Background
Improvement of survival after heart transplantation (HT) brings new challenges in recognizing and managing endocrine complications in the patient population [
1]. The most dynamic period with the greatest changes in endocrine profile is the first post-transplant year [
2‐
4] resulting in vitamin D deficiency, hypogonadism, post-transplant diabetes mellitus (PTDM), and bone loss [
2‐
9].
Many factors have been associated with endocrine changes early after HT: association of glucocorticoid treatment with bone loss and vertebral fractures (VFs) is well established [
10]. Treatment with a high-dose glucocorticoid regimen leads to accelerated bone loss in the first 6 months after HT, after which the rate of decline of bone loss slows down. Bone loss is predominantly trabecular [
10]. In animal models, the calcineurin inhibitors (CNIs) cyclosporine A and tacrolimus are further associated with bone loss and increased bone turnover [
11]. There is also evidence of CNI-associated bone loss in clinical settings, yet it is difficult to assess their separate impact on post-transplant osteoporosis since, in the early period, CNIs are always administered with glucocorticoids [
12].
Furthermore, glucocorticoids and CNIs both cause hyperglycemia and PTDM [
13]. High doses of glucocorticoids are also associated with hypogonadism, with the lowest levels of testosterone being observed immediately after HT [
3]. Secondary hyperparathyroidism as a consequence of reduced kidney function, and decreased 25-hydroxycholecalciferol (25OHD) is also expected in the early post-transplant period. Low 25OHD is related to decreased dietary intake of 25OHD-containing foods, limited sun exposure and the effect of glucocorticoids on absorption and metabolism of 25OHD [
14,
15]. Reduced kidney function is related to the underlying cardiac disorder (cardio-renal syndrome) and treatment with CNI [
16]. The prevalence of secondary hyperparathyroidism after HT, ranging from 30 to 75%, has been assessed in only a few studies [
6,
17,
18]. Similarly, thyroid function in the early post-transplant period has remained largely unaddressed [
19‐
21].
Methods
Aims
The aim of this study was to assess the endocrine profile and endocrine management in a well-defined national cohort of heart transplant recipients in the early post-transplant period. We determined glucose metabolism, male hypogonadism, thyroid function, calciotropic axis, BMD, incidence of osteoporotic fracture and related treatment intervention.
Study design, setting of the study, patient population
We conducted a retrospective cohort study on 123 consecutive HT recipients who received transplants at the Advanced Heart Failure and Transplantation Programme, Department of Cardiology, University Medical Center Ljubljana between the years 2009 and 2018. After discharge, all HT recipients were referred to the Endocrine Outpatient Clinics, Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Center Ljubljana for protocol-based endocrinological screening. Patients included in this analysis had at least one endocrine follow-up visit within the 1st post HT year. We excluded patients who failed to complete the endocrine outpatient follow-up visit within the 1st post-transplant year, pediatric patients (< 18 years of age) and patients with major postoperative non-fatal complications including post-operative renal replacement therapy or tracheostomy. Altogether, 39 patients were excluded: 37 of those patients had major postoperative complications (21 non-fatal, 16 fatal); 2 patients were pediatric (< 18 years of age).
At the time of endocrine assessment, all patients were treated with standard triple immunosuppression therapy (steroids, CNI, mycophenolate mofetil). The maintenance dose of methylprednisolone in all patients was 4 mg per day. The doses of cyclosporine and tacrolimus were regularly monitored and adjusted to target serum concentrations (cyclosporine A 200–250 μg/L; tacrolimus was 6–10 μg/L). The maintenance dose of mycophenolate mofetil in patients treated with cyclosporine A was 3000 mg per day and in patients treated with tacrolimus, 2000 mg per day. Per protocol, all HT recipients additionally received vitamin D (cholecalciferol and alphacalcidiol) and Ca2+ supplementation (Ca2+ carbonate 1 g per day).
In all patients, we collected epidemiological and transplant-related data and data on patients’ medical therapy. Specifically, we focused on collecting data on vitamin D deficiency, bone mineral density (BMD), history of low energy fractures, hypogonadism (in male recipients), PTDM, thyroid and parathyroid function. Normal endocrinological profile was defined as normal testosterone in men, normal TSH, iPTH, serum calcium, glucose homeostasis, sufficient concentration of 25OHD, normal BMD, and no fractures.
Assessment of anthropometric parameters
Height was measured with an accuracy of 1 cm and body weight with an accuracy of 1 kg. Body mass index (BMI) was calculated as the weight in kilograms divided by square of height in meters.
Biochemical analysis
We collected data for testosterone, free testosterone, sex hormone binding globulin (SHBG), thyroid-stimulating hormone (TSH), thyroxine (fT4) and triiodothyronine (fT3), intact parathyroid hormone (iPTH), corrected calcium, 25OHD, collagen type 1 cross-linked C-telopeptide (CTX), N-terminal propeptide of procollagen type 1 (P1NP), glucose, creatinine and estimated glomerular filtration rate (eGFR) calculated with Modification of Diet in Renal Disease Study equation [
22]. Renal dysfunction was defined according to KDIGO guidelines [
23].
Primary hyperparathyroidism is defined as corrected calcium increased above the upper level of normal (2.65 mmol/L) or in the upper half of normal (above 2.45 mmol/L) and iPTH increased above 65 pmol/L. Secondary hyperparathyroidism is reported if calcium decreases below the lower level of normal value or in the lower half of normal (lower than 2.45 mmol/L) and iPTH increases above 65 pmol/L [
24].
Normal levels of 25OHD are defined as serum levels above 75.0 nmol/L. Insufficiency of 25OHD is defined as serum level 50.0–74.9 nmol/L, mild deficiency as 25.0–49.9 nmol/l and severe deficiency below 24.9 nmol/L. BMD was measured in the lumbar spine, hip and femoral neck by dual-energy X-ray absorptiometry (DXA) with the use of Discovery DXA System (Hologic, Bedford, MA). Measurements are provided in g/cm
2 and T-scores. Osteoporosis is defined as T-score ≤ − 2.5 SD, osteopenia as − 1.0 SD < T-score > − 2.5 SD and normal BMD as T-score ≥ − 1.0 SD at any measured site. Osteopenia and osteoporosis are defined in accordance with the International Osteoporosis Foundation [
25]. Osteoporotic VFs were confirmed by X-ray in patients with suspected fractures based on history and medical examination. Non-VFs were recorded based on history and medical records. Numbers of patients who were treated with intravenous or oral bisphosphonates (BP) or teriparatide within the 1st year post HT were recorded.
Hypogonadism assessment
Testosterone was measured using coated tube RIA (DiaSorin S. p. A., Salluggia, Italy and Diagnostic Products Corporation, LA) and SHBG with chemiluminescent immunoassay method (Immulite 2000 Analyzer, Siemens Healthcare, Erlangen, Germany). Low levels of total testosterone were defined as testosterone serum levels of less than 11.0 nmol/L [
26]. Hypogonadism was defined as testosterone deficiency with clinical signs of hypogonadism.
Assessment of thyroid function
TSH was measured with the immune method of anti-FITC monoclonal antibody (ADVIA Centaur XP, Siemens Healthcare, Erlangen, Germany), fT3 and fT4 with the chemiluminescent immunoassay method (ADVIA Centaur XP, Siemens Healthcare, Erlangen, Germany), iPTH with the chemiluminescent immunoassay method (IMMULITE® 2000, Siemens Healthcare, Erlangen, Germany) and calcium with the CPC method (ADVIA 2400, Siemens Healthcare, Erlangen, Germany). Free T4 and fT3 were measured only in cases of high or low TSH. Normal levels of TSH were defined as 0.55–4.78 mE/L, fT4 11.5–22.7 pmol/L, fT3 4.6 pmol/L. Hypothyroidism was interpreted as increased TSH with normal or reduced fT3 and fT4 and as substitution of levothyroxine [
27]. Hyperthyroidism was interpreted as treatment with thyreostatic drugs or reduced TSH with normal or increased fT3 and fT4 [
28].
Glucose was measured with the standard oxidase method (Beckman Coulter Glucose Analyzer, Beckman Coulter Inc. CA, USA). The diagnosis of post-transplant diabetes mellitus (PTDM) was defined as newly diagnosed fasting glucose ≥7.0 mmol/L on more than one occasion, random glucose ≥11.1 mmol/L with symptoms, two-hour glucose after a 75 g oral glucose tolerance test of ≥11.1 mmol/L, and HbA1c ≥6.5% [
29]. We identified patients with diabetes mellitus (DM) and defined type of DM in each individual. We acquired information about anti-diabetic medical therapy and/or any changes in the anti-diabetic medical regimen after HT from institutional patient digital records.
Statistical methods
All statistical analyses were done for descriptive purposes. Numerical variables are presented as median (interquartile range), and categorical variables with proportions. 95% binominal (Clopper-Pearson) exact confidence intervals for proportions were computed. We used the Mann-Whitney test to compare differences in gender, age and treatment with zoledronic acid (ZA). Correlation of iPTH with eGFR, level of chronic kidney disease and 25OHD was calculated with Spearman’s rank correlation coefficient. P value of < 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY).
Discussion
The current study presents a thorough assessment of the endocrine profile of HT recipients in the early post-transplant period. Our main findings suggest that the 25OHD deficiency is most prevalent, followed by low BMD and hypogonadism in males. Other disorders such as diabetes mellitus, hyperparathyroidism and thyroid dysfunction are also common. Most patients had a coincidence of two or three disorders; less than 5% had no endocrine irregularities.
In our cohort, 11.4% of the patients developed PTDM within the first 12 months post HT. These results are in accordance with those provided by Ye et al. [
5], where cumulative rates of PTDM were 11.1, 32.0 and 38.4% after 12-, 24- and 36-months post HT respectively [
5]. With the documented time-dependent increase in incidence of PTDM after HT, it is clear that other factors in addition to glucocorticoids, in particular CNIs, are involved in its pathogenesis, and that this entity differs from steroid DM [
30]. CNIs decrease insulin release by direct toxic effect on pancreatic β-cells. Cyclosporine A binds to cyclophilin D in the mitochondrial permeability transition pore and interferes with insulin stimulation. CNIs regulate the dephosphorylation of the nuclear factor of activated T-cell proteins and cAMP responsive element-binding transcription factor activity-2. This decreases β-cell survival, replication and function [
13].
Many studies have reported that solid organ transplant recipients have low 25OHD serum levels [
2,
31]. In our cohort, 25OHD deficiency was common (64 (54,7%)), despite substitution of cholecalciferol that had been started before or immediately after HT in most of the patients. In a study by Stein et al., 91% of transplant recipients had low 25OHD shortly after HT, which was more than two times higher than in our cohort. While Stein et al. used calcium supplements containing also 400–600 IU of cholecalciferol and multivitamin containing 400 IU cholecalciferol [
2], our patients received in median 2000 IU of cholecalciferol per day and in median 0.5 μg alfacalcidol per day. Based on these data, we propose that treatment with cholecalciferol and/or alfacalcidiol should be started before transplantation and 25OHD serum levels should be carefully monitored throughout the duration of substitution. This is particularly relevant since vitamin D deficiency is related to significant post HT bone loss and fractures, muscle weakness and increased risk of falls [
6] and might potentiate the immunosuppressive action of calcineurin inhibitors or prednisolone [
32].
In our cohort, about 20% of patients had secondary hyperparathyroidism. The incidence of hyperparathyroidism in other studies on solid organ recipients was even higher, ranging between 30 and 100% [
5,
17,
18]. The lower incidence of secondary hyperparathyroidism in our cohort is probably related to more intensive treatment with cholecalciferol, along with alfacalcidiol and calcium supplementation that had been started before or immediately after HT, and resulted in lower incidence of hypovitaminosis D than reported in other studies [
5,
17,
18]. Interestingly, the pathophysiological mechanisms underlining the hyperparathyroidism in this patient cohort are not completely clear, as it was not related to liver or renal failure, low 25OHD or low 1.25-dihydroxycholecalciferol [
17]. We speculate that mild impairment of kidney function is sufficient for an increase in iPTH in HT recipients when compared with the general population [
5]. This hypothesis is in accordance with our data, as in our cohort, hyperparathyroidism was weakly negatively correlated to eGFR and weakly positively correlated with the level of chronic kidney disease.
In terms of thyroid disorders, recent studies suggest that hyperthyroidism is present in 21%, hypothyroidism in 13%, and low fT3 syndrome in 18 patients within 1 month after HT respectively [
20]. Our results corroborate these data, as hyperthyrosis was established in 10% and hypothyrosis in 5% of the studied cohort. Clinical effects of thyroid disorders after HT are substantial, as hypothyroidism is linked to increased length of hospitalization, and cytomegaloviral infections occur most commonly in patients with hyperthyroidism [
20]. Atrial fibrillation could be associated with hyperthyroidism, but was not observed in our patient cohort within the first post-HT year. The most important risk factor of thyroid disfunction in the HT population is a history of treatment with amiodarone [
21]. History of treatment with amiodarone was not consistently available in our cohort, and was therefore not included in the analysis.
Hypogonadism is common in male post-transplant patients [
33]. In our cohort, the incidence of hypogonadism was 40%, which is in accordance with other published studies. In a study by Fleischer, total testosterone was decreased in 63% of men after 1 month, in 33% 2 months and in 21% 6 months after HT [
3]. It is suggested that hypogonadism persists in 14% at 1 year after transplantation [
3]. It is currently assumed that treatment with corticosteroids has the most important impact on gonadal axis, followed by an influence of recent major surgery [
34,
35]. Pathophysiological mechanisms of hypogonadism after HT remain underexplored. While the effect on direct inhibition of testicular testosterone synthesis and suppression of luteinizing hormone secretion is well established in animal studies, currently there is no data to suggest any negative effects of cyclosporine A on hypogonadism in the clinical setting. Low testosterone might also be a marker of impaired graft function and an increased incidence of low-grade rejection episode early after HT [
36‐
38].
In HT patients, the most rapid bone loss associated with fractures develops during the 1st year after transplantation [
4]. Mechanisms of bone loss in this period are multifactorial and include combinations of accelerated turnover and hypogonadism [
7], low 25OHD, secondary hyperparathyroidism, dietary calcium deficiency and medication such as corticosteroids, loop diuretics, and CNIs [
39]. Our results were in accordance with the study of Carbonare et al., where 180 HT patients had BMD measured on lumbar spine and hip. BMD was reduced in osteoporotic range in 13% on the spine and 25% on the hip [
40].
17.1% of patients in our cohort were diagnosed with fractures, and 87% of VF and all non-VFs occurred in relation to osteoporosis and osteopenia. A study by Leidig-Bruckner on 105 HT patients reported the occurrence of at least one VF in 21% of patients within the first post HT year, which is higher than shown in our data [
41]. Most of the other evidence, based on older data, shows 40–48.2% incidence of VFs in HT recipients [
40,
42,
43], with T-score below − 1.5 SD being recognized as the most important risk factor [
40]. The median T-scores in our patient cohort were − 1.1 SD for the lumbar spine, − 1.1 SD for the femoral neck and 0.5 SD for the hip. Most of our patients were treated with calcium substitution and vitamin D supplementation before or immediately after HT; 65% of patients received BP within 1st year, 54% of patients were treated with ZA, and 2 patients received teriparatide. Compared to a study published by Löfdahl, where 35, 38 and 16% received calcium supplements, vitamin D supplements and BP in the first postoperative year respectively [
44], the number of treated patients in our cohort was significantly higher. ZA was recognized as the most effective antiresorptive drug for prevention of transplantation bone loss in this population [
45]. Relatively higher BMD and intensive treatment intervention with the most efficient antiresorptive drug available for this population can explain the lower incidence of fractures in our patients when compared to previously reported observations that included patients in a comparable timeframe [
40,
42]. However, even greater reduction of fractures could likely be achieved with preoperative treatment when decreased BMD is present. In a more recent cohort of 105 HT patients, where antiresorptive treatment was initiated before solid organ transplant or immediately post-transplant, the incidence of VFs in the 1st year was 3.8% [
46]. It is thus important to follow the guidelines of The International Society of Heart and Lung Transplantation guidelines for the care of HT recipients which recommend BP treatment for all HT recipients with decreased BMD within the first post-transplant year [
47].
Several limitations of our study need to be addressed. The major limitation of this study is its retrospective, single-center design, with missing data and limited availability of X-rays of the spine in asymptomatic patients, which can result in underreported clinically silent VFs. Nevertheless, our results were derived from a relatively large and a well-defined cohort of HT recipients, who were treated according to a strict and uniform immunosuppressive protocol which can significantly decrease selection bias. Additionally, this is the first study to analyze all endocrine axes in HT recipients in the early post-transplant period. With this we are confident that our results can add to the current knowledge of endocrine disorders in this patient cohort. Additionally, our results may aid in the design of future research for the endocrine management of HT recipients.
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