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Original article

Combination of serum inhibin B and follicle-stimulating hormone levels can not improve the diagnostic accuracy on testicular sperm extraction outcomes in Chinese non-obstructive azoospermic men

HUANG, Xiang; BAI, Quan; YAN, Li-ying; ZHANG, Qiu-fang; GENG, Li; QIAO, Jie

Editor(s): CHEN, Li-min

Author Information
doi: 10.3760/cma.j.issn.0366-6999.2012.16.016
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Abstract

Intracytoplasmic sperm injection (ICSI) with spermatozoa recovered by testicular sperm extraction (TESE) has provided the possibility for the azoospermic men to reproduce.1–3 The TESE procedure has been shown to be successful even in testes with only focal spermatogenesis.4 However, unsuccessful sperm recovery occurs in as many as 50% of non-obstructive azoospermic patients, which results in both emotional and physical distress for these patients.3 In addition, it has a risk of complications such as hematoma, devascularisation and fibrosis that could adversely affect the function of the testes.5,6 A non-invasive marker predicting successful sperm recovery before TESE would be useful for evaluating azoospermic men. It would decrease the surgical risk and reduce the failed results in the patients to some extent. In previous studies, non-invasive parameters such as serum follicle-stimulating hormone (FSH) and testicular size have been investigated.7–10 Elevated serum FSH levels seem to be inversely related with the probability of sperm in the testis, although the predictive value of this relation is very weak for excluding patients from TESE.4,7,9,10 Testicular volume also has an inverse relation to the presence of spermatozoa in the testis. However, spermatozoa were also successfully retrieved from testes with a volume of less than 5 ml.11 So, a more accurate marker for predicting the successful sperm recovery before TESE would be useful for azoospermic men.

Inhibin-B is a heterodimer glycoprotein that primarily originates from the male testes. It is regulated by the presence of an advanced stage of spermatogenesis and has an inhibitory effect on gonadotropin secretion.12–14 Theoretically, inhibin B should be a direct indicator of spermatogenesis, and a more sensitive marker of the functional state of the seminiferous tubule than FSH.15–17 But it is still controversial whether serum inhibin B determination is a sensitive predictor of successful TESE in azoospermic men.11,18–25

The study was to investigate the diagnostic accuracy of the serum inhibin B levels and the success of TESE outcome in a large population of Chinese non-obstructive azoospermic men, and to compare the results with other parameters such as serum FSH levels and testicular volume.

METHODS

Patients

This study was approved by the Ethics Committee of Peking University Third Hospital, China. Written informed consents were obtained from each participant before the initiation of the investigation. Totally, 305 men with nonobstructive azoospermia were analyzed between November 2008 and February 2012. All the subjects underwent a diagnostic infertility evaluation that included medical and reproductive history, physical examination, and three semen analyses according to WHO guidelines. The testicular volume (defined here as the mean of the testis volumes) was calculated by ultrasound examination. Patients with obstructive azoospermia, for which microsurgical epididymal sperm aspiration (MESA) could be done to retrieve the sperm, were excluded from the analyses. Moreover, patients with karyotype abnormalities or testosterone/gonadotropin therapy within 3 months of the pre-study assessments were also excluded.

Hormone analysis

Peripheral blood samples were drawn for hormone measurements. Serum FSH, luteinizing hormone (LH), estradiol (E2) and testosterone levels were determined by a competitive immunoassay, employing the Immulite analyser (Diagnostic Products Corporation, Los Angeles, CA90045, USA). The normal ranges were 0.7-11.1 mIU/L for FSH, 0.8-7.6 mIU/L for LH, 0-206 pmol/L for E2 and 8.49-55.4 nmol/L for testosterone respectively. The intra- and inter- assay coefficients of variation were <5% and <8%, respectively. Serum inhibin B levels were measured using the inhibin B Gen II ELISA kit according to the manufacturer's instructions (Beckman Coulter, A81301, USA ). The detection range of the assay was 0-1000 pg/ml.

Testicular sperm extraction

Patients underwent regional anaesthesia and both testes were exposed through a single midline median raphe incision. A tissue wedge (about 25 mg) was incised and isolated by surgical scissors.8 Surgery was discontinued when sperm were found on wet preparations of the biopsies. For TESE, testicular biopsies were immersed in sperm culture medium (SAGE IVF inc. ART-1006, USA) in a Petri dish and sent to the reproductive biology laboratory. Testicular tissue was minced and shredded in the culture medium with two scalpels. The presence of spermatozoa was examined directly under an inverted microscope at ×400 magnification.17 The tissue suspension was then centrifuged for 5 minutes at 1600 r/min and resuspended in 500 μl of sperm culture medium. It was incubated (5% CO2, 5% O2, 90% N2, 37°C) overnight (more than 12 hours) and checked again under the microscope. The TESE outcome was interpreted as successful or unsuccessful, according to the presence or absence of spermatozoa, respectively.

Testicular biopsies for histological evaluation

In addition to the biopsy for TESE, another testicular tissue was immediately immersed in Bouin's solution and fixed for 48 hours for histological examination. Serial sections were prepared from paraffin blocks and stained with haematoxylin-eosin. The results were classified as the following spermatogenic phenotypes: hypospermatogenesis (reduction in the number of spermatogenic cells including spermatid) or germ cell mature arrest (MA, the absence of later stages of spermatogenesis), sertoli cell only syndrome (SCOS, the absence of germ cells and were lined with sertoli cells in the seminiferous tubules.26

Statistical analysis

Statistical analysis was performed using SPSS 18.0 software (SPSS Inc., USA). The data were presented using mean ± standard deviation (SD). Proportion was presented with percent and actual number. Differences of characteristics and predictors between the successful and unsuccessful TESE groups were assessed in the Mann-Whitney U test. The characteristics of patients with different histological phenotypes were compared by the Kruskal-Wallis test, and the Mann-Whitney U test was used for further comparison between two groups. The area under the receiver operating characteristic (ROC) curve was used to assess the predictive accuracy of selected parameters for the likelihood of retrieving sperm (with a value of 0.5 indicating no predictive power and 1.0 indicating perfect prediction).27 Paired χ2 test and χ2 partition were used for diagnostic accuracy comparison among different predictors. Correlation between different variables was analyzed using the Pearson r coefficient. P <0.05 was considered statistically significant.

RESULTS

The patients' clinic characteristics and serum hormones levels and the outcome of TESE are summarized in Table 1. Testicular sperm was successfully retrieved in 137 of 305 patients (44.9%), and the serum inhibin B concentrations were significantly higher in the successful TESE group than that in the unsuccessful group (P <0.001; Table 1). The serum FSH levels were significantly lower in the successful TESE group than that in the failed TESE group (P <0.001). The mean testicular volumes were significantly larger in the successful TESE group than in the failed TESE group (P <0.001). There were no significant differences in serum LH and testosterone levels between the two groups (P=0.641, P=0.143, respectively).

Table 1
Table 1:
Patients' clinical characteristics and serum hormones on the outcome of TESE

All the patients were then divided into three groups (hypospermatogenesis group, SCOS group and MA group) according to histological analysis. Table 2 showed values of serum hormone levels and testicular volumes of the three histological groups. Successful sperm retrieval achieved in the hypospermatogenesis group was significantly higher than in the SCOS and MA groups (P <0.001 for both groups). Mean inhibin B concentrations in the SCOS and MA groups were significantly lower than in the subjects with hypospermatogenesis (P <0.001). The SCOS patients had the lowest mean serum inhibin B concentration compared with the other groups (P <0.001). An inverse correlation between serum inhibin B and FSH levels was observed in all subjects (r=-0.567, P <0.05). There were differences in the testicular volumes between the hypospermatogenesis group and the other groups, while, no significant differences were found in serum LH levels between the normal and the other groups. And no differences were observed in the serum testosterone levels among the three groups (P=0.108).

Table 2
Table 2:
Patients' characteristics in different of the histological phenotypes

The predictive value of basal serum inhibin B, 1/FSH, inhibin B/FSH ratio and testicular volumes to discriminate between successful and unsuccessful TESE were assessed by calculating the area under the ROC curve (Figure 1).

Figure 1.
Figure 1.:
Receiver operating characteristic (ROC) curves of basal serum inhibin B, 1/FSH, testicular volume and basal inhibin-B/FSH ratio for testicular sperm retrieval.

The characteristics of the ROC curve (AUC) for each parameter are summarized in Table 3. Comparing the diagnostic accuracy of serum inhibin B and FSH on the outcome of TESE, we saw no significant differences between these two assays (P=0.312). And there were no differences between the combination of the two parameters (inhibin-B/FSH ratio) and inhibin B or FSH alone (P=0.706 and P=0.205, respectively). However, the diagnostic accuracy of testicular volumes was significantly lower than other parameters (P=0.001).

Table 3
Table 3:
Diagnostic accuracy of serum inhibin B, FSH and testicular volumes on the outcome of TESE

DISCUSSION

Our study presents the outcome of 305 Chinese non-obstructive azoospermic patients who had TESE. Similar with most previous studies, our data showed significant differences in the mean levels of inhibin B and FSH as well as in the mean testicular volume among the groups of successful and unsuccessful patients.18–20,26,28 But this was different from some other studies, and our sperm retrieval rate using TESE (44.9%) was slightly lower than these studies (about 50%).21,23 Since 1998, enzymatic digestion of testicular biopsy has been successful at reducing sperm recovery failures in patients with non-obstructive azoospermia.29 Even motile spermatozoa have been observed in some of the enzymatically digested residual tissues when no sperm were detected after mechanical treatment of these biopsies. However, enzymatic digestion has not been included in our procedures. The discrepancy between the studies might have been due to the selection of patients and the methods used in the TESE procedures.21

The differences in the inhibin B levels among the three histological groups demonstrated that inhibin B is a marker of seminiferous tubule function. In fact inhibin B has been reported to be associated with late stages of spermatogenesis.19,30,31 Furthermore, the inverse correlation between inhibin B and FSH in our patients reflects the negative feedback mechanism reported in other studies.15,16,18,19 In this study, a minority of patients with SCOS and MA underwent successful TESE. TESE outcomes were inconsistent with testicular histology as these patients might have focal spermatogenesis in their testes. Recently, microdissection testicular sperm extraction (Micro-TESE) has been recommended as a first line treatment for men with non-obstructive azoospermia, especially with severely impaired spermatogenesis, which has a higher successful retrieve rate compared to conventional TESE.32 However, Micro-TESE is a technical procedure requiring a significant learning curve and longer operative time. And the chance of sperm retrieval during Micro-TESE can be associated with the duration of the operation and the repeated operation.32,33 One of the current issues is that the decision to retrieve tubules is based only on appearance, as there is no guarantee that the removed tubules contain sperm. Multiphoton microscopy, a novel laser imaging technology, enables label-free immediate visualization of many biological processes in living tissue at subcellular resolution.34 It has the potential to facilitate real-time visualization of spermatogenesis and to prevent testicular injury and prevent long or repeat procedures in humans, such as testicular sperm extraction for men with severely impaired spermatogenesis.34

Different from a recent meta-analysis of diagnostic accuracy studies which demonstrated serum inhibin B cannot serve as a stand-alone marker of TESE (sensitivity: 65% and specificity: 83%), the present study showed a higher sensitivity of 83.5% with a little lower specificity of 79.1%.35 In agreement with former studies, the area under the ROC curve for serum inhibin B was similar with that of FSH.20,22–24 Although significant differences in the serum inhibin B levels between successful and failed groups of TESE were found, inhibin B was not a more sensitive predictor of the successful TESE than FSH in azoospermic patients.22,24 It was confirmed that the combination of the two parameters did not improve the diagnostic accuracy of TESE outcome in our study, which was consistent with many previous studies.22,24,26 But a controversy still exists as a few studies have reported that the combination of the two parameters was better.20,30 The Micro-TESE operation and enzymatic digestion of testicular biopsy were not included in our procedure, which may have improved the sperm recovery rate in previous studies. The diagnostic accuracy of inhibin B as a predictor of TESE outcome needs to be investigated in the further studies with different TESE procedures.

In conclusion, basal serum inhibin B level might be a predictor of the presence of sperm in TESE in Chinese men with non-obstructive azoospermia. However, inhibin B is not superior to FSH in predicting the presence of sperm in TESE. And the combination of them does not improve the diagnostic accuracy of TESE outcome.

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Keywords:

azoospermia; follicle-stimulating hormone; infertility; inhibin B; testicular sperm extraction

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