AND IDENTIFICATION OF SPERMATOZOA AND SPERMATIDS IN THE EJACULATE OF NON-OBSTRUCTIVE
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ODIVAL TIMM JR,
AGNALDO P. CEDENHO, DEBORAH M. SPAINE, MARCIA H.P. BUTTIGNOL, RENATO FRAIETTA,
VALDEMAR ORTIZ, MIGUEL SROUGI
of Human Reproduction, Division of Urology, Paulista School of Medicine,
University of São Paulo, UNIFESP, São Paulo, Brazil
To search and to identify spermatozoa and spermatids, present in the ejaculate
of non-obstructive azoospermic patients.
Materials and Methods: 27 patients, aged
between 18 and 48 years, with initial diagnosis compatible with non-obstructive
azoospermia, underwent up to 3 seminal samples, with assessment of macroscopic
and microscopic parameters differentiated for each sample. In the first
sample, 5 mL of semen were analyzed in a Horwell chamber in order to assess
the presence or absence of spermatozoa. The procedure was repeated with
2 other aliquots. In the absence of spermatozoa, the entire sample was
transferred to a conic tube and following centrifugation the sediment
was freshly analyzed. The second seminal sample was collected only when
no spermatozoa were found in the first sample and the research was performed
in the same way. In cases where spermatozoa were not seen, the sample
was centrifuged and the obtained sediment was stained by the panoptic
method and observed under common light microscopy (1250X). The third seminal
sample was collected only in cases when patients had not shown spermatozoa
in the first and second seminal samples.
Results: 4/27 (14.8%) patients presented
spermatozoa in the first seminal sample and 6/23 (26.1%), in the second
seminal sample. No spermatozoa were seen in the third sample, however,
11/17 (64.7%) presented spermatids.
Conclusion: In clinical situations where
the initial diagnosis is non-obstructive azoospermia, one single routine
seminal analysis is not enough to confirm this diagnosis and the analysis
of the centrifuged sediment can have relevant clinical consequences. Among
patients considered non-obstructive azoospermic, when duly assessed, 37%
presented spermatozoa and 64.7%, spermatids.
words: male infertility; azoospermia; sperm capacitation; spermatozoa;
Int Braz J Urol. 2005; 31: 42-48
male factor in conjugal infertility is expressive and, thus, must receive
close attention from all health professionals who develop activities in
the human reproduction field. Alone it is responsible for 30% of causes
of conjugal infertility and associated with the female factor in more
than 20%, the male component has been targeted in many studies and paradigm
For a long time, the search and identification
of germ cells in their various developmental stages in ejaculated semen
have received little or no attention. Reasons for this fact are numerous
and we can mention that in fresh exam of ejaculated semen, the identification
of such cells is not an easy process and demands great expertise of the
examiner, is time-consuming and, above all, until recently it did not
change prognosis in terms of treatment. For all these reasons, these cells
often appear in reports under the generic term of round cells, with no
other connotation. The fresh analysis of such cells does not allow accurately
distinguishing round spermatids from leukocytes neither from other germ
cells that can appear in the semen. However, with the introduction and
increasing indications intracytoplasmic sperm injection in human oocytes,
the search of germ cells in the ejaculate, especially in non-obstructive
azoospermic patients, has gained major importance. Firstly, the presence
of such cells can allow the injection of this haploid material in oocytes
from this patient’s partner in an in vitro fertilization program,
which has already enabled the birth of normal children. The first term
pregnancy achieved with the use of round spermatids occurred in 1995 (2,3).
Secondly, with the technical advance that we have witnessed in the last
few years, the identification and isolation of such cells could enable
in-vitro culturing, propelling them to more developed stages, that is,
round spermatids becoming elongated spermatids with more resilient DNA
bands and less subjected to fragmentation during chromosomal pairing (4).
In the near future, the characterization of these cells in the ejaculate
can allow the use of genic therapy for correcting eventual defects in
When considered, these new perspectives
warrant the current concern in exhaustively search for the presence of
spermatozoa or young elements of the germ lineage, in the ejaculate of
non-obstructive azoospermic patients. This work aims to search and identify
spermatozoa and spermatids in the ejaculate of patients classified as
study was prospectively performed in the period from January to December
2002 and was approved by the institutional Research Ethics Committee.
Twenty-seven patients aged between 18 and
48 years, with initial diagnosis compatible with non-obstructive azoospermia,
were duly enrolled and attended. The inclusion criteria for this study
were patient classified as non-obstructive azoospermic, with no relevant
antecedents concerning the reproductive system, and the exclusion criterion
was non-obstructive azoospermic patient with leukocytospermia (> 1.0
x 106 neutrophils/mL) (5).
Testicular volume was measured using the
Prader orchidometer. Hormonal assessment was performed through dosing
of follicle stimulating hormone (FSH), in serum, by the immunometric technique
(Immulite, DPC, USA) (normal = 0.7 to 11.1 mIU/mL).
All patients participating in this research
underwent up to 3 seminal analyses (Figure-1). In an area annex to the
laboratory, patients collected semen samples through masturbation, in
one-way sterile propylene vials (Pleion, Brazil), from a batch that had
been previously tested for plastic toxicity to spermatic motility. Samples
were collected following a 2- to 3-day period of absence of ejaculation,
with a one-week interval between collections.
The macroscopic assessment, identical for
all 3 seminal samples, was performed according to the criteria in the
manual of World Health Organization (5). The following parameters were
assessed: coagulation, liquefaction time, color, aspect, volume, viscosity
The microscopic assessment was performed
differently for each collected sample:
1) First seminal sample: after assessing
the macroscopic parameters, the sample was homogenized by manually agitating
the collecting vial and, with the aid of an automatic pipette (MLA, USA)
a 5-µL drop of semen was placed in the center of a Horwell counting
chamber (Arnold R. Horwell Limited, London). The drop was covered by a
glass coverslip and observed under a common light microscope (Nikon, model
Eclipse 200, Japan), at 400X magnification, in order to verify the presence
of spermatozoa and round cells, as well as to assess spermatic motility.
Similarly, this procedure was repeated with 2 additional 5-µL aliquots
of semen. In cases where spermatozoa were found, the spermatic concentration
was determined through volumetric dilution associated with hematocytometry,
using an optimized Neubauer chamber. Values were expressed in millions
per milliliter (5). In cases where no spermatozoa were seen on the Horwell
counting chamber, the entire seminal sample was transferred to a 15-mL
graded conic tube (Corning, reference 430791, USA). The tube was centrifuged
at 1200 rpm for 30 minutes. The supernatant was discarded, maintaining
only 0.02 mL of sediment in the tube that was observed between glass slide
and coverslip with a common light microscope, at 400X magnification, in
order to verify presence or absence of spermatozoa.
2) Second seminal sample: using the Horwell
chamber, the search for spermatozoa was performed similarly to the first
seminal sample. In cases where spermatozoa were not seen in the Horwell
chamber in the second sample, the entire seminal material was transferred
to a 15-mL graded conic tube (Corning, reference 430791, USA). The sample
was centrifuged at 1200 rpm for 30 minutes. The supernatant was discarded,
maintaining only 0.02 mL of sediment in the tube, with the addition of
0.5 mL of Biggers, Whitten and Whittingham culture medium (BWW medium).
A new centrifugation was performed with identical velocity and time. The
supernatant was discarded and the sediment was used for preparing a smear
on a clean and labeled glass slide. The slide containing the smear was
left to dry at room temperature and, subsequently, was placed in an air
stove (Fanem, model 002CB, Brazil) at temperature of 36.5ºC for 6
hours, so that complete fixation of the material would take place. Each
slide was stained by the panoptic method, and the observation of spermatozoa
and germ epithelial cells was performed through common light microscopy
(Nikon, Microphot FXA, Japan) with 1250X magnification, under immersion
3) Third seminal sample: spermatozoa were
initially searched in 3 5-µL drops of semen, similarly to the procedure
used for the first seminal sample. In this third sample, when no spermatozoa
were observed on the Horwell chamber, the entire seminal material was
transferred to a 15-mL grade conic tube (Corning, reference 430791, USA).
This material underwent centrifugation at 1200 rpm for 30 minutes. The
supernatant was discarded, the sediment was re-suspended in 1 mL of BWW
culture medium and transferred to a 15-mL graded conic tube, containing
discontinuous Percoll® density gradient (Amershan, Pharmacia Biotech,
reference 17-0891-01, Uppsala, Sweden) with 3 layers (45%, 70% and 90%),
with 1.5 mL of 45% layer, 1 mL of 70%, and 1 mL of 90%. The tube was centrifuged
again at 1200 rpm for 30 minutes. Each layer was aspirated separately,
laid in a graded conic tube, containing 5 mL of culture medium (BWW) and
centrifuged at 1200 rpm for 10 minutes. Following this period, the supernatant
was discarded, maintaining 10 µL of sediment in the tube, which
were used for preparing a smear in a clean and labeled slide. The slides
containing the smears from each layer were left to dry at room temperature
and were subsequently placed in an air stove (Fanem, 002CB, Brazil), at
36.5ºC for 6 hours, so that fixation of the material could occur.
Each slide was stained by the panoptic method. The observation of spermatozoa
and germ epithelial cells was performed through common light microscopy
(Nikon, Microphot FXA, Japan) with 1250X magnification under immersion
clinical and laboratorial data of the patients enrolled in this study
are presented in Table-1.
After collection and analysis of the 1st
seminal sample, of the 27 patients who participated in this study with
presumed diagnosis of non-obstructive azoospermia, 3 presented spermatozoa
during observation in the Horwell chamber (mean spermatozoa of 5,000/mL)
and another patient presented spermatozoa on the fresh analysis of the
seminal material following centrifugation. Thus, 4/27 (14.8%) patients
presented spermatozoa on the analysis of the 1st semen sample performed
at the laboratory.
The remaining 23 patients were referred
for collection and analysis of the 2nd seminal sample according to the
protocol established in this research. While no spermatozoon was identified
with the Horwell chamber, 6 patients evidenced spermatozoa on the analysis
of the stained sediment, totalizing 26.1% of patients with spermatozoa
on the analysis of the 2nd seminal sample.
The remaining 17 patients underwent collection
of the 3rd seminal sample. In these patients, no spermatozoon was detected
with the Horwell chamber, neither in fresh sediment, in the sediment stained
by panoptic method or in the separated and stained Percoll fractions.
However, fractions stained by panoptic method revealed that 11/17 (64.7%)
patients presented young elements of the germ lineage, more specifically,
spermatids. Data are summarized in Table-2.
patient with no relevant clinical antecedents, no abnormalities on the
genital physical examination, spermiogram with volume equal or superior
to 2 mL and absence of spermatozoa could be considered as having non-obstructive
azoospermia (5). However, if this patient undergoes collection and analysis
of a second seminal sample and some spermatozoa are detected, how should
this patient be considered? As someone with severe oligozoospermia and
not non-obstructive azoospermia anymore? Having virtual azoospermia, as
proposed by Tournaye et al. (7), in contrast with absolute azoospermia,
when no spermatozoon is found in the ejaculate or in the post-centrifuged
sediment. This condition should be considered as cryptozoospermia or intermittent
azoospermia as other authors advocate (8). Could it be only a categorization
issue, a question of semantics, or simply a motive for debates among experts?
The slightest such difference could be, would it bring relevant clinical
Considering the history and progress achieved
in the field of reproductive medicine during the 90s, we must differentiate
2 periods: before and after 1992, when the technique of intracytoplasmic
sperm injection (ICSI) was clinically applied for the first time, leading
to the birth of the first child generated by this kind of biotechnology
(9). Until 1991, under a clinical, therapeutic and prognostic perspective,
the discussion between non-obstructive azoospermia and severe oligozoospermia
was meaningless, and this difference had more academic than clinical interest.
On the other hand, with the advent of ICSI, this difference gains a new
dimension, because non-obstructive azoospermic patients could not be genetic
fathers, while patients with severe oligozoospermia could constitute a
family with their own genes (10).
Due to this dramatic change, the concept
of azoospermia itself, as issued by the manual of the World Health Organization,
should be reconsidered. When should we consider a patient as azoospermic?
Searching for spermatozoa in a single seminal sample or in several samples?
Should we always consider fresh research following centrifugation? Which
time and centrifuge force applied to the semen contained in the tube would
reassure us about the final result? Would the laboratories be prepared
for and warned about these differences and their consequences? Additionally,
would the patients and physicians themselves be prepared? All this serves
to promote greater reflection about a spermiogram report showing previous
azoospermia and seminal volume over 2 mL.
How can we be sure if a patient with non-obstructive
azoospermia has or not spermatozoa in the ejaculate or within his testes?
We have recently learned, through several published works, that the patient’s
age, testicular volume and serum FSH level are not reliable parameters
for predicting, in last instance, what happens inside the testis (11,12).
Apparently, the most reliable parameter
is the histopathological examination of testicular fragments obtained
from testicular biopsy (13). Despite having higher predictive value, it
would constrain the patient to undergo, in practical terms, at least 2
biopsies, one diagnostic and another therapeutic, with all risks and inconveniences
derived from these procedures (14).
Many investigators have searched the answer
through a more attentive analysis of germ elements present in the ejaculate,
thus considering not only the presence of spermatozoa, but immature elements
of the germ lineage as well (15,16). Through an exhaustive research in
the seminal fluid, the investigator tries to detect any haploid cell that
could better reflect what is happening inside the seminiferous tubules.
In this work, we addressed these 2 aspects, that is, the presence of spermatozoa
or young cells (spermatids) in the ejaculate of patients considered as
having non-obstructive azoospermia, with prognostic purposes, considering
the ICSI technique.
It is worth to stress that all 27 patients
enrolled in this study had been initially diagnosed with non-obstructive
azoospermia, as confirmed by a spermiogram performed at other centers.
The first seminal analysis performed at our laboratory revealed already
4 patients with spermatozoa. In the second sample, other 6 patients were
added, revealing that 10/27 (37%) could no longer be considered azoospermic
strictly speaking. On the other hand, among the 17 patients who underwent
the third sample, 11 did not shoe spermatozoa, but had spermatids. The
importance of finding such cells has not been completely established yet.
However, since the maturation stop is considered infrequent during this
stage, it allow us to assume that some areas inside the testes can present
further differentiated elements, such as elongated spermatids, or even
islets containing spermatozoa (14,17). Even with experimental character,
spermatids might be injected into oocytes, producing pre-embryos and pregnancy,
as the literature has already shown (4).
The present data allow us to conclude that
one routine seminal analysis is not enough to establish the diagnosis
of non-obstructive azoospermia. Additionally, in clinical situations where
the initial diagnosis is non-obstructive azoospermia, the analysis of
the centrifuged sediment can have relevant clinical consequences. In this
study, the majority of patients with initial diagnosis of non-obstructive
azoospermia presented at least round spermatids in the ejaculate (64.7%).
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Received: September 12, 2004
Accepted after revision: January 4, 2005
Dr. Deborah M. Spaine
Rua Leandro Dupret, 204 / 43
04025-010, São Paulo, SP, Brazil
Fax: + 55 11 5573-0014