The effects of desflurane on male rat reproductive hormones, testicular tissue, and sperm morphology: an experimental study

Abstract

Background

Desflurane is a widely used inhalational anesthetic known for its advantageous properties in clinical settings. This study aimed to investigate the effects of desflurane inhalation on male reproductive hormones, testicular tissue integrity, and sperm morphology in a rat model.

Methods

Thirty male rats were allocated into six experimental groups:

Control group (C): Administered 2 L/min of O₂ for 18 minutes daily over seven days.

Group D1: Exposed to 1 minimum alveolar concentration (MAC) of desflurane and 2 L/min of O₂ for 18 minutes daily over seven days.

Group D2: Received the same treatment as Group 1 for seven days, followed by a seven-day recovery period without intervention.

Group D3: Administered 1 MAC desflurane and 2 L/min of O₂ for 18 minutes daily over 14 days.

Group D4: Received the same treatment as Group 3 for 14 days, followed by a seven-day recovery period without intervention.

Group D5: Administered the same treatment as Group 3 for 14 days, followed by a 14-day recovery period without intervention.Biochemical analyses were conducted to measure serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and inhibin B. Histopathological evaluations were performed to assess testicular tissue integrity, and sperm morphology was examined to identify abnormalities.

Results

Significant histopathological damage was observed in all experimental groups compared to the control group (p < 0.05). The proportion of morphologically abnormal spermatozoa was significantly higher in Groups D2, D3, D4, and D5 compared to the control group (p = 0.030, p = 0.002, p < 0.001, and p = 0.016, respectively). Compared to the control group, serum FSH levels showed a slight decrease across desflurane-exposed groups (ranging from −1.4% to +4.02%). The LH levels demonstrated a gradual reduction of approximately 0.32%–7.38%, while serum testosterone concentrations increased markedly, reaching up to 178% of the control level in the D4 group. Inhibin-B levels exhibited a progressive elevation of nearly 23–95% compared to control group.

Conclusion

Chronic inhalation of desflurane, a modern inhalational anesthetic, was found to adversely affect testicular histology, sperm morphology, and the regulation of male reproductive hormones in rats. These findings highlight potential reproductive toxicity associated with prolonged desflurane exposure.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12871-026-03668-4.

Introduction

Male fertility is closely linked to the production of normal spermatozoa, which is the result of a highly regulated and complex process known as spermatogenesis. This process encompasses three key phases: mitosis, meiosis, and spermiogenesis [1]. Spermatogenesis is tightly regulated by a delicate balance of endocrine and paracrine signaling pathways. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH), secreted by the pituitary gland, play pivotal roles in this regulation. FSH acts on Sertoli cells, while LH stimulates Leydig cells, both of which provide essential factors necessary for the proper progression of spermatogenesis [2]. The intricate network of signals governing spermatogenesis remains an active area of research. Importantly, external environmental factors have the potential to disrupt this process, leading to impaired fertility and abnormalities in sperm production [3, 4].

With the rapid advancements in medical technology, the frequency of surgical procedures has increased significantly in recent years. This has inevitably led to a corresponding rise in the use of volatile anesthetic agents. However, the routine administration of inhalation anesthetics has been associated with various adverse effects on multiple organ systems, including the liver, kidneys, lungs, and nervous system, as documented in both experimental and clinical studies [4–8]. Of particular concern is the evidence suggesting that repeated exposure to volatile anesthetics may induce DNA damage in germ cells, contribute to congenital anomalies in offspring, and cause morphological alterations in sperm cells [7, 9–11]. Despite these findings, the potential effects of modern inhalation anesthetics on the male reproductive system remain underexplored.

Desflurane (2-difluoromethoxy-1,1,1,2-tetrafluoroethane; C₃H₂F₆O) is a halogenated, fluorinated ether that is colorless, non-flammable, and liquid at room temperature. It has been widely used in clinical practice for the maintenance of general anesthesia since its introduction in 1989 [12, 13]. Desflurane is characterized by several advantageous pharmacological properties, including minimal metabolism (0.02%) and a low blood/gas partition coefficient (0.42), which contribute to its rapid induction and recovery profiles. Additionally, desflurane anesthesia has been associated with reduced postanesthesia care unit stay durations, facilitating earlier patient discharge [14]. However, desflurane exhibits a distinct molecular profile characterized by its high fluorine content and minimal hepatic metabolism (< 0.02%), resulting in different systemic and cellular effects compared to other agents. These pharmacokinetic and metabolic properties may influence its impact on oxidative stress, endocrine regulation, and testicular physiology. Therefore, specific evaluation of desflurane’s reproductive toxicity is warranted to elucidate whether its physicochemical uniqueness translates into differential biological consequences, especially in the context of chronic occupational exposure [15, 16]. Despite its widespread use, a systematic review of the literature reveals a lack of experimental studies investigating the potential effects of desflurane on the male reproductive system.

Given this gap in knowledge, the present study was designed to investigate the effects of repeated desflurane exposure on sperm morphology, testicular tissue integrity, and reproductive hormone levels in a rat model.

Materials and methods

Chemicals

Ketamine and xylazine were procured from Alfasan International B.V. (Woerden, Netherlands). Hematoxylin Harris and Eosin Y (1% alcoholic) were obtained from Atom Scientific Ltd (Manchester, UK). Modified Human Tubal Fluid Medium was purchased from Irvine Scientific (California, USA). Bouin’s solution was sourced from Tek-Path Medikal (Izmir, Turkey).

Animals

The study was conducted following approval by the Tokat Gaziosmanpasa University Local Ethics Committee for Animal Experimentation (Approval No: 2013-HADYEK-12, Approval date: 29.02.2013). A total of 30 adult male Wistar-Albino rats (90 days old, 250–300 g) were obtained from the Experimental Medicine Unit at Gaziosmanpasa University, Turkey. The animals were housed under controlled conditions, including a 12-hour light-dark cycle (lights on at 07:00 a.m.), room temperature maintained at 20–24 °C, and relative humidity of 40–50%. Rats were housed in polycarbonate cages with ad libitum access to tap water. Each cage contained 2–3 animals to allow for social interaction while minimizing overcrowding. Cage cleaning and monitoring were performed regularly to maintain hygiene and animal welfare throughout the experimental period, in accordance with institutional and national guidelines for laboratory animal care.

A glass anesthesia chamber (40 × 50 × 60 cm) was used for desflurane exposure. The chamber was equipped with an anesthetic gas inlet at the top left and an outlet at the top right, as previously described in the literature [13]. An airtight connection was established between the chamber and the anesthetic circuit using an anesthesia machine (Prima SP Alpa, Penlon Limited, Oxon, UK). The exposure dose of 1 MAC desflurane administered with 2 L/min of O₂ for 18 min daily was selected based on previous studies investigating occupational exposure levels in controlled experimental settings. This dosing protocol was designed to simulate the cumulative low-dose exposure experienced by health care workers during repeated, short-term contact with inhalational anesthetics in poorly ventilated or inadequately scavenged operating room environments, rather than the higher, continuous exposure a patient would receive during a surgical procedure. The duration and concentration were chosen to reflect a realistic occupational risk model while minimizing excessive stress or toxicity in the animal subjects [9–11].

The rats were randomly divided into six groups (n = 5 per group) using a computer-generated random number sequence:

• Control group (C): Administered 2 L/min of O₂ for 18 min daily over seven days.

• Group D1: Exposed to 1 minimum alveolar concentration (MAC) of desflurane and 2 L/min of O₂ for 18 min daily over seven days.

• Group D2: Received the same treatment as Group D1 for seven days, followed by a seven-day recovery period without intervention.

• Group D3: Administered 1 MAC desflurane and 2 L/min of O₂ for 18 min daily over 14 days.

• Group D4: Received the same treatment as Group D3 for 14 days, followed by a seven-day recovery period without intervention.

• Group D5: Administered the same treatment as Group D3 for 14 days, followed by a 14-day recovery period without intervention.

At the end of the experimental periods, euthanasia was performed via cervical dislocation under anesthesia induced by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Intracardiac blood samples (5 mL) were collected immediately for hormonal analysis, and bilateral testes were dissected. The right testis was fixed in Bouin’s solution for 24 h, dehydrated, and embedded in paraffin blocks for histopathological examination. The left testis was placed on ice and stored at − 70 °C for subsequent biochemical analyses.

Hormone assays were performed on samples labeled with anonymous codes to ensure that laboratory personnel were blinded to group allocation. Similarly, histopathological evaluation was conducted in a blinded manner by two independent histologists using coded slides. This approach was implemented to minimize observer bias and to enhance the reliability of the measurements.

Histopathological analysis of testicular tissue

Paraffin-embedded testicular tissues were sectioned at 5 μm thickness, mounted on glass slides, and stained with hematoxylin and eosin (H&E) for light microscopic examination (Nikon Eclipse E600W, Tokyo, Japan). Six sections from each testis were randomly numbered and evaluated by a blinded investigator. At least five microscopic fields per testis were assessed. Histopathological injury scores were determined using a four-level grading scale as described by Cosentino et al. [17]:

• Grade 1: Normal testicular architecture with orderly arrangement of germinal cells.

• Grade 2: Slight disorganization and non-cohesive germinal cells with closely spaced seminiferous tubules.

• Grade 3: Moderate disorganization, sloughed germinal cells, shrunken pyknotic nuclei, and less distinct seminiferous tubule borders.

• Grade 4: Severe disorganization, tightly packed seminiferous tubules, and coagulative necrosis of germinal cells.

Sperm morphology analysis

Epididymal sperm was collected by mincing the caudal part of the epididymis in 2 mL of Ham’s F10 medium. For the purpose of sperm release into the medium, caudal epididymides were cut into small pieces and then put in the CO₂ incubator of 37 °C for 30 min. Following pipetting, samples were diluted. A drop of about 10 µl of the sperm suspension was placed on a microscope slide, air-dried, then fixed by the alcohol 96% for 30 min and were stained using the haematoxylin– eosin technique. A total of 300 spermatozoa was examined under a light microscope at 400× magnification (Zeiss microscope, Oberkochen, Germany). Five random fields were assessed for morphological abnormalities, including bicephalic, amorphous, hook-less, and tail abnormalities (coiled or abnormal tails). The percentage of abnormal sperm was calculated and analyzed [18, 19].

Biochemical analysis of reproductive hormones

Blood samples were allowed to clot for 20 min, centrifuged at 1500 × g (4 °C) for 15 min, and the serum was aliquoted into Eppendorf tubes. Samples were stored at − 20 °C until analysis.

For testicular tissue analysis, tissues were weighed and homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.4) containing 0.50 mL/L Triton X-100 using an IKA Ultra-Turrax T25 Basic homogenizer (Stanfen, Germany) at 13,000 rpm for 2 min. All procedures were performed at 4 °C.

Serum levels of LH, FSH, testosterone (T), and inhibin B (Inh-B) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits specifically validated for rat samples (LH and FSH: YH Biosearch Laboratory, Shanghai, China; T: Cayman Chemical Company, Michigan, USA; Inh-B: SunRed Biotechnology Company, Shanghai, China). All assays were performed in accordance with the manufacturer’s instructions. The sensitivity, specificity, and cross-reactivity of each assay were within the ranges reported for rat specimens. To ensure analytical reliability, intra-assay and inter-assay coefficients of variation (CVs) were maintained below 10% and 12%, respectively. Hormone concentrations were expressed as mIU/mL for LH and FSH, pg/mL for T, and ng/mL for Inh-B.

Statistical analysis

Data normality and variance were assessed using the Kolmogorov-Smirnov test. Quantitative data are presented as mean ± standard deviation or median and interquartile range, while qualitative data are expressed as frequencies and percentages. Parametric or nonparametric tests were applied as appropriate. Histopathological injury scores and hormonal levels were analyzed using the Kruskal-Wallis test, with post-hoc comparisons performed using Dunn’s test. Bonferroni correction was applied to all multiple comparisons. All statistical analyses were conducted using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA), with statistical significance set at p < 0.05.

Results

Histopathological findings

The effects of desflurane on testicular tissue were evaluated using H&E staining. Histopathological findings are illustrated in Fig. 1, and the corresponding injury scores, graded on a scale of 1 to 4, are presented in Fig. 2. Significant differences in histological injury scores were observed between the control group (C) and all experimental groups (p < 0.05). Furthermore, the mean histological injury scores in Groups D3 and D4 were higher than in Group D1 (p = 0.005 and p = 0.042, respectively). Group D3 also exhibited higher injury scores compared to Group D2 (p = 0.042), while Group D5 demonstrated lower scores than Group D3 (p = 0.015).

Fig. 1.

Histological sections. Histological sections of rat testis tissues staining by H&E. A Control; (B) D1, arrow shows mild degenerative changes were observed in focal areas of the seminiferous tubule epithelium; (C) D2, arrows show mild to moderate degenerative changes in the seminiferous tubule epithelium, accompanied by mild edema and congestion in the interstitial space; (D) D3, arrows show pronounced degenerative changes in the seminiferous tubule epithelium, along with severe edema and congestion in the interstitial space; (E) D4, arrows show moderate degeneration of the seminiferous tubule epithelium and interstitial edema and evident congestion; (F) D5, arrow shows slight degenerative changes in the seminiferous tubule epithelium. Five animals were included in each group, and six testis sections from each of five animals per group were assessed (n = 30). Scale bars: 50 μm. Magnification: 100×

Fig. 2.

Histological injury scores. A four-level grading scale described by Cosentino et al., which ranged from 1 to 4 due to the injury severity, were used. The intergroup comparisons were performed by using Mann-Whitney U test. Each group contained five rats. P<0.0033 (after Bonferroni correction)Interquartile range (IQR) for groups: C: IQR (1 – 1); D1: IQR (1.5 – 2); D2: IQR (2 – 3); D3: IQR (3 – 3.5); D4: IQR (2 – 3); D5: IQR (2 – 2.5) Comparisons between groups (no significance): C-D1: p = 0.014; C-D2: p = 0.005; C-D3: p = 0.004; C-D4: p = 0.005; C-D5: p = 0.004; D1-D2: p = 0.222; D1-D3: p = 0.005; D1-D4: p = 0.042; D1-D5: p = 0.421; D2-D3: p = 0.042; D2-D4: p = 0.690; D2-D5: p = 0.690; D3-D4: p = 0.222; D3-D5: p = 0.015; D4-D5: p = 0.310

In Group D1, mild degenerative changes were observed in focal areas of the seminiferous tubule epithelium (Fig. 1B), whereas Group C displayed normal testicular architecture with orderly arranged germinal cells (Fig. 1A). Group D2 exhibited mild to moderate degenerative changes in the seminiferous tubule epithelium, accompanied by mild edema and congestion in the interstitial space (Fig. 1C). Group D3 showed pronounced degenerative changes in the seminiferous tubule epithelium, along with severe edema and congestion in the interstitial space, although no loss of spermatogenic cells was observed (Fig. 1D). In Group D4, moderate degeneration of the seminiferous tubule epithelium and interstitial edema and congestion were evident (Fig. 1E). Group D5 displayed slight degenerative changes in the seminiferous tubule epithelium (Fig. 1F).

Light microscopic analysis revealed that chronic inhalational exposure to desflurane induced structural impairments in the seminiferous tubule epithelium and caused interstitial edema and congestion (Fig. 1A and F).

Biochemical analyses

Significant differences in serum levels of FSH, LH, T, and Inh-B were observed across all groups (p < 0.05, Table 1).

Table 1.

Hormone levels in serum samples

	C	D1	D2	D3	D4	D5	p
	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)
	N = 5	N = 5	N = 5	N = 5	N = 5	N = 5	P
FSH (mIU/mL)	9.94 (9.905–10.125)	9.8 (9.445–10.090)	9.72 (9.560–9.805)	9.54 (9.315–10.145)	9.92 (9.680–9.965)	10.36 (9.820–10.490)	p = 0.128
PoC	–	-1.4%	-2.21%	-4.02%	-0.2%	+ 4.22%
LH (mIU/mL)	6.09 (5.935–6.260)	6.11 (5.855–6.160)	5.76 (5.635–5.880)	5.64 (5.355–5.815)	5.91 (5.535–5.930)	5.96 (5.665–6.120)	p = 0.028*
PoC	–	-0.32%	-5.41%	-7.38%	-2.95%	-2.13%
T (pg/mL)	325.8 (176.55–573.80)	531.1 (475.55–622.20)	721.4 (517.10–1018.45)	668.2 (588.65–765.80)	906.3 (612.55–1003.95)	609.8 (554–710.75)	p = 0.025*
PoC	–	+ 63.01%	+ 121.42%	+ 105.09%	+ 178.17%	+ 87.17%
Inh-B (ng/mL)	0.389 (0.330–0.476)	0.479 (0.467–0.494)	0.485 (0.472–0.544)	0.663 (0.637–0.818)	0.714 (0.685–0.831)	0.756 (0.682–0.805)	p = 0.001*
PoC	–	+ 23.13%	+ 24.67%	+ 70.43%	+ 83.54%	+ 94.34%

C control group, D1 desflurane 1 group, D2 desflurane 2 group, D3 desflurane 3 group, D4 desflurane 4 group, D5 desflurane 5 group, FSH follicle stimulating hormone, PoC percentage of change compared to C; LH luteinizing hormone, T testosterone, Inh-B inhibin B

*p < 0.05

Kruskal-Wallis test. All intergroup comparisons were performed by using Dunn’s test

Intergroup comparisons in FSH levels:

C-D2: p = 0.009; D2-D5: p = 0.028

Intergroup comparisons in LH levels:

C-D2: p = 0.019; C-D3: p = 0.007; C-D4: p = 0.044; D1-D2: p = 0.041; D1-D3: p = 0.015

Intergroup comparisons in T levels:

C-D2: p = 0.012; C-D3: p = 0.030; C-D4: p = 0.003; D1-D4: p = 0.023

Intergroup comparisons in Inh-B levels:

C-D3: p = 0.002; C-D4: p = 0.001; C-D5: p = 0.001; D1-D3: p = 0.020; D1-D4: p = 0.008; D1-D5: p = 0.006; D2-D4: p = 0.021; D2-D5: p = 0.018

LH Levels: Serum LH levels in Groups D2, D3, and D4 were lower than in Group C (p = 0.009, p = 0.028, and p = 0.028, respectively). LH levels in Groups D2 and D3 were also lower than in Group D1 (p = 0.028 for both).

FSH Levels: FSH levels in Group D2 were lower than in Group C (p = 0.009), whereas FSH levels in Group D5 were higher than in Group D2 (p = 0.028).

T Levels: Serum T levels in Groups D2, D3, and D4 were higher than in Group C (p = 0.036, p = 0.047, and p = 0.016, respectively). T levels in Groups D3 and D4 were also elevated compared to Group D1 (p = 0.047 and p = 0.036, respectively).

Inh-B Levels: Serum Inh-B levels in Groups D2, D3, D4, and D5 were higher than in Group C (p = 0.047, p = 0.009, p = 0.009, and p = 0.009, respectively). Inh-B levels in Groups D3, D4, and D5 were also elevated compared to Group D2 (p = 0.009 for all).

Testicular tissue hormone levels

Significant changes in testosterone (T) and inhibin B (Inh-B) levels were observed in testicular tissue across all groups (p < 0.05, Table 2).

Table 2.

Hormone levels in testicular tissue samples

	C	D1	D2	D3	D4	D5	p
	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)
	N = 5	N = 5	N = 5	N = 5	N = 5	N = 5	P
T (pg/mL)	2307.3 (1907.9–2958.5)	2600.2 (2150.6–2925.9)	2521.5 (2243.80–2726.40)	1470.4 (744.5–2360.6)	2779.35 (2431.5–3293.2)	2769.9 (2726.4–2864.2)	p = 0.051
PoC	–	+ 12.69%	+ 9.28%	-39%	+ 20.45%	+ 20.04%
Inh B (ng/mL)	0.006 (0.005–0.008)	0.005 (0.002–0.011)	0.038 (0.029–0.046)	0.089 (0.054–0.096)	0.093 (0.067–0.129)	0.244 (0.199–0.292)	p = 0.000116*
PoC	–	-16.66%	+ 533.33%	+ 1383.33%	+ 1450%	+ 3966.66%

C control group, D1 desflurane 1 group, D2 desflurane 2 group, D3 desflurane 3 group, D4 desflurane 4 group, D5 desflurane 5 group, T testosterone, Inh-B inhibin B, PoC percentage of change compared to C

*p < 0.05, Kruskal-Wallis test. All intergroup comparisons were performed by using Dunn’s test

Intergroup comparisons for T levels:

D1-D3: p = 0.046; D2-D3: p = 0.047; D2-D5: p = 0.040; D3-D4: p = 0.037; D3-D5: p = 0.008

Intergroup comparisons for Inh-B levels:

C-D3: p = 0.014; C-D4: p = 0.009; C-D5: p = 0.000082; D1-D3: p = 0.010; D1-D4: p = 0.007; D1-D5: p = 0.000051; D2-D5: p = 0.012

T Levels: Testicular T levels in Group D3 were higher than in Group D1 (p = 0.046) but lower than in Group D2 (p = 0.047). In contrast, T levels in Group D5 were higher than in Group D3 (p = 0.040). Additionally, T levels in Groups D4 and D5 were elevated compared to Group D3 (p = 0.037 and p = 0.008, respectively).

Inh-B Levels: Testicular Inh-B levels in Groups D2, D3, D4, and D5 were higher than in Group C (p = 0.009, p = 0.009, p = 0.014, and p = 0.009, respectively). Inh-B levels in these groups were also elevated compared to Group D1 (p = 0.009, p = 0.009, p = 0.014, and p = 0.009, respectively). Furthermore, Inh-B levels in Groups D3, D4, and D5 were higher than in Group D2 (p = 0.036, p = 0.014, and p = 0.009, respectively). Inh-B levels in Group D5 were also elevated compared to Groups D3 and D4 (p = 0.009 and p = 0.014, respectively).

Sperm morphology assessment

Significant differences in sperm morphology were observed among all groups (p < 0.05, Table 3). The percentages of abnormal sperm in Groups D2, D3, D4, and D5 were higher than in Group C where Group D3 and D4 were showed significance (p = 0.030, p = 0.002, p = 0.0011, and p = 0.016, respectively). Additionally, the percentages of abnormal sperm in Groups D3 were raised in which D4 were significantly elevated compared to Group D1 (p = 0.017 and p = 0.0011, respectively).

Table 3.

Abnormal sperm morphology

	C	D1	D2	D3	D4	D5	p
	Median (IQR)	Median (IQR	Median (IQR	Median (IQR	Median (IQR	Median (IQR
	N = 5	N = 5	N = 5	N = 5	N = 5	N = 5	P
PAS	5.66 (5.01–5.75)	4.71 (4.67–5.14)	6.91 (6.56–7.27)	7.46 (7.03–7.48)	8.8 (7.09–9.05)	6.63 (6.49–7.48)	p < 0.001*
PoC	–	-16.7%	+ 22.08%	+ 31.8%	+ 55.47%	+ 17.13%

C control group, D1 desflurane 1 group, D2 desflurane 2 group, D3 desflurane 3 group, D4 desflurane 4 group, D5 desflurane 5 group, PAS percentage of abnormal sperms, PoC percentage of change compared to C

*p < 0.05

Kruskal-Wallis test. All intergroup comparisons were performed by using Dunn’s test

Intergroup comparisons:

C-D2: p = 0.007; C-D3: p = 0.000022; C-D4: p = 0.002; C-D5: p = 0.023; D1-D3: p = 0.005

Discussion

The findings of this study demonstrate that chronic exposure to desflurane induces testicular tissue injury and increases the percentage of morphologically abnormal sperm in rats. Furthermore, significant alterations were observed in the levels of reproductive hormones, including FSH, LH, T, and Inh-B, which correlated with the intensity and duration of desflurane exposure.

The delicate balance between fertility and infertility is influenced by various environmental and pharmacological factors [20, 21]. Numerous studies have investigated the effects of chemicals and drugs on the male reproductive system, both in humans and animal models [9–11, 20, 21]. For instance, Ceyhan et al. reported that inhalational anesthetics such as sevoflurane and isoflurane can induce testicular damage in rabbits [7]. Similarly, Kaya et al. demonstrated that chronic exposure to sevoflurane adversely affects testicular tissue structure and disrupts the hypothalamic-pituitary-gonadal axis, leading to alterations in FSH, LH, T, and Inh-B levels [13].

Spermatogenesis, the process by which male gametes are produced, involves a series of tightly regulated stages, including proliferation, differentiation, and maturation. This process transforms round, immotile germ cells into elongated, motile spermatozoa capable of fertilization [22, 23]. Spermatogenesis is regulated by the hypothalamic-pituitary-gonadal axis, wherein gonadotropin-releasing hormone from the hypothalamus stimulates the anterior pituitary to secrete FSH and LH. LH acts on Leydig cells to produce testosterone, which, along with FSH, stimulates Sertoli cells to support germ cell development [20]. Sertoli cells, often referred to as “nurse cells,” provide essential nutrients and metabolic intermediates, such as amino acids, carbohydrates, lipids, vitamins, and metal ions, to developing germ cells [24, 25]. Additionally, Sertoli cells secrete inhibin B in response to FSH stimulation, which negatively regulates pituitary FSH secretion [26]. Thus, FSH, LH, testosterone, and inhibin B play critical roles in spermatogenesis and serve as key indicators of male reproductive health.

Beyond the documented impact on the hypothalamic-pituitary-gonadal (HPG) axis, some studies have suggested that volatile anesthetics such as isoflurane and desflurane may also influence other endocrine axes. Isoflurane, for instance, has been associated with alterations in the hypothalamic-pituitary-adrenal (HPA) axis, potentially reducing ACTH and corticosterone levels in animal models due to suppressed synaptic transmission in the hypothalamus [24–28]. While data on desflurane are more limited, its structural and functional similarity to isoflurane raises the possibility of broader endocrine disruption [28]. These findings suggest that the observed hormonal changes may not be strictly confined to the gonadal axis but may reflect a more general downregulation of hypothalamic and pituitary function. However, the normalization of hormone levels after withdrawal supports the idea that these effects could be transient and reversible. Further research evaluating a wider range of hormones, including those of the thyroid and adrenal axes, is warranted to clarify whether the observed endocrine suppression is axis-specific or part of a more global functional decrease.

The hormone-dependent nature of spermatogenesis makes it susceptible to disruption by external factors, including drugs and chemicals. For example, Xu et al. demonstrated that isoflurane exposure disrupts FSH, LH, and testosterone levels by inhibiting hypothalamic and pituitary function, likely through the suppression of synaptic transmission or nerve signaling [20]. In the present study, significant changes in FSH and LH levels were observed. While FSH levels decreased in Groups D1, D2, D3, and D4, only the reduction in Group D2 was statistically significant. Similarly, LH levels decreased significantly in Groups D2, D3, and D4, with the lowest levels observed in Group D3, which corresponded to the highest intensity of desflurane exposure. The lack of recovery in LH levels after a seven-day withdrawal period in Group D2 suggests that chronic desflurane exposure may impair hypothalamic or pituitary hormone production. However, the normalization of hormone levels during short- and long-term recovery periods indicates that the adverse effects of desflurane may be reversible.

Unexpectedly, testosterone levels in both serum and testicular tissue exhibited an irregular pattern that did not correlate with the severity of desflurane exposure. This anomaly, coupled with the progressive increase in serum and testicular inhibin B levels, suggests that desflurane may influence testosterone and inhibin B through mechanisms involving sex hormone-binding globulin or albumin, although the exact pathway remains unclear.

An apparent inconsistency was observed in the hormonal profile, where testosterone levels increased despite a concomitant decrease in LH and FSH concentrations. This paradoxical finding may be explained by several physiological mechanisms. First, local intratesticular regulation can modulate steroidogenesis independently of pituitary input, as Leydig cells possess autocrine and paracrine control systems that respond to oxidative stress and inflammatory mediators [29]. Second, transient activation of stress pathways, including the hypothalamic–pituitary–adrenal axis, may influence gonadotropin secretion while temporarily stimulating adrenal or testicular androgen production. Additionally, the temporal dynamics of feedback regulation between the hypothalamus, pituitary, and gonads may result in asynchronous fluctuations, particularly under short experimental exposure periods [30]. In addition, The apparent paradox of increased serum testosterone and inhibin B levels despite suppressed LH and FSH concentrations likely reflects a dissociation between systemic hormone measurements and intratesticular endocrine dynamics induced by chronic desflurane exposure. In the present study, this discrepancy is most evident in Group D3, where serum testosterone remained significantly elevated while intratesticular testosterone showed a marked reduction of approximately 39%. This finding suggests that circulating testosterone levels may not accurately reflect local Leydig cell steroidogenic capacity under conditions of anesthetic-induced testicular stress.

One plausible mechanistic explanation is that desflurane alters testosterone distribution, metabolism, or clearance rather than uniformly enhancing androgen synthesis. Volatile anesthetics have been shown to influence hepatic enzyme activity and plasma protein binding. A desflurane-induced reduction in testosterone clearance or an increase in binding to circulating carrier proteins (e.g., albumin or sex hormone–binding globulin–like proteins in rodents) could result in elevated serum testosterone concentrations despite reduced intratesticular production. In this context, serum testosterone would appear elevated while the bioavailable fraction within the testicular microenvironment declines, as observed in Group D3. Additionally, intratesticular testosterone is tightly regulated by paracrine and autocrine mechanisms that are partially independent of pituitary LH stimulation. Oxidative stress, interstitial edema, and microvascular alterations—clearly demonstrated histopathologically in Group D3—may impair Leydig cell steroidogenesis locally, even in the presence of relatively preserved or redistributed circulating testosterone. This concept is supported by the recognized compartmentalization of testosterone, where intratesticular concentrations are normally several-fold higher than serum levels and are essential for spermatogenesis. A reduction in intratesticular testosterone, therefore, may contribute to impaired sperm morphology despite elevated systemic levels.

The concomitant increase in serum and testicular inhibin B further supports a compensatory or dysregulated Sertoli cell response. Inhibin B secretion may be upregulated as a local reaction to germinal epithelial injury or altered Sertoli–germ cell signaling, rather than reflecting normal FSH-driven feedback regulation. This interpretation is consistent with the observed suppression of FSH alongside rising inhibin B levels and suggests disruption of the classical hypothalamic–pituitary–gonadal feedback loop.

Taken together, these findings indicate that desflurane exposure may induce a state of endocrine uncoupling, characterized by preserved or elevated serum hormone levels but impaired intratesticular hormonal milieu. This dissociation underscores the importance of evaluating both systemic and tissue-specific hormone levels when assessing reproductive toxicity. The marked decline in intratesticular testosterone in Group D3, despite high serum testosterone, highlights a potential mechanism by which desflurane adversely affects spermatogenesis and testicular integrity independent of pituitary gonadotropin stimulation. Therefore, these findings should be interpreted with caution, acknowledging that hormonal responses to anesthetic agents can be complex and context-dependent.

Sperm morphology serves as a sensitive indicator of disturbances in the male reproductive system. However, limited studies have explored the effects of inhalational anesthetics on sperm morphology [7, 9, 11, 23]. Campion et al. reported that isoflurane exposure reduces sperm motility in rats [31], while Zanin et al. observed structural abnormalities in sperm following chronic exposure to isoflurane [9]. In contrast, Kaya et al. found no significant changes in the percentage of abnormal sperm despite reductions in sperm concentration and motility following sevoflurane exposure [13]. In the current study, a significant increase in the percentage of abnormal sperm was observed in Groups D2, D3, D4, and D5 compared to the control group. This suggests that desflurane may disrupt the differentiation or maturation of spermatogonia, leading to abnormal sperm morphology. The reduction in abnormal sperm percentage in Group D5 compared to Group D4 indicates that a 14-day recovery period may be necessary to mitigate the structural impairments caused by desflurane exposure.

The structural integrity of the testis is critical for normal reproductive function, and numerous factors can compromise testicular tissue. Xu et al. demonstrated that isoflurane exposure induces dose-dependent damage to seminiferous tubules, characterized by disorganized epithelium, sloughed germ cells, and interstitial edema [21]. Similarly, Kaya et al. reported progressive degenerative changes in seminiferous epithelium, including germ cell loss and interstitial edema, following chronic sevoflurane exposure [13]. In the present study, histopathological examination revealed significant testicular tissue damage in all experimental groups, with the most severe changes observed in Group D3. These changes included degenerative alterations in seminiferous epithelium, interstitial edema, and congestion, although no loss of spermatogenic cells was observed. The correlation between histopathological injury scores and hormonal changes in Group D3 further underscores the adverse effects of desflurane on testicular function. Additionally, the incomplete recovery of tissue damage after a seven-day withdrawal period in Groups D2 and D5 suggests that longer recovery periods may be required for tissue repair.

Current evidence indicates that opioids exert multifaceted adverse effects on the male reproductive system through mechanisms involving oxidative stress, disruption of androgen-dependent pathways, and DNA damage. Kosal et al. demonstrated that transdermal fentanyl administration leads to marked deterioration of testicular architecture, including disorganization of the seminiferous epithelium and increased interstitial connective tissue, suggesting opioid-induced oxidative injury within the testis [32]. Consistently, Yazdani et al. reported that heightened oxidative stress and germ-cell–specific apoptosis following testicular ischemia/reperfusion were associated with significant reductions in epididymal sperm quality, supporting the notion that oxidative imbalance plays a central role in germ cell vulnerability and impaired spermatogenesis [33]. Moreover, Zhang et al. showed that fentanyl modulates apoptosis through the Sirt1-dependent inhibition of NF-κB signaling, indicating that opioids not only induce oxidative stress but may also alter nuclear regulatory pathways involved in DNA integrity and cell survival [34]. Taken together, these findings suggest that opioid exposure may contribute to reproductive toxicity through the convergence of oxidative stress, androgen receptor dysregulation, and opioid-mediated interference with DNA repair and apoptotic signaling.

Environmental chemical exposure remains a significant public health issue, and infertility is increasingly recognized as a potential consequence of toxicant exposure [35]. Although concerns have been raised regarding the reproductive risks associated with inhalational anesthetics in clinical settings, evidence from animal studies provides only preliminary insights into these effects. Previous reports—including meta-analytic data suggesting increased rates of spontaneous abortion among healthcare workers exposed to anesthetic gases—highlight the need for continued investigation; however, such findings cannot be directly extrapolated to humans due to differences between experimental and occupational exposure conditions [36]. Research in animal models has demonstrated that waste anesthetic gases may disrupt germ cell development, sperm morphology, and motility, but these observations should be interpreted cautiously as they represent early preclinical data rather than confirmed human outcomes [11 [23, 37, 38].

This study is the first to investigate the effects of chronic desflurane exposure on male reproductive hormones, testicular tissue, and sperm morphology in rats. The findings suggest that desflurane may induce testicular tissue damage, disrupt reproductive hormone levels, and impair sperm morphology. These results highlight the potential risks associated with occupational exposure to inhalational anesthetics among health care workers.

There are some limitations for this study. First one is the limited duration of the follow-up period used to assess the reversibility of the observed effects. While changes in reproductive hormone levels, testicular histology, and sperm morphology were noted, the timeframe may not have been sufficient to determine whether these alterations are fully reversible after cessation of desflurane exposure. Longer-term studies are necessary to evaluate the potential for recovery of reproductive parameters over time. Additionally, as this study was conducted in an animal model, caution must be exercised when extrapolating the results directly to humans. Further research is needed to validate these findings in clinical settings and to establish safe exposure thresholds for health care professionals regularly exposed to inhalational anesthetics. Third one is the small sample size, with only five rats included in each experimental group. Such a limited number of animals reduces the statistical power of the analysis and increases the risk of both type I and type II errors, potentially obscuring subtle but biologically relevant differences between groups. Although small animal numbers are often used to minimize ethical and logistical burdens, the limited sample size may restrict the generalizability and reproducibility of the findings. Future studies should include a larger cohort and, ideally, perform an a priori power analysis to ensure adequate statistical sensitivity for detecting meaningful biochemical and histological changes. Finally, testicular tissue evaluation was based solely on H&E staining and a semi-quantitative injury scoring system. While H&E provides valuable information on general morphology, it does not allow detailed assessment of specific cellular or molecular alterations such as apoptosis, oxidative stress, or fibrosis. Incorporating additional histochemical or immunohistochemical stains—such as TUNEL, Masson’s trichrome, or markers of oxidative injury—would provide more comprehensive insights into the underlying mechanisms of tissue damage.

Conclusion

Chronic exposure to desflurane adversely affects testicular tissue integrity, sperm morphology, and the regulation of reproductive hormones in rats. These findings underscore the potential reproductive toxicity of inhalational anesthetics and emphasize the need for further research to elucidate the underlying mechanisms and mitigate the risks associated with occupational exposure.

Authors’ contributions

SD, HYD, HT, AA and MS—performed the research. SD and HYD—designed the research study. AA—performed the pathological analysis. SD, HYD, HT and MS—analyzed the data and wrote the draft of the manuscript. All authors read and approved the final manuscript. All authors were involved in the collection of experimental data.

Funding

This work was supported by Gaziosmanpasa University Scientific Research Projects Unit (Grant Number: 2013/94).

Data availability

The data presented in this study are available on reasonable request from the corresponding author.

Declarations

Ethics approval and consent to participate

This experiemental study was approved by Tokat Gaziosmanpasa University Animal Experimentations Local Ethics Committee (Grant number: 2013-HADYEK-12).

Competing interests

The authors declare no competing interests.