Effects of 5G frequencies on sperm parameters, mating success, and offspring outcomes in rats

Animals

A total of 36 (N=36) Sprague Dawley rats consisting of 18 males and 18 females (∼8 wk old, 200±50 g) were obtained from the animal unit. Male rats were obtained one month earlier to allow for their exposure to the 5G technology. All animals were housed in individually ventilated cages with food and water provided ad libitum, a 12-h light-dark cycle, and an ambient temperature of 22±5°C.

The male rats were randomly divided into three groups (n=6) in each group: a sham-exposed Control group and two exposure groups receiving 3.5 GHz and 24 GHz signals. The sample size was determined based on a Test Guideline OECD 407.⁹ The exposure procedure was conducted in the radiation room for 7 h daily over 60 d. No blinding was applied during the experimental procedures. All assessments were conducted with knowledge of the group assignments due to logistical constraints.

At the end of the experimental procedures, all animals were euthanized via intraperitoneal overdose (>0.1 mL/100g) of ketamine (3.34 mg/kg, Ilium, USA), xylazine (3.34 mg/kg, Ilium, USA), and zoletil-50 (1.66 mg/kg, Virbac, Australia) (KTX) cocktail. Death was confirmed by the absence of corneal reflex, loss of tail pinch reflex, forelimb, and hindlimb pedal reflex.

Exposure settings

The exposure model was designed to simulate realistic 5G environmental conditions¹⁰ using frequencies of 3.5 GHz (mid-band) and 24 GHz (high band) spectra allocated for 5G communication.¹⁰ Both frequencies were chosen to enable a comparative evaluation of their biological effects, as no prior study investigated pregnancy outcomes following paternal exposure at these frequencies.

The Control group was placed in the radiation room with the antenna or device used for the exposure set to inactive mode. The 3.5 GHz group was exposed using an omnidirectional microstrip antenna (Supplementary Fig. 1), operating at 22 dBm output power. The antenna provided by the Faculty of Electronics and Computer Technology and Engineering, Universiti Teknikal Malaysia Melaka. The 24 GHz exposed group utilized a 24 GHz Tuya WiFi Smart Human Presence Detector (Shenzhen, China), equipped with an LD2420 24G mmWave antenna capable of emitting a 24 GHz signal. This antenna operates at an 11 dBm output power and represents the higher end of the 5G technology spectrum, operating within the mmWaves radiation range.

The exposure device was positioned 20 cm from the animal cages (Supplementary Fig. 2), consistent with previous rodent studies.⁶ Additionally, this distance was also aligned with international safety guidelines from the United States Federal Communications Commission (US FCC) and International Commission on Non-Ionizing Radiation Protection (ICNIRP).^11,12 Metal food holders were replaced with plastic containers to minimize electromagnetic interference, and a radiation meter, EXA Signal Analyzer N9010A (Keysight, USA), was placed inside the cage before exposure to verify the presence and strength of the signal.

Determination of the female oestrus cycle

Female rats’ oestrus cycle was monitored by vaginal smear analysis before the mating procedure. Vaginal lavage samples were collected between 7:00 AM and 10:00 AM.¹³ and examined under a field microscope, Olympus BX51, at 20x objective magnification to determine the stage of the oestrus cycle. The oestrus cycle is characterized by four phases: proestrus, oestrus, metestrus, and dioestrus ( Fig. 1). These phases are identified based on the presence of three types of cells: nucleated epithelial cells (round and nucleated), leukocytes (small and round), and cornified keratinized cells (anucleate and irregular in shape). Female rats in the proestrus or oestrus phase were considered ready to mate.¹⁴

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Fig. 1. The appearance of the cells in the vaginal cytology during the evaluation of the oestrous cycle under a light microscope, with objective magnification of 20X. (A) proestrus, (B) oestrus, (C) metestrus, (D) dioestrus. Here, K refers to keratinized cells; E, epithelial cells; L, leukocytes. The image was captured using Olympus BX51 (Olympus, Japan).

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Mating procedure

The mating procedure begins at week 7 of exposure, one week before the end of the exposure period, to reduce the likelihood of recovery upon withdrawal of the exposure. Males and females cohabitated on a 1:1 basis, and daily vaginal swabs were collected and examined microscopically for the presence of sperm.¹³ The detection of a copulatory plug or sperm in the vaginal smears was considered gestation day 0,¹⁵ and all female rats were separated individually in monitoring cages until the delivery or for up to 30 d.

Evaluation of sperm parameters

After mating, male rats were euthanized, and their testes were harvested. The cauda epididymis was carefully separated and minced in 2 mL of pre-warmed phosphate-buffered saline for the sperm sample. The mixture was incubated at 37°C for 40 min, allowing the sperm to swim out for collection.

Sperm concentration

A drop of 10 µL of the sperm suspension was placed on a Makler chamber. Sperm concentration was counted as an average of five rows under 10x magnification.

Sperm motility

A drop of 10 μL sperm suspension was placed on a microscope slide, and 200 spermatozoa were counted in duplicate at 40x magnification. Sperm motility was determined following the WHO guidelines, which are: A: rapidly progressive motile (RP), B: slow progressive (SP), C: non-progressive motile (NP), and D: immotile (IM).¹⁶ Motility was calculated as the percentage of total motile sperm (A + B+ C/total counted sperm) x 100.

Sperm viability

The hypoosmotic swelling test (HOST) was used to determine sperm viability. Sperm suspension was mixed with a hypo-osmotic solution (1:10), incubated at 37°C for 30 m. About 10 μL of the mixture was applied to a microscope slide, smeared, air-dried at room temperature, and stained with a Diff-Quik Stain Kit (Epredia, USA) to improve sperm visibility under a bright-field microscope. Viable sperm cells were counted in duplicate under 40x magnification, examining 200 sperm cells per sample.

Pregnancy outcomes

All female rats were monitored throughout their gestation periods, and the gestation lengths for each animal were recorded. Mating index¹⁷ and pregnancy rate¹⁸ were calculated using the following formulas: $\text{Mating index (\%)=}\frac{\text{No}\text{. of females mated}}{\text{No}\text{.of females placed with males}}\text{x 100}$ $\text{Pregnancy rate (\%)}=\frac{\text{No}\text{. of pregnant female rats}}{\text{Total success fully mated female rats}}\times \text{100}$

Birth metrics

Birth weight, live birth rate, and sex ratio of the pups: After 24 h post-natural delivery, each dam was assessed to record the total number of pups per litter, still births, and any observed anomalies. Furthermore, all the pups were weighed individually using a balance with 0.01 g sensitivity, and the live birth rate was determined by using the following formula.¹⁹ $\text{Live birth rate}\left(\text{\%}\right)\text{=}\frac{\text{Number of live pups}}{\text{Number of total pups born}}\text{x 100}$

The sex ratio was also recorded after 24 h of delivery and calculated using the following formula: $\text{Sex ratio=}\frac{\text{No}\text{. of live male pups}}{\text{Number of live female pups}}$

All the pups and the dams were euthanized after the data collection.

Statistical analysis

All results were expressed as mean±standard deviation (mean±SD). One-way ANOVA was used to compare the significance of sperm parameters and the birth weight of pups. Kruskal-Wallis was used to analyze the gestation period. Statistical analysis was performed using GraphPad Prism 10 software (GraphPad Software, USA), with P<0.05 considered significant.

Sperm parameters

The findings showed that the 3.5 GHz (119.2 x 10⁶/mL ± 4.22, P<0.001) and 24 GHz (131.3 x 10⁶/mL ± 8.55, P<0.001) exposed groups demonstrated a significant decrease in sperm concentration compared to the Control group (175.5 x 10⁶/mL ± 2.31) ( Fig. 2A). The differences between exposed groups were not statistically significant (P=0.31).

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Fig. 2. Analysis of sperm parameters in each group. Data were generated using GraphPad Prism 10 software and presented as mean ± SEM. (A) sperm concentration in each group. ^****Represents a significant different between 3.5 GHz and Control group with P<0.0001. ^***Represents a significant different between 24 GHz and Control group with P=0.0002. ns, represent no significant difference between exposed groups. (B) Percentage of total motile sperm in each group. ^**Represents a significant different between 3.5 GHz and Control group with P=0.0010. ^****Represents a significant different between 24 GHz and Control group with P<0.0001. (C) Percentage of viable sperm in each group. No significant difference was recorded.

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The 3.5 GHz (43.6%±2.77, P=0.001) and 24 GHz (27.3%±7.58, P<0.001) exposed groups also showed a significant decrease in the percentage of sperm motility compared to the Control group (75.6%±3.12) ( Fig. 2B) but no significant difference between the two exposed groups (P=0.0822). The percentage of sperm viability remained unchanged across the group ( Fig. 2C).

Mating outcomes

Two females in the control group exhibited irregular oestrus cycles. However, the variability is a recognized biological phenomenon in rodents.²⁰ Consequently, the Control group achieved a mating index of only 66.7%, whereas both exposed groups achieved a 100% mating index ( Table I).

The mating period for the Control group recorded the highest (3.00±1.16 d), followed by the 24 GHz group (2.5±1.22 d), and the 3.5 GHz group (2.17±0.41 d), with no significant difference between the groups (P=0.50).

Both the Control and the 3.5 GHz groups showed 100% pregnancy rates. However, the 24 GHz group had a reduced pregnancy rate of 83.3%, with one female rat failing to become pregnant despite the presence of sperm in the vaginal plug. The gestation period showed no significant difference across groups (P=0.05).

Birth metrics