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Department of Endocrinology, University Hospital Ghent, De Pintelaan 185, 9000 Gent, Belgium
¹ Department of Endocrinology, Free University Amsterdam, 1007 MB, The Netherlands
² Department of Hematology, University of Liège, 4000 Sart Tilman, Belgium

(Requests for offprints should be addressed to G G T’Sjoen; Email: guy.tsjoen{at}ugent.be)

	Abstract

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The objective of this investigation was to study the effects of sex steroids on levels of haemoglobin (Hb) and haematocrit (Hct) and to analyse whether these effects can be related to levels of the soluble transferrin receptor (sTfR), a marker of erythropoietic activity. Nineteen male-to-female transsexuals were randomly assigned to either oral ethinyl oestradiol (EE) (n=12) or transdermal 17ß-oestradiol (E2) (n=7); both treatments included the anti-androgen cyproterone acetate (CA). Six male-to-female transsexuals were treated with CA only. Fifteen female-to-male transsexuals were treated with i.m. testosterone esters. The Hct, and levels of Hb, IGF-I, GH and sTfR were measured before and after 4 months of hormone administration. Androgen administration significantly increased the sTfR concentration by 31.5% (P=0.008) and increased levels of Hct, Hb and IGF-I. Both regimens of CA with oral EE and transdermal E2 reduced plasma testosterone similarly to castrate values and decreased Hb and Hct. The CA+oral EE combination induced a decrease in sTfR of 19.0% (P=0.002) which was not the case with CA+transdermal E2 (P=0.27). This cannot be explained by the profound decline in plasma testosterone which was similar with both regimens, but this difference could be related to the different effects of the two regimens on plasma IGF-I. This assumption is supported by the positive correlation that was found to exist between plasma sTfR and IGF-I after the interventions (P<0.05).

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Men have higher haematocrit (Hct) and haemoglobin (Hb) concentrations than women (Morris et al. 1956), in all likelihood due to their higher plasma testosterone. Men who are hypogonadal have lower than normal Hct and Hb concentrations and testosterone treatment increases those variables to normal (Tenover 1992, Jockenhovel et al. 1997). In support of a role of testosterone is the observation that, in hypogonadal men, administration of transdermal testosterone dramatically increases Hct, from mildly anaemic to midnormal within 3 months (Snyder et al. 2000). Also, in healthy young men changes in circulating testosterone concentrations induced by gonadotrophin-releasing hormone (GnRH) agonist and testosterone administration are associated with testosterone dose- and concentration-dependent changes in Hb (Bhasin et al. 2001). The effects of testosterone on the Hct have been documented in several small and short-term studies, as reviewed by Hajjar et al.(1997). The mechanisms by which androgens exert these effects have not been elucidated. It has been suggested that androgen action on erythropoiesis is mediated by a nuclear androgen receptor (Claustres & Sultan 1988).

Administration of testosterone has been successfully used for the treatment of refractory anaemia in males (Piedras et al. 1998). It has been postulated that the administration of androgens to man and laboratory animals results in an increase in plasma erythropoietin activity. However, it has also been shown in recent years that in androgen-induced erythrocytosis, there is no increase in erythropoietin levels (Dickerman et al. 1998, 1999). Induction of androgen deprivation with a luteinizing hormone-releasing factor (LHRH) agonist did not result in changes in serum erythropoietin levels (Weber et al. 1991). Interestingly, there is no difference in serum erythropoietin levels between men and women, even though there is a significant difference in their testosterone and Hb levels (Miller et al. 1985). So, it seems that the androgen-mediated increases in Hb levels and Hct are not exclusively mediated by erythropoietin, and testosterone may have a direct effect on bone marrow stem cells (Shahidi 1973, Krabbe et al. 1978, Mooradian et al. 1987, Krauss et al. 1991). Our study monitored the effects of sex steroids on Hb and the Hct in transsexuals undergoing cross-sex hormone administration, making use of a quantitative assay of bone marrow erythropoietic activity, the soluble transferrin receptor (sTfR).

Iron transport in the plasma is carried out by transferrin. The interaction with a specific membrane receptor, the TfR allows iron to be included in cells. In both animal and human serum a soluble form of the TfR (sTfR) has been identified (Beguin 2003). Marrow erythropoietic activity appears to be the most important determinant of sTfR levels (R’Zik & Beguin 2001). In situations characterized by diminished erythropoietic activity, sTfR levels are decreased. When erythropoiesis is stimulated sTfR is increased. Measurements of sTfR are useful for monitoring the erythropoietic response to various forms of therapy. Predicting an early therapeutic response by measuring sTfR is possible when changes in Hb are not yet apparent (Beguin 2003).

To our knowledge, this is the first paper describing the effects of exogenous oestrogens and (anti-) androgens on the levels of the sTfR.

	Materials and Methods

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Subjects

We included 25 male-to-female (MF) and 15 female-to-male (FM) Caucasian transsexuals. Additional details of this study group can be found in Giltay et al.(2000). Psychological criteria for the diagnosis and treatment followed the guidelines provided by the Harry Benjamin International Gender Dysphoria Association (Walker et al. 1985). Nineteen MF transsexuals were open-label-randomised to receive either oral ethinyl oestradiol (EE) (Lynoral, 100 µg/day; Organon, Oss, The Netherlands; n=12) or transdermal 17ß-oestradiol (E2) (Estraderm TTS 100, 100 µg twice a week; CIBA-Geigy, Basel, Switzerland; n=7), both in combination with cyproterone acetate (CA) (Androcur, 100 mg/day; Schering, Berlin, Germany) which is a progestational compound with androgen receptor-blocking properties. Because the effects of the administration of CA alone on sTfR levels are unknown in men, we also studied six MF transsexuals who received CA alone during the first 4 months of cross-sex hormone administration. FM transsexuals were treated with testosterone esters (Sustanon, 250 mg/2 weeks, i.m.; Organon). All FM transsexuals had had regular menstrual cycles (28–31 days) before cross-gender sex hormone administration. There was no evidence of iron deficiency, hypertension, cardiovascular disease, thrombo-embolism, diabetes mellitus or use of sex hormones in any of the subjects tested. Due to the availability of blood samples, the number of participants in this freezer study is smaller than in the study of Giltay et al. 2000 where 30 MF transsexuals receiving CA+oral or transdermal oestrogens, 10 MF transsexuals receiving CA alone, and 17 FM transsexuals were described.

Smoking status (yes or no) and body mass index (BMI, weight/height²) were also assessed. Informed consent was obtained from all subjects, and the study was conducted according to the principles of the Declaration of Helsinki and approved by the Ethical Review Board of the University Hospital Ghent and the Free University Amsterdam.

Assays

Each subject served as his or her own control, with samples drawn before and during hormone administration. In FM transsexuals, blood was drawn at baseline between days 5 and 9 of the follicular phase of the menstrual cycle. During testosterone treatment, blood was drawn within 5–9 days after the previous testosterone injection. An intravenous catheter was placed in the antecubital vein of supine subjects after an overnight fast and 10 min of bed rest. Because of the travel time to the clinic, the time of blood sampling was between 0830 h and 1330 h. Within-subject time of sampling was, however, comparable before and after 4 months of hormone administration: mean 1055 h (95% confidence interval (CI), 1037–1112 h) versus mean 1037 h (95% CI, 1019–1055 h) respectively, P=0.10. The mean intraindividual variation was 50 min (95% CI, 40–70 min). Blood was collected without a tourniquet into evacuated tubes (Diatube H CTAD, i.e. citrate, theophylline, adenosine and dipyridamole; Becton Dickinson, Rutherford, NJ, USA). Samples were immediately placed on ice and centrifuged at 3500 g for 30 min at 4 °C to obtain platelet-poor plasma. Plasma was separated and snap-frozen within 1 h and stored at –70 °C until analysis. The samples underwent one freeze–thaw cycle before serum sTfR, iron, transferrin and ferritin were measured. Serum sTfR was measured by a commercially available ELISA (R&D, Minneapolis, MN, USA). Serum ferritin was measured by the nephelometry method (Behring Nephelometer Analyzer; Dade Behring Marburg Co. Ltd, Marburg, Germany) following the manufacturer’s instructions. Iron and transferrin were measured by standard methods (Modular; Roche). Standardized radio-immunoassays were used to measure serum levels of E2 (Double Antibody; Sorin Biomedica, Saluggia, Italy) and testosterone (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA, USA). Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by immunometric luminescence assays. Commercial assays were used for determinations of growth hormone (GH) (Immulite 2000; Diagnostic Products Corp.) and insulin-like growth factor (IGF-I) (extraction method, DSL-5600; Diagnostic System Laboratories, Webster, TX, USA). The effects of transdermal administration of E2 were compared with those of oral EE, both in combination with oral CA.

Statistics

Most variables in the analysis turned out to be positively skewed. In order to meet the necessary model assumptions, a natural logarithmic transformation in these analyses was used for hormonal parameters and sTfR. All data are given as (geometric) means (± S.D. or 95% CI). Student’s t-tests for independent samples or ² tests were used to compare baseline differences. ANCOVA was used for intergroup comparisons after adjusting for possible confounders. Baseline values were correlated with the Pearson correlation coefficient.

An ANOVA for repeated measurements or a Student’s t-test for paired samples was used to analyse the effects of cross-gender sex hormones. An ANCOVA was used to compare effects of different treatment regimens and to adjust for potential covariates. Proportional changes, after 4 months of cross-sex hormone administration were correlated with the Spearman correlation coefficient. If values were below the lower limit of detection, the value of that lower limit was used for statistical analysis (1.0 nmol/l for testosterone, 0.3 IU/l for LH and 0.5 IU/l for FSH). A two-tailed P<0.05 was considered statistically significant. The software used was SPSS 10.0 for Windows 8.0.

	Results

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Pre-treatment values

At baseline, all subjects were eugonadal by clinical and laboratory criteria. Iron deficiency as judged from serum iron, ferritin and transferrin serum levels was not present in any of the participants. Baseline characteristics are presented in Table 1. There was at baseline an expected difference between men and women for Hb, Hct and ferritin (P<0.001), but no significant difference for sTfR, iron or transferrin levels. In MF transsexuals at baseline, there were no significant differences between the two groups except for BMI that was significantly (P=0.035) higher in males randomised to treatment with oral EE compared with males randomised to treatment with transdermal E2. Between MF transsexuals treated with oral EE and MF treated with CA only there was a difference in sTfR and serum iron levels at baseline (both P<0.05). There was no significant difference in sTfR between MF transsexuals treated with transdermal E2 and MF transsexuals treated with CA only.

In the group treated with CA alone, there were small but significant decreases in serum levels of testosterone and E2 (P<0.05) after 4 months treatment, and no changes in LH and FSH (P=0.72 and 0.22 respectively). Hb, Hct and sTfR levels did not change, while there was a trend for serum IGF-I to increase (P=NS). No significant relation between IGF-I and sTfR was established.

Oral and transdermal oestrogen administration, both with CA, decreased serum levels of testosterone (both P<0.001), LH (P<0.001 and P=0.007 respectively) and FSH (P<0.001 and 0.003 respectively). The combination of CA+EE administration (the latter not measured by the oestradiol assay used) suppressed endogenous oestradiol levels; whereas percutaneous administration of E2 increased plasma levels of E2 up to values typical of the midfollicular phase in women (Table 2).

View this table: [in this window] [in a new window]   Table 2 Laboratory data before and after 4 months of cross-gender sex hormone administration in MF transsexuals, randomised for administration of oral ethinyl oestradiol in combination with CA or transdermal 17ß-oestradiol in combination with CA

After testosterone administration to FM transsexuals, serum levels of testosterone increased markedly (P<0.001), whereas serum levels of E2, LH and FSH decreased (P=0.03, 0.01 and 0.005 respectively) (Table 3). The FM transsexuals had significantly higher testosterone levels after 4 months of treatment, compared with the 3 groups of MF transsexuals at baseline (P<0.001). Hb, Hct, sTfR and IGF-I rose significantly after testosterone administration in FM transsexuals. There was no significant relation between IGF-I and sTfR in this group at month 4. However, the relation between IGF-I and sTfR at month 4 was confirmed when including all participants, both female and male (r=0.689, P=<0.01). Plasma iron concentrations were unaffected by the different hormonal treatments (data not shown).

View this table: [in this window] [in a new window]   Table 3 Laboratory data before and after 4 months of cross-gender sex hormone administration in FM transsexuals receiving testosterone and in MF transsexuals receiving CA only

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A clear example of an effect of androgens was provided by testosterone administration to females. The dose of testosterone administered to females induced plasma testosterone levels in the high-normal or above-normal range for men and after 4 months increased Hb, Hct and sTfR, together with higher serum IGF-I levels (Table 4), but there was no demonstrable correlation between levels of IGF-I and sTfR. However, a reduction in circulating testosterone to approximately 50% of baseline by CA only in males – which also has antiandrogenic effects at the level of the androgen receptor – had no effect on Hb, Hct and sTfR values. A non-significant increase in serum IGF-I was seen in this group, with a non-significant decrease of serum GH levels. In this group, no correlation could be established between levels of IGF-I and sTfR.

Androgen therapy leading to supraphysiological plasma testosterone levels produced a stimulation of bone marrow erythropoietic activity, possibly through a direct effect of testosterone on erythropoiesis, but possibly in part mediated through IGF-I induction. The administration of CA only resulted in an approximately 50% reduction in plasma testosterone but had no effect on Hb, Hct or sTfR, possibly explained by the concomitant rise of IGF-I. There was no correlation between levels of IGF-I and of sTfR in this group. Transdermal E2 combined with CA had similar increasing effects on IGF-I levels as CA only, without a demonstrable effect on sTfR, but there was a correlation between levels of IGF-I and sTfR. The decrease in Hb and Hct must probably be ascribed to the profound fall in plasma testosterone. However, administration of oral EE combined with CA reduced sTfR levels significantly, with a correlation between levels of IGF-I and of sTfR. The effects of the decrease in testosterone and IGF-I levels may be responsible for the significant decrease in sTfR levels upon administration of CA with oral oestrogens, while after transdermal oestrogens plus CA – in spite of a decline of plasma testosterone – there was no decrease of sTfR; this can probably be explained by the rise of IGF-I with the latter regimen. This speculation is further substantiated by the significant correlation of plasma sTfR and IGF-I following both oestrogen regimens.

A larger number of participants and the measurement of other potential confounders, such as erythropoietin levels, would have strengthened the interpretation of these data. This study paves the way for other larger studies that are needed to assess these interactions further. Evaluating the various effects in shorter intervals and for a longer period of time would be interesting in order to see if described effects do or do not persist.

In conclusion, our results show that profound alterations in plasma testosterone levels from male to female values, and vice versa, are associated with significant changes in Hb and Hct of the order of 10%. A reduction of plasma testosterone of the order of 50% had no impact on Hb and Hct. We tested whether these changes in Hb and Hct could be related to levels of sTfR, a marker of diminished/increased erythropoietic activity. Indeed, the increase in Hb/Hct upon testosterone administration was associated with an increase in sTfR, while there was no change in sTfR levels with lack of change of Hb/Hct upon administration of CA. The fall in Hb/Hct levels upon administration of CA combined with either oral EE or transdermal E2 had disparate effects on levels of sTfR: a decline with CA+oral EE, but no effect on levels of sTfR was observed with CA+transdermal E2. This disparity cannot be explained by the effects these two hormone regimes had on plasma testosterone. Earlier studies have indicated that administration of oral EE reduces plasma IGF-I, which is not the case with transdermal E2. In addition, indeed, in our study a correlation between plasma IGF-I and sTfR could be demonstrated after the intervention with the two modes of oestrogen administration and for the total group of patients. This assumption is not fully supported by the results of testosterone administration: this resulted in a rise of both plasma IGF-I and sTfR but these increases did not correlate.

	Acknowledgements

 
The authors are indebted to Ms I. Bocquaert, Dr M. Bunck and Dr E. Giltay for their help and expert assistance in the realization of this study. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received 22 March 2005
Accepted 15 April 2005

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