The role of hormones on Toxoplasma gondii infection: a systematic review

Background: Toxoplasma gondii is the causal agent of toxoplasmosis in which one third of the world's population has been infected. In pregnant women, it may cause abortion and severe damage to the fetal central nervous system. During pregnancy, the prevalence of toxoplasmosis increases throughout the second and third quarter of gestation, simultaneously progesterone and 17β-estradiol also increase. Thus, it has been suggested that these hormones can aggravate or reduce parasite reproduction. The aim of this study was reviewing the relationship between hormones and infection caused by T. gondii in several experimental animal models and humans, focused mainly on: (a) congenital transmission, (b) parasite reproduction, (c) strain virulence, (d) levels of hormone in host induced by T. gondii infection, and (e) participation of hormone receptors in T. gondii infection. Are the hormones specific modulators of T. gondii infection? A systematic review methodology was used to consult several databases (Pub Med, Lilacs, Medline, Science direct, Scielo, Ebsco, Sprinker, Wiley, and Google Scholar) dated from September, 2013 to March, 2014. Results: Thirty studies were included; eight studies in humans and 22 in animals and cell cultures. In the human studies, the most studied hormones were testosterone, progesterone, prolactin, and 17β-estradiol. Type I (RH and BK) and Type II (Prugniaud, SC, ME49, T45, P78, and T38) were the most frequent experimental strains. Conclusions: Thirty-five years have passed since the first studies regarding T. gondii infection and its relationship with hormones. This systematic review suggests that hormones modulate T. gondii infection in different animal models. However, given that data were not comparable, further studies are required to determine the mechanism of hormone action in the T. gondii infectious process.


INTRODUCTION
Toxoplasma gondii (T. gondii) is the causal agent of toxoplasmosis and one third of the world population has been affected by this parasite (el-On and Peiser, 2003). In immunocompetent adults, 80% of the cases can be asymptomatic. On the other hand, in immunocompromised patients, T. gondii is an opportunistic parasite that has been held responsible for mortal encephalitis (Cabrera-Muñoz et al., 2010).
Congenital transmission of T. gondii causes severe consequences in which the degree of damage depends on the time when the mother is infected (Speroff et al., 1999). Infection during early pregnancy can result in apoptosis of placental cells and fetal resorption (Senegas et al., 2009). When pregnant females infected during latter stage of pregnancy and inflammatory responses are low, congenital transmission is likely to occur (Roberts et al., 2001;Pfaff et al., 2008). The transmission frequency of T. gondii is high (80%) at end of pregnancy.

PREGNANCY AND T. gondii INFECTION
During pregnancy, maternal hormones alter the immune responses of the mother in the presence of fetal antigens. The increases in the susceptibility to infection and a diminished proinflammatory response have critical anti-parasitic properties that cause an unfavorable development of toxoplasmosis (Craig et al., 2001;Roberts et al., 2001;Prigione et al., 2006;Dionne et al., 2012). In the second and third trimester of gestation, there is a significant increase of 17β-estradiol and progesterone levels and it is during this period, when the prevalence of Toxoplasma infection increases (Montoya and Remington, 2008;Al-warid and Al-qadhi, 2012).

17β-ESTRADIOL AND T. gondii INFECTION
17β-estradiol (E2) is synthetized mainly in the ovary, breast, endometrial tissue, and brain. E2 plays a vital role in the menstrual cycle and human reproduction. In the nervous system, the estrogens are neuroprotective (Duenas et al., 1996;Arevalo et al., 2010). It has been reported that the administration of pharmacological doses of 17β-estradiol increases the susceptibility to Toxoplasma infection (Pung and Luster, 1986).

PROGESTERONE
Progesterone is present in the ovary and corpus luteum where it is primarily involved in the second phase of the menstrual cycle and reproductive processes of women. Progesterone is synthetized in breast, endometrial, and brain too (Speroff et al., 1999). In cells infected with tachyzoites of T. gondii, progesterone did not regulate the replication of parasites (Gay-Andrieu et al., 2002). Progesterone levels are reduced during pregnancy in sheep after infection by T. gondii (Aiumalamai et al., 1990;Fredriksson et al., 1990).

TESTOSTERONE LEVELS REGULATION BY T. gondii INFECTION IN HUMAN BEINGS AND MICE
Testosterone and their derivatives (dihydrotestosterone and dehydroepiandrosterone) are androgens produced mainly in male gonads, adrenal glands and the brain. Testosterone can act directly as a ligand of androgen receptors (AR) found in several target tissues. Androgens stimulate the development of the secondary sexual characters in males, participate in human reproduction and maturation of human fetal testes (O'Shaughnessy and Fowler, 2014). In the brain, it is considered as a neuroprotective hormone (Kurth et al., 2014). IgG anti-Toxoplasma antibodies were significantly correlated to testosterone (Shirbazou et al., 2011), and results are different accord type strain (Kaňková et al., 2011). T. gondii produces high testosterone levels in infected animals and mRNA expression of luteinizing hormone receptor (LHR) (Oktenli et al., 2004;Abdoli et al., 2012;Lim et al., 2013).

CORTICOSTEROIDS EFFECT ON T. gondii
Cortisol is a glucocorticoid hormone secreted by the adrenal cortex. It works through a signal transduction pathway that initiates by hormone linkage to specific cell receptors. Proteins synthesized by the glucocorticoid response inhibit or stimulate the specific tissue (Gardner et al., 2011). Cortisone increased the amount of tachyzoites, cysts and cystozoite, as the breakage of cysts released a higher resistant antigen-cystozoite in mice brains infected with T. gondii (Hulínská et al., 1990).

ANTI-PARASITIC EFFECT OF PROLACTIN ON T. gondii INFECTION
PRL is capable of inhibiting multiplication of Toxoplasma in murine microglial cell cultures (Benedetto et al., 2001). PRL significantly restricted intracellular growth of Toxoplasma in mice and human cell lines (Dzitko et al., 2010(Dzitko et al., , 2012. Moreover, it been documented that women with hyperprolactinemia showed lower T. gondii prevalence (Dzitko et al., 2008). It has been reported that serum human prolactin (shPRL) has the capacity to bind to live RH tachyzoites (type I) and ME49 (type II) strains in a specific way (Dzitko et al., 2013).
The aim of this study was to review the relationship between hormones and infection by T. gondii in several experimental animal models and humans. Focusing the information on: (a) congenital transmission, (b) parasite reproduction, (c) strain virulence, (d) levels of hormone in host induced by T. gondii infection, (e) participation of hormone receptors in T. gondii infection.

DATABASE SEARCH
Reports from September 2013 to February 2014 were obtained from a total of nine databases (Pub Med, Lilacs, Medline, Science direct, Scielo, Ebsco, Sprinker, Wiley, Google Scholar). Mesh terms were "Toxoplasma or toxoplasmosis or Toxoplasma gondii" combined with progesterone, 17β-estradiol, testosterone, cortisol, cortisone, aldosterone, 11-desoxicorticosterone, dihydrotestosterone, dehydroepiandrosterone, and non-steroid hormones; growth hormone, prolactin, parathyroid hormone, corticotrophin, insulin, glucagon, luteinizing hormone, thyroid stimulating hormone, human chorionic gonadotropin, antidiuretic hormone, oxytocin, melanocyte stimulating hormone, somatostatin, thyrotropin-releasing hormone, gonadotropinreleasing hormone, noradrenaline, adrenaline, melatonin, thyroxine, and triiodothyronine. Toxoplasma and hormones and strain Toxoplasma. The criteria used for including data were: the full text of papers written in English (reviews and case reports not considered), studies performed on humans, animals, and in cell cultures.

DATA COLLECTION METHODS
Two reviewers (GRML and GMAF) carefully studied all selected studies. The full text of selected original articles were obtained and reviewed. Inclusion criteria for this analysis were explicit data of all independent variables and at least one dependent variable; data collection and criteria eligibility were established for determining the frequency or proportion of each study. The independent variables were T. gondii strain, hormones, study design, stage of infection and developmental stage of the parasite, post infection evaluation time, age, host, and technical analysis. Dependent variables were increased or decreased of infection and number of parasites. Reference lists of full-text publications were examined for identifying studies not originally selected Figure 1.
From 30 articles meeting inclusion criteria, all results were captured on an Excel database. A number of studies presented frequency distribution of dependent variables; in these cases, the sum of the products of each value by frequency was included for comparison in the database. Some articles presented ranges, mean plus standard deviation; these articles were included in the database using the median.

RESULTS
One thousand two hundred and seventy eight articles potentially related to T. gondii or hormones were found. However, only 45 were selected and of these, 30 met the inclusion criteria for this systematic review. The analysis was divided into three categories: (A) humans in Table 1, (B) several species of animals in Table 2, and (C) Cell cultures in Table 3 and studies conducted in the time period that this research included Figure 2.
Eight T. gondii strains were also analyzed: two Type I (eight RH and four BK) and six Type II (two PRU, ME49 and SC and one T45, P78, T38) and two not specified ( Table 2).
The most frequent parasite stage of development studied was the tachyzoite (n = 11), followed by cyst (n = 8), ooquiste (n = 2), and bradizoite (n = 1). The number of parasites used for each experiment depended on the stage of parasite development and the host. In the murine model, tachyzoites from 1 × 10 4 to 1 × 10 7 were used (Benedetto et al., 2001;Abdoli et al., 2012;Dzitko et al., 2013). The number of cysts used in different rodent species was from 8 to 100 (Stahl and Kaneda, 1998b;Liesenfeld et al., 2001). In an experiment with goats, 1250 bradyzoytes were used (Engeland et al., 1996) and in another study with sheep infected with ooquistes, the number of ooquistes was not indicated (Aiumalamai et al., 1990) ( Table 2).
Concerning non-steroid hormones, prolactin and thyroxine hormone have been studied. In this study, other non-steroid hormones such as growth hormone, parathyroid, corticotrophin, insulin and glucagon, luteinizing and follicle hormone, thyroid stimulating, human chorionic gonadotropin, antidiuretic, oxytocin, melanocyte stimulating, somatostatin, thyrotropinreleasing hormone, gonadotropin-releasing hormone, noradrenaline, adrenaline, melatonin, and triiodothyronine were not associated to Toxoplasma infection.

DISCUSSION
Congenital toxoplasmosis is one of the most significant burdens of T. gondii infection in humans. Both the maternal-fetal transmission and hormonal levels during pregnancy are poorly understood and yet, may play an important role during the course of the disease. In pregnant women with acute toxoplasmosis, low levels of progesterone and low levels of estrogens can induce severe infection caused by T. gondii (Al-warid and Al-qadhi, 2012). The changes in endocrine phenomena occurring during pregnancy, as well as the size and maturity of the placenta and the embryonic/fetal immune response definitely affect the ability to be protected from invasion or to fight infection (Ortiz-Alegría et al., 2010).
In pregnant women with toxoplasmosis, low levels of progesterone and estrogen can induce severe infection. Nevertheless, the mechanism is unknown (Al-warid and Al-qadhi, 2012). Current studies show that there weren't any statistically significant differences in progesterone levels between infected and uninfected women with T. gondii, although higher progesterone levels were observed in uninfected women compared to low level in infected women. Moreover, estrogen levels in both chronic and uninfected women did not exhibit significant differences, although www.frontiersin.org October 2014 | Volume 5 | Article 503 | 9    1979 1980 1986 1990 1998 2001 2002 2004 2005 2007 1996 2008 2010  infected women had a higher level, compared to uninfected women.
The study of 17β-estradiol in T. gondii infection began in 1979, when hexoestrol was administered to mice and increased the number of T. gondii cysts in muscle (Kittas and Henry, 1979). At the same time, the susceptibility to T. gondii infection increased in mice when pharmacological estrogen concentrations were used (Pung and Luster, 1986). Nevertheless, 35 years have passed since these experiments were performed and no further studies regarding 17β-estradiol mechanism in T. gondii infection have been reported.
Progesterone levels are reduced during pregnancy in sheep after infection by T. gondii (Aiumalamai et al., 1990;Fredriksson et al., 1990). This hormonal change could be contributing to the susceptibility to T. gondii infection in sheep.
In RAW 264.7 cells infected with tachyzoites of T. gondii, progesterone did not regulate the replication of parasites (Gay-Andrieu et al., 2002). However, bone marrow stem cells activated with Lippolysaccharide (LPS) and treated with progesterone, while infected with T. gondii tachyzoites, cells exhibited a significant reduction in parasite death compared to activated controls (Jones et al., 2008). These results suggest that progesterone can modulate the survival of parasites in vitro.
The results of this study showed that steroid hormones are the most studied toxoplasmosis interaction. However, the information has a great heterogeneity and is not comparable, due to their different experimental designs. For example, the progesterone has been studied in mice (Pung and Luster, 1986), sheep (Aiumalamai et al., 1990), goats (Engeland et al., 1996), and bone marrow stem cells cultures (Jones et al., 2008). Furthermore, in these experiments, different strains and parasite stage of development were used. Moreover, no study has shown how steroid hormones regulate T. gondii infection.
The first observation of T. gondii infection and its association with testosterone in humans shows that acute infection by this parasite produced temporary hypogondatrophic gonadal insufficiency (Oktenli et al., 2004). On the other hand, there are several human studies analyzing different genders, using portrait pictures of 89 male students, of which 18 were Toxoplasma infected, and 109 female students. When statistically corrected for age, men with latent toxoplasmosis were perceived as more dominant (p = 0.009) and masculine (p = 0.052). These results suggest that the higher level of testosterone could be responsible for at least some of the toxoplasmosis-associated shifts in human and animal behavior (Hodková et al., 2007). In 2008, Flegr showed that the relationship between age, gender and 2D:4D ratio in hands sharply increased with Toxoplasma infection. Infected males had higher testosterone levels, while infected females had lower levels, than Toxoplasma-free males and females, respectively. Toxoplasma-infected males had a lower left hand 2D:4D ratio than Toxoplasma-free males. These results suggest that the relationship between 2D:4D ratio is particularly strong for the left hand and 2D:4D dimorphism will probably be higher in countries with a high prevalence of toxoplasmosis (Flegr et al., 2008b). These results indicate that sexual hormones and gender are key factors determining susceptibility to Toxoplasma infection.
Significantly, lower levels of testosterone in male and female mice with latent toxoplasmosis (strain T38 of T. gondii) were compared to uninfected controls (Kaňková et al., 2011). On the other hand, Liesenfeld in 2001 described the effect of sexual steroids and gender in the susceptibility to infection by T. gondii in mice. Death occurred in female mice before males, and mortality in females was associated to an increase in the number of tachyzoites. Female mice testosterone treatment reduced the number of parasites and pathology.
5α-Dihydrotestosterone reduced the number of cysts in mice infected with T. gondii cysts strain T45. Mice treated with corticosterone increased twice the number of cysts of T. gondii (Pung and Luster, 1986;Hulínská et al., 1990). These results showed that corticosterone could exacerbate the infection process.
The prevalence of T. gondii infection was analyzed in women with hyper and hypoprolactinemia, with a significant increase in this last group (Dzitko et al., 2008). In other studies using peripheral blood mononuclear cells (PBMC) of patients with hyperprolactinemia revealed that exogenous recombinant human prolactin (rhPRL), as well as autologous shPRL from inactivated serum, significantly restricted intracellular growth of Toxoplasma in these cultures (Dzitko et al., 2012). PRL may be one of the potential humoral factors implicated in the limitation of T. gondii invasion. A physiological increase in PRL concentration during pregnancy may significantly reduce the risk of T. gondii proliferating in the expecting mother (Dzitko et al., 2012). rhPRL reduced T. gondii replication in human cells (Hs27 y HeLa) and murine cells (L929), (Dzitko et al., 2010(Dzitko et al., , 2013. Afterwards in another experimental study, the replication of parasites was reduced in L929 cells treated with prolactin. These results indicate that the inhibition of replication of T. gondii was caused by a limited capacity of the parasites to penetrate host cells, as demonstrated by the reduced number of infected cells. On the other hand, PRL stimulates T cell proliferation (Clevenger et al., 1992) and the release of various protective cytokines as TNF-α which control efficiently the course of T. gondii infection (Benedetto et al., 2001). The possible PRL action could be bidirectional, namely PRL may limit the proliferation of Toxoplasma via surface host cell receptors (Dzitko et al., 2013) leading to the release of protective type-1 cytokines, such as interleukin 12 (IL-12) and IFN-c (Matalka, 2003), and by inhibiting their penetration ability (Dzitko et al., 2010(Dzitko et al., , 2013.
In the last 35 years, researchers worldwide have made a great effort to advance in the field of knowledge on how the hormones are involved in T. gondii infection, however, a major number of studies and the use of modern molecular methods are required to define the mechanistic role of hormones in the regulation of toxoplasmosis.

IMPLICATIONS FOR RESEARCH
A crucial factor is the difference in experimental models to study of T. gondii infections and hormones. As well, type's strains and the number limited studies to comparative analysis.