Exploring the Need of Stopping Hormone Treatment in Transgender Persons Before Elective Genital Surgery

Exploring the need of stopping hormone treatment in transgender persons before elective genital surgery.

Arne Dereu Student number: 01408784 Supervisor: Prof. Dr. Guy T’Sjoen

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Medicine in Medicine

Academic year: 2017 – 2019

Exploring the need of stopping hormone treatment in transgender persons before elective genital surgery.

Arne Dereu Student number: 01408784 Supervisor: Prof. Dr. Guy T’Sjoen

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Medicine in Medicine

Academic year: 2017 – 2019

Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat. Universiteitsbibliotheek Gent, 2021.

This page is not available because it contains personal information. Ghent University, Library, 2021. Voorwoord In de eerste twee jaar van de masteropleiding geneeskunde is het de bedoeling dat de student een thesis schrijft. De keuze tussen een literatuuronderzoek of experimenteel onderzoek is vrijblijvend en is de persoonlijke beslissing van de student. Na een tijdje hierover te reflecteren, koos ik voor een literatuuronderzoek. Elke student mag een persoonlijke top tien onderwerpen opstellen, en de computer verdeelt – naargelang een algoritme – de onderwerpen onder de studenten. Ik was opgetogen toen het onderwerp “Exploring the need of stopping hormone treatment in transgender persons before elective genital surgery” mij toegewezen werd.

Kort nadien contacteerde ik mijn promotor Prof. Dr. T’Sjoen en mijn begeleidster Justine Defreyne een eerste keer om in juni 2017 een eerste gesprek te hebben. Tijdens dit gesprek gaven mijn promotor en begeleidster mij algemene informatie over endocriene en chirurgische zorg voor transgenders, en bijkomende informatie over het betreffende literatuuronderwerp. Ze vermeldden een aantal algemene bronnen die een uitstekende leidraad boden voor een goede opstart van het literatuuronderzoek. Gedurende de eerste master werd het overgrote deel van het literatuuronderzoek verricht en daarnaast schreef ik ook de eerste versie van de inleiding en methodologie, met een aanzet tot resultaten en discussie. In de tweede master werden het abstract, de resultaten en discussie uitgewerkt en werd de hele masterproef op punt gesteld. Hierbij waren de constructieve feedback en tips van zowel professor T’Sjoen als Justine Defreyne zeer cruciaal van belang. Ik zou hen dan ook zeer erg willen bedanken voor de vrijgemaakte tijd, feedback en adviezen. Daarnaast wil ik professor T’Sjoen nogmaals bedanken voor het beschikbaar stellen van dit interessante onderwerp voor de masterproef.

Ik wil ook nog enkele mensen bedanken voor hun aandeel in deze masterproef. Graag wil ik mijn vriend Kevin bedanken voor het opstellen van de bijgevoegde figuren in deze masterproef. Tevens wijs ik erop dat het schrijven van de masterproef in het Engels een aangename, maar niet altijd evidente uitdaging voor mij was. Mijn vriend Luca heeft me hierbij geholpen door het nalezen van mijn masterproef. Ik kreeg ook veel steun van mijn ouders en broer die zeer geïnteresseerd waren in het verloop en uitwerken van deze masterproef.

Arne Dereu, 28 november 2018

List of abbreviations used

ADP Adenosine diphosphate APC resistance Activated protein C resistance APPT Activated partial thromboplastin time AT III Antithrombin III ATP Adenosine triphosphate BMI Body mass index CDC U.S. Medical Eligibility Criteria COCs Combined oral contraceptives DHEA Dehydroepiandrostenedione ECS Elasticated compressive stockings EPCR Endothelial protein C receptor ERalpha & ERbeta Oestrogen receptors alpha & beta GnRH Gonadotropin-releasing hormone HDL-cholesterol High-density lipoproteine cholesterol IL-10 Interleukin 10 INR International normalized ratio IPS Intermittent pneumatic compression devices LDL-cholesterol Low-density lipoproteine cholesterol LMWH Low molecular weight heparins NICE British National Institute for Health and Clinical Excellence NO Nitric oxide PAF Platelet-activating factor PAI Plasminogen activator inhibitor PF3 Phospholipids PT Prothrombin time RCT Randomized controlled trial SHBG Sex hormone-binding globulin TAFI Thrombin-activatable fibrinolysis inhibitor TF Tissue factor = tissue thromboplastin TFPI Tissue-factor pathway inhibitor t-PA Tissue-type plasminogen activator TXA2 Thromboxane A2 UKMEC United Kingdom Medical Eligibility Criteria ULVWF Ultralarge vWF u-PA Urokinase-type plasminogen activator VTE Venous thromboembolism vWF von Willebrand factor WHO World health organization

Table of contents

1. Abstract ...... 1

1.1 English abstract ...... 1

1.2 Nederlandstalig abstract ...... 2

2. Introduction ...... 4

2.1 Transgender people: Definition and medical care ...... 4

2.2 Normal hemostasis and coagulation ...... 9

2.3 Associated problems with hemostasis and coagulation and surgical precautions ...14

2.4 Purpose of this research ...... 21

3. Methodology ...... 22

4. Results ...... 25

4.1 Effects of testosterone on the cardiovascular system, hemostasis and coagulation 25

4.2 Testosterone replacement therapy in hypogonadal cisgender men and surgery ....28

4.3 Effects of oestrogens on the cardiovascular system, hemostasis and coagulation .30

4.4 Hormonal contraception in cisgender women and surgery ...... 33

4.5 Effects of cyproterone acetate on the cardiovascular system, hemostasis and coagulation ...... 35

5. Discussion ...... 36

5.1 Thoughts and limitations regarding current literature ...... 36

5.2 Recommendations on future research ...... 40

5.3 Conclusion ...... 41

6. Reference list ...... 42

1. Abstract

1.1 English abstract Purpose: Cardiovascular morbidity and mortality in transgender men is comparable to a cisgender control population. Cardiovascular morbidity and mortality rates in transgender women are historically higher, particularly for venous thromboembolisms, compared to a cisgender control population. Currently gender affirming hormone therapy is often interrupted two weeks prior to elective major surgery in transgender women until mobilization is reobtained. This practice is sometimes seen in transgender men. The rationale behind this is preventing cardiovascular morbidity in the perioperative period, but no studies have been performed on this subject. The purpose of this thesis is exploring whether the current treatment interruption is necessary.

Methods: An extensive literature research was executed on the effects of testosterone, oestrogens, progesteron and anti-androgens on the thrombotic pathways and coagulation cascade. Research on cardiovascular morbidity and perioperative cardiovascular outcomes in surrogate groups such as cisgender women on hormonal contraception and hypogonadal cisgender men on testosterone replacement therapy was performed to find possible correlations which might be extrapolated to the transgender population.

Results: Whilst testosterone administration in transgender men generally results in higher body weight and BMI (body mass index), no rise in cardiovascular morbidities was found. Testosterone therapy results in specific alterations in the coagulation cascade and the thrombolytic pathways but despite those alterations the PT (prothrombin time), APPT (activated partial thromboplastin time), thrombin time and fibrinogen concentration are not affected in transgender men. In hypogonadal cisgender men on testosterone replacement therapy, no increased risk of postoperative mortality and cardiovascular morbidity was found compared to cisgender men without hormonal treatment. Oestrogen administration seems to alter platelet activity and the coagulation system to a more prothrombotic state, which results in a higher risk for venous thromboembolism. Oestrogen biochemical structure, dose and route of administration does play a role in thromboembolic risk. Cisgender women on combined oral contraceptives (COCs) exert a higher thrombotic risk, which is further increased when using COCs combined with undergoing surgery. These findings have led to the suggestion to stop COCs 4 weeks before major surgery with a prolonged period of immobilization, which is rarely applied to other types of surgery. Research on anti-androgens only showed a higher risk of venous thrombosis in cisgender women when using cyproterone acetate in oral contraceptives, compared to other COCs. No information on other anti-androgens or progestogens was found.

Conclusion: Currently, there is no literature on gender affirming hormone therapy and perioperative risk. As the available literature suggests an increased cardiovascular morbidity in transgender women, and given the prothrombotic effects of oestrogens in cisgender as well as transgender women, interrupting gender affirming hormone therapy in transgender women prior to major surgery with immobilization seems like a thoughtful decision. However, given that testosterone administration does not lead to increased thrombotic risk in hypogonadal cisgender men on testosterone therapy, discontinuing gender affirming hormone therapy does not seem necessary in transgender men. More studies are needed on the perioperative thrombotic risk and the thrombotic effects of oestrogens, anti-androgens and progestogens in transgender women.

1.2 Nederlandstalig abstract Doelstelling: Cardiovasculaire morbiditeit en mortaliteit bij transgender mannen is vergelijkbaar met een cisgender controle populatie. Cardiovasculaire morbiditeit en mortaliteit bij transgender vrouwen ligt historisch hoger, vooral voor veneuze trombo-embolieën, vergeleken met een controlepopulatie. Momenteel wordt gender affirmerende hormoontherapie twee weken voor electieve majeure chirurgie gestopt bij transgender vrouwen tot mobilisatie terug verworven is. Deze praktijk wordt soms toegepast bij transgender mannen. De motivering hiervoor is het voorkomen van cardiovasculaire morbiditeit in de perioperatieve periode, alhoewel hier nog geen specifieke studies over gedaan zijn. Het doel van deze thesis is het exploreren of de huidige onderbreking van de behandeling noodzakelijk is.

Methoden: Een uitgebreid literatuuronderzoek werd uitgevoerd op de effecten van testosteron, oestrogenen, progestagenen en antiandrogenen op de trombotische pathways en de stollingscascade. Daarnaast werd er onderzoek ondernomen naar cardiovasculaire morbiditeit en perioperatieve cardiovasculaire uitkomsten in gelijkwaardige groepen, zoals: cisgender vrouwen die hormonale contraceptie nemen en hypogonadale cisgender mannen op testosteron vervangingstherapie, om eventuele correlaties te vinden die naar de transgender populatie zouden kunnen geëxtrapoleerd worden.

Resultaten: Terwijl testosteron toediening bij transgender mannen over het algemeen resulteert in een stijging van het lichaamsgewicht en BMI (body mass index), is er geen verhoging in cardiovasculaire morbiditeit teruggevonden. Testosteron geeft specifieke wijzigingen in de coagulatiecascade en de trombotische pathways, maar ondanks deze wijzigingen zijn de PT, APPT, trombine tijd en fibrinogeen concentraties niet beïnvloed bij transgender mannen. Bij hypogonadale cisgender mannen die testosteron vervangingstherapie nemen, wordt er geen stijging in de postoperatieve mortaliteit en

2 cardiovasculaire morbiditeit teruggevonden in vergelijking met cisgender mannen zonder hormoonbehandeling. Oestrogeentoediening lijkt de plaatjesactivatie en coagulatiecascade te wijzigen naar een meer protrombotische staat, met een hoger risico voor veneuze trombo- embolieën. De biochemische structuur, dosis en toedieningswijze van het oestrogeen spelen hierbij een rol. Cisgender vrouwen die gecombineerde orale contraceptiva gebruiken hebben een hoger trombotisch risico, dat verder wordt verhoogd wanneer gecombineerde orale contraceptiva gebruikt wordt terwijl chirurgie wordt ondergaan. Deze ondervindingen hebben geleid tot de suggestie om gecombineerde orale contraceptiva 4 weken voor majeure chirurgie met een verlengde periode van immobilisatie te stoppen. Bij chirurgie zonder verlengde periode van immobilisatie wordt dit zelden gedaan. Bij het onderzoek naar antiandrogenen werd enkel een hoger risico voor veneuze trombose gevonden bij cisgender vrouwen wanneer cyproterone acetaat in orale contraceptiva werd gebruikt, vergeleken met cisgender vrouwen op orale contraceptiva zonder cyproterone acetaat. Er werd geen informatie gevonden over antiandrogenen of progestagenen.

Conclusie: Momenteel is er geen literatuur over gender affirmerende hormoontherapie en het perioperatieve risico. Aangezien de beschikbare literatuur een verhoogde cardiovasculaire morbiditeit suggereert bij transgender vrouwen, en gezien de protrombotische effecten van oestrogenen in cisgender en transgender vrouwen, lijkt het onderbreken van gender affirmerende hormoontherapie bij transgender vrouwen voor majeure chirurgie gevolgd door een periode van immobilisatie, een doordachte beslissing. Testosterontoediening leidt echter niet tot een verhoogd trombotisch risico bij hypogonadale cisgender mannen op testosteron therapie, waardoor het onderbreken van gender affirmerende hormoontherapie niet noodzakelijk lijkt bij transgender mannen. Meer studies zijn nodig op het perioperatieve trombotische risico en de trombotische effecten van oestrogenen, antiandrogenen en progestagenen bij transgender vrouwen.

2. Introduction

2.1 Transgender people: Definition and medical care 2.1.1 Definition and epidemiology We assign a newborn to the male or female sex based on the phenotypic appearance at birth. This is where the sex-chromosome largely determines the outcome. Gender identity differs from biological sex and is defined as the way an individual perceives their own gender. A person’s feeling of gender identity can be measured on a continuous scale ranging from male to female and one can identify anywhere on that scale. In transgender people, the feeling of gender identity differs from the birth-assigned sex. This gender incongruence can express itself with a distress, called gender dysphoria (1-3). If desired, options for transition include social, psychological, legal, hormonal and surgical transition.

Transgender people are a very diverse group and difficult to count. Some live with gender incongruence without transitioning, some stick with a social transition without demanding gender-affirming health care, some buy hormones on the internet or on other non-medical platforms, and some don’t make their transgender status known to others or don’t demand health care due to stigma and other social problems (4). Because of this difficulty, studies assessing the number of transgender people were mainly based on clinic-based data, for example ranging from the number of people who underwent gender affirming surgery (5), to the number of people attending clinical services in general (4). Van Caenegem et al (6) examined the prevalence of gender ambivalence (identifying equally with the sex assigned at birth as with the opposing sex) and gender incongruence (identifying stronger with the other sex than the one assigned at birth) in the general population of Belgium by using surveys in 2015. In the population survey they found that 2,2% of men and 1,9% of women identify as gender ambivalent, while 0,7% of men and 0,6% of women identify as gender incongruent. In a bigger sample in the Netherlands, 1,1% of men and 0,8% of women identify as gender incongruent (7). Only a minority of people who reported gender incongruence or ambivalence stated a desire for gender affirming care, which explains the discrepancy of these numbers with the often used prevalence rate of 1 in 12 900 transgender women and 1 in 33 800 transgender men in Belgium (6).

2.1.2 Gender affirming endocrine care If desired, endocrine treatment of transgender people consists of gender affirming hormone treatment. This implies the administration of testosterone in transgender men and oestrogens plus anti-androgen therapy in transgender women, aimed at serum sex hormone levels within the cisgender physiological range (8). Both over- and undertreatment are undesirable, as supraphysiological levels can induce undesired events such as thrombosis, while subphysiological levels can induce a hypogonadal state (2, 3, 9). Therefore, gender affirming hormone therapy is a chronic therapy, and long-term administration of gender affirming therapy post gender affirming surgery is indicated. However, to date, there is no consensus regarding how long gender affirming hormone therapy should be given, as both serum testosterone levels and serum oestradiol physiologically decrease in menopausal women and (to some extent) in aging cisgender men (10). Gender affirming hormone therapy can be initiated after the confirmation of the diagnosis by a mental health professional (2).

2.1.2.1 Transgender men If gender affirming hormone therapy is desired in transgender men, testosterone therapy can be initiated. Preferred options include parenteral testosterone injections or testosterone gel. Commonly used injectable testosterone agents include testosterone enanthate or cypionate (100 – 250 mg intramuscular every 2 to 4 weeks), testosterone undecanoate (1000 mg intramuscular every 10 to 12 weeks) and mixed testosterone ester preparations with testosterone propionate, testosterone phenylpropionate, testosterone isocaproate and testosterone decanoate (250 mg every 2 – 3 weeks or 500 mg every 3 – 6 weeks) (2). There is a big fluctuation between the supraphysiological testosterone level observed right after the injection and the lower level of testosterone the days before the next administration. Less fluctuations are observed with testosterone undecanoate compared to testosterone enanthate or cypionate. Stable levels of testosterone are important to prevent mood swings (2, 11).

The administration of testosterone in transgender men induces masculinization characterized by increasing facial and body hair, bulkier musculature, increased sexual desire, increased oiliness of skin with possible development of acne, redistribution of fat mass, deepening of the voice, clitoral growth and male pattern hair loss (8).

2.1.2.2 Transgender women Transgender women receive a combination of oestrogens and anti-androgens (including GnRH analogues) to obtain the female secondary sexual characteristics, while suppressing the endogenous testosterone production. Breast growth, reduction of masculine hair growth and a more female fat distribution are desired effects of the gender affirming hormones (10). Oestrogens can be administered orally, transdermally or intramuscularly. There are some

5 beliefs that the oral route of administration contains a higher thromboembolic risk, due to the first pass effect, but this has not been tested in randomized controlled trials. It has been observed that using ethinyl oestradiol as an oestrogen means a strongly increased risk of thromboembolic events compared to the other oestrogens in general. This is why the Endocrine Society Clinical Practice Guideline discourages using ethinyl oestradiol and encourages using another oestrogen such as oestradiol valerate (8). Transdermal oestrogen preparates show a lower thromboembolic risk and more stable serum levels of oestradiol, but can cause skin reactions or (in case of transdermal patches) skin adhesion problems. Due to not being directly handled by the hepatic metabolism, and reaching the liver in small quantities through the portal circulation, the transdermal route results in a more favorable lipid, inflammatory and coagulative profile and may be preferable in persons with other risk factors for thrombosis (3).

For the suppression of endogenous testosterone production, there are multiple options. Cyproterone acetate, a progestin, is the most commonly used anti-androgenic substance. The action mechanism of cyproterone acetate is twofold: First, it suppresses the gonadotrophins, thereby suppressing testicular androgen secretion. Second, it binds competitively to the androgen receptor. A daily dose of 25 mg orally is reported in the recent literature (12). Another option is the use of GnRH agonists such as leuprolide, triptorelin or goserelin, which block the GnRH receptors at the pituitary, thus inhibiting gonadotropin secretion and downstream testicular testosterone production. Leuprolide, triptorelin or goserelin are injected subcutaneously once a month or once every three months. A third option is spironolactone which has both an anti-androgenic and an oestrogen-receptor agonistic action (3). Spironolactone directly inhibits the interaction of androgens with the androgen receptor (8). The administration consists of 100 – 200 mg/day orally (3). Once orchidectomy is performed, there is no more endogenous testosterone production, and anti-androgens can be stopped (13).

Some transgender women report an improvement of breast development, mood or sexual desire when adding progestogens to their hormone scheme. Hembree et al (8) report that there are no well-designed studies on the role of progestogens in feminizing hormone regimens. Current evidence does not prove or disprove the above called anecdotal effects. However, adding progestogens to the hormone scheme might increase the thrombo-embolic risk, as seen in premenopausal cisgender women on oral contraceptives including progestogens. No research has been done in transgender women (14).

Transgender males: Parenteral testosterone: - Testosterone enanthate or cypionate 100 – 200 mg sequentially intramuscularly every 2 weeks, or 50 – 100 mg sequentially subcutaneously every week - Testosterone undecanoate 1000 mg every 12 weeks Transdermal testosterone - Testosterone gel 1,6% 50 – 100 mg/day - Testosterone transdermal patch 2,5 – 7,5 mg/day Transgender females: Oestrogens: - Oral oestradiol 2,0 – 6,0 mg/day - Transdermal oestradiol patch 0,025 – 0,2 mg/d (with a new patch every 3 to 5 day) - Parenteral oestradiol valerate or cypionate 5 – 30 mg intramuscularly every 2 weeks or 2 – 10 mg intramuscularly every week Anti-androgens: - Spironolactone 100 – 300 mg/day - Cyproterone acetate 25 – 30 mg/day - GnRH agonist 3,75 mg sequentially subcutaneously every month or 11,25 mg sequentially subcutaneously every 3 months Table 1. Options for gender affirming hormone therapy in transgender persons. Adapted from Hembree WC, Cohen-Kettenis PT, Gooren L, Hannema SE, Meyer WJ, Murad MH, et al. Endocrine Treatment of Gender-Dysphoric/Gender-Incongruent Persons: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2017;102(11):3869-903 (8).

2.1.3 Surgical gender affirming care Possible surgical options for transgender men include: bilateral mastectomy, chest contouring, pectoral implants, metoidioplasty, phalloplasty, urethral lengthening, scrotal reconstruction with testicular prostheses with or without hysterectomy and/or bilateral salphingo- oophorectomy (1).

Possible surgical options for transgender women are: breast augmentation, facial feminization surgery, voice feminization surgery, thyroid cartilage reduction, gluteal augmentation, bilateral orchiectomy, penectomy, clitoroplasty, vaginoplasty and/or vulvoplasty (1).

2.1.4 Risks and safety of gender affirming hormonal therapy Gender affirming hormonal therapy has different risks for both transgender men and transgender women. While testosterone administration in transgender men is perceived safe in short- and medium-term studies on cardiovascular health, the current literature on anti- androgen and oestrogen administration in transgender women reports an increased risk of venous thrombosis and pulmonary embolism (9, 10), which resulted in a slightly higher cardiovascular mortality rate in transgender women compared to transgender men and the general population (9). However, these studies reported on transgender women using mainly oral ethinyl oestradiol. New studies assessing cardiovascular and thromboembolic risk in transgender women adhering to current treatment regimens are needed to assess the risk.

Wierckx et al (9) found a prevalence of 60,7 cases of venous thrombosis and/or pulmonic embolism per 1000 persons in transgender women on gender affirming hormone therapy compared to 9,2 per 1000 persons in transgender women before gender affirming hormone therapy. Wierckx et al (10) also found an estimated incidence of venous thrombosis of (21/10.000) transgender women, which is 20 times as much compared with a control group of cisgender men and 7 times higher compared to cisgender women on oral contraceptives (3,01/10.000). The same study also reported a higher incidence of cerebrovascular disease in transgender women compared to the general population. Asscheman et al (15) published a meta-analysis in 2014 about the risk of venous thromboembolism in transgender women, also confirming higher risk in this group compared to others. Older literature did not correct for variables such as diabetes, obesity and ethinyl oestradiol use, which influences the available meta-analyses on the subject. However, abandoning the use of ethinyl oestradiol also reduced venous thromboembolism rates. Arnold et al (16) confirms this. In their study on 676 transgender women, all using oral oestradiol and 634 using spironolactone as anti-androgen, there was only one event of venous thromboembolism, resulting in an incidence rate of 7,8/10 000 person years. This was lower than the incidence of 8 – 27/10 000 person years in the general population and lower than the 30/10 000 person years in postmenopausal women on oestrogen therapy. Due to the small sample size this could be an underestimation.

2.2 Normal hemostasis and coagulation 2.2.1 Definition To have an understanding on the influence of sex hormones on coagulation, one must understand the basics of coagulation. When a blood vessel gets damaged, a complex chain of events takes place, to make sure the blood vessel gets repaired and bleeding stops. Indispensable in this chain of events is coagulation, which must operate quickly, only when needed and locally. This also explains why it is a strongly regulated system, with counteractive opponents such as fibrinolytic anticoagulants. Initially, a hemostatic plug of platelets arises by pegging them down to collagen. This process is called aggregation or the primary hemostasis. Thereafter this plug of aggregated platelets will convert to a firmer fibrin clot. This process is called the coagulation or secondary hemostasis (17, 18).

2.2.2 Primary hemostasis: platelet adhesion and aggregation

Figure 1: Platelet adhesion and aggregation. Adapted from Bonhomme F et al (18).

Non-damaged endothelium secretes specific antithrombotic agents such as nitric oxide (NO), prostacyclin and endothelin-1, which also regulate the vascular tone. NO is the most important antithrombotic agent due to its specific regulatory function of vascular homeostasis and protection of the vessels against thrombosis and atherosclerosis. NO causes blood vessel relaxation, prevents platelet aggregation and blood cell adhesion to the endothelium surface, and decreases the expression of proinflammatory genes (19). When the endothelial barrier gets damaged, a local vasoconstriction occurs to reduce the blood flow and the extravascular blood loss, and hemostasis takes off by the blood being exposed to the subendothelium. The von Willebrand factor (vWF), a glycoprotein circulating in the blood, binds to exposed subendothelial collagen. This link induces conformational changes in specific sites on the vWF,

9 which now allow platelets to bind the vWF to their vWF receptors in a glycoprotein (GP) GPIb/V/IX complex. This initial adhesion of the platelets to the subendothelial collagen matrix is then further stabilized through direct contacts between specific platelet receptors and subendothelial components, for example the linking of platelet collagen receptors GP Ia-IIa and GP VI with collagen. Platelet adhesions leads to platelet activation, with the excretion of coagulation factors, calcium and adenosine diphosphate (ADP). In addition, thromboxane A2 (TXA2) and platelet-activating factor (PAF) are also released, they are prothrombotic agents that further activate platelets and promote. A change of shape from a discoid to a globular shape also occurs with platelet activation, and the platelets will reveal a negatively charged phospholipid layer, which will be important for the process of coagulation and thus the secondary hemostasis. Multiple platelets start to cluster together due to cross-linking the GP IIb/IIIa aggregation receptors, which are highly expressed in activated platelets (17, 18).

2.2.3 Secondary hemostasis: Fibrin clot formed due to the coagulation process

Figure 2: The coagulation cascade: intrinsic and extrinsic pathway. Adapted from Johari V et al (20).

Theoretically, there are two pathways to form this solid coagulated clot: the intrinsic and the extrinsic one. They converge to the common pathway that begins by activating factor X. The activation of specific coagulation factors will ensure the initiation of the cascade, resulting in the activation of other coagulation factors due to a protease function of the coagulation factors, with finally the formation of fibrin, which forms the blood clot around the loose aggregation of platelets. (20).

All the needed components for the intrinsic pathway are already present in the blood. The initiating element for the intrinsic cascade is the contact of factor XII with a negatively charged surface, more precisely collagen laying underneath the endothelium and thus being exposed when there is vessel damage. Prekallikrein or high molecular weight kininogen (HMWK) catalyzes this activation of factor XII. Activated factor XII activates factor XI, which activates factor IX. Furthermore, factor X gets activated by the combination of IXa and factor VIIIa, supported by calcium and phospholipids (PF3) from the aggregated platelets. Activated factor VIII arises when factor VIII is separated from the von Willebrand factor (17). The extrinsic pathway needs the presentation of tissue factor (TF, synonym tissue thromboplastin) on the subendothelial cell membrane of fibroblasts and smooth muscle cells. Damage to the endothelium can initiate expression of TF. The TF interacts with factor VII and together they induce the conversion of factor X to activated factor X. Activated factor X leads to the conversion of prothrombin to thrombin, and this thrombin induces the conversion of soluble fibrinogen to insoluble fibrin, which then is able to form the clot (21). The clot consists of fibrin monomers linked to each other, due to the formation of covalent cross-linkages. The coagulation factors XIII and calcium catalyze this formation. (17)

Recent views on hemostasis point out that the intrinsic pathway does not have a true physiologic role in hemostasis, as deficiencies of HMWK, prekallikrein and factor XII only cause a longer activated partial thromboplastin time (APTT) but not a bleeding disorder. Besides that it has been proven that the TF/factor VII complex, which activate the extrinsic pathway, can also activate factor IX of the intrinsic pathway. This makes the activation of the TF/factor VII complex the most important trigger in the coagulation system. Furthermore, thrombin also activates factor XI, which results in another link between the extrinsic and intrinsic pathway. (21)

To summarize, hemostasis should be seen as a cell-based model with initiation, amplification and propagation. The initiation begins with blood being exposed to TF expressed on fibroblasts and smooth muscle cells lying under the damaged endothelium. Factor VII binds to it and activates the coagulation cascades. The small amount of thrombin then goes through amplification due to activating platelets with increasing platelet adhesion and activating factors V, VIII and XI, so that the activated platelets wear factor Va, VIIIa and XIa on their surface. The propagation is defined by producing tenase and prothrombinase complexes. The tenase complex consists of factor VIIIa and factor IXa, and is formed on the moment that factor IXa moves from the TF-bearing cell to the binding receptor, expressed on activated platelets. This tenase complex activates factor X which, when activated, forms a complex with factor Va to form the prothrombinase complex. This complex ensures a propagating production of thrombin

11 and in that way also of the conversion of fibrinogen to fibrin. Factor XIII, known as the fibrin stabilizing factor, also gets activated by this complex to firm up the clot (21).

To assess the performance of the coagulation system, two laboratory tests are mainly used: the prothrombin time (PT) and the activated partial thromboplastin time (APTT). The PT assesses the functioning of the extrinsic pathway and the common pathway, while the APTT assesses the functioning of the intrinsic pathway and the common pathway. The PT is the time in seconds it takes to form a clot from a patient’s plasma after the adjunction of calcium and the activator of the extrinsic pathway thromboplastin. Measurements of the PT on a same sample of plasma can vary between multiple laboratories due to the use of thromboplastin from different companies with different sensitivities. Therefore, the international normalized ratio (INR) forms a solution, converting the PT to a normalized ratio by taking the differences in sensitivity into account. The APTT is the time in seconds it takes to form a clot from patient’s plasma after the adjunction of phospholipid (an intrinsic pathway activator) or another surface- activating agent, and calcium. Deficiencies or inhibitors of clotting factors of the extrinsic and common pathway will result in a prolongation of the PT, while those of the intrinsic and common pathway will result in a prolongation of the APTT (18, 22).

2.2.4 Anticlotting mechanisms Multiple mechanisms help to keep clotting local and only operable when needed. These are mainly the fibrinolytic system, the protein C/S system, tissue-factor pathway inhibitor (TFPI) and antithrombin (20). Antithrombin III (AT III) is a serine protease inhibitor, circulating in the blood, that blocks multiple clotting factors (IXa, Xa, XIa, XIIa and thrombin) in their working, the most important one being thrombin. AT III ensures that coagulation stays located at the site of injury instead of forming little blood clots in uninjured vessels due to neutralizing the thrombin that is distributed throughout the circulatory system from the injury site (17, 18).

The initiation of coagulation is regulated by the tissue-factor pathway inhibitor TFPI. This TFPI circulates for 10% in an active free form in the blood, the other 90% is bound to lipoproteins and has little function. Heparin and shear stress upregulate the expression of TFPI. TFPI has two Kunitz domains, one mimicking the substrate of factor Xa, the other mimicking the TF- FVIIa complex, and thus both inhibiting those. This limits thrombus formation to the site of TF- exposure (23).

The protein C/S system is the system that will eventually stop the clotting process. Formed thrombin also binds to endothelial thrombomodulin, in a complex which also involves the endothelial protein C receptor EPCR. This complex activates protein C. Combined with his cofactor protein S, activated protein C can cleave the activated coagulation factors Va and VIIIa. This suppresses the thrombin formation and will eventually lead to a stop of fibrin

12 formation (18). This mechanism also proves that healthy endothelium is antithrombotic due to the presentation of thrombomodulin on every endothelial cell. It also shows that the coagulation process is indeed very complex because, for instance, thrombin can work both procoagulant in the blood and anticoagulant in a complex with thrombomodulin (17).

The fibrinolytic system is important in degrading the fibrin clot to fibrin degradation products. Essential is plasminogen. This is a circulating plasma protein, activated by plasminogen activators (PA) to become plasmin. There is a tissue-type plasminogen activator t-PA and a urokinase-type plasminogen activator u-PA, found in urine (18). Plasmin lyses fibrin and fibrinogen to the fibrin degradation products. These fibrin degradation products inhibit thrombin (17). This fibrinolytic system is closely monitored to not be overactive by inhibitors such as plasminogen activator inhibitor (PAI) (inhibits t-PA and u-PA) and thrombin-activatable fibrinolysis inhibitor (TAFI), which inhibits the function of plasmin. (18)

Figure 3: The fibrinolytic system. Adapted from: Bonhomme et al (18).

If any of these mechanisms know impairments in a specific patient, then this patient predisposes for thrombotic events due to a prothrombotic condition. For example, deficiencies of protein C, protein S, antithrombin III. Factor V Leiden, a mutation in factor V which prevents cleavage by protein C, also predisposes for thrombotic events (18).

2.3 Associated problems with hemostasis and coagulation and surgical precautions 2.3.1 Abnormal thrombosis and venous thromboembolism A simplified pathophysiologic mechanism of venous thrombosis is found in Virchow’s triad. Virchow states that three factors influence the formation of a venous thrombosis: changes in blood composition, the blood flow or in the blood vessel wall (24-26). The first factor, the blood composition, is interpreted by changes in plasma levels of both procoagulatory and anticoagulatory substances. Both the activation of the intrinsic and the extrinsic coagulation pathway promotes the formation of a clot, except if there is a counterbalance in the anticoagulation system. This explains why an elevation in the plasma levels of factor VIII, factor IX, factor XI and fibrinogen are linked to venous thromboembolisms. As mentioned before, deficiencies and thus decreases in the plasma levels of anticoagulant proteins such as antithrombin, protein C, protein S and activated protein C resistance (APC resistance) are also linked to venous thromboembolism (27).

The second factor is the blood flow. When the blood flow stalls, the risk on venous thrombosis rises, illustrated by a higher risk when a patient is bedridden, compared to a mobile patient. This stasis causes an accumulation of prothrombotic substances. These would normally flow downstream and get inactivated. For example, thrombin would normally flow further into the capillaries, where endothelial thrombomodulin and heparin sulfate proteoglycans would inhibit the thrombin. Thrombomodulin converts thrombin to an anticoagulant due to formation of APC, and heparin sulfates inactivate thrombin by accelerating the thrombin-antithrombin interaction by allosteric and other interactions. Besides that, stasis of blood causes desaturation of hemoglobin in the local erythrocytes. Hypoxic responses in multiple cell types such as platelets, endothelial cells and leukocytes will take place. This hypoxic state is linked to thrombotic processes after 2 hours of nonpulsatile flow. Hypoxia activates the endothelium with exocytosis of Weibel-Palade bodies (granules containing vWF and P-selectin on their membranes), with an increase of P-selectin on endothelial cells and more secretion of vWF. The vWF secreted at the moment of hypoxia is called ultralarge vWF (ULVWF). ULVWF is a hyperadhesive form of vWF and more procoagulant than regular vWF, and it can also bind leukocytes and erythrocytes besides platelets. (24). Stasis on its own is an important contributing factor but not enough to produce thrombosis. One could see stasis as the approving factor for other events to make thrombosis happen (25).

The third factor is the vessel wall. Under normal conditions, the endothelium causes a local fibrinolytic and vasodilatory state, in which platelet adhesion, platelet activation, inflammation and coagulation are suppressed. Multiple mechanisms contribute to this anticoagulatory state

14 such as the endothelial production of thrombomodulin, heparin sulfate, TFPI, t-PA, u-PA and their precise working mechanisms, explained before. NO, prostacyclin and IL-10 are also produced by the endothelium, and these inhibit the adhesion and activation of leukocytes and cause vasodilating responses. When the endothelium gets troubled by e.g. a vascular trauma or sepsis, it turns into a proinflammatory and prothrombotic source. Endothelial cells will now secrete PAF and endothelin-I which cause vasodilatation. Also vWF, TF, PAI-1 and factor V will be produced, inducing thrombosis (25). It has also been observed that some thrombi are caused by rupture of an atherosclerotic plaque. This plaque exposes adhesives for platelets such as vWF, collagen but also the coagulant TF. The origin of atherosclerosis is linked to negative changes in the lipid profile, and can show a link between the lipid status and thrombus formation (24).

Inflammation in particular can trigger thrombosis due to increasing plasma levels of PAI-1, TF, fibrinogen and platelet activity, while decreasing thrombomoduline. Inflammation activates the endothelium, with release of Weibel-Palade bodies. The vWF and P-selectin that the Weibel- Palade bodies contain are important in binding leukocytes but also platelets (24). Venous stasis and the following ischemia also upregulate P-selectin. Two specific selectins, P- and E- selectins, function as cell adhesion molecules and allow leukocytes to transmigrate. Besides that, P-selectin can localize and bind prothrombotic microparticles. These microparticles are phospholipid vesicles, exfoliated from platelets, leukocytes and endothelial cells. These microparticles can interact with activated platelets, inducing expression of TF and initiation thrombosis. They also inhibit fibrinolysis because microparticles, derived from platelets, express PAI-I. They are bound to the thrombus by P-selection and thus delay thrombus resolution and promote thrombus growth, after initiating thrombosis (25).

During the 1990’s, multiple hereditary risk factors for venous thromboembolism were discovered. These were classified by their level of impact. Deficiencies of antithrombin, protein C and protein S cause a 5- to 10-fold increased risk for venous thromboembolism (28). Hereditary risk factors for venous thromboembolism include: - Antithrombin deficiency is seen in 1,1% of patients with venous thromboembolism. Patients with this deficiency are at greater risk than those with protein C or S deficiency. One study observed that 85% of these patients have had a thromboembolic event before the age of 50, in comparison with only 50% of the patients with protein C or S deficiency. The prevalence of protein C or protein S deficiency is low and is diagnosed in less than 1% of the patients with venous thromboembolism (26). - Factor V Leiden and mutations in prothrombin and fibrinogen cause a 2- to 5-fold increased risk and are moderately strong risk factors (28). Important to note is that 20 to 60% of the

patients with recurrent venous thromboembolism have the factor V Leiden mutation, and show APC resistance. - The prothrombin G20210A mutation is another mutation that causes a three times increased risk of thrombosis (26). - There are multiple other weak genetic risk factors with relative risks between 1 and 1,5. - An increased thrombotic risk can also be acquired during life, by altering the concentrations of proteins of importance in hemostasis. For example, elevation of the concentrations of prothrombin, factor VIII, factor IX, factor XI, fibrinogen; or decrease of the concentrations of antithrombin, TFPI, protein S or protein C (28).

Besides the hereditary risk factors, multiple non-hereditary risk factors have also been discovered. Strong risk factors for venous thromboembolism are major general surgery (abdominal or thoracic operations longer than 30 minutes, coronary artery bypass, urological surgery and surgery for gynecological malignancies), major orthopedic surgery, spinal cord injury causing chronic paralysis and thus blood stasis, fracture of the pelvis, hip or long bones causing immobilization, multiple trauma, malignancy, myocardial infarction, congestive heart and respiratory failure. All these risk factors are strong enough to justify thromboprophylaxis. Additional risk factors are having an age above 40, obesity, immobility, varicose veins, pregnancy and the puerperium and the use of oral contraceptives. Their separate risks are not big enough to justify thromboprophylaxis, but multiple of these risk factors or one of the risk factors in combination with a strong risk factor from the summary above, may justify the use of thromboprophylaxis (26).

Malignancy is a special risk factor. Arman Trousseau was the first to notice an alteration of the blood, causing a hypercoagulable state in patients with cancer. Although other risk factors may be present in patients with cancer, such as concomitant infectious illness, bed rest with stasis, central venous catheters, vascular compression or tumoral invasion, one can see that tumors release procoagulant substances or feedback to other cells to release these substances. Pancreatic adenocarcinomas, stomach cancer, colon cancer, ovarian cancer, breast cancer, lung cancer and brain cancer are associated with TF for example. Another remarkable risk factor are non-O blood groups. These people have higher levels of vWF than those of the O blood group, because of increased clearance of vWF in the O-group. Correlations have been found between vWF and higher levels of factor VIII. This factor binds with vWF to dodge rapid degradation and thus survive in the circulation. Due to higher amounts of factor VIII and more vWF-mediated systemic endothelial activation, non-O blood groups are at a greater risk for venous thromboembolism (24).

2.3.2 Thromboprophylaxis and the association with surgery Thromboprophylaxis does not only include pharmacological interventions, but also fast-track anesthesia, patient mobilization and mechanical interventions help to reduce the chance of thrombotic issues. Regional anesthetic techniques are associated with less perioperative blood loss and venous thromboembolism. Regional anesthetics are preferred over general anesthetics when feasible. Mechanical prophylaxis consists of elasticated compressive stockings (ECS) and intermittent pneumatic compression devices (IPC). ECS reduce the risk of venous thromboembolism with approximately 50 - 66% and are recommended in all hospitalized surgical patients, until they return to a normal level of mobility. IPC are inflatable sleeves connected to a pump around the whole leg or only the lower leg. These can inflate alternately or sequentially so that pressure stows the blood from the venous circulation in the direction of the heart. The risk is reduced with approximately 60% (18).

Of all the pharmacological interventions, aspirin, vitamin K antagonists, heparin are the most important ones. Other parenterally administered anticoagulants such as hirudin analogues and argatroban can be used if the patient is known with heparin-induced thrombocytopenia and/or antithrombin deficiency. Aspirin alone is not recommended, because it is less effective than heparin derivates in preventing venous thromboembolism. Aspirin inhibits the cyclooxygenase irreversibly, therefore lowering the production of thromboxane A2, which results in an anti- aggregating action on the platelet for the full lifetime of the platelet. Vitamin K antagonists such as warfarin, phenprocoumon and acenocoumarol, inhibit the liver enzyme epoxide reductase, thereby lowering the liver-produced coagulation factors prothrombin, factor VII, factor IX, factor X, but also protein C and S. Last but not least are the heparins. Nowadays the low molecular weight heparins (LMWH) are much more in use than the unfractioned heparin. Heparins inactivate thrombin and factor Xa due to an antithrombin-dependent mechanism. Heparin binds antithrombin, thereby stimulating its action. Due to inhibition of thrombin, activation of factor V, factor VIII, factor IX and platelets decrease, which prevents the augmentation and propagation of the thrombotic process. If we compare unfractioned heparin and LMWH, it becomes apparent that LMWH have a less strong effect on thrombin, but also have a smaller effect on platelets and thereby interfere less with primary hemostasis, which makes them preferred (18).

LMWH are easy to use and have a favorable risk-benefit ratio, making them the first choice in thromboprophylaxis during major surgery. In very low risk patients, early mobilization will suffice, and no pharmacological or mechanical interventions are needed. In low risk patients, mechanical prophylaxis during the period of immobilization should suffice. Moderate risk patients can use heparins or mechanical prophylaxis, and those with a high risk should receive both mechanical prophylaxis and heparins. A number of medical organizations streamlined guidelines for thromboprophylaxis during surgery, such as the British NICE (National Institute for Health and Clinical Excellence) (18).

The thrombotic risk is increased in surgery due to exposing tissue factor following the endothelial disruption, venous stasis due to immobilization and alterations in coagulation proteins (29). The risk of thromboembolic incidents is substantially increased up to 12 postoperative weeks. This varies by type of surgery, with hip or knee replacement having the biggest risk followed by cancer surgery, vascular surgery, surgery for fractures and other orthopedic surgery and gastrointestinal surgery. All other types of surgery carry a less significant risk (30). The type of surgery performed is thus important to comprehend and assess the factual risk in a particular patient (31). To keep the risk as small as possible, Tangpricha et al also noted the importance of modifying other risk factors before surgery, by applying lifestyle interventions on bodyweight, smoking and drug misuse, especially for plastic surgery procedures in which microcirculation is involved (14).

The NICE guidelines confirm the different prophylactic approaches according to the risk profile and to the type of surgery. They advise to stop oestrogen-containing oral contraceptives and hormone replacement therapy 4 weeks before elective surgery. NICE has one overlapping advice for gynecological, thoracic and urological surgery – the type of surgery closest to the surgery transgender women and transgender men undergo. Thromboprophylaxis has to start at admission, by using mechanical interventions, added with pharmacological thromboprophylaxis for patients with a low risk of major bleeding. Pharmacological prophylaxis can stop when the patient starts to regain mobility, usually after 5 to 7 days (32).

The American College of Chest Physicians use different guidelines for orthopedic and nonorthopedic surgery. In the non-orthopedic guideline, they give different recommendations based on the risk rate (see table 2) (33).

Risk rate VTE Rogers Caprini Recommendations Type of surgery score* score* Very low <7 0 No specific pharmacologic or Most outpatient or (<0,5%) mechanical prophylaxis other than same-day surgery early ambulation Low (1,5%) 7-10 1-2 Mechanical prophylaxis, preferably Spinal surgery for with IPC nonmalignant disease Moderate (3%) >10 3-4 - If not at high risk for major Gynecologic bleeding complication: LMWH, noncancer surgery low-dose unfractioned heparin or Cardiac surgery mechanical prophylaxis, Most thoracic surgery preferably with IPC Spinal surgery for - If at high risk for major bleeding malignant disease complication: mechanical prophylaxis preferably with IPC High risk (6%) >= 5 - If not at high risk for major Bariatric surgery bleeding complication: LMWH Gynecologic cancer and mechanical prophylaxis surgery preferably with IPC or elastic Pneumonectomy stockings Craniotomy - If at high risk for major bleeding Traumatic brain injury complication: Mechanical Spinal cord injury prophylaxis, preferably with IPC, Other major trauma until the risk of bleeding diminishes and pharmacologic prophylaxis may be initiated. - If LMWH and unfractioned heparin are contraindicated in someone with no high risk for bleeding complications: Low- dose aspirin, fondaparinux, or mechanical prophylaxis with IPC Table 2: The risk assessment and recommendations for surgery patients. * The Rogers score & Caprini score are tools to assess the risk on a VTE. The Rogers score is based on variables that are independent predictors of VTE risk such as type of operation, laboratory values and patient characteristics, while the Caprini score estimates the VTE risk by various VTE risk factors. Adapted from Gould MK, Garcia DA, Wren SM, Karanicolas PJ, Arcelus JI, Heit JA, et al. Prevention of VTE in Nonorthopedic Surgical Patients. CHEST.141(2):e227S-e77S (33)

2.3.3 Current policy regarding interruption of gender affirming hormone treatment in transgender people before elective major surgery The UZ Gent (University Hospital of Ghent) mainly uses two different preparations in the treatment of transgender men. These are Nebido ® (1000 mg testosterone undecanoate in 4 ml) and Sustanon 250 ® (a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg isocaproate and 100 mg testosterone decanoate). Nebido is typically administered once every three months, resulting in a stable testosterone concentration throughout those three months. Sustanon is administered every two to three weeks. The administration of sustanon results in a high testosterone peak in the beginning, while the concentration declines during the following weeks. Nebido does not give a high testosterone peak in the beginning because of a delayed release of the product throughout the three months. Theoretically, a high testosterone peak (as observed after Sustanon administration) may result in an increase of serum oestradiol levels due to aromatase enzyme activity, which converts testosterone into oestradiol in adipose tissue. Hypothetically, this might increase thromboembolic risk. This is the reason for interruption Sustanon administration one week before elective major surgery in transgender men, but not administration of Nebido. In transgender women, gender affirming hormone treatment is always interrupted two weeks prior to elective major surgery, until mobilization is regained (15).

2.4 Purpose of this research Sex steroids are known to influence the hematological system of hemostasis and coagulation. Historically, the use of ethinyl oestradiol in transgender women has been associated with an increased risk for thromboembolism, although recent studies in transgender women on oestradiol valerate have shown none to moderate increases in the incidence of thromboembolic events (14). Furthermore testosterone therapy in transgender men leads to an increase in serum hematocrit levels, which is related to an increased incidence of thromboembolism (2). However, in the study by Defreyne et al (34), serum hematocrit levels increased to levels within cisgender reference ranges. None of the transgender men presented with serum hematocrit levels >54% and no thrombo-embolic events were documented in the study cohort of transgender men. In addition, large cohort studies have concluded that gender affirming hormonal therapy is not associated with increased mortality in transgender persons (35, 36).

There have been case reports of postoperative deep venous thrombosis in transgender women, undergoing elective gender affirming surgery, even when adhering to the usual thrombosis prophylaxis (37). Due to this fact and the thrombophilic alterations of gender affirming hormonal therapy, this hormonal therapy is usually stopped a variable number of weeks prior to elective surgery, and is reinitiated once the patient regained his/her mobility (10, 37). Usually the gender affirming hormonal therapy is interrupted two weeks before the elective surgery, and resumed after mobilization is regained or after hospital discharge (15). No studies have confirmed that this recommendation does help in lowering the occurrence of postoperative venous thrombosis (10, 37). Transgender women also receive postoperative prophylaxis for deep vein thrombosis (14). This practice is also seen in transgender men on Sustanon, following a suggested increase in serum oestradiol levels, following testosterone injections.

The purpose of this research paper is to explore whether it is indeed essential to stop the gender affirming hormonal therapy a number of weeks in advance or whether it is an unnecessary measure with no significant influence on the outcome of complications after elective gender affirming surgery, when adhering to standard prophylactic treatment. Furthermore, the timing of interruption of hormonal treatment remains unknown. No controlled trials have evaluated the need for interrupting oestrogen therapy perioperatively in transgender persons. In absence of controlled trials in transgender women, literature on the effects of hormone treatment on coagulation and thrombosis parameters will be explored. Data will be interpreted from surrogate groups, who also use hormonal treatments, such as cisgender women on contraceptive hormones or hypogonadal cisgender men on testosterone, to search for parallels and possibly identify risks of hormonal therapy prior to surgery.

3. Methodology For this literature research, articles were searched using three online scientific paper libraries/search engines: PubMed, Google Scholar and Embase. A time filter was not used when looking for articles, because of the relative scarcity when looking for articles about transgender people. Articles were searched for multiple times throughout the period between August 2017 and October 2018, with the most sources and papers being searched for in October & November 2017.

My promotor gave me some explorative information to get on the right track about transgender people care with articles such as the papers of Meriggiola et al and with the book “Testosterone: Action, Deficiency, Substitution” written by Nieschlag et al. Cross-referencing brought me to articles such as those from Shatzel et al and others, which were very useful sources used in the segment “Transgender people: Definition and medical care“. For the segment “Effects of testosterone on the cardiovascular system, hemostasis and coagulation”, some sources came from cross-referencing from the book by Nieschlag et al, other articles were found by search strings as written underneath.

At first, I did some explorative searches on PubMed with MeSH-terms such as “Transgender Persons”, “Sex Reassignment Surgery”, “Thrombosis”, “Hemostasis”, “Blood Coagulation” and “Platelet Activation”. Searches where “Transgender Persons” were included, such as the search string – "Thrombosis"[Mesh] AND "Transgender Persons"[Mesh] AND "Sex Reassignment Surgery"[Mesh] – delivered little results, this search string even had 0 results. This illustrates that little research has been done towards the specific thrombotic risk transgender persons have during sex reassignment surgery, and confirms the necessity of this research paper.

Using the above MeSH-terms in different combinations together with hormone-related terms such as "Testosterone"[Mesh], "Cyproterone Acetate"[Mesh] and hormone therapy brought me to some interesting articles being used in the segment ““Transgender people: Definition and medical care“.

There was a lot of information on Google Scholar about the surgical thromboprophylaxis. Searches such as “Mechanism of anticoagulants”, “Mechanism of heparin”, “Risks of venous thromboembolism” and “Surgery thromboprophylaxis” together with the NICE guidelines concerning venous thromboembolism, gave the appropriate sources to consult for the section called “Thromboprophylaxis and the association with surgery”

As seen in figure 4, to find articles about the effects of hormones on the coagulation system, terms referring to transgender people needed to be excluded to find results. In the specific segments about the links between testosterone/oestrogen/cyproterone acetate and the hemostasis/coagulation system, this is reflected by many experiments and evidence coming from cisgender persons on replacement or add-on hormonal therapy, or even from animal experiments. The search strings – “ "Platelet Activation"[Mesh] AND "Transgender Persons"[Mesh] - "Blood Coagulation"[Mesh] AND "Transgender Persons"[Mesh] - "Hemostasis"[Mesh] AND "Transgender Persons"[Mesh] – all brought no results. Even when performing an open search, without the MeSH-terms, there were no results. This indicates little specific research on those topics have been done on transgender people.

Literature on oestrogens, testosterone and anti-androgens was also searched using the different scientific paper libraries. An overview of the search strategy is shown in figure 4. The two other used articles in the testosterone segment, from Carol et al and Anderson et al are sources which are referred to in the above called book about testosterone.

Figure 4: Searching for articles about cyproterone acetate (I), testosterone (II) and oestrogens (III) and the coagulation system.

In the search for articles on the surrogate groups and their correlation with hormone use to surgery, search strings without MeSH-terms were used such as “stopping contraceptive pill surgery” – “hormonal contraception and thrombotic risk” – “testosterone replacement therapy surgery” – “testosterone replacement therapy cardiovascular risk surgery” resulting in a limited amount of usable articles. Looking in the references of these articles brought forward one more usable article. Using search strings with MeSH-terms such as "Contraception"[Mesh] and "General Surgery"[Mesh] - "Venous Thromboembolism"[Mesh] AND "Estrogens"[Mesh]) AND "General Surgery"[Mesh] - "Surgical Procedures, Operative"[Mesh] AND "Venous Thromboembolism"[Mesh] AND "Hormones" [Pharmacological Action] - "Surgical Procedures, Operative"[Mesh]) AND "Venous Thromboembolism"[Mesh])) AND "Estrogens"[Mesh] - "Venous Thromboembolism"[Mesh] AND "General Surgery"[Mesh] AND "Testosterone"[Mesh] - "Venous Thromboembolism"[Mesh] AND "Surgical Procedures, Operative"[Mesh] AND "Testosterone"[Mesh] - "Surgical Procedures, Operative"[Mesh]) AND "Venous Thromboembolism"[Mesh] – and different combinations of the above used MeSH-terms did not result in any usable articles or any usable information, showing already that information on these surrogate groups is scarce as well. A different surrogate group, cancer patients on anti- hormone treatment, was not further investigated after an exploratory search did not reveal many useful articles.

An extensive search was done on papers describing what happens when gender affirming hormone therapy is interrupted. Different search strings such as “stopping hormones transgender” – “interrupting gender affirming hormone therapy transgender” – “stop therapy transgender” and searches including the associated MeSH-terms for transgender and words emphasizing the interruption of therapy, didn’t bring forward any usable paper.

4. Results

4.1 Effects of testosterone on the cardiovascular system, hemostasis and coagulation Testosterone administration is considered safe in transgender men, without increased morbidity risks. Most studies agree on the fact that administration of testosterone in transgender men increases body weight and body mass index (BMI). Jarin et al (38) studied 72 transgender men starting with testosterone administration, starting with a weekly dose of 25 mg, with subcutaneous doses of 25, 50 or 100 mg at subsequent visits. They started with a mean baseline BMI of 26 kg/m², which raised to 27,3 kg/m² after 6 months of therapy (a mean value raise of 1,3 kg/m²). Higher BMI values result in a significantly higher risk of cardiovascular morbidity and mortality compared with healthy BMI values (BMI 18,5 kg/m² to 24,9 kg/m²), and also results in a longer period of life living with cardiovascular morbidity. Hazard ratios for incident cardiovascular diseases were 1,21 and 1,32 in respectively middle- aged men and women with overweight (BMI of 25,0 kg/m² to 29,9 kg/m²); 1,67 and 1,85 for obesity (BMI 30 kg/m² – 39,9 kg/m²) and 3,14 and 2,53 for morbid obesity (BMI 40 kg/m² and higher) (39)

Testosterone administration also consistently increases systolic blood pressure and possibly diastolic blood pressure, whilst possibly negatively influencing the lipid profile by increasing triglycerides and LDL-cholesterol (low-density lipoproteine cholesterol) and decreasing HDL- cholesterol (high-density lipoproteine cholesterol) values. These alterations are also undesired in the context of cardiovascular risk (40). However, this seems to have no influence on the morbidity and mortality rates in transgender men compared to controls (41).

4.1.1 Effects of testosterone on platelet adhesion & aggregation Campelo et al (19) reported in 2012 that testosterone promotes vasodilatation in rat aortic strips. Lower concentrations of testosterone induce vasorelaxation, based on an endothelium dependent process, by synthesis of NO. Higher concentrations of testosterone have a direct, endothelium independent, vasodilating effect on vascular muscle cells. Besides that, dehydroepiandrostenedione DHEA, a precursor of testosterone, stimulates endothelial cell proliferation and angiogenesis. DHEA also triggers NO synthesis.

Banerjee et al (42) reported in 2014 an increase in ADP-induced platelet aggregation, in relationship with testosterone. They researched what happened when adding testosterone to platelet-rich plasma from both cisgender men and women. Administrating supplementary testosterone results in an acute amplification in the ADP-induced platelet aggregation in cisgender males, but not in cisgender females. Activation of testosterone receptors in platelets

25 might explain this phenomenon. In addition to that, a double-blind, placebo-controlled, randomized, parallel-group study of 16 healthy cisgender men (43) showed that administration of testosterone replacement therapy was associated with an increased TXA2-receptor density on the platelets, and thus an increased maximum platelet aggregation as well. This could be genetically explained, as the gene encoding for this receptor has a glucocorticoid-responsive element that may be altered by testosterone. This mechanism could contribute to the thrombotic complications in cisgender male athletes abusing androgens.

In contradiction, a prospective study performed in 2004 by Rhoden et al (44) showed no change in platelet activity upon initiation of testosterone therapy in 32 cisgender men, treated for 52 weeks with supraphysiological doses of testosterone (200mg of intramuscular testosterone enanthate weekly). Malan et al (45) suggested a negative association between testosterone and vWF, after analyzing cardiovascular variables of 173 cisgender men. This decrease of vWF opposes the observed increased platelet aggregation, when testosterone is administrated.

4.1.2 Effects of testosterone on the coagulation system Testosterone causes many different alterations in the coagulation system. Brodin et al (46) states that testosterone has a positive association with t-PA. Meanwhile the precursor of testosterone, dehydroepiandrosterone, reduced both t-PA and plasminogen activator 1. Caron et al (47) reported that plasminogen activator inhibitor in plasma is negatively related to testosterone in normogonadal cisgender men. They found the association when examining blood values in 54 normogonadal cisgender men in 1989. Rhoden et al (44) found a decrease in prothrombotic factors, prothrombinase activity and protein C and S, but their effects are said to be equalized by an increase in antithrombin III and fibrinolytic activity. In a study of 32 hypogonadal cisgender men treated for 52 weeks, they investigated the effects of supraphysiologic doses of 200 mg testosterone injections. Malan et al (45) found an association between low testosterone and an increase of fibrinogen and plasminogen activator inhibitor type I when analyzing cardiovascular variables of 173 cisgender men. Andreotti et al (23) also observed this reduction in plasminogen activator inhibitor type I and found that it was linked to the reduction in expression of that substance. Besides that, they also found an increased level of mRNA and protein expression of t-PA and TFPI, under influence of testosterone. These effects got diminished by adding flutamide, a testosterone receptor antagonist, and can be seen as proof that the effect is indeed caused by testosterone. On the other hand, Andreotti et al observed that supraphysiological concentrations of testosterone reduce t-PA and TFPI expression. This could be a second mechanism contributing to thrombotic complications in male athletes abusing androgens. Research on the effect of testosterone on human umbilical vein endothelial cells revealed these effects (23, 46).

In a study of Mueller et al (41) with long-term administration of long-acting 1000 mg testosterone undecanoate every 3 months, for 1 year, in 35 transgender men, it has been observed that the four monitored coagulation parameters (PT, APPT, thrombin time and fibrinogen concentration) showed no significant differences with baseline values. Furthermore, Pelusi et al (11) reports that APTT and fibrinogen did not change while PT slightly increased significantly at week 54 of testosterone treatment in transgender men. Although testosterone causes alterations in concentrations of prothrombotic and antithrombotic coagulation substances, it should be noticed that overall, testosterone causes little to no changes on the action of the coagulation system. This is also deduced by the fact that no increased rate of venous thrombosis is observed in transgender men on gender affirming hormone therapy (37).

4.1.3 Effects of testosterone on the hemoglobin, hematocrit and lipid system The most important effect of testosterone is an increase in hemoglobin and hematocrit. Multiple studies confirm this. Mueller et al (41) reported that long-term administration of testosterone in transgender men causes a significant increase in hematocrit and hemoglobin, with a mean increase of 5% in hematocrit. They observed 35 transgender men during one year, taking a long-working 1000 mg testosterone undecanoate injection every three months. Jarin et al (38) confirmed these findings for adolescent transgender men.

A study in 18 hypogonadal cisgender men by Snyder et al (48) confirms that testosterone plays an important role in stimulating erythropoiesis. Hypogonadal cisgender men have lower hemoglobin levels than age-matched controls, which increased from subnormal to a normal range after the initiation of testosterone therapy.

Supraphysiologic levels of hematocrit can have serious consequences because the rise in blood viscosity can harm the vascular circulation (44). That’s why the Endocrine Society recommended to temporarily halt testosterone therapy in hypogonadal men with hematocrit levels over 0,54 g/l (37). In the latest guidelines this rule is no longer included (8). Although there is an increase in blood viscosity, Shatzel et al (37) noted that the secondary erythrocytosis does not particularly lead to an increased thrombotic rate. In addition Defreyne et al (34) stated that there are no reasons to assume that the observed mild increase in serum hematocrit levels is associated with an increased thrombotic risk on short term, as the study only saw a maximum measured hematocrit level of 54,0% in transgender men, without observing any thromboembolic events during follow up. This prospective study also showed lower erythrocytosis rates when using testosterone undecanoate, compared to testosterone esters and testosterone gel. When concerned about hematocrit levels in transgender men, changing therapy to testosterone undecanoate may prevent unnecessary interruptions in hormonal treatment.

Testosterone influences the lipid profile as well. Pelusi et al (11) reported that with testosterone therapy in transgender men, plasma HDL declines significantly, and LDL increases significantly. Total cholesterol and triglycerides did not change. However, there is no significant increase in cardiovascular risk reported. Mueller et al (41) observed a significant decline in the sex hormone-binding globulin SHBG and in HDL in transgender men. Triglycerides and total cholesterol levels did not change. These alterations in lipid status, together with the stimulated erythropoiesis, could be an argument for increased risk of cardiovascular events. Nevertheless, the morbidity and mortality rates in transgender men are not different from controls (10).

Concluding, one could say that low and normal endogenous testosterone are no risk factors for cardiovascular disease. However, supraphysiological testosterone, e.g. androgen abuse in athletes, is associated with acute vascular events. No risks are included for transgender men during gender affirming hormonal therapy because the aim is a physiological male testosterone concentration (46).

4.2 Testosterone replacement therapy in hypogonadal cisgender men and surgery Martinez et al (49) studied the risk of venous thromboembolism in hypogonadal men receiving testosterone treatment in an randomized controlled trial (RCT). Starting the treatment showed an increased risk of venous thromboembolism of 63% during the first six months, with a decrease thereafter. During the first six months of testosterone use, the increased risk results in 10.0 additional venous thromboembolisms above the base rate of 15.8 per 10000 person years. Although this increased risk is still relatively low, it’s comparable to the risk of venous thromboembolism in women and the increased risk among women using oral contraceptives or oestrogen replacement therapy for menopause. The transient increased risk due to testosterone might be explained due to the effects on thrombosis via decreased fibrinolysis. This transient increase might be later nullified due to a provoked secondary response with more fibrinolysis, explaining why it only occurs in the first 6 months. Another study of Cole et al (50) followed 3422 hypogonadal men on testosterone replacement therapy during 17 months to conclude that there is no increased risk of thromboembolism and cardiovascular disease.

The example above illustrates how multiple studies on the subject might come to conflicting conclusions. Clavell et al (51) acknowledged this controversy regarding testosterone replacement therapy in cisgender hypogonadal men in relation with cardiovascular events and reviewed the available literature in 2018. The conclusion was that the overall benefit and safety of exogenous testosterone was yet to be established for testosterone replacement therapy.

The relationship between testosterone replacement therapy and cardiovascular risks is still unclear as some studies find a positive association, while others find a negative association and yet others find no association at all.

Hypogonadal cisgender men treated with higher than approved doses of testosterone replacement therapy, thus having higher serum testosterone concentration, showed an increased risk in cardiovascular events. Other studies report an association between low testosterone levels and increased cardiovascular mortality. There is an optimal testosterone level associated with reduced cardiovascular mortality, with concentrations above and below this level increasing cardiovascular mortality (31). A possible explanation for this testosterone optimum is in the fact that testosterone has both positive and negative effects on mechanisms linked to cardiovascular health and diseases. Testosterone replacement therapy decreases fat mass, reduces insulin resistance and thus modifies components of the metabolic syndrome (31). Conversely, testosterone affects mechanisms and molecules in the coagulation and hemostasis system that can possibly increase cardiovascular risk as mentioned in the previous paragraphs.

No literature mentions the interruption of testosterone replacement therapy before elective surgery. Not much information was found about being on testosterone replacement therapy before the period of surgery and its effects on postoperative outcomes. Argalious et al (31) were the first ones to investigate if cisgender men on testosterone replacement therapy undergoing noncardiac surgery have an increased risk of postoperative mortality and cardiovascular morbidity. 947 patients on testosterone replacement therapy and 4598 control patients were included. No significant difference was observed on postoperative in-hospital mortality and cardiovascular morbidity, including myocardial infarction, stroke, pulmonary embolism and deep venous thrombosis.

4.3 Effects of oestrogens on the cardiovascular system, hemostasis and coagulation Sex hormones influence the risk of developing cardiovascular disease and explain the gender differences seen in epidemiologic data. This data suggests that oestrogen is protective against coronary heart disease. Fifty percent of this protective effect could be assigned to the beneficial effect of oestrogens on the lipid profile (52). Miller et al (53) report that the incidence of adverse thrombotic events increases significantly at the menopause, which implies that the absence of female sex steroid hormones plays an important role in obtaining the disease. Oestrogen treatments for postmenopausal women show a delay in these age-related cardiovascular events. This contradicts other RCTs where one could see an increase in thrombotic events in older asymptomatic post-menopausal cisgender women with oestrogen treatments. Those thrombotic events include strokes, myocardial infarction and blood clots (52). One meta- analysis demonstrated an increased risk for venous thrombosis, four times higher compared with controls in the first year of therapy. Cisgender women with other cardiovascular risk factors show an even higher rate (53).

Data from Wierckx et al (9) demonstrate an increased prevalence of thromboembolic events in transgender women. Before starting gender affirming hormonal treatment, the prevalence of thromboembolic events is 9,2/1000 persons. This is 60,7/1000 persons in transgender women. Half of the thromboembolic events in transgender women occurred during the first year of treatment. One or more of the typical risk factors (smoking, immobilization and/or clotting disorder) were almost always present in those events. In transgender women, the risk of thrombosis also seems virtually higher during the first year of therapy, because thrombosis is most likely to occur in the first year after starting gender affirming hormonal treatment, but is due to the before mentioned risk factors. After the first year, only the people with a low chance on thromboembolic events remain (37).

4.3.1 Effects of oestrogen on platelet adhesion & aggregation Oestrogens influence platelets, as platelets and their precursors, the megakaryocytes, express oestrogen receptors. The oestrogen receptors ERalpha and ERbeta, are activated by oestrogens and then modulate nuclear gene transcription of the oestrogen receptors, other receptors and proteins important in hemostasis such as tissue factor pathway inhibitor, matrix metalloproteinase and nitric oxide synthase. This defines the platelet phenotype and influences the coagulation state (53). In animal models, oestrogen reduces platelet aggregation and ATP secretion. In humans, however, they seem to induce mechanisms involved in platelet activation and thus could lead to a prothrombotic state. More information has to be gathered on this subject (27).

4.3.2 Effects of oestrogen on the coagulation system Explanation for the rise in thrombotic events is found in the changes in the plasma levels of coagulation substances. Shatzel et al (37) reported that cisgender women receiving menopausal hormone replacement therapy have procoagulant shifts, with increased levels of prothrombin, factor VII, factor VIII, factor X and fibrinogen, and decreased levels of antithrombin and protein S. Furthermore, they develop an activated protein C resistance. There are no specific studies done on the effects of gender affirming hormonal therapy in transgender women on the hemostatic variables, but we expect the same results as with the menopausal hormone replacement therapy in cisgender women.

Tchaikovski et al (28) reported about the association of oral contraceptives and the coagulation system, and also found an increased plasma level of fibrinogen, prothrombin, coagulation factors VII, VIII and X, but decreased levels of factor V in cisgender women. This prothrombotic shift was observed even after progressively decreasing the oestrogen dose to the lowest amount of 15-30 µg of ethinyl oestradiol, necessary for effective contraception. Oral contraceptives also alter the plasma levels of substances important in the antithrombotic system such as decreasing the plasma level of antithrombin, protein S and TFPI. Oral contraceptives also enhance fibrinolytic activity. On the other hand, this is partially neutralized by increase in thrombin-activatable fibrinolysis inhibitor (TAFI). They also result in an increase in acquired APC resistance.

Sandset et al (54) report on the same hemostatic alterations as Tchaikovski et al and Shatzel et al. Sandset et al also adds that plasma levels of SHBG increase dose-dependently with oestrogen administration. These levels decrease with progestagen administration. The levels of SHBG show a linear relationship with the risk for venous thrombosis and thus could be used as a marker for risk of venous thrombosis. SHBG has also shown a correlation with APC resistance.

4.3.3 Effects of oestrogen on the hemoglobin, hematocrit and lipid system Komesaroff et al (55) reported that oestrogens in cisgender women alter the serum lipid concentrations, coagulation and fibrinolytic systems, anti-oxidant systems and the production of other vasoactive molecules such as nitric oxide and prostaglandins, all of which can influence the development of vascular disease. Oestrogens act as vasodilators through an endothelium-dependent effect, an increase in nitric oxide production. This results in lower blood pressure and a positive influence on the lipid profile, which leads to a decrease in cardiovascular risk profile.

Jarin et al (38) reports that gender affirming therapy in transgender women enhances cholesterol transport by raising triglycerides and HDL, while at the same time lowering LDL. In addition, a decrease in blood pressure in transgender women on gender affirming therapy has been observed.

4.3.4 Biochemical structure, dose and route of administration of the oestrogen Canonico et al (56) reviewed which factors in postmenopausal oestrogen administration contribute to the increased risk of venous thromboembolism. The route of oestrogen administration, the daily doses and the chemical structure play a role. Gender affirming hormone therapy with ethinyl oestradiol appears to have a greater impact on hemostatic values compared to oral contraceptives containing ethinyl oestradiol, which is explained by the higher dose of ethinyl oestradiol in gender affirming hormonal therapy compared to oral contraceptives, showing a dose-dependent relationship. However, ethinyl oestradiol is no longer used in the hormone treatment of transgender women (57). Studies in transgender women confirm that ethinyl oestradiol carries a higher risk for venous thrombosis and cardiovascular mortality (37) because of a much stronger prothombotic effect of ethinyl oestradiol compared to other oral oestrogens such as oestradiol valerate (36), following the higher resistance to metabolic degradation of ethinyl oestradiol (37). Research on the effect of oestradiol valerate on hemostatic values remains inconclusive (57).

Different routes of administration are coupled with different oestrogens and could thus lead to confounding. The oestrogenic compound of oestrogen therapy is either 17 beta-oestradiol, the bio-identical oestrogen, or synthetic ones such as oestradiol valerate and conjugated equine oestrogens. 17 beta-oestradiol can be given orally and transdermally. The synthetic ones are only administered orally (56).

Overall one could say that there is an increase of activation markers of coagulation and, in response, an increased fibrinolysis, confirmed by increased plasma levels of prothrombin fragments 1 and 2 and D-dimers. An increased APC resistance and baseline thrombin generation have also been associated with oral oestrogens. Oral oestrogens induce blood coagulation activation and a hypercoagulatory state, consistent with the increased thrombotic risk of using oral oestrogens. Administration of transdermal oestrogens in postmenopausal women show inconsistent reports on their effects on coagulation factors. Some say that there are no changes, others find alterations in fibrinogen, factor VII or factor VIII. No changes have been observed in promotors and inhibitors of the fibrinolytic system. Most of the studies confirm that the transdermal oestrogens have no effects on markers of coagulation and fibrinolysis, and thus do not show an effect on hemostasis (56).

Tchaikovski et al (28) doubts the big influence of difference in route of administration, they state that alternative routes of administration to oral administration, such as transdermal administration, do not decrease the risk of venous thrombosis. Besides many coagulation factors being synthetized in the liver, a big part of the proteins involved in coagulation, such as large fractions of protein S and TFPI, are synthetized in endothelial cells. This could explain why there is no big influence of the first-pass liver effect, namely because of difference in systemic concentration of the oestrogens, which influences the synthesis of the proteins in the endothelial cells. Further nuance is brought by a study of Toorians et al (57) who reported no significant change in the levels of protein C or protein S in transgender women treated with cyproterone acetate only or with transdermal 17-beta-oestradiol and cyproterone acetate combined. However, the incidence of venous thrombosis in transgender women on oral ethinyl oestradiol was higher compared to therapy with transdermal 17-beta-oestradiol. When using oral ethinyl oestradiol, one could see a significant alteration of protein C and protein S. Using oral 17-beta-oestradiol did not show differences in levels of protein C, protein S and prothrombin compared to oral ethinyl oestradiol, thus confirming first-pass liver effects as the cause of differences with transdermal 17-beta-oestradiol. Shatzel et al (37) confirmed that transdermal oestrogens indeed have less effects on the hemostatic variables compared to oral administration, which was also observed in transgender women.

4.4 Hormonal contraception in cisgender women and surgery When cisgender women are on combined oral contraceptives (COCs) and exposition to trauma or surgery, the thrombotic risk rises 5 – 12,5 times. Trenor et al. (29) recommends to discontinue COCs 4 to 6 weeks before surgery and an adjusted thromboprophylaxis if within the first year of COC use. The rationale behind this is that COCs alter plasma concentration of factor X, fibrinogen and antithrombin III, which are normalized again after stopping administration of COCs between 2 – 6 weeks (58).

Further on, the WHO (World Health Organization), UKMEC (United Kingdom Medical Eligibility Criteria for contraceptive use) and the CDC (U.S. Medical Eligibility Criteria for contraceptive use) give some extra nuance by mentioning that, in major surgery with prolonged immobilization, it is an unacceptable health risk to take COCs, with the UKMEC suggesting to discontinue COCs at least 4 weeks prior to surgery, whilst in major surgery without prolonged immobilization the advantages generally outweigh the risks, thus not having to stop the contraception per se (29). Progestin-only and intrauterine contraceptives are not expected to be associated with an increased perioperative thrombotic risk (59). An aesthetic surgeon reported two cases of postoperative venous thromboembolism in cisgender women using a Nuvaring, a vaginal ring releasing a lower dose of hormones than COCs carry. A suggestion is made to also halt vaginal ring use 4 weeks prior to surgery until 2 weeks after surgery (60).

After an extensive literature search, no RCTs were found in which the perioperative event rate of thromboembolic events is compared in cisgender women on hormonal contraceptives or COCs in particular, compared to cisgender women not taking them.

A study of Getahun et al (61) illustrates the differences in how venous thromboembolism (VTE) occurs in cisgender women on hormones compared to transgender women. In a big cohort study on the incidence of cardiovascular events in transgender persons, with 2842 transgender women and 2118 transgender men included and 48686 cisgender men and 48775 cisgender women as control populations, the sample is of a statistically powerful size, compared to earlier studies. In the cisgender women, VTE rates increased relatively rapidly after the administration of hormones began, to then decrease and plateau by 5 years of follow-up. In transgender women who initiate hormone administration, venous thromboembolism rates only increased after 2 years of follow-up, and continued to rise for another 5 to 6 years. The study reported cross-sex oestrogen as a risk factor for venous thromboembolism, as transgender women had a higher incidence compared to cisgender women and cisgender men, and this difference became bigger the longer the follow-up. The risk-differences after 2 years were 4,1 per 1000 persons compared to cisgender men and 3,4 per 1000 persons compared to cisgender women, to increase to 16,7 per 1000 persons compared to cisgender men and 13,7 per 1000 persons compared to cisgender women after 8 years of follow up. The pattern of venous thromboembolism in transgender women is also different from cisgender women receiving hormone replacement therapy.

4.5 Effects of cyproterone acetate on the cardiovascular system, hemostasis and coagulation Cyproterone acetate, the progestagin with anti-androgenic effects, is often used in treatment of transgender women and research on the influence on the cardiovascular system and coagulation are scarce.

Andersen et al (62) reported in 1999 that cyproterone acetate does not reduce the oestrogen effects on hemostatic variables.

Later, in 2003, Toorians et al (57) concluded that cyproterone acetate caused a slight but significant increase of the normalized APC sensitivity rate, thus causing a mild APC resistance, which could be explained by the antiandrogenic effects of cyproterone acetate. This could theoretically be interpreted as an increased risk for venous thrombosis in persons taking cyproterone acetate, although no study or reports on this association are available.

In 2014, Van Hylckama Vlieg et al (63) stated that several studies observing oral contraceptives containing cyproterone acetate (the so-called Diane ®, Claudia ®, Daphne ®, Elisamylan ® pills, containing 2 mg of cyproterone acetate, with a high dosage of ethinyl oestradiol 35 µg) show an increased risk of venous thrombosis, compared to other oral contraceptives, although other studies deny this. Their analysis confirmed an 1,7-fold increased risk of venous thrombosis when comparing an oral contraceptive containing cyproterone acetate with an oral contraceptive containing levonorgestrel as the progestin, both having the lowest amount of oestrogen of 20 µg. More specific studies on specific target audiences could be useful to further on investigate if and how cyproterone acetate influences the risk on having venous thrombosis.

5. Discussion

5.1 Thoughts and limitations regarding current literature As mentioned before, literature on the need to stop hormone treatment prior to surgery in transgender people is scarce to nonexistent, only advising to stop gender affirming hormone treatment 2 weeks prior to surgery until regaining mobilization in transgender women (15). Literature about surrogate groups such as cisgender women on hormonal contraception and cisgender men on testosterone replacement therapy were investigated to look for parallels. However, data in relation to these groups was limited as well.

5.1.1 Studies concerning oestrogens and cisgender women on hormonal contraceptives Shatzel et al (37) noticed in a review on thrombotic issues in transgender people that no specific studies on the effects of gender affirming hormonal therapy in transgender women on the hemostatic variables are done, expecting the same results as oestrogen administration in cisgender women have (e.g. with the menopausal hormone replacement therapy in cisgender women). To extrapolate this information to the situation of transgender women taking gender affirming hormone therapy multiple factors have to be considered. Firstly, the composition in COCs and gender affirming hormone therapy might exist of both oestrogens and progestogens, yet the concentration and specific molecules might not be the same. Secondly, effects of these compounds might differ or alter between cisgender women and transgender women. Unknown factors might contribute to the different VTE profile seen in the study of Getahun et al (61) about cisgender women on hormones compared to transgender women on gender affirming hormone therapy. The VTE profile differs in transgender women compared to the cisgender women by VTE rates only increasing after 2 years of follow-up, continuing for another 5 to 6 years, showing a higher risk-difference the longer the follow up.

The interruption of COCs administration in cisgender women is advised 4 weeks prior to major surgery with prolonged immobilization (29), due to the 2 to 6 weeks needed after stopping COCs to wash out alterations in plasma concentrations of molecules involved in thrombotic pathways (58), but not in major surgery without prolonged immobilization, if no particular cardiovascular comorbidities exist (29). This raises the question if the interruption period of 2 weeks in transgender women is not too short, and should become a 4-week period (see 5.2 Recommendations on future research), bearing the above called factors in mind.

Figure 5 resumes the effects of oestrogen on the coagulation & fibrinolytic system. On a platelet-level, more platelet aggregation & platelet mediated ATP secretion was found. Coagulation factor VII, factor X, prothrombin and fibrinogen increase in concentration. Anti- coagulant substances such as TFPI, anti-thrombin III and protein C decrease in concentration.

Paradoxically, a decrease in factor V and an increase in TAFI were observed. All these alterations together result in an oestrogen-induced pro-thrombotic state, resulting in the thrombotic risk in cisgender women on COCs (29).

Figure 5: Scheme of the observed oestrogen effects on the coagulation & fibrinolytic system. Red arrows and text indicate changes in concentration of the concerned substance. Adapted from Bonhomme F et al (18) and Johari V et al (20).

Comparing different oestrogen treatments, ethinyl oestradiol showed a strong prothrombotic effect, especially when compared to oestradiol valerate (36). However, ethinyl oestradiol is discontinued, with oestradiol valerate being the standard care in transgender women. Still, more research on the precise effect of oestradiol valerate on hemostatic values is desired (57). Shatzel et al (37) also confirms that transdermal administration of oestrogens has less effects on the hemostatic variables compared to oral administration, which has also been observed in transgender women. Recommending the use of transdermal administration of oestrogens,

37 rather than oral administration might be positive regarding cardiovascular risks, with only skin adhesion problems and skin reactions as side effects (3).

Besides oestrogens, anti-androgens and sometimes progestogen are administered in transgender women. No clear research on these compounds in transgender women has been done yet. Tangpricha et al (14) suggests that progestogens added to the gender affirming hormone therapy scheme might increase the thrombo-embolic risk, as this is seen in premenopausal cisgender women on COCs. Van Hylckama Vlieg et al (63) saw an increased risk of venous thrombosis in cisgender women on oral contraceptives containing cyproterone acetate, compared to those without, which points towards the use of cyproterone acetate being a cardiovascular risk factor. Further research on cyproterone acetate, other anti-androgens and progestogen administration in transgender women is needed.

5.1.2 Studies concerning testosterone and hypogonadal men on testosterone replacement therapy Some studies concerning the molecular effects of testosterone on the coagulation and fibrinolytic system show limitations. Campelo et al (19) found that testosterone promotes vasodilatation on rat aortic strips. Research should be done on human specimens to be sure this conclusion is not contradictory to what happens in humans such as Artero et al (27) mentioned. The increase in ADP-induced platelet aggregation due to testosterone, found by Banerjee et al (42), was found only in cisgender males, not in cisgender females, resulting in the question what the precise effect in transgender people is. Rhoden et al (44) studied the testosterone effect on platelet activity, with supraphysiological doses of testosterone of 200 mg intramuscular testosterone enanthate weekly, while the dose used in transgender men is 100 – 200 mg intramuscularly every 2 weeks or 50 – 100 mg subcutaneously every week (8). The study of Rhoden et al is thus not representative for the gender affirming hormone treatment in transgender men. These three cases show that effects of testosterone are not consistent.

Figure 6 resumes the effects of testosterone on the coagulation & fibrinolytic system. On a platelet-level, more platelet aggregation is explained due to activation of testosterone receptors and a higher density of TXA2-receptors. Testosterone promotes endothelial cell proliferation and increases NO concentration, contributing to angiogenesis. The decrease in vWF, plasminogen activator inhibitor, protein C and protein S confront the increase in TFPI, antithrombin III and tPA. Overall however, it was seen that testosterone causes little to no changes in the outcome of the coagulation system measured by the PT, APPT, thrombin time and fibrinogen concentration in transgender men (41) thus explaining the unaltered risk of venous thrombosis in transgender men receiving gender affirming hormone therapy (37).

Figure 6: Scheme of the observed testosterone effects on the coagulation & fibrinolytic system. Red arrows and text indicate changes in concentration of the concerned substance. Adapted from Bonhomme F et al (18) and Johari V et al (20).

Currently, gender affirming hormone therapy is only interrupted before surgery in transgender men on Sustanon, because of the rise of serum oestradiol following the testosterone peak right after Sustanon injection. Whilst there is still debate on the cardiovascular risks in cisgender men on testosterone replacement therapy (51), a cohort study of Defreyne et al (34) showed no thrombo-embolic events in transgender men. The cohort study of Getahun et al (61) confirms no increased risk for thromboembolic events in transgender men. The limited literature on hypogonadal cisgender men taking testosterone replacement therapy before surgery showed no increased risk of postoperative mortality or cardiovascular morbidity (31). Yet again, caution is recommended in drawing parallels between transgender men on gender affirming hormone therapy and hypogonadal cisgender men on testosterone replacement therapy.

5.2 Recommendations on future research Specific literature search on the perceived mental and physical effects of interrupting gender affirming hormone therapy administration in transgender women yielded no results. Studies on these perceived effects of hormone interruption during the two weeks before surgery are desired, to be able to consider whether or not interrupting gender affirming hormone therapy prior to surgery is advantageous or not and whether there are perceived side effects of interrupting therapy.

The rationale behind stopping administration of COCs 4 weeks prior to major surgery with immobilization in cisgender women is that it takes 2 – 6 weeks for concentrations of antithrombin III, fibrinogen and factor X to normalize again (58). However, this is an intermediate endpoint predicting the hard endpoint, occurrence of venous thromboembolic events, to decrease. No RCT with patients stopping COCs 4 weeks prior to major surgery wjth immobilization and controls not stopping COCs 4 weeks prior to surgery, which would be ethically problematic, have been performed.

New studies on the influence of interrupting gender affirming hormone treatment is needed in transgender women and in transgender men on Sustanon who have an increased level of oestradiol following the testosterone peak just after administration. Due to the increased thromboembolic risk seen with oestrogens, priority is on a study assessing the thromboembolic risk in transgender women.

A possible study design would be a randomized controlled trial consisting of three age-matched groups. One group consists of transgender women who continue to take their gender affirming hormone therapy preoperatively, a second group consists of transgender women who stop gender affirming hormone therapy two weeks prior to major surgery (thus following current policy), and a third group consists of transgender women who stop gender affirming hormone therapy four weeks prior to major surgery. The suggestion for this third group follows the fact that in cisgender women the administration of COCs is stopped already 4 weeks instead of 2 weeks prior of major surgery with prolonged immobilization (29). The study samples should thus be followed starting from 4 weeks prior to elective major surgery, until 3 months after surgery, to be able to assess the perioperative thromboembolic risk correctly. A power calculation should be performed to determine the sample sizes of the three groups in accordance with the thromboembolic risk.

Multiple endpoints should be observed. Besides a hard endpoint, the perioperative occurrence of venous thromboembolic events, intermediate endpoints such as thrombotic parameters PT, APPT, thrombin time, factor X concentration, antithrombin III concentration and fibrinogen concentration should be observed as well. These intermediate endpoints should be measured

40 on multiple moments (4 weeks prior to surgery, 2 weeks prior to surgery, at the time of surgery, 2 weeks post-surgery, 4 weeks post-surgery, 2 months post-surgery and 3 months post- surgery). Also important is to take psychological questionnaires about mental health on these exact same points of time, to be able to investigate a possible effect of treatment interruption on mental health. Exclusion criteria for the study are patients of cardiovascular diseases which have an influence on the cardiovascular profile, thrombosis and coagulation processes such as diabetes, hypertension, clotting diseases, bleeding diseases etc.

With this proposed study design, a more evidence-based approach on interrupting hormone treatment in transgender women prior to elective surgery is possible by comparing multiple interruption regimes and a non-interruption regime, in accordance with a possible influence on mental health.

5.3 Conclusion Taking in account that the risk of cardiovascular disease is increased in transgender women, and that oestrogens show prothrombotic qualities, and taking in account that the risk on venous thromboembolism is higher perioperatively in cisgender women taking COCs (29), discontinuing gender affirming hormone therapy in transgender women seems a thoughtful decision awaiting elective major genital surgery with a period of immobilization.

In transgender men, discontinuing gender affirming hormone therapy before major surgery with immobilization is not always implemented (37), backed-up by testosterone administration in transgender men showing no alterations in thrombotic parameters such as PT, APPT, thrombin time and fibrinogen concentration (41), and because hypogonadal cisgender men on testosterone replacement therapy show no additional perioperative risks (31). Recommending interruption of gender affirming hormone therapy before major surgery in transgender men seems unnecessary when on Nebido. More research is needed to assess if interruption of gender affirming hormone therapy in transgender men on Sustanon is useful.

Concluding, more studies are needed on the perioperative thrombotic risk of transgender men on Sustanon and especially transgender women, the thrombotic effects of oestrogens and the anti-androgens in transgender women, whilst also studying the perceived mental and physical effects of interrupting gender affirming hormone treatment.

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