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ISSN: 2047-2919 ANDROLOGY

ORIGINAL ARTICLE

Correspondence:
Ettore Caroppo, Asl Bari, PTA F Jaia, UO The seminiferous tubule caliber
Fisiopatologia della Riproduzione Umana e PMA,
Via de Amicis 15, 70014 Conversano (Ba), Italy. pattern as evaluated at high
E-mail: ecaroppo@teseo.it
magnification during
a
Presented at ESHRE 2018 – Barcelona, Spain, Oral
Presentation Session 05 ‘Emerging data in clinical microdissection testicular sperm
andrology’.
extraction predicts sperm retrieval
Keywords:
microTESE, non-obstructive azoospermia, in patients with non-obstructive
seminiferous tubule pattern, sperm retrieval, testis
histology azoospermiaa
Received: 14-Apr-2018
1
Revised: 14-Aug-2018 E. Caroppo , 2E. M. Colpi, 3G. Gazzano, 2L. Vaccalluzzo, 2,4E. Piatti, 1G.
Accepted: 16-Aug-2018
D’Amato and 2,4G. M. Colpi
1
doi: 10.1111/andr.12548 Department of Maternal and Child Health, Reproductive and IVF Unit, Asl Bari, Conversano, Italy,
2
Andrology and IVF Unit, Clinica San Carlo, Paderno Dugnano, Italy, 3Division of Anatomic
Pathology, Istituto Auxologico Italiano IRCCS, Milano, Italy, 4Andrology Unit, Procrea Institute,
Lugano, Switzerland

ABSTRACT
Background: microTESE proved to be the gold standard surgical approach for patients with non-obstructive azoospermia (NOA),
but sperm retrieval rates (SRRs) vary considerably among centers. Some authors compared their SRRs with the pattern of seminifer-
ous tubule caliber found at high magnification, but none provided diagnostic accuracy measures.
Objective: The present retrospective study sought to verify the diagnostic accuracy of the pattern of seminiferous tubule caliber in
predicting the sperm retrieval in NOA patients.
Materials and Methods: Data from 143 infertile NOA men undergoing unilateral (64) or bilateral (79) microTESE (222 testes) were ret-
rospectively evaluated. During microTESE, if present, dilated tubules (DTs) were retrieved, otherwise tubules with slightly larger caliber
(SDT) (924) than that of the surroundings were removed. When no DT or SDT were found, not dilated tubules (NDTs) were excised.
Results: Spermatozoa were retrieved in 95 of 222 testes (42.8%); sperm retrieval was successful in 90% of testes with DTs, in 47% of
those with SDTs, and only in 7% of those with NDTs (p < 0.0001). Stepwise binary logistic regression revealed that the combination
of seminiferous tubule pattern and testis histology was significantly predictive of SSR, being able to classify 86.8% of testes, with an
excellent diagnostic accuracy (AUC 0.93). The median number of spermatozoa retrieved was significantly higher in DTs compared
with SDTs and NDTs.
Discussion: The results of the present study provide reliable accuracy measures in support of the relationship between seminifer-
ous tubule caliber pattern and SSR in patients with non-obstructive azoospermia. We are proposing for the first time that spermato-
zoa may be retrieved even from slightly dilated tubules in about half of cases. The pattern of tubules retrieved, together with
histology, may represent an additional outcome measure of microTESE.
Conclusion: The pattern of seminiferous tubules together with testis histology predicts sperm retrieval with an excellent diagnostic
accuracy.

INTRODUCTION gold standard surgical approach to NOA patients, as it was

Retrieving testicular spermatozoa in patients with non-
obstructive azoospermia (NOA) is challenging, as seminiferous
tubules containing intact spermatogenesis may be present
only in focal areas of the testis. Among the surgical tech-
niques used to retrieve testicular spermatozoa, microdissec-
tion testicular sperm extraction (microTESE) proved to be the
1.5 times more likely to result in successful sperm retrieval
(SSR) as compared to conventional TESE (Bernie et al., 2015).
Its superior ability in providing viable spermatozoa for intra-
cytoplasmic sperm injection (ICSI) is because of the optical
magnification (15–249) of the testicular parenchyma that
allows the identification of dilated and sometimes opaque

8 Andrology, 2019, 7, 8–14 © 2018 American Society of Andrology and European Academy of Andrology

SEMINIFEROUS TUBULE PATTERN PREDICTS SPERM RETRIEVAL
tubules, which are presumed to contain mature germ cells
(Schlegel, 1999).
Although the microTESE technique has been introduced two
decades ago, still the reported sperm retrieval rates differ
between authors: Such a variability could be ascribed to several
factors, not the least being the surgeon’s skill and experience
(Ishikawa et al., 2010). Very recently, a letter to the Editor (Song,
2017) and then two Editorials (Jensen et al., 2017; Carrell &
Simoni, 2017) appearing in Andrology pointed out that the
results of microTESE could be affected by several confoundings,
including different tissue processing methods and the time,
the skill and effort dedicated to the identification of spermatozoa
in the testicular specimen, and patient characteristics, and
therefore may account for the broad range of reported sperm
retrieval rates, so that they asked for improved outcomes mea-
sures for microTESE success, in order to address the remaining
questions in using such a technique more effectively in patients
care.
Among the potential factors able to explain the outcome of
microTESE in individual NOA subjects, the evaluation of the
prevalent seminiferous tubule caliber pattern found at high
magnification has attracted the attention of few authors in the
past decade (Okada et al., 2002; Tsujimura et al., 2002; Amer
et al., 2008), but none provided an evaluation of its diagnostic
accuracy in predicting the sperm retrieval success in NOA
patients.
The present study sought to determine, by providing diagnos-
tic accuracy measures, whether the pattern of seminiferous
tubule caliber found at high magnification during microdissec-
tion testicular sperm extraction (microTESE) may represent a
reliable outcome measure of such a surgical procedure.
MATERIALS AND METHODS
Ethical approval
All procedures were carried out in accordance with the inter-
national and national guidelines and regulations on the diagno-
sis and treatment of non-obstructive azoospermia. No additional
diagnostic or therapeutically procedure was carried out for
research purposes.
This study has been approved by the Ethics Committee Ticino,
Switzerland (email dss-ce@ti.ch), protocol no. 2018-00213.
Inclusion criteria
All patients with NOA who underwent microTESE whose
tubule caliber pattern was described in the case history and
stored in electronic format were included in the study.
Exclusion criteria
Patients with NOA with an history of testicular torsion,
mumps, or bacterial orchitis were excluded from the study.
Subjects
We reviewed the clinical data of all infertile male patients with
non-obstructive azoospermia referring to the IVF unit from Jan-
uary 2015 through July 2017 to undergo microdissection testicu-
lar sperm extraction. During all microTESE procedures,
following our consolidated clinical procedure, we carefully
recorded the caliber pattern of all tubules retrieved and took
some pictures (about 20–409 patient) of the tubules themselves.
© 2018 American Society of Andrology and European Academy of Andrology
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Every patient’s tubule caliber pattern as observed at high magni-
fication was annotated in the patients’ case history and stored in
an Excel dataset; data collection and analysis were retrospec-
tively conducted.
The criteria for the diagnosis of NOA were similar to those
applied in a previous study from our group (Caroppo et al.,
2017). Briefly, patients were defined to have NOA if semen analy-
sis showed azoospermia without spermatozoa in the pellet, ejac-
ulate volume and pH were higher than 1 mL and 7.2,
respectively, and no sign of obstruction of the seminal tract was
detected on physical examination, scrotal, and transrectal
ultrasound.
All patients underwent a careful physical evaluation, and
blood samples were obtained to assay their FSH, LH, and total
testosterone (TT) levels. Testis volume was measured by scrotal
ultrasound. An additional semen sample was obtained on the
day of planned surgical procedure in order to confirm the
absence of spermatozoa in the pellet.
Patients who gave their informed consent to the surgical
procedure were included in the study.
Surgical procedure and sample processing
microTESE was performed under general anesthesia through a
transversal incision of the testis covering from two-thirds to
three-quarters of its circumference, according to a line preserv-
ing as much as possible the transversal subalbugineal vessels.
Intra- or subalbugineal vessel bleeding, if any, was cauterized
with a microsurgical bipolar thermal device (with 0.3 mm wide
tips). The testis was opened like a book by a gradual and very
gentle separation of the lobular tissue of both sides with a spat-
ula or a Vannas micro-forceps, in order to avoid tissue stretching
and to preserve the caliber of the underlying seminiferous
tubules. Next, the tissue was observed under the operating
microscope at 10–249 magnification in the search for areas con-
taining the tubules that appeared clearly dilated, that is, those
tubules with a diameter larger than all the surrounding ones
constituting most of the parenchyma (Fig. 1A). These tubules
can be found grouped in a tiny heap, sometimes lie solitary sur-
rounded by smaller size tubules, or more rarely occupy a small
part of a lobule. Dilated tubules (DTs) were removed and given
to the embryologists in the surgical theater for the immediate
identification of spermatozoa. If no normal, dilated, tubules
were identified, then any tubule whose caliber appeared slightly
larger than that of the surroundings (at 924 magnification) was
removed (slightly dilated tubules—SDTs; Fig. 1B). After examin-
ing the surface, if necessary, the deeper part of the testicular par-
enchyma was explored, and tubules were examined both along
the septa and by delicately detaching groups of them from the
adjacent ones by Vannas micro-forceps, again retrieving all
those areas with clearly dilated or only apparently dilated semi-
niferous tubules, according to the protocol described by Sch-
legel, (1999). When such a procedure did not yield spermatozoa,
and because of the volume of the testis, a moderate part of the
gland had remained unexplored, a second albugineal incision,
parallel to the preceding one, was performed. When no dilated
or slightly dilated tubules were found in the different areas of the
testis, not dilated tubules (NDTs) were excised according to a
sort of mapping, by removing tiny fragments of testicular tissue
from the two separated surfaces, at different depths from the
albuginea to the hilum (Fig. 1C). The surgical procedure lasted
Andrology, 2019, 7, 8–14 9

E. Caroppo et al.
Figure 1 Pattern of seminiferous tubules as observed at high magnification during microTESE (A) Dilated tubules (white arrow). (B) Slightly dilated tubules
(blue arrow) as compared to not dilated tubules (white arrow) at the highest magnification (924). (C) Uniformly not dilated tubules. [Colour figure can be
viewed at wileyonlinelibrary.com]
(A)
(B)
for 1.5–4 h, bilateral microTESE being more time-consuming
than unilateral.
The embryologists were blinded to the pattern of tubules
retrieved during microTESE. The removed tubules were
washed in Hepes-buffered medium to remove the blood. The
tiny fragments of testicular tissue were placed in sterile petri
dishes with 0.5 mL sperm washing medium and meticulously
minced using microscissors. The resulting fragments were
then passed through a intravenous angiocatheter for 3–5 min,
until a homogeneous pulverized suspension was obtained.
Small aliquots of the suspension were directly examined
under an optical microscope. The meticulous search for sper-
matozoa could last up to 4 h. Only afterward, the testicular
tissue surfaces were irrigated for antisepsis with Ringer solu-
tion (plus 80 mg gentamycin/100 mL). Hemostasis was then
performed with the patient’s blood pressure being normalized
by the anesthetist, by gently pressing the testicular tissue for
4 min using gauze wet with the antiseptic solution, and even-
tually (but rarely) using the microsurgical bipolar thermal
device. The albuginea incision was closed with a continuous
suture of Vicryl 5/0 or 4/0, involving only its external layer in
order to avoid any additional damage to the subalbugineal
vessels. The tunica vaginalis opening was repaired by a con-
tinuous Vicryl 4/0 after instillation into the vaginalis cavity of
1.5 mg betamethasone (a 2 mL ampoule) to prevent pain and
tunica vaginalis adhesions. Dartos and skin were closed by
separate stitches with Vicryl 3/0 suture.
If an appropriate sperm yield was obtained, dissection of the
contralateral testicle was avoided, otherwise dissection of the
contralateral side proceeded.
All surgical procedures were performed by the same urologist
(GMC), with about 900 microTESE surgical procedures per-
formed in the past twenty years. Processing of all testis
10 Andrology, 2019, 7, 8–14
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(A)
(C)
specimens was made by the same two embryologists with
17 years of experience in the field.
Testicular histology
All the histopathology specimens were evaluated by an unique
pathologist (GG) with about 17 years of experience in the field.
A fragment of one or more of the tubules of the same diameter
(DT, SDT, or NDT) found in each single testis was fixed in
Bouin’s solution and sent to the pathologist. Histological analy-
sis was conducted by examining at least 100 different tubule sec-
tions. The histopathological results were defined as: (i) Sertoli
cell-only syndrome (SCO) when the seminiferous tubules were
exclusively populated by Sertoli cells; (ii) focal SCO when tubules
with SCO were interspersed with rare tubule containing germ
cells; (iii) early and late maturation arrest (MA), characterized
with an arrest of the spermatogenetic maturation sequence at
spermatogonia/spermatocytes or round spermatid level respec-
tively; (iv) hypospermatogenesis (HYPO), when all stages of germ
cells (spermatogonia, spermatocytes, and spermatids) were pre-
sent although reduced in number; (v) hyalinosis when peritubu-
lar fibrosis with peritubular and intratubular hyalinosis were
found together with absence of Sertoli and germ cells; and (vi)
intraepithelial neoplasia (IN) when malignant germ cells were
identified within the seminiferous tubules.
Statistical analysis
The difference in serum hormonal level and testicular size in
patients stratified according to sperm retrieval success was eval-
uated by Mann–Whitney U-test, as the variables were not nor-
mally distributed according to one-sample Kolmogorov–
Smirnov test. The frequencies of histopathological patterns in
patients with and without SSR and per tubule pattern were com-
puted by chi-squared test. The distribution of tubule caliber
© 2018 American Society of Andrology and European Academy of Andrology
 ANDROLOGY

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SEMINIFEROUS TUBULE PATTERN PREDICTS SPERM RETRIEVAL

pattern in patients with and without SSR was computed by with 45X/46XXY mosaicism, and in all patients with transloca-
Kruskal–Wallis test. tions. Three patients had deletion of AZFc region and one of
The predictive ability of clinical parameters, tubule caliber AZFb and AZFc of the Y chromosome: Sperm retrieval was suc-
pattern, and histology on sperm retrieval success was evaluated cessful in all three patients with AZFc deletion following unilat-
by binary logistic regression with stepwise selection method, the eral microTESE, but not in the patient with AZFb and AZFc
entry testing being based on the significance of the statistic deletion despite bilateral microTESE.
score: SSR was set as binary-dependent variable, while tubule Spermatozoa were retrieved in 95 of 222 testes (sperm retrieval
pattern and testicular histology as categorical (nominal) vari- per testes 42.8%). For the purpose of this study, from now
ables utilizing the simple contrast coding, with ‘DT’ and ‘SCO’ as onward data will be reported as per testes rather than per
reference category, respectively. The obtained predictive proba- patients.
bilities were used as the test variable to obtain area under curve Table 2 displays the results as obtained in testes with or with-
(AUC) estimates derived from receiving operator characteristic out SSR. No difference was seen in testis volume, FSH and LH
(ROC) curve. serum levels between cases with SSR or sperm retrieval failure
The predictive ability of clinical parameters and histology on (SRF); individual AUC estimates confirmed that none of the
tubule caliber pattern was evaluated by binary logistic regression above parameters was predictive of SSR (0.48, 0.50, and 0.55 for
using two dummy binary-dependent variables (DT plus SDT or FSH, LH, and testis volume, respectively). On the other hand,
SDT plus NDT). The obtained predictive probabilities were used total testosterone serum level was significantly lower in testes
as the test variable to obtain AUC estimates. with SRF compared to those with SSR (p = 0.007), but its predic-
Statistical significance was set at p < 0.05 for all analyses. All tive value was poor (AUC 0.61).
computations were performed using IBM SPSS for WINDOWS Histopathological findings differed significantly among testes
(Chicago, IL, USA). with SSR or SRF (Pearson’s chi-squared test 54.170, p < 0.0001).
SCO and hyalinosis were the prevalent finding in testes with
Sample size estimation SRF, while focal SCO was found only in testes with SSR, and SSR
The sample size requested for binary logistic regression was was associated with HYPO in the 96.4% of cases.
calculated according to Peduzzi and coworkers (Peduzzi et al., The tubule caliber pattern differed significantly among testes
1996). Given that the proportion of positive cases (p) was 42.8% with or without SSR (Pearson’s chi-squared test 114.690,
(sperm retrieval per testes) and the number of independent vari- <0.0001; Table 2). About 90% of dilated tubules were found in
ables (k = testis histology and tubule seminiferous caliber pat- testes with SSR, while only 7% of non-dilated tubules contained
tern) was two, the minimum number of cases to include in this viable sperm cells. On the other hand, the percentage of slightly
study to obtain a statistical power of 80% with a set at 0.05 was dilated tubules were roughly comparable among testes with or
46.7 (N = 10 k/p). without SSR. The number of spermatozoa retrieved was signifi-
cantly higher when dilated tubules were retrieved compared
RESULTS with the other two caliber categories (p < 0.001; Table 2).
One hundred and forty-three male patients with non-obstruc- Binary logistic regression with stepwise selection for the
tive azoospermia were included in this study: 64 underwent uni- prediction of sperm retrieval excluded FSH, LH, testosterone,
lateral and 79 bilateral microTESE. Patients clinical, hormonal, and testicular volume, as they were not significant to the model.
and histopathological findings are displayed in Table 1. On the other hand, the combination of histology plus tubule cal-
Sperm retrieval was successful for 79 of 143 patients (55.2%), iber pattern was significantly predictive of SSR with a diagnostic
23 of 79 (29%) after bilateral, and 57 of 64 (89%) after unilateral accuracy of 86.8% (Table 3). The tubule caliber pattern, when
microTESE, respectively. Sperm retrieval was successful in one computed as the sole predictive factor, allowed the correct clas-
of six patients with non-mosaic Klinefelter syndrome, in patient sification of 82.4% of testes, while histology classified 72.7% of
them. Individually, AUC estimates as obtained from the com-
Table 1 Clinical parameters of 143 patients undergoing unilateral or bilat- puted predictive probabilities demonstrated that histology had a
eral microTESE fair diagnostic accuracy (0.7), while tubule caliber pattern accu-
Parameter Results racy was good (AUC 0.89); the combination of both variates had
an excellent discriminatory power (AUC 0.93). Table 4 displays
Age (years) 36 (33–39) [26–62] the combination between tubule caliber pattern, histology, and
Non-mosaic Klinefelter S (%) 6 (4)
45X/46XXY (%) 1 (0.7)
successful sperm retrieval.
Robertsonian translocations (%) 4 (2.8) Binary logistic regression for the prediction of tubule caliber
Testis volume (mL) 7.3 (6–9) [1.5–17] pattern was run with two dummy binary-dependent variables
FSH mIU/mL 21 (14.8–28) [1.47–68.7]
(dummy 1 = DT plus SDT and dummy 2 = SDT plus NT); the
LH mIU/mL 6.3 (4.4–10.8) [0.2–48]
Total testosterone (ng/dL) 390 (300–525) [134–890] dummy variable that performed better was the dummy 2. Step-
Testis histology 220 reports (per testis) wise selection excluded all variates but histology, which cor-
SCO (%) 143 (65) rected classified 75.9% of testes (Table 3), although with a poor
Focal SCO (%) 9 (4.1)
diagnostic accuracy (AUC 0.67).
MA 29 (13.1%)
HYPO 28 (12.8)
Hyalinosis 9 (4.1%) DISCUSSION
IN 2 (0.9) The results of the present study confirm that the seminiferous
Data are displayed as median and interquartile range with ranges in squared tubule caliber pattern as found at high magnification during
parentheses or as count with percentages in parentheses. microTESE is predictive of sperm retrieval in patients with NOA.

© 2018 American Society of Andrology and European Academy of Andrology Andrology, 2019, 7, 8–14 11
 ANDROLOGY

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E. Caroppo et al.

Table 2 Comparison of clinical, histological,

Successful sperm retrieval Sperm retrieval failure p and tubular caliber parameters in testes with or
without successful sperm retrieval
Patients age (years) 36 (33–40) [26–62] 35 (33–38) [26–46] 0.23
Testis volume (mL) 7.5 (6–9) [1.5–17] 7.1 (5.5–8.5) [1.6–17] 0.15
FSH mIU/mL 21 (16–28) [1.8–47.6] 20.8 (14.7–29.5) [1.47–68.7] 0.96
LH mIU/mL 6.9 (4.4–10) [0.2–27.4] 6.2 (4.4–10.9) [0.5–48] 0.944
Total testosterone 422 (350–550) [220–890] 380 (277–497) [134–860] 0.007
(ng/dL)
Testis histology
SCO (%) 45 (31.5%) 98 (68.5%) <0.0001a
Focal SCO (%) 9 (100) 0 (0)
MA (%) 11 (38) 18 (62)
HYPO (%) 27 (96.4) 1 (3.6)
Hyalinosis (%) 2 (22.2) 7 (77.8)
IN (%) 1 (50) 1 (50)
Tubule caliber pattern
Dilated (%) 63 (90) 7 (10) <0.0001b
Slightly 25 (47%) 28 (53%)
dilated (%)
Not dilated (%) 7 (7%) 92 (93%)
Sperm count per tubule caliber pattern
Dilated 50,000 (9000–300,000) [500–5.2 9 106] / <0.0001a
Slightly dilated 1000 (500–1000) [500–450,000] /
Not dilated 500 (500–500) [up to 500] /
tubules

Data are displayed as median and interquartile range with ranges in squared parentheses or as count with
percentages in parentheses. aPearson’s chi-squared test. bKruskal–Wallis test.

Table 3 Binary logistic regression analysis and

Model Chi-square p Cox and Snell Nagelkerke % cases Intercept AUC AUC estimates derived from ROC curve
R-square R-square correctly odds ratio
predicted

Prediction of sperm retrieval success

Histology 63.329 <0.0001 0.25 0.336 72.7 38.9 0.7
Tubule pattern 133.731 <0.0001 0.45 0.608 82.4 0.84 0.89
Tubule pattern 156.749 <0.0001 0.51 0.68 86.8 28.4 0.93
plus histology
Prediction of tubule caliber patterna
Histology 33.843 <0.0001 0.143 0.2 75.9 0.85 0.67
a
A dummy dependent binary variable was set incorporating SDT and NDT tubules.

Table 4 Combination of tubule pattern, histology, and successful sperm tubule caliber pattern had a good diagnostic accuracy in predict-
retrieval
ing SSR. Since the first report on microTESE procedure (Schlegel,
Histology Dilated Slightly dilated Not dilated Total 1999), it was suggested that the recovery of opaque and dilated
seminiferous tubules would result in an higher chance of obtain-
SCO (SSR) 32 (30) 33 (13) 78 (2) 143 (45)
Focal SCO (SSR) 7 (7) 2 (2) 0 9 (9)
ing viable spermatozoa for ICSI. Three years later, Okada and
MA (SSR) 7 (4) 10 (4) 12 (3) 29 (11) coworkers (Okada et al., 2002) evaluated 74 patients with NOA
HYPO (SSR) 20 (19) 6 (6) 2 (2) 28 (27) undergoing microTESE and found dilated tubules in 20 of 33
Hyalinosis (SSR) 3 (2) 2 (0) 4 (0) 9 (2)
patients with SSR (60%), while Tsujimura (Tsujimura et al.,
IN (SSR) 1 (1) 0 (0) 1 (0) 2 (1)
Total 70 (63) 53 (25) 97 (7) 220 (95)a 2002) evaluated 56 patients with NOA and found homogeneous
dilated tubules in seven, all of them with SSR (100%), 23 patients
SSR, successful sperm retrieval. aHistology was not available in two cases. with homogeneous non-dilated tubules, all with failed sperm
retrieval, and 26 with heterogeneous tubule thickness (the pat-
This finding, being already anticipated by previous studies (Sch- tern of seminiferous tubules retrieved was not specified), 17
legel, 1999; Okada et al., 2002; Tsujimura et al., 2002; Amer et al., (65%) of whom had SSR. Amer and colleagues (Amer et al., 2008)
2008), is now fully supported by the diagnostic accuracy mea- provided a measure of tubule caliber using a micrometer fixed to
sures provided by the present study. the eyepiece of the operating microscope. They reported that
We found that sperm retrieval was successful in 90% of cases sperm retrieval rate was significantly higher in cases with tubule
when dilated tubules were found at high magnification, but only diameter >150 lm (SSR 49.2% vs. 31.4% respectively), reaching
in 7% of cases when not dilated tubules were retrieved. The bin- 84.2% when tubule diameter exceeded 300 lm.
ary logistic regression analysis, as well as the resulting predictive Our data show that sperm retrieval was unsuccessful only in
probabilities used to obtain AUC estimates, confirmed that the seven cases when dilated tubules were retrieved, showing

12 Andrology, 2019, 7, 8–14 © 2018 American Society of Andrology and European Academy of Andrology

SEMINIFEROUS TUBULE PATTERN PREDICTS SPERM RETRIEVAL
heterogeneous histological characteristics (MA in three cases,
SCO in two cases, and HYPO and hyalinosis in the two remain-
ing cases). As shown by a morphometric study (Volkman et al.,
2011), sometimes highly dilated tubules (diameter >400 lm)
may be found in the testes of patients with NOA, showing
impaired spermatogenesis together with thickened lamina pro-
pria because of the increased extracellular matrix: The differen-
tial diagnosis between these tubules and those with intact
spermatogenesis could be hard even for an expert surgeon, espe-
cially when such tubules are found by chance in testes with
otherwise predominantly non-dilated tubules.
Notably, the present study suggests that it could be worthless
to retrieve not dilated tubules, as the chance of finding sperma-
tozoa is around 7%. On the other hand, in the absence of clearly
dilated tubules, the retrieval of tubules with a larger diameter
compared with that of the surroundings (NDT) may result in
SSR in about half of cases, according to our results. It has to be
specified, however, that the identification of such tubules, which
requires the highest optical magnification (924), can result
easier to skilled and experienced surgeons. Dabaja and Schlegel
(Dabaja & Schlegel, 2013) argued that the identification of
dilated tubules becomes easier, the operative time decreases,
and the SSR increases when a surgeon exceeds experience with
more than 500 microTESE procedures.
Our data suggest also that when testis histology is coupled to
the tubule pattern, SSR is predicted with an excellent diagnostic
accuracy (AUC 0.93). Histology has been previously found to be
the unique parameter able to predict the outcome of microTESE
(Abdel Raheem et al., 2013; Bernie et al., 2013; Althakafi et al.,
2017; Li et al., 2018), though with a poor/fair diagnostic accuracy
(Li et al., 2018). The only limit of testis histology is that it may be
available when conventional TESE or, in rare cases, testis biopsy
has been performed prior to microTESE, but this is not a rare
event for NOA patients.
On the other hand, histology showed a poor diagnostic accu-
racy in predicting the tubule caliber pattern. As displayed in
Table 4, SCO was more prevalently found in not dilated tubule,
while HYPO was associated with dilated tubules in 71.4% of
cases. Okada and coworkers found dilated and opaque seminif-
erous tubules in all cases with HYPO, but only in two of nine and
12 of 19 patients with MA and SCO, respectively (Okada et al.,
2002), while Tsujimura et al. found that all testes showing an
homogeneous pattern of dilated tubules had HYPO, while those
with homogeneous thin tubules had SCOS (95.6%) or MA (0.4%)
(Tsujimura et al., 2002). The discrepancy between these data
and ours may be explained by the study selection criteria: we, in
fact, included patients with different clinical conditions, includ-
ing those with known cytogenetic abnormalities. Data from mor-
phometric studies suggest that several clinical conditions
(varicocoele, cryptorchidism, Klinefelter syndrome) may have an
impact on tubule structure and size, regardless of testicular his-
tology (Sato et al., 2008).
Indeed, the predictive factors evaluated in the present study
are intraoperative (tubule pattern) and post-operative (testis his-
tology) and therefore require surgery to be obtained. Yet, pre-
operative clinical parameters are hardly predictive of microTESE
success. Our data confirm that the microTESE outcome cannot
be predicted on the basis of patients FSH, LH and testicular vol-
ume; relevantly, microTESE was found to be successful in
patients with very high serum FSH levels (Ramasamy et al.,
© 2018 American Society of Andrology and European Academy of Andrology
ANDROLOGY
20472927, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.12548 by Cochrane Romania, Wiley Online Library on [19/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2009) and/or severe testicular atrophy (Bryson et al., 2014). We
found that such parameters did not even predict the tubule pat-
tern (Table S1). In spite of the lack of pre-operative predictive
factors, we are providing an intraoperative predictor of micro-
TESE success that may assist the surgeon during the surgical
procedure: The knowledge that even slightly, apparent dilated
tubules may harbor spermatozoa in about half of cases would
compel him or her to extensively search for those tubules in
the case of failure to detect dilated tubules at high magnifica-
tion. We would highlight that the present study is providing
for the first time ever sperm retrieval rates in tubules which
are neither ‘dilated’ nor ‘not dilated’. In addition, we are sug-
gesting to add the seminiferous tubule caliber pattern to the
microTESE outcome report in order to provide the patients
(and the referred urologist/clinical andrologist as well) with
valuable information about the degree of their spermatogene-
sis impairment, given that the classical yes or not microTESE
report is poorly informative about that, being the chance of
retrieving spermatozoa not fully related to the spermatogene-
sis integrity, but also to the surgeon skill and experience, and
to the time spent in the laboratory in the identification of the
spermatozoa in the testicular specimens. Such a report would
help the counseling of patients referring for a redo microTESE:
If the previous surgical evaluation at high magnification, in
the hand of a skilled urologist, identified only not dilated
tubules, the patient would be counseled about the relatively
scarce chance of having his spermatozoa retrieved with
another microTESE attempt.
This study has some limitations. The tubule pattern was sub-
jectively evaluated. Najary and coworkers performed an experi-
mental pilot study aiming at evaluating the accuracy of
multiphoton microscopy (MM) in detecting tubules with intact
spermatogenesis, and found a concordance rate of 86% between
MM and histology, but acknowledged that MM needs to be
miniaturized and its safety has to be ensured before its intro-
duction in the clinical practice (Najari et al., 2012). In the lack
of such a potentially innovative diagnostic tool, the surgeon’s
skill and experience remains the only factor that can account
for the reliability of the results: In this light, the circumstance
that all surgical procedures were performed by the same highly
experienced urologist may represent a methodological
strength, as both the identification of tubule caliber and the
sperm retrieval rate could not have been affected by interopera-
tor variability. Another limitation of the study is that that data
were retrospectively collected and analyzed; however, data col-
lection was easy as all data were annotated in patients’ clinical
history and in a Excel dataset, following our consolidated clini-
cal procedure, and for the same reason, the risk of selection
bias was negligible.
In conclusion, the present study suggests that the seminifer-
ous tubule caliber pattern as observed at high magnification dur-
ing microTESE is strictly correlated with the chances of
retrieving spermatozoa in patients with NOA. We are providing
an intraoperative predictor of microTESE success and suggest to
add the tubule pattern and testis histology to the microTESE
outcome report in order to render it more informative to the
patient.
CONFLICT OF INTEREST
The authors have no conflict of interests to disclose.
Andrology, 2019, 7, 8–14 13

E. Caroppo et al.
FUNDING
No funds were needed for this study.
AUTHORS’ CONTRIBUTIONS
E. C. designed the study, analyzed and interpreted the data,
and drafted the article. E.M.C. contributed to the acquisition of
data and critically revised the manuscript. G.G. performed all the
testis histology evaluation, substantially contributed to the con-
ception and the design of the study, and critically revised the
manuscript. L.V. contributed to the acquisition of data, prepared
the figure, and critically revised the manuscript. E.P. contributed
to the acquisition of data and critically revised the manuscript.
G.D. critically revised the manuscript. G.M.C. conceived and
designed the study, performed all the surgical procedures, iden-
tified the seminiferous tubule pattern and collected the results,
contributed to the manuscript drafting, and critically revised the
manuscript.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Support-
ing Information section at the end of the article.
Table S1 Clinical parameters as stratified per tubules caliber pattern.
© 2018 American Society of Andrology and European Academy of Andrology