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J Adv Med Biomed Res 2026, 34(2): 98-109 |
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Ethics code: IR.ZUMS.REC.1399.419
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Khavasi N, Saboory E, Najmi Z, Ghassemifard L, Ghanbari A M, Sardari S. Protective Effects of Cuscuta epithymum Extract on Reproductive Health in a Pre-Pregnancy Stress Rat Models. J Adv Med Biomed Res 2026; 34 (2) :98-109
URL: http://journal.zums.ac.ir/article-1-7733-en.html
URL: http://journal.zums.ac.ir/article-1-7733-en.html
Narjes Khavasi1 
, Ehsan Saboory2 , Zahra Najmi3 , Leila Ghassemifard1 , Amir Mohammad Ghanbari4 , Saeed Sardari *5
, Ehsan Saboory2 , Zahra Najmi3 , Leila Ghassemifard1 , Amir Mohammad Ghanbari4 , Saeed Sardari *5
1- Department of Persian Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
2- Department of Addiction Studies, School of Medicine, Zanjan University of Medical Science, Zanjan, Iran
3- Department of Clinical and Translational Science, Tufts Medical Center, Boston, MA, USA & Department of Obstetrics and Gynecology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
4- School of Medicine, Zanjan University of Medical Science, Zanjan, Iran
5- Department of Persian Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran ,ssardari504@zums.ac.ir
2- Department of Addiction Studies, School of Medicine, Zanjan University of Medical Science, Zanjan, Iran
3- Department of Clinical and Translational Science, Tufts Medical Center, Boston, MA, USA & Department of Obstetrics and Gynecology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
4- School of Medicine, Zanjan University of Medical Science, Zanjan, Iran
5- Department of Persian Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran ,
Keywords: Immobility stress, Pre-gestation, Reproductive functions, Sex hormones, Cuscuta Epithymum, Animal study
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ABSTRACT
Background & Objective: Presentational stress attenuates circulating levels of sex hormones and potentially affects reproductive function. This study investigated the effects of simultaneous administration of Cuscuta epithymum (CE) extract on stress-related reproductive parameters in parental Wistar rats and on the serum sex hormone levels of their offspring.
Materials & Methods: In this experiment, 64 female rats and 36 male Wistar rats were divided into various categories: Control (no stress or CE extract), CE Extract (received only the extract), Stress (exposed only to stress), Stress + CE Extract (exposed to both stress and received the extract). Parental reproductive parameters were assessed across groups, including vaginal epithelial thickness and vaginal pH in female rats, sperm parameters in males, and circulating sex hormone levels in both sexes. Offspring were evaluated for sex ratio, litter size, and serum sex hormone levels in all groups.
Results: Presentational stress significantly reduced sperm parameters in adult males and decreased the thickness of the vaginal epithelium in adult females (P< 0.01). However, co-administration of Cuscuta epithymum extract in the stress-exposed groups significantly improved these parameters compared with the stress group and restored them toward control levels. Additionally, stress exposure led to a significant reduction in the number of male offspring per litter (P < 0.01), an effect that was significantly mitigated by CE extract administration (P < 0.01).
Conclusion: Concomitant administration of Cuscuta epithymum extract attenuated the adverse effects of stress on parental reproductive parameters, offspring sex ratio, litter size, and serum sex hormone levels. These findings suggest that CE extract may serve as a promising natural supplement for preserving reproductive function under stress before conception.
Keywords: Cuscuta epithymum, Reproductive health, Pre‑pregnancy stress, Rat model, Herbal extract, Oxidative stress, Fertility, Persian Medicine
Background & Objective: Presentational stress attenuates circulating levels of sex hormones and potentially affects reproductive function. This study investigated the effects of simultaneous administration of Cuscuta epithymum (CE) extract on stress-related reproductive parameters in parental Wistar rats and on the serum sex hormone levels of their offspring.
Materials & Methods: In this experiment, 64 female rats and 36 male Wistar rats were divided into various categories: Control (no stress or CE extract), CE Extract (received only the extract), Stress (exposed only to stress), Stress + CE Extract (exposed to both stress and received the extract). Parental reproductive parameters were assessed across groups, including vaginal epithelial thickness and vaginal pH in female rats, sperm parameters in males, and circulating sex hormone levels in both sexes. Offspring were evaluated for sex ratio, litter size, and serum sex hormone levels in all groups.
Results: Presentational stress significantly reduced sperm parameters in adult males and decreased the thickness of the vaginal epithelium in adult females (P< 0.01). However, co-administration of Cuscuta epithymum extract in the stress-exposed groups significantly improved these parameters compared with the stress group and restored them toward control levels. Additionally, stress exposure led to a significant reduction in the number of male offspring per litter (P < 0.01), an effect that was significantly mitigated by CE extract administration (P < 0.01).
Conclusion: Concomitant administration of Cuscuta epithymum extract attenuated the adverse effects of stress on parental reproductive parameters, offspring sex ratio, litter size, and serum sex hormone levels. These findings suggest that CE extract may serve as a promising natural supplement for preserving reproductive function under stress before conception.
Keywords: Cuscuta epithymum, Reproductive health, Pre‑pregnancy stress, Rat model, Herbal extract, Oxidative stress, Fertility, Persian Medicine
1. Introduction
A state of stress occurs when the body experiences a negative stimulus that disrupts its natural balance (1). The likelihood of infertility increases significantly as a result of prolonged stress, which has pronounced effects on fertility (2, 3).
Stressful conditions can decrease the quantity, mobility, and progressive movement of sperm by adversely affecting the epididymis and inhibiting spermatogenesis (4, 5). Moreover, a reduction in the efficiency and quantity of Y sperm, along with a change in the acidity of the vaginal milieu, leads to a higher likelihood of delivering a female child (6), as well as causing shifts in the plasma concentration of testosterone (7) and estradiol (5, 8).
On the other hand, pre-pregnancy stress can affect the fetus and have harmful effects such as, alterations in sex hormone levels in male and female offspring. When an individual experiences stress, the hypothalamic-pituitary-adrenal (HPA) axis becomes activated. The sexual behavior of mice is impacted by immobility stress, a form of stress that combines physical and psychological factors, as it triggers the HPA axis (3, 9).
While a range of pharmaceutical and synthetic agents are routinely employed to manage stress, their use is frequently associated with adverse side effects. Consequently, there is an increasing emphasis on investigating medicinal plants and their derivatives as potentially safer and more tolerable alternatives (10).
One such plant, widely studied and utilized in Persian medicine to alleviate reproductive disorders and stress-related complications, is Cuscuta epithymum. Recent studies have reported significant roles of Cuscuta epithymum in decreasing the adverse effects of stress on the liver and spleen immune system, as well as remarkable anti-neuroinflammatory and anti-seizure effects in rats (11,12). The genus Cuscuta epithymum belongs to the Convolvulaceae family (Note: Cuscutaceae is now often merged with Convolvulaceae) and is distributed worldwide, comprising approximately 170 species. In Iran, 18 species of Cuscuta epithymum have been identified across various regions (13). Cuscuta epithymum is rich in phenolic compounds and flavonoids, including cuscutin, amarbelin, β-sitosterol, stigmasterol, myricetin, quercetin, cuscutamine, luteolin, and bergenin, which contribute to its antioxidant properties (14, 15). In Persian medicine, Cuscuta epithymum is prescribed for the treatment of excessive coldness and blood accumulation in the reproductive systems of males and females, support spermatogenesis, and for the treatment of infertility in women. Cuscuta epithymum is also used to prevent infertility (16, 17). Hence, taking into account the impact of stress on parental fertility and circulating sex
hormone levels, along with stress effects before conception on hormonal shifts in their offspring, and considering the reported effects of Cuscuta epithymum species on stress and reproductive function in both sexes this study was designed to evaluate the potential protective role of Cuscuta epithymum extract in a rat model of stress. This study aimed to assess CE's influence on parental reproductive parameters, including vaginal epithelial thickness and vaginal pH in female rats, sperm parameters in male rats, and serum sex hormone levels in both sexes. Furthermore, offspring were assessed for sex ratio, litter size, and sex hormone levels across different groups.
2. Materials and Methods
2.1 Subjects
The percolation technique was employed for obtaining the extraction of the CE extract in this experimental study (herbarium code: 5501). The Cuscuta epithymum plant was crushed and then subjected to two standard sieves (160 and 250 micrometers) for proper sanitation. Afterwards, the 1500 g powder was transferred to the percolator, where 70% ethanol was used as the solvent.
The dark color and pungent odor indicated the purity of the CE extract. The solvent was separated using a Rotary Evaporator (IKA RV 05-B, Germany) at 45 to 50 °C, then subjected to vacuum drying at 45 °C (53 OT, Iran). The drying procedure persisted until a stable weight was achieved. This method was repeated three times at 24-hour intervals. After filtering the extract, concentration was performed in a water bath under vacuum conditions, followed by lyophilization (FD-5N; Eyela, Tokyo). Ultimately, the product was preserved at 4 degrees Celsius. (19, 20).
2.3 Restraint stress procedure
A period of immobility stress was experienced by rats, as described by Nakhjiri et al. (21). Briefly, this process included moving the rats to the designated testing area. They were housed in a restraining device under standard room conditions. Each day, the rats were placed within the confinement chamber (a cylindrical plastic container measuring 16 cm in length and 6 cm in width) for a duration of 2 hours (12).
2.4 Sperm analysis
Male rats (n = 8 in each group) were anesthetized with isoflurane on the first morning following the completion of the stress period. One epididymis was randomly selected for evaluation of sperm parameters. The caudal part of the epididymis was dissected, then floated in 5 ml of pre-heated Ham's F10 solution (Sigma, USA), then incubated up to 15 minutes at 37°C in order to allow the sperms to diffuse into the buffer.
A total of 10μl of the sperm suspension was smeared on a Neubauer chamber, finally, it was prepared to analyze sperm motility with a light microscope (Olympus BX51, Japan). After 15 minutes, 10 microscopic fields with magnifications of 400 times, counting more than 200 sperms, were observed (5). Sperm motility was defined as: a) progressive (P) b) non-progressive (NP), and c) immotile (IM) when sperms had no movements. Progressive and non-progressive sperms were presented as a percentage of total sperm motility. Sperms having abnormalities in the tail, neck and head sections were considered abnormal. After diluting the suspension, one drop was placed on an erythrocytometer, and sperm count was performed with a light microscopy (Olympus, Japan) (22). To assess the morphology of sperm, its suspension was smeared on a glass slide and then stained with Eosin. Finally, sperms were categorized into two major classes: a) normal and b) abnormal, sperms with dark pink are considered dead sperm as eosin penetrated into the sperm because of the breakdown of the cell membrane.
2.5 Vagina sampling
The pH of the vagina was measured using pH indicator paper (23). For this purpose, the pH meter paper was inserted up to one-third of the length of the vagina using forceps, kept inside for 1 minute, and the pH change was recorded. Finally, the rats were anesthetized with isoflurane and tissue samples were prepared from the rats' vaginas. Obtained tissues fixation was performed in 10% buffered formalin, then it was sectioned and finally stained with H&E. Motic images were used to measure the thickness of vaginal Cuscuta epithymum. For each section, 10 different lengths were measured to obtain the average thickness (5).
2.6 Mating and offspring
Twenty-four hours after the cessation of the immobilization stress protocol, each male rat was housed individually with two female rats to assess reproductive performance. After a 3-day cohabitation period, the males were removed, and the females remained in the cages until the completion of gestation. Upon delivery, offspring were evaluated for litter size, sex ratio, and serum estradiol and testosterone levels.
2.7 Blood sample collection
After the end of the stress phase for parents and at PND 25, under general anesthesia with isoflurane, blood samples were obtained at 8:00 a.m. and 11:00 a.m., through cardiac puncture.
The samples were poured into EDTA-containing tubes, and then centrifuged at 9000 rpm. The obtained plasma samples of offspring, and serum samples of adult male and female rats were placed at -80 °C. The blood samples were used for quantification of testosterone and 17-beta estradiol, using commercial ELISA kit including Zellbio GmbH (Germany, Cat. No: ZB-10259D-R9648) and Zellbio GmbH (Germany, Cat. No: ZB-10174M-R9648), respectively. The amounts were presented as pg/mL for 17-beta estradiol and ng/mL for testosterone.
2.8 Statistical analysis
The analysis was conducted using SPSS software (SPSS Inc., Chicago, IL, USA) edition 22 and presented as mean ± SEM. The Kolmogorov–Smirnov test was utilized to check the distribution of control data. As a result, data analysis was done parametrically with the use of normal distributions and one-way analysis of variance (ANOVA) for comparisons of results for multiple groups.
When applicable, Tukey post hoc test was used. Sex ratio was analyzed using K2 test. A p < 0.05 was considered significant in all instances.
3. Result
Analysis was performed on the complete data of 100 adult rats and their 131 offspring.
3.1 Sperm parameters
There was a statistically significant difference in sperm parameters including count, motility viability, and morphology between the groups (Figure 1).
Figure 1. Sperm Parameters in Control Adult Rats, CE extract, stress and stress + CE extract groups: Panel a; Total count: *** p<0.001, ** p<0.01, with control group, # indicates p<0.05, ## indicates p<0.01 with CE Extract group. Panel b; Motility: ** indicates p<0.01, *** indicates p<0.001with control group, ### indicates p<0.001 with CE Extract group, $$ indicates p<0.01, $$$ indicates p<0.001 with Stress group. Panel c; Viability: * indicates p<0.05, *** indicates p<0.001with control group, ### indicates p<0.001 with CE Extract group, $$$ indicates p<0.001 with Stress group. Panel d; Morphology: ** indicates p<0.01 with control group, ## indicates p<0.01 with CE Extract group, $$$ indicates p<0.001 with Stress group. (Prepared by Authors, 2026).
3.2 Sperm count
The results demonstrated that the number of sperm in the stress group was significantly lower compared to the control, CE extract, and stress + CE extract groups (1.68±0.42, 3.78±0.9, 3.03±0.64, and 2.12±0.3; P= 0.002) (Table1). Administration of Cuscuta epithymum extract to the stress-exposed group resulted in an increase in sperm count from 1.68±0.42 to 2.12±0.3. However, this difference was not statistically significant (Table2).
3.3 Sperm motility
According to table 1 sperm motility revealed that the percentage of sperms with progressive movements in the stress group was significantly lower compared to the control, CE extract, and stress + CE extract groups (7.26±1.21, 33.88±11.25, 35.02±7.62, and 33.22±9.65; P= 0.001). However, no significant difference was found among the groups in the percentage of sperm with non-progressive movements. The percentage of immotile sperm in the stressed rats was significantly higher in comparison with control, CE extract, and stress + CE extract groups (60.08±8.89, 35.48±10.64, 30.85±10.54, and 39.01±6.97; P< 0.004). The percentage of progressive sperms in CE extract group significantly reached the baseline level in control group (7.26±1.21 vs. 33.22±9.65; P< 0.001); and the number of immotile sperms was significantly decreased from 60.08±8.89 to 39.01±6.97 (P= 0.008) (Table2).
3.4 Sperm viability
The percentage of sperms with abnormal morphology in the stress group was significantly higher compared to the control, CE extract, and stress + CE extract groups (60.13±10.46, 36.37±9.34, 37.64±7.46, and 37.15±11.18; P= 0.004) (Table 1).
As it has been shown in table 2, CE extract administration to the stress group significantly decreased the percentage of sperms with abnormal morphology from 60.13±10.46 to 37.15±11.18, (P< 0.001).
Table 1. Comparison of Sperm parameters between the groups
Note: Control (neither stress nor CE extract), CE Extract (just received extract), Stress (just received stress), Stress+ CE Extract (received both stress and extract). * Indicates significant difference with other groups, one-way ANOVA and Tukey post hoc test.
3.6 Effects of pre-gestational stress and CE extract on plasma concentrations of estradiol and testosterone in parents and offspring
Significant differences in plasma testosterone and estradiol levels were observed among the experimental groups in both parents and offspring at postnatal day (PND) 26, as illustrated in (Figure 2).
3.7 Testosterone level in adult rats
In adult male rats, plasma testosterone concentration (ng/dl) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (0.82±0.15, 2.12±0.48, 0.95±0.5, and 3.08±0.57; P= 0.002) (Table 3).As shown in Table 4, CE extract administration to the stress group significantly increased adult male rats’ serum testosterone level from 0.82±0.15 to 3.08±0.57 ng/dl, P= 0.001).
3.8 Testosterone level in offspring
Figure 2. Serum testosterone and estradiol levels of parents (a and c) and pups (b and d) represented in the charts a, b, c, and d in the control, CE extract, stress and stress + CE extract groups: Panel a; Testosterone: * indicates p<0.05 with control group, ## indicates p<0.01 with CE Extract group, $$$ indicates p<0.001 with Stress group. Panel b; Testosterone Offspring: * indicates p<0.05 with control group, # indicates p<0.05 with CE Extract group, $$ indicates p<0.01 with Stress group. Panel c; Estradiol: # indicates p<0.05 with CE Extract group, $ indicates p<0.05 with Stress group. Panel d; Estradiol Offspring: * indicates p<0.05, ** indicates p<0.01 with control group. (Prepared by Authors, 2026).
3.9 Estradiol level in adult rats
In the adult female rats, plasma estradiol concentration (pg/ml) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (26.8±3.18, 44.2±36.77, 67.62±37.31, and 72.26±13.15; P= 0.048) (Table 3). According to table 4, administration of CE extract to the stress group significantly increased adult female rats’ serum estradiol level from 26.8±3.18 to 72.26±13.15 pg/mL, (P= 0.027).
3.10 Estradiol level in offspring rats
Similar to adults, in female offspring rats, plasma estradiol concentration (pg/ml) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (30.56±9.72, 66.92±37.82, 44.27±23.96, and 13.62±7.42; P= 0.029) (Table3). However, administration of CE extract to the stressed group did not result in a statistically significant increase in serum estradiol levels (P = 0.28; Table 4)
Table 3. Hormone Levels in Parents and Offspring rats
3.11 Effects of pre-gestational stress and CE extract on vaginal thickness, vaginal pH and Offspring Number
3.11.1 Vaginal Thickness
The measurement of the vaginal epithelium thickness in mature rats revealed that the stress group exhibited a notably reduced thickness in comparison to the control, CE extract, and stress + CE extract groups (P=0. 005; table 5). In the group subjected to stress, the application of CE extract led to a significant rise in the thickness of the vaginal epithelium in adult rats. (P<0.001; Table 6 and Figure3).
3.11.2 Vaginal PH
Vaginal pH assessment in adult female rats revealed a significant reduction in the stress group compared to the other groups (P < 0.001; Table 5). CE extract in the stress group resulted in a significant increase in vaginal pH, rising from 4.5 ± 0.109 to 5.00 ± 0.000 (P < 0.001; Table 6). These findings indicate that CE extract effectively counteracts the stress-induced reduction in vaginal pH.
3.11.3 Offspring Number and Sex Ratio
The total number of offspring in the control group was 29 (12 males, 17 females); in the CE extract group, 45 (26 males, 19 females); in the stress group, 20 (6 males, 14 females); and in the stress + extract group, 37 (25 males, 12 females). Although the overall number of offspring did not differ significantly among the groups (P = 0.08; Table 5), a significant difference was observed in the male-to-female ratio.
The stress group showed a markedly lower percentage of male offspring (30%) compared with the control (41.38%), CE extract (57.78%), and stress + CE extract (71%) groups (P = 0.018; Table 5). Furthermore, treatment with CE extract in the stress group significantly increased the total number of offspring (P = 0.041) and the number of male offspring (P = 0.002; Table 6).
Table 5. Comparison of vaginal epithelial thickness, vaginal pH, the number of offspring, and sex ratio between the groups
Note: Control (neither stress nor CE extract), CE Extract (just received extract), Stress (just received stress), Stress+ CE Extract (received both stress and extract). Vaginal epithelial and pH of Vagina were analyzed using one way ANOVA and Tukey post hoc test; sex ratio was analyzed using K2.
Table 6. Comparison of vaginal epithelium thickness, vaginal pH, the number of offspring, and sex ratio between stress + CE Extract groups
5. Conclusion
The findings may not be directly translatable to humans due to inherent interspecies biological differences, and the pathophysiology of certain diseases in animal models may not fully replicate human conditions. Therefore, additional studies involving human subjects are essential to accurately evaluate the effects of Cuscuta epithymum extract on pregestational stress and to assess its potential for clinical application, still, it is plausible to infer that Cuscuta epithymum exerts a beneficial influence on reproductive function.
6. Declarations
6.1 Acknowledgments
We appreciate Fatemeh Amiri (Persian medicine expert) for assist in animal dissection and thanks to Zanjan University of Medical Sciences for financial support.
6.2 Ethical Considerations
All experiments followed the guidelines of the Medical Ethics Committee of the Iranian Ministry of Health and Medical Education (IR.ZUMS.REC.1399.419).
6.3 Authors' Contributions
All authors contributed equally to this work.
6.4 Conflict of Interest
The authors have no financial conflicts of interest and no funding sources to disclose.
6.5 Fund or Financial Support
This work was supported by the Zanjan University of medical sciences.
6.6 Using Artificial Intelligence Tools (AI Tools)
No AI tools were used in this manuscript
Stressful conditions can decrease the quantity, mobility, and progressive movement of sperm by adversely affecting the epididymis and inhibiting spermatogenesis (4, 5). Moreover, a reduction in the efficiency and quantity of Y sperm, along with a change in the acidity of the vaginal milieu, leads to a higher likelihood of delivering a female child (6), as well as causing shifts in the plasma concentration of testosterone (7) and estradiol (5, 8).
On the other hand, pre-pregnancy stress can affect the fetus and have harmful effects such as, alterations in sex hormone levels in male and female offspring. When an individual experiences stress, the hypothalamic-pituitary-adrenal (HPA) axis becomes activated. The sexual behavior of mice is impacted by immobility stress, a form of stress that combines physical and psychological factors, as it triggers the HPA axis (3, 9).
While a range of pharmaceutical and synthetic agents are routinely employed to manage stress, their use is frequently associated with adverse side effects. Consequently, there is an increasing emphasis on investigating medicinal plants and their derivatives as potentially safer and more tolerable alternatives (10).
One such plant, widely studied and utilized in Persian medicine to alleviate reproductive disorders and stress-related complications, is Cuscuta epithymum. Recent studies have reported significant roles of Cuscuta epithymum in decreasing the adverse effects of stress on the liver and spleen immune system, as well as remarkable anti-neuroinflammatory and anti-seizure effects in rats (11,12). The genus Cuscuta epithymum belongs to the Convolvulaceae family (Note: Cuscutaceae is now often merged with Convolvulaceae) and is distributed worldwide, comprising approximately 170 species. In Iran, 18 species of Cuscuta epithymum have been identified across various regions (13). Cuscuta epithymum is rich in phenolic compounds and flavonoids, including cuscutin, amarbelin, β-sitosterol, stigmasterol, myricetin, quercetin, cuscutamine, luteolin, and bergenin, which contribute to its antioxidant properties (14, 15). In Persian medicine, Cuscuta epithymum is prescribed for the treatment of excessive coldness and blood accumulation in the reproductive systems of males and females, support spermatogenesis, and for the treatment of infertility in women. Cuscuta epithymum is also used to prevent infertility (16, 17). Hence, taking into account the impact of stress on parental fertility and circulating sex
hormone levels, along with stress effects before conception on hormonal shifts in their offspring, and considering the reported effects of Cuscuta epithymum species on stress and reproductive function in both sexes this study was designed to evaluate the potential protective role of Cuscuta epithymum extract in a rat model of stress. This study aimed to assess CE's influence on parental reproductive parameters, including vaginal epithelial thickness and vaginal pH in female rats, sperm parameters in male rats, and serum sex hormone levels in both sexes. Furthermore, offspring were assessed for sex ratio, litter size, and sex hormone levels across different groups.
2. Materials and Methods
2.1 Subjects
Sixty-four adult female and 36 male Wistar rats (180–200 g) were obtained from the Pasteur Institute, Tehran, Iran (Rats had not mated before the start of the experiment). They were maintained under standard conditions in a 12-hour light/dark cycle, 22±2 °C, relative humidity of 45–55%, pathogen-free, food and water ad libitum in the animal house at Zanjan University of Medical Sciences, Zanjan, Iran. All experiments followed the guidelines of the Medical Ethics Committee of the Iranian Ministry of Health (IR.ZUMS.REC.1399.419). The sexual cycle of female rats was equalized by being placed next to sterilized males. Subsequently, male and female subjects were divided into four groups: 1. Control (no exposure to stress or CE extract), 2. CE Extract (only received the extract), 3. Stress (only underwent stress), 4. Stress + CE Extract (exposed to both stress and the extract), as illustrated in the study diagram. Males experienced immobility stress for 2 hours each day over a period of 50 consecutive days (the duration needed for a full spermatogenesis cycle), while females were subjected to stress for 15 days (encompassing three estrous cycles). During this period, the appropriate groups were given oral doses of 100 mg/kg of CE extract. (18). The vaginal pH of female rats was also measured, and a biopsy of their vaginas was taken to assess the thickness of the vaginal epithelium. Semen samples from male rats was collected via the epididymis to assess sperm parameters. The blood of male and female rats was collected by heart puncture to investigate sex hormone levels in the serum. After the end of the stress phase, the rats entered the mating phase. The Trichus method (2 females and one male) was used for mating (all the female rats were mated on day 51 with males). The rats that were placed in a cage for mating are as follows: McFc, McFc+Ex, MsFs, MsFs+Ex (M: male, F: female, C: control, S: stress, Ex: extract). Reproductive indices in pregnant rats after delivery (offspring sex ratio and litter size) were investigated in all groups. After the pups grew up on postnatal day (PND) 25, a male and a female pup from each mother in all groups were selected for hormonal examination.
2.2 Preparing extractThe percolation technique was employed for obtaining the extraction of the CE extract in this experimental study (herbarium code: 5501). The Cuscuta epithymum plant was crushed and then subjected to two standard sieves (160 and 250 micrometers) for proper sanitation. Afterwards, the 1500 g powder was transferred to the percolator, where 70% ethanol was used as the solvent.
The dark color and pungent odor indicated the purity of the CE extract. The solvent was separated using a Rotary Evaporator (IKA RV 05-B, Germany) at 45 to 50 °C, then subjected to vacuum drying at 45 °C (53 OT, Iran). The drying procedure persisted until a stable weight was achieved. This method was repeated three times at 24-hour intervals. After filtering the extract, concentration was performed in a water bath under vacuum conditions, followed by lyophilization (FD-5N; Eyela, Tokyo). Ultimately, the product was preserved at 4 degrees Celsius. (19, 20).
2.3 Restraint stress procedure
A period of immobility stress was experienced by rats, as described by Nakhjiri et al. (21). Briefly, this process included moving the rats to the designated testing area. They were housed in a restraining device under standard room conditions. Each day, the rats were placed within the confinement chamber (a cylindrical plastic container measuring 16 cm in length and 6 cm in width) for a duration of 2 hours (12).
2.4 Sperm analysis
Male rats (n = 8 in each group) were anesthetized with isoflurane on the first morning following the completion of the stress period. One epididymis was randomly selected for evaluation of sperm parameters. The caudal part of the epididymis was dissected, then floated in 5 ml of pre-heated Ham's F10 solution (Sigma, USA), then incubated up to 15 minutes at 37°C in order to allow the sperms to diffuse into the buffer.
A total of 10μl of the sperm suspension was smeared on a Neubauer chamber, finally, it was prepared to analyze sperm motility with a light microscope (Olympus BX51, Japan). After 15 minutes, 10 microscopic fields with magnifications of 400 times, counting more than 200 sperms, were observed (5). Sperm motility was defined as: a) progressive (P) b) non-progressive (NP), and c) immotile (IM) when sperms had no movements. Progressive and non-progressive sperms were presented as a percentage of total sperm motility. Sperms having abnormalities in the tail, neck and head sections were considered abnormal. After diluting the suspension, one drop was placed on an erythrocytometer, and sperm count was performed with a light microscopy (Olympus, Japan) (22). To assess the morphology of sperm, its suspension was smeared on a glass slide and then stained with Eosin. Finally, sperms were categorized into two major classes: a) normal and b) abnormal, sperms with dark pink are considered dead sperm as eosin penetrated into the sperm because of the breakdown of the cell membrane.
2.5 Vagina sampling
The pH of the vagina was measured using pH indicator paper (23). For this purpose, the pH meter paper was inserted up to one-third of the length of the vagina using forceps, kept inside for 1 minute, and the pH change was recorded. Finally, the rats were anesthetized with isoflurane and tissue samples were prepared from the rats' vaginas. Obtained tissues fixation was performed in 10% buffered formalin, then it was sectioned and finally stained with H&E. Motic images were used to measure the thickness of vaginal Cuscuta epithymum. For each section, 10 different lengths were measured to obtain the average thickness (5).
2.6 Mating and offspring
Twenty-four hours after the cessation of the immobilization stress protocol, each male rat was housed individually with two female rats to assess reproductive performance. After a 3-day cohabitation period, the males were removed, and the females remained in the cages until the completion of gestation. Upon delivery, offspring were evaluated for litter size, sex ratio, and serum estradiol and testosterone levels.
2.7 Blood sample collection
After the end of the stress phase for parents and at PND 25, under general anesthesia with isoflurane, blood samples were obtained at 8:00 a.m. and 11:00 a.m., through cardiac puncture.
The samples were poured into EDTA-containing tubes, and then centrifuged at 9000 rpm. The obtained plasma samples of offspring, and serum samples of adult male and female rats were placed at -80 °C. The blood samples were used for quantification of testosterone and 17-beta estradiol, using commercial ELISA kit including Zellbio GmbH (Germany, Cat. No: ZB-10259D-R9648) and Zellbio GmbH (Germany, Cat. No: ZB-10174M-R9648), respectively. The amounts were presented as pg/mL for 17-beta estradiol and ng/mL for testosterone.
2.8 Statistical analysis
The analysis was conducted using SPSS software (SPSS Inc., Chicago, IL, USA) edition 22 and presented as mean ± SEM. The Kolmogorov–Smirnov test was utilized to check the distribution of control data. As a result, data analysis was done parametrically with the use of normal distributions and one-way analysis of variance (ANOVA) for comparisons of results for multiple groups.
When applicable, Tukey post hoc test was used. Sex ratio was analyzed using K2 test. A p < 0.05 was considered significant in all instances.
3. Result
Analysis was performed on the complete data of 100 adult rats and their 131 offspring.
3.1 Sperm parameters
There was a statistically significant difference in sperm parameters including count, motility viability, and morphology between the groups (Figure 1).
Figure 1. Sperm Parameters in Control Adult Rats, CE extract, stress and stress + CE extract groups: Panel a; Total count: *** p<0.001, ** p<0.01, with control group, # indicates p<0.05, ## indicates p<0.01 with CE Extract group. Panel b; Motility: ** indicates p<0.01, *** indicates p<0.001with control group, ### indicates p<0.001 with CE Extract group, $$ indicates p<0.01, $$$ indicates p<0.001 with Stress group. Panel c; Viability: * indicates p<0.05, *** indicates p<0.001with control group, ### indicates p<0.001 with CE Extract group, $$$ indicates p<0.001 with Stress group. Panel d; Morphology: ** indicates p<0.01 with control group, ## indicates p<0.01 with CE Extract group, $$$ indicates p<0.001 with Stress group. (Prepared by Authors, 2026).
3.2 Sperm count
The results demonstrated that the number of sperm in the stress group was significantly lower compared to the control, CE extract, and stress + CE extract groups (1.68±0.42, 3.78±0.9, 3.03±0.64, and 2.12±0.3; P= 0.002) (Table1). Administration of Cuscuta epithymum extract to the stress-exposed group resulted in an increase in sperm count from 1.68±0.42 to 2.12±0.3. However, this difference was not statistically significant (Table2).
3.3 Sperm motility
According to table 1 sperm motility revealed that the percentage of sperms with progressive movements in the stress group was significantly lower compared to the control, CE extract, and stress + CE extract groups (7.26±1.21, 33.88±11.25, 35.02±7.62, and 33.22±9.65; P= 0.001). However, no significant difference was found among the groups in the percentage of sperm with non-progressive movements. The percentage of immotile sperm in the stressed rats was significantly higher in comparison with control, CE extract, and stress + CE extract groups (60.08±8.89, 35.48±10.64, 30.85±10.54, and 39.01±6.97; P< 0.004). The percentage of progressive sperms in CE extract group significantly reached the baseline level in control group (7.26±1.21 vs. 33.22±9.65; P< 0.001); and the number of immotile sperms was significantly decreased from 60.08±8.89 to 39.01±6.97 (P= 0.008) (Table2).
3.4 Sperm viability
The percentage of sperm viability in the stress group was significantly lower compared to the control, CE extract, and stress + CE extract groups (44.75±5.78, 65.04±12.43, 77.08±3.99, and 69.6±5.49; P< 0.001)(Table1). According to table 2, administration of CE extract to the stress group significantly increased the percentage of viable sperms from 44.75±5.78 to 69.6±5.49, P< 0.001).
3.5 Sperm morphologyThe percentage of sperms with abnormal morphology in the stress group was significantly higher compared to the control, CE extract, and stress + CE extract groups (60.13±10.46, 36.37±9.34, 37.64±7.46, and 37.15±11.18; P= 0.004) (Table 1).
As it has been shown in table 2, CE extract administration to the stress group significantly decreased the percentage of sperms with abnormal morphology from 60.13±10.46 to 37.15±11.18, (P< 0.001).
Table 1. Comparison of Sperm parameters between the groups
Note: Control (neither stress nor CE extract), CE Extract (just received extract), Stress (just received stress), Stress+ CE Extract (received both stress and extract). * Indicates significant difference with other groups, one-way ANOVA and Tukey post hoc test.
3.6 Effects of pre-gestational stress and CE extract on plasma concentrations of estradiol and testosterone in parents and offspring
Significant differences in plasma testosterone and estradiol levels were observed among the experimental groups in both parents and offspring at postnatal day (PND) 26, as illustrated in (Figure 2).
3.7 Testosterone level in adult rats
In adult male rats, plasma testosterone concentration (ng/dl) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (0.82±0.15, 2.12±0.48, 0.95±0.5, and 3.08±0.57; P= 0.002) (Table 3).As shown in Table 4, CE extract administration to the stress group significantly increased adult male rats’ serum testosterone level from 0.82±0.15 to 3.08±0.57 ng/dl, P= 0.001).
3.8 Testosterone level in offspring
Consistent with the findings in adult rats, male offspring in the stress group exhibited significantly lower plasma testosterone concentrations (ng/dL) compared to the control, CE extract, and stress + CE extract groups (0.24±0.09, 0.37±0.12, 1±0.24, and1.37±0.18; P= 0.031) as presented in Table3. Administration of CE extract to the stress-exposed group significantly increased serum testosterone levels in male offspring from 0.24±0.09 to 1.37±0.18ng/dl, P= 0.009) as shown in (Table 4).
Figure 2. Serum testosterone and estradiol levels of parents (a and c) and pups (b and d) represented in the charts a, b, c, and d in the control, CE extract, stress and stress + CE extract groups: Panel a; Testosterone: * indicates p<0.05 with control group, ## indicates p<0.01 with CE Extract group, $$$ indicates p<0.001 with Stress group. Panel b; Testosterone Offspring: * indicates p<0.05 with control group, # indicates p<0.05 with CE Extract group, $$ indicates p<0.01 with Stress group. Panel c; Estradiol: # indicates p<0.05 with CE Extract group, $ indicates p<0.05 with Stress group. Panel d; Estradiol Offspring: * indicates p<0.05, ** indicates p<0.01 with control group. (Prepared by Authors, 2026).
3.9 Estradiol level in adult rats
In the adult female rats, plasma estradiol concentration (pg/ml) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (26.8±3.18, 44.2±36.77, 67.62±37.31, and 72.26±13.15; P= 0.048) (Table 3). According to table 4, administration of CE extract to the stress group significantly increased adult female rats’ serum estradiol level from 26.8±3.18 to 72.26±13.15 pg/mL, (P= 0.027).
3.10 Estradiol level in offspring rats
Similar to adults, in female offspring rats, plasma estradiol concentration (pg/ml) was significantly lower in the stress group when compared to control, CE extract, and stress + CE extract groups (30.56±9.72, 66.92±37.82, 44.27±23.96, and 13.62±7.42; P= 0.029) (Table3). However, administration of CE extract to the stressed group did not result in a statistically significant increase in serum estradiol levels (P = 0.28; Table 4)
Table 3. Hormone Levels in Parents and Offspring rats
Note: Control (neither stress nor CE extract), CE Extract (just received extract), Stress (just received stress), Stress+ CE Extract (received both stress and extract).
3.11 Effects of pre-gestational stress and CE extract on vaginal thickness, vaginal pH and Offspring Number
3.11.1 Vaginal Thickness
The measurement of the vaginal epithelium thickness in mature rats revealed that the stress group exhibited a notably reduced thickness in comparison to the control, CE extract, and stress + CE extract groups (P=0. 005; table 5). In the group subjected to stress, the application of CE extract led to a significant rise in the thickness of the vaginal epithelium in adult rats. (P<0.001; Table 6 and Figure3).
3.11.2 Vaginal PH
Vaginal pH assessment in adult female rats revealed a significant reduction in the stress group compared to the other groups (P < 0.001; Table 5). CE extract in the stress group resulted in a significant increase in vaginal pH, rising from 4.5 ± 0.109 to 5.00 ± 0.000 (P < 0.001; Table 6). These findings indicate that CE extract effectively counteracts the stress-induced reduction in vaginal pH.
3.11.3 Offspring Number and Sex Ratio
The total number of offspring in the control group was 29 (12 males, 17 females); in the CE extract group, 45 (26 males, 19 females); in the stress group, 20 (6 males, 14 females); and in the stress + extract group, 37 (25 males, 12 females). Although the overall number of offspring did not differ significantly among the groups (P = 0.08; Table 5), a significant difference was observed in the male-to-female ratio.
The stress group showed a markedly lower percentage of male offspring (30%) compared with the control (41.38%), CE extract (57.78%), and stress + CE extract (71%) groups (P = 0.018; Table 5). Furthermore, treatment with CE extract in the stress group significantly increased the total number of offspring (P = 0.041) and the number of male offspring (P = 0.002; Table 6).
Table 5. Comparison of vaginal epithelial thickness, vaginal pH, the number of offspring, and sex ratio between the groups
Note: Control (neither stress nor CE extract), CE Extract (just received extract), Stress (just received stress), Stress+ CE Extract (received both stress and extract). Vaginal epithelial and pH of Vagina were analyzed using one way ANOVA and Tukey post hoc test; sex ratio was analyzed using K2.
Table 6. Comparison of vaginal epithelium thickness, vaginal pH, the number of offspring, and sex ratio between stress + CE Extract groups
Note: Vaginal epithelial and pH of Vagina were analyzed using one way ANOVA and Tukey post hoc test; sex ratio was analyzed using K2 test.
Figure 3. Histological analysis of vaginal epithelium, H&E staining with ×20 magnifications: A. Control group, B. CE extract group, C. Stress group, and D. Stress + CE extract group. The thickness of the vaginal epithelium is marked in the figure and labeled with the letter 'e' representing the epithelial layer. (Prepared by Authors, 2026).
4. Discussion
Our findings indicate that immobilization stress markedly reduced sperm parameters and administration of Cuscuta epithymum extract resulted in an increase in sperm motility, motile sperm, progressively motile sperm, viable sperm, and sperm with normal morphology. Notably, the proportion of immotile sperm was significantly elevated in the stress group and was substantially reduced following CE extract administration. These results suggest that CE extract mitigates the deleterious effects of pre-gestational stress on sperm quality, particularly in terms of motility and viability. The findings of the present study are consistent with previous studies, which suggest that sperm quality and testicular parameters decreased remarkably after 50 days of exposure to immobility stress (24, 25). The findings of Safavi et al. confirmed the adverse effects of immobility stress on epididymal tissue with increasing duration of stress exposure (26). There have also been reports about the undesirable effects of stress on the quality of animal or human semen, including a decreased level in sperm volume, sperm density and
motility, and normal sperm count (27). Almeida et al., have shown that adult male rats, subjected to long-term immobilization, demonstrate a reduction in sperm concentration and spermatid generation and also decreased plasma testosterone content (28). Kolbasi et al., (2020) showed that in the chronic stress group, sperm density and overall motility rate were not remarkably different among other experimental groups, although normal sperm morphology and progressive motility were significantly decreased (29). In this study, we observed that the extract of CE was able to reduce the complications in sperm parameters caused by stress. Studies have also shown that reproductive organ weights, sperm count, sperm motility, the ratio of live to dead sperms and the percentage of sperms with normal morphology in the groups that received the CE extract increased significantly (30, 31). These findings highlight the negative effects of stress on oogenesis and spermatogenesis which leads to impaired reproductive organ function in parental subjects.
Consequently, these stress-induced disruptions also affected offspring sex ratio and contributed to a reduction in the total number of offspring. This study also investigated testosterone and estradiol levels in parents and offspring. The findings demonstrated that testosterone diminished remarkably in the stress group compared to the control group. However, the group that received CE extract along with stress experienced an increase level of testosterone significantly compared to the stress group and even the control group. The level of estradiol in the stress + CE extract groups increased significantly compared to the stress group, while in offspring, estradiol dramatically decreased in the stress and stress + CE extract groups. The hypothalamic-pituitary-adrenal axis has a preventive role in the reproductive system of females. The corticotropin-releasing hormone prevents the secretion of the hypothalamic gonadotropin-releasing hormone, and causes resistance of target tissues to estradiol (16) and plasma estradiol contents in female rats are lowered during chronic stress (17). Mahmoudkhani et al. reported findings consistent with the present study, demonstrating that serum levels of testosterone and 17β-estradiol were significantly reduced in stressed parental subjects (32) and Ozegbe et al. (2012) reported that the seed and stem of C. australis, are effective in reducing male and female reproductive disorders. C. australis extracts induced significant effects on the blood plasma concentrations of follicle exciting hormone(FSH), luteinizing hormone(LH), and testosterone of the adult male rats (30). This stress remarkably elevated the number of female offspring in each litter (32). The present findings demonstrated a significant reduction in offspring number in the stress group compared to the CE extract and stress + CE extract groups. Additionally, the stress group exhibited a higher proportion of female offspring, whereas the stress + CE extract group showed a reversal of this trend, with a superiority of male offspring. These results suggest that Cuscuta epithymum extract mitigates stress-induced reproductive impairments and enhances male fertility and offspring number. One of the mechanisms related to stress on fertility is that paternal exposure to environmental hazards diminishes the number of semen Y-bearing sperms or decreases their ability to get to or fertilize the oocyte (33). Also, estradiol levels are lowered during chronic stressors (34). Maternal predatory stress and immobility stress throughout the pre-maturation phase of oocytes reduces the developmental potential of oocytes both before and after implantation (33, 35). Therefore, this may lead to a disproportionate loss of XY embryos (33). Furthermore, male fetuses grow larger and therefore require a greater maternal resource and it is possible not to adapt with stressful intrauterine environment developmentally. Instead, female fetuses consume fewer resources during their development and demonstrate lower growth and needs in response to maternal stress (36). These results suggest that Cuscuta epithymum may partially mitigate the detrimental effects of stress on reproductive outcomes. Consistent with our study, stress has also been demonstrated to impact the thickness of the vaginal epithelium across all stages of the estrous cycle (32). The stress group exhibited a significant reduction in vaginal epithelial thickness compared to the control group. However, treatment with Cuscuta epithymum extract in the stress-exposed group resulted in a marked increase in vaginal thickness, approaching levels observed in the control group. Similarly, Kim et al. in 2016 also observed an increase in vaginal thickness with Cuscuta epithymum extract (37). The probable mechanism of Cuscuta's action is attributed to its abundance of antioxidant compounds. The study of Ma et al. showed similar results. They reported that Cuscuta epithymum chinensis extracts and isolates (including flavonoid antioxidant compounds) had several impacts on male and female reproductive systems such as better fertility. The potential mechanisms of this action is the antioxidant effect, so that total flavonoids of Cuscuta epithymum control apoptosis and prevent abortion (38, 39), since increased ROS level injures oocyte and sperm DNA directly, it contributes to lipid peroxidation (17, 40, 41), inflammation and induce sperm apoptosis rapidly increased circulating glucocorticoid during stress, leads to testicular involution and subsequent remarkable decrease of testosterone release as well (42, 43).Our findings indicate that immobilization stress markedly reduced sperm parameters and administration of Cuscuta epithymum extract resulted in an increase in sperm motility, motile sperm, progressively motile sperm, viable sperm, and sperm with normal morphology. Notably, the proportion of immotile sperm was significantly elevated in the stress group and was substantially reduced following CE extract administration. These results suggest that CE extract mitigates the deleterious effects of pre-gestational stress on sperm quality, particularly in terms of motility and viability. The findings of the present study are consistent with previous studies, which suggest that sperm quality and testicular parameters decreased remarkably after 50 days of exposure to immobility stress (24, 25). The findings of Safavi et al. confirmed the adverse effects of immobility stress on epididymal tissue with increasing duration of stress exposure (26). There have also been reports about the undesirable effects of stress on the quality of animal or human semen, including a decreased level in sperm volume, sperm density and
motility, and normal sperm count (27). Almeida et al., have shown that adult male rats, subjected to long-term immobilization, demonstrate a reduction in sperm concentration and spermatid generation and also decreased plasma testosterone content (28). Kolbasi et al., (2020) showed that in the chronic stress group, sperm density and overall motility rate were not remarkably different among other experimental groups, although normal sperm morphology and progressive motility were significantly decreased (29). In this study, we observed that the extract of CE was able to reduce the complications in sperm parameters caused by stress. Studies have also shown that reproductive organ weights, sperm count, sperm motility, the ratio of live to dead sperms and the percentage of sperms with normal morphology in the groups that received the CE extract increased significantly (30, 31). These findings highlight the negative effects of stress on oogenesis and spermatogenesis which leads to impaired reproductive organ function in parental subjects.
5. Conclusion
The findings may not be directly translatable to humans due to inherent interspecies biological differences, and the pathophysiology of certain diseases in animal models may not fully replicate human conditions. Therefore, additional studies involving human subjects are essential to accurately evaluate the effects of Cuscuta epithymum extract on pregestational stress and to assess its potential for clinical application, still, it is plausible to infer that Cuscuta epithymum exerts a beneficial influence on reproductive function.
6. Declarations
6.1 Acknowledgments
We appreciate Fatemeh Amiri (Persian medicine expert) for assist in animal dissection and thanks to Zanjan University of Medical Sciences for financial support.
6.2 Ethical Considerations
All experiments followed the guidelines of the Medical Ethics Committee of the Iranian Ministry of Health and Medical Education (IR.ZUMS.REC.1399.419).
6.3 Authors' Contributions
All authors contributed equally to this work.
6.4 Conflict of Interest
The authors have no financial conflicts of interest and no funding sources to disclose.
6.5 Fund or Financial Support
This work was supported by the Zanjan University of medical sciences.
6.6 Using Artificial Intelligence Tools (AI Tools)
No AI tools were used in this manuscript
Type of Study: Original Research Article |
Subject:
Medical Laboratory and Animal Investigation
Received: 2025/10/25 | Accepted: 2026/03/17 | Published: 2026/05/20
Received: 2025/10/25 | Accepted: 2026/03/17 | Published: 2026/05/20
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