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Serum Inhibin B and Follicle-Stimulating Hormone Levels as Tools in the Evaluation of Infertile Men: Significance of Adequate Reference Values from Proven Fertile Men

Department of Growth and Reproduction (A.-M.A., J.H.P., N.J., T.K.J., N.E.S.), Copenhagen University Hospital, DK-2100 Copenhagen, Denmark; Department of Biostatistics (J.H.P.) University of Copenhagen, DK-2200 Copenhagen, Denmark; and Department of Environmental Medicine (T.K.J.), University of Southern Denmark, DK-5000 Odense, Denmark

Address all correspondence and requests for reprints to: Anna-Maria Andersson, M.Sci., Ph.D., Department of Growth and Reproduction, Copenhagen University Hospital, Section GR 5064, Blegdamsvej 9, DK-2100 Copenhagen OE, Denmark. E-mail: anna{at}rh.dk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Inhibin B and FSH levels in 289 idiopathic infertile men were compared with reference materials consisting of 303 proven fertile men (reference group 1) and 307 healthy men from the general population with unknown fertility status (reference group 2). The diagnostic power of these two serum markers of spermatogenesis was evaluated by the use of receiver operating characteristic plot analysis, and an example of how both markers can be used simultaneously in a bivariate reference chart is presented.

Inhibin B levels were significantly lower and FSH levels were significantly higher in the infertile men, compared with either reference group, but with significant overlap, especially with reference group 2. Nevertheless, approximately 50% of the infertile men had an inhibin B or FSH, respectively, below the 2.5 percentile or above the 97.5 percentile of reference group 1, whereas only approximately 25% of the infertile men had an inhibin B or FSH, respectively, below the 2.5 percentile or above the 97.5 percentile of reference group 2. Fourteen and 11% of reference group 2 had an inhibin B or FSH, respectively, below the 2.5 percentile or above the 97.5 percentile of reference group 1, suggesting that a significant number of individuals from the general population with unknown fertility but otherwise healthy may actually be subfertile.

In conclusion, 1) proven fertile men constitute the most appropriate reference group in the evaluation of the FSH-inhibin B axis; the sensitivity of these markers to identify infertility increased by approximately 20% when fertile men rather than men from the general population were used as control group; 2) FSH alone had a slightly higher positive predictive value than inhibin B alone, but the positive predictive value were highest when both markers of spermatogenesis were used in an inhibin B/FSH ratio; and 3) a bivariate reference chart is a valuable objective tool in the simultaneous evaluation of FSH and inhibin B as two interrelated markers.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
REFERENCE RANGES FOR hormone levels are important clinical tools in the evaluation of endocrine disorders. However, they should be based on a normal, healthy reference population. Thus, it is important to exclude from the reference population individuals with known acute or chronic illness, medicine use, or other conditions that might affect a given hormone level.

Accordingly, reference ranges for reproductive hormones preferably should be based on a normal, fertile population. However, traditionally subnormal fertility has not always been considered as an exclusion criterion for reference populations, partly because subfertility is not always considered an illness and partly because information on fertility status may be difficult to obtain. Therefore reference ranges for reproductive hormones are often based on reference populations for which no knowledge of the fertility is available. In the evaluation of patients’ hormone levels, it is equally important to have an estimate of a possible overlap between the normal ranges and ranges observed in known pathological cases.

Inhibin B is a testicular polypeptide hormone responsible for the negative feedback regulation of pituitary FSH secretion in men (1). Since the development of specific inhibin immunoassays in the mid-1990s (2, 3), which established inhibin B as the physiologically relevant circulating inhibin form in men (4), several studies of serum inhibin B in relation to male reproductive health have been published (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Inhibin B has been shown to be a marker of spermatogenesis (6, 16) and subfertile men generally have decreased or even undetectable serum levels of inhibin B (5) with the exception of men with obstructive forms of azoospermia or spermatogenic arrest at the level of spermatocytes, who may have normal levels of inhibin B (8, 9). However, most of these studies have been based on a relatively small number of infertile men with a very heterogeneous etiology and small, poorly defined, or lacking control groups. Only limited evaluations of the diagnostic significance of serum inhibin B measurements have thus been performed.

We therefore measured serum inhibin B and FSH in 289 idiopathic infertile men with sperm concentration less than 20 million/ml and in two control groups consisting of 307 healthy men with unknown fertility from the general population and 303 proven fertile men with a sperm concentration 20 million/ml or greater.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study populations

Idiopathic infertile men were consecutive patients referred to our andrology clinic during the period 1997–1999. They had a history of at least 1 yr unexplained childlessness, in which an obvious problem in the female partner was excluded. A male infertility problem was often already identified at referral from the infertility clinics to our andrology clinic. Eligibility criteria was a sperm concentration less than 20 million/ml and no evidence or suspicion of obstructive forms of azoospermia or secondary oligozoospermia due to orchitis or iatrogenic causes. All included subjects had a normal 46, XY karyotype, an androgen receptor gene with a normal CAG repeat number, and no microdeletions on the Y chromosome. Blood and semen samples were available from 289 eligible patients. Information on body mass index (BMI) and self-reported history of cryptorchidism was available from, respectively, 78 and 81% of the infertile men. Sixty-six of 242 infertile men reported a history of cryptorchidism. Infertile men with a history of cryptorchidism were included because the group of fertile men also included men with a history of cryptorchidism, and although men with a history of cryptorchidism cannot strictly be called idiopathic infertile, the cryptorchidism itself was idiopathic.

Fertile men included in this study consisted of 303 proven fertile men from the same geographical area as the infertile men. They were part of a study of reproductive health in partners of pregnant women previously described in detail (17). In brief, the eligibility criteria for the participating men were age range from 20–45 yr and the pregnancy of the female partner had been achieved by normal sexual relations. In the present study, the men should in addition have a sperm concentration 20 million/ml or greater. Information on BMI and self-reported history of cryptorchidism was available from, respectively, 100 and 75% of the fertile men. Thirty-one of 227 fertile men reported a history of cryptorchidism.

Healthy men from the general population, with unknown fertility constituted our laboratory reference material for the 20–45 age group and included 307 men. Data from these men have previously partly been published (18). Ninety-nine of the men were 40-yr-old men from the general population participating in a population survey conducted at the Research Center for Prevention and Health (MONICA 10) after exclusion of men with chronic or acute disease. Details of the MONICA 10 population are described elsewhere (19). Forty-nine of the 307 men were medical students, laboratory personnel, and spouses of laboratory personnel who did not have any chronic or acute illness or used any form of medicine around the period of blood sampling. These men were aged 20–44 yr (evenly distributed). The remaining 159 men were originally enrolled from the same geographical area in a time-to-pregnancy study of couples trying to conceive for the first time. The study population was described in detail elsewhere (16). Briefly, couples with no prior knowledge of fertility status were enrolled from four labor unions. The couples were followed up for 6 months and time to pregnancy was recorded. The men were between 20 and 35 yr and delivered a semen and blood sample at entry to the study. Because these men were recruited as having no a priori knowledge of their fertility, they were considered representative of the general male population.

Characteristics of the three study populations are given in Table 1.

Blood samples were drawn from an antecubital vein and centrifuged after clotting. Serum was stored at –20 C until analysis. Serum inhibin B was measured in a double-antibody immunoenzyme-metric assay (Oxford Bio-innovation, Oxfordshire, UK) using a monoclonal antibody raised against the inhibin ß_B-subunit in combination with a labeled antibody raised against the inhibin -subunit, as previously described (3). The inhibin B assay has a detection limit of 20 pg/ml, and the intra- and interassay coefficients of variation were less than 12% and less than 17% in the full range, respectively. Serum FSH was measured by time-resolved immunoflourometric assays (DELFIA, Wallac, Turku, Finland). The detection limit was 0.06 IU/liter, and the intra- and interassay coefficient of variation was less than 8% in the full range. Detection limit was defined as the dose corresponding to the value that is 2 SDs above the mean of the zero standard measurement.

Calculations and statistics

Hormone levels in the different groups and subgroups are presented as median and 2.5, 25, 75, and 97.5 percentiles. The Mann-Whitney U test was used to test for difference in hormone levels between the different groups and subgroups. Correlations between serum hormone levels and sperm concentration were tested by Spearman’s rank correlation coefficient. The relationships between serum inhibin B and FSH levels, and covariates age and BMI were tested in a general linear model. Inhibin B levels were square root transformed, and FSH levels were transformed by the use of the natural logarithm to obtain homoscedacity and an approximate normal distribution. Age and BMI entered the model linearly. For each subject an inhibin B/FSH ratio was calculated as inhibin B (picograms per milliliter)/FSH (international units per liter). Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivities and specificities of the two serum markers, inhibin B and FSH, alone or combined as either one being outside the cutoff level or expressed as an inhibin B/FSH ratio with regard to discriminate infertile patients from men in the general populations as well as fertile men. For inhibin B and inhibin B/FSH ratio, the sensitivity was defined as percent of infertile men with a measurement below the cutoff level, and the specificity was defined as percentage of men from the reference group with a measurement at or above the cutoff level. For FSH the sensitivity was defined as percent of infertile men with a measurement above the cutoff level, and the specificity was defined as percentage of men from the reference group with a measurement below or at the cutoff level. For inhibin B and FSH combined, the sensitivity was defined as percent of infertile men with either an inhibin B measurement below or a FSH measurement above the respective cutoff levels, and the specificity was defined as percent of men from the reference group with an inhibin B measurement above or at and a FSH measurement below or at the respective cutoff levels. The positive predictive value (the proportion of men in a population with a positive test, e.g. inhibin B below the cutoff, that is correctly diagnosed, i.e. they are infertile) is defined as:

and the negative predictive value (the proportion of men in a population with a negative test, e.g. inhibin B above the cutoff, that is correctly diagnosed, i.e. they are fertile) was defined as:

where prev_I is the prevalence of male infertility in the given population. Bivariate percentile reference charts with an estimator of the 97.5 percentile curve were constructed based on either the men from the general population or the proven fertile men as previously described in detail (18).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Serum levels of inhibin B, FSH, and inhibin B/FSH ratios in the general population and fertile and infertile men are shown in Table 1 and Fig. 1. In addition, data for the subgroups of fertile men with a sperm concentration 40 million/ml or greater [corresponding to a previously published estimated cutoff of an optimal sperm concentration (20)] and for the subgroup of infertile men with a sperm concentration less than 1 million/ml are also shown in Fig. 1.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Inhibin B (A), FSH (B), and inhibin B/FSH ratio (C) levels in the general population, in proven fertile men, and in idiopathic infertile men (median, 2.5, 25, 75, and 97.5 percentiles). Data for the subgroup of fertile men with a sperm concentration of 40 million/ml or greater and the subgroup of infertile men with a sperm concentration of less than 1 million/ml are also shown.

In relation to the men from the general population 21.5 and 26.3% of the infertile men had a serum inhibin B level and inhibin B/FSH ratio, respectively, below the 2.5 percentile of the general male population and 24.9% had a FSH level above the 97.5 percentile of the general male population.

Correlation between serum inhibin B, FSH, and inhibin B/FSH ratio and sperm concentration and total sperm count

As has been shown several times previously, we also found a significant positive correlation between serum inhibin B and sperm concentration and total sperm count; both in the group of proven fertile men (r = 0.218, P < 0.001 and r = 0.224, P < 0.001 for sperm concentration and total sperm count, respectively) and the group of idiopathic infertile men (r = 0.480, P < 0.001 and r = 0.456, P < 0.001, respectively). Sperm concentration also correlated positively with the inhibin B/FSH ratio and negatively with serum FSH in the fertile men (r = 0.240, P < 0.001 and r = –0.202, P < 0.001, respectively) as well as in the infertile men (r = 0.509, P < 0.001 and r = –0.496, P < 0.001, respectively).

Influence of age, BMI, and history of cryptorchidism on serum inhibin B and FSH levels

The influence of age and BMI on serum inhibin B and FSH levels was tested by a general linear model in the men from the general population and in fertile men separately. Within the age range (20–44 yr), FSH, but not inhibin B, correlated with age (P < 0.001 and P = 0.006 for men from the general population and fertile men, respectively). In both men from the general population and fertile men, FSH increased approximately 1% per year of age. No correlation between BMI and inhibin B or FSH could be found within the BMI range of 18–35 kg/m² of the fertile men (data on BMI were not available for the majority of the men from the general population).

In the infertile men, neither age nor BMI seemed to have any influence on serum inhibin B and FSH levels within the age (21–56 yr) and BMI (17–39 kg/m²) range of this group.

The infertile men with a history of cryptorchidism had significantly lower inhibin B levels (median 58 pg/ml vs. 122 pg/ml, P < 0.001) and higher FSH levels (median 11.85 IU/liter vs. 7.65 IU/liter, P < 0.001) than the infertile men with no history of cryptorchidism, although they did not differ with regard to sperm concentration (median 1.2 million/ml vs. 1.9 million/ml, P = 0.254). In contrast, among the fertile men, there was no significant difference in serum inhibin B levels, FSH levels, or sperm concentrations between the formerly cryptorchid men and those with no history of cryptorchidism.

Diagnostic efficiency of serum inhibin B and FSH levels

The efficiency of serum inhibin B and FSH measurements and the inhibin B/FSH ratio to discriminating between idiopathic infertile men and the reference group was evaluated by ROC plot analysis both in relation to men from the general population and in relation to the proven fertile men. The result of the ROC plot analysis is presented in Fig. 2. The area under the ROC plot is a measure of the efficiency of the marker to categorize samples from the infertile men and the respective reference groups correctly: the higher the area, the better the efficiency.

View larger version (16K): [in this window] [in a new window]   FIG. 2. ROC plot analysis of inhibin B, FSH, and inhibin B/FSH ratio for discriminating idiopathic infertile men from men from the general population and proven fertile men, respectively.

When the infertile men were evaluated against the men with proven fertility, the efficiency of FSH and inhibin B alone and the ratio of the two were almost identical, although best for the inhibin B/FSH ratio. The efficiency of discriminating between the infertile men and the reference group was, however, significantly increased when the proven fertile men were used as reference group indicated by an approximately 10% increase in the area under the curve of the ROC plot, compared to when the men from the general population were used as reference group.

ROC plot analysis can also be a help in defining cutoff levels. However, definitions of cutoff levels are always the result of a compromise between a high sensitivity and a high specificity that includes considerations on how many false-positive and false-negative cases one will accept. The cutoff levels of inhibin B, FSH, and inhibin B/FSH ratio, respectively, which corresponded to a specificity of 95% is presented in Table 2 together with the corresponding sensitivities. In addition, the sensitivity and specificity when using inhibin B and FSH measurements combined as either one being outside the respective cutoff levels are shown in Table 2. By choosing a specificity of 95%, a false-positive result will be expected in 5% of the cases. However, other cutoff levels might be chosen, which give a lower specificity (more false positive) but a higher sensitivity (less false negative).

View this table: [in this window] [in a new window]   TABLE 3. Positive and negative predictive values of serum inhibin B and FSH alone or combined as markers of male infertility in populations with different prevalence of infertility at the cutoff levels defined in Table 2 for the proven fertile men as reference group

An alternative way of exploiting two interrelated serum markers is to produce a bivariate (two-dimensional) reference chart, which incorporates the values of both. Figure 3A shows the data of the proven fertile men plotted in this way and the corresponding 97.5 percentile reference curve. The reference curve (drawn line) is constructed in the following way: all combinations of inhibin B and FSH on the line (i.e. inhibin B:112 and FSH:4.96) are found or estimated such that exactly 2.5% of the fertile men simultaneously have a lower inhibin B and a higher FSH. An inhibin B and FSH measurement from a new individual can be plotted as a point in Fig. 3A and has a normal inhibin B-FSH profile if the point is inside the curve, i.e. is in the upper left corner of the plot. The individual is abnormal if the point is outside, i.e. to the right or below the curve. Based on the data from the proven fertile and the idiopathic infertile men, it can be estimated that the 97.5 percentile fertile reference curve has a specificity of 95% and a sensitivity of 60% corresponding to a positive predictive value and a negative predictive value, assuming a prevalence of infertility of 5%, of, respectively, 39 and of 98%.

View larger version (13K): [in this window] [in a new window]   FIG. 3. A, Data from the proven fertile men plotted in a bivariate chart for inhibin B and FSH with the corresponding estimated 97.5 percentile. B, Data from the idiopathic infertile men plotted in a bivariate reference chart for inhibin B and FSH showing the estimator of the 97.5 percentile for the proven fertile men (full drawn line) as well as the estimator of the 97.5 percentile for the men from the general population (dotted line). Dots below and to the right of both estimated 97.5 percentiles represent individuals with hormone levels outside the normal range, irrespective of which reference group used, and dots above and to the left of both 97.5 percentiles represent individuals that have normal inhibin B and FSH levels in relation to both reference groups. Dots placed between the two 97.5 percentiles represent individuals who are identified as having normal hormone levels in relation to men from the general population but identified as having hormone levels outside the normal range in relation to proven fertile men.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This is the first study in which the diagnostic efficiency of serum inhibin B as a marker of spermatogenesis in idiopathic infertile men was evaluated against a large, well-characterized reference material consisting of fertile men. Our data confirm that serum inhibin B and FSH levels correlate well with sperm concentration and thus support their role as serum markers of spermatogenesis. The importance of using a correct reference material in reproductive endocrinology is emphasized by our finding that approximately 10% of the men from a normal reference material consisting of healthy men with unknown fertility had serum inhibin B and FSH levels that were out of range in relation to a normal reference range based on men with proven fertility and a normal sperm concentration. Not surprisingly, the general healthy male population with unknown fertility apparently included a subpopulation of subfertile but otherwise healthy men, which may constitute up to 10–13% of the general population. Consequently, greater overlap in inhibin B and FSH levels was observed when the idiopathic infertile men were compared with the men from the general population with unknown fertility than when compared with the proven fertile men. Accordingly, the efficiency of the two serum markers of spermatogenesis inhibin B and FSH to discriminate between the infertile men and the reference group increased by 6–11% when proven fertile men rather than men from the general population with unknown fertility were used as reference group.

Neither age nor BMI seemed to affect serum inhibin B within the ranges studied. In the reference groups, serum FSH levels increased only slightly with age and did not seem to be affected by BMI. Thus, normal adult male reference materials for inhibin B and FSH need to be related to neither BMI nor age, at least not within the age (20–45 yr) and BMI range (18–35 kg/m²) of our reference group. This is in line with a recent cross-sectional study of inhibin B showing comparable inhibin B levels in adult men at all ages from 16– 89 yr (21).

In accordance with Brazao et al. (22), we observed significantly lower serum inhibin B levels and higher FSH levels in the formerly cryptorchid infertile men, compared with the infertile men with no history of cryptorchidism. Lee et al. (23) have showed that serum inhibin B levels among formerly cryptorchid men are related to their fertility index with lower inhibin B levels in infertile vs. fertile formerly cryptorchid men. Although we observed a relation between inhibin B and FSH levels and former cryptorchidism among the infertile men, no relation between former cryptorchidism and sperm concentration could be found in our study. This is likely due to the fact that only infertile men with a significantly impaired spermatogenesis (sperm concentration < 20 million/ml) were included in this study.

Inhibin B has been evaluated as a serum marker of spermatogenesis in a number of studies (6, 8, 10, 11, 15, 16), but proper reference materials have been lacking, thus hampering a statistical evaluation of the diagnostic value of this serum marker. The number of patients and controls included in this study allowed us to perform a thorough evaluation of serum inhibin B and FSH levels, alone or combined, as markers of spermatogenesis using ROC plot analysis to define cutoff levels. It should, however, be emphasized that the definition of a cutoff value always is a compromise between a high sensitivity and a high specificity and therefore is partly based on a subjective decision on how many false-negative and false-positive samples is acceptable. The cutoff levels presented here were those that allowed for maximum 5% false-positive cases (specificity 95%). In the clinic, other cutoff values that give a lower specificity but a higher sensitivity might be chosen. Likewise, choosing a different cutoff level might increase the positive predictive value (less false positives) but do so at the expense of a lowering of the negative predictive value resulting in more false-negative cases.

We found that the efficiencies of inhibin B alone and FSH alone to discriminate between the infertile men and the reference group were almost identical but best when the proven fertile men were used as references group. The positive predictive value of FSH alone was higher than that of inhibin B alone. In contrast, Pierik et al. (8) in a study of infertility patients found by ROC analysis that higher diagnostic efficiency was obtained with inhibin B than FSH. This discrepancy may appertain to the reference groups used. Whereas we used either men from the general population or fertile men with a normal sperm concentration as the reference group, Pierik et al. used infertility patients with a adequate Johnsen’s score (based on the histology of a testicular biopsy) as the reference group to patients with an inadequate Johnsen’s score.

Expressing both markers in an inhibin B/FSH ratio also increased the positive and negative predictive values, compared with using the two markers alone. However, although using the two markers in combination (as a positive test, either hormone being outside the respective cutoff levels and a negative test, neither hormone being outside the respective cutoff levels) increased the sensitivity, it did not increase the positive predictive value due to an increased number of false-positive cases.

The observed prediction values of inhibin B and FSH alone or in combination makes it clear that these two markers can never stand alone in the diagnosis of male infertility. Even with a well-defined reference material, a proportion of the infertile men will still fail to be identified if the diagnosis is based on these markers alone. However, in our clinical practice, they have proven to be extremely valuable additional tools, adding to the information obtained by clinical examinations and semen analysis. In most cases they correlate well with both sperm concentration and testicular volume in line with their role as markers of spermatogenesis. Perhaps most added value of these serum markers comes when they do not correlate with the result of semen analysis. Thus, a high inhibin B serum levels in conjunction with azoospermia may be suggestive of an obstructive form of azoospermia, although spermatogenic arrest at a late spermatocyte stage could also be an explanation (9).

Although cut-off values and reference ranges are widely used in the clinical practice, they may not be the most informative way of evaluating patient results, especially when two or more markers may be combined. An alternative way to evaluate two related markers could be by a bivariate reference charts as presented here. This way of presenting data may be particularly informative in relation to inhibin B and FSH serum levels because such charts can discriminate between low inhibin B levels due to a primary hypogonadism and low inhibin B levels due to a secondary hypogonadism because FSH levels will be increased in the first situation but decreased in the second situation. Furthermore, this way of exploiting a reference material is not affected by subjective decisions on cut-off levels.

In conclusion, proven fertile individuals proved to be the most appropriate reference group in the evaluation of reproductive hormone levels because a significant number of otherwise healthy men from the general population with unknown fertility may actually be subfertile. Using an appropriate fertile reference group, relatively high predictive values were obtained with either inhibin B or FSH alone. However, the predictive values were increased by combining the two markers in an inhibin B/FSH ratio. Bivariate reference charts with nonparametric estimators are informative tools in the evaluation of two interrelated serum markers.

	Acknowledgments

 
We thank the Research Centre for Prevention and Health for the samples they provided from men from the general population.

	Footnotes

 
This work was supported by the European Commission under the 5th framework program (Environmental Reproductive Health, Contract QLK4-1999-01422) and the Danish Medical Research Council.

Abbreviations: BMI, Body mass index; ROC, receiver operating characteristic.

Received December 15, 2003.

Accepted March 4, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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