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Probability
to retrieve testicular spermatozoa in azoospermic patients
H.-J.
Glander1 , L.-C. Horn2, W. Dorschner3,
U. Paasch1, J. Kratzsch4
1Department
of Dermatology/Andrology Unit, 2Department of Pathology,
3Department
of Urology 4Department of Clinical Chemistry and Pathological
Biochemistry,
University of Leipzig, D-04103 Leipzig, Germany
Asian J Androl 2000 Sep; 2: 199-205
Keywords:
sperm
extraction; testicular histology;
azoospermia; male
infertility; follicle stimulating hormone
Abstract
Aim: The degree of probability to retrieve spermatozoa from testicular tissue for intracytoplasmic sperm injection into oocytes is of interest for counselling of infertility patients. We investigated the relation of sperm retrieval to clinical data and histological pattern in testicular biopsies from azoospermic patients. Methods: In 264 testicular biopsies from 142 azoospermic patients, the testicular tissue was shredded to separate the spermatozoa, histological semi-thin sections of which were then evaluated using Johnsen score. Results: The retrieval of spermatozoa correlated significantly (P<0.001) with the testicular volume (r=0.49), the FSH concentration (r=-0.66), the maximum score (r=0.85) and the mean Johnsen score (r=0.81). In the multivariate regression analysis the successful testicular sperm extraction showed the closest relationship to the maximum score. The testicular volume correlated significantly with the mean Johnsen score (r=0.64, P<0.001), and the basal serum FSH concentration mainly with the maximum score (r=-0.77; P<0.001). Patients with a history cryptorchidism showed a significantly lower Johnsen score compared to the patients who did not have any testicular disease in the past (3.72.4 vs. 5.92.5; P<0.01). Conclusion: In a limited range, the testicular volume and the FSH concentration in serum were related to the Johnsen score which correlated significantly with the sperm retrieval. The successful sperm retrieval can be expected in all azoospermic patients irrespective of the results of clinical examination. However, the probability of retrieval of spermatozoa decreased significantly in patients with a FSH level >18 U/L, testicular volume <5 mL, mean Johnsen score <5, and maximum Johnsen score <7.1 Introduction
Testicular sperm extraction (TESE) can provide spermatozoa for intracytoplasmic sperm injection into oocytes (ICSI) to achieve a pregnancy. TESE has been used to overcome infertility of azoospermic patients who could not be successfully treated by other methods[1,2]. Every azoospermic patient is entitled to surgical sperm search, but during counselling patients often ask for the probability to retrieve spermatozoa from testicular biopsy. The examination of prognostic indicators achieving this aim is therefore needed[3,4]. Mostly, the testicular volume, the serum FSH level and the histological pattern of testicular biopsy are used for prediction, but several studies on TESE applying various laboratory methods and clinical design lead to different opinions[3-11]. We investigated the probability to retrieve testicular spermatozoa dependent on the testicular volume, the serum FSH concentration, and the testicular histology evaluated by the Johnsen score[12-14] after processing by semithin cutting technique[15]. The examination was based on 264 testicular biopsies from 142 azoospermic patients. Carcinoma in situ(CIS) were screened by a monoclonal antibody to placental-alkaline phosphatase[16].2 Materials and methods
2.1
Patient group
The
patient group consisted of 142 azoospermic men, aged 24-62 (mean 33.1)
years (Table 1), attending
an ICSI programme using testicular spermatozoa. All patients suffered
from primary infertility for 1 and 9 years and failed to respond to routine
infertility treatment. Men with congenital bilateral absence of vas deference
(CBAVD) or patients who had undergone previous vasectomy were excluded
from the study. Thirty
two (22.5%) of the patients had a history of cryptorchidism, 13 patients
of testicular trauma, 8 of an inflammation of testis or/and epididymis,
and 24 on mumps virus infection. Karyotyping of the patients revealed
no chromosomal abnormalities except three patients: two with Klinefelter's
syndromes (47,XXY) and one with Robertsonian translocation (13q14q).
The testicular volume was
estimated with Prader's orchidometer[17]. One hundred and
twenty two patients were biopsied bilaterally. Unilateral testicular biopsy was
performed in 20 cases with atrophy of a testis, ablatio testis or if the
patient refused a bilateral biopsy. The open biopsy technique was performed
under general anaesthesia after detailed explanation to the patient and
obtaining a written and informed consent. The biopsy included a scrotal
exploration after a median raphe incision and examination of the testis
and epididymis with microscopic assistance using
a 10 magnification. An avascular region near the mid portion of the
testis was chosen for puncture to prevent injury to the testicular blood
supply. Measurements of serum concentrations of testosterone, LH and FSH
were performed by immunofluorescence
assays from Delfia assay system (Pharmacia, Freiburg, Germany). Azoospermia
was confirmed in at least two ejaculates by examination of a semen pellet
at 1000 magnification after centrifugation at 2300g.
Table
1. Patient group, 142 patients, 264 biopsies, means (range).
|
Age
(years) |
33.35.4(24-62) |
| Testicular
volume (mL) |
9.15.8(0.5-22) |
| FSH
(U/L) |
12.711.6(0.8-67.9) |
| LH
(U/L) |
5.33.6(0.9-24.3) |
| Testosterone
(nmol/L) |
14.25.1(6.7-32.5) |
2.2
Handling of testis biopsies
Testicular
specimen of about 200-300 mg was placed in a Petri-dish containing about
2 mL Ham's F-10 medium with
2% bovine serum albumin[18]. Dividing the specimen into
2 parts one part was shredded with microscopic glass slide and the supernatant
was checked for the presence of spermatozoa. The sperm extraction was
regarded as positive if at least a single vital spermatozoa could be seen
in 5 l of the culture
medium of bioptates (five tries). The vitality of the cells was evaluated
by mixing one droplet of the cell suspension with one droplet of eosin-Y
solution. Spermatozoa stained with the dye were considered dead. The other
part of the specimen was used for histological and immunohistological
evaluations, i.e.
for semithin and paraffin sections, respectively. The specimens were fixed in
5.5% glutaraldehyde and then postfixed in 1% OsO4 . After dehydration
the tissue was embedded in Epon 812[15]. Semithin sections
with a thickness of 1
m were stained with Toluidine as well as with hematoxylin/eosin and
evaluated under light microscope at 400 and 1000 magnification. The
germinal epithelium of at
least 50 tubules was assessed according to a modified Johnsen score[12-14]
(Table 2). Moreover, the tubular diameter, the structure of lamina
propria, the interstitial tissue, especially the Leydig cells were evaluated.
Carcinoma in situ (CIS) were screened by enhanced polymer one-step staining[19,20]
using a monoclonal antibody to placental-alkaline phosphatase (16; DAKO,
Hamburg, Germany; antibody code no. M 7191, clone 8A9) and the DAKO EnVisionTM
detection system (code-no. K4016) after deparaffinization. Isotype controls
were performed using mouse IgG1 antibodies besides the controls substituting
the primary antibody by buffer. The positive controls were based on sections
of human placenta and testis with
known CIS, which is shown in
Figure 1.
Table
2. Johnsen score (12-14).
|
10 |
full
spermatogenesis |
|
9 |
many
late spermatids, disorganized tubular epithelium |
|
8 |
few
late spermatids |
|
7 |
no
late spermatids, many early spermatids |
|
6 |
no
late spermatids, few early spermatids, arrest of spermatogenesis
at the spermatid stage, disturbance of spermatid differentiation |
|
5 |
no
spermatids, many spermatocytes |
|
4 |
no
spermatids, few spermatocytes, arrest of spermatogenesis at the
primary spermatocyte stage |
|
3 |
spermatogonia
only |
|
2 |
no
germ cells, Sertoli cells only |
|
1 |
no
seminiferous epithelial cells, tubular sclerosis |
Figure
1. Positive control of immunohistochemistry. Labelling of a carcinoma
in situ using monoclonal antibody to placental-alkaline phosphatase (Dako,
Hamburg, Germany;
antibody code no. M 7191, clone 8A9) as primary antibody and the Dako
EnVisionTM detection system (code-no. K4016).
Figure 2. Focal
spermatogenesis in a Sertoli cell-only pattern.
2.3
Statistical analysis
3
Results
Retrieval
of spermatozoa was successful in 52.3% of the biopsies, which showed a
Figure 3. The frequency (%) of bioptates with retrieval of spermatozoa (A) within the FSH concentration group, (B) the testicular volume group, (C) the group of a defined mean Johnsen score, and (D) in dependence on the maximum Johnsen score; the numbers in the figure mean positive samples per total within the group.
Furthermore,
the basal FSH concentration and the testicular volume correlated significantly
with the mean as well as the maximum Johnsen score ( P<0.001;
Figure 4 and
5, Table 3).
The mean Johnsen score decreased from 6.91.9 to 1.90.7 if the FSH
concentration increased from normal to 3 times normal (Figure
4). The mean Johnsen-Score of
the biopsies of 139 right testicles amounted to 5.12.6, and of the 125
left testicles, 5.22.6. Twenty six (21.3%) of the 122 patients biopsied
bilaterally showed a difference of more than 1 score between the 2 testicles
(range 1-7). Within the biopsies a wide intertubular variation of the
Johnsen scores was obvious (Table 4) including focal spermatogenesis
in Sertoli cell-only patterns (Figure
3). About half of the specimen showed more than 20% of the mean as
standard deviation. Testes after a mal-descensus were characterised by
a signif
Table
3. Correlation coefficients of the mean and maximum score with the basal
concentration of FSH, testicular volume and sperm retrieval (P<0.001;
Spearman's rank correlation test).
|
|
Mean |
Maximum |
retrieval
of |
|
FSH
(U/L) |
-0.72 |
-0.76 |
-0.66 |
|
Testicular
volume (mL) |
0.64 |
0.55 |
0.49 |
|
Retrieval
of |
0.81 |
0.85 |
|
Table
4. Intertubular variations of the Johnsen score (14) within a specimen.
|
Standard
deviation, expressed |
n
(%) |
| 10 |
39
(14.8) |
| >1020 |
99
(37. 5) |
| >20
40 |
94(35.6) |
| >40 |
32
(12.1) |
| Total |
264
(100) |
Figure
4. Correlation of mean Johnsen score with FSH level in serum (Spearman's
test).
Figure 5. Correlation
of mean Johnsen score with testicular volume (Spearman's test).
4
Discussion
On
examining 264 testicular biopsies from 142 azoospermic men, we estimated
the degree of probability for spermatozoa retrieval to be dependent on
the serum FSH level,
the testicular volume, and the mean as well as the maximum spermatogenic score.
In a principle, successful sperm retrieval can be expected in all azoospermic
patients irrespective of the results of clinical examination. However,
the probability of sperm retrieval depended on the parameters examined.
In two patients sperm retrieval was also successful where the FSH concentration
was more than three times the normal value and the testicular volume less
than 2 mL. Thus, only the probability or rate of successful sperm retrieval could
be predicted but not the sperm retrieval result of an individual subject. Our
results do not represent a standard for the selection of the azoospermic
patients to do testicular sperm extraction (TESE) for the ICSI procedures,
but they may be helpful for the pre-operative counselling to the couples.
The
values of the FSH concentration, testicular volume and morphology to predict the
result of TESE are controversial[21,22] and uncertainties exist
concerning the probability
in a group with defined parameters. The inverse correlation between spermatogenesis
and serum FSH level is a well-known phenomenon[23]. However,
with the serum FSH in a given individual, it may not be reliable to predict
the presence of functional spermatozoa in the testis[24] or
the sperm retrieval succesfulness[4,25]. In men with complete
maturation arrest[13,26] or
focal Sertoli cell only syndrome[24],
normal FSH levels may be found. On the other hand, testicular spermatozoa
could be retrieved in men with 3 times elevated FSH level[9].
Therefore, the serum inhibin B level in combination with the FSH concentration
was considered to be a more sensitive marker for impaired spermatogenesis
than FSH alone[4]. However, even this combination could not
reliably predict the presence of sperm in testicular samples. Several
studies have shown that the FSH
is not always a reliable criterion for identifying patients with unsuccessful
sperm extraction from testicular sample[4,5,7]. Ostad et
al[26] were
not able to determine a statistically significant difference for serum
FSH levels between cases with sperm retrieval success and failure. In
contrast, a statistically significant trend occurred with the increase
in plasma FSH and failed attempts in sperm retrieval[5,22].
We confirmed this relationship by finding a decreased probability of sperm
retrieval with increased FSH levels but a precise prediction of TESE result
for an individual patient was not possible.
Testicular
volume was shown to be closely related to testicular sperm production and
sperm retrieval[5,22], but there are papers denying this relationship[3,9].
Our findings support the former results: the probability of retrieving
spermatozoa in testes increased significantly with testicular volume >5
mL.
Contrary
results were also presented concerning the correlation between testicular
biopsy evaluations and the success rate of sperm retrieval. The results
of Tournaye et al[10].
Who found high sperm retrieval rates for all histological categories,
could not be confirmed by others[5,7]. A significantly decreased
success of sperm retrieval were described from hypospermatogenesis to
pure Sertoli-cell-only pattern[5]. Moreover, the spermatogenesis
classified by Johnsen score
was positively correlated to sperm retrieval[7]. The visualization
of spermatids on testicular
histology showed a strong association with successful TESE[5].
In our patient group the identification of spermatozoa in the semi-thin
sections and in the wet\|preparations of biopsies showed a conformity
of 76% similar to the result of Schulze et al[21].
In our multivariate regression analysis the maximum Johnsen score showed
the closest relationship to sperm retrieval. Thus, the most advanced score
of spermatogenesis rather than the predominant pattern determines the
outcome of TESE, because the retrieval of
spermatozoa depends on the existence of sperm in only one area[3].
Multiple
biopsies have been recommended[6,25] since spermatogenesis
may be focal in non-obstructive azoospermia[3]. We preferred
single testicle biopsies to have comparable amounts of tissue samples
independent of testicular volume. Furthermore, small testicular volumes
are inadequate for multiple biopsies and even multiple biopsies may be
unable to find focal spermatogenesis. We performed open biopsies which
enables the identification of spermatogenically active regions of the
testicle by direct examination of the individual seminiferous tubules
[27]. Frequently, bilateral biopsies may reveal different histological
results[7,25]. More than a fifth of our patients biopsied bilaterally
differed between the two testicles by more than 1 score. Other authors
indicated a difference between the left and right testes in more than
30% of the patients[21].
Patients
with a cryptorchidism in the history showed a significantly lower Johnsen-score
compared with the patients who did not have any testicular disease in
the past (3.72.4 vs 5.92.5; P<0.01). Furthermore, it is
noteworthy that in our
patient group there was a prevalence of cryptorchidism (23.1%), which
was significantly higher than that of 1-3% as reported for the general
population[28]. These facts underline the negative effects
of cryptorchidism on subsequent fertility, probably, by germ cell loss
through apoptotic pathways[29].
In principle, every azoospermic patient, independent of classification[30], is entitled to surgical sperm search, but the probability of successful testicular sperm retrieval may relate to the testicular volume, the FSH level in serum, and the mean as well as the maximum Johnsen score of testicular biopsy. In counselling, a rough likelihood for a successful TESE can be estimated but a sure identification of patients with failing testicular sperm extraction is very unlikely due to a considerable overlap in values of the variables.
AcknowledgementThe authors gratefully acknowledge Mrs. G. Kersten for her excellent technical assistance and Dr. J. Kleine-Tebbe for editorial assistance.
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Correspondence
to: Dr.
H.-J. Glander, Department of Dermatology/Andrology Unit, University
of Leipzig, Liebigstrasse 21, D-04103 Leipzig, Germany.
Tel: +49-341-9718 640, Fax: +49-341-9718 649
Received
2000-05-31 Accepted 2000-07-21
