The Profertility and Aphrodisiac Activities of Tribulus terrestris L.: Evidence from Meta-Analyses

Tribulus terrestris L. (TT) is a traditional medicinal plant, which belongs to the Zygophyllaceae family. TT extracts have been widely used for diuretic, analeptic, aphrodisiac, and profertility properties. To quantitatively evaluate the profertility and aphrodisiac e ﬀ ects of Tribulus terrestris L ., we undertook the present meta-analyses on published data. A thorough literature screening was performed to identify articles evaluating the e ﬀ ect of TT on spermatogenesis, male fertility, reproductive, and aphrodisiac parameters. We shortlisted 30 relevant studies conducted on humans and rodents. Meta-analyses were conducted to evaluate the quantitative impact of TT on various fertility parameters. In case of humans, the pooled analysis on 133 subjects showed signi ﬁ cant improvements in sperm concentration ( SDM = 0 : 624 , 95% CI = 0 : 13 to 1.117, p = 0 : 013 ) and sperm motility ( SDM = 0 : 742 , 95% CI = 0 : 331 to 1.152, p = 0 : 001 ). TT resulted in nonsigni ﬁ cant increases in testosterone and LH and a nonsigni ﬁ cant decrease in FSH. Similar to the above, TT improved sperm count, sperm motility, and sperm viability in rodents with normal or compromised fertility. The e ﬀ ect on hormone levels was less credible with frequent variations across studies and animal models. The aphrodisiac activity was studied in castrated animal models or normal rodents, both of which showed signi ﬁ cant improvements in mounting frequency and intromission frequency and signi ﬁ cant declines in mounting latency and intromission latency. These meta-analyses suggested that TT possesses profertility and aphrodisiac activities.


Introduction
Infertility is defined as the failure to achieve a clinical pregnancy after 12 months or more of unprotected regular sexual intercourse [1].Approximately 40% of infertility cases worldwide are due to the male factor infertility [2,3].A large number of the infertile cases are still classified as idiopathic, and diagnosis tends to be descriptive, often leading to ineffective medical approaches for treatment [2].Semen quality is the main predictor of male fertility and is used as the primary checkpoint to assess the causes of infertility.As per the WHO 2010 guidelines, sperm count and motility are important criteria to establish infertility in males [4].However, not all cases of infertility are explained by a decline in semen parameters.This leads to the failure of establishment of an appropriate cause of infertility in a large number of patients.Plant products offer an attractive alternative for the treatment of such cases.Plants products are often a complex mixture of natural products, exerting multipronged effects required for improvement in fertility.
Till date, a variety of traditional medicinal plants from India, China, Africa, and other countries have been claimed to have aphrodisiac and profertility effects [5].Tribulus terrestris L. (TT), known as Gokshura in Ayurveda, has been claimed to be effective in treating urogenital diseases including the loss of libido [6,7].In addition to the profertility effects in males, it is often prescribed for the treatment of infertility, impotence, erectile dysfunction, and low libido [8][9][10][11].Tribulus terrestris L., also known as Gokshura, caltrop, Maxican sandbur, and goathead, is a small leafy plant that belongs to the family Zygophyllaceae, largely inhabitant to the tropical and temperate 2.3.Data Extraction.Using a spreadsheet, data were collected to document research design, number of participants, dose and duration, quantitative outcomes, and the primary findings.Whenever the data were available in the form of graphs, the values were extracted using web plot digitizer (https://apps.automeris.io/wpd//).The data were divided in four major groups: (i) pre-or postdata of human infertile males treated with TT, (ii) control vs. TT-treated animals, (iii) chemically induced/castrated infertile rat/mouse models vs. TT treatment (therapeutic effect), (iv) coadministration of Tribulus terrestris and infertility-inducing agents in rat/ mouse (prophylactic effect).Quantitative evidence, therapies given, and other information were collected by two authors independently (AA and RV).The inconsistencies were overcome through discussion with the senior author, leading to a consensus.
2.4.Quantitative Data Analysis.Meta-analysis was performed as detailed in our previous study [30].Standard difference in mean (SDM) was used as the 'effect size' statistic in human studies and Hedges 'g' was used as the 'effect size' statistic in case of animal studies.Q and I 2 represent the heterogeneity taking into account that I 2 value < 25% means low heterogeneity, 50 percent means moderate heterogeneity and 75 percent suggests considerable heterogeneity [31,32].Both the fixed effect and the random effects models were used to measure the pooled effect size value.The fixed effect model was used for drawing inference where the heterogeneity was not significant but in the case of significant heterogeneity, the random effects model was used.Treatment protocols for the dose and length of TT therapy were significantly heterogeneous across studies (Table 1).The methodological information and other details for the studies included in this analysis are given in Table 2.For human studies, pre-and postdata were taken, while in rat/mouse studies, control/TT-treated (therapeutic), control/TT-treated (preventive), and infertile model/TTtreated were considered for qualitative analysis, irrespective of the dose and duration of treatment.
2.5.Sensitivity Analysis.Sensitivity analysis was performed by exclusion of one study at a time, followed by reestimation of the effect size after exclusion of each study.A significant change in the overall conclusion was used to identify a sensitive study.
2.6.Publication Bias.Publication bias analysis was performed qualitatively on the basis of asymmetry in the funnel plot and quantitatively using Egger's intercept test value.In case of significant bias, Duval and Tweedie's trim and fill method was used to compute unbiased estimates.

Results
3.1.Literature Screening.Literature search was performed using the keywords, such as Tribulus terrestris L., reproduction, male infertility, and aphrodisiac property in various

Tribulus terrestris L. Improves Sperm Concentration.
The effect of TT on sperm concentration was analysed by conducting meta-analyses on human and animal studies.In case of humans, four studies administered TT in a total of 133 infertile oligozoospermic/idiopathic infertile/erectile dysfunction/androgen deficient individuals [10,25,27,33].These studies used TT dose from 250 mg to 750 mg (tablet, dried extract or ethanolic/methanolic extract).The studies showed significant heterogeneity (Q = 10:655, I 2 = 71:843, p = 0:014), suggesting the use of the random effects model for drawing inference.The pooled effect size estimate showed a significant improvement in sperm concentration upon TT administration (SDM = 0:624, 95% CI = 0:13 to 1.117, p = 0:013; Figure 2).The comparison of quantitative data showed an average of 66.36% improvement in sperm concentration in infertile subjects.
Another set of animal studies was conducted on rodent models of infertility created by the administration of sodium valproate, cypermethrin, copper overload, bisphenol A, cyclophosphamide, malathion, morphine, strepozotocin, TCDD, diabetes, castration, or sulphasalazine.TT in these studies was administered either subsequent to the creation of model to score its therapeutic impact or along with the method of model creation to investigate the prophylactic effect of TT against a decrease in sperm count.The therapeutic effect of TT on sperm concentration was determined through pooled analysis of data from four studies with nine datasets [9,41,42,45].Meta-analysis showed significant heterogeneity (Q = 27:305, I 2 = 70:701, p = 0:001), suggesting the use of the random effects model for drawing inference.The pooled effect size estimate showed significant improvement in sperm concentration upon TT administration (Hedges' g = 15:003, 95% CI = 11:198 to 18.808, p = 0:001; Figure 2), suggesting a significant therapeutic effect.
The prophylactic effect of TT was studied using data pooled from two studies with four datasets [38,39].Pooled analysis showed significant heterogeneity (Q = 26:976, I 2 = 88:879, p = 0:001), suggesting the use of the random effects model for drawing the inference.The pooled effect size estimate showed a significant impact of TT in preventing a drop in sperm count; in fact, TT was able to not only prevent the decline but also improve sperm concentration (Hedges' g = 3:938, 95% CI = 1:500 to 6.376, p = 0:002; Figure 2).
Animal studies were conducted on rodent models of infertility generated by various means.The therapeutic effect of TT was determined through a pooled analysis on four studies [9,11,41,42] with nine datasets.The test of heterogeneity showed significant heterogeneity (Q = 75:129, I 2 = 89:352, p = 0:001), suggesting the use of the random effects model for drawing inference.The pooled effect size estimate showed a significant improvement in sperm motility upon TT administration (Hedges' g = 10:29, 95% CI = 6:502 to 14.091, p = 0:001; Figure 3).

Tribulus terrestris L. Improves Sperm Viability.
The effect of TT administration on sperm viability was studied in animals only.In these studies, TT was given to rodents with or without fertility compromise.
Animal studies were conducted on rodent models of infertility generated by various means.TT in these studies was administered either afterwards to evaluate the therapeutic effect or along with the method of model creation to investigate the prophylactic effect.Nine studies with seventeen datasets had administered TT in therapeutic mode in animal models of infertility generated by various means [9,11,18,[40][41][42][43][44][45].The data across these studies were heterogenous (Q = 183:855, I 2 = 91:298, p = 0:001), favoring the random effects model.The pooled effect size estimate showed significant improvement in testosterone upon TT administration (Hedges' g = 3:523, 95% CI = 2:164 to 4.822, p = 0:001; Figure 5).
Four studies with seven data sets evaluated the effect of TT on intromission frequency in control animals [16,17,46,52].Meta-analysis showed significant heterogeneity (Q = 29:64, I 2 = 79:759, p = 0:001), suggesting the use of the  14 Andrologia random effects model for drawing inference.The pooled effect size estimate showed significant improvement in IF upon TT administration (Hedges' g = 7:580, 95% CI = 5:329 to 9.832, p = 0:001; Figure 11).Sperm concentration, LH, ML, MF, IL, and IF in control animal groups; sperm concentration, sperm motility, sperm viability, T, FSH, ML, MF, IL, and IF in therapeutic animal groups; sperm concentration, sperm motility, sperm viability, T, FSH, and LH in prophylactic groups showed publication bias.For these parameters, the trim and fill method was used to generate bias-free estimates (Figure 12).After adjustment, the conclusions for different parameters in normal animal study (sperm concentration, LH, ML, IL, and IF), therapeutic study (sperm concentration, motility, testosterone, FSH, ML, and IF), and prophylactic study (motility and viability) were changed.Therefore, this data must be taken with caution.
3.6.Sensitivity Analysis.Sensitivity analysis was undertaken with the exclusion of one study at a time to identify sensitive studies.The effect size estimates along with 95% CI after omission of each study are presented in Figure 13 for the parameters where a sensitive study was identified.We found Ismail et al. [25] on sperm concentration in humans, Salahshoor et al. and Munir et al. [9,36] on testosterone estimation in control rats, Sharma et al. and Tag et al. [39,45] in FSH estimation in control rats, to be sensitive.In therapeutic studies, Hemalatha and Hari and Tag et al. [42,45] in FSH, Moghaddam et al. [43] in LH and Tyagi et al. [18] sub-group with 5 mg/kg dosage in MF estimation, were sensitive.

Discussion
Tribulus terrestris L. is a traditional medicinal plant that has been claimed to have aphrodisiac and profertility effects in the Ayurvedic and Chinese medicinal systems [6,7].Apart from its curative effects in infertility, the impact on sperm parameters in normal individuals and its effect on libido and  15 Andrologia hormones are equivocal across studies.Therefore, we undertook the present meta-analyses to evaluate the effects of TT supplementation on sperm parameters, libido, and sex hormones by pooling data from animal and human studies.

Tribulus terrestris L. Improves Sperm Parameters
Irrespective of Fertility Status.The findings of this metaanalysis suggested that TT administration improves sperm parameters (sperm count, motility, and viability) significantly, irrespective of the status of fertility.TT administration resulted in a significant improvement in sperm concentration, sperm motility, sperm viability in infertile human patients, infertile animal models, and normal rodents.It also provided a prophylactic effect in animals exposed to various fertility compromising agents, further endorsing its profertility effect.Pooled analysis on animal experimentation suggested that TT may also improve sperm parameters under normal conditions, but the effects were more pronounced under stress conditions or infertility.This is supported by the studies on humans as well as infertile animal models.This metaanalysis supports the profertility effects of TT in more than one ways.4.2.Tribulus terrestris L. Exerts Aphrodisiac Effect.TT showed capability to improve the male sexual functions, including erectile function and libido.The most interesting data in this regard was generated on the castrated rats, in which TT showed significant aphrodisiac activity [18,40].Since castration leads to low androgen status, affecting structural, biochemical, pharmacological, and erectile function [53,54], improvement in libido parameters in these animals provides the strongest evidence in support of its aphrodisiac activity.Physiologically, low level of androgen, as seen in hypogonadism, is associated with decreased sexual desire and activity [55,56], which was counteracted by TT.We found that TT administration led to improvement in mounting frequency, intromission frequency with a reduction in mounting latency and intromission latency, suggesting a strong aphrodisiac effect of TT.

Mechanism of Action of Tribulus terrestris L.
It has been suggested that protodioscin works by increasing the conversion of testosterone into the more potent androgen, i.e., dihydrotestosterone [33]; however, this meta-analysis suggested that the effect on testosterone may account for its biological effects only to a little extent.TT is believed to be a scavenger of free radicals due to its active constituents (saponins, alkaloids, flavonoids), which might improve sperm parameters, particularly under stress conditions [57].The effect of TT on the Ca 2+ channel could be a possible reason for its positive impact on sperm motility [58].TT may improve semen parameters because of its strong antioxidant  16 Andrologia activity by regulating Nuclear factor erythroid 2-related factor (Nrf-2) and Heme-oxygenase-1 (HO-1) signalling.TT increases Nrf-2 and HO-1 in reproductive tissue that restore antioxidant enzyme activity, which might account for its aphrodisiac activity.Nrf2, a transcriptional factor for the expression of various antioxidant genes, may explain its key role in oxidative stress response.TT increases HO-1, which is induced by an increase in factors like oxidative stress and reactive oxygen species in order to exert its antioxidant property [16].Among possible reasons for its aphrodisiac effect may be the enhanced conversion of protodioscin to dehydropepiandrosterone (DHEA), a neurosteroid.DHEA is an antagonist to Gama-amino butyric acid (GABA), which has an  17 Andrologia inhibitory effect on intromission activity, thus exerting an aphrodisiac effect [15,46].However, the aphrodisiac effect of TT does not appear to be because of its effect on testosterone levels, as it resulted in only a marginal improvement in testosterone level.While protodioscin has been suggested to act on hypothalamus to stimulate LH and FSH secretion that further improves testosterone production by the Leydig cells [10,33,40], we found a mild effect of TT on T, FSH, and LH levels, suggesting that TT may provide such benefits in infertility or in adverse exposures but not under normal conditions.Ca 2+ inhibits enzyme phosphodiesterase that may explain its impact on sperm motility by preventing the degradation of cAMP and its aphrodisiac activity by preventing the degradation of cGMP [59].cGMP regulates contractile state of corporal smooth muscles through its cGMP-dependent protein kinase activity [60].Oxidative stress might also result in increased cytotoxic effect of nitric oxide, a crucial player in controlling sperm viability and aphrodisiac effect.Apart from the above, there may be other mechanisms by which TT exerts aphrodisiac effect, such as its effect on cavernous smooth muscles, NO release, and cGMP degradation.

Protodioscin May Account for the Profertility and
Aphrodisiac Activity of Tribulus terrestris L. Tribulus terrestris L. is rich in a number of chemical constituents such as steroids, saponins, flavonoids, alkaloids, unsaturated fats, vitamins, and tannins [57], of which protodioscin has been investigated in particular.Interestingly, protodioscin has also been clinically tested for its usefulness or benefits in treating male infertility, especially oligozoospermic infertility [22,33]).Adimoelja et al. debated over TT for improving fertility in idiopathic oligoasthenoteratozoospermia (OTA) patients by conversion of its phytochemical derivative, protodioscin to dehydroepiandrosterone (DHEA) [61,62].However, protodioscin has also been shown to exert aphrodisiac activity by acting on neurotransmitters like nitric oxide that help in the relaxation of corpus cavernosum smooth muscles [63].Although we cannot rule out the fact that other phytochemicals present in the TT extract could contribute to the observed effects, the results obtained in these studies suggest that protodioscin present in this extract could account for the beneficial effects of TT. 4.5.Other Evidence That Supports the Profertility Effect of Tribulus terrestris L. In an in vitro study, treatment with TT improved the Leydig and spermatogonia cell numbers significantly [47].TT saponin root extract also showed favorable effect in illnesses like edema, leucorrhea, ascites, inflammations, and urinary tract infections.On the other hand, some studies showed the beneficial effect of TT in male infertility, whereas Adimoelja et al. concluded that Tribulus terrestris L. improves acrosome morphology of spermatozoal cells and enhanced the acrosome reaction, which contributes to improved fertility [22].18 Andrologia

Conclusions
In conclusion, these meta-analyses showed a significant impact of TT on sperm parameters (sperm concentration, sperm motility, and sperm viability) in humans and animal models.The profertility effect was seen not only in infertility but also in normal animals.Significant improvements in sperm parameters under stress and normal conditions suggests that TT may be an excellent profertility medicine.The most commonly used dosage was 750 mg/day, split in one or two doses, though rigorous studies on the variation of dosage are required.Similarly, these meta-analyses support that TT has significant aphrodisiac activity, characterized by significant increases in the MF and IF and significant decreases in the ML and IL.However, no consistent pattern of change (improvement) was seen in hormones (LH, FSH), except for testosterone where nonsignificant improvement upon TT administration was seen.Therefore, the aphrodisiac activity of TT may be because of its impact on other parameters, such as cGMP, protein kinases, and contractile state of corporal smooth muscles through as yet unknown mechanisms.With regard to its mechanism of action, further studies are required.While protodioscin appears to be one of the active constituents of TT, other compounds have been seldom experimented with.Therefore, we encourage more number of studies on TT in order to get a better picture of its mechanism of action.The small number of studies pooled in these meta-analyses was a limitation of this study.Subgroup analysis with regard to the type of extract could not be conducted due to the availability of a limited number of studies.

Figure 1 :
Figure 1: The PRISMA flow diagram showing the process of literature screening and study selection.

Figure 2 :
Figure 2: The forest plot showing the effect of TT on sperm concentration.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 3 :
Figure 3: The forest plot showing the effect of TT on sperm motility.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 5 :
Figure 5: The forest plot showing the effect of TT on Testosterone.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

3. 3 .
Tribulus terrestris L. Regulates Hormone Levels 3.3.1.Tribulus terrestris L. Improves Testosterone Level Marginally.The effect of TT on testosterone was analysed by conducting meta-analyses on human and animal studies.

11 Andrologia 3 . 4 .
Tribulus terrestris L. Possesses Aphrodisiac Activity.Tribulus terrestris L. has also been claimed to possess aphrodisiac activity.Aphrodisiac activity across most of the studies was assessed in animals by quantitative analysis of the mounting latency (ML), mounting frequency (MF), intromission latency (IL), and intromission frequency (IF).

Figure 6 :
Figure 6: The forest plot showing the effect of TT on FSH.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 7 :
Figure 7: The forest plot showing the effect of TT on LH.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 8 :
Figure 8: The forest plot showing the effect of TT on the mounting latency.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 9 :
Figure 9: The forest plot showing the effect of TT on the mounting frequency.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in centre.The overall association is shown by a diamond-shaped box (blue).

Figure 10 :
Figure 10: The forest plot showing the effect of TT on the intromission latency.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in the centre.The overall association is shown by a diamond-shaped box (blue).

Figure 11 :Figure 12 :
Figure 11: The forest plot showing the effect of TT on the intromission frequency.The p value represents the significance, the horizontal black bar represents 95% CI with Hedges' g/SDM in centre.The overall association is shown by a diamond-shaped box (blue).

5 MFFigure 13 :
Figure 13: Representation of sensitivity analysis of parameters where sensitivity was found.For visual comparison, the overall effect size and the computed effect size after exclusion of one study at a time are presented.The diamonds represent the SDM (Hedge's g) value and the pink zone represents 95% CI.

Table 1 :
Methodological details of the studies included in the meta-analysis.

Table 2 :
Summary of the meta-analyses performed on various parameters.