurane used as an inhalation anesthetic on spermmorphology and reproductive hormones in rat testes.Materials and methods: Forty Wistar-Albino male rats were divided into five groups of eight ratseach. The control group received 2 L/min oxygen for seven days, 2 h/day while sevofluranetreatment S1 received 1 minimal alveolar concentration (MAC) sevofluraneþ 2 L/min oxygenfor seven days, 2 h/day, and sevoflurane S2 received 1 MAC sevofluraneþ 2 L/min oxygen forseven days, 2 h/day followed by seven days of no treatment. Sevoflurane treatment S3 received1 MAC sevofluraneþ 2 L/min oxygen for 14 days, 2 h/day and sevoflurane treatment S4received 1 MAC sevofluraneþ 2 L/min oxygen for 14 days, 2 h/day, with no treatment for thefollowing seven days. All rats were examined histologically after experimental procedures.Rat luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and inhibinlevels were measured.Results: Histological injury scores were significantly higher in S2, S3, and S4 receivingsevoflurane in comparison to the control group (p¼ 0.001, 50.001, and 0.001, respectively).Sperm motility and concentration decreased in S3 and S4 compared to the control group(p¼ 0.03 and 0.02, respectively). Significant differences were detected among all groups forserum LH, FSH, T, and inhibin serum concentrations (p50.05).Conclusion: Testicular and sperm morphology, and reproductive hormones were affected bychronic exposure to sevoflurane. However, more randomized, controlled, and well-designedclinical studies with larger population are needed to confirm of these results.

Keywords

FSH, inhibin, LH, sevoflurane, sperm, testis

History

Received 12 September 2012Revised 28 January 2013Accepted 31 January 2013Published online 13 March 2013

Introduction

Spermatogenesis in humans, performed by germ cells in the

seminiferous tubules found in testicular tissue, is a complex

process. The basic process includes stimulation of Sertoli

cells by follicle-stimulating hormone (FSH) secreted from the

pituitary and the stimulation of testosterone (T) synthesis

following stimulation of Leydig cells by luteinizing hormone

(LH) secreted from the pituitary (Sofikitis et al., 2008).

Abnormalities or disruptions in sperm production are

indicated by deterioration in sperm number and movement

and may result in infertility. There are many reasons for sperm

number and motility deterioration, including testicular torsion

or trauma, infection, sexual and immunologic factors, crypt-

orchidism, gonadal dysgenesis and obstruction of the repro-

ductive channels, varicocele, and drug use, all of which

adversely affect spermatogenesis (Bilban et al., 2005; Ceyhan

et al., 2005; Cosentino et al., 1986; Jungwirth et al., 2012;

Kaymak et al., 2012; Kharasch, 1996; Rezvanfar et al., 2008;

Whitney, 2012; Yang, 2011).

In many clinics, the number of surgical procedures is

increasing at present, due to a variety of factors. It is well

known that inhaled anesthetics are used routinely in many

surgical procedures all over the world. In the past 10 years,

several studies have shown that various side effects may occur

with the routine use of inhaled anesthetics. Experimental and

clinical studies indicate that nephrotoxic, hepatotoxic, neuro-

toxic, and genotoxic effects may result from inhalation of

anesthetics (Bilban et al., 2005; Kaymak et al., 2012;

Kharasch et al., 1996; Land et al., 1981; Szyfter et al.,

2004). However, the number of studies on this issue is

extremely limited. It has been reported that continuous

exposure to the inhalation anesthetics causes infertility,

spontaneous abortion, congenital anomaly, germ cell

damage, DNA damage, and morphological changes in

sperm cells (Arena & Pereira, 2002; Ceyhan et al., 2005;

Cosentino et al., 1986; Gauger et al., 2003; Land et al., 1981;

Oropeza-Hernandez et al., 2002; Rezvanfar et al., 2008;

Szyfter et al., 2004; Whitney, 2012; Yang, 2011). Indeed,

evaluation of the pharmacological and chemical effects of

Address for correspondence: Dr. Ziya Kaya, Department of Anesthesiol-ogy and Reanimation, Gaziosmanpasa University School of Medicine,Tokat 60100, Turkey. E-mail: [email protected]

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inhalation anesthetics on sperm is extremely important for

men of reproductive age. Sevoflurane is a highly fluorinated

methyl-isopropyl ether (2,2,2-trifluoro-1-ethyl(trifluoro-

methyl) fluoromethyl ether, C4H3F7O), colorless, non-flam-

mable, liquid in room temperature and has been available in

clinical use since 1995. The elimination of sevoflurane is

primarily by lung; however, some metabolism does occur. It is

very useful for induction and maintenance of anesthesia and

has an inoffensive odor, non-irritant to the airway. It is suitable

for outpatient and prolonged procedures due to its quick

recovery (Kaymak et al., 2012; O’Keeffe and Healy, 1999;

Reichle & Conzen, 2003; Young & Apfelbaum et al., 1995).

However, there have been very few studies on the effects of

sevoflurane on sperm morphology or reproductive hormones.

The aim of this study is to investigate the effects of

repeated exposure of sevoflurane on rat testicular tissue and

reproductive hormones.

Materials and methods

After approval of local ethics committee (2010 HADYEK-

44), a total of 40 adult male Wistar-Albino (5 months) rats

with weights ranging from 250 to 350 g were selected. The

rats were housed at room temperature 20–24 �C, 50� 10%

humidity, and 12-h light and 12-h dark. The rats were put into

polycarbonate cages 40� 50� 60 cm in diameter. The anes-

thetic gas inlet was supplied by the top right of the cage as

previously described in the literature (Ceyhan et al., 2005).

Holes were opened for anesthetic gas output in the upper left

side of the cage. After connecting the anesthesia machine

(Siemens Kion; Siemens, Solna, Sweden) to the anesthesia

circuit, a tubing head was placed on the entrance hole in the

cage. Gas supply minimal alveolar concentration (MAC) was

set as 1 and was allowed to flow freely with 100% oxygen.

The gas outlet was provided from other holes. Rats were

divided into five groups each containing eight animals: the

control group – seven days for 2 h, 2 L/min O2; the first group

– sevoflurane 1 (S1) for 2 h/day for seven days, 1 MAC

sevofluraneþ 2 L/min O2; the second group (S2) – 2 h/day for

seven days, 1 MAC sevofluraneþ 2 L/min O2, with nothing

given for the following seven days; the third group (S3) – 2 h/

day for 14 days, 1 MAC sevofluraneþ 2 L/min O2; and the

fourth group (S4) – 2 h a day for 14 days, 1 MAC

sevofluraneþ 2 L/min O2 with nothing given for the follow-

ing seven days. Euthanasia was performed in the control and

S1 at the end of day 7, S2 and S3 at the end of the day 14, and

S4 at the end of day 21 by 50 mg/kg intraperitoneal injection

of ketamine. After ketamine injection, 5 cc of blood was

collected for hormonal evaluations. After the experimental

procedures, all rats were sacrificed and bilateral testes were

removed. One testis from each animal was fixed in Bouin

fluid for 24 h immediately upon collection, then dehydrated

and embedded in paraffin for histo-chemistry. The contralat-

eral testis from each animal was frozen for biochemical

analysis. The testicular tissue was stored at �80 �C until

biochemical analyses.

Histological evaluation

Paraffin blocks were cut in 5-mm-thick sections, and the

sections were stained with hematoxylin and eosin (H&E)

and examined under the light microscope. The histological

evaluation was done in a blind, randomly numbered fashion.

Eight animals and six sections were evaluated for each

animal. A four-level grading scale similar to that of

Cosentino et al. was used to quantify histological injury

(Cosentino et al., 1986; Whitney, 2012). Grade 1 showed

normal testicular architecture with orderly arrangement of

germinal cell. Grade 2 (slight effect) injuries showed less

orderly, non-cohesive germinal cells and closely packed

seminiferous tubules. Grade 3 (moderate effect) injuries

exhibited disordered, sloughed germinal cells with shrunken,

pyknotic nuclei and less distinct seminiferous tubule border.

Grade 4 (severe effect) injuries involved seminiferous

tubules closely packed with coagulative necrosis of the

germinal cells.

Sperm assessment

The epididymis spermatozoa were also examined for mor-

phological abnormalities following exposure to sevoflurane.

Diff-Quick staining was applied, and morphology of rat

sperm was evaluated under the light microscopy with a minor

modification (Rezvanfar et al., 2008; Wyrobek et al., 1983).

Epididymal sperms were collected by opening the epididymis

in 2 mL of Ham’s F10 medium and incubating for 5 min at

37 �C in an atmosphere of 5% CO2 to allow sperm to swim out

of the epididymis tubules. One drop of sperm suspension was

placed on a microscope slide; five microscopic fields were

randomly selected and observed at 400� magnification using

a phase contrast microscope; and the percentage of motile

sperm was evaluated microscopically within 2–4 min of their

isolation from the epididymis and was expressed as a

percentage of motile sperm out of the total sperm counted.

Epididymal sperm counts were obtained by the method

described in the WHO manual (World Health Organization,

1999). Briefly, a 5-mL aliquot of epididymis sperm was

diluted with 95 mL of diluent, and approximately 10 mL of this

diluted specimen was transferred to each of the counting

chambers of the hemocytometer. Two hundred sperms were

counted for each animal, and total abnormality was expressed

as incidence/200 sperms/animal. For the analysis of morpho-

logical abnormalities, sperm smears were drawn on clean

and grease-free slides and allowed to air-dry overnight. The

slides were examined at 400� for morphological abnormal-

ities such as amorphous, hook-less, bicephalic, coiled, and

abnormal tails.

Biochemical evaluation

After about 20 min to allow clotting, blood samples from rats

were separated by centrifugation for 15 min (at 4 �C, 1500 g)

and serum was separated. Later, the serum separated into

eppendorf tubes was stored at �40 �C until analysis. In all

groups, serum LH, FSH, T, and inhibin B (beta sub-unit, Inh

B) levels were measured from separated serum by using LH,

FSH and Rat T ELISA kits (Cusabio Biotech, Wuhan, Hubei,

China) and de Rat Inh B Enzyme Immunoassay Kit

(RayBiotech, Inc., Norcross, GA) according to the manufac-

turer’s instructions. IU/L was calculated for LH and FSH,

ng/mL was calculated for T and Inh B.

DOI: 10.3109/08958378.2013.773109 Sevoflurane exposure on rat testicular tissue 193

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Statistical analysis

All data were expressed as mean� standard deviation. All

statistical calculations were performed with SPSS 15.0

program (SPSS Inc., Chicago, IL). Compliance with the

normal distribution of data was analyzed using the

Kolmogorov–Smirnov test. For comparison in biochemical

values of data between all groups, one-way analysis of

variance (one-way ANOVA) and Kruskal–Wallis H test were

used. Multiple comparisons between the groups were made by

Tukey’s HSD and Tamhane’s test. Multiple comparison in

histological groups were evaluated by using ‘‘All Pairwise

Multiple Comparison’’ followed by Duncan’s test. Data were

represented as mean� SEM from eight animals per group.

P values50.05 were considered significant statistically.

Results

Histopathological findings

To assess the effect of sevoflurane on rat testicular tissue

pathology, the testicular tissues from each group were

analyzed by H&E staining. Figure 1 represents the histo-

logical findings used for injury scoring (injury score of 0–4,

respectively). The mean histological injury scores were

significantly higher in all sevoflurane-treated groups, except

S1, compared to control (Figure 1F). In addition, the mean

histological injury scores of S3 were significantly higher than

S1. The testicular tissues from S1 displayed slight degenera-

tive changes in the seminiferous epithelium; however, no

significant differences were found between control and S1

(Figure 1B). The control rats showed normal seminiferous

tubule morphology (Figure 1A). Seminiferous tubules, germ

cells, Sertoli, and Leydig cells appear complete, without

infiltration and hemorrhagic signs. Light microscopy revealed

that chronic sevoflurane exposure resulted in interstitial space

dilatation, germ cell loss, and edema in the testicular tubule of

rats (Figure 1C–E). S2 displayed moderate disruption of the

seminiferous epithelium (Figure 1C). The seminiferous epi-

thelium in S3 showed severe degeneration with disordered,

sloughed germinal cell with shrunken, psychotic nuclei,

interstitial space dilatation, and presence of edema

(Figure 1D). In S3, there were no spermatogenetic cells in

some tubules. The histological grade of S4 was significantly

higher than control (p¼ 0.01), but no significant difference

was detected between S4 and other groups (p40.05).

Sperm characteristics

No significant differences were found in sperm morphology

between groups. However, sperm concentration and sperm

motility were reduced in S3 and S4 in comparison to controls

(Table 1).

Biochemical results

Significant differences were detected among all groups for

serum LH, FSH, T and Inh B (p50.01, Table 2). Serum

LH levels were significantly higher in S2, S3 and S4 than in

the control (p¼ 0.026, 50.001 and 0.001, respectively).

In addition, S3 LH values were significantly higher in

comparison to control, S1 and S2 (p50.001, 50.001 and

0.020, respectively). The mean LH value of S4 was signifi-

cantly higher than S1 (p¼ 0.004); however, it has a signifi-

cant lower value than S3 (p¼ 0.026). S2 had significantly

lower values for serum FSH in comparison to the control

(p¼ 0.015) and has significantly lower mean value than S4

(p¼ 0.011). S3 had significantly higher T value than the

control, S1 and S4 (p50.001, 0.003 and 0.04, respectively).

Serum Inh B levels were significantly higher in S2, S3 and S4

than the control (p¼ 0.014,50.001 and50.001, respectively).

S3 Inh B values were significantly higher in comparison to S1

(p¼ 0.027). S4 Inh B levels were found to be significantly

higher than S1, S2 and S3 (p50.001, 50.001 and 0.016,

respectively).

Discussion

This study showed that chronic exposure of inhalation

anesthetics resulted in a decrease in sperm motility and

concentration. In addition, histological findings reveal that

sevoflurane may damage the testicular tissue in rats.

Similarly, reproductive hormones such as FSH, LH, T and

Inh B displayed significant changes during the study.

Infertility is defined as an inability to achieve pregnancy

following one year of unprotected intercourse and it is

considered an important public health issue (Greenhall &

Vessey, 1990). In the etiology of infertility, male factors

contribute to 50% of cases, either directly or indirectly

(Jungwirth et al., 2012). It is well known that FSH, LH and

T play an important role in the spermatogenetic process.

Sertoli cells are stimulated by FSH, which is secreted from

the pituitary, resulting in initiation of spermatogenesis.

Testosterone is synthesized by Leydig cells in the interstitium

following stimulation by LH, and this synthesized T is

utilized for spermatogenesis in the seminiferous tubule

(Sofikitis et al., 2008). Moreover, Inh B plays an important

role in the spermatogenesis. Inhibin B is produced in the

Sertoli cells in the testis due to the stimulation of FSH.

Inhibin B has been defined as a gonadal hormone that exerts a

specific negative feedback action on the secretion of FSH

from the gonadotropic cells of the pituitary gland (De Jong,

1988). For this reason, it is accepted that Inh B is an

important marker of Sertoli cell activity in males with

impaired spermatogenesis (Cai et al., 2011). In clinical

practice, all of these hormones are used for the evaluation

of male infertility. Spermatogenesis is a complex process and

several factors affect on the spermatogenesis. In the literature,

the effect of numerous drugs on spermatogenesis has been

evaluated (El-Harouny et al., 2010; Khaki et al., 2009; Ragni

et al., 1988; Vaisheva et al., 2007). In these studies, the

alterations of hormone parameters in the hypothalamo–

hypophyseal–testicular axis have been shown. Similarly, in

the present study, we found that LH levels were increased in

S1, S2 and S3 associated with testicular damage; however,

this increase was statistically significant only in S2 and S3.

The decreased LH level in S4 compared to S3 showed that

negative effect of sevoflurane on Leydig cells was decreased

in chronic period. Relation to this, decreased FSH levels in

S1, S2 and S3 suggested that sevoflurane has an effect on

FSH centrally (hypothalamic or hypophyseal). In S4, the FSH

levels were close to baseline, and this finding suggested that

194 Z. Kaya et al. Inhal Toxicol, 2013; 25(4): 192–198

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negative effects of sevoflurane on FSH were decreased in

chronic period. In addition, Inh B levels showed an increase in

each group, which is an interesting result of this study.

However, Inh B, which is a new hormone and has a complex

mechanism, may be affected by multiple factors. Similarly, T

levels in S1, S2 and S3 were increased. In contrast, T levels in

S4 were lowered to a non-significantly closer value in

comparison to controls. For this reason, it can be suggest that

the effect of sevoflurane on T might be realized over the

structures such as sex hormone binding globuline and

albumine except LH.

In addition to hormone levels, semen parameters affected

several factors. Sperm motility, morphology, and concentra-

tion are very important parameters for the assessment of

Figure 1. Representative figures of histology of rat testicular tissues as indicated by hematoxylin and eosin staining. (A) Control, (B) Group S1, (C)Group S2, (D) Group S3 and (E) Group S4. Scale bars: 50mm. Eight animals were used for each group and six slides per animal were evaluated(n¼ 48). Histologic injury scores after exposure of sevoflurane. The scoring range was 0 to 4 using the modified scoring system reported by Cosentinoet al. (1986) based on depth of injury. The mean score for each group (n¼ 48) was analyzed by analysis of ‘‘All Pairwise Multiple Comparison’’followed by Duncan’s test. a: Control–S2 (p¼ 0.001); b: Control–S3 (p50.001); c: Control–S4 (p¼ 0.001); d: S3–S1 (p¼ 0.007). No significantdifferences were found in Control–S1 (p¼ 0.053), S1–S4 (p¼ 0.097), S1–S2 (p¼ 0.121), S2–S3 (p¼ 0.073), S2–S4 (p¼ 0.71) and S3–S4 (p¼ 0.084).


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damage to the male reproductive system. The effects of drugs

on testicular tissue have been reported in many studies

(Campion et al., 2012; Ceyhan et al., 2005; Madhubabu &

Yenugu, 2012; Tartarin et al., 2012). There are few studies

examining sperm morphology and movement and the effects

of sevoflurane on reproductive hormones (Kaymak et al.,

2012; Szyfter et al., 2004; Wang et al., 2008). In this context,

Wang et al. (2008) investigated the effects of sevoflurane and

isoflurane on sperm motility in 20 human volunteers and they

identified that sevoflurane does not affect the viability and

motility of human sperm at clinically relevant high concen-

trations. On the other hand, exposure to isoflurane at

clinically relevant concentrations increases sperm motility

and viability, while both motility and viability slowly decrease

at higher concentrations. However, at the end of the study,

they specified that these effects were reversible when the

anesthetic agent was withdrawn. Similarly, Stutler et al.

(2007) specified that high doses and long-term exposure

reduced sperm motility and velocity through the direct effects

of circulating agents, but at low concentrations, these effects

were not seen. Chapin et al. (1992) proposed that isoflurane

does not affect sperm motility directly, but prevent the

successful emergence of sperm from the vas deferens,

reducing the number of sperm but not affecting fertility.

Another study on the effect of isoflurane on sperm morph-

ology specifies that isoflurane does not decrease motility

directly, despite a demonstrated effect on the integrity of

sperm. Instead, the study specified that isoflurane reduces the

number of sperm by inhibiting smooth muscle contraction in

the vas deferens and by preventing ejaculation of the sperm

from the vas deferens (Campion et al., 2012). After

subchronic exposure to the inhalation anesthetic halothane,

sexual behavior and effects on sperm motility were investi-

gated. Fifteen and 30 days after exposure, it was observed that

both sperm motility and male sexual behavior were inhibited.

Forty-five and 60 days after the exposure, the absence of these

findings was attributed to the tolerance developing over time

(Oropeza-Hernandez et al., 2002). In contrast to the studies

mentioned above, in the present study, the mean histological

injury scores were significantly higher in all sevoflurane

treated groups, except S1, compared to control. However, the

seminiferous epithelium in S3 showed the severe degeneration

including disordered, sloughed germinal cells with shrunken,

psychotic nuclei, interstitial space dilatation, and presence of

edema. In S3, there were no spermatogenetic cells in some

tubules. The histological grade of S4 was significantly higher

than control, but statistically significant difference was not

detected between S4 and other groups. In addition, serum

Table 2. Biochemical findings in all groups.

Variable Control S1 S2 S3 S4 p

LH (IU/L) 7.87� 2.21c,d,e 7.96� 1.10f,h 12.15� 4.14c,g 18.85� 2.10d,g,h,i 13.83� 4.23e,f,i 50.001a

FSH (IU/L) 55.1� 12.3j 43.7� 8.96 33.9� 10.8j,k 41.6� 15.2 58.2� 19.6k 0.007a

Testosterone (ng/mL) 4.13� 0.87m 4.52� 0.96n 5.67� 1.64 7.94� 2.60m,n,o 5.41� 1.84o 0.001a

Inh B (ng/mL) 0.36� 0.14p,q,r 0.49� 0.20s,t 0.63� 0.12p,u 0.76� 0.23q,s,v 1.06� 0.18r,t,u,v 50.001b

LH, luteinizing hormone; FSH, follicle-stimulating hormone; Inh B, inhibine beta subunit. Values are given as mean� SD. S, Sevoflurane.aOne-way ANOVA test was used.bKruskal–Wallis H test was used. Multiple comparisons between groups were made by Tukey HSD and Tamhane’s test.Significant differences for intergroup comparisons (LH):cControl–S2 (p¼ 0.026); dControl–S3 (p50.001); eControl–S4 (p¼ 0.001); fS1–S4 (p¼ 0.004); gS2–S3 (p¼ 0.02); hS3–S1 (p50.001); iS3–S4(p¼ 0.016).Significant differences for intergroup comparisons (FSH):jControl–S2 (p¼ 0.015); kS2–S4 (p¼ 0.011).Significant differences for intergroup comparisons (testosterone):mControl–S3 (p50.001); nS1–S3 (p¼ 0.003); oS3–S4 (p¼ 0.04).Significant differences for intergroup comparisons (Inh B):pControl–S2 (p¼ 0.014); qControl–S3 (p50.001); rControl–S4 (p50.001); sS1–S3 (p¼ 0.027); tS1–S4 (p50.001); uS2–S4 (p50.001); vS3–S4(p¼ 0.016).

Table 1. Effects of sevoflurane on the sperm concentration, motility and abnormal sperm morphology of male rats.

Control S1 S2 S3 S4

Sperm concentration (106/mL) 168.87� 2.1a,b 127.96� 5.50 102.15� 7.4 88.5� 2.9a 73.83� 8.3b

Sperm motility (%) 76.1� 2.3c,d 63.7� 8.9 83.9� 6.8 41.6� 4.2c 58.2� 9.6d

Abnormal sperm count (%) 4.3� 0.7 5.52� 0.6 5.7� 1.9 7.4� 1.6 6.1� 2.4

Data are represented as mean� SEM from 8 animals/group. 200 sperm was counted per animal. S, sevoflurane. Inter-group differences were evaluatedby using ‘‘All Pairwise Multiple Comparison’’ followed by Duncan’s test.aSignificant differences between sperm concentration of Control and S3 (p¼ 0.03).bSignificant differences between sperm concentration of Control and S4 (p¼ 0.02).cSignificant differences between sperm motility of Control and S3 (p50.001).dSignificant differences between sperm motility of Control and S4 (p50.001).No significant differences were found in sperm concentration of Control versus S1 (p¼ 0.051), sperm concentration of Control versus S2 (p¼ 0.053),sperm motility of Control versus S1 (p¼ 0.056) and sperm motility of Control versus S2 (p¼ 0.06).No significant differences were found in abnormal sperm count (%) of Control versus S1 (p¼ 0.056), abnormal sperm count (%) of Control versus S2(p¼ 0.052), abnormal sperm count (%) of S1 versus S2 (p¼ 0.31), abnormal sperm count (%) of S1 versus S3 (p¼ 0.052) and abnormal sperm count(%) of S3 versus S4 (p¼ 0.152).


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hormone levels were increased in S3, compatible with the

histological changes.

As illustrated with histological and hormonal studies,

7 and 14 days exposure of sevoflurane have significant effect

on the testicular morphology. In regards of the duration of

anesthesia, the severe testicular damage was observed after

longer exposure of sevoflurane. Interestingly, recovery was

defined after 14 days exposure of sevoflurane in S4; however,

S2 did not exhibit recovery. We assumed that toxic degrad-

ation or elimination of sevoflurane seems to be delayed and

not effective in S2. In this situation, several complex

mechanisms may play a role. It might also be possible to

detect recovery in S2 if the longer durations were admini-

strated. Further studies need to elucidate the exact mechanism

of duration and elimination of sevoflurane.

Similar to our study, in multiple studies, it has been

shown that anesthetic agents increase infertility, among health

personnel working in the operating room environment,

causing damage to germ cells and changes in sperm

morphology and motility (Arena & Pereira, 2002; Boivin,

1997; Gauger et al., 2003; Izdes et al., 2009; Land et al.,

1981; Oropeza-Hernandez et al., 2002). In a clinical study,

it was shown that spontaneous abortions among pediatric

anesthesiologists occur with higher prevalence than among

non-pediatric anesthetists. They attributed this to occupational

exposure to inhalation anesthetics during induction and use of

un-cuffed endotracheal tube (Gauger et al., 2003). Kaymak

et al. (2012) have demonstrated in their in vitro study that

exposure to different concentrations on inhalation anesthetics

(sevoflurane, desflurane, isoflurane and halothane) may cause

DNA damage in sperm cells and increasing the dose or

exposure may increase this risk. At the end of this study,

researchers expressed that studies on the risk of repeated

drug exposure are lacking and that this effect should be

investigated in future. In connection with this, in present

study, the effects of chronic exposure were examined and the

effects of repeated exposure of the testis to sevoflurane were

severe. Similarly, the study by Ceyhan et al. (2005) in rabbits

reveals that chronic exposure to inhalation anesthetics

decreases sperm motility and concentration and increases

the number of abnormal sperm. In the same study, they

specified that spermatogenesis was also depressed and the

observation of immature sperm formation in the weeks

following supports their hypothesis.

As a result, we can suggest that chronic exposure to

inhalation anesthetics may negatively affects testicular

tissue, associated sperm count, motility and presence of

abnormal forms, and the production of hormones that play a

direct role in spermatogenesis such as FSH, LH, T and Inh

B. From the findings of the present study, it can be

speculated that anesthesiologists in the operating room may

be affected by the exposure of inhalation anesthetics.

However, to elucidate the exact mechanism by which

sevoflurane and other inhalation anaesthetics affects sperm,

there is a need for experimental studies and larger

prospective randomized trials.

Declaration of interest

The authors declare that there is no conflict of interest.

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evaluation of effects of repeated sevoflurane exposure on rat testicular tissue and reproductive...

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