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Original Article |
University Department of Growth and Reproduction, Rigshospitalet (L.F.-L., H.L., A.M.A., E.C., N.E.S., E.R.-D.M.), DK-2100 Copenhagen, Denmark; Andrology Unit, Department of Clinical Physiopathology, University of Florence (C.K.), Florence, Italy; The Fertility Clinic, Herlev University Hospital (S.B.), DK-2730 Herlev, Denmark; and Reproduction, Fertility, and Populations, Institut Pasteur (C.K., K.M.), 75724 Paris, France
Address all correspondence and requests for reprints to: Dr. Lone Frydelund-Larsen, University Department of Growth and Reproduction, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: lfl{at}biobase.dk.
Abstract
Testicular production of inhibin B is believed to be dependent on the presence of germ cells within the seminiferous tubules. However, this association has recently been questioned in patients with deletions of azoospermia factor (AZF) on the Y chromosome. We have addressed this problem in 442 unselected infertile/subfertile patients (excluding obstructive and iatrogenic forms) who were analyzed for Yq microdeletions. AZFc microdeletions were found in 16 patients (3.8% of the total infertile group, but 9% of the subgroup with azoospermia or severe oligozoospermia with sperm concentration <1 x 106/ml). The reproductive hormone profiles in patients with AZFc microdeletions were analyzed and compared with those in infertile patients without microdeletions and those in fertile control individuals. The mean serum inhibin B concentration in the patients with AZFc microdeletions (39.5 ± 36.0 pg/ml) was significantly lower than that in the group of infertile patients without microdeletions (134.6 ± 88.5 pg/ml). However, no significant difference was found compared with that in a matched group of infertile patients with comparably low sperm counts (72.6 ± 75.5 pg/ml). Bilateral testicular biopsies in the AZFc-deleted patients revealed a variable histological pattern suggestive of a progressive depletion of seminiferous epithelium. An association between testicular pathology and the reproductive hormone profile was found; the more severe forms had lower inhibin B and higher FSH levels. Importantly, if Sertoli cell-only tubules were prevalent in the biopsy, inhibin B was invariably undetectable. In patients with bilateral spermatocytic arrest, inhibin B remained within the normal range, which is consistent with a role of spermatocytes in the maintenance of inhibin B secretion. Our data support the view that, in contrast to recently published data, in patients with AZF microdeletions the serum concentration of inhibin B is dependent upon the functional interaction between Sertoli cells and spermatocytes and/or spermatids.
MICRODELETIONS OF THE long arm of the human Y chromosome, found in males with idiopathic infertility, are associated with spermatogenic failure and have been used to define three regions of Yq [azoospermia factor a (AZFa), AZFb, and AZFc] that contain genes with a function in germ cell development and/or regulation of spermatogenesis (1, 2). Microdeletions in infertile men most frequently occur in the AZFc region, whereas deletions in the AZFb and especially the AZFa regions are relatively rare (3). Previous studies reported spermatogenic alterations varying from severe hypospermatogenesis to spermatogenic arrest and Sertoli cell-only syndrome (SCOS) in patients with apparently similar microdeletions. A genotype-phenotype correlation has been found for the complete forms of AZFa and AZFb deletions that are associated with SCOS and spermatogenic arrest, respectively (4, 5). Partial AZFa and AZFb deletions and AZFc deletions present variable phenotypes ranging from azoospermia to oligozoospermia (2, 6). We have addressed this problem in a group of patients with AZFc deletions, but in addition to testicular histology and sperm concentrations, we have focused our attention on another important aspect of the phenotype, which is the reproductive hormone profile.
It is established that the reproductive hormone profile in general and especially the serum concentrations of inhibin B and FSH reflect the functional state of the seminiferous epithelium in the adult testis (7, 8). Studies in which the serum inhibin B level was related to the histopathological pattern of the testicular biopsy have confirmed that inhibin B is reduced in men with more severely affected spermatogenesis, particularly those with SCOS or spermatogenic arrest at an early stage, e.g. at the level of spermatogonia (9, 10, 11). In contrast, normal or near-normal inhibin B levels are observed in azoospermic men with spermatogenic arrest at a later stage (e.g. of spermatocytes or spermatids) or in men with obstructive azoospermia (8, 11). Studies of hormonal regulation and the diurnal rhythm of inhibin B secretion provided additional evidence that the serum concentration of inhibin B reflects a functional interaction between germ cells and Sertoli cells as well as Leydig cells and the pituitary (12, 13).
The relationship among inhibin B, FSH, and spermatogenesis was recently questioned in a study by Foresta et al. (14) in which reproductive hormones in infertile patients with Yq microdeletions were investigated. These researchers found relatively high inhibin B levels in a group of 27 AZF-deleted patients compared with those in similar patients without microdeletions. They observed also a trend characterized by lower (but not statistically significant) FSH levels in the microdeleted patients. As deletion of the AZFc region affects only germ cell-specific genes, these researchers concluded that the Sertoli cell function may be partially altered in these patients, but as inhibin B levels were high, the production of this hormone was maintained by Sertoli cells independently from neighboring germ cells (14).
In our study we analyzed the serum concentrations of reproductive hormones in infertile patients with AZFc microdeletions and compared these to concentrations in a matched group of infertile patients without Yq microdeletions and to those in a group of fertile control individuals. In contrast to the study by Foresta et al. (14), we have found low serum inhibin B and elevated FSH levels in the majority of 16 patients with AZFc microdeletions compared with the fertile control subjects. Moreover, these parameters did not differ from those in the matched group of infertile men without Yq microdeletions. In addition, we made histopathological assessment of spermatogenesis in 10 of the AZFc-microdeleted patients. We found an association of the hormonal profile and testicular phenotype and propose that the reported lack of genotype-phenotype association in previous studies might be due to a progressive natural history of testicular impairment in AZFc patients.
Subjects and Methods
Subjects
To limit any selection bias, a group of 650 unselected consecutive patients referred to our andrology clinic because of male infertility/subfertility were enrolled in a study of Yq microdeletions. The men were partners in couples with a history of at least 1-yr unexplained childlessness, in which any problem in the female partner was excluded. The patients had sperm counts ranging from azoospermia to normospermia. All patients with evidence or suspicion of obstructive forms of azoospermia or secondary oligozoospermia due to orchitis or iatrogenic causes were excluded from this study. A total of 442 patients, most of them with idiopathic infertility, were included in the study. The mean age in this group was 33 (ranging from 20–45). The breakdown of this group according to sperm concentration was as follows: azoospermia, n = 69; cryptozoospermia (<0.2 x 106/ml), n = 50; very severe oligozoospermia (0.2–1.0 x 106/ml), n = 58; severe oligozoospermia (1.01–5.0 x 106/ml), n = 85; moderate oligozoospermia (5.01–20.0 x 106/ml), n = 104; and normospermia (>20.0 x 106/ml), n = 76. As a control group, we included 100 healthy recent fathers who were recruited via their pregnant partners from an obstetric clinic for a study of reproductive fitness of European populations (15). All patients and control subjects underwent a routine andrological examination, including detailed semen and hormonal analyses. The semen and blood samples for analysis of reproductive hormones were obtained from all patients in the morning. In addition, a molecular analysis of the Yq region was performed on DNA isolated from a peripheral blood sample. The local ethical committee approved the study, and informed consent was obtained from the patients.
Semen and hormonal analyses
Semen analyses were performed according to WHO guidelines (16) in our laboratory, which is under rigorous internal and external quality control (17, 18). In nearly all patients, semen analysis was repeated at least three times; in a few patients with azoospermia only two analyses were performed. Median values of sperm concentrations and semen volumes were used in this study.
Analysis of reproductive hormones was performed in our certified laboratory using rigorously validated assays. The laboratory is enrolled in a systematic external quality control program. Normal values and ranges were established on the basis of large cohort studies of unselected healthy Danish men (7). Serum concentrations of FSH, LH, and SHBG were measured using time-resolved immunofluorometric assays (DELFIA, Wallac, Inc., Turku, Finland). The detection limits, mean values, and normal ranges (5th and 95th percentiles) were: for FSH, less than 0.06 and 4.2 IU/liter (1.4–9.4 IU/liter); for LH, less than 0.05 and 3.6 IU/liter (1.5–6.4 IU/liter); and for SHBG, less than 0.23 and 30 nmol/liter (13–81 nmol/liter), respectively. In all three DELFIA assays the intra- and interassay coefficients of variation (CV) were less than 7% in the full range. Total testosterone and estradiol were measured by RIAs (Coat-a-Count, Diagnostic Products, Los Angeles, CA; and Pantex, Santa Monica, CA, respectively). The detection limits, mean values, and the normal range were less than 0.23, 16.2, and 7.7–24.5 nmol/liter for the testosterone assay and less than 18, 80, and 44–202 pmol/liter for the estradiol assay, respectively. In the testosterone assay the intra- and interassay CV were less than 10% in the normal range; in the estradiol assay the intraassay CV was less than 8%, and the interassay CV was less than 13% in the normal range. Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay [from Oxford-Bioinnovation (Oxford, UK), which is identical to the commonly used Serotec (Oxford, UK) assay], using a monoclonal antibody raised against the inhibin ßB-subunit in combination with a labeled antibody raised against the inhibin 
-subunit, as previously described (9, 19). This assay has been extensively validated in a number of our previous studies (7, 10, 12). The inhibin B assay in our laboratory has a detection limit of 20 pg/ml (which was used for undetectable levels to calculate mean and median values), the mean and normal range were 210 and 80–406 pg/ml, respectively. The intra- and interassay CV were less than 12% and less than 17% in the full range, respectively.
Statistical analysis
Statistical analysis of hormone data was performed using the SPSS statistical program (version 10, SPSS, Inc., Chicago, IL). Levene’s test was used for assessing the equality of variances, and t test was used to compare mean and median values between groups. Two-sided values of P < 0.05 were considered statistically significant.
Molecular analysis
Screening for microdeletions was performed by PCR analysis of genomic DNA extracted from lymphocytes. A total of 5 anonymous sequence-tagged sites markers and 5 Yq-specific genes, spanning the 3 AZF regions, were screened. The loci were: AZFa: sY84 and the DBY gene; AZFb: sY114, sY134, and eIF-1AY; and AZFc: sY152, sY158, and the genes BPY2 and DAZ (sY254). In addition, we analyzed the SRY gene, which is located on Yp. Each locus was analyzed separately in a duplex reaction with ZFX/ZFY as an internal control. For each duplex reaction the PCR conditions were 95 C for 5 min, followed by 35 cycles of 95 C for 1 min, 62 C for 1 min, and 72 C for 1.5 min, followed by 72 C for 10 min. The analyses were performed with a male control sample (positive control), a female sample (negative control), and a blank sample (containing no DNA). If a microdeletion was detected, the analysis was repeated 3 times. The third time the analysis was performed using genomic DNA from a second, independently obtained blood sample.
Testicular biopsies
Testicular biopsies were performed only in selected patients: 1) candidates for intracytoplasmic sperm injection or 2) patients with abnormalities in the ultrasound examination that raised a suspicion of carcinoma in situ. Of the 16 patients with AZFc microdeletions, 10 had bilateral testicular biopsies performed for 1 of these reasons. In all instances biopsies were performed bilaterally, using a standard open surgical technique. Tissue fragments of approximately 3 x 3 x 3 mm were fixed immediately in Stieve’s fluid (which is preferred because of its excellent morphological detail) or in buffered formalin. The tissues were dehydrated, paraffin embedded, and subsequently sectioned. At least 100 serial sections were made for each biopsy. The sections were stained with hematoxylin-eosin, and for each block an immunostaining for placental-like alkaline phosphatase was performed to facilitate detection of carcinoma in situ cells, if present. Histological examination was performed using a light microscope (Carl Zeiss, New York, NY). The following histological terms were used to describe prevalent patterns: SCOS, if no germ cells were detected in the biopsy; spermatogenic arrest of germ cell maturation at the spermatocyte level [also called spermatocytic arrest (SPA)]; and heterogeneous mixed patterns. All biopsies were examined independently by two of the authors (N.E.S. and E.R.-D.M.).
Results
Molecular analysis
Yq microdeletions were detected in 16 of 442 (3.6%) unselected patients with infertility/subfertility. The frequency of microdeletions in the subgroup with azoospermia and severe oligozoospermia (sperm concentration, <1 x 106/ml) was 9.0%. Twelve of the 16 patients with microdeletions had azoospermia or cryptozoospermia, and no microdeletions were detected in the patients with sperm counts of more than 1 x 106/ml (Table 1
). The highest sperm concentration detected in a Yq deleted patient was 0.13 x 106/ml. Thus, the frequency of microdeletions among the infertile men with sperm concentrations less than 0.2 x 106/ml was 13.4% (16 of 119). These frequencies are in agreement with our previous study that examined the presence of Yq microdeletions in unselected subfertile patients (20). All identified microdeletions encompassed the AZFc region, removing markers sY152 and sY158 and the genes BPY2 and DAZ (sY254). No isolated gene-specific microdeletions were detected. No deletions in the AZFa or AZFb regions were found.
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), who had a visible deletion of chromosome Y (45X/46XdelYq). Hormone analysis
The results of the hormone analysis in the patients with Yq microdeletions are listed in Table 1. All patients, except one with bilateral SPA, had markedly decreased serum concentrations of inhibin B; in 7 of 16 (44%) inhibin B was undetectable, in 5 additional cases the concentration was barely above the detection threshold, which is 20 pg/ml. Most of the patients had increased FSH levels, and some also had higher LH levels. Normal FSH concentrations were observed in patients with relatively higher inhibin B levels. Testosterone levels were markedly decreased in 2 patients with apparent hypogonadism (no. 9 and 11 in Table 1). Three other patients (no. 10, 12, and 13) had testosterone levels at the lowest values of our laboratory’s normal range (8.1–8.6 nmol/liter). In the remaining patients serum testosterone was within the normal range, but significantly lower than in the fertile control individuals. As a matter of fact, all of our infertile groups, with or without microdeletions, had, on the average, lower total testosterone levels than the fertile control individuals, in agreement with earlier observations in our clinic (Andersson et al., manuscript in preparation) and other centers (21). For comparison, Table 2 shows average hormone values (medians and means) for the group with microdeletions, for all other infertile men without microdeletions (excluding obstructive forms), and for a control group of men with normospermia and proven fertility. In addition, to provide a group with impairment of spermatogenesis directly comparable to the microdeleted patients, the average values for 103 infertile men with idiopathic azoospermia or severe oligozoospermia with sperm concentrations below 0.2 x 106/ml were calculated. For calculation of average concentrations of inhibin B, an arbitrary value of 20 pg/ml (a detection limit) was used in cases with undetectable levels.
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Bilateral testicular biopsies available for 10 patients with AZFc microdeletions revealed variable histological abnormalities (Table 1 and Fig. 1). However, detailed analysis demonstrated a remarkable consistency; SPA, SCOS, or a combination of both was present in nearly all patients with microdeletions. Three patients had a bilateral homogenous pattern of SPA or SCOS. Two patients had a homogeneous pattern of SCOS in 1 testicle and SPA in the other. Three patients showed a bilateral mixed pattern of SCOS and SPA. Finally, 2 patients had a mixed pattern of SCOS and SPA on 1 side and a homogenous pattern of either SPA or SCOS on the other side. Only 2 patients had a few tubules with some spermatids present in the biopsy. In biopsies with SPA a remarkably increased sloughing of seminiferous epithelium was noted, with many degenerating spermatocytes visible in lumen of tubules. Other histological abnormalities included occasional presence of partially undifferentiated Sertoli cells (1 case), atrophic hyalinized tubules (3 cases), and micronodules of Leydig cells (in 1 case with bilateral SCOS).
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Our study has provided new evidence for the interaction of Sertoli cells and germ cells to maintain inhibin B production in the testis, based on analysis of the phenotype of patients with microdeletions of the AZFc region of the Y chromosome that have a specific impairment of spermatogenesis. We found an excellent association of their hormonal profiles with testicular pathology assessed in the biopsy; the more severe forms had lower inhibin B and higher FSH levels. The mean inhibin B concentration in the patients with AZFc microdeletions (39.5 ± 36.0 pg/ml) was significantly lower than that in the whole group of infertile patients without microdeletions (134.6 ± 88.5 pg/ml; P < 0.001), and conversely the opposite was true for FSH levels. However, these differences virtually disappeared when compared with 103 patients with comparably low sperm counts (Table 2); inhibin B in this subgroup was nearly as low as that in the microdeleted group (72.6 ± 75.5 pg/ml; P = 0.12), and FSH was equally high (18.0 ± 15.9 IU/liter). Thus, the concentrations of inhibin B and FSH, although they reflect quite well the status of spermatogenesis, are not a predictive indicator of the presence or absence of a microdeletion in the AZFc region.
Our results are in disagreement with a recently published study that investigated reproductive hormones in patients with Yq microdeletions and found relatively high inhibin B levels (14). In that study the mean inhibin B and FSH concentrations in 14 cases with AZFc deletions were 163.8 ± 45.3 pg/ml and 9.3 ± 3.5 IU/liter, respectively. The inhibin B value was in the lower normal range compared with that in normospermic control individuals (229.7 ± 81.6 pg/ml) and was significantly higher than that in nondeleted infertile men (76.5 ± 46.1). This high mean concentration in azoospermic patients is very difficult to comprehend, considering that the majority of the patients with microdeletions had an SCOS pattern in a testicular biopsy (14). A difference in specificity of the inhibin B assay cannot be a reason for the discrepancy, because the Oxford-Bioinnovation assay used in our laboratory is identical to the Serotec assay used in the Foresta’s laboratory. We speculate that the discrepancy may be due to the fine needle aspiration procedure used by Foresta et al. (14), which perhaps led to overlooking areas of spermatocytic arrest or a false overrepresentation of SCOS. The needle aspiration technique is a very useful method; however, for reliable quantitative analysis of spermatogenesis, several aspirates should be used (22). We stress the importance of the presence of spermatocytes, because among our patients with microdeletions, the two with inhibin B levels within normal range (patients 2 and 3 in Table 1) had bilateral homogeneous spermatocytic arrest without SCOS tubules visible in testicular biopsies.
This observation supports the hypothesis that the presence of spermatocytes may be necessary for inhibin B production in the adult testis (11, 23). In other patients who had tubules without germ cells (spermatocytes or spermatids) in the biopsy (SCOS or a prevalence of SCOS with very few SPA tubules), inhibin B was invariably undetectable. We believe that this phenomenon is caused not by an inherent problem with Sertoli cells, but by a lack of interaction between Sertoli cells and meiotic or postmeiotic germ cells, which in the adult testis are necessary to maintain inhibin B production.
Such a functional interaction is apparently not required by immature Sertoli cells in the prepubertal testis. Thus, the presence of a large number of undifferentiated Sertoli cells in a dysgenetic adult testis could also explain a high inhibin B level in a patient with SCOS. There is, however, no information concerning the maturation of Sertoli cells in Foresta’s study. The hypothesis of a developmental change in inhibin B regulation is consistent with our previous observations in both adult patients and children (10, 23). The pubertal switch in the regulation of inhibin B production was illustrated in a study of men with testicular cancer, whose inhibin B levels became undetectable after testicular irradiation that eradicated germ cells but preserved Sertoli cells (10), and by a study of survivors of leukemia, who had undetectable serum inhibin B after puberty, but had normal levels in early childhood despite SCOS (confirmed by testicular biopsies) caused by cytotoxic treatment of leukemia (23).
In this study and our previous study (20), all patients with AZFc deletions had severe testiculopathy in histological examination, accompanied in some cases by signs of testicular atrophy, such as the presence of partially hyalinized tubules or micronodules of Leydig cells. We suggest that the variable histological picture may be related to a progressive nature of the testicular defect caused by a deletion of the AZFc region. In some patients with the SPA we noted a marked depletion of seminiferous epithelium, probably due to the failure of progression through the checkpoint of the first meiotic division. We hypothesize that this failure would cause a progressive degradation of the seminiferous epithelium, which would then lead to an increased occurrence of Sertoli cell-only tubules, resulting in SCOS. The lack of germ cells would disrupt the Sertoli-germ cell interaction and inhibit the normal production of inhibin B. This, in turn, may lead to an overstimulation of the testis by gonadotropins and formation of Leydig cell micronodules. Finally, an atrophy of tubules with a gradual hyalinization of basal membranes may occur.
The hypothesis of a progressive process of testicular atrophy associated with a deletion of the DAZ genes within the AZFc region was previously proposed based on histological analysis in four patients (24). Moreover, a progressive decrease in sperm concentration from severe oligozoospermia to azoospermia over a period of time has been reported in infertile men with AZFc deletions (25, 26, 27). Very recently, spontaneous regression of the germinal epithelium over time, assessed by testicular biopsies, was described in a patient with an AZFc microdeletion (28). Taken together these findings strongly support the hypothesis of a progressive process and may explain the poor correlation between genotype and phenotype in the patients with AZFc deletions reported in previous studies.
In conclusion, our data support the view that the serum concentration of inhibin B is not just a simple secretion product of Sertoli cells, but also reflects the integrity of germ cells, especially the presence of spermatocytes and spermatids. Patients with impaired spermatogenesis due to deletion of the AZFc region retain this functional relationship and provide additional strong evidence for the existence of germ cell-Sertoli cell cross-talk.
Acknowledgments
We thank Drs. Niels Jørgensen and Anne-Grethe Andersen for help with collection of control samples, Jane Hinrichsen and Lene G. Pedersen for excellent technical assistance, as well as other members of the staff of the andrological and hormone laboratory for performing routine tests.
Footnotes
This work was supported by grants from the Council of Copenhagen University Hospitals, the Danish Medical Research Council, the European Union, Andersen’s Foundation, and the Danish Cancer Society.
Abbreviations: AZF, Azoospermia factor; CV, coefficient of variation; SCOS, Sertoli cell-only syndrome; SPA, spermatocytic arrest.
Received May 10, 2002.
Accepted August 29, 2002.
References
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