Foreword

Thank you for choosing GlycanAge as a partner on your journey to promote healthy practices and establish longevity routines. GlycanAge is a science-backed test of biological age which, as of November 2022, has over 400 partners including leading longevity clinics, medical doctors and other specialists with diverse backgrounds (nutritionists, mental health coaches, personal trainers, and others). This guide is intended to help you understand and interpret the GlycanAge test results and prepare for common and sometimes challenging situations that you will encounter as a GlycanAge partner or advisor specialist.

The guide is separated into three sections:

1. General information and guidelines for doing the GlycanAge test;
2. Guidelines for understanding GlycanAge results and indexes;
3. Guidelines for consultations with your clients.

By becoming a GlycanAge partner specialist or advisor, you agree to follow general guidelines for interpretation of GlycanAge results. Please make sure to read carefully every one of these sections before taking on your first client and keep this document handy in case you want to consult it before consultations.

General information and guidelines for doing the GlycanAge test

This section covers general information about the GlycanAge test and its inventors, and contains guidelines regarding its use, testing steps, contraindications, and other frequently asked questions.

Who invented the GlycanAge test?

GlycanAge is a patented science-backed test that determines biological age by analyzing a unique profile of complex carbohydrates (glycans) attached to the most abundant antibody in our blood: Immunoglobulin G (IgG). The first study connecting IgG N-glycosylation pattern to aging was made available online in 2013, at the same time as the first Horvath's clock of epigenetic age, and it was published in The Journals of Gerontology in 2014 (1). The test patent was first registered by Genos Ltd, a private research institute founded by Prof. Gordan Lauc, PhD, with headquarters in Osijek and the main scientific facility in Zagreb, Croatia. The ownership of the patent was later transferred to GlycanAge Ltd, a UK-based biotech start-up (and Genos' sister company) that turned the scientific findings into the commercial GlycanAge test.

How does GlycanAge measure biological age?

GlycanAge analyzes immunoglobulin G (IgG) N-glycan profile using a method called capillary electrophoresis. IgG is the most abundant antibody in the human blood, whilst glycans are complex carbohydrates that get attached to the constant region of the IgG, affecting its structure and function as well as interactions with other cells and molecules of the immune system (2). Some IgG molecules also have glycans attached to their variable region which, for example, affects its binding properties to bacteria or cells (2). There are over 30 different N-glycan structures typically present on IgG molecules of every individual, but the ratios of different structures differ depending on age and health (1, 2).

What is the age limit for doing the GlycanAge test?

GlycanAge is intended for adults aged 20 to 80. It should not be done in children since their IgG glycosylation profile very much differs from adults', which is also why 20 is set as the lowest measurable biological age. Our baseline of biological aging is adjusted to the age limit of 80 which is also the highest biological age the test can measure. Whilst an obvious limit to the test, most individuals who live to extreme older ages only do so with less inflammation than an average individual (3) and as such, delay or escape the common diseases that associate with aging (4). Hence, in such individuals, one can expect to observe glycan profiles as seen in chronologically younger population. Individuals over the age of 80 can do the GlycanAge test should they wish to. However, for anyone aged 80 or above, the test is imprecise as it can only measure lower, but not higher biological age.

Are there any risks and contraindications to doing the GlycanAge test?

GlycanAge is a simple and safe pin-prick test that requires only four drops of blood, and if instructions are followed carefully, it does not pose any danger to the client. However, individuals suffering from bleeding disorders such as hemophilia and von Willebrand disease, as well as those on anticoagulant therapy for blood clotting disorders, should consult their family physician or chosen specialist before taking the test. Recent surgery, blood transfusion or severe case of an infection (e.g. sepsis), are some examples of contraindications to testing. In such cases, individuals should allow 2 to 3 months of recovery before testing their GlycanAge.

Why is the GlycanAge test done from blood?

GlycanAge is based on the analysis of glycans attached to immunoglobulin G. The function of IgG is to defend our body from foreign and dangerous substances through activation of various immunological pathways, mistakes of which can also lead to overactivation and autoimmunity (5). Glycans attached to IgG can change the effector function of IgG and mediate pro-inflammatory or anti-inflammatory type of reactions (6, 7). Both functions are needed for a strong immune system, but the balance between pro- and anti- inflammatory state is necessary for homeostasis and will ultimately determine the level of chronic inflammation, the extent of molecular damage to cells, and consequently, the rate of inflammaging that drives the biological age (8).

How does GlycanAge sample collection look like?

The GlycanAge test kit is designed to be a simple finger prick testing tool used at home. The kit contains sterile wipes, lancets for skin puncture, plasters, a blood collection card with a desiccant, a plastic bag and a return envelope. Prior to testing, rings and tight bracelets should be removed from the sampling hand, and sleeves unbuttoned. After warming up the extremities by rubbing the hands together or holding them underneath a warm stream of water for a few seconds, the skin of one of the ring fingers should be cleaned with an alcohol wipe and left to dry. A single lancet puncture on the side of the ball of the chosen ring finger should be enough to gather four drops of blood (one per circle), but if needed, the procedure can be repeated on a different finger of the same hand or on the other hand. If blood drops collected in each circle are less than 0.5cm in diameter, it is advisable to collect additional drops within the same circle, making sure that the edges of blood drops in each circle do not overlap. Once collected, samples need to air dry for 2 to 6 hours, after which are to be closed and sealed in the plastic bag provided.

Are there any critical steps in GlycanAge sample collection that can affect test results?

Sealing the sample in the bag provided after drying is crucial for sample stability. The exact duration of drying will depend on the room temperature and air moisture, but it is generally accepted that leaving the sample unprotected for longer than 8 hours after testing will have a negative effect on sample stability. Only sealing the sample immediately after drying will ensure result reliability.

My client was recently sick. Can this affect their GlycanAge?

Due to a relatively long half-life of IgG (9), markers of inflammaging as measured by IgG N-glycans are much less sensitive to acute diseases and conditions in comparison to C-reactive protein (CRP) that has a half-life of a couple of days only (10). Mild and short-lasting acute infections don't affect the entirety of the IgG N-glycome, but some severe acute diseases can affect IgG glycosylation (11) and consequently GlycanAge as well. For example, if your client suffered from a severe pneumonia, severe COVID-19 syndrome (12), or other severe acute infection complicated by sepsis, their GlycanAge might reflect such pathological states for up to 3 months following resolution of the infection. The best recommended course of action in such cases is using the GlycanAge test once full recovery has been achieved, typically after 3 to 6 months.

Are there medications and surgical procedures that can interfere with the GlycanAge test results?

Certain medications and surgical procedures can affect IgG N-glycosylation patterns. For instance, weight loss as a result of bariatric surgery (13) and hormone replacement therapy (unpublished data) are expected to affect GlycanAge test results. However, if therapy taken is long-term and/or permanent, this is less likely to affect GlycanAge test results. To utilize the GlycanAge test to the fullest and assess efficacy of a medical or surgical intervention, it is recommended that GlycanAge is done prior to and redone 3 months after the intervention. If your client has received a one-off blood transfusion or gamma globulin blood derivatives, such as IVIG (14), a minimum of 3 to 6 months should be allowed between the treatment and the GlycanAge test.

Can recent vaccination affect the GlycanAge test results?

Though vaccination can and does affect IgG N-glycosylation, studies published to date looking at the SARS-CoV-2 vaccination (15) as well as influenza and tetanus vaccination (16) revealed IgG N-glycosylation differences on antigen-specific antibodies, with the total IgG1 N-glycosylation remaining unaffected (16). Since GlycanAge analyzes total and not antigen-specific IgG N-glycans, vaccination is not expected to affect the GlycanAge test results.

Can pregnant or breastfeeding women do the GlycanAge test?

Pregnancy, postpartum recovery and breastfeeding are life stages involving many changes to the female body that will affect your client's IgG N-glycosylation (17) and consequently their GlycanAge result. Bearing this in mind, it is possible to do the GlycanAge test during pregnancy, but it will capture pregnancy-related changes rather than the typical, chronic low-grade inflammation. Hence, in order to find out the true biological age, the test should be repeated after the end of pregnancy and upon re-establishment of menstrual cycle.

How do different life stages affect GlycanAge results?

There are different life stages that can affect GlycanAge results. Firstly, childhood is a period of growth and maturation of the immune system and is therefore characterized by a changing profile of IgG N-glycosylation that differs from the typical profile of an adult (18). In women, GlycanAge is strongly affected by sex hormones (19). Thus, the above mentioned states (pregnancy, postpartum recovery and breastfeeding) produce atypical GlycanAge results, warranting that the test is repeated once physiology of the menstrual cycle is restored. When a woman reaches perimenopause, she experiences a period of rapid change in the IgG glycome (20) that manifests as a rapid increase in her GlycanAge. On the other hand, hormone replacement therapy (HRT) seems to be able to halt the progress of and even reverse biological age (unpublished data).

Does the menstrual cycle affect the GlycanAge test?

Due to a long half-life of IgG, under normal circumstances, the menstrual cycle should not (and according to some preliminary data does not appear to) affect IgG glycosylation and thus should not have a significant effect on GlycanAge (21). Menstrual cycle-associated pathologies and states such as endometriosis and polycystic ovary syndrome would in theory be expected to significantly affect GlycanAge in a cycle-dependent manner, but scientific data tp support (or oppose) this is lacking.

Does menopause affect GlycanAge?

Since GlycanAge is affected by sex hormones (19), perimenopause is accompanied by a period of rapid change in IgG glycans (20). Consequently, women in perimenopause experience a sharp increase in their biological age, sometimes in a period of only several months. In menopause, women continue to have a higher GlycanAge. Higher biological age in menopause, as mentioned above, can be challenged bioidentical HRT (unpublished data).

Is it possible to do the GlycanAge test in transgender individuals?

Though data is yet to be collected on IgG N-glycans in transgender individuals, it has been shown that estrogens regulate glycosylation of IgG in both women and men (22), and it would therefore be reasonable to assume that a change in sex would affect GlycanAge, though it is not known how and to what extent. While the absolute biological age will be affected, GlycanAge may still be used in transgender population to determine the relative effect of lifestyle or medical interventions, as long as the sex hormone levels are controlled and stable.

How often can my client repeat their GlycanAge test?

Since the GlycanAge test is as simple as a regular blood test, there are no limitations as to how many times it can be repeated. What limits the frequency of testing, in theory, is a relatively long half-life of IgG of several weeks. In reality, it is the intervention in question and its effectiveness that should guide the frequency of testing. For instance, while a moderate change in diet might have an effect on GlycanAge after 6 to 12 months following implementation, a pharmacological intervention, such as bioidentical HRT, may produce an impact after as little as 3 months of treatment. Therefore, we would generally recommend allowing 3 to 6 months before retesting once a lifestyle or pharmacological intervention has been implemented.

How does GlycanAge compare to epigenetic and other biological age tests?

There is no standard definition of what biological age means, and therefore, different aging tests use different biomarkers of aging or different aging clocks. This has resulted in the development of different biological age clocks based on different biological aging theories (e.g. inflammaging, changes in methylation, telomere shortening). Such tests may provide different biological age values, depending on specific parameters measured. The GlycanAge test is glycan-based biological age clock derived from age-associated IgG N-glycans (1), central molecules of the adaptive immune system. In comparison to Horvath's and Hannum epigenetic age clocks, GlycanAge has associations with a number of chronic diseases, as well as disease risk factors such as body mass index (BMI), C-reactive protein (CRP), forced expiratory volume (FEV1) and systolic blood pressure (23).

Guidelines for understanding GlycanAge results and indexes

This section is dedicated to the basic scientific principles underpinning the GlycanAge test that you ought to be familiar with. Before becoming a GlycanAge consultation specialist or partner, you must meet the minimum education requirement on IgG glycans and the test itself. This involves a 40 minute educational call with one of the GlycanAge scientists as an introduction into the world of glycans. That will however, not cover everything you need to know in order to provide a high-quality result interpretation. As a GlycanAge consultant or consulting partner, you will be required to keep up-to-date with progress and science behind the GlycanAge test. All of the lectures intended for our partners and specialists are made available on our partner platform, whilst the most important aspects of result interpretation are covered here. Before going into the specifics of GlycanAge results, the following section introduces important aspects of the GlycanAge test that you should be familiar with before commencing consultation work.

What is IgG N-Glycosylation?

The GlycanAge test is a patented science-backed test that analyzes immunoglobulin G (IgG) N-glycan profile using capillary electrophoresis. IgG is the most prevalent antibody in the human blood and glycans are complex carbohydrates that get enzymatically attached to the IgG in the process of secondary modification of the IgG protein backbone – this process is referred to as glycosylation. Glycosylation is controlled by many enzymes, mainly glycosidases and glycosyltransferases that participate in glycosylation machinery forming complex carbohydrates in a series of steps. An example is an enzyme called galactosyltransferase that is found in the Golgi apparatus and regulates addition of a glycan building block called galactose (24). While DNA serves as a template for mass protein production, there is no simple template for glycan production. Instead, glycosylation is controlled by a complex network of different genes and their epigenetic regulation that affects gene expression, as well as by other factors such as substrate availability, enzyme activity and location within the organelles (25). IgG glycosylation occurs within B cells where IgG is produced, and it is modified by different stimuli that B cells receive during their activation and differentiation (26). Additionally, depending on the molecule (e.g. proteins, lipids, RNA) and the site to which glycans bind glycosylation may occur in different ways, but in humans two main glycosylation processes are distinguished: N-linked glycosylation (linked to amino acid asparagine, as occurs on IgG protein) and O-linked glycosylation (linked most frequently to amino acid serine or threonine, as part of the glycocalyx) (25). All IgG molecules have an N- glycosylation site in their constant region, which affects their structure and function, as well as interactions with other cells and molecules of the immune system. Some IgG molecules also have glycans attached to their variable region, which affects their binding properties to bacteria and/or cells (2).

What is IgG glycome?

Glycome represents an entirety of all the glycans found in a single unit (e.g. human glycome is the entirety of all human glycans, B cell glycome is the entirety of all glycans found on B cells). There are over 30 different N-glycan structures present on IgG molecules of every person, but the ratios of different IgG glycan structures differ depending on age and health between different individuals. The collection of all IgG N-glycans is called IgG N-glycome, sometimes referred to as IgG glycome.

Glycation versus glycosylation

Glycans are one of the four basic building blocks of life. Due to their complexity and lack of tools that would make them easier to understand, they have been ignored until very recently even by the scientists and researchers working in glycoscience-related fields. As a GlycanAge advisor or partner, you are likely to encounter some misconceptions concerning glycans. The most frequent misconception is that IgG glycans are formed by attachment of free blood glucose. It is important to clarify that unlike glycation (a well-known example of which is a glycated protein HbA1c), glycosylation, which enables glycan production, is a process controlled by many enzymes. IgG glycosylation occurs within B cells and is modified by different stimuli received by B cells during their activation and differentiation (26), though there are also some reports about B cell independent sialylation in mice (27). This is very much unlike glycation that occurs in random, uncontrolled fashion and is not mediated by enzymes.

Building blocks of IgG N-glycans

IgG glycans are composed of different building blocks assembled into tree-like structures that have two branches (or arms) on top of an N-acetylglucosamine (GlcNAc) and mannose base, but they may also contain a third branch (arm) – bisecting N-acetylglucosamine (GlcNAc). 5 building blocks (i.e. 5 distinct simple carbohydrates) that are encountered as part of IgG N-glycans in humans are N-Acetylglucosamine, core fucose, mannose, galactose and sialic acid (also called N-acetylneuraminic acid). All IgG N-glycans contain the same backbone that consists of two N-Acetylglucosamine molecules and three mannose molecules. Additional carbohydrates may get attached to this backbone. For example, one glycan structure might have two galactoses, one sialic acid and a core fucose on top of the common backbone, while another glycan might have no additions at all. To help group and classify different glycans, we separate them into groups with similar structural properties. Glycans that contain no galactoses are called agalactosylated glycans (G0), glycans with one galactose are monogalactosylated (G1), and glycans with two galactoses are referred to as digalactosylated glycans (G2). Glycans with at least one sialic acid are called sialylated glycans (S), those with a core fucose are fucosylated glycans (F), whilst those with a third middle arm are bisected glycans (B).

Changes in IgG glycome associated with age

After years of research and hundreds of published papers, the original research publication that found an association between IgG N-glycosylation, aging and health was made available online on Bioarchive server in 2013 (at the same time when Horvath's epigenetic test was published). This publication reported a distinct pattern of IgG glycan trends during aging and recognized two distinct groups of glycan structures: structures associated with aging and pro-inflammatory states whose proportion increases with the advancing chronological age (agalactosylated glycans G0, bisecting GlcNAc B), and structures whose proportion decreases with the advancing chronological age and ill-health, thought to be mainly anti-inflammatory (digalactosylated glycans G2, sialylated glycans S). Other glycan structures (monogalactosylated G1 and fucosylated F) are not as strongly associated with aging, though they do show association with general health. Structures most strongly associated with aging (found among G0, G2 and S glycans) together make the glycan clock of aging — the GlycanAge test.

Changes in IgG glycome associated with ill-health

Aside from aging-related changes in biology, GlycanAge is associated with well-known clinical biomarkers of health including waist circumference, BMI, HbA1C, triglycerides, blood glucose, calcium, D-dimer, cholesterol, low-density lipoprotein cholesterol (LDL-C), uric acid, fibrinogen, insulin (1) and CRP (28). Many diseases and conditions (including, but not limited to inflammatory diseases, alloimmune diseases and autoimmune diseases), show abnormal IgG N-glycosylation, often characterized by a low level of galactosylation (29). Such changes in galactosylation are mostly observed as an increase (high level) in agalactosylated glycan structures (G0), and a decrease (low level) in digalactosylated (G2) and sialylated (S) glycan structures. Additionally, decreased sialylation (S) and increased bisecting GlcNAc (B) associate with advanced age (1), as well as obesity (13). Interestingly, IgG N-glycosylation profile can turn pro-inflammatory years before the occurrence of any disease symptoms (29). For profiles of IgG N-glycosylation associations with diseases described by the existing scientific literature, please consult table 1.

Disease-specific changes in IgG glycosylation

GlycanAge measures the biomarkers of inflammaging that can be a trigger or a consequence of many different aging-related conditions. In fact, many different diseases show signatures of abnormal IgG N-glycosylation that we see with aging. Furthermore, pro-inflammatory changes in IgG glycosylation can appear years before any symptoms (29).

It is hard to pin down disease-specific glycosylation changes within IgG only, since chronic inflammation plays a role in the etiology of many diseases (30, 31). However, certain structures of IgG N-glycans show different trends of association with various diseases. For instance, in aging, autoimmune and infectious diseases, we would generally observe low sialylation and galactosylation, while in alloimmune diseases, high sialylation and galactosylation. On the other hand, some malignancies display high amounts of highly branched sialylated glycans, while others are characterized by low sialylation. A comprehensive list of IgG glycosylation and disease associations reported in published scientific literature to date is provided in table 1.

It is important to state that the changes in IgG N-glycosylation are a reflection of the changes in the immune system and not necessarily a consequence of ill-health. Chronic inflammation can appear years before the occurrence of any disease symptoms.

GlycanAge is a wellness test, not a medical or diagnostic tool, so when considering IgG N-glycosylation in the context of any disease process, other data (including but not limited to individual's personal medical history, family history, blood test results and lifestyle assessment) should be considered and carefully evaluated so that the best holistic care may be offered.

Global variability of human glycome

Different individuals show different profile of IgG N-glycosylation, but these differences are not random. A study from 2020 that looked at 27 different worldwide populations from Europe, Asia, Africa and Oceania, found that IgG N-glycosylation associates with country or residence, and has a strong association with the development index of a country. Those individuals living the developing countries had higher levels of chronic low-grade inflammation. Out of all analyzed glycan traits, it was monogalactosylation that had the strongest association with country of residence/development index (63).

Heritability and environmental effect on GlycanAge results

A study published in 2022 looked at the heritability of the glycan clock and the role that the environment plays in the glycan clock of biological age. The study concluded that heritability of the glycan clock of age was 39% on average, and that a majority of environmental contributions came from shared environmental factors (chronological age and molecular aging), while 14-19% were to do with unique environmental effects that may be attributed to lifestyle (64).

Understanding the GlycanAge test result

GlycanAge is a patented test that determines one's biological age based on the glycan clock (1). While there are many glycan structures on IgG, the GlycanAge test takes into consideration only those structures that were scientifically proven to have a significant association with age and age-related health. Application of a multivariable polynomial regression model results in the calculation of glycan-based biological age. Since glycosylation is affected by factors such as sex, geography and ethnicity, there are multiple baselines used for calculation of GlycanAge, most notably female and male baseline of adults aged 20-80. A client's GlycanAge signature is hence plotted to a baseline of population that corresponds to their chronological age, sex and the ethnicity baseline. It is also important to note that the GlycanAge biological age result is not calculated through a combination of all indexes presented in the report. It is, in fact, a separate measure taking into consideration only the structures that are most relevant for the glycan clock.

Understanding different indexes presented in the GlycanAge result report

In addition to GlycanAge, every client also receives results of five GlycanAge indexes in their personal report: Glycan Youth, Glycan Median, Glycan Shield, Glycan Mature, and Glycan Lifestyle. Indexes are generated by grouping the glycans with similar structural properties and weighing them against the same baseline of adults aged 20-80. Since some glycans contain multiple structural properties, the same structure can be included in multiple indexes. For instance, a structure containing a bisecting GlcNac, which is also monogalactosylated, is included in both Glycan Lifestyle and Glycan Median index. It is also important to note that described indexes provide an additional layer of information about analyzed IgG N-glycan groups, but are not necessarily components of the final biological age result. GlycanAge biological age result is not calculated through a combination of all indexes presented in the report; it is a separate measure taking into consideration only the structures that are most relevant for the glycan clock. A single glycan structure can be simultaneously included in multiple indexes and in GlycanAge. To sum up, indexes are generated by grouping glycans with similar structural properties together, while glycan structures used for the final GlycanAge calculation are grouped together based on their association with aging.

Glycan Mature

Glycan Mature is an index describing IgG N-glycan structures that are agalactosylated, meaning they do not contain any galactose units (these structures are sometimes also referred to as G0). Low level of galactosylation, and therefore an increased or high ratio of agalactosylated (G0) structures in the entire IgG glycome, is a hallmark of aging (1), as well as many diseases and states including autoimmune aand inflammatory diseases, cancers, cardiometabolic syndrome and others (29). Low galactosylation may precede the disease onset. This is best demonstrated in individuals with high agalactosylation (G0) who have a higher risk of developing rheumatoid arthritis (32). In light of reported disease associations to date, G0 glycans are often characterized as pro-inflammatory. It is important to note that since these glycan structures do not contain galactose, they cannot be sialylated (S) either, but might have some other structural features such as bisecting GlcNAc (B).

Insight into the relevance of G0 glycans for the immune system and disease pathogenesis:

A deeper mechanistic insight into the immune system pathways activated by agalactosylated glycans is still required. It was previously proposed that agalactosylated (G0) IgG N-glycans activate complement cascade, a danger-sensing part of the immune system that clears microbes and damaged cells from the body, though that proposition was not without the controversies (reviewed in 29). A more recent publication identified IgG4 agalactosylation as a trigger for complement activation via lectin pathway leading to unwanted cellular damage in an autoimmune kidney disease called primary membranous nephropathy (65). IgG4 is considered to be an anti-inflammatory and tolerance-inducing subclass of IgG antibody. However, in IgG4-related allergies and IgG4 autoimmune-related diseases such as primary membranous nephropathy, chronic inflammatory demyelinated polyradiculoneuropathy (CIDP), thrombotic thrombocytopenic purpura (TTP) and muscle-specific kinase myasthenia gravis (MuSK-MG), it seems to plays a pro-inflammatory role (66). Most publications exploring IgG N-glycosylation and disease associations report a high G0 in disease, with the exception of Parkinson's and some alloimmune diseases.

Glycan Median

Glycan Median describes a subset of structures that are monogalactosylated, meaning they have one galactose (also called G1). G1 glycans do not change with age (1) and therefore do not contribute to the final GlycanAge result. However, they have a shared genetic causal variant (pleiotropy) with many biomarkers, conditions and diseases including asthma, high-density lipoprotein (HDL), primary biliary cirrhosis, inflammatory bowel disease, rheumatoid arthritis and ulcerative colitis (67). Additionally, specific monogalactosylated (G1) glycan structures are predictive of future cardiovascular diseases and events (68), so a decreased Glycan Median index in women might be used as a hint to investigate their Glyco Cardio index (see a specific section dedicated to this index). A study published in 2020 found that monogalactosylation was strongly associated with an individual's country of residence – that is, 38% of G1 glycans variability was explained by the socio-economic development level of a country (63).

Insight into the relevance of G1 glycans for the immune system and disease pathogenesis:

A recent in-vitro study (69) found that IFN-γ, a cytokine that plays a role in viral response and whose overexpression triggers tissue-specific and systemic autoimmunity (70), significantly increases the proportion of monogalactosylated (G1) N-glycans. Increase in monogalactosylated IgG N-glycans in disease has been observed in some but not all publications to date (see table 1). It is important to note that in-vivo and in-vitro IgG glycosylation are not exactly the same. Hence, the mentioned finding needs to be taken with reservation.

Glycan Youth

Glycan Youth describes a subset of structures that are digalactosylated, meaning they have two galactoses (G2 glycans). These glycan structures are observed in high amounts in young and healthy individuals (1), and are thought to be anti-inflammatory. Again, it is important to note that many digalactosylated glycans will also have other structural features, such as the presence or absence of sialic acid or bisecting GlcNAc that can modify the end effect of such galactosylated structures on the immune system.

Insight into the relevance of G2 glycans for the immune system and disease pathogenesis:

One study performed in mice found that IgG1 Fc galactosylation is necessary for inhibition of pro-inflammatory signalling cascade by binding to the inhibitory receptor FcγRIIB (71). FcγRIIB is the receptor for IgG Fc domain that is involved in the maintenance of immune tolerance, and consequently, it is also involved in autoimmune disease susceptibility (72). Highly galactosylated IgG molecules have been reported to inhibit the pro-inflammatory activity of the complement component C5a, a fragment released upon cleavage of C5 complement component (73). Complement formation on cell membrane results in loss of water that can lead to death of the affected cells. Though this mechanism is required for defence against pathogens, an increase in the pro-inflammatory component C5a has been reported to contribute to a number of diseases, including systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, sepsis and cancer (74).

Contrary to these findings, several other studies report a pro-inflammatory effect of IgG galactosylation, especially terminal galactosylation – glycans that end with galactose and are not further extended by sialic acid(s). This occurs through enhancement C1q complement component and pro-inflammatory Fcγ receptors, both of which boost pro-inflammatory processes such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) (29). There are some diseases in which an increase of galactosylation in comparison to healthy controls has been reported. One example is fetal neonatal alloimmune thrombocytopenia (FNAIT) in which increased galactosylation is accompanied by increased sialylation and decreased fucosylation (75). Another example is Parkinson's disease in which neutral agalactosylated (G0) glycans are decreased, while neutral (non-containing sialic acid) monogalactosylated (G1) and digalactosylated glycans (G2) are increased in comparison to healthy controls (the latter change was not statistically significant, however). Most papers reporting on associations between disease and IgG N-glycosylation observe a decrease in digalactosylation (G2) in disease states (table 1) (29).

Glycan Shield

Glycan Shield describes structures that contain at least one galactose and are further extended with one or more sialic acids (S glycans). Sialylated IgG N-glycans represent the most complex structures present on IgG, and can also be characterized by other structural features such as bisecting GlcNAc and/or fucosylation. S glycans are highly present in young and healthy individuals, and are generally thought to be anti-inflammatory (1, 29).

Insight into the relevance of S glycans for the immune system and disease pathogenesis:

Sialic acid units are thought to exert an anti-inflammatory effect on the immune system by modulating interaction affinity between Fcy receptors and Fc region of the IgG. For instance, sialic acids lower the affinity for FcyRIIIa, a receptor on natural killer cells whose binding leads to antibody-dependent cellular cytotoxicity (ADCC) (76). ADCC kills antibody-coated cells and, though crucial for removal of the cells infected by viruses as well as tumor cells, it can also cause unwanted cellular damage (e.g. in autoimmune diseases (77)). In addition, there are some conflicting results pointing to the possible pro-inflammatory function of the sialylated N-glycan IgG Fc region that regards its binding to the C1q component of the complement and complement-dependent cytotoxicity (29). Nonetheless, most publications reporting on associations between diseases and IgG N-glycosylation, report a decrease in sialylation (S) in disease states when compared to healthy controls (table 1) (29). An exception to this rule seem to be some alloimmune diseases, such as fetal neonatal alloimmune thrombocytopenia (FNAIT),in which increased sialylation is accompanied by increased galactosylation and decreased fucosylation (75), as well as some malignancies (lung cancer (78), thyroid cancer (79), multiple myeloma (80)).

When it comes to more mechanistic insights on the effect of sialylation on disease etiology, one study performed in mice reported that a hyposialylated IgG (low S glycans) was enough to cause hypertension in mice, while supplementation with a precursor of sialic acid, N-acetyl-D-Mannosamine, breaks the link between hypertension and obesity (81).

Glycan Lifestyle

Glycan Lifestyle describes structures analyzed the GlycanAge test that contain bisecting GlcNAc (B glycans). Though it is affected by ageing, increased Glycan Lifestyle is also associated with signs of unhealthy lifestyle and/or certain environmental factors, including obesity (13), chronic obstructive pulmonary disease (COPD) and specifically COPD in smokers (51). Another study found that a decreased level of agaloctosylated glycans containing bisecting GlcNAc (G0, B) in middle age was an early feature associated with familiar longevity (families having at least two siblings living beyond 89 (men) or 91 (women) years of age) (82).

Insight into the relevance of B glycans for the immune system and disease pathogenesis:

Bisecting GlcNAc can trigger antibody-dependent cellular cytotoxicity (ADCC) through the enhanced binding of IgG Fc domain to FcγRIIIa (also known as CD16a), which is a receptor found on mast cells, macrophages and natural killer cells (83). ADCC is an immune system process responsible for killing antibody-coated cells, and it is crucial in removing the cells infected by viruses as well as tumor cells. In case of some autoimmune diseases, ADCC can, however, also cause unwanted cellular damage (77).

Glyco Cardio

Glyco Cardio is an index currently available in beta version as an add-on extension to the GlycanAge test in women only, iand it can be generated upon request. As the Glyco Cardio becomes commercially available, this information will be updated. Some of the monogalactosylated structures strongly associated with a future risk of cardiac events, and a poor Glycan Median may be a sign of an unfavorable Glyco Cardio index.

Using GlycanAge indexes in GlycanAge result interpretation

Though categorised as a wellness rather than a diagnostic test, the GlycanAge test and indexes can still provide useful insights into their client's overall health if users are familiar with limitations of the test. For instance, while Glycan Youth, Glycan Mature, Glycan Shield and Glycan Lifestyle indexes change with age (1), genetics and environment explain the inter-individual differences in monogalactosylated glycans (G1) which are presented in form of the Glycan Median index. For detailed information about every index, please read the chapters dedicated to individual indexes and refer to table 1.

Troubleshooting and getting support

As science moves forward, this guide will be periodically updated with the latest research findings relevant for the GlycanAge test understanding and interpretation. For early access to published research and preliminary results of ongoing research studies, make sure to attend GlycanAge educational webinars which will also be recorded and available to you at all times through your partner platform. For questions that are not covered in this guideline or in webinars, please contact our GlycanAge support team.

Guidelines for in-person and/or online consultations

This section is dedicated to in-person and/or online consultations intended to deliver the GlycanAge result and offer individualized lifestyle or health recommendations. In case of partner clinics where standard protocols for result delivery and health advice are already in place, it is recommended that the same approach used to deliver results of any prognostic or diagnostic test which might cause a strong emotional reaction is used upon GlycanAge result delivery as well. GlycanAge specialist video calls or in-person consultations should include an introduction, emotion management, personalized recommendations and a final (written) report.

Introduction

Start your consultation by introducing yourself, finding out what encouraged your client to test and offering a brief explanation of the GlycanAge test. Give some detail about your background and expertise.

Example:

Hi, my name is Jane Doe. I have a PhD in molecular biology and work as a head of research and development team at GlycanAge. Today I will be your GlycanAge advisor and reveal your GlycanAge result to you. Is this your first GlycanAge test? What was the main reason you decided to measure your biological age? Allow me to shortly introduce our test first. GlycanAge is a science-backed test that looks at complex sugar molecules that are attached to immunoglobulin G, the most prevalent antibody in our blood. These sugary extensions are called glycans, and they act as antennae mediating the conversation between our antibodies and the rest of the immune system, stirring either a pro- inflammatory ("attack") or an anti-inflammatory ("rest") signal. It is important our body is able to emit both signals. Our body should be able to both attack pathogens when they are present, but also keep the immune system in "rest" phase when danger is absent in order to prevent collateral damage.

Emotional impact of the GlycanAge test result

Receiving results of the GlycanAge test can be a highly emotional experience for your clients. Strong or negative emotional reactions may occur even when the GlycanAge test result is favorable (e.g. biological age is much lower than chronological age) and in situations when result is only partly unfavorable (e.g. GlycanAge is overall favorable, but one of the indexes is very unfavorable). Though data on psychological reactions to biological age tests is lacking, cases concerning other predictive tests have been described whereby even favorable results triggered strong reactions manifesting as disbelief, or loss of old and development of new beliefs about self in those with previously strong identity-related beliefs (84).

As the bearer of news, good or bad, you will often witness your clients' emotional reactions in real time. One of your responsibilities as an advisor or specialist will be to help your GlycanAge clients process the news in the first few minutes of the call/in-person consultation. A study published in 2017 concluded that distraction from stress was associated with better cortisol recovery after an acute stress episode, in contrast to denial and stress-devaluation (85).

Additionally, you will see that clients might have different coping techniques when faced with their GlycanAge result. Their socio-economic, cultural and educational background, as well as their personality, will affect how they react. While some clients might immediately seek rational explanation and actionable steps, others might experience sadness, denial or anger. A study about coping with grim news that come with genetic test result reveal found that knowing and recognizing stress response syndromes can help clinicians respond and determine when counselling is not enough, and when evaluation and psychotherapy for stress response might be needed (table 2) (86). Though biological age is not as deterministic as receiving results from genetic tests, it could still trigger a pathological stress response which should be recognized and addressed early. If you recognize severe or pathological stress response but do not have means or knowledge to address it, please inform our GlycanAge support team.

Observe your client's emotional reaction and if necessary, allow a few minutes for them to process the result. In order for this process to go as smoothly as possible, here are some tips you may find useful, as well as actions to avoid.

Do:

1. Mention GlycanAge statistics and stories as a means of distraction to reduce acute stress and regain client's sense of control. Example: Biological age can be changed with targeted interventions; on average, our clients reduce their biological age by six years, and we have many examples of individuals who managed to reduce their biological age by 15 or 20 years in one or two years of implementing lifestyle improvements. We had one situation in which our client changed their job...
2. Express compassion and support in regaining control over biological aging. Example: I am here to support you in this process and help you identify areas you can improve in.
3. Make sure your client is ready to process additional information. Example: Are you ready to review the rest of your results and discuss what you may do to improve, or do you want to clarify some things before we proceed?

Do not:

1. Increase acute stress by shaming. Example: With your BMI, GlycanAge this high is not surprising.
2. Increase acute stress by mentioning diseases. Example: It does not mean you will get cancer.
3. Promote denial or avoidance strategies through self-resolution or other means. Example: Everything happens for a reason. Everything will work out.
4. Devalue stress and/or stress-associated emotions. Example: How you feel is not objective. Your GlycanAge result shows that you do not have any chronic inflammation and you should not be disappointed with your result.
5. Give promises of success. Example: We will reduce your biological age in no time - you will be 10 years younger next time you test!

Individual recommendations

As a specialist in your own field, you are free and encouraged to use your own knowledge and expertise as long as it complies with GlycanAge general guidelines. To make sure your advice is up-to-date, please follow our monthly GlycanAge webinars. All the lectures are recorded and available on your partner platform. When providing individual recommendations:

Do:

1. Encourage science-backed lifestyle changes.
2. If needed, encourage your client to consult a specialist and/or consider further health testing.
3. Encourage taking another GlycanAge test to re-assess the success of lifestyle interventions when possible.

Do not:

1. Encourage giving up or changing any existing disease therapy if you are not their chosen physician for disease management.
2. Encourage significant lifestyle change in people with chronic illness without consulting their chosen healthcare specialist, unless you are a specialist in the relevant field.
3. Give advice based on rumors or scientifically unfounded ideas (e.g. crystals or astrology).
4. Give advice that ignores or goes directly against one of the GlycanAge verified IgG glycome findings.

Challenging interpretation situations with examples of GlycanAge case studies

As a GlycanAge specialist, you will most likely encounter some of the listed challenging situations with regards to the GlycanAge test interpretation. To help you provide an adequate response, these guidelines also provide case studies with known outcomes.

Example: Excellent GlycanAge result, poor Glycan Median index

A frequent situation that can be disturbing to the client is a case in which their GlycanAge is excellent, but their Glycan Median index is poor. Glycan Median represents a relative contribution of selected monogalactosylated glycan structures to the total IgG N-glycome. Though monogalactosylated glycans are mostly anti-inflammatory, they are not as tightly associated with advancing chronological age and age-associated health, which is the case with Glycan Shield, Glycan Mature and Glycan Youth indexes. Low Median index is not necessarily a poor result if combined with favorable Glycan Youth and Glycan Mature, as it can be a reflection of the fact that the vast majority of IgG glycans are digalactosylated and therefore even more anti-inflammatory. However, significantly lower monogalactosylation in combination with a higher bisecting GlcNAc has been observed in individuals with chronic obstructive pulmonary disease that can be caused by exposure to many forms of air pollution. In case of a suboptimal or poor Glycan Median, particularly when combined with suboptimal Glycan Lifestyle index and regardless of their overall GlycanAge result, exposure to air pollution should be considered, and if needed, spirometry should be recommended as a follow-up diagnostic test. An extremely low Glycan Median, particularly when combined with strong family history of cardiac events or cardiometabolic issues might be an indication for obtaining a Glyco Cardio index. Some of the monogalactosylated structures strongly associate with a future risk of cardiac events, and a poor Glycan Median may be a sign of an unfavorable Glyco Cardio. In the case study presented below, the client had a very favorable GlycanAge of 20 years, but a very poor Glycan Median and a family history of cardiac events. Such a result was an indication for generating a Glyco Cardio report which turned out unfavorable. Due to the poor Glyco Cardio index, this individual decided to visit a chosen cardiologist, and after a careful examination by the cardiologist, it was found that the client already had minor vascular blockages that could have posed danger in the future and required medical treatment.

Example: Excellent GlycanAge result, poor Glycan Lifestyle index

Glycan Lifestyle index represents a relative contribution of selected bisected glycan structures in the total IgG N-glycome. Bisecting GlcNAc is mostly pro-inflammatory, especially if the structures are agalactosylated as well. Structures with bisecting GlcNAC can also be galactosylated and sialylated, meaning they can have complex combinations of pro- and anti- inflammatory traits. A high (unfavorable) Glycan Lifestyle index, particularly when combined with an overall favorable GlycanAge, is not an immediate cause for concern. However, one possible explanation for such a result could be chronic obstructive pulmonary disease and connection to tobacco use, more specifically, cigarette smoking, since individuals with COPD/smokers tend to have a significantly increased amount of bisected IgG N-glycans, but not a significant decrease in digalactosylation (G2) and sialylation (S) (51). A study published in 2022 estimated that prevalence of COPD in the world in 2019 was 10.3%, and listed tobacco smoking, exposure to indoor air pollution, ambient air pollution and occupational pollutants as the leading risk factors for COPD (87). Urban environment with polluted air, the effect of indoor air pollution and passive as well as active exposure to air pollutants should be considered, and if needed, spirometry should be recommended as a follow-up diagnostic test, especially when both Glycan Lifestyle and Glycan Median are suboptimal regardless of the GlycanAge test result.

Example: Uptrending GlycanAge and/or poor GlycanAge result that does not seem to be improving after favorable lifestyle interventions

Consistently poor GlycanAge result despite favorable lifestyle improvements, especially in case of strong family history of a certain group of diseases and/or complex personal medical history, might indicate an undiagnosed underlying pathology. In such cases, it is recommended that advisors who are not medical experts themselves refer their clients to GlycanAge advisors who are medical specialists, their medical partner institutions or their client's chosen healthcare practitioner for further assessment. Additionally, in women who are in perimenopause, a rapid increase in biological age will occur regardless of the many positive lifestyle habits and changes, which has to do with sex hormone deficiency that occurs during that period in life (20, 21). In such cases, it is recommended your client is assessed by a (peri)menopause specialist.

Further recommended testing

In situations where there is a strong family history of a certain disease or a group of diseases combined with an unfavorable GlycanAge test result, please consider referring your client to a medical specialist for a health check-up, or if you are a medical specialist yourself recommend further testing relevant to your client's case. The same should be considered in cases where repeated GlycanAge test results do not show improvement after implementation of positive lifestyle changes. Some (but not all) laboratory (blood) tests recommended in such cases include:

• Full blood count
• Blood clotting tests
• Urea and electrolytes (renal function: Na+, K+, urea and creatinine)
• Bone profile (Ca2+, phosphate, ALP)
• Serum and intracellular Mg+
• Thyroid function (TSH, Total or free T4 or T3, anti-TPO antibodies)
• Liver function test (ALT, AST, bilirubin, albumin)
• HbA1c and/or fasting glucose, OGTT
• Lipid profile fasting or non-fasting (total cholesterol, LDL-C, HDL-C, triglycerides, total cholesterol: HDLcholesterol ratio, non-HDL cholesterol)
• Iron studies, B12, folate
• Stool or blood serum zonulin

Writing the conclusion

Partner clinics typically have their own format of consultation conclusion that their clients are familiar with and are encouraged to use those, provided they include specifics of the GlycanAge test. For GlycanAge specialists, the final part of the consultation will be writing the summary report. You are encouraged to create a template that you could later adjust to the individual needs of your clients. To help you create such templates, we are providing several examples of consultation conclusions.

Conclusion example 1

Dear XXX,

It was nice meeting you virtually.

Your result is: GlycanAge: XX,

Additional results of glycan indexes: Glycan Youth XX, Glycan Median XX, Glycan Shield XX, Glycan Mature XX, Glycan Lifestyle XX.

About your GlycanAge result:

Your biological age as measured by GlycanAge closely resembles your chronological age which is a good result. As we discussed, GlycanAge is a measure of chronic systemic inflammation, and is affected as much by genetics, as is by lifestyle choices, as well as natural passage of time. A good result (biological age being equal to or better than chronological age) indicates that anti-inflammatory glycans dominate in IgG glycoprofile and it is these that protect one against inflammaging which is a common denominator to a range of chronic diseases, as well as physiological changes associated with aging. Research done to date shows that GlycanAge associates well with prediction of high blood pressure and pre-diabetes incidence, as well as future risk of hospitalisation, performing better than the majority of other "omics" clocks.

Lifestyle:

It is great to hear that you have recently started implementing healthier lifestyle choices. The idea is to have a healthy relationship with food and exercise, amongst other lifestyle factors, and try to live a healthier life for longer. In order to keep inflammation level low and obtain such a good (or even better) GlycanAge next time you test, there is some room for improvement in your case which you can act on - details with advice are explained below.

Weight maintenance:

Keeping a healthy weight for height, i.e. achieving a normal body mass index and more importantly, healthy proportion of body fat (for a man your age, healthy body fat % is in the range from 8% to 19%), is an important factor in keeping your systemic inflammation levels low. Abdominal (tummy) fat correlates well with the risk of developing metabolic syndrome and I am happy you addressed it already with a 5-kilogram weight loss in the last month (which now puts you closer to a normal range weight for height). Do make sure to lose weight at not too quick a pace - up to 0.5kg weight loss per week is considered sustainable in the long run.

Diet:

Diet-wise, GlycanAge research showed that no single type of diet is the best one in all circumstances and applicable to all individuals. Inter-individual differences are to be blamed, though in a way that is good news as it allows our genetic and microbiome-related differences to stand out. What does apply to all individuals and helps with reducing systemic inflammation are 1) sufficient fibre intake and 2) avoidance of overly processed foods (sugary, savoury, pre-made meals and so on). Low-calorie diet seems to be associated with significant improvement across all GlycanAge indices in obese people. On the other hand, calorie restriction, particularly when combined with high intensity exercise, may have the opposite (negative) effect on inflammation, leading to a higher GlycanAge.

On the contrary, high intensity exercise coupled with sufficient rest and adequate nutrition favours a good GlycanAge. You will find fibre in plants (fruit, veg, seeds, spices, grains) and it is the variety of plants you eat a week (20+ and more) that counts the most. Since you are on a high protein diet, make sure to check where your meat comes from and try to choose the best quality, organic, lean meat. You may also find good protein sources in seafood, dairy and plants, too. Continue cycling in some carbohydrates as you already do, and try to include some available fermented foods (examples are kefir, soya-based products, kimchi, natto, fermented pickles, etc.) as these diversify your gut microbiome. Once you are back in the UK, I would highly recommend you go to your local bookshop and get a copy of Spoonfeed by Tim Spector. It is an insightful book on common diet myths, packed with evidence-based facts.

Exercise:

Your regime of alternate day high intensity training, and long walks coupled with gym-based training, is a good one, though pay attention to how your body reacts to your high frequency/high intensity workouts.

Overtraining may have a negative effect on your GlycanAge and thus it is important to incorporate adequate rest. Similar goes for intermittent fasting which seems to help with blood sugar control, weight loss/maintenance and bringing inflammation levels down. What it does for our body in the long run is unknown and so feel free to experiment and take a break from it every now and then if it feels too strenuous, particularly since you engage in a fairly strict regime of 16/8.

Habits and stress:

I am happy you sleep well, since sleep is one of the keys to a healthy immune system. Looking after your mental health should not be ignored either. A good way to work both on physical and mental health is spending time outside. It has been scientifically proven that spending just 2 hours per week somewhere in nature does us a lot of good (https://www.nature.com/articles/s41598-019-44097-3).

Additionally, have a think about whether you would benefit from addressing any emotions that do not have a chance to come out, since burying things deep down never does us any good. That could be something you could do on your own (e.g. through meditation or mindfulness) or through a chat (formal or less formal) with a professional. I think it is also important to address the fact that having a parent go through a cancer and chemotherapy journey is not easy at all and that it is important you have someone close to talk to about these things.

Well done for quitting smoking and keeping away from tobacco consumption - we see current as well as ex-smokers' GlycanAge results being negatively affected by smoking.

Further recommendations:

Moving on, I suggest you repeat your GlycanAge after you complete the biohacking plan that you are following at the moment. Since it is a fairly long lifestyle improvement plan, it might be a good idea to retest 3 to 6 months after you started, simply to assess how all the changes you are implementing are affecting your biological age. Remember, glycans do not change daily, but are rather stable for about 4 to 6 weeks, and change in response to a lifestyle intervention 3 to 6 months into it or sooner in case of pharmacological interventions.

I hope you found our consultation useful and that some of these insights will come in handy when thinking about lifestyle factors that affect your biological age.

Conclusion example 2

Dear XXX,

It was a pleasure meeting you virtually.

Your result is: GlycanAge: XX,

Additional results of glycan indexes: Glycan Youth XX, Glycan Median XX, Glycan Shield XX, Glycan Mature XX, Glycan Lifestyle XX.

About your GlycanAge result:

Your biological age as measured by GlycanAge is excellent. GlycanAge is a measure of chronic systemic inflammation, and is affected as much by genetics, as is by lifestyle choices, as well as natural passage of time.

A good result (biological age being equal to or better than chronological age) indicates that anti-inflammatory glycans dominate in IgG glycoprofile and it is these that protect one against inflammaging which is a common denominator to a range of chronic diseases, as well as physiological changes associated with aging.

Research done to date shows that GlycanAge associates well with prediction of high blood pressure and pre-diabetes incidence, as well as future risk of hospitalisation, performing better than the majority of other "omics" clocks.

Lifestyle:

Your healthy weight for height, active lifestyle, varied and balanced diet, adequate sleep and absence of any major chronic illnesses all contribute to your low level of systemic inflammation. Your positive lifestyle interventions have so far compensated very well for the loss of oestrogen over the last decade since your menopause onset. Bear in mind that genetics also influences your glycoprofile - indexes that most positively correlate with favourable genetics are Glycan Youth and Glycan Mature.

Life stages:

Lacking systemic oestrogen exerts numerous negative effects on the body, as you are aware. It negatively affects your mental health and cognitive function, makes your bones more fragile (causing osteopenia in your case), has a negative overall effect on your cardiovascular system and may potentially cause a range of debilitating symptoms (such as hot flushes, genital atrophy, urinary system issues).

We know now that oestrogen is crucial for glycosylation of the IgG antibody (which is exactly what we measure via the GlycanAge test) and lack of oestrogen will cause a shift to pro-inflammatory (bad) glycans. Physiologically, oestrogen levels drop suddenly when women reach menopause and this kicks off the process of accelerated aging.

The good news is that with replacement of oestrogen (and progesterone where indicated), biological age, as well as common menopausal symptoms, can and do improve. It is never too late to start HRT, though evidence suggests that women see the best progress and gain the most benefit if HRT is started within 10 years of menopause (or before the age of 60).

Nevertheless, even if started after those time points, most women do gain health benefits associated with HRT. As we discussed, given your relative lack of any major menopausal symptoms and a good quality of life overall, you may wish to think of starting hormone replacement therapy more as an investment into a longer healthspan in order to further optimise your health. HRT protects your bones, cardiovascular system, lowers LDL (bad cholesterol) and raises HDL (good cholesterol) (as per meta-analysis paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039020/).

There is also some speculation it could help prevent obesity and type 2 diabetes. As with any pharmacological drug, there are potential side effects and risks associated with HRT use, and thus these need to be weighed against potential benefits for each patient individually. Dr Newson is one of the experts in the field who would be able to advise you on all the details, go through the risks and benefits, and if deemed appropriate, find a good HRT combination for you. Testosterone replacement is increasingly being prescribed, too. It helps with brain fog symptoms as well as reduced libido, which are both relevant in your case. More info on HRT is available in the attached leaflet. It is great you got your baseline GlycanAge prior to potentially starting HRT. That way we could assess how being on HRT may affect your GlycanAge next time you test. Combining that with your own subjective perception of HRT (side effects, symptom improvement), you will have enough information to make further decisions.

You may use GlycanAge that way to assess efficacy of any lifestyle or pharmacological intervention. GlycanAge is stable for about 4 to 6 weeks, and changes in 3 months in response to a pharmacological intervention and 3 to 6 months in response to a lifestyle intervention (e.g. diet or exercise change).

Diet:

Diet-wise, GlycanAge research showed that no single type of diet is the best one in all circumstances and applicable to all individuals. Inter-individual differences are to be blamed, though in a way that is good news as it allows our genetic and microbiome-related differences to stand out. What does apply to all individuals and positively affects inflammation are 1) sufficient fibre intake and 2) avoidance of overly processed foods (sugary, savoury, pre-made meals...).

It is hence advisable to experiment with different diets and see which one fits you best, though in your case, your current diet seems to suit you and certainly plays a part in your great GlycanAge result.

There is a strong case for having a varied diet, since as a human race, we have historically relied on different food groups to sustain our calorie needs. Fresh fruit, vegetables and other plants are advisable to make a big part of your daily intake. Plants have a known low-inflammatory potential which makes the gut happy and it looks like you are doing very well in this domain. For a reference, on the following link you will find more details about an experiment of one of our clients and biohacker himself who managed to improve his GlycanAge by introducing 30+ different plants each week (https://glycanage.com/blog/journeys/dr-nick-engerer-self-experiment-with-diet/, if you scroll down, you will also find his own web blog where his experiment was explained in more detail).

Meat and fish if you consume them, on the other hand, should be of known origin - it is worth thinking about what the farmed animals were fed with and whether meat is imported or not, for instance. With fish, it is advisable to avoid farmed fish due to widespread antibiotic use (to prevent infection) and use of different chemicals to make fish look more appealing. Protein that comes from meat, fish, eggs, dairy or plants is crucial for muscle mass maintenance. Carbohydrates are also important as they represent the primary source of energy, especially during exercise. I highly recommend Professor Tim Spector's book called Spoonfed which gives a lot of evidence-based insight into common diet myths and food industry lobbying.

Exercise:

Following on what we talked about in terms of exercise, frequency and intensity, again, an individualised approach here is the best one. Optimal type and amount of exercise will differ between individuals, but generally both aerobic and muscle strengthening type of exercise are recommended. The former may be categorised according to intensity level which may be determined by heart rate (https://www.cdc.gov/physicalactivity/basics/measuring/heartrate.htm) - minimum 75 minutes of high intensity (or minimum 150 minutes of moderate intensity) aerobic activity is advised weekly (as per the World Health Organisation). Again, such advice is just a guide. Running, brisk walking, swimming, cycling are some examples of aerobic activity.

Muscle strengthening workout has a range of positive health outcomes (https://bjsm.bmj.com/content/56/13/755). Two focused muscle strengthening sessions a week (~45 mins each) are good enough and that includes working with own or external weights, resistance bands as well as some vigorous forms of yoga/pilates.

Habits and stress:

Achieving adequate amounts of sleep, both quantity (7 to 9 hours a night) and quality-wise (no interruptions, reducing sleep-onset insomnia) is crucial for the normal function of the immune system.

Those who chronically lack sleep tend to have higher risk of developing immunodeficiency as well as dementia, cardiovascular disease and diabetes. What affects sleep and vice versa is indeed very complex, and mood, cognitive functioning, hormonal (im)balance all play important roles.

It is always good to stress the importance of looking after your mental health. Chronic stress (at work or home), burnout at work or major life events, may all have a negative impact on your biological age. Your body and mind work together and so neither should be not looked after. A good way to work both on physical and mental health is spending time outside - since you live in the countryside, you have a great opportunity to do just that. It has been scientifically proven that spending just 2 hours per week somewhere in nature does us a lot of good (https://www.nature.com/articles/s41598-019-44097-3).

Further recommendations:

In regards to keeping your bones healthy, muscle strengthening (weight-bearing) exercise, diet and keeping your weight healthy all play a part. What calcium supplementation does is questionable. A systematic review from 2015 concluded that calcium supplementation (dietary, isolated calcium supplements or combined vitamin D and calcium supplements) does not help prevent fragility fractures, unless one indeed is deficient in either calcium or vitamin D (https://www.bmj.com/content/351/bmj.h4580, scroll all the way down for a concise summary).

Increasing calcium intake (via diet or supplements) has a minor positive effect on increasing bone mass density that is unlikely to be sustainable and overall beneficial (https://www.bmj.com/content/351/bmj.h4183, find concise summary at the very end). Medications widely used in treatment of osteoporosis are bisphosphonates. You may explore this option with your GP and see if that is something that may be suitable for you/you may have indication for (given you are osteopenic and had a recent wrist fracture).

Lastly, yearly blood tests, regular blood pressure check and dental health check are good ways of easy and affordable health prevention. Blood tests I would recommend you get next time round are full blood count, clotting, iron studies, B12, folate; urea and electrolytes, liver function tests, Vitamin D, bone profile (calcium, phosphate, ALP, albumin, total protein); thyroid function tests, HbA1c (diabetes test), lipid profile; C-reactive protein.

I hope you found our consultation useful and that some of these insights will come in handy when thinking about lifestyle factors that affect your biological age.

Tables

Table 1: Disease-specific changes in IgG glycosylation

High = high amounts of   |   Low = low amounts of
Strong association   Weak association   Insignificant association
* neutral glycoforms of this group containing sialic acid

Table 2: Stress responses to receiving results

References: