The Role of Probiotics and Synbiotics on Hirsutism

: Probiotics and synbiotics are known to have beneﬁcial effects on human health and disease. Hirsutism, a disorder that is characterised by the presence of coarse terminal hairs in a male-like pattern, is usually caused by elevated androgen levels in blood plasma. This disorder is usually observed in PCOS women and it is linked to insulin resistance (IR). Although idiopathic hirsutism (IH) is not shown to have excess androgen production from the ovarian and adrenal glands, increased 5 α -reductase in peripheral tissues and insulin resistance are common observations. The effect of probiotics and synbiotics have been recently studied on PCOS women; androgens were also included in the hormonal groups that were investigated. Only a few studies focus on hirsutism and the potential effect of the beneﬁcial microbes mentioned, whereas the increasing interest on insulin resistance and synbiotics indicate a potential beneﬁcial effect on hirsutism through the management of insulin resistance. effects of IR are directly linked with hirsutism through excess androgen production. This relationship between IR and hirsutism was studied in healthy women, where adipose tissue was collected and in-vitro treatments with testosterone and/or anti-androgens were conducted on insulin stimulated adipose cells [100].


Introduction
Fermented food, such as yoghurt, bread, bear and wine, has been widely known since ancient times, and some have been used since then, for therapeutic purposes [1][2][3]. Fermented dairy products (yoghurt, kefir) were used to treat diseases like diarrhea and other infections of the intestinal tract [4,5]. Interaction between microorganisms and human health was first reported in 1907, where yoghurt microflora was described by Elie Metchnikoff [6]. Fermented food is still used as an additional treatment for disease, such as wounds [7][8][9], gastroenteric disorders and infections [7,10], as well as an overall support for health [7,8,[10][11][12]. The term probiotic was introduced by Werner Kollath [13] and according to the World Health Organisation (WHO), this term refers to the microorganisms that ''when administered in adequate amounts, confer a health benefit on the host" [14]. Probiotics are found to have multiple beneficial roles on human health and disease, through restoration of gut microbiota (GM) [15,16], symptom improvement on intestinal disease, such as irritable bowel syndrome (IBS) [17], modulation of hormonal profile in animals and humans [18][19][20][21][22] and skin infections and healing [23][24][25]. Along with the beneficial microorganisms, prebiotics, which are fermented compounds that support and promote modifications in the activity and composition of GM [26], have also been studied. It is shown that a controlled combination of probiotics and prebiotics, called synbiotics, can provide a more enhanced beneficial effect on human health and disease [16,[27][28][29].
Although the effect of probiotics and synbiotics are extensively studied, there has been limited research conducted on their effect on sex hormones and sex hormone imbalances, such as hirsutism, a disorder that is mainly characterised by elevated androgen levels in women. The aim of this review is to focus on the role of probiotics and synbiotics and their metabolic process on hirsutism.

Probiotics
Lactobacillus, Streptococcus, Bifidobacterium, Lactococci and Saccharomyces are some of the probiotic species that are known [30]. Microorganisms that are mostly used as treatments

Hirsutism
Hirsutism is a condition that appears in 5-10% of women and it is recognized by the presence of coarse terminal hairs in a male-like pattern [88]. Excess hair growth that consists of terminal hairs are present in areas were women normally have thinner hair. The clinical diagnosis of hirsutism is completed based on Ferriman and Gallwey criteria ( Figure 1) [88] and the areas that are scored include the face, chest, thighs, upper arms, abdomen and back. Scores (m-FG) from 1-4, with 1 describing minimal terminal hair and 4 describing frank virilization, are given to the mentioned areas whist total scores less than 8 are considered normal [88]. patients suffering from ulcerative colitis [77]. Another study on patients with ulcerative colitis showed that B. breve and galacto-oligosaccharide (GOS)-containing beverage (Yakult) managed to clinically improve the condition of the patients, through reduction of UC markers, such as myeloperoxidase, and by lowering the faecal pH [87].

Hirsutism
Hirsutism is a condition that appears in 5-10% of women and it is recognized by the presence of coarse terminal hairs in a male-like pattern [88]. Excess hair growth that consists of terminal hairs are present in areas were women normally have thinner hair. The clinical diagnosis of hirsutism is completed based on Ferriman and Gallwey criteria ( Figure 1) [88] and the areas that are scored include the face, chest, thighs, upper arms, abdomen and back. Scores (m-FG) from 1-4, with 1 describing minimal terminal hair and 4 describing frank virilization, are given to the mentioned areas whist total scores less than 8 are considered normal [88]. Hirsutism scoring system presenting scores ranking from 1-minimal hirsutism, to 4 virilization in 9 body parts. A total score of less than 8 is considered normal, whereas higher scores indicate mild to severe hirsutism [88].
Hirsutism is a quite complex condition that can occur due to other syndromes or disease, such as PCOS ( Figure 2). Although the main reason for this condition is the presence of excess androgens, such as testosterone (T) and dihydrotestosterone (DHT), IH shows normal levels of androgens [89,90]. The most common reason for the excess androgen levels is PCOS and 60-80% of PCOS women suffer from hirsutism [91]. The diagnosis of this condition is conducted according to the Rotterdam criteria, by which two of the three criteria have to be met: (a) evidence of hyperandrogenism, (b) oligo-and/or anovulation and (c) polycystic ovaries [92]. Other clinical features are ovarian enlargement and IR, which is found to be very common amongst PCOS women, whilst the syndrome has also been linked with cardiovascular disease and inflammation. The heritability of this disorder has been studied and it is possible that genetic changes on the androgen receptor gene can lead to hirsutism [90]. During this study, a repeatability of the trinucleotide CAG Figure 1. Hirsutism scoring system presenting scores ranking from 1-minimal hirsutism, to 4 virilization in 9 body parts. A total score of less than 8 is considered normal, whereas higher scores indicate mild to severe hirsutism [88].
Hirsutism is a quite complex condition that can occur due to other syndromes or disease, such as PCOS ( Figure 2). Although the main reason for this condition is the presence of excess androgens, such as testosterone (T) and dihydrotestosterone (DHT), IH shows normal levels of androgens [89,90]. The most common reason for the excess androgen levels is PCOS and 60-80% of PCOS women suffer from hirsutism [91]. The diagnosis of this condition is conducted according to the Rotterdam criteria, by which two of the three criteria have to be met: (a) evidence of hyperandrogenism, (b) oligoand/or anovulation and (c) polycystic ovaries [92]. Other clinical features are ovarian enlargement and IR, which is found to be very common amongst PCOS women, whilst the syndrome has also been linked with cardiovascular disease and inflammation. The heritability of this disorder has been studied and it is possible that genetic changes on the androgen receptor gene can lead to hirsutism [90]. During this study, a repeatability of the trinucleotide CAG in exon 1 was observed and more frequent repeats in the Nterminal domain of the androgen gene were linked with the development of the disorder. However, other researchers have not shown a significant role of CAG in the pathogenesis of hirsutism [93,94]. Hirsutism has also been linked with IR ( Figure 3) [91,[95][96][97][98][99][100][101], whilst excess insulin production leads to hyperinsulinemia, which increases luteinizing hormone (LH) through insulin receptor stimulation [97]. LH, along with follicle-stimulating hormone (FSH), is a hormone that regulates androgen production through the secretion of them from ovarian and adrenal glands. Moreover, high levels of insulin inhibit sex hormonebinding globulin (SHBG), a hormone that binds with plasma T and is considered its major determinant along with 17-β hydroxysteroids from plasma [102]. Consequently, both effects of IR are directly linked with hirsutism through excess androgen production. This relationship between IR and hirsutism was studied in healthy women, where adipose tissue was collected and in-vitro treatments with testosterone and/or anti-androgens were conducted on insulin stimulated adipose cells [100]. sulin receptor stimulation [97]. LH, along with follicle-stimulating hormone (FSH), is a hormone that regulates androgen production through the secretion of them from ovarian and adrenal glands. Moreover, high levels of insulin inhibit sex hormone-binding globulin (SHBG), a hormone that binds with plasma T and is considered its major determinant along with 17-β hydroxysteroids from plasma [102]. Consequently, both effects of IR are directly linked with hirsutism through excess androgen production. This relationship between IR and hirsutism was studied in healthy women, where adipose tissue was collected and in-vitro treatments with testosterone and/or anti-androgens were conducted on insulin stimulated adipose cells [100].

Figure 2.
Hirsutism can originate from adrenal or ovarian disorders, or both. Various syndromes and disorders related to the adrenal glands, such as Cushing's syndrome, adrenogenital syndrome, 21-hydroxylase-deficient adrenal hyperplasia, insulin-resistant acanthosis nigricans and androgen secreting tumour, show high levels of cortisol hormone and hirsutism. Ovarian hirsutism is caused by PCOS that is characterised from high levels of free testosterone and/or polycystic ovaries, insulin resistance and hyperandrogenemia. Idiopathic hirsutism is caused by peripheral increase of androgens, and women that suffer from it show increased levels of 5α-reductase and insulin resistance, whilst they do not show any abnormalities in their ovarian or adrenal function.
Adipose tissue contains adipocytes, also known as fat cells and are responsible for fat energy storage. This study showed that the exposure of these cells to testosterone led to insulin resistance, suggesting there is a link between hirsutism, androgen presence and insulin resistance development.
Other disorders that are known for the increased production of androgens are hyperandrogenic insulin-resistant acanthosis nigricans syndrome [103,104], 21-hydroxylase-deficient non-classic adrenal hyperplasia [90,105], androgen-secreting tumor [106], and rarely androgenic drug intake [107]. On the other hand, the causes of IH are not well known. However, when hormonal profiles of PCOS women and IH women were compared, IR was found to be significant in both groups compared to the control group. IR Figure 2. Hirsutism can originate from adrenal or ovarian disorders, or both. Various syndromes and disorders related to the adrenal glands, such as Cushing's syndrome, adrenogenital syndrome, 21-hydroxylase-deficient adrenal hyperplasia, insulin-resistant acanthosis nigricans and androgen secreting tumour, show high levels of cortisol hormone and hirsutism. Ovarian hirsutism is caused by PCOS that is characterised from high levels of free testosterone and/or polycystic ovaries, insulin resistance and hyperandrogenemia. Idiopathic hirsutism is caused by peripheral increase of androgens, and women that suffer from it show increased levels of 5α-reductase and insulin resistance, whilst they do not show any abnormalities in their ovarian or adrenal function.
Adipose tissue contains adipocytes, also known as fat cells and are responsible for fat energy storage. This study showed that the exposure of these cells to testosterone led to insulin resistance, suggesting there is a link between hirsutism, androgen presence and insulin resistance development.
Other disorders that are known for the increased production of androgens are hyperandrogenic insulin-resistant acanthosis nigricans syndrome [103,104], 21-hydroxylasedeficient non-classic adrenal hyperplasia [90,105], androgen-secreting tumor [106], and rarely androgenic drug intake [107]. On the other hand, the causes of IH are not well known. However, when hormonal profiles of PCOS women and IH women were compared, IR was found to be significant in both groups compared to the control group. IR and hyperinsulinemia increase the insulin-like growth factor (IGF) which affects hair follicles [95,96], and therefore it is suggested that IR is potentially a cause of IH. This disorder is also characterised as an increase in 5α-reductase in peripheral tissues, an enzyme that converts T to DHT, and is generally responsible for the metabolism of steroids [95]. Potential causes of IH include increased sensitivity of hair follicles to androgens, and androgen receptor gene polymorphism [96]. and hyperinsulinemia increase the insulin-like growth factor (IGF) which affects hair follicles [95,96], and therefore it is suggested that IR is potentially a cause of IH. This disorder is also characterised as an increase in 5α-reductase in peripheral tissues, an enzyme that converts T to DHT, and is generally responsible for the metabolism of steroids [95]. Potential causes of IH include increased sensitivity of hair follicles to androgens, and androgen receptor gene polymorphism [96]. The relationship between insulin resistance and hirsutism. Hyperinsulinemia is caused when insulin resistance is left unmanaged, causing an increase in LH and FSH, which regulate the production of ovarian and adrenal androgens. This increase results in excess androgens, and eventually hirsutism. Moreover, insulin resistance reduces SHBG, which leaves plasma testosterone unbound leading to increased free testosterone and hirsutism. Chronic androgen exposure leads to adipose and skeletal muscle insulin resistance. The graph above presents the circular relationship between the two conditions, leading to a vicious cycle of effects.

Therapeutic Index
Hirsutism treatments are usually combinational treatments, and they include peripheral androgen blockage, androgen suppression and cosmetic intervention. Cosmetic and mechanical interventions are more effective and long lasting when used in combination with either androgen suppression treatment or peripheral androgen blockage. Moreover, alternative treatments have been investigated in recent years and naturally derived additional treatments have been used for the improvement of hirsutism, as well as live organisms that are shown to have a significant role in regulating and reducing androgen production on PCOS women.

Chemically Derived Therapies
Current drug therapies for hirsutism include androgen suppression and peripheral androgen blockage, also known as anti-androgens. For a more effective treatment these drugs are usually administered in combination [108][109][110][111][112].
Androgen suppression treatments include insulin sensitisers, long acting gonadotrophin releasing hormone (GnRH) analogue and estrogen-progestin oral contraceptive (OC) pills [98,[112][113][114]. OC action can differ in composition, but it is not shown to significantly influence the effectiveness of the treatment. Their compositions can be monophasic, biphasic or triphasic and the action of the OC is through the suppression of LH and FSH and therefore a reduced androgen production from the ovaries [90]. Although this treatment is shown to reduce SHBG and hyperandrogenemia in general, it does not have an Figure 3. The relationship between insulin resistance and hirsutism. Hyperinsulinemia is caused when insulin resistance is left unmanaged, causing an increase in LH and FSH, which regulate the production of ovarian and adrenal androgens. This increase results in excess androgens, and eventually hirsutism. Moreover, insulin resistance reduces SHBG, which leaves plasma testosterone unbound leading to increased free testosterone and hirsutism. Chronic androgen exposure leads to adipose and skeletal muscle insulin resistance. The graph above presents the circular relationship between the two conditions, leading to a vicious cycle of effects.

Therapeutic Index
Hirsutism treatments are usually combinational treatments, and they include peripheral androgen blockage, androgen suppression and cosmetic intervention. Cosmetic and mechanical interventions are more effective and long lasting when used in combination with either androgen suppression treatment or peripheral androgen blockage. Moreover, alternative treatments have been investigated in recent years and naturally derived additional treatments have been used for the improvement of hirsutism, as well as live organisms that are shown to have a significant role in regulating and reducing androgen production on PCOS women.

Chemically Derived Therapies
Current drug therapies for hirsutism include androgen suppression and peripheral androgen blockage, also known as anti-androgens. For a more effective treatment these drugs are usually administered in combination [108][109][110][111][112].
Androgen suppression treatments include insulin sensitisers, long acting gonadotrophin releasing hormone (GnRH) analogue and estrogen-progestin oral contraceptive (OC) pills [98,[112][113][114]. OC action can differ in composition, but it is not shown to significantly influence the effectiveness of the treatment. Their compositions can be monophasic, biphasic or triphasic and the action of the OC is through the suppression of LH and FSH and therefore a reduced androgen production from the ovaries [90]. Although this treatment is shown to reduce SHBG and hyperandrogenemia in general, it does not have an effect in low-grade inflammation and other hormonal disorders. Third generation OCs are not as effective at reducing the levels of SHBG when compared to older combined OC [115]. OCs containing sex hormones progestin and estrogen are shown to have significant effect on the hormonal profiles of hirsute women, reducing the levels of androgens, such as T, dehy- droepiandrosterone sulfate (DHEAS) and DHT [116]. GnRH is a hormone produced in the hypothalamus and it stimulates the production of LH/FSH which stimulates the ovaries to produce androgens [97]. GnRH analogues are antagonists of GnRH and, when used on PCOS women, were shown to reduce the total and free T, DHEAS and androstenedione sex hormone. This treatment is usually combined with OCs. Moreover, the combination of insulin sensitisers with OC has also been studied. Insulin sensitisers, such as metformin and thiazolidenediones, are shown to reduce androgen production through the regulation of blood glucose and the increase of insulin sensitivity [108,112,117,118]. Metformin has been widely used, alone or in combination with other OCs or anti-androgens, for the treatment of PCOS as it is shown to decrease levels of LH, testosterone, and androstenedione, and improve the ovulation and decrease IR [108,109,112,113,[119][120][121][122]. Metformin is shown to be equally but moderately effective on reducing the hair diameter in PCOS women when compared to anti-androgen combined with OC [112]. Although there was no decrease of androgen levels, the effect is suggested to appear due to the reduction of hyperinsulinemia. This suggests that managing insulin resistance and increasing insulin sensitivity might be a better therapy treatment and approach, than administrating anti-androgens. Overall, the combination of metformin with other drugs appears to be more effective on the treatment of hirsutism, decreasing m-FG scores, levels of androgens and further drop of free androgens through the enhancement of SHBG [109].
Peripheral androgen blockage drugs include cyproterone acetate, spironolactone, flutamide and bicalutamide. 5α-reductase inhibitors, such as finasteride, have also been used for the treatment of hirsutism [123]. These drugs bind to the androgen receptors in selective tissues and therefore reduce the synthesis of androgens [124][125][126]. Cyproterone acetate is shown to be effective in treating excessive hair growth and reducing androgen levels [100,110,112,123,124,127,128], although side effects, such as weight gain, tiredness, loss of libido and adrenal disfunction were reported [127][128][129]. The effect of this progestogenic drug is through the inhibition of gonadotropin response to reduce T levels, decrease hair growth and androgen levels [127,128,130]. Spironolactone was originally used as a diuretic drug. However, it was shown to reduce T, through cytochrome P450 inhibition, without having any effect on SHBG or free T [131]. It is shown to be an effective and tolerable drug for the treatment of hirsutism, especially when combined with OC [132][133][134]. Flutamide is reported to be a better choice of treatment for the disorder studied when compared to the previously mentioned drugs [134]. It is shown to have a significant effect on hirsutism, acne and male-pattern hair loss in hirsute women. However, severe side effects are reported, such as embryotoxicity, thus it is suggested to use in combination with other drugs, such as insulin sensitisers or OC [98,108,111,122,134]. Bicalutamide was shown to have similar effectiveness with the above drugs on reducing m-FG scores and lower androgen levels, although it did not have any effect on insulin resistance [135][136][137]. Despite the effectiveness of these drugs, many subjects are sceptical when it comes to the administration of such treatments due to the potentially severe side effects. Thus, the potential therapeutic effect of naturally derived therapies is also studied.

Naturally Derived Therapies
Many natural remedies have been studied and used for the management of hirsutism and acne, however, extensive research has not been conducted for many of them [138][139][140][141]. Some of the naturally derived anti-androgens are spearmint tea, green tea, licorice, Chinese peony, marjoram, and red reishi [141]. In a study where the effects of spearmint tea as an anti-androgen were investigated, forty two patients were asked to consume two cups of spearmint tea every day for thirty days [142]. Their gonadotrophin levels and androgen hormones were tested before and after the treatment and a significant reduction of total and free T was observed. Increased LH and FSH were also observed after short administration with spearmint tea [140]. Marjoram is shown to significantly reduce levels of DHEAS and improve IR, and therefore inhibit the production of androgens [143]. Diet has also been investigated on the role of hormonal imbalances especially since the production of androgens is related to IR. Studies are still at early stages, looking at the potential effect of dairy and starch on the hormonal profile of PCOS women [144][145][146]. Studies where women were asked to follow a low starch and low dairy diet presented mixed results and the role of these foods on hormonal profiles requires more research. In addition to plantbased remedies and diet for the treatment of hirsutism and other hormonal imbalances, live organisms have also been studied on their potential effect on the hormonal profile of hirsute women [147][148][149][150][151].

Probiotics and Hirsutism
Microbes have been widely used for the production of various chemicals in the pharmaceutical world. In a recent study, after metabolically engineering Rhodococcus ruber Chol-4, it was shown to produce high amounts (61%) of testosterone through the conversion of 4-androstene-3,17-dione (AD) to the mentioned hormone [152]. There is increasing literature on how probiotics can affect the hormonal profiles in animals and humans and how probiotic supplementation can be used for the management and improvement of metabolic disorders and conditions. When the GM taxa of boards and gilts was analysed, host sex hormones were shown to significantly interact with the GM, which indicates the relationship between hormones and microbes [20]. Another research study that presents the relationship between the gut microbiota and the hormonal balance, is when the androgen levels of normal GM mice were compared to germ-free mice and findings showed exceptionally low unconjugated DHT detected in the later subject's distal intestine in contrast with the former subjects [153]. Interestingly, in the same study, human male feces samples with normal GM were tested and similar findings were observed compared to the normal GM mice samples. For the mice samples, male and female subjects were included. It is therefore proposed that treatment with probiotics could potentially affect the levels of DHT and T.
As mentioned earlier, PCOS is the most common cause of Hirsutism and most women that suffer from this syndrome show symptoms of increased hair growth in a male-like pattern. There is growing literature on the role of probiotics on PCOS and how the mentioned microbes are potentially involved in the management of this condition. Tremellen and Pearce in 2012 presented a detailed hypothesis on the development of PCOS starting from dysbiosis of gut microbiota (DOGMA) and how this causes a series of effects that eventually leads to hirsutism along with other symptoms of PCOS [154]. More specifically, it is hypothesised that the disruption of the balance of GM, and in combination with a diet high-in-sugar and saturated fat, leads to increased gut mucosa permeability. This allows the gram-negative bacteria to enter the systemic circulation and stimulate the immune system causing mild chronic inflammation, leading to damage in insulin receptors that further develops into IR and hyperinsulinemia in the long term. The latter condition affects the ovaries to produce excess androgens and disrupts their normal function, causing further disturbance in the development of follicles. Therefore, it is possible to hypothesise that these conditions are less likely to occur if a well-balanced gut microbiota is maintained. The above hypothesis was further investigated a few years later, where the potential gut permeability, and the level of inflammation and GM in PCOS women was studied [155]. Even though the GM was indeed found to be altered, compared to healthy subjects, there was no significant changes in gut permeability and function suggesting that "leaky" gut is only potentially a symptom in women with PCOS. In another study where the relationship of PCOS and gut microbiota was investigated, PCOS rats' GM was compared to control groups and a decreased number of Lactobacillus, Clostridium and Ruminococcus were detected, whereas Prevotella, a gram-negative bacteria, was found in higher numbers than for the healthy subjects [156]. The rat's PCOS was induced using an aromatase inhibitor called letrozole and the subjects showed increased levels of androgens along with symptoms of abnormal hormonal levels and cycles with their ovaries morphologically altered. To further investigate this relationship, faecal microbiota and Lactobacillus were transplanted in the gut of PCOS rats and restoration of the gut microbiota was observed along with improvement of PCOS symptoms. Interestingly, the androgen levels were reduced, and the ovaries' morphology was improved. Such effects are in agreement with more recent studies where androgen levels and hirsutism are shown to improve after supplementation with probiotics [148,149,157]. Possibly the first investigation on hormonal profiles of PCOS women after the supplementation of probiotics, was presented in 2018 in a study where 60 women participated [157]. Probiotics Lactobacillus and Bifidobacterium, two of the most common species used in probiotic treatments, were supplied to the subjects for 12 weeks after which time significant increase in SHBG was observed, whilst m-FG scores and total testosterone were decreased. Combinational treatments of vitamin D and probiotics also showed a significant response when it comes to androgen level reduction [149]. Although this suggests that probiotics might be more efficient on managing hirsutism when combined with vitamin D, other researchers have not shown any significant effect of the vitamin alone to the referred condition [158][159][160][161], despite its significant anti-inflammatory and insulin sensitivity effect [162][163][164]. Therefore, it is possible that the effects of the combinational treatment are due to the improvement of IR from vitamin D, along with the probiotics effect [149]. It is also reported that PCOS-rats showed improvement in reproductive function after the treatment with probiotics [165]. Although only a few studies have looked at the effects of probiotics on hirsutism, the literature found on the role of the former on IR and sensitivity has been explored in several studies [27,[165][166][167][168][169][170][171][172][173]. Moreover, it is now well established from a variety of studies that hirsutism is linked with IR [21,[95][96][97]104,[174][175][176][177]. As described earlier IR and hyperinsulinemia enhances the production of androgens such as T and DHT, whilst reduces SHBG levels leading to increased free testosterone levels [102].
Considering the above it is necessary to briefly present the role of probiotics on IR through which a potential improvement of hirsutism can occur. Research has been conducted on PCOS subject as well as prediabetics and healthy participants. In 2015, Shoaei et al. studied PCOS women for 8 weeks while the subjects were under probiotic supplementation, observing decreased FBS, insulin levels and IR [173]. The same observations were made in another study conducted on PCOS women where probiotics were supplemented for 12 weeks [167]. Glucose levels decreased during pregnancy and insulin sensitivity increased during the 12 month postpartum period, in a study where subjects were treated with Lactobacillus and Bifidobacterium [168]. Lower glucose levels were also observed when post-menopausal women were provided with Lactobacillus plantarum-containing beverages [170]. It is reported that Lactobacillus salivarius UBL22, when supplemented on healthy subjects, showed a significantly reduced fasting insulin compared to the control group (placebo) [27]. Another study conducted on prediabetic participants showed a reduction in fasting glucose and fasting insulin levels after treatment with probiotics (Table 1) [166]. Table 1. Probiotic treatments and the hirsutism-relevant outcomes.

Treatment Study Population Relevant Outcomes Reference
Lactobacillus PCOS 1 rats Improvement on estrous cycles, reduction of androgen biosynthesis, normalization of ovaries, GM 2 restoration, reduction of Prevotella [156] L. acidophilus, L. casei, B. bifidum PCOS women Reduction of total testosterone, increase of SHBG 3 , decrease of m-FG scores [157] L. acidophilus, L. reuteri, B. bifidum and Vitamin D PCOS women Reduction of total testosterone and hirsutism [149]

Synbiotics and Hirsutism
In reviewing the literature, very few studies were found on the association between synbiotics and hirsutism. A recent study looked at the effects of synbiotics on PCOS women using a mix of pomegranate juice, inulin and Lactobacillus [178]. Testosterone levels were significantly reduced after the treatment with synbiotics, compared to the control group. These findings were accompanied with an increase in insulin sensitivity and improved anthropometric measurements. Inulin is a prebiotic that has been shown to improve IR and has a beneficial effect on SHBG [147,179,180]. In a 12 week study, Lactobacillus bifidum, Lactobacillus acidophilus, Lactobacillus casei and inulin were used for the treatment of PCOS women and their hormone levels were studied along with other biomarkers, such as serum high sensitivity C-reactive protein [19]. Despite the statistical significance of the decrease of m-FG scores, the clinical picture of the subjects showed little improvement and longer treatment with prebiotic inulin was suggested. Selenium, known for its prebiotic activities [181][182][183] was also used in a combinational treatment with probiotics on women and the hormonal profiles were studied [150,184,185]. Although initial studies have not shown an effect of the treatment on free [184] and total [185] testosterone, a decrease on m-FG scores was observed [184]. On a 12-week study conducted two years later, 60 PCOS subjects were treated with Lactobacilllus, Bifidobacterium and selenium-containing synbiotics and, in contrast with the above findings, a statistically significant reduction of total testosterone and hirsutism was shown [150]. There is abundant room for further progress in determining the exact effect of synbiotics on hirsutism, whilst there is an increasing literature on the beneficial effects of synbiotics on IR on PCOS women.
Brief investigation of the role of synbiotics on IR is necessary to understand how synbiotics could potentially improve hirsutism through the improvement and management of IR. A year after Nasri et al. (2018) published their work as described earlier, Samimi et al. (2019) investigated the effects of the same synbiotics supplementation on biomarkers of IR and sensitivity along with cholesterol and triglycerides on PCOS women [180]. In this study, it was shown that Lactobacillus bifidum, Lactobacillus acidophilus, Lactobacillus casei and 0.8 g of inulin, significantly improved insulin metabolism markers but not FBS, after 12 weeks of treatment, suggesting that metabolic disorders can benefit from the mentioned treatment while managing symptoms. Longer treatment periods (24 weeks) were also investigated and prediabetic adults were treated with Lactobacillus and Bifidobacterium in a inulin-containing prebiotic [166]. There was no significant improvement on fasting insulin levels and plasma glucose between the two studied time points, 12 and 24 weeks. The use of a single probiotic along with inulin is found in a study that was conducted on diabetic rats and their insulin markers were observed [80]. The synbiotic containing Lactobacillus plantarum and inulin managed to increase insulin sensitivity. All the above studies described were conducted on subjects that were either diagnosed as prediabetics or suffered from conditions (PCOS) for which IR is known to be a symptom. However, the beneficial effect of synbiotics was also observed when healthy subjects were treated with Lactobacillus salivarius UBL S22 and FOS and the treatment was shown to improve insulin sensitivity significantly more compared to the probiotics alone [27]. Interestingly, despite the higher variation of probiotic bacteria in the synbiotic mix (Bifidobacterium breve, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus thermophilus), in combination with the fructo-oligosaccharide inulin on PCOS women, it does not show a significant effect on the FBS and insulin sensitivity [179]. Lactobacillus synbiotics and 200 µg/day selenium [186] were used for the treatment of PCOS subjects for 12 weeks and it was found to improve IR and insulin levels, findings that were not observed when selenium alone was used for the treatment of diabetic participants [187].
Other studies show similar observations on fasting plasma glucose [188] and insulin levels, IR and insulin sensitivity ( Table 2) [189]. Table 2. Synbiotic treatments and the hirsutism-relevant outcomes.

Treatment Study Population Relevant Outcomes Reference
Lactobacillus and pomegranate juice PCOS 1 women Reduced testosterone levels, increased insulin resistance and improved anthropometric measurements [178] L. bifidum, L. acidophilus, L. casei and inulin PCOS women Reduced m-FG scores but did not affect the clinical picture of hirsutism [19] Lactobacillus, Bifidobacterium and Selenium

PCOS women
Reduced total testosterone and hirsutism [150] L. bifidum, L. acidophilus, L. casei and inulin PCOS women Improved insulin metabolism markers but did not influence FBS 2 [180] Lactobacillus, Bifidobacterium and inulin Prediabetics Decreased fasting insulin levels and fasting plasma glucose [166] L. plantarum and inulin Diabetic rats Improved insulin resistance, hypothalamic levels of insulin and leptin [80] L. salivarius UBL S22 and fructooligosaccharides Healthy participants Improved insulin sensitivity [27]

Conclusions
Probiotics and synbiotics have been widely used for the treatment of several conditions and diseases [54,155,186,[190][191][192]. The potentially healing properties of natural products are presented more often in recent years and people tend to turn to more natural ways of treating symptoms or even aiming towards them providing a reversing effect. On the other hand, endocrinology is a very complex area of study. Although hirsutism, a disorder characterised of excess terminal hair in a male-like pattern [88,193], has been generally studied, not much research was conducted on the effects of probiotics/synbiotics on this condition. The complexity of the disorder is also shown through its relationship with lipid metabolism [194]. Main markers for the determination of lipid metabolism, such as low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglycerides are shown to be altered in women suffering from PCOS and other related disorders [195]. Elevated levels of triglycerides and lower levels of LDL, a group of lipoproteins that are the main source of cholesterol, are observed in hirsute women. Moreover, insulin is known to increase the activity of tissue lipoprotein lipases (LPL), an enzyme that takes part in the metabolism of lipoproteins and is regulated by plasma hormones. IR, on the other hand, can lead to the elevation of triglycerides, an observation that indicates an indirect relationship of hirsutism with high levels of lipids through the increase of IR through the elevated levels of androgens and vice versa. However, this complex connection between hormones and lipids appears to be independent, as shown in a study where hirsute women were treated with GnRH and, despite the unaffected IR and lipid metabolism of the subjects, estrogens and androgens were decreased [195]. The vicious circle between increased androgens and IR, as shown in Figure 3, supports the hypothesis that hirsutism can affect the metabolic process of lipoproteins and the related enzymes, through the enhancements of IR. Therefore, treatments that provide an overall improvement to IR and steroid levels could potentially alter the metabolic process of lipids and enzymes. Although there is an extensive research on the effects of androgens on cardiovascular disease risk on men [196][197][198][199], not many studies exist on the effects of hirsutism on lipid metabolism. There is a mixed association between the effects of elevated androgen levels on women and their independent relationship with lipids and cardiovascular disease [200]. The purpose of the current study was to investigate the role of probiotics and synbiotics on hirsutism.
The limited literature found on this topic, shows that probiotics can decrease the production of androgens, and specifically total testosterone, and improve m-FG scoring on hirsute women, increasing the levels of SHBG that bind and regulate free testosterone [102,153,201]. Moreover, synbiotics are shown to have a similar effect with probiotics but more enhanced [27]. Therefore, it is suggested that a combination of probiotics and prebiotics can improve and help on the management of hirsutism and other conditions. Additionally, considering the direct relationship of hirsutism with IR [91,95,96,174], the effects of the beneficial compounds on IR was also examined. Growing literature shows a reduction of FBS, insulin levels, insulin resistance and insulin sensitivity after treatments with probiotics and/or synbiotics supports the beneficial effects on hirsutism through the mentioned mechanisms [178][179][180]202]. Taken together, these findings suggest a role for probiotics and synbiotics in reducing androgens and decreasing m-FG scores, directly and through managing and decreasing IR. Moreover, treatments with probiotics and/or synbiotics are shown to improve the lipid metabolism in hirsute women, reducing serum triglycerides levels [27,167,203], reducing levels of LDL and increasing HDL [27,186,203], although it is not clear whether hirsutism is independently correlated with lipoprotein elevation. The majority of studies found in the literature refer to research conducted on PCOS women, since most hirsute women also suffer from this metabolic syndrome. Current drug therapies on hirsutism including insulin sensitisers, antiandrogen monotherapies and OCPs, can have severe side effects [98,[112][113][114] and a potential therapeutic role of probiotics along with prebiotics, can be a significant relief for women suffering from the disorder.
The effect of the studied microorganisms on the metabolic profile of women is shown to be significant and interestingly, correlated with many abnormalities. The combinational effect of them on IR, steroids and lipids profiles, shows a multifunctional character of these treatments that could potentially support the metabolic processes to such degree that conventional treatments with drugs could be replaced, especially when combined with lifestyle changes, such as exercise and diet. Although there is evidence for the beneficial role of probiotics and synbiotics on hirsutism and the related disorders, more studies need to be conducted on their direct effects, mechanisms, length of treatment and dosage.
Author Contributions: The author confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.
Funding: This research received no external funding.