The need to minimize immunosuppressive intensity after kidney transplantation is particularly acute in pediatric recipients, who face a lifetime of immunosuppression-related complications. Attention has recently focused on the need to reduce exposure to calcineurin inhibitors (CNIs) in an attempt to reduce CNI-related graft nephropathy1 and other toxicities. Use of the mammalian target of rapamycin (mTOR) inhibitors everolimus and sirolimus to facilitate CNI sparing has shown considerable promise.2-6 However, evidence from the adult setting is not necessarily applicable to pediatric recipients since there is a greater propensity to nonadherence during adolescence,7 and infants and children require body surface-adjusted dosing.8 Nevertheless, although randomized controlled trials of mTOR inhibitors in pediatric kidney transplant recipients are lacking, there are prospective reports of small series of children treated de novo with an mTOR inhibitor and reduced-exposure CNI,9-12 or converted successfully from a conventional CNI-based therapy to an mTOR inhibitor13,14 in response to graft nephropathy.
Contemporary mTOR inhibitor dosing regimens do not seem to impair longitudinal growth velocity in children compared to conventional regimens.10,11,15-18 Indeed, use of an mTOR inhibitor within a steroid-free regimen may improve the rate of growth.19 Concerns have arisen, however, about the effect of mTOR inhibitors on gonadal function.20 Single-center, retrospective analyses have reported suppressed testosterone levels in adolescent21 and adult22-24 male kidney transplant recipients. In these series, most of the patients were receiving sirolimus treatment in the early 2000s, at a time when loading doses were frequently applied and target thresholds were higher than at present. Evidence regarding effects on pubertal development and reproductive hormone levels under contemporary mTOR inhibitor regimens remains sparse. One prospective10 and one retrospective study18 of children receiving an mTOR inhibitor after kidney transplantation have reported normal levels of sex hormones, but data were available from no more than 20 patients in either study. Data regarding the onset of puberty in children receiving mTOR inhibitor therapy are very limited.18
Our center has routinely measured hormone levels and recorded Tanner stages in pediatric kidney transplant patients for over 20 years. We report here a retrospective analysis which examines pubertal development and reproductive hormones in a large series of pediatric kidney transplant patients receiving mTOR inhibitor therapy.
MATERIALS AND METHODS
This was a retrospective analysis of children undergoing kidney transplantation during the period from 1993 to 2012 at the Department of Pediatric Nephrology, Hannover Medical School, Germany. Patients were eligible for analysis if they were <14 years at time of transplantation, of known gender, received cyclosporine A (CsA; Neoral®, Novartis Pharma, Basel, Switzerland) as the primary immunosuppressant, and had a minimum follow-up period of 1 year posttransplant. Observations ended at the age of 18 years, when patients transferred to the adult nephrology department.
Basiliximab induction therapy, when used, was administered on days 0 and 4 (20 mg in patients weighing >35 kg, 10 mg in patients weighing <35 kg; Simulect®, Novartis Pharma AG, Basel, Switzerland). Prednisolone (300 mg/m2) was administered during transplant surgery and in the first week posttransplant (60 mg/m2/day), then decreased successively every 7 days to 30, 15, 12, 9, and 5 mg/m2/day. From 2006 onwards, steroids were discontinued if protocol biopsy at month 6 posttransplant was normal. Total steroid dose was calculated from the start date (even if this was before transplantation, as was the case in six patients) to the date of final discontinuation or last date of follow-up, with any periods of interruption assigned a value of zero.
CsA was administered at an initial dose of 400 mg/m2/day with target trough levels of 200 to 250 ng/mL. From 1993 to 2006, the primary immunosuppression regimen at our center consisted of CsA, MMF, and steroids. Tacrolimus was only given if specified by a clinical trial protocol. Some patients were changed to low-dose CNI (at half the standard CsA dose), with MMF discontinuation and initiation of everolimus in the event that fibrosis was detected on clinically indicated biopsies. From 2006 onwards, the primary immunosuppression regimen was changed to low-dose CNI and everolimus. In this cohort of patients, the CsA dose was reduced at 2 weeks posttransplant (2006-2008) or 4 weeks posttransplant (from 2008 onwards) to 200 mg/m2/day targeting trough levels of 75-100 ng/mL, then reduced again at 6 months to target 50-75 ng/mL. From 2006 onwards, only a few patients initially received an MMF-based immunosuppressive regimen (combined with tacrolimus or CsA), who had been referred to our center in Hannover, when they were referred for transplantation to Hannover from other centers with no experience with EVR, and long-term follow-up was performed locally. There was no selection for EVR treatment according to the immunological risk before transplantation. In patients receiving conventional immunosuppression, the CsA target trough level was 150-200 ng/mL during the first 6 months after transplantation and 100-150 ng/mL thereafter. Everolimus was started at 2 weeks posttransplant at a dose of 0.8 mg/m2 b.i.d., targeting trough concentrations of 4-6 ng/mL until month 6 and 3-5 ng/mL thereafter. Mycophenolate mofetil (MMF) was administered at 1200 mg/m2 in 2 doses, adjusted to reach an area under the curve concentration of 40-60 ng*h/mL. Sirolimus was not administered de novo but given in cases where with CNI toxicity or posttransplant lymphoproliferative disease (PTLD) developed. In the event of CNI-related toxicity, sirolimus (target trough level 5-8 ng/mL) was combined with CsA (target trough level 40-60 ng/mL). In children who developed PTLD, sirolimus (target trough level 5-8 ng/mL) was combined with prednisolone (3 mg/m2 body surface area) and MMF (1200 mg/m2). All patients who developed PTLD were treated with only rituximab and steroids, and not with cyclophosphamide.
Clinical and laboratory data collected at annual follow-up visits were obtained from medical records. Additionally, a nonstandardized questionnaire was developed and issued for completion by patients and their parents (Table S1, SDC, http://links.lww.com/TP/B228). Information requested on the questionnaire included age at first menstruation (females) or first ejaculation (males). Age at onset of puberty was assessed by Tanner stages.25,26 In girls, stage P2 demarcates the first pubic hair and stage B2 demarcates breast budding. Puberty was defined as achievement of both P2 and B2 stages. In boys, stage P2 indicates first pubic hair and stage G2 indicates first pubertal changes of genitalia. Puberty in boys was defined as the first age at which both P2 and G2 were reached. Calculations of the mean age at which Tanner stages were achieved excluded patients who had already reached the stage at the time of kidney transplantation and patients who had received less than 1 year’s treatment with an mTOR inhibitor before reaching Tanner stage 2.
Total exposure to mTOR inhibitor therapy before the time of hormone measurements (ie, 16 years of age) was calculated by an index including exposure based on mean trough levels and time, as follows: everolimus: mean trough level [ng/mL] × time [years|; sirolimus: mean trough level [ng/mL] × 0.5 × time [years]. The factor 0.5 was selected because in standard of care, sirolimus target trough levels are approximately twice as high as those for everolimus.
Other evaluations included the age of spermarche and menarche (as reported on the questionnaire, with additional data on menarche requested at annual follow-up visits), and serum levels of reproductive hormones (lutenizing hormone [LH], follicle-stimulating hormone [FSH], androstendione, dehydroepindrosterone sulfate [DHEAS], testosterone, and estradiol). Developmental status and hormone levels were compared to published reference data from healthy children27,28 and age-matched data from the local laboratory at which patients’ samples were measured.29 “Early” or “late” events were defined as >2 SD below or above the mean age in a healthy normal population.27,28 Reproductive hormone levels were evaluated at age 16 because published data27,28 show that puberty has typically been achieved by that age in most of the patients and hormone levels would be expected to be comparable to adult patients. “High” and “low” hormone levels were defined as >2 SD above or below the mean values for an age-matched population measured at the local laboratory.29 Patients who received mTOR inhibitor therapy for less than 1 year before the age of 16 were excluded from analysis. Graft function was assessed by estimated glomerular filtration rate (eGFR) based on the new complete Schwartz formula.30
Patients were analyzed according to whether they received an mTOR inhibitor for any period of time posttransplant, or received no mTOR inhibitor. Continuous variables are presented as means (standard deviation [SD]) or medians with 95% confidence intervals (CIs). Categorical parameters were compared between groups using t test or Fisher’s exact test. The Kaplan-Meier method was used to estimate time to Tanner stages, with between-group comparisons based on the log-rank test. Patients were censored (1) if they had not achieved the Tanner stage 2 by the last follow-up visit; (2) if there was no record of the Tanner stage in more than 2 successive years then, in the first subsequent documentation, there was a marked progression in Tanner stage (because the point of change could not be identified accurately); and (3) if mTOR inhibitor therapy had been received for less than 1 year before reaching Tanner stage 2.
This study was approved by the ethics committee of Hannover Medical School.
In total, 108 children were eligible for analysis. Of these, 67 (62.0%) received an mTOR inhibitor and 41 received no mTOR inhibitor therapy (38.0%). The mean (SD) age of the study cohort at time of transplant was 7.0 (4.2) years, ranging from 0.7 to 13.8 years, and over half the population (n = 66, 61.1%) were boys. Patient demographics and other characteristics were similar between the mTOR inhibitor and control groups. Age at time of transplant was higher and duration of follow-up was shorter in the cohort given an mTOR inhibitor, but otherwise the 2 groups were similar (Table 1). The time on dialysis did not differ significantly between patients who did or did not receive mTOR inhibitor therapy, either for peritoneal dialysis (P = 0.209) or hemodialysis (P = 0.422). Overall, the median observation time was 7.0 (range 1.0–16.0) years from time of transplant.
No deaths occurred during follow-up. Ten grafts were lost: eight in boys receiving an mTOR inhibitor, who were switched late to mTOR inhibitor treatment in response to CNI toxicity observed on kidney biopsies undertaken due to progressive loss of graft function, and 2 in boys who did not receive an mTOR inhibitor.
Immunosuppressive therapy is summarized in Table 2. Of the 67 patients who received an mTOR inhibitor during the follow-up period, 56 were given everolimus and 11 were given sirolimus. The mean (SD) age at the start of mTOR inhibitor therapy was 10.3 (3.6) years for everolimus and 9.5 (4.5) years for sirolimus. Treatment with an mTOR inhibitor was started during the first year posttransplant in 35 cases (32 everolimus, 3 sirolimus). The everolimus patients were given primary mTOR inhibitor therapy 2-4 weeks after transplantation and the three sirolimus patients were switched from CNI therapy due to the onset of PTLD during the first year after transplantation. The remaining 32 patients started mTOR inhibitor therapy between 1 and 15 years posttransplant in response to CNI-related toxicity or onset of PTLD. The median time to starting everolimus was 1 year after kidney transplantation (range 9 months–15 years), whereas the median time to starting sirolimus was 2 years (range 1-9 years). At last follow-up, all 67 patients were still receiving mTOR inhibitor therapy. Mean (SD) everolimus trough concentration was 4.3 (1.3) ng/mL and mean sirolimus trough concentration was 5.6 (1.4) ng/mL. The median exposure index was 17.4 (range 1.9-62.1) ng/mL for everolimus and 17.3 (12.3-47.5) ng/mL for sirolimus. All except three patients were given concomitant CNI therapy while receiving an mTOR inhibitor; 32 received MMF (Table 2). In the cohort with no mTOR inhibitor treatment, all except four patients were given MMF with CNI therapy (37/41).
Mean doses and trough levels of immunosuppressive medications are given in Table 2b.
All patients received primary steroid therapy, with a mean duration of 5.1 years. The minimum duration of steroid therapy was 10 months. Steroids were discontinued in 29/108 patients between months 10 and 12 posttransplant, in those cases where the primary immunosuppression was based on mTOR inhibitor therapy and a protocol biopsy at month 6 posttransplant was normal.
Thirty-nine patients (15 girls, 24 boys) were excluded from the analysis of pubertal development because they had already reached Tanner stage >2 at first follow-up, because they had received less than 1 year’s treatment with an mTOR inhibitor before reaching Tanner stage 2, or because follow-up data were missing. The disposition of all patients is summarized in Figure 1.
Of the 27 girls (64%) in whom information on menarche was available, 27 attained menarche (15 in the mTOR inhibitor group, 12 without mTOR inhibitor therapy). The median age at onset of menarche was 12.5 years, ranging from 10 to 16 years. This was consistent with data from the general female population in Germany showing a median (95% CI) age of 12.8 years (range 12.8-12.9).31 The median age of menarche was similar in girls treated with an mTOR inhibitor (13.0 [range 10-15] years) or without an mTOR inhibitor (12.3 [range 10-16] years) (Table 1). Premature menarche (defined as >2 SD below the mean of the German female population, ie, <10.9 years ) occurred in three girls, 2 of whom received mTOR inhibitors. Menarche was delayed (defined as >2 SD above the mean, ie, >15 years ) in 1 girl, who did not receive an mTOR inhibitor. The age at which girls reached Tanner stage P2 was similar with mTOR inhibitor (median 11.6 [95% CI 11.3-12.0] years) or without (median 11.1 [95% CI 9.6-12.6] years), as was age at stage B2 (median 11.6 [95% CI 10.3-12.9] years vs. 11.2 [95% CI 10.6-11.9] years). Kaplan-Meier estimates showed no significant differences between groups (log-rank P = 0.262 for stage P2; P = 0.753 for stage B2) (Figure 2a). The median duration of mTOR inhibitor therapy was 2.0 (range 1.0-8.0) years before reaching Tanner P2 and 2.0 (1.0-9.0) years before reaching Tanner B2.
There was no difference in the development of Tanner stage P2 or Tanner stage B2 according to the use or length of dialysis before kidney transplantation in girls with or without mTOR inhibitor treatment, based on Kaplan-Meier estimates (log-rank P values ranged from 0.252 to 0.800) (Figure S1a, SDC, http://links.lww.com/TP/B228).
In total, 23 boys or their parents (21.3%) returned the questionnaire, of whom 14 (eight in the mTOR inhibitor group, six without mTOR inhibitor therapy) stated that they had reached spermarche and stated their age at onset. The median age (range) at spermarche was 13.0 (11.7-16.0) years, compared to 13.8 (range 11.8-15.7) years in the non-transplanted population.28 The median age of first spermarche was 13.0 years in the groups with or without mTOR inhibitor therapy (Table 3). Early spermarche (either stated as “early” in the questionnaire or >2 SD below the median age in healthy German boys, ie, <11.8 years ) was reported in 2 boys, both of whom received mTOR inhibitor therapy. One boy had delayed onset (>2 SD after the median age in healthy German boys, ie, >15.7 years ), who was not given an mTOR inhibitor. The age at which boys reached Tanner stage P2 was similar in the mTOR inhibitor-treated group (median 12.9 [95% CI 12.1-13.7] years) or the non-mTOR inhibitor group (median 13.0 [95% CI 12.8-13.2] years) (log-rank P = 0.796) (Figure 2b). The median duration of mTOR inhibitor therapy before reaching Tanner P2 was 4.0 (range 1.0-8.0) years. The age at which Tanner stage G2 was achieved was also similar (median 13.1 [95% 11.8-14.3] years with mTOR inhibitor, median 12.9 [95% CI 11.7-14.0] years without) (log-rank P = 0.344) (Figure 2b). The median duration of mTOR inhibitor therapy before reaching Tanner G2 was 3.0 (1.0-8.0) years.
There was no difference in the development of Tanner stage P2 or Tanner stage G2 according to use or duration of dialysis before kidney transplantation in boys with or without mTOR inhibitor treatment (log-rank P values ranged from 0.073 to 0.096) (Figure S1b, SDC, http://links.lww.com/TP/B228).
As would be expected, almost all patients had passed through puberty by age 16 years, so reproductive hormones were evaluated at this age. Patients in whom these hormones could be evaluated had received mTOR inhibitor therapy for a mean of 3.6 (range 1.0-7.0) years by the age of 16 years. Patients who started mTOR inhibitor therapy after the age of 16 years were excluded from the analysis.
Data on reproductive hormones were available in 14 girls, at age 16.0-16.9 years. Levels of LH, FSH, androstendione, DHEAS, and estradiol were similar between patients treated with mTOR inhibitor or no mTOR inhibitor (P values between 0.39 and 0.85). Abnormal hormone levels compared to the local age-matched population were infrequent, and there were no apparent differences in the incidence of such abnormalities between girls who did or did not receive mTOR inhibitors (Table 3).
Data on reproductive hormones were available in 19 boys, at ages 16.0–16.9 years. The groups treated with mTOR inhibitor or no mTOR inhibitor showed no statistical differences in levels of any hormone: LH, FSH, androstendione, DHEAS, or testosterone (P values between 0.28 and 0.85). As observed in the girls, most boys had reproductive hormone levels within the normal ranges for the local age-matched population, and there were no marked differences in the rate of abnormalities in the mTOR inhibitor or non-mTOR inhibitor groups.
Mean (SD) eGFR was 52.2 (15.5) mL/min/1.73 m2 in the non-mTOR inhibitor group, 55.2 (24.4) mL/min/1.73 m2 in the patients who received everolimus, and 60.3 (27.8) mL/min/1.73 m2 in the few patients (n = 11) who received sirolimus. Mean eGFR was not significantly different between the cohort who received an mTOR inhibitor versus those given no mTOR inhibitor (P = 0.068). The correlation between eGFR and abnormal hormone levels at the age of 16 years was significant only for dehydroepindrosterone sulfate (DHEA-S) (P = 0.013) and nonsignificant for all other hormones. The correlation between eGFR and pubertal development at the age of 16 years was also nonsignificant.
The incidence of PLTD was 9% (10/108 patients). Of these 10 patients, nine were treated with a conventional immunosuppression regimen before PTLD was diagnosed and one child was receiving the mTOR inhibitor everolimus. Thus, the incidence of PTLD was 3% (1/32) in those patients who received a primary mTOR inhibitor-based regimen.
Patients With Cystinosis
Five patients in the mTOR inhibitor group and no patients receiving conventional immunosuppression suffered from cystinosis, a disease which can have a negative influence on the development of puberty. All of these patients were girls. In a subgroup analysis of these patients, no abnormal hormone levels were found. Due to the low number of children with cystinosis, no statistical comparisons for Tanner stages versus age at menarche/spermarche could be performed.
In this, the largest series so far to examine adolescent development in patients receiving an mTOR inhibitor after kidney transplantation, use of low-exposure mTOR inhibitors—mainly in combination with low-dose CNI—showed no clinically relevant effect on the age of puberty or production of reproductive hormones in virtually all patients. Most patients showed normal pubertal status for age, and hormone levels were typically within the normal range for the general age-matched population. Pubertal milestones and hormone production were generally similar to that seen in patients managed at the same center who did not receive any mTOR inhibitor therapy. Because most patients in our population received an mTOR inhibitor in combination with CNI therapy, trough levels of everolimus and sirolimus were far lower than in older studies in which patients received an mTOR inhibitor with concomitant MMF.5,6,16,19
We are not aware of previous studies assessing the age of onset for menarche or spermarche, or specific stages of puberty, in kidney transplant patients receiving mTOR inhibitor therapy. In the 27 and 14 girls, respectively, for whom data were available on menarche or hormone levels, we found no evidence that menarche or the onset of puberty (as defined by Tanner stages P2 and B2) differed between girls receiving mTOR inhibitor or not. Perhaps of more potential concern is a possible effect of mTOR inhibitors on pubertal development in boys. Preclinical data suggest that mTOR inhibition may reversibly disrupt the early stages of spermatogenesis,31,32 and there are data showing reduced sperm count and quality in sirolimus-treated adult males33,34 although the mechanisms are not yet fully clarified20,34 and the matter remains controversial. In our population, information on spermarche was available from 14 boys and on hormone levels in 19 boys, and spermarche or onset of puberty was not delayed under mTOR inhibitor treatment compared to controls. However, this represents a small pool of patients for analysis, and the quality of semen was not assessed. Maintenance immunosuppression in men can potentially affect testicular function. It was recently shown that the total sperm count of 24 young men transplanted a mean of 18.6 years previously was significantly lower than in age-matched controls, and only 22% showed normspermia.35 The majority of patients had received CsA-based immunosuppression. It is clearly important to determine any effect of mTOR inhibitors on semen quality in future studies.
End-stage renal disease is usually associated with gonadal hormone disturbances,36,37 which typically improved after kidney transplantation.38 In vitro data from cultured pituitary cells, which express the gonadotropic-releasing hormone (GnRH), have shown that treatment with sirolimus diminishes the response to GnRH stimulation by 49%, an effect accounted for by involvement of the mTOR pathway in GnRH signaling.39 Previous studies of pediatric kidney transplant patients treated with an mTOR inhibitor, however, have not detected any effect on reproductive hormones (LH, FSH, and estradiol), which were normal for age although numbers were low (≤10).10,18 In our cohort, hormone levels in eight girls treated with mTOR inhibitor were similar to those of six girls who did not receive mTOR inhibitors. Rates of abnormal hormone levels were low, and we did not detect any difference between the 2 treatment groups.
Of particular interest was testosterone production in the male patients, given previous data suggesting that levels are suppressed under sirolimus therapy after kidney21-24 or heart40 transplantation. There is also limited evidence to suggest that gonadotropic hormones (LH and FSH) are increased under sirolimus versus patients without mTOR inhibitor treatment in adult male transplant recipients.22,23,40 A recent evaluation of 15 adolescent male patients (median 15 years) who were switched to CNI-free high-dose sirolimus therapy reported a lower-than-expected increase in testosterone levels (with a decrease in some patients), and observed that time on sirolimus was independently associated with lower testosterone (P = 0.010).24 In contrast, Kranz et al. documented an age-appropriate increase of serum testosterone concentrations in each of seven boys during puberty, other than in one patient who had concomitant liver fibrosis and PTLD; no abnormalities of LH or FSH were reported.18 Pape and colleagues also observed testosterone levels within normal range in a cohort of 10 boys, accompanied by normal levels of LH and FSH who were also part of this study.10 In our larger series described here, where data on reproductive hormones were available from 19 boys, mean levels were similar to those of a reference nontransplanted adult population.
This series offers a relatively high number of patients for analysis, monitored for a mean of over 6 years, with annual data collection on reproductive hormone levels and, uniquely, the age at which pubertal stages were attained. Unlike a randomized trial, we did not apply exclusion criteria other than age at baseline and a minimum duration of 1 year’s follow-up, such that the results reflect a typical pediatric kidney transplant population. Information about adverse events experienced by the patients at our center given primary mTOR inhibitor treatment compared to a group of matched patients given conventional immunosuppression has recently been published.41
Interestingly, graft losses occurred only in boys. Given the absence of evidence in the literature for a gender difference in graft survival after pediatric kidney transplantation,42 it seems reasonable to assume that this finding is an anomaly arising from the relatively low number of patients and the preponderance of boys in our population.
However, there are notable limitations. First, we included patients in the mTOR inhibitor treatment group regardless of the time of onset of duration of mTOR inhibitor therapy. Slightly more than half the patients in the mTOR inhibitor group (37/67) started treatment during year 1, but although the median time to initiation was 1 year for everolimus and 2 years for sirolimus, a small number of patients started late, for example in response to onset of PTLD, following detection of fibrosis on transplant biopsy, and in some cases after onset of puberty. Also, the follow-up time in patients without an mTOR inhibitor was somewhat longer. We recognize that this may have led to under-reporting of the long-term effects of mTOR inhibition. Second, the questionnaire about age of pubertal events was completed by only 39% of patients or their parents and a further 10% of patients had not reached puberty at final follow-up. Although we do not envisage that this skewed the results, it did reduce the data pool substantially. Third, as noted above, we did not collect data on the function or number of spermatocytes, which is likely to be highly challenging in adolescents, and due to the cut-off of 18 years, fertility was not assessable. It should be borne in mind that although the date of first menarche is usually remembered accurately, the date of first ejaculation can be more difficult to recall and these data may be less reliable. Lastly, pubertal status was documented only once a year, a relatively long duration between assessments.
In conclusion, we observed comparable sexual maturation ages and reproductive hormone levels in adolescent kidney transplant patients receiving low-exposure mTOR inhibitors and reduced CNI therapy or conventional CNI-based immunosuppression. Long-term data on fertility are still lacking.
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