Medical Applications of Molecular Biotechnologies in the Context of the Hashimoto’s Thyroiditis

1. Introduction

Hashimoto’s thyroiditis (HT) is a chronic destructive inflammatory process that develops by autoimmune mechanisms [1]. TH falls within autoimmune thyroid diseases (AIDT) precisely because of inflammatory response to immune alterations [2,3].

Basically, morphological features of HT include four signatures such as lymphoid infiltrates, fibrosis, oxyphilic changes of follicular cells and varying degree of destruction of glandular tissue [4].

Immunological hallmarks of HT enclose serum antibodies raised against various thyroid antigens encompassing from thyroid peroxidase and antithyroglobulin to thyroid-stimulating hormone receptor [5,6]. Morphological and immunological HT traits don’t emerge concurrently. In fact, there is a small proportion of patients that show cytological features of HT whereas thyroid antibodies are low detectable in their serum [7].

Reductions of serum thyroid hormones levels are noted in HT patients: this is in case no glandular cells secrete enough thyroid hormones able to meet the needs of body. However, hypothyroidism (hy-T) is diagnosed based on serum levels of several biochemical markers such as thyroid stimulating hormone (TSH) and other thyroid hormones used to confirm the diagnosis [8,9]. HT hormonal indicators report different degree of hy-T, independently by entity of morphological damages [10]. Therefore, HT may clinically present either by prominent or mild hy-T symptoms [1]. These appear related with atypic activity of muscle and nerve fibers, alteration of glucose-lipid metabolism, cognitive and psychological disorders [11,12,13].

Currently, a biochemical “grading” system is used to identify latent hy-T forms (see Table 1 in Ref. [4]) [4]. Above of all, this system is designed for HT treatment by levothyroxine (L-T4). This system is built by confrontation between serum levels of TSH and free thyroxine (T4). At the time that L-T4 replacement was indicated as the first choice for treatment of hy-T, the method of doing this hormonal comparison became essential. In fact, since 2014 the guidelines of American thyroid Association recommend L-T4 treatment strategy for hy-T forms [14,15,16]. Further, this is in according to nationwide data from the National Health Service in the United Kingdom and European thyroid association, too [17]. However, basic science and clinical evidence are inducing to development investigations on LT4/LT3 combination therapy [18,19]. New data come up about limitations of serum TSH biochemical marker because of it partially reflects total thyroid status [20,21,22]. Lastly, 10–15% of hy-T patients voice their discontent because of L-T4 treatment outcomes [23,24]. In fact, this recent evidence urges to involve in the care of hy-T above all hy-T patients themselves [25,26].

1.1. HT biomarkers and epidemiological data

At large, HT diffusion can be reported by considering two distinct types of serum biomarkers. Firstly, HT epidemiological information can be compiled based on autoimmune biomarkers of thyroid inflammation. Secondly, to archive HT epidemiological data, HT diffusion can be related to biochemical markers of hy-T and then, to onset of hy-T symptoms.

By focusing on serum immunological biomarkers, HT is considered a gender functional disorder [27]. This is because of mechanisms underlying the appearance of autoantibodies [28,29]. In fact, the disruption of immune tolerance is genetically driven [29]. Particularly, HT autoimmune anomalies are genetically based on gender and pre-existing susceptibility individual [28,30,31]. In turns, environment plays a critical role on altered genetic background by doing influence the disease development. [27,31]. Hence, HT is reported in women 10-15 times more often than men by an incidence peak around 30-50 years [32]. Conversely, in men the HT incidence increases with aging and then, incidence peak is reached 10-15 years later [32].

When HT diffusion is related to hy-T incidence, substantial differences emerge between hy-T that spreads in endemic area of iodine deficiency and what goes accompanied by HT. Zimmermann et colleague had already observed that hy-T typically emerges in HT patient independently by iodine nutrition status [33,34]. This is because of hy-T develops even in HT patients living in area with sufficient iodine intake. Instead, when population are resident in iodine-deficient localities, it is quite common to find endemic hy-T [35].

Gender differences come up even when HT is related to onset of hy-T signs and symptoms. In fact, distinctive clinical courses and different outcomes are observed in women in respect with men. Further, difference of gender is a key determinant even in therapeutic responses to L-T4 [27,36]. Usually, hy-T symptoms include fatigue, cold intolerance, and constipation. However, there is a large variation in clinical presentation of symptoms [16].

In women, hy-T develops more frequent at a later age than HT, especially, after 60 years of age [27]. In addition, hy-T symptoms have not determinant role for identification of endocrine disorder. This is because of hy-T symptoms may occur in healthy women subjects, too. Lastly, L-T4 therapy may be associate with residual symptoms despite normal thyroid tests [18,24].

In men, hy-T symptoms that accompany overt HT are more recurrent, last longer and usually less treatable. [27]. Therefore, the presence or absence of symptoms may be contributing factors to identification of hy-T. Lastly, L-T4 therapy is less frequently accompanied by other side effects in men.

1.2. Prevalence of HT diagnoses

The methods used to diagnose HT have a long history related to description of morphological alterations of thyroid gland, recognition of autoimmune pathogenesis and identification of thyroid hormones [4]. For a proper diagnosis of HT, several methods are involved, further, different biomarkers are assessed independently or in combination with each other [4]. Mainly, serum, ultrasound and pathological examinations are considered how HT diagnostic methods [4,32]. Current research has reported the global prevalence of diagnoses of HT according to different diagnostic methods [32]. Moreover, data about the prevalence of methods useful to confirm HT diagnosis have been provided [32]. Therefore, HT is prevalently diagnosed by ultrasonography (13.2%) and pathological examination (12.5%) [32]. When serum autoantibodies profile is considered, the prevalence rate of HT diagnosed stands at 7.8% (see Figure 9 in Ref. [32]) [32]. The combination of two methods, including serum antibody titres and color Doppler ultrasound, is used for HT diagnosed with a prevalence of 10.4% [32]. This prevalence is considerably lower (4.7%) if three methods such as autoantibody titres, color Doppler ultrasonography and fine needle aspiration are associated. To confirm HT diagnosis is prevalently used thyroid tissue alone (14.1%) [32].

1.3. Molecular biotechnologies (MB) and HT

MB are the pivot for new biomedical methodologies because of their capability of revealing molecular pathogenetic pathways as well as genetic susceptibility of population to develop AIDT [6]. Above all now that use of genetic analysis is turning out to be a key tool for clinical genomic investigations owing to its high accuracy, reproducibility, and reliability of results.

The effective clinical application of MB can be assessed in accordance with the advice of qualified clinical trial studies (CTSs) [37]. These investigations are the basis of genomic screenings designed to detect viral genetic material involved in pathogenesis of diseases. That too, but especially CTSs can test how well genomic screenings work to identify susceptibility to develop diseases in subgroups of populations belonging to a specific continent.

Molecular alterations occurring in the context of HT play crucial roles to promote cellular proliferation of both lymphocytes and glandular tissue. Indeed, mucosa-associated lymphoid tissue (MALT) lymphomas can originate to the site of HT [38,39]. On the other hand, it is long since HT is reported concurrent with cancerous follicular lesions such as nodular goiter, adenoma, and carcinoma [39,40,41,42]. MB are currently employed on pathotyping of MALT lymphoma [43,44]. Further, these analyses are applied in dubious diagnoses of thyroid glandular cancerous lesions [45]. Mainly, these are part of thyroid innovative medicine that point trough biomarkers to early molecular diagnose, personalized treatment, prediction of cancerous risk and prognostic information [46].

Two were the main objectives of the study: firstly, to perform a systematic analysis of CTSs conducted on HT populations living at different geophysical latitude (HT-CTSs). This was done to establish the frequency by which these CTSs were concluded in different continents and when they were planned. Secondly, to identify samples in which MB were applied.

Therefore, wide-ranging search was conducted on CTSs provided at https://beta.clinicaltrials.gov/ web site through the files covered by “autoimmune thyroiditis Hashimoto” keywords [47]. To follow, some of these findings were selected as they were referring to HT-CTSs that planned to apply molecular technologies (mHT-CTSs).

In the context of hygiene hypothesis (HH), divergences among geographic diffusion of HT and molecular fingerprint of HT patients were also considered.

Current applications of MB for pathological practices were discussed separately. Mainly, these concerned molecular aspects for diagnosis of malignant thyroid lesions associated with HT.

2. Material and Methods

2.1. Data Sources

A systematic review of CTSs for HT was performed by surveying all results of search for terms “autoimmune thyroiditis Hashimoto” at place namely, “Condition or disease” of https://beta.clinicaltrials.gov/ web site [47].

2.2. Study Selection

A number of 75 CTSs was found for these keywords that included also three synonyms of conditions or disease such as “autoimmune thyroiditis”; “thyroiditis Hashimoto” and “Hashimoto” [47]. Mainly, 29 related terms were found of which ten pertained to “autoimmune thyroiditis” synonym (Hashimoto; thyroiditis autoimmune; Hashimoto Disease; HASHIMOTO THYROIDITIS; Hashimoto's thyroiditis; Hashimoto's Disease; chronic thyroiditis; Hashimotos Disease; Chronic lymphocytic thyroiditis; Lymphocytic thyroiditis); ten to “thyroiditis Hashimoto” (Hashimoto; Hashimoto Disease; Autoimmune Thyroiditis; HASHIMOTO THYROIDITIS; Hashimoto's thyroiditis; Hashimoto's Disease; Chronic lymphocytic thyroiditis; chronic thyroiditis; Lymphocytic thyroiditis; Hashimotos Disease) and nine to “Hashimoto” (Hashimoto Disease; Autoimmune Thyroiditis; HASHIMOTO THYROIDITIS; Hashimoto's thyroiditis; Hashimoto's Disease; Chronic lymphocytic thyroiditis; chronic thyroiditis; Lymphocytic thyroiditis; Hashimotos Disease) [47].

2.3. Inclusion criteria

Two were the inclusion criteria adopted for this investigation. The enrollment of HT patients was designed as the first criteria to select CTSs, whereas the molecular analyses were used as the second.

In the first instance, the above CTSs were scrutinized to enucleate the full set of trials carried on HT population. Secondly, to assess the effective application of MB, “Study Plan” section was analyzed for all CTSs [47]. Here, there were details on how a single CTS was planned and what the study was measuring. Specially, “Outcome Measure” sub-section provided insight into the use of molecular analysis to realize the aim of CTS.

2.4. Exclusion criteria

By reviewing and studying the 75 CTSs, 30 of them were considered irrelevant. Mainly, five CTSs were eliminated from this study because of they did not recruit participants with HT: i.e., HT was an “Medical Subject Headings” term or a collateral effect to a therapy (NCT05077865, NCT04239521, NCT04349761, NCT05680376, NCT03957616).

Twenty-four CTSs remained outside because of these concerned other autoimmune, inflammatory, or lymphocytic diseases (NCT03872284, NCT04823728, NCT05225883, NCT03993262, NCT05177939, NCT04339205, NCT04175522, NCT03530462, NCT03835728, NCT03542279, NCT03004209, NCT05198661, NCT04106596, NCT04708626, NCT01456416, NCT04875975, NCT05280600, NCT05682482, NCT05605223, NCT05503264, NCT03941184, NCT05422664, NCT05711563, NCT05772611).

One’ of CTS was omitted because of “Hashimoto” keyword indicated the location of study (NCT04339127).

2.5. Data Extraction

According to the first inclusion criteria, data extraction was performed. Basically, 45 CTSs were extracted for evaluation and included in this study because of they had effectively investigated on HT populations (Table 1). These HT-CTSs were entered into systematic analysis by evaluating seven variables such as continental and geo-location, start date, primary completion date, completion date, last verified and conclusion of study (Table 1).

2.6. Data Synthesis

According to both inclusion criteria, data were synthetized. Finally, 6 mHT-CTSs were recorded because of they had scheduled to use MB to realize their aims (Table 2). To evaluate each mHT-CTSs, six additional variables were added to previous seven. These were corresponding to: target sequences, analysis, and methods, biospecimen genetic retention and description, type and model of study, time perspective, and enrollment of subjects, respectively. Responsible party and results overview were shown, too (Table 2).

3. Results

3.1. CTSs conducted on HT population.

Forty-five CTSs enrolled HT patients. Thirty-seven of them provided information about geolocations by specifying where studies have been conducted (Table 1). In fact, there were not items in 17.7% of HT-CTSs (Table 1).

HT-CTSs were geographically assigned to four continents with different distribution. Then, 0.04% of HT-CTSs were conducted in Africa, 15.5% in America, 13.3% in Asia and 48.8% in Europe (Table 1).

In Africa, HT-CTSs were planned between 2011-2018. In America, HT populations were listened in CTSs since 2006, whereas, in Asia from 2011 onwards. In Europe, the first CTS on HT population was arranged in 2004 (Table 1).

Both HT-CTSs planned in Africa were completed (Table 1). Out of a total of 7 HT-CTSs mapped in American continent, around 85.7 per cent were completed. Conclusions were found only in one of 6 Asian HT-CTSs (16.6%) and in fourteen of 22 European HT-CTSs (63.6%) (Table 1).

These data indicate HT-CTSs specially provide large amount of information about populations living at European latitude. This is due to the hugest number of planned and concluded HT-CTSs in Europe in respect with other continents.

3.2. Clinical application of MB in HT-CTSs

In the list of mHT-CTSs, six trials were included. Two of them (33.3%) were finalized to display viral sequences. For the four remaining HT-CTSs, two were designed to identify bacteria and two to set genetic polymorphisms to associate with susceptibility for HT (Table 2).

Two DNA viruses were investigated from mHT-CTSs in the French population: these corresponded to parvo and polyoma viruses (Table 2). Both viruses were identified by polymerase chain reaction (PCR) method. Specially, genetic strands of polyomavirus were detected in different biospecimen such as blood, urine, and thyroid tissues. Based on study model, the spread of parvovirus was screened through an observational study (NCT03114267). Conversely, polyoma virus was approached by an interventional CTS (NCT03103776). A cohort model with retrospective analysis was followed for the observational study. Contrariwise, a model of parallel assignment was assigned to interventional HT-CTSs (Table 2). Consequently, both mHT-CTSs were planned to have knowledge about viral pathogenesis of HT by PCR analysis. However, NCT03114267 CTS investigated viral cause and effects by longitudinal analysis that evaluated retrospectively the outcome in HT population. Differently, NCT03103776 CTS investigated viral cause and effects on several populations affected of autoimmune diseases among which also an HT population.

The mHT-CTSs investigating bacteria aimed to identify microbiotas. Oral and fecal microbiota were examined in the Chinese population by measuring PCR sequencing of 16S rRNA gene (Table 1 and Table 2). Human feces were used to pick up microbiota genetic materials for the investigation namely NCT03390582 (Table 2). Both mHT-CTSs were observational studies; however, oral microbiota was evaluated by a case-control study whereas, fecal microbiota through a cohort study (Table 2).

Among mHT-CTSs programmed for interception of HT susceptibility, NCT00958113 investigation was performed in Colorado (USA); whereas NCT02491567 CTS was set for the Greek population (Table 1 and Table 2). DNA was examined on biospecimens such as saliva and blood leucocytes. Both mHT-CTSs pertained to observational, case-control studies with cross-sectional examination.

4. Discussion

HT may appear through different clinical and histological aspects and thus, morphological and serum diagnosis of HT are not interchangeable [4]. In addition, HT may be associated to benign and malignant follicular lesions as well as lymphomatous proliferations [39,42,43]. MB are promising surveying methods to apply on HT population.

Totally, 75 CTSs were examined in this study to assess the effective clinical use of MB for planning of trials. By examination of mHT-CTSs is emerged that MB have been employed for two unique scopes. Firstly, to reveal infective etiopathogenesis of HT; and secondly, to determine molecular fingerprinting of HT in populations. Mostly, in this investigation were isolated four trials in which clinical applications of MB served to display viral or bacterial genomes. This is demonstrating how these methos are functioned properly for exploring the complexity of infective HT pathogenesis.

Viral and bacterial infections are currently involved in HT pathogenesis, by multiple and often intertwined pathways.

Based on the old Th1/Th2 paradigm, the so-called hygiene hypothesis (HH) has been adapted to infective etiology of AIDT at the end of the last century [48,49,50,51]. Briefly, this hypothesis postulates that early infections in childhood protects against establishment of autoimmunity. [48,51,52,53,54]. Further, a reduced exposures to microbial environment in childhood is considered as element conducive to increase of autoimmune diseases in adults [55]. This is because of immune system educated by pathogens exposition may better suppress autoimmunity. However, the extension of HH to support of HT pathogenesis has not reported complete agreement [51].

Closely related to HH there are socio-demographic profiles of HT population, data come from migration survey and biographic info of HT patients.

By different concentrations, HT subjects are geographically distributed on the continental territories. A geographical map created on the bases of demographic observations reveal higher concentrations of HT subjects in Africa and Oceania (Figure 1) [32]. On the bases of socio-demographic observations, two divergent findings have been recorded. In low- and middle-income countries, the highest prevalence of HT patients is found among low-middle-income subjects (11.4%) (see Figure 8 in Ref. [32]) [32]. However, HT patients are prevalently concentrated in high-income countries. [32]. Therefore, the HH pathogenetic concepts can be applied to the last phenomena, whereas the first evidence seems limited only to infectious etiology of HT.

For over fifty years, surveys on transmigration of populations are persistently reporting that subjects migrating from a country with low incidence of autoimmune disorders develop immune-related diseases by the same frequency of the original inhabitants of the host country [52,56,57,58,59,60,61]. These data suggest an environmental effect at beginning of autoimmune diseases.

By reporting biographic info of HT patients, several investigations have focused a surprising association occurring between birth month of individuals and HT. Mostly, HT patients were born in winter and autumn [62]. This data suggests that cold weather protect against TPO-Ab development [63]. Nevertheless, this evidence is consistent with infective etiology of HT due to the abundant spread of infectious agents in winter. Further, these findings support HH because of children born in winter have early exposure to infectious agents facilitating the development of autoimmune disease. However, moving from these premises, it is possible even to affirm that incidence of HT for the individual subject may be predicted based on birthday information. Summing up these phenomena, HH seems jarring with genetic features observed in autoimmune disorders, especially in HT.

Molecular analyses have mapped on the short arm of chromosome 6 (6p) a super-region of 7.6 Mb including the extended major histocompatibility complex (eMHC) [64,65]. This region lengthens telomerically from RPL12P1 to HIST1H2AA and it is composed by six clusters and six super-clusters [65]. At 6p21.3 of eMHC are localized human leukocyte antigen (HLA) genes that are highly polymorphic. HLA expressions are strongly related to infection, immunity, and inflammation [66].

In HT, genetic polymorphisms of HLA changes depending on ethnicity [67]. This is because of different expressions of haplotypes in Caucasians (DR3, DR5, DQ7, DQB1*03, DQw7 or DRB1*04-DQB1*0301) in respect with Japanese (DRB4*0101, HLA-A2, DRw53) and Chinese (DRw9) HT patients [67]. Together, these data suggest that non-genetic factors trigger on onset of autoimmune disorders through an unidentified genetic background that is common to entire HT population. Therefore, among phases composing HT pathogenesis, genetic individual susceptibility enters at a later stage in respect with environment factors.

Genetic disparities of HLA profiles are established through use of molecular techniques. These methods have the advantage of arranging systematically HLA haplotypes by symbols. The complexity of nomenclature of HLA haplotypes has been organized by multiple molecular techniques [68]. The first molecular approach to display HLA alleles concerned application of Sanger sequencing-based typing (PCR-SBT) methods [68]. High-throughput sequencing (HTS) methods, including next-generation "short-read" (NGS) and third-generation "long-read" sequencing methods, are the natural evolution of PCR-SBT. Lastly, Oxford Nanopore Technology MinION is progressively reorganizing the number of HLA alleles [69]. Genotyping investigations on Graves’s disease (GD) have identified novel HLA alleles through high-resolution NGS [70,71]. Further, the use of methods based on machine learning are useful to predict HLA subtypes in GD [72]. These investigations suggest of matching different medical biotechnologies to better explain pathogenetic stages involving HLA haplotypes for development of autoimmune disorders.

By focusing on available molecular sources for CTSs appears that parvo and polyoma viruses were investigated from mCTSs.

The role of viruses in inducing HT has been explored but it is still not completely determined [51,73,74]. New data are coming up about roles of DNA and RNA viruses to trigger HT [75,76,77]. DNA viruses namely, parvovirus 19 (B19V), human hepatitis C virus and human herpes virus-6 have been associated to viral pathogenesis of HT [75,76,77,78,79,80]. Among RNA viruses, human immunodeficiency virus (HIV) has been related with HT as it is able to activate the immune inflammatory response through IL-6 [81,82]. Specially, in HIV patients this cytokine plays an important role by orchestrating the inflammatory cascade associated with HT [82]. The importance of IL-6 has been recognized even in animal model of DNA virus infection. In fact, IL-6 amounts are incremented in lung tissues of naïve Balb/c mice that received parvoviruses [83].

Parvoviruses are widespread in different countries of American, Europe, and Asian continent. [77]. Among DNA viruses, parvoviruses display highest levels of replication and recombination [84]. These viruses can replicate autonomously or conversely, they recombine with a helper-virus to be perpetuated [84]. The International Committee on Taxonomy of Viruses (ICTV) has reported members of Parvovirinae family as small (~20 nm in diameter), icosahedral, non-enveloped viruses that have a small single-stranded DNA of 4–6 kb [85]. In 2020, the Executive Committee of the ICTV has approved a revision for taxonomic of the family Parvoviridae [86]. Although the definition to describe these viruses remained, genetic criteria used to demark members composing this family have been updated. The proposal criteria proceed from discoveries of new members of the family Parvoviridae through application of HTS methods. Basically, the classification based on the association with host has been abandoned because of these viruses infect phylogenetically disparate hosts (see Table 1 in Ref. [86]) [86]. In this family have been incorporated infectious agents for animals showing a host range large. In fact, this is enough vast to include many phyla ranging from primates, mammals, avian species to invertebrates [86]. Beyond this, the family Parvoviridae embraces pathogens for arthropods clades, namely arachnids of Chelicerata, that molecular clock estimates go back to marine fossils of the late Cambrian period [87,88,89]. In 1975, Cossart and colleagues detected for the first-time B19V in serum sample of subject screened for hepatitis B virus [90]. Thirty years later, Allander and colleagues discovered bocavirus 1 (HBoV1) in human sample of nasopharyngeal aspirates belonging to children with respiratory tract infection [91]. B19V1 may cause a widespread and self-limiting infections in children and adults, known as erythema infectiosum or fifth disease [92]. Both, B19V and HBoV1 are pathogens for humans and have been detected in cancerous thyroid cells and HT lesions [75,76,93,94].

B19V and HBoV1 exhibit a particular tropism for nuclear compartment. The host machinery for nuclear import of viral capsid is a critical step in the early phase of infection [95,96,97]. The capsid binding protein namely, cleavage and polyadenylation specificity factor 6 plays a dominant role in directing integration to euchromatin of HBoV1 and lentivirus HIV-1, too [95,96,97]. At later stages of infection, the replication of B19V leads to morphological changes of nucleus. These are due to spatial reorganization of chromatin that appears marginalized to the nuclear periphery by super-resolution microscopic examination [98].

In this investigation, MB have proved their worth for composing the future genetic makeup of individuals suffered from HT. This is because these methodologies were employed to disclose genetic susceptibility for HT in two molecular CTSs. Currently, several microsatellites have been proposed as significant elements to build up the molecular HT phenotypes. Specially, heterozygous genotype Arg/Pro of rs 1042522 located on TP 53 gene, polymorphism of IL-23R gene rs17375018, polymorphisms of IL-6 gene promoter (-572) C/G and IL-6 rs1800795 have been associated with HT susceptibility [99,100,101,102].

With the introduction of precision medicine in 2015, MB are considered instrumental to management of cancerous lesions [103]. Molecular medicine has a key role for diagnosis and treatment of thyroid cancers associated to HT by isolating molecular alterations in histological and cytological samples. On histological fragments, the application of MB concerns the diagnosis of MALT lymphoma that develops around the primary HT alterations (see Table 1 in Ref. [104]) [104]. Genomic dissections of lymphomatous cells are employed to reveal molecular phenotypes of MALT lymphoma.

5. Conclusion

Decades of biomedical research on polymorphisms of HLA have revealed many genetic regions associated with HT. However, epidemiological evidence related to HT diffusion cannot be fully explained by HLA genetic differences. This study sheds a light on the require of new linkage between MB and production of data on demographic events such as births and migrations. This is because HH has not yet been proven, this has been widely criticized but not clearly disproved.

Besides, in HT tissues are detected DNA viruses that cause mild manifestation of inflammatory disease but produce nuclear DNA damages. Therefore, DNA viruses have relevance on HT pathogenesis and would offer important opportunities to develop antiviral strategies able also to treat HT. Mostly, viral infections should be considered in future for the development and refinement of HT therapies to use as an alternative or in conjunction with hormone replacement.

Lastly, MB have enormous potential to promote precision medicine by development of robust biomarkers to use for diagnosis and personalized therapies.