Printer Friendly

Effects of sevoflurane on female reproductive functions in Wistar rats.

Byline: Serkan Dogru, Hatice Yilmaz Dogru, Ilknur Butun, Akgul Arici, Ismail Benli, Tugba Karaman, Hakan Tapar, Aynur Sahin, Serkan Karaman, Semih Arici and Asker Zeki Ozsoy


Objective: To determine the effects of sevoflurane by inhalation on female reproductive hormones and ovarian tissues.

Methods: This experimental study was conducted at the Gaziosmanpasa University, Tokat, Turkey, and comprised Wistar-Albino female rats. The rats were divided into six groups; one control and five study groups. The control group (C) received 2 L/min O2 in 18 min/day for seven days; the first study group (S1) received 1 minimum alveolar concentration sevoflurane + 2 L/min O2 in 18 min/day for seven days; the second group (S2) received 1 minimum alveolar concentration sevoflurane + 2 L/min O2 in 18 min/day for seven days and no treatment for the following seven days; the third group (S3) received 1 minimum alveolar concentration sevoflurane + 2 L/min O2 in 18 min/day for 14 days; the fourth group (S4) received 1 minimum alveolar concentration sevoflurane + 2 L/min O2 in 18 min/day for 14 days and no treatment for the following seven days; and the fifth group (S5) received 1 minimum alveolar concentration sevoflurane + 2 L/min O2 in 18 min/day for 14 days and no treatment for the following 14 days.

The duration of the study was 28 days in February 2015. Reproductive system hormone levels were analysed and histological assessment of the ovaries was performed. SPSS 20 was used for data analysis.

Results: Of the 30 rats, there were 5(16.7%) in each group. Histological injury scores in S2, S3, S4, and S5 were significantly higher than in C (p=0.016, p=0.008, p=0.016 and p=0.032, respectively). The hormone levels belonging to follicle stimulating hormone, luteinising hormone, estradiol and progesterone revealed significant alterations in all groups (p<0.05).

Conclusion: Chronic exposure to sevoflurane negatively affected the histological structure of the ovary and hormonal regulation.

Keywords: Follicle stimulating hormone, Luteinising hormone, Estradiol, progesterone, Sevoflurane. (JPMA 67: 877; 2017)


Along with the technological advancements in industry, chemicals are becoming an indispensable part of our daily lives. In contrast with the advantages that make life easier for human beings, chemical compounds may interfere with the organs or the systems of the body and lead to undesirable effects.

There are several chemicals in the environment that can have adverse effects on the human body. Many of these chemicals are integral factors in the production of substantial substances. Of the 87,000 chemicals registered for commerce in the United States (US), unfortunately only 10% of these compounds have been analysed for potential health effects.1 Of these, only a portion has been assessed for reproductive health effects. Inhalation anaesthetics are one of the subgroups of these substances which have been widely used in medical field. Various experimental and clinical studies showed that the routine use of inhalation anaesthetics may have nephrotoxic, hepatotoxic, neurotoxic and genotoxic effects.2-8 In contrast with the excessive amount of the data on toxicity, limited studies exist on the effects of volatile anaesthetics on the reproductive system.

Sevoflurane, which is a clear, non-flammable liquid at room temperature with no pungent odour and is well-suited for use in ambulatory anaesthesia, is a highly fluorinated substance named fluoromethyl hexafluoroisopropyl ether (C4H3F7O) that was first studied in humans in the US in the late 1970s.9 It has a low blood-gas partition coefficient of 0.69, providing faster balance on the alveolar concentration with the inspired (delivered) concentration that results in rapid induction of, and recovery from, anaesthesia. Although the main elimination occurs in the lungs, a 5% of administered dose of sevoflurane undergoes metabolism by the liver.10 Literature review revealed that there has been no data collected on the effects of sevoflurane on the female reproductive system.

The present study was planned to determine the effects of repeated exposure to sevoflurane on rat ovary tissue and reproductive hormones.

Material and Methods

This experimental study was conducted at the Gaziosmanpasa University, Tokat, Turkey, and comprised rats. Exposure to sevoflurane was performed in an anaesthesia chamber measuring 40 x 50 x 60 cm. Ketamine and xylazine were obtained from Alfasan International B.V. (Woerden, Netherlands). Formaldehyde was purchased from Histomed (Montenegro, BR). Harris Haematoxylin and Eosin (HandE) Y 1% alcoholic were purchased from Atom Scientific Ltd (Manchester, United Kingdom [UK]).

After obtaining approval from the institutional ethics committee, adult female Wistar-Albino rats were obtained from the experimental medicine unit of the university. All animals were 90 days old, weighed 250-300 grams, and were selected in the same period of oestrus cycle as assessed through vaginal smears. The animals were kept in a room maintained at 20-24degC with a 12-hour light-dark cycle (lights on from 0600 to 1800 hours) and a constant humidity of 40-50%. All rats were housed in polycarbonate cages with tap water ad libitum.

For sevoflurane administration, rats were placed into a glass anaesthesia chamber measuring 40 x 50 x 60 cm, which was connected to an anaesthesia system (Prima SP Alpa, Penlon Limited, Oxon, UK). As previously described by Ceyhan et al., two holes, one at the top left side of the chamber and the other at the upper right side of the chamber, were opened for anaesthetic gas inlet and outlet.11 Rats were randomly divided into six groups, including one control and five study groups.

The control group (C) received 2 L/min O2 in 18 min/day for seven days; the first group (S1) received 1 minimum alveolar concentration (MAC) sevoflurane + 2 L/min O2 in 18 min/day for seven days; the second group (S2) received 1 MAC sevoflurane + 2 L/min O2 in 18 min/day for seven days and no treatment for the following seven days; the third group (S3) received 1 MAC sevoflurane + 2 L/min O2 in 18 min/day for 14 days; the fourth group (S4) received 1 MAC sevoflurane + 2 L/min O2 in 18 min/day for 14 days and no treatment for the following seven days; the fifth group (S5) received 1 MAC sevoflurane + 2 L/min O2 in 18 min/day for 14 days and no treatment for the following 14 days. The duration of the study was 28 days in February 2015.

All rats were anaesthetised by intra-peritoneal injection of ketamine 90 mg/kg and xylazine 10 mg/kg (Alfasan International B.V.), and were killed performing cervical dislocation at the end of the 7th day in C and S1 groups, the 14th day in S2 and S3 groups, the 21st day in S4 group, and the 28th day in S5 group. Intra-cardiac blood samples (5cc) were collected for hormonal biochemical analysis. Bilateral ovaries were subsequently removed, and the right ovary of all animals was fixed quickly upon collection in a 10% neutral buffered formalin solution. Tissues were dehydrated and embedded in paraffin for histopathological evaluation. The left ovary of each animal was placed on ice and then transferred to a -70degC freezer where they remained frozen until biochemical analysis.

Five mm thick sections were prepared from paraffin-embedded ovary tissue and mounted on glass slides. For histological assessment by light microscope (Nikon Eclipse E600W, Japan), the mounted sections were stained with HandE. Ovary sections from each of the five animals per group were randomly numbered. Thereafter, all slides were coded to perform a blind semi-quantitative analysis on the ovary sections. Histopathological injury was evaluated by an investigator who was initially blinded to the experiment. At least five microscopic fields of each ovary in each group were randomly selected for the evaluation. A modified five-level grading scale based on oedema, follicular cell degradation, vascular congestion, haemorrhage and infiltration by inflammatory cells was used to determine the histopathological injury score of the ovary.

Normal ovarian architecture was defined as grade 0; mild oedema, mild follicular cell degradation, mild vascular congestion, no haemorrhage and no leukocyte infiltration was grade 1; moderate oedema, moderate follicular cell degradation, moderate vascular congestion, no haemorrhage and no leukocyte infiltration was grade 2; severe oedema, severe follicular cell degradation, severe vascular congestion, minimal haemorrhage and minimal leukocyte infiltration as grade 3, and severe oedema, severe follicular cell degradation, severe vascular congestion, haemorrhage and leukocyte infiltration as grade 4,12,13

The blood samples were given 20 minutes to form clotting, and the serum was separated by centrifugation at 1,500g (4degC) for 15 minutes. Then, the serum was transferred into separate eppendorf tubes and stored at -20degC until analysis. Serum luteinising hormone (LH), follicle stimulating hormone (FSH), progesterone (PG) and oestrogen (ES), and ovarian tissue LH, PG and ES levels in all groups were obtained by using LH (YH Biosearch, Shanghai, China), FSH (YH Biosearch, Shanghai, China), ES (Cayman Chemical Company, Michigan, US), and PG (Cayman Chemical Company, Michigan, US) rat enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. The outcomes of follicle stimulating hormone and luteinising hormone were calculated as mIU/mL, and estradiol and progesterone as pg/mL.

With a standard deviation difference of 1.06 in FSH levels with a two-sided type I error of 0.05, and a power of 0.80, five rats in each group were required to find a significant difference.

SPSS 20 was used for data analysis. Normality and variance were evaluated using the one sample Shapiro-Wilk test for each variable. Quantitative data was presented as means and standard deviation and qualitative data as frequency and percentage. The hormonal levels and histopathological injury scores of all groups were analysed using the Kruskal-Wallis analysis of variance (ANOVA) and post-hoc comparisons were conducted by Tukey's honest significant difference (HSD) test with Bonferroni correction. P0.05). There were mild follicular cell degradation and mild vascular congestion in S4 (Figure 1E). In addition, mild oedema and mild vascular congestion was found in S5 (Figure 1F).

The evaluation under light microscopy revealed that chronic sevoflurane exposure led to follicular cell oedema, degeneration, congestion and bleeding in the ovaries of rats (Figure-1B-1F).

The mean histopathological injury scores were significantly higher in all groups, except S1, compared to C (Figure-2).

There were significant differences found among all groups for serum FSH, LH, estradiol ES and PG (p<0.05). Serum FSH levels were significantly higher in S5 compared to C, S1, S2, S3, and S4 (p=0.009, p=0.009, p=0.009, p=0.009, p=0.009, respectively). In addition, serum LH levels in S5 were significantly higher than in C, S1, S2, S3, and S4 (p=0.012, p=0.009, p=0.009, p=0.009, p=0.009, respectively). The ES levels in S2, S3, and S5 were significantly higher than C (p=0.026, p=0.015, p=0.026, respectively). In S4, the PG levels were detected as significantly higher than in S1 (p = 0.009) (Table-1).

Table-1: Serum hormone levels.






Additionally, there were significant alterations in all groups of ovarian tissue LH, ES and PG levels (p<0.05). The LH levels in S2 were significantly lower compared to C and S3 (p=0.021, p=0.020, respectively). In S3, the LH levels were significantly higher than S5 (p=0.049). S3 and S4 had significantly higher ES values compared to C (p=0.009, p=0.028, respectively). In S3, the mean PG level was significantly higher than C (p=0.036). In addition, the PG levels in S2, S3 and S4 were significantly higher compared to S1 (p=0.021, p=0.021, p=0.047, respectively) (Table-2).

Table-2: Ovarian tissue hormone levels.






The present study revealed that acute and sub-chronic exposure to sevoflurane may lead to several significant hormonal changes in the reproductive system and may lead to ovarian tissue damage, as supported by the histological findings. It is well described that the oestrous cycle of rats lasts four or five days and consists of proestrus, oestrus, metoestrus, and dioestrus, and includes various alterations in the concentrations of gonadal steroids and gonadotropins.14-17 Briefly, in the proestrus phase, the oestradiol level rises and ovarian follicles grow fast. This stage takes approximately 12 hours and is similar to the follicular phase of humans. After LH surge, ovulation occurs in the night of oestrus. Associated with the absence of a fertilisation during the time of ovulation, the corpora lutea is temporarily functional and excretes a low quantity of progesterone.18

As mentioned above, the regulation of reproductive functions is controlled by a finely tuned balance between androgens and sex steroids, which can be affected by numerous bio-chemical agents. Various studies have demonstrated the impacts of these agents on the hormone levels of the hypothalamo-hypophyseal axis.19-23 Similar findings were revealed in the present study. For instance, serum FSH and LH values were significantly increased in S1, S2, S4 and S5. The FSH and LH rise in S1 and S2 suggested that seven days (acute) of exposure may lead to ovarian tissue damage, causing responsiveness to FSH and LH. In addition, the increase of FSH and LH in S4 and S5 with the decrease in S3 suggested that 14 days of exposure (sub-chronic) could damage the hypotalamic-hypophyseal axis and be followed by a progressive rise of hormone levels during the recovery period.

The ES showed an increase in S1, S2, S4 and S5, and a decrease in S3. Elevated levels of ES in S1 and S2 suggested that acute exposure may disturb the integrity of follicles containing ES, leading to a hormonal release. In relation, the ES decline in S3 suggested that sub-chronic exposure could disrupt the ES synthesis of theca cells in the ovarian follicles, and the following increase in S4 and S5 confirmed that ES production improved during the recovery period. The ovary tissue levels of ES in S3, followed by an increase in S4 and S5, confirmed this phenomenon. The PG levels showed an inconsistent route compared to the degree of exposure. However, PG has complex production and secretion mechanisms, which can be influenced by many factors.

A systematic review of the literature revealed that no study existed on the effects of sevoflurane on the female reproductive system. In contrast, several studies have been conducted on the effects of sevoflurane on males.11,24-27 In accordance with the study conducted by Ceyhan et al., chronic sevoflurane exposure in male rabbits may lead to testicular dysfunction and directly affects the sperm morphology, resulting in abnormalities in the sperm shapes.11 In addition, Kaymak et al. used semen samples of 28 volunteers and exposed the samples to various concentrations of halothane, isoflurane, sevoflurane and desflurane, searching for deoxyribonucleic acid (DNA) damage in sperm cells. The investigators reported that the inhalation anaesthetics, with the exception of desflurane, have the potential to cause genotoxicity.25

Similarly, Wang et al. demonstrated that sevoflurane with a dose from 1.4% to 5.6% shows minimal effect on motility and vitality of human sperm, while expecting impairment in the motor function of human sperm.26 A recent study conducted by Kaya et al., investigating seven days and 14 days of sevoflurane exposure on the male rat reproductive system, revealed that chronic exposure to sevoflurane can result in testicular tissue damage, decreased sperm count and motility, occurrence of abnormal sperm forms, and disruption in reproductive hormones having a direct role in fertility such as FSH, LH, ES and PG.24

Moreover, the mean histological injury scores showed significantly higher values in all study groups, except S1, compared to C. The S3 revealed severe deterioration including severe oedema, severe follicular cell degradation, severe vascular congestion, and bleeding sites. Lower histological injury scores in S4 and S5 suggested that there can be an improvement in ovarian tissue associated with the recovery period (seven days and 14 days). The diminished serum hormone levels in S3 after an increase in S4 and S5 provides a symmetric pattern with the histological scores. In terms of the duration of sevoflurane inhalation, most damage appeared with longer exposure. The existence of a recovery period in S2 caused a decrease in histologic injury score after S1.

There can be two speculative explanations for this phenomenon; first, perhaps seven days of exposure (acute) was not sufficient to trigger the repair systems of the tissues or simply mild damage was not enough to initiate a response or recovery in ovary tissues. Second, perhaps seven days of exposure led to higher levels of injury; however, it could not be detected in S1 associated with the euthanasia of rats at the end of seven days in S1, thus no increased level of injury scores was detected. In S2, the injury level could be raised to a higher level and then decreased in seven days of recovery to the present value.

Currently, several investigators are focused on the single issue that anaesthetic agents may cause infertility among health workers.28-31 In this context, Guirguis et al. emphasised the association between exposure to anaesthetic gases and abnormal pregnancy outcomes.28 In addition, Gauger et al. demonstrated that there is a higher incidence of spontaneous abortion in paediatric anaesthesiologists compared to non-paediatric, which might be associated with the occupational exposure to inhalation anaesthetics during induction and the use of un-cuffed endotracheal tubes in paediatric anaesthesia.29 Various studies attested to the findings of previous authors, reporting that occupational exposure to unscavenged waste and anaesthetic gases may lead to adverse outcomes, or that inhalation exposure can lead to first-born female offspring.30,31

This study had several limitations as well. First, the effect of sevoflurane on ovarian cell DNA could not be included in the study design, which might provide more detailed information about cell damage. Second, a new hormone named anti-mullerian hormone level could not be investigated, which shows the functional status of the ovaries and might support the findings of the present study.

To our knowledge, this study was the first experimental investigation on the effects of sevoflurane on the female rat reproductive system.


Acute and sub-chronic exposure to sevoflurane may negatively alter the hormonal regulation and histological structure of the female reproductive system. Through the current study, our understanding of inhalation anaesthetics has developed considerably; however, there is scarcity of useful clinical outcome data to guide this issue. Further studies are required to elucidate the exact mechanisms of the inhalation anaesthetics.


We are grateful to Erkut Somak, Yilmaz Ozcan and Serkan Kavak for their contribution to the study.

Disclaimer: None.

Conflict of Interest: None.

Source of Funding: The study was funded by Gaziosmanpasa University Scientific Research Projects Unit.


1. US Government Accountability Office. Actions are needed to improve the effectiveness of EPA's chemical review program. Testimony before the Committee on Environment and Public Works, US Senate. Report No. GAO-06-1032T; 2009. [online] [cited 2015 May 15]. Available from: URL: Accessed February 27, 2015.

2. Nishiyama T, Yokoyama T, Hanaoka K. Liver and renal function after repeated sevoflurane or isoflurane anaesthesia. Can J Anaesth 1998; 45: 789-93.

3. Nishiyama T, Yokoyama T, Hanaoka K. Effects of sevoflurane and isoflurane anesthesia on arterial ketone body ratio and liver function. Acta Anaesthesiol Scand 1999; 43: 347-51.

4. Nishiyama T. Effects of repeat exposure to inhalation anesthetics on liver and renal function. J Anaesthesiol Clin Pharmacol 2013; 29: 83-7.

5. Martin JL. Volatile anesthetics and liver injury: a clinical update or what every anesthesiologist should know. Can J Anaesth 2005; 52: 125-9.

6. Orhan H, Sahin A, Sahin G, Aypar U, Vermeulen NP. Urinary lipid and protein oxidation products upon halothane, isoflurane, or sevoflurane anesthesia in humans: potential biomarkers for a subclinical nephrotoxicity. Biomarkers 2013; 18: 73-81.

7. Pellegrini L, Bennis Y, Velly L, Grandvuillemin I, Pisano P, Bruder N, et al. Erythropoietin protects newborn rat against sevoflurane-induced neurotoxicity. Paediatr Anaesth 2014; 24: 749-59.

8. Zizek D, Ribnikar M, Zizek B, Ferlan-Marolt V. Fatal subacute liver failure after repeated administration of sevoflurane anaesthesia. Eur J Gastroenterol Hepatol 2010; 22: 112-5.

9. Young CJ, Apfelbaum JL. Inhalational anesthetics: desflurane and sevoflurane. J Clin Anesth 1995; 7: 564-77.

10. Smith I, Nathanson M, White PF. Sevoflurane--a long-awaited volatile anaesthetic. Br J Anaesth 1996; 76: 435-45.

11. Ceyhan A, Cincik M, Bedir S, Ustun H, Dagli G, Kalender H. Effects of exposure to new inhalational anesthetics on spermatogenesis and sperm morphology in rabbits. Arch Androl 2005; 51: 305-15.

12. Kurt A, Isaoglu U, Yilmaz M, Calik M, Polat B, Hakan H, et al. Biochemical and histological investigation of famotidine effect on postischemic reperfusion injury in the rat ovary. J Pediatr Surg 2011; 46: 1817-23.

13. Guven S, Muci E, Unsal MA, Yulug E, Alver A, Kadioglu Duman M, et al. The effects of carbon dioxide pneumoperitoneum on ovarian blood flow, oxidative stress markers, and morphology during laparoscopy: a rabbit model. Fertil Steril 2010; 93: 1327-32.

14. Paccola CC, Resende CG, Stumpp T, Miraglia SM, Cipriano I. The rat estrous cycle revisited: a quantitative and qualitative analysis. Anim Reprod 2013; 10: 677-83.

15. Freeman ME. The ovarian cycle of the rat. In: Knobil, E., Neil, J., ed. Physiology of Reproduction. New York: Raven Press Ltd, 1994; 1893-928.

16. Faccio L, Da Silva AS, Tonin AA, Franca RT, Gressler LT, Copetti MM, et al. Serum levels of LH, FSH, estradiol and progesterone in female rats experimentally infected by Trypanosoma evansi. Exp Parasitol 2013; 135: 110-5.

17. Nephew KP, Long X, Osborne E, Burke KA, Ahluwalia A, Bigsby RM. Effect of Estradiol on Estrogen Receptor Expression in Rat Uterine Cell Types. Biol Reprod 2000; 62: 168-77.

18. Hegazy R, Hegazy A. DMPA-Induced Changes in Estrogen and Progesterone Receptors of Ampulla of Rat-oviducts: An Immunohistochemical Study. Universal J Med Sci 2015; 3: 33-40.

19. Osmanagaoglu MA, Usul H, Yulug E, Kesim M, Karahan SC. Hormonal and histological changes in the ovaries with high-doses of methylprednisolone administration for acute spinal cord injury: an experimental study. J Obstet Gynaecol 2013; 33: 585-90.

20. Fattore L, Spano MS, Altea S, Fadda P, Fratta W. Drug-and cue-induced reinstatement of cannabinoid-seeking behaviour in male and female rats: influence of ovarian hormones. Br J Pharmacol 2010; 160: 724-35.

21. Illera JC, Silvan G, Martinez MM, Blass A, Pena L. The effect of dexamethasone on disruption of ovarian steroid levels and receptors in female rats. J Physiol Biochem 2005; 61: 429-38.

22. Quignot N, Bois FY. A Computational Model to Predict Rat Ovarian Steroid Secretion from In Vitro Experiments with Endocrine Disruptors. PLoS ONE 2013; 8: e53891.

23. Hardt DJ, James RA, Gut CP Jr, McInturf SM, Sweeney LM, Erickson RP, et al. Evaluation of submarine atmospheres: effects of carbon monoxide, carbon dioxide and oxygen on general toxicology, neurobehavioral performance, reproduction and development in rats. I. Subacute exposures. Inhal Toxicol 2015; 27: 83-99.

24. Kaya Z, Sogut E, Cayli S, Suren M, Arici S, Karaman S, et al. Evaluation of effects of repeated sevoflurane exposure on rat testicular tissue and reproductive hormones. Inhal Toxicol 2013; 25: 192-8.

25. Kaymak C, Kadioglu E, Coskun E, Basar H, Basar M. Determination of DNA damage after exposure to inhalation anesthetics in human peripheral lymphocytes and sperm cells in vitro by comet assay. Hum Exp Toxicol 2012; 31: 1207-13.

26. Wang LJ, Wang XH, Sun HJ, Xu B. Effects of inhalation anaesthetics on human sperm motility and vitality in vitro. Br J Anaesth 2008; 101: 883-4.

27. Szyfter K, Szulc R, Mikstacki A, Stachecki I, Rydzanicz M, Jaloszynski P. Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo. J Appl Genet 2004; 45: 369-74.

28. Guirguis SS, Pelmear PL, Roy ML, Wong L. Health effects associated with exposure to anaesthetic gases in Ontario hospital personnel. Br J Ind Med 1990; 47: 490-7.

29. Gauger VT, Voepel-Lewis T, Rubin P, Kostrzewa A, Tait AR. A survey of obstetric complications and pregnancy outcomes in paediatric and nonpaediatric anaesthesiologists. Paediatr Anaesth 2003; 13: 490-5.

30. Gupta D, Kaminski E, McKelvey G, Wang H. Firstborn offspring sex ratio is skewed towards female offspring in anesthesia care providers: A questionnaire-based nationwide study from United States. J Anaesthesiol Clin Pharmacol 2013; 29: 221-7.

31. Hoerauf KH, Wallner T, Akca O, Taslimi R, Sessler DI. Exposure to sevoflurane and nitrous oxide during four different methods of anesthetic induction. Anesth Analg 1999; 88: 925-9.
COPYRIGHT 2017 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Journal of Pakistan Medical Association
Article Type:Report
Date:Jun 30, 2017
Previous Article:Comparing the effectiveness of Betamethasone Gel with Lidocaine Gel local application on endotracheal tube in preventing post-operative sore throat...
Next Article:Depression and anxiety in patients undergoing elective and emergency surgery: Cross-sectional study from Allama Iqbal Memorial Teaching Hospital,...

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |