Toxoplasma gondii infection and testosterone alteration: A systematic review and meta-analyses

Background Toxoplasma gondii (T. gondii) is a worldwide distributed protozoan parasite which has infected a wide range of warm-blooded animals and humans. The most common form of T. gondii infection is asymptomatic (latent); nevertheless, latent toxoplasmosis can induce various alterations of sex hormones, especially testosterone, in infected humans and animals. On the other hand, testosterone is involved in behavioral traits and reproductive functions in both sexes. Hence, the purpose of this systematic review is to summarize the available evidence regarding the association between T. gondii infection and testosterone alteration. Methods In the setting of a systematic review, an electronic search (any date to 10 January 2023) without language restrictions was performed using Science Direct, Web of Science, PubMed, Scopus, and Google Scholar. The PRISMA guidelines were followed. Following the initial search, a total of 12,306 titles and abstracts were screened initially; 12,281 were excluded due to the lack of eligibility criteria or duplication. Finally, 24 articles met the included criteria. A mean±standard deviation (SD) was calculated to assess the difference of testosterone between T. gondii positive and T. gondii negative humans. The possibility of publication bias was assessed using Egger’s regression. P-value < 0.05 was considered statistically significant. Results This systematic review identified 24 articles (18 studies in humans and six studies in animals). Most human studies (13 out of 19) reported an increased level of testosterone following latent toxoplasmosis in males, while three studies reported decreased levels and two studies reported an insignificant change. Eleven articles (seven datasets in males and seven datasets in females) were eligible to be included in the data synthesis. Based on the random-effects model, the pooled mean± SD of testosterone in T. gondii positive than T. gondii negative was increased by 0.73 and 0.55 units in males and females, respectively. The Egger’s regression did not detect a statistically significant publication bias in males and females (p = value = 0.95 and 0.71), respectively. Three studies in male animals (rats, mice, and spotted hyenas) and two studies in female animals (mice and spotted hyenas) reported a decline in testosterone in infected compared with non-infected animals. While, one study in female rats reported no significant changes of testosterone in infected than non-infected animals. Moreover, two studies in male rats reported an increased level of testosterone in infected than non-infected animals. Conclusions This study provides new insights about the association between T. gondii infection and testosterone alteration and identifies relevant data gaps that can inform and encourage further studies. The consequence of increased testosterone levels following T. gondii infection could partly be associated with increased sexual behavior and sexual transmission of the parasite. On the other hand, declining testosterone levels following T. gondii infection may be associated with male reproductive impairments, which were observed in T. gondii-infected humans and animals. Furthermore, these findings suggest the great need for more epidemiological and experimental investigations in depth to understand the relationship between T. gondii infection and testosterone alteration alongside with future consequences of testosterone alteration.


Introduction
Toxoplasma gondii (T.gondii) is a worldwide prevalent intracellular protozoan parasite which infects about one-third of human and animal populations [1,2].The cat family (Felidae) as the definitive hosts and a wide spectrum of warm-blooded vertebrates including humans serve as intermediate hosts [1,2].Humans get the infection through ingestion of contaminated foods and water containing oocytes which shed in the cat feces, or by consumption of raw/undercooked meat containing parasite tissue cysts [2].Other routes of transmission include organ transplantation and blood transfusion from infected to uninfected individuals, as well as congenital transmission from infected mothers to their fetus [1,3].Recent studies also suggested that the parasite could transmit via sexual intercourse in humans [4] and rats [5].
According to estimations, more than one-third of the human population has been infected with the parasite worldwide [2].Nevertheless, most human infections are asymptomatic in immunocompetent individuals [6].In immunocompromised individuals, the infection could have life-threatening sequels, such as toxoplasmic encephalitis, myocarditis, or disseminated infections [7,8].Congenital toxoplasmosis is also a life-threatening condition which may lead to abortion, stillbirth, and preterm birth [9][10][11][12].The intensity of T. gondii infection depends on several factors, including genetic background [13], immunity status [14], and the parasite virulence [15,16].T. gondii consists of three main strains (Types I, II, and III), which have some differences in virulence factors and epidemiological patterns [17][18][19].While type I strains (such as RH and GT-1) are highly virulent, type II strains (e.g., ME49 and PRU) and type III (e.g., VEG, NED, and CEP) have lower virulence than type I strains [19,20].
Testosterone is the primary male hormone that is responsible for male sex characteristics and reproductive functions, such as spermatogenesis and fertility.Females also need certain levels of testosterone.In females, most testosterone converts into the sex hormone estradiol [21].Testosterone is primarily produced in the testes and ovaries in males and females, respectively.A small amount of testosterone is produced in the adrenal glands in both sexes [21].
Several studies in humans and animal models revealed that T. gondii infection influenced testosterone levels.While some studies reported an increased level of testosterone, others reported a decline level following T. gondii infection [22].It seems that several factors, such as the parasite strain and intensity of infection could influence this variation [22].Inasmuch as testosterone is important in different physiological processes (e.g., reproductive function and sexual behavior), this systematic review is aimed to summarize data regarding the effects of T. gondii infection on testosterone levels in humans and animals and discusses their influential factors.

Materials and methods
The present study was conducted following the guideline of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement [23] (S1 Checklist).

Strategy search
The search was performed in international databases (Science Direct, Web of Science, PubMed, and Scopus) and the search engine, Google Scholar, published from any date to 10 January 2023.The following search terms were selected using Medical Subject Heading (MeSH) terms alone or in combination: ("Toxoplasma gondii" OR "T.gondii" OR "toxoplasmosis") AND ("testosterone" OR "hormone" OR "androgen").Additionally, to avoid ignoring the reference lists of all included studies were reviewed.As such, the citations of all selected articles were hand-searched in Google Scholar for potentially eligible articles.

Eligibility criteria, study selection, and data extraction
Two independent reviewers (AA and AT) selected the articles.After the initial search, all selected articles were screened by title and abstract, then the relevant articles were imported into the EndNote X8 software (Thomson Reuters, New York, USA).Duplicated articles were checked and removed in the next step.Then, if the articles met the following criteria, they were included in the systematic review: (1) papers with full-text or abstract in English, and (2) original research articles, short reports or letters to the editors that studied the association between T. gondii infection and testosterone.Articles were included if they fulfilled the following Population, Intervention, Comparison and Outcomes (PICO) criteria [24]: Participants/ Population: animals or humans, Interventions/exposure: T. gondii infection, Comparison or control: compared with uninfected human or animals, Outcomes: levels of testosterone.
The data extracted and tabulated from each study, including: (1) First Author, (2) Publication Year, (3) Country, (4) Study Design, (5) Type of Population (case and control), and (6) Findings.All extracted data was entered into the respective tables (for humans and animals) by the primary researcher and verified by another researcher.Any discrepancies were reviewed and resolved by consensus.

Quality assessment
Quality assessment of the included articles was done by The Joanna Briggs Institute (JBI) Critical Appraisal Checklist [25], which contains eight questions with four options including Yes, No, Unclear, and Not applicable.For including and excluding papers, each paper takes a maximum of one star for each numbered item and the total score of 4-6 and 7-10 points were specified as moderate and high quality, respectively.Based on the obtained score, the authors have decided to include (4-10 points) and exclude (�3 points) the papers.

Data synthesis and statistical analysis
Data was analyzed using comprehensive meta-analysis software version 2. To assess the association between T. gondii with testosterone in humans, a mean±standard deviation (SD) using the random effects model and corresponding 95% confidence intervals (CI) were calculated for each study.Egger's regression (Qualitative method) was applied to assess the possibility of publication bias during the analysis.P-value < 0.05 was considered statistically significant.

Study selection
As shown in the PRISMA flowchart (Fig 1 ), a total of 12,306 titles and abstracts were screened initially; 12,281 were excluded due to the lack of eligibility criteria or duplication.Finally, 24 articles (18 studies in human and six animal studies) met the included criteria.Tables 1 and 2 summarize the information of the included articles regarding the association between T. gondii infection and testosterone in humans and animals, respectively.

Evidence for increased testosterone in human infected with T. gondii. Fig 2 summarizes the included studies and Table
3.3.1.3Evidence for increased cortisol levels in human infected with T. gondii.Three studies in males [26,29,38] and two studies in females [26,38] reported an increased level of cortisol in T. gondii seropositive than seronegative counterparts.

Description of human studies.
The first studies regarding T. gondii and testosterone in humans were conducted by Flegr et al in 2008.In the Czech Republic [40,41].They conducted case-control studies among T. gondii IgG seropositive female and male students.The results showed that T. gondii-seropositive men have a higher concentration of testosterone and T. gondii-seropositive women have a lower concentration of testosterone compared with Toxoplasma-free subjects [40,41].An article was published by a group of researchers in Iran https://doi.org/10.1371/journal.pone.0297362.g002[26].They found that T. gondii seropositive women and men had a higher concentration of serum cortisol and testosterone than seronegative individuals.As such, a significant association was found between T. gondii seropositivity with hair loss in women, hirsutism in women, and height increase in women and men.Stress and anxiety indices were also increased in T. gondii seropositive men and women, whereas the depression index increased only in seropositive men compared with the control group [26].Abdul-Lateef et al. [32] found a significant correlation between T. gondii IgG seropositivity with an increase in serum testosterone, IL-12, and IFN-γ among an Iraqi population [32].Eslamirad et al. [28] found an association between T. gondii IgG seropositivity with decreased testosterone levels in healthy men than the seropositivity control group [28], but they did not find an association between T. gondii seropositivity and serum lipid levels [27].Mahbodfar et al. [29] found that T. gondii seropositive individuals had significantly higher levels of testosterone and cortisol than seronegative individuals.As such, the rates of alopecia and acne were significantly increased in seropositive men than seronegative men, and the rate of hirsutism was significantly increased in seropositive women than seronegative women [29].Colosi et al. [42] found no statistically significant difference in serum testosterone, follicle-stimulating hormone (FSH), and sperm characteristics among T. gondii seropositive men compared with seronegative individuals.Zghair et al. [33] demonstrated that the levels of total and free testosterone, but not FSH, were significantly higher in T. gondii-seropositive men compared with the seronegative control group.Zouei et al. [30] found a statistically significant increase in the level of serum testosterone among T. gondii-seropositive men and women compared to non-infected men and women in an Iranian population.Borra ´z-Leo ´n et al. [43] showed a significantly positive relationship between T. gondii IgG seropositivity with higher testosterone levels, interpersonal sensitivity, and psychoticism symptoms in seropositive men, but not women, than non-infected control groups [34].A study among T. gondii seropositive and seronegative women revealed an increased level of testosterone, but not progesterone and prolactin, in seropositive women compared with seronegative control groups.Al-Masoudi et al. [35] found a decreased level of testosterone and an increased level of luteinizing hormone (LH) in T. gondii seropositive individuals compared to controls in a healthy Iraqi population.Al-Kurdy et al. [36] found no statistical differences in the concentration of testosterone among the T. gondii seropositive men than seronegative controls.AL-Asady et.Al. [37] found a very slightly higher serum level of testosterone and LH and insignificant lower levels of FSH in seropositive women compared to controls.El-Gebaly et al. [38] demonstrated that schizophrenic patients showed higher T. gondii antibody titer, cortisol, and free testosterone levels in both genders and lower Glutathione (GSH) than controls.As such, T. gondii seropositive schizophrenic patients had higher testosterone levels and lower glutathione levels than seronegative patients.Bayani et al. [31] investigated the relationship between toxoplasmosis with testosterone, prolactin, dehydroepiandrosterone (DHEA), FSH, LH, and thyroid stimulating hormone (TSH) among T. gondii seropositive and seronegative infertile couples.Although some alterations were observed, no statistically significant differences were detected in these hormones among T. gondii seropositive and seronegative groups [31].In an interesting report, Hagag et al. [39] found a positive association between T. gondii seropositivity and a significant elevation of free testosterone levels among patients with androgenic alopecia and acne vulgaris compared with the seronegative group.There are also some case reports regarding the association of acute toxoplasmosis with lower testosterone levels in males with hypogonadotrophic hypogonadism [44] as well as a case with intracranial toxoplasmosis presenting as panhypopituitarism [45].

Meta-analysis of human studies.
As shown in Table 3, eleven papers (seven datasets in males and seven datasets in females) on the association between T. gondii and testosterone were eligible to include in the data synthesis.Based on the random-effects model, the pooled mean± SD of testosterone in T. gondii positive than T. gondii negative were calculated to be 0.73 and 0.55 in males and females, respectively (Figs 3 and 4).It means that, testosterone increased by 0.73 and 0.55 units in T. gondii positive compared to T. gondii negative males and females, respectively.The publication bias was not statistically significant in males (p = 0.95) and females (p = 0.71), respectively.

Animal studies. 3.3.2.1 Evidence for increased levels of testosterone in animals infected with T. gondii.
Two studies in rats [46,47] reported an increased level of testosterone in infected than non-infected animals (Fig 2 and Table 2).

Evidence for decreased or unchanged levels of testosterone in animals infected with T. gondii.
Three studies in male animals (rats [48], mice [49], and spotted hyenas [50]) and two studies in female animals (mice [49], and spotted hyenas [50]) reported a decline level of testosterone in infected animals compared with non-infected animals.While, one study in female rats [51] reported no significant changes of testosterone in infected than non-infected animals (Fig 2 and Table 2).

Evidence for alteration of cortisol levels in animals infected with T. gondii.
One study reported a declined level of cortisol in T. gondii-infected male rats [52], while one study [50] reported no significant change of cortisol levels in T. gondii-infected male and female spotted hyenas.
3.3.2.4 Description of animal studies.Kanˇkova ´et al. [49], reported that T. gondii-infected mice (both females and males) had significantly lower concentration of testosterone.Abdoli et al. [48] reported that male rats with T. gondii infection had a temporary decline in serum and intratesticular testosterone and fructose in seminal vesicles.As such, the percentage rates of sperm motility, viability, and concentration were significantly decreased and sperm abnormality was significantly increased after infection, but it reverts to the normal level on days 60 and 70 post infection [48].Lim et al. [46] observed that T. gondii infection in male rats enhances testicular expression of genes involved in the synthesis of testosterone (LHR, StAR, and P450scc), resulting in greater testicular testosterone production.Afshari et al. [47] showed significantly increased levels of serum alkaline phosphatase and testosterone in T. gondiiinfected male rats compared with the uninfected control group.Laubach et al. [50] found a negative association between T. gondii infection and plasma testosterone among female (cubs and subadults) and adult male hyenas, which means that the infected animals have lower testosterone levels than uninfected animals.Indeed, no associations were found between T. gondii infection and cortisol in any age class or sex group of hyenas [50].

Discussion
Testosterone is involved in a variety of physiological functions, such as behavioral traits and reproductive functions in both sexes [21].In this study, we reviewed data regarding T. gondii infection and testosterone variations in human studies and animal models (Fig 2 and Tables 1  and 2).We observed that most of the included studies in humans reported an increased level of testosterone [26, 29-34, 37-41, 43], while some studies reported a decreased level [27, 28,  35] or insignificant changes [36,42] (Fig 1).As such, these variations were different in males and females in some studies [40,41].In animal models, some studies reported a declining level of testosterone [48][49][50], while others reported an increased level [46,47] or insignificant changes [51].Notably, variations in testosterone levels are most probably due to infection with different parasite strains, or a difference in host variations, which consequently influence the intensity of infection [15,16,22,53].Host variations also influence the intensity of T. gondii infection [54].Among animals, mice and New and Old-World monkeys are highly sensitive to T. gondii infection; while sheep are intermediately sensitive, and goats, cattle, deer, horses, and pigs are resistant to the infection [54].In humans, immunocompromised patients and pregnant women are at high risk of severe T. gondii infection, while T. gondii infection is usually asymptomatic (latent) among immunocompetent individuals, [7,12].Like humans, the laboratory rat (Rattus norvegicus) is resistant to T. gondii infection and is a suitable model for the study of chronic T. gondii infection [55,56].
Testosterone plays an important role in sexual behavior and mating success [57][58][59][60].On the other hand, recent evidence revealed that T. gondii infection augments sexual behavior and attractiveness in humans [61] and experimentally infected rodents [5].In this regard, Borra ´z-Leo ´n et al. [61] assessed several factors related to attractiveness among T. gondii-infected and non -infected individuals.They found that both T. gondii-infected men and women had lower facial fluctuating asymmetry, while infected women had lower body mass index, higher number of sexual partners, and a higher self-perceived attractiveness than non-infected control groups.They also assessed the attractiveness and perceived health of facial pictures of T. gondii-infected and non-infected subjects by an independent group of raters and found that both infected women and men were rated as more attractive and healthier than non-infected individuals [61].Increased testosterone could enhance sexual behavior and attractiveness in infected subjects and could increase mating opportunity and transmission of T. gondii through sexual intercourse.In this regard, Lim et al. [46] reported that T. gondii infection (induced by Prugniaud strain) enhances testicular expression of genes that are involved in the synthesis of testosterone in experimentally infected male rats.Dass et al. [5] demonstrated that T. gondiiinfected male rats had higher sexual attractiveness to non-infected females, resulting in increased mating of infected males with non-infected females.They also confirmed sexual transmission of T. gondii through intercourse, whereas T. gondii cysts were detected in the epididymis of infected males, vaginal lavage of naïve females that mated with infected males, as well as in brains of pups which born from these matings [5].As such, secretion of T. gondii in semen and sexual transmission of the parasite have been reported in dogs [62], goats [63][64][65], sheep [66,67], cattle [68], and pigs [69].Notably, T. gondii transmission in sheep was reported by artificial insemination of contaminated frozen semen [70].There is also indirect evidence that suggests sexual transmission of T. gondii in humans.In this regard, a recent study by Tong et al. [4] confirmed the presence of T. gondii tissue cysts in human semen by immunofluorescence staining and molecular methods.Furthermore, it is proposed that unprotected sex and oral sex could be an important route of T. gondii transmission in humans [71,72].Hlava ´čova ´et al. [73] performed a two-year study to compare the seropositivity to T. gondii in couples and analyzed the serological status of sexual partners.The results indicated that the prevalence of T. gondii infection was higher in women who had infected male partners than in women with uninfected male partners (25.6% vs 18.2%, respectively; P = 0.045).This study also suggests that a partner's seropositivity may be a risk factor for infection in women (prevalence ratio = 1.418;P = 0.045) but not in men (prevalence ratio = 1.058;P = 0.816) [73].This evidence was also supported by studies among female sex workers [74] and individuals with a history of sexual promiscuity [75] in Mexico.In this regard, Alvarado-Esquivel et al. [74] found a significantly higher incidence of latent toxoplasmosis among female sex workers compared with age-and sex-matched control groups (15.44% vs 3.67% in case and control groups, respectively, P = 0.0001).As such, female sex workers had significantly higher anti-T.gondii IgG titers (>150 IU/mL) than the control group (9.6% vs 2.9%, respectively P = 0.007) [74].Another study by the same group of researchers in Mexico [75] revealed a significantly higher prevalence of anti-T.gondii IgG antibodies among individuals with sexual promiscuity than individuals without this practice (18.1% vs 10.3%, respectively; OR: 1.91; 95% CI: 1.41-2.60;P< 0.0001).Indeed, higher titers of anti-T.gondii IgG antibodies (>150 IU/mL) were significantly increased in participants with sexual promiscuity than participants without this history (9.2% vs 4.6% respectively; OR: 2.09; 95% CI: 1.38-3.16;P = 0.0003).Additionally, the association of T. gondii seropositivity and serointensity with sexual promiscuity was observed in men but not in women [75].Collectively, it seems that T. gondii infection could manipulate the mate choice of their host to increase their transmission rates.This phenomenon could be mediated partly by enhancing testosterone levels, which consequently increase sexual behavior and mating success [22,76].
Testosterone has a pivotal role in spermatogenesis and male reproductive functions.A declined level of testosterone was reported following T. gondii infection in mice [49] and rats following infection with a T. gondii type I strain [48], as well as male and female spotted hyenas (Crocuta crocuta) which were naturally infected with T. gondii [50].On the other hand, T. gondii infection could induce male reproduction impairment by interfering in spermatogenesis and testicular damage [44,48,[77][78][79][80], which may be partly mediated by declining testosterone levels.In this regard, Abdoli et al. [48] showed that T. gondii infection (induced by RH strain) induced a temporary decline in serum and intratesticular testosterone levels, fructose in seminal vesicles, as well as declining of sperm motility, viability, concentration, and increased of sperm abnormality in male rats.Hlava ´čova ´et al. [81] compared the prevalence of latent toxoplasmosis in men with and without semen abnormalities and found that T. gondii-infected men had significantly lower sperm concentration and motility compared with T. gondii-negative men.Although another human study did not find a significant association between latent toxoplasmosis and semen abnormalities [42].Considering the possible role of T. gondii in male reproductive impairment, it is recommended that populations with high prevalence of male infertility be examined for T. gondii infection.
There are some limitations to this systematic review.The lack of published articles from many countries were infertility is common is a major limitation.The observed association should be interpreted with caution, because the timeline of T. gondii infection and disease process could not be evaluated from the available data.Importantly, T. gondii seroprevalence has been associated with many different risk factors, which were not evaluated in this work.As such, such confounding factors, including environmental toxins [115][116][117] and coinfections with other pathogens [100] may also affect the levels of sex hormones.
The results of this work can provide useful guidance for planning future studies.It would be important to focus on those parts of the world in which there is a lack of data on this subject.Moreover, including all pertinent risk factors would allow to better clarify the epidemiological aspects of T. gondii infection in infertility individuals and testosterone alterations.Optimally, prospective cohort studies and using more comprehensive serology panels (e.g., including IgG avidity testing) for estimating the timing of T. gondii infection could elucidate the timeline of risk factors of infection.

Conclusion
This study indicated that latent toxoplasmosis is associated with increased testosterone levels in most studies in humans and some studies in non-human animals.This change could be associated with increased sexual attractiveness in infected subjects which lead to sexual transmission of the parasite.On the other hand, some studies demonstrated a decreased level of testosterone in T. gondii-infected animals and humans.This change could partly be associated with male reproductive impairments, which were observed in T. gondii-infected human and non-human animals.These findings suggest the great need for more epidemiological and experimental studies in depth understanding the relationship between T. gondii infection, testosterone alteration, and further consequences.

Fig 2 .
Fig 2. A summary of studies on the relationship between T. gondii infection, testosterone, and steroid hormone levels in males and females.

gondii antibodies ►A significant increase in testosterone and cortisol was found in T. gondii seropositive individuals, but not for DHEA. ►A significantly increased in the rates of alopecia and acne in seropositive men than
•15 men with T. gondii IgG antibody •45 men without T. gondii IgG antibody ►Serum testosterone concentration in seropositive men was lower than seronegative individuals, but it was not statistically significant (P = 0.62).

Table 1 .
(Continued) [35]ase-control study to examine the relationship between T. gondii infection with serum testosterone, progesterone and prolactin levels in pregnant women with chronic toxoplasmosis•A total number of 55 T. gondii-IgG seropositive pregnant women and 51 seronegative pregnant women were enrolled as case and control groups, respectively ►Toxoplasma-seropositive women had a significant higher level of testosterone, but not progesterone and prolactin than Toxoplasma-seronegative women.8 13 Al-Masoudi et al.[35]2018 Iraq•A case-control study to examine the relationship between toxoplasmosis and, testosterone and LH hormones•66 healthy subjects were enrolled •20 (male = 15 and female = 5) seropositive individuals were enrolled as case and the same number of healthy subjects were enrolled as control group►A decreased level of testosterone and an increased level of LH were found in T. gondii seropositive individuals compared to control.3.3.1 Human studies.In human studies, 18 articles were included (Table