Combined evaluation of inhibin B, follicle stimulating hormone and luteinizing hormone improve sperm retrieval prediction in patients with non-obstructive azoospermia

doi:10.4236/arsci.2013.11001

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Vol.1, No.1, 1-5 (2013) Advances in Reproductive Sciences

http://dx.doi.org/10.4236/arsci.2013.11001

Combined evaluation of inhibin B, follicle stimulating

hormone and luteinizing hormone improve sperm

retrieval prediction in patients with non-obstructive

azoospermia*

Bing Wang, Xunbin Huang#

Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;

#Corresponding Author: [email protected]

Received 2 April 2013; revised 3 May 2013; accepted 15 May 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Introduction: Non-invasive methods that can

predict sperm recovery in patients with non-ob-

structive azoospermia (NOA) arouse interest of

clinicians. The aim of this study was to evaluate

the predictive value of sperm retrieval in NOA.

Materials and Methods: In the retrospective stu-

dy, fine needle aspiration (FNA) was performed

on 306 patients with NOA at the department of

andrology, Wuhan Tongji Reproductive Medical

Hospital. Inhibin B, FSH and LH plasma levels

were analyzed and sperms were retrieved in 67

of 306 cases (21.89%). Results: There were sta-

tistically signiﬁcant differences between suc-

cessful and unsuccessful sperm recovery groups

in terms of mean serum inhibin B, FSH and LH

levels. The areas under the curve (AUC) of inhi-

bin B, FSH and LH were 0.696, 0.729 and 0.747

respectively, and the AUC for the combined

value of the three hormones is 0.832. The cut-off

points were 27.31 pg/ml, 11.68 IU/L and 4.04 IU/L

for inhibin B, FSH and LH respectively. Conclu-

sions: This study suggests that the combined

evaluation of inhibin B, FSH and LH is a more

effective predictor for s uccessful s perm retrieval

in patients with NOA before decision making of

an invasive procedure than any single factor.

Keywords: Non-Obstructive Azoospermi a;

Sperm; Hormones; Prediction; Non-Invasive

1. INTRODUCTION

Before the introduction of intracytoplasmic sperm in-

jection (ICSI [1], as to patients with NOA, there was no

available treatment for them. Therefore, testicular sperm

extraction has become a popular surgical procedure to

obtain sperm in NOA patients [2]. The successful sperm

recovery is observed in 40% - 60% of these patients by

Testicular Epididymal Sperm Extraction (TESE) [3,4]. In

addition, TESE is an invasive procedure and may bring

some side effects [5,6] to the patients of sperm retrieval

failure, and it may result in physical and financial com-

plications. In order to avoid these unsuccessful results,

some reasonable predictions are important to offer.

Fine needle aspiration (FNA) is a less invasive method.

Bettella, A. et al. reported that the sensitivity and speci-

ficity of FNA were 44.6% and 100%, respectively. They

concluded that FNA might be a reliable diagnostic tool

for successful sperm retrieval [6]. Although FNA can

minimize damages, it is not wise to practice in all azo-

ospermia patients. Other factors such as finance, side ef-

fects and compliance should be considered before deci-

sion making.

In order to prevent these complications, some reliable

markers are needed to give a clue to clinicians that the

cases for whom TESE or FNA would be suitable. In re-

cent years, non-invasive exams such as hormonal pa-

rameters have been proposed. Inhibin B consisting of α-

subunit and β-subunit is a glycoprotein hormone mainly

produced by the Sertoli cells [7]. Inhibin B has been pro-

posed as a marker of spermatogenesis. Men with severe

oligozoospermia and idiopathic azoospermia have signi-

ficantly lower serum inhibin B levels than men with nor-

mospermia [8]. Spermatogenesis is also dependent on the

balanced endocrine interaction [9]. FSH acts on receptors

in Sertoli cells and initiate spermatogenesis, while lu-

teinizing hormon e (LH) stimulates the production of tes-

tosterone, which in turn may act on the seminiferous tu-

*Conflict of Interest: Authors declare no conflict of interest.

B. Wang, X. B. Huang / Advances in Reproductive Sciences 1 (2013) 1-5

bules and influence spermatogenesis [10]. It is indicated

that both FSH and LH are necessary for the initiation of

spermatogenesis [11].

Inhibin B, FSH and LH are all close related to sper-

matogenesis, no matter whether they can predict the suc-

cessful sperm recovery in testis in patients with NOA.

Ziaee, S.A. et al. suggested that Serum FSH and inhibin

B are useful predictors of the presence of sperm in pa-

tients with NOA. Combination of two parameters can

improve the predictive power [12], though another study

fails to conform this finding [13]. With a retrospective

study using data from our hospital in recent five years,

we assess the predictive va lues of the combined effect of

inhibin B, FSH and LH before testicular sperm retrieval.

2. MATERIALS AND METHODS

2.1. Patients

This retrospective study included 306 patients with

NOA. Patients with obstructive azoospermia were diag-

nosed by physical examination, while Seminal glycosi-

dase test, or Vas Deferens Radiography or epididymal

FNA were excluded. Patients with abnormal karyotypes

were also excluded from the data. The diagnosis of azo-

ospermia is on the basis of at least three semen analyses

after centrifugation. The ages of the patients ranged from

21 - 42 yrs. Clinical, paraclinical, and histological infor-

mation of patients were gathered and each subject was

evaluated with fully physical examination.

2.2. Hormonal Analyses

Serum levels of inhibin B were measured in all pa-

tients by a solid-phase sandwich enzyme-linked immuno-

sorbent assay (ELISA) (Serotec, Oxford, UK). FSH and

LH were evaluated by immunofluorometric assays (Au-

todelfia, Wallac, Inc., Freiburg, Germany).

2.3. Fine Needle Aspiration of Testes

To perform testicular FNA, the area around the sper-

matic cord was locally anesthetized by injecting 5 ml of

2% lidocaine after skin iodine disinfection. The aspira-

tion was then performed in the center as well as in the

upper and lower poles of each testis using a 20 Gauge

needle with a 5 ml syringe attached to it. A constant ne-

gative pressure was applied to the syringe when the nee-

dle reached the center of the testis and aspiration was

done with gentle back and forth movements of the needle

at different angles in each puncture location. The aspi-

rated tissue from each location was placed on a separate

slide, air-dried, and stained with May-Grünwald Giemsa.

FNA was performed in 306 patients with NOA just as

previously described [14]. On the basis of FNA analysis,

the subjects were divided into three cytological groups:

1) SCOS: Complete absence of spermatogenic cells in

both testes; 2) Maturation arrest: Sperm maturation

stopped in every stage of spermatogenesis; 3) severe hy-

pospermatogenesis: The remarkable reduction in the

number of germ cells. In a few cases, sperms can be re-

trieved in grou ps 2) and 3 ).

2.4. Statistics

Statistical analysis was performed with the SPSS 17.

Mean values of hormones were compared with student-t

test. Two-sided P values less than 0.05 were regarded as

statistic significant. The two groups of patients with or

without sperm recovery were assessed by Receiver oper-

ating characteristic (ROC) analysis. A cut-off point for

each hormonal was found by ROC. The area under the

ROC curve, sensitivity and specificity were obtained from

each model. The area under the ROC curve is a measure

of the efficiency of the marker to predict sperm recovery.

3. RESULTS

Sperm extraction was successful in 67 and unsuccess-

ful in 239 cases. There were statistically signiﬁcant dif-

ferences between successful and unsuccessful sperm re-

covery groups in terms of mean serum inhibin B, FSH

and LH levels (Table 1). Serum inhibin B levels were

significantly higher in the group of the successful sperm

recovery (64.59 ± 26.05 pg/ml) than the group of sperm

recovery failure (25.88 ± 9.85 pg/ml) (P = 0.019). The

mean value of serum FSH in the group of patients with

sperm recovery was 11.41 ± 3.45 IU/L and in the group

of without sperm recovery was 19.38 ± 6.95 IU/L (P =

0.006). Serum LH in different groups were 4.40 ± 1.86

IU/L and 7.53 ± 2.72 IU/L, respectively (P = 0.000).

The predictive value of inhibin B, FSH, LH and three

of them were evaluated by ROC curve (Figure 1). The

combination ROC curve represented combination of in

hibin B, FSH and LH. As shown in Table 2, the areas

under the curve (AUC) were 0.696 for inhibin B, 0.729

for FSH, and 0.747 for LH. The sensitivities and speci-

ficities were 66.7% and 67.5% for inhibin B, 74.3% and

Table 1. Mean serum hormone concentrations according to the

outcome of FNA in men with non-obstructive azoospermia.

There was statistically signiﬁcant difference between successful

and unsuccessful sperm recovery groups in terms of mean se-

rum inhibin B, FSH and LH levels.

sperm recovery P

successful unsuccessful

inhibin B 64.59 ± 26.05 25.88 ± 9.85 0.019

FSH 11.41 ± 3.45 19.38 ± 6.95 0.006

LH 4.40 ± 1.86 7.53 ± 2.72 0.000

B. Wang, X. B. Huang / Advances in Reproductive Sciences 1 (2013) 1-5

Figure 1. Receiver operating characteristic (ROC) curve of inhibin B, FSH, LH and combination of inhibin B, FSH and LH

were evaluated. The areas under the curve (AUC) were 0.696 for inhibin B, 0.729 for FSH, 0.747 for LH and 0.832 for combi-

nation.

Table 2. Predictive value of inhibin B, FSH and LH for suc-

cessful sperm retrieval in men with non-obstructive azoosper-

mia. AUC: The area under the curve.

66.7% for FSH, and 66.7% and 67.5% for LH, respec-

tively. The cut-off points w ere 27.31 pg/ml f or inhib in B,

11.68 IU/L for FSH, and 4.04 IU/L for LH. When the

predictive value of the combination of the three was as-

sessed, we found that the area under the curve was 0.832

and the sensitivity and specificity were 93.30% and

66.30%.

AUC sensitivity specificity

inhibin B 0.696 66.70% 67.50%

FSH 0.729 74.30% 66.70%

LH 0.747 66.70% 67.50%

inhibin B + FSH

+ LH 0.832 93.30% 66.30%

4. DISCUSSION

Recovery of sperms for ICSI is a crucial treatment for

patients with NOA [2,15]. However, sperm retrieval tech-

niques such as TESE and FNA are invasive procedures

and associated with potential complications [16]. Finan-

cial, emotional stress and damage to testes due to unsuc-

cessful biopsy have stimulated many researchers to find

non-invasive methods to predict sperm recovery. In addi-

tion, a biopsy may not be representative of whole testis

[16,17].

these three parameters can strengthen the power of sperm

recovery predict in patients with NOA. Because different

views can be found in the literature, for example, Bal-

lesca JL et al concluded that inhibin B and FSH are use-

ful non-invasive predictors of sperm recovery and thus,

all azoospermic males should have serum inhibin B and

FSH concentrations measurement prior to undergoing

TESE [19]. Whereas, Tunc, L. et al. failed to conform

this finding [20]. Because inhibin B, FSH and LH are

closely related to spermatogenesis, we have good reason

to b elieve that those cases obtained sperms have relatively

higher inhibin B and lower FSH and LH. Compared to

Even though it has been proposed that inhibin B be a

marker of the functional state of the seminiferous epithe-

lium [18], and FSH and LH are necessary for the initia-

tion of spermatogenesis [11], the combined evaluation of

OPEN ACCESS

B. Wang, X. B. Huang / Advances in Reproductive Sciences 1 (2013) 1-5

the traditional open testicular biopsy, FNA is a quick,

low cost, less invasive and reliable procedure [21]. We

employed this method in 306 patients with NOA and had

67 cases of successful sperm recovery. Th e probability o f

sperm retrieval in our study was 21.89% that was lower

than other study (such as 26.4% in the study of Bettella,

A.). If we make a pre-operation evaluation of sperm re-

covery in terms of the combined parameters of inhibin B,

FSH and LH, the probability of sperm retrieval in FNA

may increase.

In our study, there was statistically signiﬁcant differ-

ence between successful and unsuccessful sperm recov-

ery groups in terms of mean serum inhibin B, FSH and

LH levels. But individual hormone is not reliable in pre-

diction of sperm recovery before invasive procedures. In

order to obtain more reliable prediction, ROC curve is

used to evaluate the results. We found that the area under

the curve of inhibin B, FSH and LH were 0.696, 0.729

and 0.747 respectively. When three of them were as-

sessed together, we found that the area under the curve

increased to 0.832. The predictive power was signifi-

cantly improved by the combination of inhibin B, FSH

and LH. The cut-off points were 27.31 pg/ml for inhibin

B, 11.68 IU/L for FSH, and 4.04 IU/L for LH. We noted

that in the successful sperm recovery group, there were

58 of 67 (86.5%) patients with a serum level of inhibin B

> 27.31 pg/mL, FSH < 11.68 IU/L, and LH < 4.40 IU/L.

While in the unsuccessful sperm recovery group, only 4

of 239 (1.67%) reached that level. The result can give a

clue to clinical counseling. When the patients have a

serum level of inhibin B > 27.31 pg/mL, FSH < 11.68

IU/L, and LH < 4.40 IU/L at the same time, the probabil-

ity of sperm recovery is quite high. However, in other

individuals with a serum level of inhibin B < 27.31

pg/mL, FSH > 11.68 IU/L, and LH > 4.40 IU/L are not

able to predict absence of sperm due to multifactor in-

fluence spermatogenesis in testes. So the cut-off points

of these three hormones may be set up as a powerful pre-

dictor for successful sperm retrieval for patients with

NOA. For patients whose values of inhibin B, FSH and

LH are over the cut-off points, if sperm retrieval failed,

we advise them to take open microsurgical biopsies.

5. CONCLUSION

In conclu sion, though inhibin B, FSH and LH ar e use-

ful predictors of sperm retrieval in patients with NOA,

the combined evaluation of them would be more power-

ful in the predicting of successful sperm recovery for

patients with NOA before decision making of FNA pro-

cedure.

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